MXPA99004550A - Methods for preventing graft rejection in transplantation and for producing a universal gene therapy host cell using lymphocyte activation (lag-3) - Google Patents

Abstract

A method to prevent graft rejection of transplanted cells, tissues or organs without general immunosuppression is described. The method employs a newly discovered protein, LAG-3. When allogeneic or xenogeneic cells are engineered to express LAG-3 on their surface and transplanted, immune destruction of the implanted cell, tissue or organ is prevented, while the host's immune system remains functional. A particular application of this method allows the preparation of a universal gene therapy host cell expressing LAG-3 on its surface for protection from graft rejection by a host's immune system.

Description

METHODS TO AVOID THE REJECTION OF GRAFTING IN TRANSPLANT AND TO PRODUCE A GUEST CELL OF UNIVERSAL GEN THERAPY USING LYMPHOCYTE ACTIVATION (LAG-3) Field of Invention The present invention relates to methods for preventing graft rejection of transplanted organs, tissues or cells, in particular to such methods comprising engineering a cell type to express a LAG-3 protein when transplanted into a host. More particularly, the invention relates to the production of a universal gene therapy host cell that expresses the LAG-3 protein on its supe rf i cie Description of the Prior Art The lymphocyte activation gene (LAG-3) is a member of the immunoglobulin superfamily ie it is selectively transcribed in human activated T cells (both CD4 + and CD8 +) and NK cells (Triebel et al, 1992 ). Sequence data, comparative exon / intron organization, and chromosomal localization revealed that LAG-3 is closely related to CD4 (Baixeras et al, 1992). A close relationship between LAG-3 and CD4 was further strengthened through that they both share the same ligand, ie MHC class II molecules (Baixeras et al, 1992). However, in contrast to CD4, LAG-3 does not bind the gp 120 human immunodeficiency virus (Baixeras et al, 1992). In addition, the expression of LAG-3 was not found in primary lymphoid organs such as a vessel, lifoid tissue associated with mucosa or normal lymph nodes. However, it is easily detected in inflamed tonsils, or nodes of follicular hyperplasia, supporting the view that even though LAG-3 is expressed after activation (Huard et al, 1994A). The antigen-specific stimulation of T clones in the presence of monoclonal anti-LAG-3 antibody (mAb) led to the incorporation of increased thymidine, to the highest expression of the CD25 activation marker and to an improved cytokine production (Huard et al. , 1994B) Therefore, the sight of a soluble recombinant form of LAG-3 inhibited T-cell proliferation specifying the antigen, suggesting a role of regulation of LAG-3 in the activation of CD4 + T lymphocytes (Huard, 1996) and its implication to quell immune responses that arise. "J Recently, it has been shown that LAG-3 also acts as a co-receptor for NK cells and defines different modes of tumor cell annihilation controlled through the innate immune system (Miyazaki et al. al., 1996) The mechanics through which a T cell response to a foreign protein (allogenic or xenogenic) or cell or organ is prepared, are clearly understood. Antigen presenting cells (APCs) are attracted to areas of inflammation or damage (which can be induced through surgical transplantation) the T cell repertoire in the periphery is constantly monitoring tissues for the evidence of pathogens or the presence of tissues strangers (allo- or xenogenic). Once any of these warning signals is recognized, the APCs absorb the protein, digest it and present it to an immune host system. Allogenic or syngeneic tumor cells have been engineered to express viral IL-10, which induces local anergy to tumors. Such treatment did not affect the rejection of a non-translucent tumor at a distant site (Suzuki et al., 1995). Locally supplied IL-10 is thought to displace the repertoire of reactive T cells to t r ansp 1 cells against the Th2 phenotype that is not cytolytic and may even be protective. The cells that naturally express the Fas ligand have been tested using allogeneic or exogenous barriers without immunosuppression. Monitoring the implant site through host T cells results in its annihilation when contacted with the Fas ligand (Bellgrau et al., 1995). In addition, the rejection of pancreatic islet allografts has been avoided by the co-response of syngenic myoblasts genetically engineered to express the Fas ligand (Lau et al., nineteen ninety six) . The immune system is well equipped to quickly identify a foreign, diseased or inflamed tissue and quickly destroys it. This has always been a major barrier for organ and cell tissue transplantation as well as for gene therapy. The main problems are usually associated with chronic immunosuppression, encapsulation or immunoabsorption. Undesirable side effects of chronic immunosuppression include increased susceptibility to opportunistic infection and tumor formation. -5- The desire for long-term acceptance of grafted tissue for continuous immunosuppression is a very insistent and long-term goal in human medicine. The citation of any document herein is not intended as an admission that said document is pertinent to the prior art, or material considered for the purpose of any claim of the present application. Any statement to the content or date of any document is based on the training available to the applicant at the time of filing and does not constitute an admission to correct said statement.

Brief Description of the Invention It has now been found that transplantation of cells expressing a LAG-3 protein onto its surface results in protection against "graft rejection by the host's immune system." The present invention thus provides a genetically engineered cell, which may be part of a tissue or organs to be transplanted, comprising DNA encoding a transmembrane LAG-3 protein on its surface, resulting in protection against graft rejection by the host immune system, the DNA being genomic DNA or cDNA, said DNA may be exogenous or, in a particular embodiment of the invention, the endogenous DNA, whose expression is activated or modified through the target insertion of a regulatory sequence and / or a gene that can be amplified through homologous recombination.The LAG-3 protein is a protein that is recognized by direct anticorp When the cell is part of the tissue or organ that will be transplanted, the transfection of LAG-3 DNA can be achieved directly in the tissue or organ that will be transplanted. In particular, the cell is a universal gene therapy host cell, suitable, for example, for any type of gene therapy s omá t i-c-a or "ex vivo". In a specific embodiment, the gene therapy host cell further comprises exogenous DNA that encodes a therapeutic agent of interest, and genetically engineered cells are used as a therapeutic agent.

The term "therapeutic" as used herein, includes treatment and / or prophylaxis. In a further embodiment, the gene encoding a therapeutic agent of interest is present in the genome of the cell and the cell further comprises exogenous DNA encoding a regulatory response or a gene that can be amplified to activate or modify the expression of the endogenous gene of interest. The genetically engineered cell of the inversion can in any way contain the exogenous LAG-3 DNA only, which will be used in a mixture with other gene therapy host cells containing the therapeutic DNA of interest. The cell of the present invention is preferably selected from myoblasts, fibroblasts, hematopoietic stem cells, embryonic stem cells, fetal liver cells, umbilical vein endothelia cells, and CHO cells. Cells like the one above, which are derived from transgenic animals, are awithin the scope of the present invention.

• - Another object of the present invention is the use of a transmembrane LAG-3 protein, including muteins and variants thereof, expressed on the surfaces of cells, in the manufacture of drugs to induce protection against graft rejection by a host immune system. In addition, the present invention provides the use of a cell comprising DNA encoding a transmembrane LAG-3 protein, expressed on the surface of the cell, in the manufacture of a medicament for inducing protection against graft rejection by an immune system. Guest. The use of said cell that expresses LAG-3 on its surface, in the manufacture of a drug that will be mixed with cells, tissues or organs that will be transplanted, to induce protection against graft rejection by a host immune system, is awithin the scope of the present invention.

Brief Description of the Drawings Figure 1- Cytotoxic activity against splenocyte LH-CHO cells of mice initiated with LAG-3-CHO or LH-CHO cells. The mean value (± SD) of 5 r started as indicated in the legend; 2 uninitiated mice were also evaluated. Figure 2- Cytotoxic activity against LAG-3-CH0 cells from splenocytes of mice initiated with LAG-3-CH0 or LH-CHO cells. The mean value (± SD) of 5 mice initiated as indicated in the legend; 2 uninitiated mice were also evaluated. Figure 3- Map of mammalian expression vector Da. Abbreviation used: DHFR, dehydrofolate transcription unit (Subraimani et al, .1981); pML, derived from pBR322 (Lusky and Botchan, 1981); h-AIVSA, fragment of the intron A of the subunit a of hormones of Gl i cop ro t e ina human (Fiddes and Goodman, 1981); MMT-1, promoter of the me t a lo ti i i na 1 of mouse (Hamer and alling, 1982).

Detailed Description of the Invention Hundreds of thousands of people die each year as a result of a heart, kidney, liver, lung or pancreas failure. The only most effective therapy is transplantation. Therapy associated with the transplantation of cells, tissues or organs induces a general immunoprotection state in the host relative to the cell, tissue or grafted organ. It is desirable to establish a specific graft protection against rejection by the host immune system particularly in an allogeneic transplant, a xenogeneic transplant and gene therapy. Also, it is desirable to inhibit the tolerance to tumor tissue or otherwise allow the host immune system to attack the tumor tissue. Accordingly, the present invention is directed to all of these methods described above to utilize the finding that transplantation of cells or tissues expressing a transmembrane LAG-3 protein results in the protection of graft rejection by the host immune system. . The invention employs the gene and protein, recently discovered LAG-3, which is normally expressed in activated T cells and activated NK cells. The definition "transmembrane LAG-3 protein", as used herein, refers to any transmembrane protein that contains the extra cytoplasmic domain of LAG-3, its salts, functional derivatives, precursors and active fractions, as well as its active mutants and their active variants, which are expressed on the surface of a cell. The definition also refers to a transmembrane protein expressed in its natural state or can be fused, for example, through genetic engineering, to another protein, such as a glycosyl anchor fo s fa t idil ino si to 1 or any Relevant fragment of another transmembrane protein, for example the TNF receptor, the MPL ligand or a transmembrane immunoglobulin. The definition "salts" as used herein refers to both the salts of the carboxyl groups and the salts of the amino functions of the compound obtainable by known methods. The salts of the carboxyl groups comprise inorganic salts such as, for example, sodium, potassium, calcium salts and salts with organic bases such as those formed with an amine, such as triethanolamine, arginine or lysine. The salts of the amino groups comprise, for example, salts with inorganic acids such as hydrochloric acid and with organic acids such as acetic acid.

# The definition "functional derivatives" as used herein, refers to derivatives that can be prepared from the functional groups present in the side chains of the amino acid portions or in the N- or C-terminal groups according to with known methods and are included in the invention when they are pharmaceutically acceptable, that is, when they do not destroy the activity of the protein or do not impart toxicity to the pharmaceutical compositions they contain. Said derivatives include, for example, esters or aliphatic amines of the carboxyl groups and N-acyl derivatives of free amino groups or O-acyl derivatives of free hydroxyl groups and are formed with acyl groups such as, for example, alkanoyl or aroyl groups. "Precursors" are compounds that are converted to LAG-3 in the human or animal body. As "active fractions" of the protein, the present invention refers to any fragment or precursor of the chain of the same compound, alone or in combination with molecules or related residues bound to, for example, residues. of sugars or phosphates, or aggregates of the polypeptide molecule when said fragments or precursors show the same activity of LAG-3 as a medicament. Preferred "active fractions" are soluble fractions of the extracellular portion of the LAG-3 protein, including one or more of the four domains, DI, D2, D3, D4, of the ex tr ac it op 1 opic domain of LAG-3. . The definition "active mutants", as used herein, refers to other proteins or polypeptides wherein one or more amino acids in the structure are deleted or replaced by other amino acids, or one or more amino acids are added to that sequence with the In order to obtain polypeptides or proteins that have the same LAG-3 activity. For example, Arg 73 and / or Arg 75 and / or Arg 76 may be substituted by a different amino acid, preferably with Glu. The "active variants" of LAG-3 are differentially divided variants, as well as all primary gene transcripts, which are derived from alternative cleavage mechanisms at different cleavage sites of the gene. Preferred variants are soluble or transmembrane proteins lacking the D3 and / or D4 domain of the extracellular portion of LAG-3, optionally containing some additional amino acids after the D2 or D3 domain. The expression of the transmembrane LAG-3 protein on the surface of a cell is verified by immunoreactive methods. The trimemembrane protein is recognized, for example, by anti-LAG-3 antibodies 11E3 (Deposit No. CNCM 1-1612), 17B4 (Deposit No. CNCM 1-1240) or 15A9 (Deposit No. CNCM 1-1239). The present invention also relates to a mixture of polypeptide derivatives as mentioned above. When used in the present specifications and claims, the terms "AG-3", "LAG-3 protein", or "LAG-3 molecule" are intended to include natural, synthetic and recombinant forms of the polypeptide as well as all definitions reported previously. The cells of the present invention may be selected from primary or secondary cells. As used herein, the term primary cell includes cells present in a suspension of cells isolated from a vertebrate tissue source (before being plated, i.e., attached to a tissue culture substrate such as a box or flask), the cells present in a tissue-derived explant, both previous types of cells are plated for the first time, and the suspensions of cells derived from these cells in plaques. The term "secondary cell" or "cell strains" refers to cells in all subsequent steps in culture. That is, the first time a plaque primary cell is removed from the culture substrate and plated back (passage), it is referred to herein as a secondary cell, as are all cells in subsequent passages. Secondary cells are cell strains consisting of secondary cells which have passed one or more times. A cell strain consists of secondary cells that: 1) have passed one or more times; 2) receive a finite number of doublets of average population in the crop; 3) exhibit the properties of anchor-dependent growth, of inhibited contact (anchoring dependence does not apply to cells that are prepared in the suspension culture); and 4) are not immortalized. A "cloned cell strain" is defined as a cell strain that is derived from an individual founder cell. A "heterogeneous cell strain" is defined as a cell strain that is derived from two or more founding cells. The present invention includes primary and secondary somatic cells, such as fibroblasts, keratinocytes, epithelial cells, endothelial cells, normal cells, neural cells, elements formed from blood, muscle cells, other somatic cells, which can be cured and precursor cells. somatic cell, which have been transfected with exogenous DNA that is stably integrated in their genomes or is expressed in episormous cells. The resulting cells are designated as, respectively, transfected primary cells and transfected secondary cells. When the gene encoding a LAG-3 molecule is inserted into mammalian cells and the cells are annealed to an allogeneic or xenogeneic host, they are recognized by the host immune system but an immune response is not assembled. The host's immune system becomes unable to reject cells that might otherwise have been re-chased if they had not been engineered to express the LAG-3 molecule on its cell surface. LAG-3 can also be expressed on the cell surface of an unrelated cell type and mixed with the cells or tissues that will be transplanted with results similar to those described above. Thus, this invention relates to the transplantation of cells, tissues or organs without general immunosuppression. These cells, tissues or organs are transplanted to provide proteins or perform certain functions to treat certain diseases. They are accepted by the host through the use of a technique where LAG-3 is presented to the host's immune system. The prevention of rejection of the graft from specific cells, tissues or organs can also be achieved in recipients or through co-administration of fibroblasts or other primary or secondary cells that have been engineered to express LAG-3. This protective state may be due to the local or general inhibition of mechanisms that mediate immune responses. This protective state may be due to anergy, elimination, or insensitivity, tolerance or prevention of cell-mediated cytotoxicity. Once the protective state is established, it lasts for extended periods of time, even permanently due to a phenomenon such as an infectious tolerance (Quin et al., 1983). The invention can be used for the transplantation of tissues from cells, tissues, organs or host cells to deliver genes or gene products for a variety of human medical needs. The expression of the LAG-3 gene can be induced through standard ecombinant DNA techniques or through techniques that employ homologous comb ination to activate the endogenous LAG-3 gene. Cell types can be obtained from transgenic animals having the LAG-3 gene expressed in specific tissues or in unrelated cells through any method. Said cells can then be mixed with the cells where protection against graft rejection is desired. This suggests that the local secretion of the immuno-pro tector molecule LAG-3 does not act systematically. T r ansduced, untransformed fibroblasts of LAG-3 produce similar responses in vivo. The immuno-protective effects of the inoculum of LAG-3 transduced fibroblasts are dose dependent but independent of the source of the LAG-3 molecule. Co-administration of cells expressing LAG-3 inactivates donor T cells, while at the same time avoiding the attack of the host's immune system. This treatment induces "specific energy, tolerance or other protection of cell-mediated cytotoxicity in the recipient, which can then result in a long-term change in the immune environment allowing protection against autologous T cells. This can be done through the co-administration of a small number of allogeneic spinal cord cells from a healthy donor together with human allogeneic cells that have been engineered to express LAG-3. a reduction in self-reactive T cells through the development of my chimericism (Delaney et al., 1996). Humans with specific diseases or deficiencies can benefit from the allogeneic transplantation of many different cells, tissues or organs. For example, organs such as liver, kidney, heart, pancreas, small intestine, are commonly transplanted into cells such as islets, neural tissue for the treatment of Parkinson's disease or focal epilepsy, haematopoietic stem cells as a treatment for chemotherapy or radiation therapy, normal epatocytes for treating hypercholesterolemia, cardiac cells for myocardial infarction, muscle cells for muscular dystrophy, are suitable for the transplant. Allogenic spinal cord transplants have been difficult to achieve for a variety of reasons. These include: graft rejection, infection due to opportunistic infections as a result of graft contamination or immunosuppressive drugs, or other reasons. The rejection may be due to "the resistance of the recipient to the spinal cord graft by donors and the tendency of competent immune cells to attack the recipient, ie, graft-versus-host disease." GVHD can be controlled through elimination of the T cell graft or the co-administration of immunosuppressive drugs Graft is reduced when T cells are removed As a result of T cell elimination, there is a high incidence of graft failure, it is thought that they can provide an important function For grafting such as cytokine processing (Kernan et al., 1987), it has been shown that only small numbers of allogeneic cells but healthy bone marrow can reduce or even prevent the occurrence of autoimmune disease in experimental models. treatments such as these result in a graft-versus-host disease.The bone marrow transplant has been also used as a method to eradicate certain tumors. This is supposedly due to the ability of the allogeneic T cell to recognize and annihilate the tumor tissue, for example, in graft against leukemia. In all the above cases, general immunosuppression is prevented and the cells or organs to be transplanted are engineered with a gene encoding LAG-3, in order to express a LAG-3 protein when transplanted into a host and induce graft protection. A first problem in allogeneic transplantation is the lack of human tissue t r ansp 1 ant ab 1 e and currently the demand grows every day for humans. Although it is still considered, as an experimental procedure, xeno tr ansp 1 ant ac ion is considered as a viable alternative to the a 1 or t r ansp 1 ant ation. Animals such as pigs or baboons have now been considered as organ or cell donors. The graft rejection protection may be essential for the successful clinical use of organs of different species. The resistance of the host can be overcome, at least partially, for example, by using antibodies against CD4, CD8 cells; Human NK, or micro encapsulating the cells of the animal that will be transplanted. In accordance with the present invention, animals can be genetically altered to express the LAG-3 gene in certain cell types, such as islet cells through the use of the insulin gene promoter and the target system or another specific marked tissue system. The expression of LAG-3 in tumor tissue is believed to play an important role in resistance to cell-mediated attack against tumor tissue of the host immune system. According to a particular embodiment of the present invention, through the use of a gene therapy or ex vivo treatment due to a small amount of tumor tissue and reimplantation, the tumor tissue can be engineered to express LAG molecules. 3 anti-sense or ribosimals specific to the LAG-3 message. This could allow the immune system to react to the tissue and learn to destroy it. A small amount of tumor tissue can also be treated with an antibody to LAG-3 to prevent the inhibition of T cell induced by LAG-3 and allow the induction of cellular and humoral immunity against it. Gene therapy is now highly desirable for the treatment of a variety of diseases, including but not limited to deficiency of adenosine deaminase (ADA), sickle cell anemia, thalassemia, hemophilia, diabetes, deficiency a-ant itr ip s ina , brain disorders such as Alzheimer's disease and other diseases such as developmental disorders and heart diseases, for example, those associated with alterations in the way in which cholesterol is metabolized, and defects of the immune system. Different cells can be used for transplantation in individuals with this need, for example, my goal for the supply of diostrofine, cells that secrete material such as, TPO, GH, EPO, factor IX or other factors, blood cells for the treatment of hereditable disorders in the blood, and other human cells or primary animals such epithelial cells, connective tissue, fibroblasts, cells s in imal es, mesothelial cell and paranimal cells. The results of gene therapy have not been very helpful due to a number of problems. Even the most advanced analyzes in which a young woman has been treated with the adenosine deaminase (ADA) gene, the patient continues to receive the PAG-ADA injection weekly for fear that gene therapy alone will not be effective. One of the disadvantages of the gene therapy protocols at present is the requirement of individual production of host cells to try to avoid rejection of the cell by the host's immune system. Gene therapy must be performed on an individual basis. In addition, the expression of trans-genes is usually found to be transient due to the expression of other viral proteins, which couple the host's immune system through the use of autologous cells for gene therapy.

Disabled adenoviral vectors are used but these present problems due to the other viral proteins that are expressed that evoke an immune response. Large concentrations of viruses, even those incapacitated, stimulate an inflammatory response and an immune attack. The host cell immune system will remember the viral vector so that future administrations will be even less effective. Defective replication adenoviruses removed from viral protein are routines that are used in gene therapy protocols. Unfortunately, they have only transiently effective adult hosts in adults, presumably as a result of an immune response directed against adenoviral or recombinant proteins (Kozarsky and Wilson, 1993, Barr et al., 1992, Stratford et al., 1992; Rosenfeld. et al., 1992; Lemarchand et al., 1992). Therefore, there is a great need to develop new vectors that are not as immunogenic, or methods that allow for the immunological protection of engineered and engineered cells. These vectors are prepared at a high titration of up to 1011 plaque forming units per ml and infect many replication and non-replication cells. Use defective replication adenoviruses to deliver physiological levels of recombinant protein to the systemic circulation. The LAG-3 gene is preferably under the transcriptional control of the ubiquitously active cellular EFla promoter and the 4F2HC enhancer (Tripathy et al., 1994). Attempts have been made to avoid this problem by encapsulating the cells and through immunosuppression of the host. With the methods described herein, xenogeneic or haloggenic cells can be used as gene therapy hosts by expressing a LAG-3 molecule on their surface, in order to induce protection against graft rejection through the host's immune system. For example, these are myoblasts, neoblasts, fibroblasts, hematopoietic stem cells, embryonic stem cells, fetal liver cells, umbilical vein endothelial cells, or CHO cells. The gene therapy host cells can also be engineered to express the herpes simplex thymidine kinase gene. Said cells can be specifically destroyed through the addition of ganci lo ir. Cells sensitive to t k-gane i c 1 or vi r have an important advantage over non-sensitive cells, since they can be eliminated at any time (Bi et al., 1993). The universal host cell that is prepared according to the present invention to express LAG-3 and the Hsv-tk host on its cell surface in this manner allows the generation of a universal gene therapy host cell that can be implemented without immunosuppression and it can be destroyed at any point if its activity is no longer required. The allogeneic or xenogeneic gene therapy host cells of the present invention can be engineered to express a transgene and / or the LAG-3 gene through the use of any method of gene transfer, such as, but not limited to, , replication defective virus, adenoid virus, high efficiency retrovirus, direct injection of DNA into the spinal cord, electroporation, calcium phosphate transfection, myc-injection, encapsulation in liposomes or erythrocyte hosts. Cells expressing LAG-3 such as myoblasts or CHO cells can be co-administered with cells expressing a protein of interest that will be permanently grafted. If the passing LAG-3 exposure is adequate, then the transfected LAG-3 myoblasts or CHO cells may contain a suicide gene such as tk, as previously reported, so that they can be removed through treatment with ganciclovir. This method can be used to restore normal function to through the administration of a gene or gene product or the removal or activation of a gene that is dangerous to the body (such as an oncogene). This ctive can also be achieved by "implanting ribosomal delivery cells or antisense cDNAs to inhibit the production of unwanted protein such as HIV protein or growth factors." The method can be used to correct enzyme deficiencies such as Gaucher's disease or ADA disease. According to one more modality, the present invention is directed to a method for gene therapy comprising: (i) inserting to the human cells - or - animals of choice, the gene necessary for the therapy and the gene encoding a LAG-3 molecule; and (ii) introducing the cells resulting from step (i) within the patient. Alternatively, the above genes can be infected to a tissue organ of a patient, in vivo, through direct transfection of the genes as such, in a vehicle targeting said tissue or organ. For example, the claimed expression system allows the injection of a natural DNA or viral vector directly into a group of muscle cell-like cells or administered directly to the respiratory tract (eg, to treat cystic fibrosis). The construct contains not only the interest gene (such as the conductance regulator (CFTR cDNA) but also the LAG-3 gene which will be co-expressed in the cell type to avoid possible humoral immune responses to, for example , the adenovirus capsid proteins that could limit the efficacy of repeated administrations.The transfer of gene to hematopoietic stem cells can be used for the administration of multiple drug resistance genes to combat one of the side effects of chemo therapy. suppresses ion to rapidly divide immune cells. 'Retroviral vectors can be used in combination with cytokines such as Steel factor, team ligand, 113, Gm-CSF, IL6, G-CSF, LIF, IL12 to encourage division of Stem Cells The cells of choice can be cotransfected with genes that encode other immunosuppressive agents such as IL10, TGFβ, Fas ligand, in addition to the LAG-3 gene As a particular embodiment of the present invention, the methods described above are used to treat the recipient with a small number of cells of interest and engineered to express the LAG-3 gene that will make the host tolerant to the next administration of cells, tissues or organs due to tolerance of infection through the host's immune system. The invention will now be described by way of illustration only with reference to the following examples: EXAMPLE 1 Methods Generation of CHO cells expressing LAG-3 from t r ansmembr a. LAG-3 cDNA was excised as a Xho fragment of 1620 bp from plasmid pCDM8 (Invitrogen San Diego CA) and purified through e 1 ec t ropo r e s s in agarose gel. The fragment was subcloned into the mammalian expression vector pCLH3AXSV2 DHFRhal VSA (Da) (Fig. 3) digested with Xho. The CH0-DUKX (DHFR ") cells were transfected with the Da LAG-3 construct through the CaP0 precipitation method.The transfected cells were developed in a selection medium (MEM medium without deoxy and r ibonuc 1 eotted s + 10% dialyzed fetal bovine serum plus 1% L-glutamine + 0.02 μM methotrexate.) LAG-3 expression was verified through a western stain on lymph cell membrane preparations and periodically through cytometric analysis of flow using the monoclonal antibody anti-LAG-3 17B4.

Transplanting CHO cells into mice Chinese hamster ovary cells (CHO), either untransfected (wild-type) or transfected with full length human LAG-3 or human LH cDNA, were separated from the plastic flasks and suspended in a Dulbecco modification of a minimal essential medium (DMEM) to a concentration of 1.75x10 'cells / ml. Twenty-six C57BL / 6 female mice aged 7-9 weeks in 7 groups were distributed as indicated in Table 1 and 200ml of the indicated cell suspension, containing 3.5x10"cells, were injected subcutaneously into the right flank of each animal. In groups of 3, 6 and 7, the same mice received transfected LAG-3 cells in the right flank and control cells (transfected or non-transfected LH) in the other.4 Four days after injection, the mice were sacrificed through inhalation of CO. and the p was opened to examine the injection site.

Table 1. Experimental groups Identification No. Group CHO cells CHO cells mouse mice injected in the injected on the right flank left flank 1 5 1 to 5 wild type - 2 5 6 to 10 LAG-3 - 3 5 11 to 15 LAG-3 Wild type 4 2 16 and 17 LH - 5 5 1 8 to 22 LAG-3 - 6 2 23 and 24 LAG-3 Wild type 7 2 25 and 26 LAG-3 LH Evaluation of cytotoxicity against CHO cells Five C57BL / 6 female mice per group were injected s.c. with 4xl05 CHO cells transfected with either human LH or human LA-G-3 cDNA. After 14 days, the mice were sacrificed and the vessels removed to obtain splenocyte suspensions. Splenocyte suspensions (effectors) were diluted in a culture medium (RPMI 1640 + 10% fetal bovine serum) + antibiotic) at 107 cells / ml and plated in triplicate in different dilutions to obtain the various effectors for target ratios; 2 plates were prepared for each suspension. The target cells at 5x10 'cells / -l 00 μl (either cells, transfected LH or LAG-3), labeled with olCr, were added to the plates (one plate for each objective). After 20 hours at 37 ° C, 20 μl of each of each cavity was taken and evaluated for the release of 'Cr through liquid synthesis. The cytotoxic activity was calculated as the percentage of lysis according to the following formula: (CPMsampJe-CPMspont)% = xlOO (CPM-pax-CPMspont) where spont and max indicate the cavities with the culture medium (spontaneous release of Cr from the target cells) and Triton X 1 (maximum release of Cr) replacing the suspension of the effector, respectively.

Resul ates Transplanting CHO cells into mice Most mice received LAG-3 t ansfected cells and showed a white nodule - at the injection site that did not appear in the animals treated with the wild type CHO or in those that received CHO cells transfected with LH. Injection of wild-type CHO cells caused the appearance of diffuse hemorrhage at the injection site in the majority of mice. This phenomenon was less evident in the animals that received transfected LH cells. In mice injected with both the wild-type CHO cell and transfected LAG-3 at different sites, this nodule was evident only at the site of injection with the latter, while a hemorrhage appeared at the other site (Table 2). Histological analysis previously performed in a similar experiment showed the presence of heterologous cells in the nodules. The results of the experiment are shown in the attached graphs (see Table 1 for the identification of the mice) and are summarized in Table 3.

Table 2 Mouse No. Frequency Injected Cells (number of positive findings / total number of injected flanks): hemorrhage Nodule LAG-3 10 1/10 9/10 wild type 5 3/5 0/5 LH 2 1/2 1 / 2 LAG-3 + type 7 0/7 (a) 5/7 (c) 7/7 (a) 1/7 (c) wild * LAG-3 + LH * 2 0/2 (b) 1/2 (a) 0/2 (b) 2/2 (a) * Each type of cell is only injected in a lateral line. (a) = lateral LAG-3 (b) = lateral LH (c) = lateral WT Table 3 Cytotoxicity against CHO cells The expression of LAG-3 on the CHO cell surface did not affect the ability of mice to be immunologically and initiated against xenogeneic cells. In fact, the splenocytes from mice injected with LAG-3-CHO cells liposized both the target cells as efficiently as those from mice initiated with cells from LH-CHO mice and better than the uninitiated mice. However, the expression of LAG-3 on the cell surface was associated with a reduced sensitivity to the cytotoxic activity induced by immunization of the mice against the target as can be seen by comparing the percentage of lysis in Figure 1 and Figure 2. The "natural" cytotoxicity, exerted by splenocytes from uninitiated mice, was not reduced through the surface expression of LAG-3. This indicates that surface expression of LAG-3 reduces efficient branching of cytotoxic T lymphocyte (CTL) activity. CTL is one of the effectors that plays an important role in the rejection of transplanted organs (G. Berke, 1993) in this way the inhibition of its function can prolong the survival of the allografts.

REFERENCES 1. Triebel et al., J. Exp. Med., 171: 1393, 1990 2. Baixeras et al., J. Exp. Med., 176: 327, 1992 3. Huard et al., Immunogenetics, 39: 213, 1994A 4. Huard et al., Eur. J. Immunol., 24: 3216, 1994 B. 5. Huard et al., Eur. J. Ipununol., 26: 1180-1186, 1996 6. Miyazaki, et. al., Science, 272: 405-408, 1996 7. Susuki et al., J. Exp. Med., 182: 477-486, 1995 8. Bellgrau et al., Nature, 377: 630-632, 1995 9 Lau et al., Science, 273: 109-112, 1996 10. Subraimani et al., Mol. Cell. Biol., 1: 854-864, 1981 11. Luski and Botchan, Nature 293: 79-81, 1981 12. Fiddes and Goodman, J. Mol. Appl. Genetic 1: 3-18, 1981 13. Ha-mer and alling, J. Mol. Appl. Genetic 1: 273-288, 1982 14. Qin et al., Science, 259: 974, 1993 15. Delaney et al., J. Clin. Inyes., 97: 217-225, 1996 16. Kernan et al., Transplantation, 43: 842, 1987. 17. Kozarsky and Wilson, Current Opinions Genet. Dev., 3: 499, 1993 18. Barr et al., Gene Therapy, 1: 51, 1992 19. Stratford et al., J. Clin. Inves., 90: 626, 1992 . Rosenfeld et al., Cell 68: 143, 1992 21. Lemarchand et al., PNAS, 89: 6482, 1992 22. Tripathy et al., PNAS, 91: 11557, 1994 23. Bi et al., Human Gene Therapy 4: 725, 1993 24. Berke, The functions and mechanisms of action of cytolytic lymphocytes. Chapter 28 pages 972-974 in: Fundamental Immunology edited by W.E. Paul New York, 1993.

Claims (22)

1. A genetically engineered cell comprising DNA encoding a transmembrane LAG-3 protein on its surface resulting in the protection of graft rejection through a host immune system.

2. The cell as claimed in claim 1, wherein the DNA encoding a LAG-3 protein is exogenous.

3. The cell as claimed in claim 1, wherein the DNA encoding a protein is endogenous and its expression is activated or modified through the target insertion of a regulatory sequence and / or an amplifiable gene through homologous recombination.

4. The cell as claimed in any of claims 1 to 3, which is part of a tissue or organ to be transplanted.

5. The cell as claimed in any of claims 1 to 3, which is a gene therapy host cell.

6. The cell as claimed in the r e i vindi ca c i-o-n 5, wherein the gene therapy is a somatic gene therapy or "ex vivo".

7. The cell as claimed in any of the above indications 1 to 6, which further comprises an additional immunosuppressive agent, such as IL-10, TGFβ, or Fas ligand.

8. The cell as claimed in any of claims 1 to 7, further comprising the thymidine kinase (tk) gene which is sensitive to the suicide system of t k-gane ic lo vir.

9. The cell as claimed in any of claims 1 to be derived from transgenic animals.

10. The cell as it is. claimed in any of claims 1 to 9, selected from myoblasts, fibroblasts, hematopoietic stem cells, embryonic stem cells, fetal liver cells, umbilical vein endothelial cells or CHO cells.

11. The cell as claimed in any of claims 5 to 10, further comprising an exogenous DNA encoding a therapeutic agent of interest.

12. The cell as claimed in any of claims 5 to 10, further comprising exogenous DNA encoding a regulatory sequence or an amplifiable gene for activating or modifying the expression of an endogenous gene of interest.

13. A use of a transmembrane LAG-3 protein on the surface of a cell in the manufacture of a medicament for inducing graft rejection protection through a host immune system.

14. The use of a cell as described in any of claims 1 to 12, in the manufacture of a medicament for inducing graft rejection protection by a host immune system.

15. The use of a cell as described in any of claims 1 to 10, in the manufacture of a medicament that will be mixed with cells, tissues or organs to be transplanted, to induce the protection of graft rejection by an immune system of a Guest.

16. The process for inducing the specific protection of graft rejection of cells, tissues or organs transplanted by a host immune system, comprising treating the host patient with a cell according to any of claims 1 to 12.

17. The process as claimed in claim 16, wherein the cell is part of a tissue or organ that will be transplanted.

18. The process as claimed in claim 16, wherein the cell is a gene therapy host cell.

19. The process as claimed in claim 18, comprising: (i) inserting into the host cell of choice the gene necessary for the therapy and the gene encoding the LAG-3 molecule and (ii) introducing cells resulting from the step ( i) to a guest patient with the need of the same.

20. The process as claimed in any of claims 16 to 19, which comprises the treatment of a host patient with the need for a small number of cells engineered to express the LAG-3 gene to induce graft rejection protection by the host immune system, for the next administration of the cells, tissues or organs that will be tr ans p anned.

21. A process for allowing rejection of tumor tissue, comprising engineering engineering said tumor tissue cells to express LAG-3 antisense or ribosomal molecules specific to the LAG-3 message.

22. A genetically engineered cell comprising DNA and encoding a transmembrane LAG-3 protein on its surface to be used as a medicine.