Agents suppressing rejection of transplant

FIELD: transplantology.

SUBSTANCE: invention discloses pharmaceutical compositions containing substance effective as modulators of biological activity "induced by activation of lymphocytic immunomodulating molecule (AILIM)" (known also as "induced common stimulator (ICOS)"), in particular modulating transduction of AILIM-mediated signal.

EFFECT: achieved suppression, treatment, or prevention of rejection of transplant arising in case of transplantation of organ, a part thereof or tissue.

11 cl, 7 dwg

 

The technical field

The present invention relates to pharmaceutical compositions containing a substance which has an activity modulation of biological activity, "activation-induced lymphocytic immunomodulatory molecule" (AILIM) (also known as "induced joint stimulator" (ICOS), in particular the modulation of signal transduction mediated by AILIM.

Specifically, the present invention relates to pharmaceutical compositions comprising a substance which has an activity of the modulation (e.g. inhibition) of proliferation of cells expressing AILIM, or the modulation (e.g. inhibition) production of cytokines (e.g. interferon-γor interleukin-4) cells expressing AILIM.

More specifically, the present invention relates (1) to pharmaceutical compositions for the inhibition, treatment or prophylaxis of transplant rejection (immune exclusion), accompanying the organ part, or tissue; and (2) to pharmaceutical compositions for amplification inhibition, therapeutic or preventive effect of immunosuppressive agents on graft rejection (immune exclusion)accompanying the organ part, or tissue.

Prior art

In view of the recent is the Fund laws on organ transplantation in Japan made a number of organ transplants from patients with brain death. In one case, such benefits received from a single donor seven patients. In the following the expected increase in the number of organ transplants.

On the other hand, it is estimated that in Japan the number of patients affected severe cardiovascular and other diseases such as liver disease (acute liver failure, liver cirrhosis and so on), heart disease (severe heart failure, myocardiopathy, cardiac hypertrophy, etc.), renal diseases (renal failure, chronic glomerulonephritis, diabetic nephropathy, pyelonephritis, and so on), pulmonary disease (impaired function of both lungs etc) and diseases of the pancreas (treatment of diabetes), in which the transplantation of organs vital for treatment each year approximately 600 of cardiac patients, approximately 3000 patients with liver disease and approximately 500 patients with pulmonary diseases. Although the legal aspects of developing, the absence of organs that can be transplanted, also is really the current problem. Similarly, the lack of bodies is also a serious problem in the United States, which is an advanced country in terms of transplantation. In the USA, approximately 4300 people the EC (1999) waiting for a heart transplant and approximately 43,000 people (1999) waiting for a kidney transplant. In fact, approximately 800 and approximately 2,300 people die every year, not being able to receive respectively the transplant heart and kidneys.

Under the transplantation of tissue (such as skin, cornea and bone) or organ (such as liver, heart, kidney, lung and pancreas) imply: (1) autologous transplantation (autologous transplantation), (2) istranslatable, (3) allotransplantation and (4) xenotransplantation.

Autologous transplantation refers to the transplantation of parts of an individual in another part of the same individual and is, for example, in the case of treatment of burns by transplanting one's own healthy skin on the affected area.

Istranslatable is made between homogeneous animals. In humans, this transplantation between monozygotic twins (for example, transplantation of one kidney or liver tissue).

Allotransplantation is made between two different individuals with different genetic background, and in humans, this transplantation is performed between dizygotic twins or between individuals who have no blood relationship with each other.

Xenotransplantation is made between individuals of different species. An example is the case when the tissue or organ chimpanzees or pigs pereceive the camping person.

As stated above, it is expected that the number of cases of allotransplantation from patients with brain death will increase due to the development of the law relating to organ transplantation. However, to resolve the problem of the complete lack of organs that can be transplanted, at present, actively carried out various research aimed at practical applications of xenotransplantation, more specifically on the transplantation of human tissues or organs from non-human mammals such as the pig.

Although it is expected that the issue of the lack of transplantable tissues and organs will be resolved by the development of laws on brain death and transplantation and to improve methods of xenotransplantation, there is another very big obstacle in the treatment of diseases by allotransplantation and xenotransplantation. More specifically, the obstacle is a severe immunological rejection (graft rejection) recipients, which occurs after transplantation of tissues or organs from donors.

Graft rejection refers to a variety of immune reactions, which are aimed at the rejection and destruction of the graft (part of a living organism, which is transplanted, the cell, tissue or organ from a donor whose genetic background otlicials is from that of the recipient (i.e. if allotransplantation or xenotransplantation), because the recipient recognizes the graft as foreign substance. Immune reactions that accompany specified transplantation can be classified into (1) Verhoture exclusion, which is a strong rejection occurring directly after transplantation; (2) acute rejection, which occurs within several months after transplantation; and (3) chronic rejection observed several months after transplantation. In addition, although cell-mediated immunity due to immunocompetent cells and presented to T-cells and humoral immunity due to antibodies is endogenous co-ordinated way, the main reaction is carried out by cellular immunity.

In the rejection of organ transplant eventually becomes necrotic and fall off. In addition, the recipient develops not only systemic symptoms such as fever, leukocytosis and fatigue, but also swelling and soreness at the site of transplantation. In addition, you may experience severe complications, such as infections.

In particular, the transplantation of xenogenic graft, such as a transplant from a pig, there is a serious problem sverhmoschnogo exclusion, in which Tr is spentat rejected within a few minutes.

To suppress immunological rejection (transplant rejection)accompanying such transplant a limited number of immunosuppressive funds, because of immunological rejection caused by allotransplantation, occurs mainly as a result of cellular immunity. Such immunosuppressive tools include cyclosporine (CsA), tacrolimus (FK-506); azathioprine (AZ); mycophenolate mofetil (MMF); mizoribine (MZ); Leflunomide (LEF); adrenocorticosteroid (also known as the adrenal cortex hormones, corticosteroids, corticoide), such as prednisolone and methylprednisolone; sirolimus (also known as rapamycin); desoxypeganine (DSG); and FTY720 (chemical name: 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propandiol hydrochloride).

CTLA4 and CD28, which are the molecules responsible for the transduction of joint stimulatory signals required for the activation of T cells (molecules transduction joint stimulating signals), and especially drugs CTLA4, using a soluble CTLA4 region and its encoding gene, is also undergoing clinical development as immunosuppressive funds.

On the other hand, recently, similar to CTLA4 and CD28, which are molecules that provide the transduction joint stimulating signals was identified Molek is a, called activation-induced lymphocytic immunomodulatory molecule (AILIM; human, mouse, and rat; Int. Immunol., 12(1), p.51-55, 2000), also called induced joint stimulator (ICOS; human; Nature, 397 (6716), R-266, 1999); J. Immunol., 166(1), p.1, 2001; J. Immonol., 165(9), R, 2000; Biochem. Biophys. Res. Commun., 276(1), R, 2000; Immunity, 13(1), R, 2000; J. Exp. MecL, 192(1), R, 2000; Eur. J. Immunol., 30(4), R, 2000), as a third molecule that converts joint stimulating signals, which converts the second signal (co-stimulatory signal required for the activation of lymphocytes such as T cells, and is associated with the signal that regulates the function of activated lymphocytes such as activated T cells.

On the basis of recent studies relating to the specified molecule, it is predicted that the molecule AILIM possibly involved in the development of various diseases (e.g. autoimmune diseases, allergic conditions and inflammation)caused by the activation of immunocompetent cells such as T cells (particularly T-lymphocytes). However, so far there are no reports about the relationship between the functional modulation of the molecule AILIM and graft rejection (immune exclusion), accompanying the transplantation of tissues or organs, and the attempts at suppression, treatment, or prevention of such rejection reactions accompanying the transplantation of tissues or of the bodies, by modulation of the activity of the molecule AILIM.

In addition, it was recently identified a new molecule, called B7h, B7RP-1, GL50 or LICOS, which as believe, is a ligand that interacts with the molecule transduction joint enabling signal AILIM (Nature, Vol.402, No. 6763, pp.827-832, 1999; Nature Medicine, Vol.5, No. 12, pp.1365-1369, 1999; J. Immunology, Vol.164, pp.1653-1657, 2000; Curr. Biol., Vol.10, No. 6, pp.333-336, 2000).

In the identification of these two types of new molecules, namely AILIM (ICOS and B7RP-1 (B7h, GL50, LICOS), it was found that in addition to the known first and second transduction of signals between CD28 and CD80 (B7-1)/CD86 (B7-2) and between CTLA4 and CD80 (B7-1)/CD 86 (B7-2) includes a new third way transduction joint enabling signal, which is essential for the above-mentioned activation of lymphocytes such as T cells, and regulation the function of activated T-cells, which works through the interaction between AILIM (ICOS and B7RP-1 (B7h, GL50, LICOS).

Conducted a thorough investigation of the biological functions of these new molecules, the regulation of these molecules function of lymphocytes such as T cells, through a third transduction joint enabling data signal molecules and communication between the new signal transduction and diseases.

Description of the invention

More specifically, the present invention is to provide methods and pharmacist is logical devices, which inhibit, treat or prevent immunological rejection (graft rejection), accompanying the transplantation of tissue or organ (allotransplantation or xenotransplantation), by applying the medical and pharmaceutical methods (for example, pharmaceutical agents, such as compounds with a low molecular weight, and antibodies) to modulate the biological functions of the new molecule AILIM, which, as it transduces the second signal (co-stimulatory signal required for the activation of lymphocytes such as T cells, and is associated with the signal that modulates the function of activated lymphocytes such as activated T cells.

Another purpose is the provision of methods of strengthening suppressor effect on graft rejection existing immunosuppressive agents (cyclosporine, azathioprine, adrenocorticosteroids, FK-506, and so on) with the use of such pharmaceutical agents that modulate the biological function of AILIM (for example, such pharmaceutical products as compounds with low molecular weight and antibodies).

Through careful studies of the biological functions of AILIM mammals and method of suppression of immunological rejection (transplant rejection), which is a serious problem accompanying the tra is splantzia (allotransplantation or xenotransplantation) grafts (cells, tissue or organ), the authors present invention have found that (1) pharmaceutical agents that modulate the function of AILIM, significantly suppress immunological rejection (graft rejection), accompanying the transplantation of tissues (tissues) or body (bodies), and (2) suppressor effect of existing immunosuppressive agents on graft rejection increases with the use of pharmaceuticals which modulate the function of AILIM; and the result was decorated with the present invention.

The pharmaceutical composition according to the invention can be used as a drug to modulate various reactions in vivo are involved in the transduction together a stimulating signal to the cells expressing AILIM, mediated by AILIM (for example, proliferation of cells expressing AILIM, production of cytokines (cytokines) cells expressing AILIM, immune cytolysis or apoptosis of cells expressing AILIM, and activity-dependent induction of antibodies cellular cytotoxicity against cells expressing AILIM), and/or as a drug for the prevention of the beginning and/or progression of various diseases that are associated with signal transduction mediated AILIM, and for the treatment or prevention of such diseases.

In particular, the pharmaceutical is Kai composition according to the invention can modulate (inhibit or promote) the proliferation of cells, expressing AILIM, or can modulate (inhibit or promote) the production of cytokines (e.g. interferon-γ or interleukin-4) cells expressing AILIM, and can prevent various pathological conditions, run various physiological phenomena in which the involved signal transduction mediated by AILIM, and provides for the treatment or prevention of various diseases.

The use of pharmaceutical compositions according to the invention enables suppression, prevention and/or treatment of immunological rejection (transplant rejection), which is a serious problem in those treatments in which the body (liver, heart, lung, kidney, pancreas etc), part or tissue (such as skin, cornea and bone) from a donor transplanted (allotransplanted or xenotransplanted) recipient affected severe cardiovascular disease.

In addition, the use of pharmaceutical compositions according to the invention provides the possibility of strengthening the overwhelming rejection of the transplant effect existing immunosuppressive funds entered to suppress immunological rejection when such kinds of treatment by transplantation.

More specifically, the present invention is clucalc as follows:

(1) a Pharmaceutical composition to inhibit, treat, cure or prevent transplant rejection, accompanying the organ part, or tissue, and the specified composition includes a substance having the activity of a modulating signal transduction mediated by AILIM, and a pharmaceutically acceptable carrier.

(2) the Pharmaceutical composition to enhance the effect of one or more immunosuppressive means to suppress, treat, cure or prevent transplant rejection, accompanying the organ part, or tissue, and the specified composition includes a substance having the activity of a modulating signal transduction mediated by AILIM, and a pharmaceutically acceptable carrier.

(3) the Pharmaceutical composition according to p.(2), in which the specified immunosuppressive agent is one or more therapeutic agents selected from the group consisting of azathioprine, adrenocorticosteroid, cyclosporine, mizoribine and tacrolimus (FK-506), mycophenolate mofetil, Leflunomide, sirolimus, doxicillin, FTY720 and drug CTLA4.

(4) the Pharmaceutical composition according to any one of paragraphs.(1)to(3), and this transplantation is allotransplantation.

(5) the Pharmaceutical composition according to any one of paragraphs.(1)to(3), and the specified Tr is splantzia is a xenotransplantation.

(6) the Pharmaceutical composition according to any one of paragraphs.(1)to(5), and this organ is the liver, heart, kidney, lung or pancreas.

(7) the Pharmaceutical composition according to any one of paragraphs.(1)to(5), and the fabric is a skin, cornea or bone.

(8) the Pharmaceutical composition according to any one of paragraphs.(1)to(7), in which the specified substance is a protein substance.

(9) the Pharmaceutical composition according to p. (8), and specified a protein selected from the group consisting of

a) an antibody which binds to AILIM or a portion of the indicated antibodies;

b) a polypeptide containing the whole or any part of the extracellular region of AILIM;

c) a hybrid polypeptide containing the whole or any part of the extracellular region of AILIM and whole or any part of the constant region of the heavy chain of an immunoglobulin; and

a) a polypeptide that binds to AILIM.

(10) the Pharmaceutical composition according to any one of paragraphs.(1)to(7), in which the specified substance is a non-protein substance.

(11) the Pharmaceutical composition according to p.(10)with the specified non-protein substance is a DNA, RNA, or chemically synthesized compound.

The present invention is described in detail below with introduction definitions of terms and methods of producing substances used in this invented the I.

In this description, the term "mammal" means a person, cow, goat, rabbit, mouse, rat, hamster and Guinea pig; preferred is human, cow, rat, mouse or hamster, and especially preferred is the man.

"AILIM" according to the invention is an acronym for "activation-induced lymphocytic immunomodulatory molecule" means a molecule to the surface of mammalian cells with the structure and function described in the previous messages (J. Immunol., 166 (1), p.1, 2001; J. Immunol., 165 (9), R, 2000; Biochem. Biophys. Res. Commun., 276 (1), p.335, 2000; Immunity, 13(1), R, 2000; J. Exp. Med., 192 (1), R, 2000; Eur. J. Immunol., 30 (4), R, 2000; Int. Immunol., 12 (1), 51, 2000; Nature, 397 (6716), R, 1999; GenBank Accesion Number: BAA82129 (person); VA (rat); VA (rat variant); VA (mouse)).

Particularly preferably, the term means AILIM obtained in humans (e.g., International Immunology, Vol.12, No. 1, 51-55, 2000).

Specified AILIM is also known as ICOS (Nature, Vol.397, No. 6716, R-266, 1999) or antigen JTT-1 antigen JTT-2 (which has not passed the examination published patent application of Japan No.(JP-A) Hei 11-29599, international patent application No. WO98/38216), and listed the names of the molecules used interchangeably and refer to the same molecule.

In addition, "AILIM", which is referred to in this invention includes a polypeptide having the amino acid sequence of AILIM from each mlekovita is it described in previously published literature, and particularly preferably the polypeptide having essentially the same amino acid sequence as the sequence of the human AILIM. Moreover, variants of human AILIM, similar to the previously identified variant AILIM obtained from rat (GenBank Accesion Number: BAA82127), also included in the term "AILIM" according to the invention.

In the present description, the expression "having essentially the same amino acid sequence" means that "AILIM" according to the invention includes a polypeptide having the amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids have been substituted, subject to deletions and/or modified, and the polypeptides having the amino acid sequence where multiple amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids have been added, while the polypeptides have essentially the same biological properties, as the polypeptide comprising the amino acid sequence shown in the previous message.

Such substitutions, deletions or insertions of amino acids can be achieved in accordance with the usual method (Experimental Medicine: SUPPLEMENT, "Handbook of Genetic Candy" (1992), etc).

Examples are synthetic oligonucleotide with site-directed mutagenesis (duplex method with gaps), point mutagenesis, by which point mutations introduced randomly by treatment with nitrite or sulfite, the way in which deletion mutant was produced using the enzyme VA etc., cassette mutagenesis, scanning method of the linker, the method of inclusion errors, method of seed erroneous mating grounds, the method of synthesis of the DNA segment, and so on

Synthetic oligonucleotide with site-directed mutagenesis (duplex method with gaps) can be performed, for example, as follows. The area you want to expose mutagenesis, clone into the vector M13 phage with amber mutation, to obtain single-stranded DNA phage. Then RF I DNA vector M13, not having amber-mutations, linearized by treatment with restriction enzyme, the DNA is mixed with the above single-stranded DNA phage, denatured, and subjected to annealing, thereby forming a duplex DNA with gaps". A synthetic oligonucleotide, which introduce mutations, hybridizing with duplex DNA with gaps, and closed the ring Dunaeva DNA gain, providing a reaction with a DNA polymerase and DNA ligase. Cells of E. coli mutS with the lack of reparative activity erroneous mating transferout specified On The K. Cells of E. coli lacking suppressor activity, infecting phages grown and conduct screening only phages that do not have amber-mutations.

In the way that introduces a point mutation nitrate use, for example, the principle, as stated below. If DNA is treated with a nitrite, nucleotides desaminase, resulting adenine becomes gipoksantin, cytosine - uracil and guanine - xanthine. If dezaminirovanie DNA is introduced into cells, "A:T and G:C" are substituted respectively "G:C and A:T", because the Foundation of the gipoksantina, uracil and xanthine mate respectively with cytosine, adenine and thymine during DNA replication. Indeed, fragments of single-stranded DNA treated with nitrite, hybridize with duplex DNA with gaps", and then the mutant strains separated by manipulating the same way as when receiving a synthetic oligonucleotide with site-directed mutagenesis (duplex method with gaps).

The term "cytokine"as in the phrase "production of cytokines by cells expressing AILIM", in the present invention refers to an arbitrary cytokine produced by cells expressing AILIM (particularly T-cells).

Examples of T-cells are T-cells of the Th1 type and Th2 type, a cytokine according to the invention, in particular, means cytokine produced by T cells of the Th1 type and/or PR is arbitrary cytokine, produced by T cells of the Th2 type.

Cytokines produced by T cells of the Th1 type, include IFN-γ, IL-2, TNF, IL-3, and the cytokines produced by T cells of the Th2 type, include IL-3, IL-4, IL-5, IL-10 and TNF (Cell, Vol.30, No. 9, pp.343-346, 1998).

The term "substance"used in the present invention, specifically "a substance having the activity of a modulating signal transduction mediated by AILIM", and more specifically "the substance having the activity of inhibiting proliferation of cells expressing AILIM, or inhibition of cytokine production by cells expressing AILIM"means a naturally occurring substance or arbitrary artificially obtained substance.

Here the expression "signal transduction mediated by AILIM" means the signal transduction through AILIM, leading to changes in any of the phenotype described above, cells expressing AILIM, or in the following examples (changing of cell proliferation, cell activation, inactivation of cells, epopteia and/or the ability to produce arbitrary cytokines from cells expressing AILIM).

"Substance" can be mainly classified into "protein" and "non-protein substance".

Examples of "proteins" include polypeptides, antibodies (polyclonal antibodies, monoclonal antibodies, or portions of monoclonal antibodies).

When the substance presented yet an antibody, it preferably is a monoclonal antibody. When the substance is a monoclonal antibody, it includes not only the monoclonal antibodies obtained from a mammal, not a person, but also the following recombinant chimeric monoclonal antibodies, recombinant humanized monoclonal antibodies and human monoclonal antibodies.

When the substance is a polypeptide, it includes the following polypeptides, fragments of polypeptides (oligopeptides), hybrid polypeptides and chemically modified polypeptides. Examples of oligopeptides are peptides comprising from 5 to 30 amino acids, preferably from 5 to 20 amino acids. Chemical modification can be designed depending on various purposes, for example, to increase half-life in blood in the case of the introduction in vivo, or to increase the stability against collapse, or to increase the absorption in the digestive tract when administered orally.

Examples of polypeptides of the following:

(1) a Polypeptide containing fully or partially extracellular region of AILIM;

(2) a Hybrid polypeptide containing fully or partially extracellular region of AILIM and fully or partially constant region of the heavy chain immunoglobulin; or

(3) a Polypeptide, the cat is which binds to AILIM.

Examples of "non-protein substances" are DNA, RNA and chemically synthesized compounds.

Here "DNA" means DNA that can be used as a drug antisense DNA comprising a partial nucleotide sequence of DNA encoding the above AILIM (preferably human AILIM), or chemically modified DNA, which can be constructed based on DNA (cDNA or genomic DNA)encoding AILIM". Specifically, the antisense DNA can inhibit the transcription of DNA encoding AILIM, mRNA, or translation of mRNA into protein by hybridization with DNA or RNA, encoding AILIM.

Used here, the phrase "partial nucleotide sequence" refers to a partial nucleotide sequence containing an arbitrary number of nucleotides in any area. Partial nucleotide sequence contains from 5 to 100 consecutive nucleotides, preferably from 5 to 70 consecutive nucleotides, preferably from 5 to 50 consecutive nucleotides, and more preferably from 5 to 30 consecutive nucleotides.

When DNA is used as a drug, antisense DNA, the DNA sequence may be partially chemically modified to extend the half-life (stability) in the blood, when DNA is injected patients, the La increase the ability of the penetration of DNA through intracytoplasmic membrane or to increase resistance to decay or absorption of oral introduced DNA in the digestive organs. Chemical modifications include, for example, modified phosphate bonds, ribose, nucleotide sugar fragment and the 3'-end and/or 5'-end of the DNA structure of the oligonucleotide.

Modifications of the phosphate linkages include, for example, the conversion of one or more links in the phosphodiester bond (D-oligo), phosphorothioate communication phosphorodithioate communication (S-oligo), methylphosphate (Mr-oligo) communication phosphoroamidite communication netstate communication or methylphosphonothioate communication or combinations thereof. Modification of the ribose includes, for example, the conversion of 2'-feribotu or 2'-O-methylribose. The modified nucleotide comprises, for example, the conversion of 5-propenylboronic or 2-aminoadenine.

Here "RNA" means "RNA, which can be used as a drug, antisense RNA, containing a partial nucleotide sequence of RNA that encodes the above AILIM (preferably human AILIM}, or chemically modified RNA, which can be constructed on the basis of RNA, encoding AILIM". Antisense RNA can inhibit the transcription of DNA encoding AILIM, mRNA, or translation of mRNA into protein by hybridization with DNA or RNA encoding AILIM.

Used here, the phrase "partial nucleotide sequence" refers to a partial nucleotide sequence including the soup in itself an arbitrary number of nucleotides in any area. Partial nucleotide sequence contains from 5 to 100 consecutive nucleotides, preferably from 5 to 70 consecutive nucleotides, preferably from 5 to 50 consecutive nucleotides, and more preferably from 5 to 30 consecutive nucleotides.

The sequence of an antisense RNA may be partially chemically modified to extend the half-life in blood, when RNA is administered to patients to increase the capacity of penetration of the RNA through intracytoplasmic membrane or to increase resistance to decay or oral suction entered RNA in the digestive organs. Chemical modifications include such modifications as modifications that relate to the above antisense DNA.

Examples of "chemically synthesized compounds are arbitrary units, with the exception of the above DNA, RNA and protein substances having a molecular weight of from about 100 to 1000 or less, preferably a compound having a molecular weight of from about 100 to 800, and preferably a molecular weight of from about 100 to 600.

The term "polypeptide", included in the definition of the above "agents"means a portion (fragment) of the polypeptide component of AILIM (preferably human AILIM), preferably all ilicali extracellular region polypeptide, average of AILIM (from 1 to 5 amino acids may be optionally added to the N-terminal and/or C-terminal region).

AILIM in accordance with the present invention is a transmembrane molecule that penetrates through the cell membrane and containing 1 or 2 polypeptide chains.

In the present description "transmembrane protein" means a protein which is connected with the cellular membrane through hydrophobic region of the peptide, which "stitches" the lipid bilayer membrane one or more times, and structure which in General consists of three main areas, i.e. the extracellular region, the transmembrane region and cytoplasmic region, as in many receptors or cell surface molecules. This transmembrane protein is each receptor or a cell surface molecule in the monomer or in the form of glycosilated, heterodimer or oligomer, a conjugate of one or more chains having the same or a different amino acid sequence(s).

Here "extracellular region" means all or part of the partial structure (partial area) of the whole structure mentioned above transmembrane protein, where the partial structure exists outside of the membrane. In other words, it means the whole or part of the transmembrane region of the protein, with the exception of the area, including the TES in the membrane (transmembrane region), and the field that exists in the cytoplasm, following the transmembrane region (cytoplasmic region).

"Hybrid polypeptide"included in above "protein"refers to a hybrid polypeptide comprising all or part of the extracellular region of the polypeptide comprising the AILIM (preferably human AILIM), and "all or part of the constant region of the heavy chain of immunoglobulin (Ig, preferably human Ig)≫. Preferably, the hybrid polypeptide is a hybrid polypeptide having the extracellular region of AILIM and part of the constant region of the heavy chain of human IgG, and particularly preferably a hybrid polypeptide extracellular region of AILIM and region (Fc) of the heavy chain of human IgG, which includes the hinge domainH2 and domainH3. As IgG preferred IgG1, and as AILIM preferred human, mouse or rat AILIM (preferably human).

Used herein, the expression "all or part of the constant region of the heavy chain of immunoglobulin (Ig)" means the constant region or Fc region of the heavy chain derived from human immunoglobulin (H-chain) or a part of it. The immunoglobulin can be any immunoglobulin belonging to any class and any subclass. In particular, the immunoglobulin comprises at SEB the immunoglobulins IgG (IgG1, IgG2, IgG3 and IgG4), IgM, immunoglobulin IgA (IgA1 and IgA2), IgD, and IgE. Preferably, the immunoglobulin is an IgG (IgG1, IgG2, IgG3 and IgG4 or IgM. Examples of particularly preferred antibodies according to the invention are those related to IgG, obtained from human (IgG1, IgG2, IgG3 and IgG4).

The immunoglobulin has a Y-shaped structural unit in which four chains composed of two homologous light chains (L-chains)and two homologous heavy chains (H-chains) linked by disulfide bonds (links S-S). Light chain is composed of a variable region (VL) the light chain and the constant region (CL) light chain. Heavy chain composed of a variable region (VH) the heavy chain and the constant region (CH) the heavy chain.

The constant region of the heavy chain is composed of some of the domains have amino acid sequences that are unique to each class (IgG, IgM, IgA, IgD and IgE) and each subclass (IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2).

Heavy chain of immunoglobulin G(IgG1, IgG2, IgG3 and IgG4) composed of VH, domainH1, the hinge domainH2 and domainH3 in this order from the N-Terminus.

Similarly heavy chain IgG1 composed of VH, domain Cγ1l, hinge, domain Cγ12 and domain Cγ13 in this order from the N-Terminus. Heavy chain IgG2 made and the V H, domain Cγ2l, hinge, domain Cγ22 and domainγ23 in this order from the N-Terminus. IgG3 heavy chain composed of VH, domainγ31, hinge, domainγ32 and domainγ33 in this order from the N-Terminus. Heavy chain IgG4 composed of VH, domain Cγ41, hinge, domainγ42 and domainγ43 in this order from the N-Terminus.

Heavy chain IgA is composed of VH, domain Cα1, hinge, domainα2 and domainα3 in this order from the N-Terminus.

Similarly, the heavy chain of IgA1 composed of VH, domain Cα1l, hinge, domain Cα12 and domain Cα13 in this order from the N-Terminus. IgA2 heavy chain composed of VH, domain Cα21, hinge, domainα22 and domainα23 in this order from the N-Terminus.

Heavy chain IgD composed of VH, domainδ1, hinge, domainδ2 and domainδ3 in this order from the N-Terminus.

Heavy chain IgM is composed of VH, domainμ1, domainμ2, domainμ3 and domainμ4 in this order from the N-Terminus and has no hinge, as seen in IgG, IgA, and IgD.

Heavy chain of IgE composed of VH, domainε1, domainε2, domainε3 and domainε4 in this order from the N-Terminus and has no hinge, as seen in IgG, IgA, and IgD.

If, for example, IgG papain, it is broken down on a lightly N-terminal side of the outside of the disulfide bonds located in the hinge, where the disulfide bonds linking the two heavy chains to generate two homologous Fab fragments, where a fragment of the heavy chain composed of VHand CH1, is connected to one light chain by a disulfide bond; and one Fc, in which two homologous fragment of the heavy chain composed of the hinge domainH2 and domainH3, connected by disulfide bonds (see "Immunology Illustrated, original 2nd ed., Nankodo, pp.65-75 (1992); and "Focus of Newest Medical Science 'Recognition Mechanism of Immune System'", Nankodo, pp.4-7 (1991); and so on).

Specifically, the portion of the constant region of the heavy chain immunoglobulin"mentioned above means a portion of a constant region of the heavy chain of immunoglobulin, having structural characteristics as mentioned above and preferably represents a constant region without the C1 domain or Fc region. In particular, a sample is a region made up of a hinge domain C2 and C3 domain of each of the IgG, IgA, and IgD, or is an area composed of domain C2 domain C3 and C4 domain of each of IgM and IgE. Especially preferred example is the region of IgG1 Fc obtained in humans.

The above-mentioned hybrid polypeptide has the advantage of possibilities it is very easy to clean using affine number of the night chromatography using properties of protein a the specific data associated with a fragment of immunoglobulin, because the hybrid polypeptide according to the invention is part of a constant region (e.g., Fc) of an immunoglobulin, such as IgG, as mentioned above, as a partner hybridization. Moreover, since there are various antibodies against the Fc of various immunoglobulins, immune analysis to identify a hybrid polypeptide can be easily performed with antibodies against the Fc.

"Polypeptide that binds to AILIM" covered by the term "polypeptide", included in the definition of the above "stuff".

Specific examples of the polypeptide that binds to AILIM", is the entire polypeptide or part thereof, including known molecule called B7h, B7RP-1, GL50 or LICOS, which is a ligand that interacts with AILIM (Nature, Vol.402, No. 6763, pp.827-832, 1999; Nature Medicine, Vol.5, No. 12, pp.1365-1369, 1999; J. Immunology, Vol.164, pp.1653-1657, 2000; Curr. Biol., Vol.10, No. 6, pp.333-336, 2000).

Preferably the polypeptide is a polypeptide comprising all or part of the extracellular region of the above ligands (B7h, B7RP-1, GL50, LICOS), or a hybrid polypeptide comprising a polypeptide, and all or part of the constant region of the heavy chain of immunoglobulin (preferably a human immunoglobulin). Here the expression "extracellular region and a constant region of the heavy chain of the immunoglobulin and out the same value, as specified above.

The above polypeptides, portions of the polypeptide (fragment) and hybrid polypeptides can be obtained not only by using recombinant DNA technology, as described below, but also by the way, is well known in this area as a means of chemical synthesis or by way of cell culture, or its modified method.

"Antibody" according to the invention can be a polyclonal antibody (anticigarette) or monoclonal antibody against defined above AILIM mammals (particularly preferably human AILIM), and preferably a monoclonal antibody.

In particular, the antibody is an antibody having the activity of inhibiting proliferation of cells expressing AILIM, by binding to AILIM or activity of inhibiting the production of interferon-γ or interleukin-4 cells expressing AILIM, by binding to AILIM.

Antibodies according to the invention can be a natural antibodies obtained by immunizing mammals such as mice, rats, hamsters, Guinea pigs and rabbits, antigen, such as cells (natural cells, cell lines, tumor cells, etc.)expressing AILIM according to the invention, transformants obtained with the use of technology recombin nteu DNA in order to redundantly Express AILIM on their surface, polypeptides comprising AILIM or above hybrid polypeptides comprising the polypeptide AILIM or extracellular region of AILIM. Antibodies according to the invention also include chimeric antibodies and humanized antibodies (CDR-transplanted antibody)that can be obtained using recombinant DNA technology, and human antibodies that can be produced using transgenic animals that produce human antibodies.

Monoclonal antibodies include antibodies having any of the isotypes IgG, IgM, IgA, IgD or IgE. Preferred IgM.

Polyclonal antibodies (antisera) or monoclonal antibody can be obtained by known methods. Namely, a mammal, preferably a mouse, rat, hamster, Guinea pig, rabbit, cat, dog, pig, goat, horse, or cow, or more preferably a mouse, rat, hamster, Guinea pig or rabbit subjected to immunization, for example, the above antigen, if necessary, adjuvant's adjuvant.

The polyclonal antibody can be obtained from sera obtained from immunized thus the animal. In addition, monoclonal antibodies obtained as follows. Get hybridoma of producing antibodies of the cells, extracting the data from immunized thus animal, and myeloma cells, which are unable to produce antibodies. Hybridoma clone and conduct screening to identify clones producing monoclonal antibodies that are specific affinity to the antigen used for immunization of a mammal.

In particular, the monoclonal antibody can be obtained as follows. Immunization is performed by single or repeated injections or implantation of the above antigen as an immunogen, if necessary, adjuvant's adjuvant, subcutaneously, intramuscularly, intravenously, through the foot pad or intraperitoneally to a mammal, except man, in particular a mouse, rat, hamster, Guinea pig or rabbit, preferably a mouse, rat or hamster (including transgenic animal generated so that vibratility antibodies derived from another animal, such as the following transgenic mouse producing human antibodies). Usually immunization perform one to four times every 1-14 days after the first immunization. Cells producing antibodies, receive from immunized thus mammal in approximately 1-5 days after the last immunization. The frequency and the interval between immunizations can be appropriately adjusted depending on, for example, from property used is immunogen.

Hybridoma, which secrete a monoclonal antibody can be obtained by way Ofő'hler and Molstein (Nature, Vol.256, pp.495-497 (1975)) or its modified method. Namely hybridoma obtained by hybridization of producing antibodies of the cells contained in the spleen, lymph nodes, bone marrow or tonsils obtained from the mammal, except man, is immunized as described above, preferably in the spleen, with myeloma cells without the ability to produce antibodies, which are preferably obtained from a mammal, such as mouse, rat, Guinea pig, hamster, rabbit, or human, or preferably a mouse, rat or human.

For example, obtained from the mouse myeloma P3/X63-AG8.653 (653), P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2), PAI, FO, NSO or BW5147, obtained in the rat myeloma 210RCY3-Ag.2.3. or obtained from human myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used as a myeloma to merge cells.

Screening for producing monoclonal antibody hybridomas can be performed in the cultivation of hybridomas, for example, in microtitration tablets, by measuring the reactivity of the supernatant culture fluids in the wells in which growth of hybridoma in respect of the above immunogen used for immunization, for example, using fer entogo immune analysis such as radioimmune assay (RIA) and immunnofermentye assay (ELISA).

Monoclonal antibodies can be obtained from hybridomas by culturing the hybridomas in vitro or in vivo, such as in the ascitic fluid of mice, rats, Guinea pigs, hamster or rabbit, preferably a mouse or rat, preferably a mouse, and selection of antibodies from the resulting supernatant liquid culture or ascitic fluid of a mammal.

Cultivation of hybridomas in vitro can be performed according to, for example, from the properties of cells, the cultivation of which is expected, the objectives of the study and the different conditions of the method of cultivation is the use of known nutrient media or any medium, comes from a known baseline environment for the cultivation, maintenance and storage of hybridomas for production of monoclonal antibodies in the supernatant of the culture.

Examples of basic environments are environments with low concentrations of calcium as the environment Ham'F12 medium MCDB152 or MEM medium with a low concentration of calcium, and environment with a high concentration of calcium, such as environment MCDB104, Wednesday MEM medium (D-MEM, RPMI1640 medium, ASF104 medium or medium RD. The base medium may contain, for example, serum, hormones, cytokines, and/or various inorganic and organic substances, depending on the purpose.

Monoclonal antibodies can be is isolated and purified from the above supernatant culture or ascitic fluid by precipitation with saturated ammonium sulfate, the method of deposition euglobulin, method, Caproic acid, by the method of Caprylic acid, ion-exchange chromatography (DEAE or DE52) and affinity chromatography using antiimmunoglobulin column or columns protein A.

"Recombinant chimeric monoclonal antibody" is a monoclonal antibody obtained by genetic engineering, and specifically means a chimeric antibody such as mouse/human chimeric monoclonal antibody variable regions which are derived from immunoglobulin mammalian, non-human (mouse, rat, hamster etc), but the constant region which is derived from human immunoglobulin.

A constant region derived from human immunoglobulin has an amino acid sequence that is unique for each isotype such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD and IgE. The constant region of the recombinant chimeric monoclonal antibodies may represent that of a human immunoglobulin relating to any isotype. Preferably it represents a constant region of human IgG.

Chimeric monoclonal antibody can be obtained, for example, as follows. Needless to say that the way to obtain them is not limited.

Mouse/human chimeric who monoclonalnoe antibody can be obtained by way described in the publication Experimental Medicine: SUPPLEMENT, Vol.1.6, No.10 (1988); and in the examined published patent application of Japan No.(JP-B) Hei 3-73280. Namely, it can be obtained by surgical insertion of a gene SN (gene, encoding the constant region of H chain)obtained from DNA that encodes a human immunoglobulin downstream from active genes VH(rearangement VDJ gene encoding the variable region of the H-chain)obtained from DNA that encodes a mouse monoclonal antibody isolated from hybridoma producing mouse monoclonal antibody, and the gene WithL(the gene encoding the constant region of L-chain)obtained from DNA that encodes a human immunoglobulin downstream from active genes VL(rearangement VJ gene encoding the variable region of the L-chain)obtained from DNA that encodes a mouse monoclonal antibody isolated from hybridoma, in the same vector or different vectors expressing in the way with the subsequent transformation of host cells with the expression vector, and then the cultivation of the transformants.

In particular, DNA is first extracted from murine hybridomas producing monoclonal antibodies in the usual way, digested with appropriate restriction enzymes (e.g., EcoRI and HindIII), subjected to electrophoresis (using, for example, 0.7% of agarosegel) and analyzed by the method of southern blotting. After staining subjected to gel electrophoresis, for example, ethidiumbromid and photographing the gel assign marker position. Washed twice with water and soaked in 0.25 M HCl for 15 minutes and Then the gel is soaked in 0,4n. the NaOH solution for 10 min with gentle stirring. The DNA is transferred onto the filter for 4 hours in the usual way. The filter is recovered and washed twice 2×SSC, after the filter has sufficiently dried, it is subjected to annealing at 75°C for 3 hours. After annealing the filter handle 0,1×SSC/0,1% SDS at 65°C for 30 minutes Then it is impregnated with a 3×SSC/0,1% SDS. The resulting filter process prehybridization solution in a plastic bag at 65°C for 3-4 hours.

Then the probe DNA labeled with32P, and hybridization solution is added to the bag and allow the reaction at 65°C for 12 hours. After hybridization the filter was washed with the appropriate salt concentration, reaction temperature and time (e.g., 2×SSC/0,1% SDS, room temperature, 10 min). The filter is placed in a plastic bag small amount of 2×SSC and exposed to autoradiography after hermetic sealing of the bag.

Rearangement gene VDJ and VJ gene encoding the H-chain and L-chain mouse monoclonal antibodies, identified above by the method of southern blotting. The area contains asuu identified DNA fragment, fractionary by centrifugation in a density gradient of sucrose and perform insertion into the phage vector (e.g., Charon 4A, Charon 28, λEMBL3 and λEMBL4). E. coli (for example, LE392 and NM539) transform the vector phage to create a genomic library. Conduct screening of the genomic library by hybridization in the plaques, such as the way Benton-Davis (Science, Vol.196, pp.180-182 (1977)), using appropriate probes (H-chain gene J, L-chain (κ) gene (J), to obtain positive clones containing rearangement VDJ-gene or VJ-gene. Making the restriction map and determining the nucleotide sequence of the obtained clones, confirm whether or not genes were obtained, including the desired rearangement gene VE(VDJ) or gene VL(VJ).

Isolated human geneEand human geneLused to chimerization. For example, upon receipt of a chimeric antibody with a human IgG1, Cγ1 gene isolated in the form of a gene WithHand gene Withκ in the form of a gene WithL. These genes can be isolated from a human genomic library using mouse gene Withγ1 and murine gene Withκcorresponding, respectively, of the human gene Withγ1 and human gene Withκas probes, using the advantage of high homology between the nucleotide consistently the authorities of the gene of mouse immunoglobulin gene and a human immunoglobulin.

In particular, the DNA fragments comprising the gene Withκ and the enhancer region, isolated from a human genomic library λ Charon 4A HaeIII-AluI (Cell, Vol.15, pp.1157-1174 (1978)), for example, with the use of fragment 3 TPN HindIII-BamHI clone Ig146 (Proc. Natl. Acad. Sci. USA, Vol.75, pp.4709-4713 (1978)) and a fragment of 6.8 TPN EcoRI clone MER (Proc. Natl. Acad. Sci. USA, Vol.78, pp.474-478 (1981)) as probes. In addition, for example, after digestion of the DNA of fetal hepatocytes HindIII and fractionation by electrophoresis on agarose gel carry out the insertion of a fragment of 5.9 TPN in λ788, and then allocate human gene Withγ1 using the above probes.

Using the thus obtained murine gene VHmouse gene VLhuman geneHand human geneLand whereas the region of the promoter and the enhancer region, carry out the insertion of the human gene WithHdownstream from the mouse gene VHand the insertion of the human gene WithLcarry out downstream from the mouse gene VLin such an expression vector as pSV2gpt or pSV2neo, using the appropriate restriction enzymes and DNA ligase in the usual way. In this case, it is possible to make insertion respectively of a chimeric gene of the mouse gene VH/human geneHand the mouse gene VL/human geneLin the same vector expr is hurt or in different expression vectors.

The resulting vector(s) expression by insertion of a gene injected into myeloma, which do not produce antibodies, for example cells RH Ad'653 or cells SP210, the way to merge with protoplasts, a method using DEAE-dextran method, using the calcium phosphate method or electroporation. Conduct screening of transformants, cultivating them in media containing the drug, the corresponding gene of resistance to the drug, which is built into the expression vector, and then get the cells producing the desired chimeric monoclonal antibodies.

The desired chimeric monoclonal antibodies obtained from the supernatant of the culture subjected to this screening cells that produce antibodies.

"Humanitariannet monoclonal antibody (CDR-transplantirovannam antibody)according to the invention is a monoclonal antibody obtained by the methods of genetic engineering and, hence, in particular, humanitariannet monoclonal antibody, in which a part or all of the complementarity determining region hypervariable region originate from the complementarity determining regions of the hypervariable region of a monoclonal antibody of a mammal other than human beings (mouse, rat, hamster, etc.), the field of antigenic determinants variable regions are from a human immun who globulin, and the constant region is of the constant region of the immunoglobulin obtained in humans.

Defining complementarity region hypervariable region is hypervariable region in the variable region of the antibody and means three areas that are directly and complementary contact with the antigen (complementarity determining residues, CDR1, CDR2 and CD3). Within the box read the sections variable regions represent the four relatively conservative region, located upstream, downstream or between the three areas that define complementarity (in the frame of the reading area FR1, FR2, FR3 and FR4).

In other words, humanitariannet monoclonal antibody means an antibody in which all areas except parts of all determine complementarity areas hypervariable region of a monoclonal antibody obtained from a mammal, except man, were replaced in their respective regions derived from a human immunoglobulin.

A constant region derived from human immunoglobulin has an amino acid sequence that is unique for each isotype, such as JgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD and IgE. Constant region gumanitarnogo monoclonal antibodies in the present invention may be the howl of a human immunoglobulin, related to any isotype. Preferably it represents a constant region of human IgG. Frame reading sections of a constant region derived from human immunoglobulin, particularly not limited.

Humanitariannet monoclonal antibody can be obtained, for example, as follows. Needless to say that the way production is not limited to them.

For example, recombinant humanitariannet monoclonal antibody derived from a murine monoclonal antibodies can be obtained by genetic engineering methods, with reference to published Japanese translation of the application for an international patent (JP-WA) No. Hei 4-506458 and JP-A Sho 62-296890. Namely, at least one mouse gene CDR H-chain and at least one mouse gene CDR L-chain, the corresponding mouse gene CDR-N-chains, isolated from the hybridomas producing mouse monoclonal antibody and the human gene N-chain encoding all regions, except for the human CDR H-chain corresponding to the above murine CDR of the H chain, and the human gene for L-chain encoding the entire area, except for the human CDR L-chain corresponding to the above murine CDR L-chain genes isolated from human immunoglobulin.

Selected thus murine gene(s) CDR H-chain and the human gene(s) N-chain promptly to stable the t in the corresponding vector with the so that they can be expressed. Similarly, murine gene(s) and CDR of a human gene(s) L-chain operatively inserted into an appropriate vector so that it(and) could (?) be expressed. Alternative mouse gene(s) CDR H-chain/ human gene(s) N-chain and murine gene(s) CDR L-chain/ human gene(s) L-chain can be expressed follows operatively inserted into the same expression vector. The host cells are transformed thus obtained expression vector to obtain transformants producing humanitariannet monoclonal antibody. The cultivation of transformants from the supernatant culture fluids get humanitariannet monoclonal antibody.

"Human monoclonal antibody" is an immunoglobulin in which all areas, including variable and constant region of H chain and the variable and constant region of L-chain comprising immunoglobulin derived from genes encoding human immunoglobulin.

Human antibody (preferably a human monoclonal antibody) can be well-known methods, for example, in the same way as the above method of obtaining polyclonal or monoklonalnyh antibodies by immunization with antigen transgenic animal obtained integrirovanie the m at least gene of human immunoglobulin in the locus of the gene of the mammal, besides humans, such as a mouse.

For example, transgenic mice that produce human antibodies produced by the methods described in Nature Genetics, Vol.7, pp.13-21 (1994); Nature Genetics, Vol.15, pp.146-156 (1997); JP-a Hei WA 4-504365; JP-a Hei WA 7-509137; Nikkei Science, No. 6, pp.40-50 (1995); WO 94/25585; Nature, Vol.368, pp.856-859 (1994) and JP-WA No. Hei 6-500233.

In addition, you can also apply a recently developed method of production obtained from the human protein from the milk of transgenic cows or pigs (Nikkei Science, pp.78-84 (April, 1997)).

The phrase "antibody"used in the present invention means a partial region of a monoclonal antibody as described above. She, in particular, means F(ab')2, Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv (Fv, stabilized by disulfide) or dAb (antibody single domain) (Exp. Opin. Ther. Patents, Vol.6, No. 5, pp.441-456 (1996)).

"F(ab')2and Fab'can be obtained by treatment of immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and means the antibody fragment generated by digestion of immunoglobulin near the disulfide bonds in the hinge regions existing between each two N-chains. For example, papain cleaves IgG upstream from disulfide bonds in the hinge regions existing between each two H chains to generate two homologous fragments of antibodies, in which L-chain, composed of VL- (in Zabelina area L-chain) and C L- (const region L-chain) and a fragment of the H chain, composed of VH- (variable region of H chain) and CHγ1 (γ1-region in the constant region of H chain) are connected at their C-terminal regions through disulfide bonds. Each of these two fragments homologous antibodies called Fab'. Pepsin also cleaves IgG downstream from disulfide bonds in the hinge regions existing between each of the two H chains to generate antibody fragment, slightly larger than the fragment in which the above two Fab' connected near the hinge region. The specified fragment of the antibody is called F(ab')2.

The term "graft rejection" according to the invention refers to a variety of immune reactions, which are involved in the rejection and destruction of the graft (part of a living organism, which transplanted the cells, tissue or organ from a donor whose genetic background is different from that of the recipient (i.e allotransplantation or xenotransplantation), because the recipient recognizes the graft as foreign substance. Immune reactions that accompany specified transplantation can be classified into (1) Verhoture exclusion, which is a strong rejection occurring immediately after transplantation, (2) acute rejection, which is observed is camping within a few months after transplantation, and (3) chronic rejection observed several months after transplantation. In addition, although cell-mediated immunity due to immunocompetent cells and presented to T-cells and humoral immunity due to antibodies occur endogenous co-ordinated way, the main reaction is carried out by cellular immunity.

The result of rejection of the transplant eventually becomes necrotic and fall off. In addition, patients develop not only severe systemic symptoms such as fever, leukocytosis and fatigue, but also swelling and soreness at the site of transplantation. In addition, you may experience severe complications, such as infections.

In particular, the transplantation of xenogenic graft, such as a transplant from a pig, there is a serious problem sverhmoschnogo exclusion, in which the graft is rejected within minutes.

The term "transplant" according to the invention refers to the "organ" or "fabric", which are transplanted to the mammal to the recipient from a mammalian donor.

The phrase "organ or part"relating to transplantation according to the invention, refers to any body or its parts that make up the living body of a mammal (preferably human or pig, and W is preferably human). A preferred example is the liver, heart, lung, pancreas, kidney, colon, small intestine or part of them. Especially preferred is the liver or part of it.

The term "tissue"related to transplantation according to the invention, refers to any tissue derived from a living body of a mammal (preferably human or pig, and particularly preferably human). The preferred sample is a tissue, such as skin, cornea, bone or heart valve; however, it is not limited to them.

The term "immunosuppressive agent" according to the invention relates to any one or more existing immunosuppressive means used to suppress immunological rejection (transplant rejection) in the recipient, which is caused by the transplantation of the graft in clinical transplantation of cells, tissues or organs, whose production and sale as pharmaceutical agents have been approved by any governmental Agency; or any one or more immunosuppressive tools that can be used currently in clinical or preclinical trials or will be applied in clinical trials in the future, whose production and sale as pharmaceutical agents may be approved rights of the governmental organization after the tests.

Such immunosuppressive remedies are used not only separately but also in combination with 2, 3 or more means. Therefore, the term "immunosuppressive agent" in accordance with this invention includes the use of one type of pharmaceutical agents or combined use of many pharmaceuticals (preferably used in combination with 2 or 3 means).

Preferably, the immunosuppressive agent is, for example, one or more pharmaceutical agents selected from cyclosporine (CsA); tacrolimus (FK-506); azathioprine (AZ); mycophenolate (MMF); mizoribine (MZ); Leflunomide (LEF); adrenocorticosteroids (otherwise referred to as the adrenal cortex hormones; corticosteroids; corticoide), such as prednisolone and methylprednisolone; sirolimus (otherwise known as rapamycin); desoxypeganine (DSG); FTY720 (chemical name: 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propandiol hydrochloride) and drug CTLA4 described below. Especially preferred is any of tacrolimus (FK-506) and cyclosporine.

The term "drug CTLA4" according to the invention relates to a medicinal product which contains as active ingredient (1) a polypeptide comprising a full length (including molecules with almost identical amino acid is the selected), or completely or partially extracellular region of human CTLA4 (associated with cytotoxic T-lymphocyte antigen 4; <amino acid sequence> Genetic Bank no access NP 005205; <share> Genetic Bank no access NM 005214); (2) a hybrid polypeptide comprising a fully or partially extracellular region of human CTLA4 and fully or partially by another protein (particularly preferably fully or partially constant region of the heavy chain of human immunoglobulin (hereinafter represented here by the abbreviation CTLA4-IgFc or CTLA4-Ig); or (3) DNA, which can provide to a mammal (particularly preferably human) polypeptide under item(1), or hybrid polypeptide under item (2), or a vector comprising DNA (particularly preferred is a plasmid, typically used in gene therapy, or a viral vector derived from a virus (retrovirus, adenovirus, adeno-associated virus, etc), or the like).

In this description, each of the terms/phrases such as "extracellular region", "part", "the constant region of the heavy chain immunoglobulin", "hybrid polypeptide" and "almost the same" have the same meaning as defined above.

There are several reports of significant immunosuppressive effect mentioned above CTLA4-Ig. For example, high immunosuppressive effect Y100F (tyrosine at position 100 is replaced with phenylalanine), developed by Bristol-Myers Squibb/Repligen, was confirmed in different the x experiments on animals, and this product is also included as one of the drugs CNLA4 of the present invention (Igaku no Ayumi, Vol.194, No. 14, R-1200, 2000; J. Clin. Invest., Vol.103, p.1223-1225, 1999; N. Engl. J. Med., Vol.335, p.1369-1377, 1996; J. Exp. Med., Vol.178, p.1801-1806, 1993; Blood, Vol.94, p.2523-2529, 1999; Nature Med., Vol.6, p.464-469, 2000; Blood, Vol.83, p.3815-3823, 1995; J. Clin. Invest., Vol.2, p.473-482, 1998; Blood, Vol.86, p.2607-2612, 1995; N. Engl. J. Med., Vol.340, p.1704-1714, 1999; N. Engl. J. Med., Vol.335, p.1369-1377, 1996; J. Clin. Invest., Vol.103, p.1243-1252, 1999).

The phrase "pharmaceutically acceptable carrier" according to the invention includes a filler, a diluent, a filler, a leavening agent, stabilizer, preservative, buffer, emulsifier, an aromatic agent, coloring agent, sweetening agent, an agent that increases the viscosity, the perfume, the agent that increases the solubility or some other additive. When using one or more such carriers, the pharmaceutical composition may be formulated into tablets, pills, powders, granules, solutions for injection, solutions, capsules, tablets, elixirs, suspensions, emulsions, syrups, etc.

The pharmaceutical composition can be administered orally or parenterally. Other forms of parenteral administration include topical solution, a suppository for rectal administration and pessaries that are assigned in the usual way, which includes one or more active ingredients.

The dosage may vary depending the spine ages, sex, body weight and symptoms of the patient, treatment effect, route of administration, duration of treatment, type of active ingredient (as specified above "substances" in accordance with the present invention)contained in the pharmaceutical composition, etc., are Usually pharmaceutical composition, you can enter an adult in a dose of from 10 mg to 1000 mg (or from 10 μg to 500 mg) in one introduction. Depending on various conditions in some cases it may be sufficient dosage less than that specified above, while others may require a dosage greater than specified above.

In the case of solution for injection, it can be obtained by dissolution or suspension of the antibody in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection, when making concentration to range from 0.1 μg antibody/ml carrier to 10 mg antibody/ml carrier. Thus obtained solution for injection can be entered to a human patient in need of treatment, at doses ranging from 1 μg to 100 mg/kg body weight, preferably in the range of from 50 μg to 50 mg/kg body weight, once or several times per day. Examples of routes of administration are appropriate from a medical point of view, routes of administration, such as intravenous injection, subcutaneous injection, wew is Icona injection, intramuscular injection, intraperitoneal injection or the like, preferably intravenous injection.

Injection can also be obtained in non-aqueous diluent (for example, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, alcohol such as ethanol), suspensions or emulsions. Injection can be sterilized by filtering through a filter to filter bacteria, by mixing with a bactericidal agent, or irradiation. The solution for injection can be obtained in such a way that it is prepared at the time of application. And it lyophilizer that he was a solid composition which can be dissolved in sterile distilled water for injection or other solvent before use.

The pharmaceutical composition according to the invention is extremely useful for suppression, prevention and/or treatment of immunological rejection (transplant rejection), which is a serious problem with such treatments, when an organ (liver, heart, lung, kidney, pancreas etc) or part or tissue (such as skin, cornea and bone) from a donor transplanted (allotransplantation or xenotransplantation) recipient affected severe cardiovascular disease.

In addition, the headlight is aseptically composition according to the invention can increase the effect of suppression of immunological rejection (transplant rejection) existing immunosuppressive means, appointed to suppress transplant rejection during such treatments transplant, when the pharmaceutical composition is used in combination with immunosuppressive agents.

Brief description of drawings

Figure 1 shows the effect of antibody against AILIM and/or immunosuppressive funds for the suppression of immunological rejection (transplant rejection), accompanying the transplantation of organs, using as an indicator of the prolongation of graft survival in recipients who were transplanted liver from a donor.

Figure 2 shows the effect of suppression of immunological rejection (transplant rejection), which occurs accompanying the organ antibody against AILIM and/or immunosuppressive agent, when used as an indicator increase in number of days of survival of liver transplant, transplanted from a donor to a recipient.

Figure 3 shows the effect of suppression of immunological rejection (transplant rejection), which occurs accompanying the organ antibody against AILIM (otherwise referred to as the antibody against ICOS) when used as an indicator increase in number of days of survival of heart transplant, transplanted from a donor to a recipient.

Figure 4 is a photograph showing the degree of infiltration cleto is, expressing AILIM, transplantirovannam heart.

Figure 5 shows the effect of suppression of immunological rejection (transplant rejection), which occurs accompanying the organ antibody against AILIM/or AdCTLA4-Ig, when used as an indicator increase in number of days of survival of heart transplant, transplanted from a donor to a recipient.

Figure 6 shows the effect of antibody against AILIM used in combination with AdCTLA4-Ig, suppression of immunological rejection (transplant rejection), accompanying the organ when used as an indicator of the presence of the presence or absence of graft survival of transplanted hearts (primary heart transplantation and secondary transplantation of the heart) and the transplanted skin (primary transplantation of skin) to the recipient.

The best way of carrying out the invention

Hereinafter the present invention will be specifically illustrated with reference to examples, but it should not be seen as limited versions of the implementation described in the examples.

[Example 1] the suppression of transplant rejection substance modulating AILIM liver transplantation

<1> Materials and methods

<1-1> Animals

Adult Lewis rats (male, 210-250 g) and rats DA (males, 210-250 g) were used respectively in quality is TBE recipients and donors.

<1-2> Monoclonal antibody against rat AILIM

Used a monoclonal antibody, purified from ascitic fluid or supernatant of the culture obtained by culturing in vitro or in vivo hybridoma called "JTT-1", which was previously reported (this hybridoma was deposited at the international level, October 11, 1996 at the National Institute of biological Sciences and technology person, Advanced scientific industry and technology Ministry of economy, trade and industry, which is an international Depository Agency, certified in accordance with the Budapest Treaty. The international access: FERM BP-5707), which produces a mouse monoclonal antibody against rat AILIM (mouse monoclonal antibody against rat antigen JTT-1) (JP-A Hei 11-29599 (examples 1 and 2) and international patent application No. WO98/38216 (examples 1 and 2). Next, the specified antibody is called simply "antibody against AILIM".

<1-3> liver Transplantation

In accordance with previously published method Kamada et al., liver donors DA rats transplanted recipients-Lewis rats (Surgery, 93, R, 1983; Transplantation, 30, R, 10980; Transplantation, 28, R, 1979).

Specifically, livers obtained from rats DA, washed with a strong jet of ice-cold sterilized distilled water through the portal in the well. Then a liver transplant recipients-Lewis rats began with stitching nadpochechnoj Vena cava. Then, using the cuff technique, has made the portal vein and subhepatic Vena cava (Transplant. Proc., 19, R, 1987; Transplantation, 43, R, 1987).

If rats-recipients died within 3 days after transplantation, it was determined as a technical failure of the transplant. As a result, the success of transplantation was 95%.

<1-4> Introduction antibody against AILIM and/or immunosuppressive tools

After transplantation, each of Lewis rats (each group consisted of 5-9 animals) was administered antibody against AILIM and/or immunosuppressive agent FK-506 in the following doses and at a specified time. The day was completed transplantation considered zero-day (0).

The group did not enter any antibody against AILIM or immunosuppressive agent FK-506, used as a control.

1. Antibody against AILIM (1 mg/kg; intravenous injection; day 0)

2. Antibody against AILIM (1 mg/kg; intravenous injection; day 0 and 6)

3. Antibody against AILIM (1 mg/kg; intravenous injection; day 0, 3 and 6)

4. Antibody against AILIM (1 mg/kg; intravenous injection; day 0, 3, 6, 9 and 12)

5. Antibody against AILIM (0.3 mg/kg; intravenous injection; day 0, 3, 6, 9 and 12)

6. FK-506 (1 mg/kg; intramuscular injection; day 0)

7. Antibody FR is in AILIM (1 mg/kg; intravenous injection; day 0 and FK-506 (1 mg/kg; intramuscular injection; day 0)

The duration of survival of the transplanted liver in the recipient has been evaluated and determined in accordance with the criteria Kaplan-Maier.

<2>

The results are shown in figure 1 and figure 2. Part of the data in figure 2 represent a modernized data figure 1.

As a result, in the group which was administered antibody against AILIM (1 mg/kg) 3 or 5 times over a period of time after transplantation was observed a significant prolongation of graft survival compared with survival in control.

In addition, the group has introduced a low dosage of antibody against AILIM (0.3 mg/kg) 5 times within a period of time after transplantation, also observed a similar, significant prolongation of the survival of the transplanted liver.

In addition, surprisingly, when the antibody against AILIM was introduced even only once in combination with FK-506, which is an immunosuppressive agent used in clinical settings for many purposes, graft survival of transplanted liver was significantly lengthened, becoming significantly longer than when once introduced only FK-506 (1 mg/kg).

Based on these results revealed the following.

1) Antibody against AILIM significantly inhibits graft rejection (immune exclusion), which resistance is ordet transplantation of the graft, such as the body.

2) graft Rejection that accompanies the transplantation of the graft, such as a body can be further suppressed by applying an antibody against AILIM in combination with an immunosuppressive agent in comparison with the use of only one of them.

[Example 2] Suppression of immunological rejection substance modulating AILIM, when heart transplantation (part 1)

<1> Reagents, animals and method of testing

<1-1> Animals

As respectively recipients and donors used adult mice SN/Not (males aged 6 weeks) and BALB/c mice (males at the age of 6 weeks).

<1-2> Obtaining monoclonal antibodies against mouse AILIM

Obtaining carried out as follows.

Using cDNA encoding the full length amino acid sequences of previously published data on mouse AILIM (Int. Immunol., Vol.12, No. 1, 51-55, 2000), were transformed cell expressing AILIM, in accordance with standard methods using genetic recombinant technology.

Transformed cell homogenized and centrifuged in an ultracentrifuge (1000000×g) and subjected to centrifugation, the residue containing the fraction of the cell membrane, collected and suspended in saline solution with phosphate buffer. Poluchenno the fraction of the cell membrane were injected with together with complete adjuvant-blockers in the foot pad Wistar rats for primary immunization (day 0). In addition, the fraction of the cell membrane was introduced as an antigen in the foot pad with intervals on the 7th, 14th and 28th days. 2 days after the final immunization took cells of the lymph node.

Cells of lymph node cells and mouse myeloma PAI (JCR No. V; Res. Dosclosure, Vol.217, p.155, 1982) was mixed in a ratio of 5:1 and hybridoma producing monoclonal antibody was obtained by merging cells using polyethylene glycol 4000 (Doehringer Mannheim) as the merge agent. The selection of hybridoma spent in cultivating containing gipoksantin-aminopterin-thymidine (GAT) ASF104 medium (Ajinomoto)containing 10% fetal bovine serum, aminopterin.

The fluorescence intensity of cells stained in the reaction supernatant fluids cultures of each hybridoma with the above transfitsirovannykh cells expressing recombinant mouse AILIM, and then ensure their reaction with labeled fluorescein-isothiocyanato anti-mouse IgG (Cappel), was measured using the flow cytometer to confirm the reactivity of monoclonal antibodies produced in the supernatant of the culture of each hybridoma against mouse AILIM. The result was several hybridomas which produced monoclonal antibodies with reactivity against mouse AILIM.

One of these hybrid called "V". This hybridoma (1 6-107cells/0.5 ml/each mouse) were intraperitoneally injected with mice ICR nu/nu (females aged 7 to 8 weeks). 10-20 days to mice under anesthesia, laparotomy was performed and carried out large-scale obtaining a rat monoclonal antibodies against mouse AILIM (IgG2a) from ascites obtained in accordance with standard procedures. Then, the antibody was just called "antibody against AILIM".

<1-3> heart Transplantation

In accordance with previously published method heart donor BALB/c mice transplanted into the abdominal cavity of mice recipient SN/No. The disappearance of ripple transplantirovannam heart was regarded as the completion of transplant rejection.

<2> Experiment 1 (introduction antibody against AILIM)

Each mouse SN/No (10 mice), which was completed transplantation, antibody against AILIM (10 mg/kg) was administered immediately after transplantation (day 0; 200 μg)on day 2 (200 mg), 4-th day (200 mcg), 7th day (200 µg) and 10th day (100 µg). The group, which did not enter the antibody against AILIM (25 mice), was used as control.

The duration of survival of the graft of transplanted heart in the recipient after transplantation has been evaluated and determined in accordance with the criterion of Kaplan-Meier.

The average duration of survival of the transplanted heart in the recipient was following the nd:

(the group which was administered antibody against AILIM)

the duration of survival of transplant: 9 days, 1 mouse, 10 days in 3 mice 13 days at 4 mice 16 days 2 mice (control group)

the duration of survival of transplant: 6 days, 2 mice, 7 days in 9 mice 8 days 7 mice 9 days in 3 mice, 10 days in 4 mice.

The duration of graft survival in the control group, which did not enter the antibody against AILIM, was 7.9 days, but in contrast she was 12.3 days in the group that was administered the antibody against AILIM, and a significant prolongation of graft survival of transplanted hearts was demonstrated in the group which was administered antibody against AILIM.

<3> Experiment 2 (introduction antibody against AILIM

Used animals (donors and recipients) and the antibody against AILIM were the same as described above.

Heart transplantation was performed in a way similar to experiment 1.

Each mouse SN/No, which was completed transplantation, antibody against AILIM (100 µg/day) was administered intraperitoneally immediately after transplantation (day 0), day 2, day 4, day 7 and day 10. The group, which did not enter the antibody against AILIM, was used as control.

The mean survival of the graft, transplanted into a recipient, amounted to approximately 7.7 days in the control group, at that time what I like in the group, which was injected antibody against AILIM, it was approximately 40.9 days (intermediate value: 29 days/maximum: 120 days) (figure 3). Namely, in the group which was administered antibody against AILIM was demonstrated significant prolongation of the survival of the graft of transplanted heart.

In addition, staining hematoxylin/eosin (staining) in accordance with standard methods, the degree of infiltration of cells expressing AILIM (ICOS), transplantirovannam heart analyzed each of the control mice (without therapy after heart transplantation) and in mice after transplantation was introduced antibody against AILIM.

As a result, in the untreated group observed a significant infiltration of cells expressing AILIM (ICOS), and necrosis of the heart muscle (the painted part). On the other hand, in transplantirovannam heart mice, which were injected antibody against AILIM, necrosis of the heart muscle is not observed, and it was confirmed a significant reduction in the infiltration of cells expressing AILIM (ICOS) (figure 4).

[Example 3] the Suppression of immunological rejection substances modulating AILIM, heart transplant and skin

<1> Reagents, animals and method of testing

<1-1> Adenoviral vector

Recombinant adenovirus containing the expression cassette or to the NC, coding hCTLA4-Ig (a hybrid protein comprising the extracellular region of human CTLA4 and human Fc)or gene β-galactosidase (LacZ) E. coli, was obtained by homologous recombination between the expression cassette of Comedy pAdex/CAhCTLA4-Ig (Transplantation, Vol.68, No. 6, R, 1999) and the genome of the parent strain of adenovirus (Proc. Acad. Sci. USA., Vol.90, No. 24, R-11502, 1993).

Then the proliferation of the recombinant virus inside 293-cell line derived from human kidney. The resulting virus was collected and stored at -80°C. the Recombinant virus containing the cDNA hCTLA4-Ig, and adenovirus containing LacZ, were named respectively AdCTLA4-Ig and AdLacZ.

<1-2> Animals and antibody

Adult males (210-250 g) Lewis rats (RT11) were used as recipients, and adult males (210-250 g) DA rats (RT1a) or BN (RT1n) were used as donors.

Used mouse monoclonal antibody against rat AILIM obtained in example 1.

<1-3> heart Transplantation and skin and the way to test

In accordance with previously published method (J. Thorac. Cardiovasc. Surg., Vol.57, No. 2, R-229, 1969) hearts obtained from DA rats, transplanted into the abdominal cavity of Lewis rats. Directly after heart transplantation in rats-recipients intravenously injected antibody against rat AILIM (1 mg/kg) and/or AdCTLA4-Ig (109nl is soobrazuya units; pfu) per dose.

The group of animals with a transplant, which was not introduced any antibody against rat AILIM or AdCTLA4-Ig, and the group of animals with a transplant, which was administered AdLacZ, were used as controls. The method of treatment of each group of animals represented, as shown below:

Group 1: Allotransplantation (Lewis/DA) without immunosuppressive treatment.

Group 2: Istranslatable (Lewis/Lewis) without immunosuppressive treatment.

Group 3: Allotransplantation (Lewis/DA) with the introduction of AdLacZ.

Group 4: Allotransplantation (Lewis/DA) with the introduction of AdCTLA4-Ig.

Group 5: Allotransplantation (Lewis/DA) with the introduction of antibody against AILIM.

Group 6: Allotransplantation (Lewis/DA) with the introduction of AdCTLA4-Ig and antibody against AILIM.

The disappearance of the pulsation of the transplanted heart was regarded as the completion of transplant rejection. Graft rejection was confirmed by histological analysis of mononuclear cells, which were unfilterable tissue transplanted heart, and necrosis of muscle cells, confirmed by staining according to standard methods.

Then in the side wall of the thoracic cavity of rats-recipients from group 4 and group 6, in which the transplanted heart has survived for a long period, 140 day after heart transplantation transplanted thick enough the first transplant of rat skin donor line DA. After transplantation of the skin immunosuppressive treatment with antibody against AILIM, AdCTLA4-Ig or similar did not. The end of the duration of survival of the skin graft was determined when the degree of visually noticeable skin graft was reduced to 10% or less of the original state.

Then on the 200-th day from the original heart transplant DA rats using the technique of cuff (Acta Pathol. Environ. Scand. [A], Vol.79, R-272, 1971) heart donor rat DA again transplanted into the neck 3 rats-recipients from group 6, which showed signs of transplant rejection after receipt of skin graft donor.

Next, on the 150th day after the original heart transplant from donor rats DA heart donors BN rats transplanted the remaining rats-recipients from group 6, which transplantirovannam heart survived for a long period.

Statistical evaluation of the degree of survival of transplant recipients was performed in accordance with the criterion of Kaplan-Meier.

<2> Results of experiments

As shown in figure 5, a significant prolongation of graft survival of transplanted heart recipients compared with the untreated group of animals who performed the xenotransplantation (group 1)were observed in the group of animals that were administered AdLacZ (group 3), and in the group of animals which were injected odnokratno the dose of antibody against AILIM (group 5).

On the other hand, in the group of animals which were injected AdCTLA4-Ig (group 4), survival of the graft of transplanted heart (original transplanted rat heart DA) was significantly prolonged (on average, approximately 64 days). Moreover, 3 rats from group 4 (10 rats) observed the survival of the graft of transplanted heart for a long period, amounting to 100 days or more (figure 5).

Moreover, in the group of animals that have been used AdCTLA4-Ig and antibody against AILIM in combination (group 6), survival of the graft of transplanted heart (original hearts of the rats DA) indefinitely lengthened (300 days or more) in all recipients (figure 5).

Rats-recipients group 4 transplantirovannam heart (original transplantirovannam heart donor rats DA) were rejected along with the rejection of the transplanted skin, whereas in rats-recipients group 6 the rejection of transplanted hearts were not observed.

As shown in Fig.6, all rats-recipients of group 4 and group 6, which was transplanted skin from the donor, transplantirovannam skin was torn away. However, in contrast to group 4 and the control group, in which the rejection of the transplanted skin was completed in 12 days or less after the date of transplantation of skin, the complete rejection of several tsachilas was observed within 16 days or less in group 6. T is some result shows that combined use AdCTLA4-Ig and antibody against AILIM may delay the rejection of transplanted skin compared with the case where AdCTLA4-Ig is applied separately.

Interestingly, in rats-recipients from group 6, which was confirmed by the long-term survival of the graft of transplanted heart (original hearts of the rats DA), transplantirovannam skin was completely rejected, as indicated above, but the survival of the graft was observed during the indefinite period of the second transplanted heart (heart of rats donor DA, transplantirovannam second time). In addition, in rats-recipients, which the original transplanted heart has survived throughout the study period.

Recipients of group 6, which transplanted rat heart donor DA, the original transplanted rat heart DA continued to pulsate and survived throughout the study period, but the hearts of rats donor BN, transplanted a second time, were rejected within a period similar to the results obtained in animals of group 1.

Industrial applicability

The pharmaceutical compositions according to the invention is extremely useful for suppression, prevention and/or treatment of immunological rejection (transplant rejection), serious problems accompanying the methods of treatment by the graft is tion (allotransplantation or xenotransplantation) organs (liver, heart, lungs, kidneys, pancreas and so on), their parts or tissues (skin, cornea, bone, etc.) from donors to recipients affected severe cardiovascular diseases.

The pharmaceutical compositions according to the invention may also stronger to suppress transplant rejection when used in combination with the existing immunosuppressive drugs, which is administered to suppress transplant rejection (immune exclusion) when such transplant treatments.

In addition, the pharmaceutical composition comprising the human antibody against AILIM included in the pharmaceutical compositions according to the invention is extremely useful drug because it does not cause any side effects such as allergies, when the antibody derived from mice injected man.

1. Pharmaceutical composition for suppression, treatment, or prevention of transplant rejection, accompanying the organ part, or tissue, and this composition includes an effective amount of a substance having the activity of a modulating signal transduction mediated activation-induced lymphocytic immunomodulatory molecule (AILIM), and a pharmaceutically acceptable carrier.

2. Pharmaceutical HDMI is required to enhance the effect of one or more immunosuppressive means to suppress, treatment or prevention of transplant rejection, accompanying the organ part, or tissue, and this composition includes an effective amount of a substance having the activity of a modulating signal transduction mediated activation-induced lymphocytic immunomodulatory molecule (AILIM), and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, in which the specified immunosuppressive agent is one or more therapeutic agents selected from the group consisting of azathioprine, adrenocorticosteroid, cyclosporine, mizoribine and tacrolimus (FK-506), mycophenolate mofetil, Leflunomide, sirolimus, desoxypeganine, FTY720 and drug CTLA4.

4. The pharmaceutical composition according to claim 1, where the specified transplantation is allotransplantation.

5. The pharmaceutical composition according to claim 1, where the specified transplantation is a xenotransplantation.

6. The pharmaceutical composition according to claim 1, where the body is the liver, heart, kidney, lung or pancreas.

7. The pharmaceutical composition according to claim 1, where said fabric is a skin, cornea or bone.

8. The pharmaceutical composition according to claim 1, in which the specified substance is a white the TV stuff.

9. The pharmaceutical composition of claim 8, where the specified protein substance selected from the group consisting of

a) an antibody which binds to AILIM or a portion of the indicated antibodies;

b) a polypeptide comprising all or part of the extracellular region of AILIM;

c) a hybrid polypeptide comprising all or part of the extracellular region of AILIM, and fully or partially constant region of the heavy chain of an immunoglobulin; and

d) a polypeptide that binds to AILIM.

10. The pharmaceutical composition according to any one of claims 1 to 7, in which the specified substance is a non-protein substance.

11. The pharmaceutical composition of claim 10, where the specified non-protein substance is a DNA, RNA, or chemically synthesized compound.

Priority items:

01.03.2001 according to claims 1, 2, 4-11;

16.01.2002 according to claim 3.



 

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FIELD: biotechnology, immunology.

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1 tbl, 1 ex

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6 cl, 1 tbl, 9 dwg, 10 ex

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EFFECT: higher efficiency of application.

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24 cl, 2 tbl

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