MXPA98002536A - Antibodies anti- - Google Patents

Abstract

Human anti-Fas antibodies that are cross-reactive with mouse and human Fas, useful in the treatment of conditions attributable to abnormalities in the Fas / F-ligand system

Description

ANTI-ANTIBODIES ANTI-FAS FIELD OF THE INVENTION The present invention relates to antibodies and fragments thereof, especially humanized antibodies that recognize the Fas antigen, to DNA encoding all or part of said antibody, and to agents comprising said antibodies, for the prophylaxis and treatment of conditions arising from Abnormalities in Fas system / Fas ligand.

BACKGROUND OF THE INVENTION The physiological death of cells in a living organism, in the natural course of events, is known as apoptosis, and is distinguished from the pathological death of cells, ie, necrosis [see Kerr et al. (1972) Br.

J. Cancer, 26, 239 et seq.]. Apoptosis is an example of programmed cell death, which is when certain cells are programmed, in advance, to die in a living organism in the natural course of events, such as when the cell in question has performed a predetermined function. Apoptosis is characterized by morphological changes such as the curved surface of the cell, condensed nuclear chromatin and fragmented chromosomal DNA, among others. Apoptosis has an important role to play in the differentiation of lymphocytes (T cells and B cells) by eliminating cells that recognize an autoantigen. In this regard, it has been shown that 9% of cells, or even more, such as those that react with autoantigens, are eliminated in the thymus during the maturation of C-T lymphocytes Shigekazu Nagata. Tanpakushitsu Kakusan Koso, (1993), 38, 2208-2218], When such cells are not eliminated by apoptosis, then this is considered to be a cause of autoimmune disease, due to the presence of mature self-reactive lymphocytes in the system [ see Nakaya and others (1995), Mebio, 12 (10) 79-86]. Several molecules involved in apoptosis have been identified, including: Fas [see Yonehara, S., et al., (1989), J. Exp. Med., 169, 1747-1756]; tumor necrosis factor receptor [see Loetscher, H., et al., (1990), Cell, 61, 351-359]; CD40 [see Tsubata, T., et al. (1993), Nature, 364, 645-648]; and perforin / granzyme A [see Jenne, D. E, "et al., (1988), Im unol. Rev. 103, 53-71]. Fas is a transmembrane protein, present on the cell surface, and the binding of its extracellular domain to a protein generally known as the "Fas ligand", expressed on the surface of other cells, induces apoptosis in the cell that expresses Fas . Abnormalities in the Fas / Fas ligand system result in several disorders, due to failure in the suppression of cells that could be harmful to homeostasis, and that should be eliminated by apoptosis or, alternatively, by induction of apoptosis in unscheduled cells otherwise for their elimination, and which could be essential to maintain homeostasis. Said disorders are referred to herein as conditions arising from abnormalities in the Fas / Fas ligand system. In the development or progression of diseases arising from Abnormalities of the Fas / Fas ligand system, it is often the case that abnormal cells, which express Fas, but which, however, are not suppressed (abnormal cells), attack tissues or normal cells or they proliferate abnormally, thereby causing disorders in tissues or cells which, in turn, lead to the respective disease symptoms. In some cases, these disorders may arise from, or may be exacerbated by, the expression of Fas on abnormal cells, thereby stimulating apoptosis in normal tissues or cells. Specific examples of diseases attributable to Abnormalities of Fas / Fas ligand are as follows.

Autoimmune diseases Relationships between several human autoimmune diseases have been reported many times (Hashimoto's disease, general lupus erythematosus, Sjögren's syndrome, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus., scleroderma, Goodpasture syndrome, Crohn's disease, hemolytic anemia is an autoimmune disease selected from the group consisting of sterility, myasthenia gravis, multiple sclerosis, Basedo's disease, thrombocytopenia purpura, rheumatoid arthritis) and abnormalities in the Fas / ligand system of Fas. In the mouse, several genetic abnormalities of the Fas / Fas ligand system are known, including the lpr (lymphoproliferation), gld (generalized lymphoproliferative disease), and lpr0 a (where the Ipr gene complements the gld gene) mutations. Mice that have such genetic abnormalities exhibit all various autoimmune symptoms, accompanied by general characteristic swelling of the lymph nodes. The mouse MRL-lpr / lpr, a mouse model of spontaneous human general lupus erythematosus, shows a marked increase in the mass of its lymph nodes and produces autoantibodies that cause nephritis due to the formation of immune complexes. It is speculated that this mouse exhibits this pathology as a result of a mutation in the Fas gene, resulting in the lack of immunological tolerance to autoantigens due to lack of Fas-induced apoptosis in the peripheral system, as well as the long-term accumulation of cells. Activated autoreactive T [see Strasser, A., Nature, 373, 385 (1995)]. In humans, several cases have been reported, including two pediatric cases including lymph node swelling, hyper-gamma globulinaemia and marked increase in CD8 CD8 T cells [see Sneller, MC, et al., (1992), J.

Clin. Invest., 90, 334]. These cases were reported based on abnormalities in the Fas gene [see Fisher, G. H., et al., (1995), Cell, 81, 935; and Rieux-Laucat, F., and others, (1995), Science, 268, 1347], and it was designated as autoimmune lymphoproliferative syndrome (ALPS). Based on these findings, it is considered that the apoptosis-inducing system, mediated by Fas, is involved to a large extent in the establishment and maintenance of self-tolerance, not only in the mouse but also in the human, and the disorders of this system. induce several autoimmune diseases. It is also known that rheumatoid arthritis has an autoimmune element, based on the fact that the vast majority of T cells that invade infected regions of patients with rheumatoid arthritis and that cause tissue destruction express Fas [see Hoa, TTM, and others, (1996), J. Rheumatol., 23, 1332-1337]. Many cases of insulin-dependent diabetes mellitus originate from a critical reduction in insulin secretion, due to the destruction of pancreatic beta cells by autoreactive T cells. In this way, the elimination of autoreactive T cells is important in the radical treatment of certain forms of insulin-dependent diabetes mellitus. In graft-versus-host disease, such as occurs after a bone marrow transplant, Fas expression increases in the affected organ, and there is a direct correlation between the degree of increase in Fas expression and damage to the target organ [see Ch. , JL, et al. (1995), J. Exp. Med., 181, 393]. Therefore, the purpose in the prevention or treatment of this disease is to block apoptosis in target organ cells and decrease the number of cells attacking the target organ.

Allergic diseases The inflammatory cells involved in allergic diseases are normally activated, and invade the lesions. Inflammatory cells accumulate locally in the lesion, and are able to continue functioning for a long time, since their lives are prolonged by suppression of apoptosis. In an experimental model in which acidophilic inflammation of the respiratory tract is induced in mice, it has been shown that the administration of anti-Fas antibody, which has apoptosis-inducing activity, through the respiratory conduit, results in disappearance of the invasion of the respiratory tract. acidophilus under the mucosa that is normally observed after inhalation of the allergen [see. Tsuyuki, S. and others, (1995) J. Clin. Invest. 96, 2924]. Therefore, it is possible to alleviate the symptoms in allergic inflammation by inducing apoptosis in inflammatory cells.

Rheumatoid arthritis Apart from the autoimmune aspect of rheumatoid arthritis described above, it is known that synovial cells that proliferate abnormally in the lesions express Fas [see Hoa, T.T.M. and others (1996), J. Rheu atol., 23, 1332]. Apoptosis can be induced by stimulating the synovial cells of said lesions with anti-Fas antibody having apoptosis-inducing activity [see Nakajima, T. et al., (1995), Arthur. Rheu., 38, 485]. In other words, the Fas / Fas ligand system is not working properly in the foci of patients with rheumatoid arthritis, and neither the autoreactive T cells nor the synovial cells that proliferate abnormally are eliminated, despite the fact that both express Fas.

Arteriosclerosis Although the final diagnosis of cell death at the center of arteriosclerosis lesions is necrosis, participation of apoptosis in the processes of progression and degeneration has been reported (see Kenji Harada (1997), Gendai Iryou, 29, 109). Electron microscopy of arteriosclerosis lesions shows apoptosis of smooth muscle cells, characterized by nucleic condensation [see Isner, J.M. and others, (1995), Circulation, 91, 2703]. In addition, it has been reported that foam cells, which are macrophages in clustering in the inner layer of the artery and that incorporate lipids in early arteriosclerotic lesions, express Fas and are provoked to undergo apoptosis with naturally occurring apoptosis-inducing anti-Fas antibodies [see Richardson, B.C. et al. (1994), Eur. J. Immunol., 24 2604], Arteriosclerotic lesions are often associated with lymphocyte infiltration, suggesting the possibility that the Fas ligand of T cells together with Fas or macrophages, are the responsible for controlling arteriosclerosis [see Kenji Harada (1997), Gendi Iry u, 29, 109].

Myocarditis and cardio iopathy The fas / Fas ligand system is probably involved in the pathogenesis of autoimmune cardiac diseases, such as ischemic heart disease, viral heart disease, dilated cardiomyopathy and chronic cardiomyopathy. Myocarditis is the inflammation of the heart muscle considered to be mainly caused by viruses, such as coxsackie virus, and is typified as chest pain, arrhythmia, heart failure or shock, after symptoms similar to a cold. Cardiomyopathy is defined as "a disease of the heart muscle of unknown cause", although its cause is also probably considered as a result of viral infection. In studies of myocarditis models in mice with heart failure, apoptotic cells (evidenced by condensation and / or fragmentation of the nuclei) are observed in the mouse heart after viral inoculation. Increased expression of Fas in the mouse muscle is also observed, which leads to speculation that the condition is a result of apoptosis induced by Fas ligand derived from infiltrating inflammatory cells, predominantly lymphocytes [see Takehiko Yamada et al. , Gendai Tryou, (1997), 29, 119]. It is known that apoptosis is induced in rat heart muscle cells cultured by ischemia, concurrently with an increase in mRNA coding for Fas in cells [see Tanaka M. et al. (1994), Circ. Res. 75, 426].

Renal diseases In many chronic kidney diseases, the reconstitution of tissue within the glomeruli results in the accumulation of extracellular substrates within the glomeruli, thereby promoting sclerosis of the glomeruli, leading to pathological loss of filtering function and finally , to chronic renal failure. In a model of progressive glomerulosclerosis, the sclerotic regions exhibit typical apoptotic appearances, at electron microscopy levels, and an increase in apoptosis in the glomeruli is observed, consistent with a decrease in the number of glomerular cells associated with the advancement of sclerosis [ see Sugiya to H., et al. (1996), Kidney Int., 49, 103], In acute glomerular nephritis, the disease is known to be relieved by apoptotic reduction in the number of mesangial cells that proliferated abnormally [see Shimizu A ., et al., (1995), Kidney Int. 47, 114 and Baker AJ et al., (1994), J. Clin. Invest. 94, 2105]. In diseases such as purpura nephritis and lupus nephritis, a marked increase in cells expressing Fas in glomeruli has been reported [see Takemura T., et al., (1995), Kidney Int. 48, 1996].

Hypoplastic anemia On the surface of hepatopoietic precursor cells of a patient with hypoplastic anemia, the expression of Fas is remarkably high in comparison with normal individuals, suggesting the participation of Fas in the decrease of hematopoietic stem cells in these patients [see Maciejewski J.P. and Otrae, (1995), Br. J. Haematol, 91, 245].

Hepatitis In fulminant hepatitis, it is known that apoptosis is induced in many hepatocytes. Extended death of hepatocytes, similar to that observed in fulminating hepatitis, was observed by intraperitoneal administration of anti-Fas JO2 antibody to mice. Thus, it is considered likely that the pathogenesis of fulminant hepatitis includes Fas-induced hepatocyte apoptosis [see Kamoga a, Y., et al., (1996), Molecular Medicine, 33, 1284; and Ogasa ara, J., et al., (1993), Nature, 364, 806]. In immunohistochemical studies, increased Fas expression was observed in the cytoplasm of hepatocytes in regions showing high levels of hepatocyte necrosis, such as within chronic hepatitis and on the cell membrane of hepatocytes from liver disease lesions, such as as fatty liver [see Hiramatsu, N, et al., (1996), International Hepatology Commun, 4, 334]. In addition, Fas is expressed in hepatitis lesions Chronic persistent C and active chronic hepatitis, both of which show scattered hepatocyte staining surrounded by infiltration lymphocytes, which are apparently cytotoxic T cells [see Mita, E., et al., (1994), Biochem, Biophys. Res. Commun, 204, 468]. The cytotoxic T cell has the function of inducing apoptosis in infected cells by the Fas ligand expressed on its surface. Similarly, they induce apoptosis in normal cells that are nearby. This condition on normal local cells, called the voyeur disorder, arises from the fact that many cells in the body express their own Fas after infection of neighboring cells. In cases of chronic hepatitis, where the ARn derived from the hepatitis C virus is substantially reduced after the administration of interferon, the expression of Fas in the liver tissue decreases markedly. Hepatocytes from patients with acute liver failure have increased amounts of Fas on the cell surface, and undergo apoptosis when exposed to Fas antibodies that induce apoptosis. In addition, in alcoholic hepatitis, the Fas ligand is expressed on the same hepatocytes within the pseudoaccine [see Galle, P. R., et al. (1995), J.

Exp. Med., 182, 1223]. In the studies that include in situ hybridization of the Fas ligand expression gene, expression was found both in hepatic infiltration lymphocytes, in case of acute liver failure, and also in the same hepatocytes in the pseudoaccine in cases of alcoholic cirrhosis (as before). In this way, it is speculated that apoptosis is induced by different mechanisms in viral cirrhosis and alcoholic cirrhosis, but both cases Fas / Fas ligand system is abnormal. In a mouse model of hepatitis, it is known that the liver disorder is inhibited by the administration of a substance capable of inhibiting Fas ligand binding to Fas [see Kondo, T., et al., (1997), Nature Medicine, 3, 409].

Acquired Immunodeficiency Syndrome Immunodeficiency in patients infected with the human immunodeficiency virus (HIV) originates, at least partially, from the apoptotic cell death of numerous immune cells not infected with HIV. The T helper cells die in contact with HIV. Since the helper T cell growth factor is essential for the suppression of apoptosis in cytotoxic T cells, the suppression of helper T cells results in the apoptosis of cytotoxic T cells. It is also considered probable that apoptosis of immune cells in patients infected with HIV is due to Fas / Fas ligand system abnormalities, based on observations that Fas expression in peripheral blood lymphocytes of HIV-infected patients it correlates well with the pathological progression of the disease [see Dhein, J., et al., (1995), Behring Inst. Mitt, 96, 13 and McCloskey, TW, et al. (1995), Cytometry, 22, 111]. Fas-positive peripheral blood lymphocytes from uninfected individuals do not rapidly undergo apoptosis by Fas stimulation, whereas peripheral blood lymphocytes from infected patients undergo Fas-induced apoptosis over a short period of time [see, O en-schaub, LB, et al., (1992), Cell Immunol, 140, 197].

Rejection after organ transplantation Rejection after organ transplantation shares certain similarities with autoimmune diseases, except that the transplanted organ is attacked by a donor's cytotoxic T cells. In this way, relief of symptoms can be expected if the functions of the cytotoxic T cells can be suppressed. For the diseases listed above, effective means for their treatment is by the elimination of abnormal cells (e.g., autoreactive T cells in autoimmune diseases, foam cells in arteriosclerosis, esanglial cells in acute glomerular nephritis, infected cells in viral infections, and synovial cells). in rheumatoid arthritis) and / or by protecting normal tissues or cells.

The problem lies in the fact that agents that are capable of inducing only Fas-mediated apoptosis are highly likely to cause disorders in normal tissues, even when the abnormal cells are removed. On the other hand, agents that are only capable of inhibiting Fas-mediated apoptosis can not eliminate abnormal cells, even though they may be able to protect normal cells. For example, the mouse anti-Fas monoclonal antibody, Jo2, has an apoptosis-inducing activity but causes fulminant hepatitis in mice [see, Ogasa ara, J. et al., (1993), Nature, 364, 806-809]. To date, an anti-Fas antibody that can be used in the treatment and / or prophylaxis of any of the above diseases, but which is not associated with any inconvenient side effects, is not known. Immunoglobulin G (IgG) is composed of two polypeptide chains (L chains), each having a molecular weight of approximately 23,000 Kd, and two heavy polypeptide chains (H chains), each having a molecular weight of approximately 50,000 KD . Both chains, H and L, consist of a repeated region of conserved amino acids consisting of approximately 110 residues. This region is referred to herein as a "domain," and constitutes the basic three-dimensional structural unit of IgG. The H and L chains consist of four and two consecutive domains, respectively.

When the antibody amino acid sequences are compared, the aminoter inal domain of the H chains and the L chains is more variable than the other domains. Therefore, it is referred to as the "variable" domain (domain V). The V domains of the H and L chains are associated with each other by their complementary nature to form variable regions at the amino termini of the IgG molecules. The other domains associate to form constant regions. The constant region sequences are characteristic for a given species. For example, the constant regions of mouse IgG differ from those of human IgG, and mouse IgG molecules are recognized as a foreign protein by the human immune system. Administration of a mouse IgG molecule to a human subject results in the production of a human anti-mouse antibody response (hereinafter referred to as "HAMA") [Schroff et al. (1985), Cancer Res. 45, 879 -885]. Consequently, a mouse antibody can not be repeatedly administered to a human subject. For effective administration, the antibody should be modified to avoid induction of the HAMA response, while maintaining the specificity of the antibody. The data from X-ray crystallographic analyzes indicate that the immunoglobulin is generally folded to form a long cylindrical structure comprising two layers of antiparallel ß sheets, each consisting of three or four chains & In a variable region, three loops of each of the V domains of the H and L chains are clustered together to form an antigen binding site. Each of these curls is called a complementarity determining region ("RDC"). The RDCs have the maximum variability in the amino acid sequence. The portions of the variable region that are not part of an RDC are called "structure regions" ("FR" regions) and generally play a role in maintaining the structure of the RDC. Kabat et al. Compared the primary sequences of many variable regions of H and L chains and identified putative RDCs or structure regions, based on sequence conservation [E.A. Kabat et al., Protein sequences of immunological interest, fifth edition, NIH Publication No. 91-3242]. Additionally, they classified the structure regions into several subgroups that share common amino acid sequences. They also identified the regions of structure that correspond between the mouse and human sequences. Studies on the structural characteristics of IgG have led to the development of methods to prepare humanized antibodies, that do not provoke a HAMA response, as described below. Initial suggestions were directed to the preparation of a chimeric antibody by binding a variable region of mouse antibody to constant regions of human origin [Morrison, S.L. and others (1984), Proc. Nati Acad. Sci. USA, 81, pages 6851-6855]. Said chimeric antibody, however, still contains many human amino acid residues and, thus, can elicit a HAMA response, especially when administered over a prolonged period. [Begent et al. (1990) Br. J. Cancer, 62, pages 487 et seq.]. Grafting of the RDC segments alone in a human antibody was then proposed in order to further reduce the number of non-human amino acid sequences that elicit the HAMA response [Jones, P.T. et al. (1986) Nature, 321, 522-525]. However, grafting of the RDC portions alone was generally found to be insufficient to maintain immunoglobulin activity against an antigen. Based on X-ray crystallography data, Chothia et al [Chothia et al. (1987), J. Mol. Biol. 196, 901-917] determined that: (1) An RDC has a region involved in antigen binding and a region involved in maintaining the structure of the RDC itself. It is possible to classify the possible three-dimensional structures for the DRC in different classes, with characteristic patterns (canonical structures); and (2) The classes of canonical structures are determined not only by the RDC sequences, but also by the nature of amino acids at the specific positions in the structure regions. As a result, it has been suggested that the RDC graft technique should also include the grafting of certain amino acid residues from the framework regions in the human antibody backbone [Queen et al., International Patent Publication W090 / 07861]. In the context of the foregoing, an antibody from a non-human mammal, from which the RDCs for the graft are obtained, is referred to hereinafter as a molecule "donor". An antibody in which CDRs are grafted is referred to below as a "receptor" molecule. When carrying out the RDC graft, the structures of the RDC region should ideally be conserved and the immunity of the immunoglobulin molecule should be maintained. Therefore, the following factors may be relevant: (1) the subgroup of the receiver; and (2) the nature of the amino acid residues that are transferred from the donor structure regions. Queen and collaborators [Patent publication International No. W090 / 07861], proposed a method for deciding whether an amino acid residue of donor FR was grafted together with the RDC sequence. According to this method, an amino acid residue of an FR region is grafted to the receptor, together with the RDC sequence, if the residue meets at least one of the following criteria: 1) The amino acid in the human structure region of the receptor is rarely found in this position in the recipient, whereas the corresponding amino acid in the donor is commonly found in this position; 2) the amino acid is located close to one of the RDC; and 3) the amino acid has a side chain atom within about 3 A of an RDC, as considered by a three-dimensional model of the immunoglobulin, and is potentially capable of interacting with an antigen or a RDC of a humanized antibody. However, no one has successfully obtained a humanized anti-Fas IgG antibody, which has apoptosis-inducing activity.

OBJECTS OF THE INVENTION It is an object of the present invention to provide an anti-Fas antibody, or similar molecule, that can be evaluated in an animal model of an animal condition related to human Fas. It is also an object of the present invention to provide a humanized anti-Fas antibody, or similar molecule, useful in the treatment and / or prophylaxis of conditions arising from abnormalities in the Fas / Fas ligand system.

BRIEF DESCRIPTION OF THE INVENTION Thus, in a first aspect, the present invention provides a molecule having a specific binding region for an epitope of the Fas antigen, the epitope being conserved between a primate and a non-primate animal. In a second aspect, the present invention provides a molecule having a specific binding region for a conserved epitope of the mammalian Fas antigen. The present invention also provides an antibody produced by the hybridoma HFE7A having the accession number FERM BP-5828, as well as a molecule having at least six RDC of antibody, the antibody being specific for human Fas, wherein the RDC they have identity with the RDC of the antibody produced by the hybridoma HFE7A having the accession number FERM BP-5828. Other objects, purposes, aspects and embodiments of the present invention will become apparent hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram that represents the construction of phFas-AIC2. Figure 2 is a diagram that represents the construction of pME-H and pME-L.

Figure 3 is a figure showing the ELISA results for the determination of the epitope recognized by the HFE7A antibody. Figure 4 is a figure showing the results of competitive testing for the determination of the epitope recognized by the HFE7A antibody. Figure 5 is a figure showing the toxicity test results of HFE7A. Figure 6 is a figure showing the results of a test with a fulminant hepatitis model. Figure 7 is a figure showing the results of a collagen-induced arthritis prevention test. Figure 8 is a summary of the first step of PCR for the production of DNA-VHH. Figure 9 is a summary of the second step of PCR for the production of VHH-DNA. Figure 10 is a summary of the third step of PCR for the production of VHH-DNA. Figure 11 is a summary of the construction of the expression plasmid carrying the VHH fragment of DNA. Figure 12 is a summary of the first step of PCR for the production of DNA-VHM. Figure 13 is a summary of the second step of PCR for the production of DNA-VHM. Figure 14 is a summary of the construction of the expression plasmid carrying the DNA VHM fragment.

Figure 15 shows a summary of the first step of PCR for the production of DNA-VMM. Figure 16 is a summary of the second step of PCR for the production of DNA-VMM. Figure 17 is a summary of the third step of PCR for the production of DNA-VMM. Figure 18 is a summary of the construction of the expression plasmid carrying the DNA VMM fragment. Figure 19 shows the positions to which the light chain sequencing primers bind. Figure 20 is a summary of the first step of PCR for the production of RV-DNA. Figure 21 is a summary of the second step of PCR for the production of VD-DNA. Figure 22 is a summary of the third step of PCR for the production of RV-DNA. Figure 23 is a summary of the construction of the expression plasmid carrying the DNA fragment VD. Figure 24 is a summary of the construction of the DNA fragment (DNA-IG5 ') comprising the CH1 region of human IgG1 and an intron. Figure 25 is a summary of the construction of the genomic DNA fragment (DNA-IG3 ') comprising the hinge region, the CH2 region, the CH3 region and the human IgG1 introns. Figure 26 is a summary of the construction of the expression plasmid pEg7AH-H. Figure 27 shows the positions to which the heavy chain sequencing primers bind. Figure 28 is a graph depicting the binding activity of humanized anti-Fas antibodies to the human Fas fusion protein. Figure 29 shows the competitive inhibition of HFE7A and humanized anti-Fas anticuefos for the fusion protein of human Fas. Figure 30 shows the cytotoxicity of humanized HFE7A for WR19L12a. Figure 31 shows the sketch of the first step of PCR for the preparation of DNA-LPDHH. Figure 32 shows the sketch of the second step of PCR for the production of DNA-LPDHH. Figure 33 shows the sketch of the third step of PCR for the production of DNA-LPDHH. Figure 34 shows the outline of the construction of an expression plasmid carrying the DNA-LPDHH fragment. Figure 35 shows the sketch of the first step of PCR for the preparation of DNA-LPDHM. Figure 36 shows the sketch of the second step of PCR for the production of PDHM-DNA. Figure 37 shows the sketch of the third step of PCR for the preparation of DNA-LPDHM. Figure 38 shows the outline of the construction of a plasmid carrying the DNA-LPDHM fragment. Figure 39 shows the sketch of the first step of PCR for the preparation of DNA-HPD1.2. Figure 40 shows the sketch of the second step of PCR for the preparation of DNA-HPD1.2. Figure 41 shows the construction of a plasmid carrying the DNA-HPD1.2 fragment. Figure 42 shows where the primers are linked for sequencing of pEgPDHV3-21. Figure 43 shows the construction of high level expression vectors for humanized light chains. Figure 44 shows the construction of high level expression vectors for humanized heavy chains. Figure 45 shows the binding activity for the fusion protein of human Fas for the supernatants of Example 12. Figure 46 shows the results of competitive inhibition of anti-HFE7A by the supernatants of Example 12. Figure 47 shows the results of induction of apoptosis in T cells by the cultures of culture supernatant of Example 12.

DETAILED DESCRIPTION OF THE INVENTION It is a striking advantage of the HFE7A antiquase of the present invention which is not only capable of inducing apoptosis in abnormal cells expressing Fas, but is also capable of inhibiting apoptosis in normal cells. This advantage extends generally to humanized anti-Fas antiquands of the present invention. No known monoclonal antibody that binds to human Fas and that has apoptosis-inducing activity is capable of binding to mouse Fas. The monoclonal antichods that bind to Mouse Fas are known, but none of them joins Fas human. In this way, anti-Fas antifog known in disease model mice can not be evaluated. In contrast, HFE7A anti-cues are capable of being evaluated in disease model mice, thereby providing means to ensure pharmaceutical efficacy and also establishing a model for the investigation of Fas function in general. It is believed that the advantages of the anti-convolutions of the present invention arise from their ability to recognize a conserved epitope on the Fas antigen. Fas is a common molecule, but it varies from species to species. Unbound by theory, it is believed that there is at least one conserved region of Fas, which is common to all mammals, and that is necessary for the Fas-inducing function. The molecules of the present invention recognize a conserved epitope of the Fas antigen. In this regard, when comparing Fas of mido and human, for example, the epitope in question does not necessarily need to be absolutely identical in the two molecules, provided that the binding region of the antigen of the molecule is capable of recognizing both. However, in general, the epitope will be exactly the same. Many anti-convolutions directed between Fas are known, including those capable of inducing apoptosis, but none have been previously obtained that bind to any type of consensus sequence. The anticuefos of the present invention, by way of contrast, are joined to a consensus sequence. Therefore, as a battery extension, instead of acting only to incapacitate or interfere by generalized binding to Fas, which can have dangerous and unpredictable effects, such as with Jo2 and fulminant hepatitis in mice (cited above), the anti-convolutions of The present invention actually acts on the active site of Fas, thereby mimicking a natural ligand, rather than only non-specifically binding to the Fae antigen. If a normal laboratory mouse, such as a BALB / c mouse, is immunized with a human Fas, anti-bud producing cells that bind both human Fas and mouse Fas will be eliminated in the thymus in the normal cure elimination of self-reactive anticuefos. Thus, in order to obtain a mouse monoclonal antibody that was targeted to a rabbit-human-mouse epitope and which, therefore, bind both human Fas and mouse Fas, it is necessary to use a mouse in which Removal has been partially or totally disabled.

It has been speculated that the Fas / Fas ligand system is involved in the process of removing autoreactive T cells in the thymus [see Shin Yonehara (1994) Nikkei Science Bessatsu, 110, 66-77]. Thus, a mouse having a mutation in the Fas / Fas ligand system is immunized (said mouse henceforth referred to as a "destroyed Fas mouse" or "Fas deficient mouse / Fas ligand"), for example, one that is unable to express the gene encoding Fas, anticuefos that bind to mouse Fas as well as human Fas can be obtained. Thus, there is also provided a method for obtaining an antiquake, or molecule, of the invention which comprises administering an immunogenically effective amount of a subetancy comprising an immunogenic epitope of heterologous Fae to a non-human animal, which is at least partially deficient in the apoptotic elimination of autoreactivae cells, and to select anti-animals from the animal later. The substance bearing the epitope of the Fas antigen can be the same Fas, or it can be another suitable substance such as a fusion pro-ine. The selection of the appropriate anti-convolutions is within the abilities of the person skilled in the art, and is exemplified below. In particular, it is preferred to use the immunized animal of the method of the invention to obtain at least one monoclonal anti-cough, which is readily obtainable using methods well known in the art. It will also be appreciated that, for ease of handling, it is preferred that the non-human animal be a mouse, although other species of rodents, such as rabbits, and other mammals in general, such as goats and macaques, can also be used; although these systems are not as well characterized as the mouse. It will also be appreciated that it is preferred that the Fas used for administration be human, although if desired, the anti-convolutions of the invention can be obtained for other mammals. However, it is generally contemplated that, although it shares a common epitope, the anti-convolutions of the present invention have universal application. Using the above method, a hybridoma was prepared which produces a novel anti-Fas anti-clump anti-Fas binding both human and mouse. A destroyed Fas mouse was immunized with human Fae and then the spleen cells were fused with mouse myeloma cells, and then monoclonal anti-convolutions were purified from the culture supernatant. The novel anti-Fas anti-clump recovered in this way has been found effective in alleviating the severity of the symptoms of the mouse autoimmune disease model. In addition, it has been shown that this anti-Fas antifungal monoclonal does not induce liver disorders, which had previously been a problem.

The respective genes for both strands of the new antibody were also cloned and sequenced, to obtain the amino acid sequences of the RDCs. Expression vectors, which comprise the respective genes for the heavy and light chains, were constructed to produce a recombinant anti-Fas antiquase. This recombinant anticuefo, obtained in fluids of culture supernatant of animal cells or transfected with these vectors, was shown to react with Fas. The anti-Fas antifungal obtained in this way, and its recombinant anti-cough clones are able to protect the liver from Fas-induced fulminating hepatitis, and are also effective in the prevention and treatment of rheumatoid arthritis. Therefore, it has now been shown that it is readily possible to provide anti-convulsants that are capable of inducing both apoptosis by Fas in abnormal cells and inhibiting Fas-induced apoptosis in normal cells, and therefore are effective in the prevention, treatment and / or prophylaxis of diseases attributable to abnormalities of the Fas / Fas ligand system. The RDC amino acid sequences of the anti-Fas anti-Fas mouse monoclonal antibody were grafted onto a human anti-cough and a non-immunogenic recombinant anti-cough was successfully obtained for human subjects., but still had Fae binding activity. The present invention allows the connection of humanized anti-convolutions that have a minimal risk of inducing a HAMA response while having an effective anti-effector effector function. As used herein, the term "human" and "humanized", in relation to anti-convolutions, refers to any anti-drug that is expected to cause little or no immunogenic response in a human subject, the subject in question being a individual or a group. It will be appreciated that, in general, it is preferred that all CDRs of a given antibody be grafted to a receptor anti-cotue, to preserve the binding region for the epitope of the Fae antigen, or the antigen binding region, as referred to in FIG. the present usually. However, there may be times when it is appropriate or desirable that not all of the RDC amount be grafted onto the donor, and this is contemplated in the present invention. It will also be understood that the graft generally encompasses the replacement, residue by residue, of one amino acid region by another. However, occasionally, especially with the transfer of a region, one or more residues may be added or omitted as desired, and said supreeione and ineercionee, as well as the appropriate replacements and investments, are within the skill of the expert in the field. matter. The antigen binding region of the present invention is a region of the molecule that corresponds to an epitope binding site of an antiquake. The binding region of the antigen does not need to be derived directly from any particular antifouling, or pair of anticuefos, and may not resemble any particular antigen binding region. The only requirement is that the antigen-binding region resembles the recognition site of an anti-cotuest as long as it is capable of binding an antigen, in this case, an epitope of the Fas antigen. Although the antigen binding region can be designed using the CDRs of a known antiquake, if these are then grafted onto a human antiquake, the binding region of the resulting antigen may not necessarily resemble that of the known antiquake, although a high degree of similarity, from the point of view of maintaining the binding specificity. It is particularly preferred that all CDRs of a nonhuman antiquake be grafted onto the human anti-cotuefo. Additionally, it is preferred that certain areas of the structure regions be incoforated in the receptor anti-trap (also referred to as the human antiquake in the preemptive), in order to maintain the three-dimensional structure of the non-human recognition site. Said areas of the structure regions typically comprise individual amino acid reagents selected by their importance, according to the following guidelines. In particular, they are refered to as rare in humans, but are common in the relevant nonhuman antiquake, and those residues that have a high probability of interacting with the epitope of the recognition site are the preferred ones to be grafted together with the DRC . When CDRs are grafted onto the human antiquake, it will normally happen that the non-human DRC replaces a relevant human DRC in its entirety, particularly when both are of the same length. However, it can also happen that only a part of the human RDC is replaced, or that only a part of the non-human RDC is grafted, usually coming from the hand between them. It can also happen that one RDC is greater than the other, but whatever the situation is, it is highly preferred to keep the region of the human structure intact, different from those that are replaced, which are written above. It will also be appreciated that the RDC of the human anti-cough should generally be used to replace the corresponding RDC in the human anti-cough. In the situation in which a light or heavy chain of human skeleton is used, that only has positions for insertion of RDC, instead of having really RDC, then similar considerations apply. It will also be understood that peeadae and light human chains do not necessarily come from the same human antiquake, not even from the same class. What is important is that the sequence of the selected receptor matches, as closely as possible, the sequence of the nonhuman antiquake. The importance of matching the two chains (light / light or peeada / peeada) ee that the anticucator reeultante must have an eitio of recognition that is as close as possible to the original non-human antiquake, to ensure the best union. Thus, the present invention also contemplates the possibility of using matches or similarities that are not the closest poeiblee, when there is reasonable expectation that the resulting recombinant antiquake will serve the required purpose. The molecules of the present invention are preferably anticuefos, although this is not necessary, provided that the antigen binding region ee binds to a Fae epitope. In this way, isolated and stabilized binding sites, for example, can be fixed to an affinity purification column support, or an administration method can comprise an adjuvant carrier molecule, for example, to which it is fixed regione epitope binding of the invention. For ease of reference, the molecules of the present invention will be generally referred to as "anti-sense" herein, but such reference encompasses all molecules of the invention, unless otherwise indicated. In the case where the molecule of the invention is an antiquase, it will be appreciated that any type of appropriate antiquake can be emulated, or employed, such as IgG, IgA, IgE and IgM, with IgG generally being preferred. When discussing molecules and anticuends herein, it will also be understood that similar considerations apply, changing what needs to be changed, to any nucleic acid sequences that encode them, as appropriate. Certain preferred embodiments of the present invention are as follows. It is preferred that the antiquase of the invention binds to a peptide comprising the amino acid sequence SEQ ID No. 1 of the sequence listing. The antiquase is preferably IgG and, most preferably, comprises a light chain polypeptide protein selected individually from amino acid sequence 1 to 218 of SEQ ID No. 50, amino acid sequence 1 to 218 of SEQ ID No. 52, amino acid sequence 1 to 218 of SEQ ID No. 54, amino acid sequence 1 to 218 of SEQ ID No. 107, and amino acid sequence 1 to 218 of SEQ ID No. 109 of the sequence listing, and wherein the heavy chain polypeptide protein preferably comprises amino acid sequence 1 to 451 of SEQ ID No. 89 or amino acid sequence 1 to 451 of SEQ ID No. 117 of the sequence listing. An antiquake of the invention, in a preferred embodiment, has a light chain and a peaked chain, the heavy chain has the following general formula (I): -FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4- (I) in which FRHi represents any amino acid sequence that comprises from 18 to 30 amino acids, CDRHi represents the sequence defined in SEQ ID No. 2 of the sequence listing, FRH_ represents any amino acid sequence consisting of 14 amino acids, CDRH2 represents the sequence defined in SEQ ID No. 3 of the sequence listing, FRH3 represents any amino acid sequence consisting of 32 amino acids, CDRH3 represents the sequence defined in SEQ ID No. 4 of the sequence listing, FRH4 represents any amino acid sequence consisting of amino acids, and each amino acid is linked to the other by a peptide bond, and the light chain has the following general formula (II): -FRLi -CDRLi -FRL2 -CDRL2 -FRL3 -CDRL3 -FRL4 - (II) in the c ual FRLi repreeenta any amino acid sequence consisting of 23 amino acids, CDRLi represents the sequence defined in SEQ ID No. 5 of the sequence listing, FRL2 represents any amino acid sequence consisting of 15 amino acids, CDRL2 represents the sequence defined in SEQ ID No. 6 of the sequence listing, FRL3 represents any sequence of amino acid consisting of 32 amino acids, CDRL3 represents the sequence defined in SEQ ID No. 7 of the sequence listing, FRL represents any amino acid sequence consisting of 10 amino acids, and each amino acid is linked to the other by a peptide bond. The invention also provides DNA and RNA encoding any of the light or heavy chain polypeptide proteins described above. Preferred is DNA comprising the nucleotide sequence 100 to 753 of SEQ ID No. 49, DNA comprising the nucleotide sequence 100 to 753 of SEQ ID No. 51, DNA comprising the nucleotide sequence 100 to 753 of SEQ ID No. 53, and / or DNA comprising nucleotide sequence 84 to 2.042 of SEQ ID No. 88 of the sequence listing. The invention also provides a recombinant DNA vector comprising the above defined DNA, as well as a host cell transformed with said vector. The host is preferably transformed with a separate vector for each encoded light and heavy chain, so that it will usually contain two vectors, although the present invention also contemplates a host transformed with only one expression vector encoding all the sequences to be expressed. Said host cell is preferably a mammal. E. coli pHSGMMó SANK73697 (FERM BP-6071), E. coli PHSGHM17 SANK73597 (FERM BP-6072), E. coli pHSGHH7 SANK73497 (FERM BP-6073), E. coli pHSHM2 SANK 70198 and E. coli pHSHHS SANK 70398 (FERM BP-6272), E. coli pgHSL7A62 (FERM BP-6274) SANK73397 (FERM BP-6074) and E. coli pgHPDHV3 SANK 70298 (FERM BP-6273) are each preferred modality of the invention. The present invention also provides a method for producing a humanized anti-Fas antishock which comprises culturing the above humanized cell, and then recovering the humanized anti-Fas anti-Fas protein. An agent for the prophylaxis or treatment of diseases attributable to abnormalities of the Fae / Fae ligand system is also provided which comprises as an active ingredient the antiquase of the present invention, especially in the case of the diseases defined above. The target diseases are autoimmune diseases (general lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjögren's syndrome, pernicious anemia, Addison's disease, eele rodérma, Goodinsture ein, Crohn's disease, anemia, autoimmune olynthesis, sterility, myasthenia gravis, multiple sclerosis, Basedow's disease, thrombocytopenia, or insulin-dependent diabetee mellitus). Separate preparations are also contemplated for: allergy; rheumatoid arthritis; rterioscleroeis; myocarditis or cardiomyopathy; glomerular nephritis; hypoplastic anemia; hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C, hepatitis B, hepatitis D) or alcoholic hepatitis); and rejection after organ transplantation. Lae FRs are present in the variable region of an H or L chain eubunit of an immunoglobulin molecule. For example, FRHi refers to the region of the structure located at the most N-terminal position of the variable region of an H chain subunit, and FRL4 refers to the fourth region of structure from the N end of the variable region of a chain subunit L. Similarly, CDRHi, for example, refers to the RDC present at the most N-terminal position in the variable region of an H chain subunit, and CDRL3 refers to the third RDC from the N-terminus. of the variable region of a L-chain subunit. The FRC bleaches the RDC regions in either the light or heavy chains. In one embodiment, a monoclonal anti-Fas antiquase suitable for preparing a humanized anti-Fas anti-tail according to the present invention can be obtained by culturing a suitable hybridoma which, in turn, can be obtained by immunizing a Fas mouse destroyed with human Fas and Subsequently fusing the spleen cells of the mouse with mouse myeloma cells. The preparation of a monoclonal anti-cough typically includes the following: a) purification of a biomacromolecule to be used as the immunizing antigen; b) preparation of anti-cotuex producing cells, after first immunizing an appropriate animal using antigen injections, bleeding the animal and analyzing the anti-cotuex titer, to determine when to remove the spleen; c) preparation of myeloma cells; d) fusing anti-cough producing cells and myeloma cells; e) selecting a hybridoma that produces an antiquake of interest; f) preparing a cell clone eola (cloning); g) optionally, culturing the hybridoma cell, or developing animals in which the hybridoma cells have been transplanted, for large-scale preparation of the monoclonal anti-clump; and h) testing the biological activities and specificity or analyzing properties of a marker agent, of the monoclonal antiquake thus prepared. The general procedure followed for the preparation of an anti-Fas anti-monoclonal antibody is described in greater detail hereinafter, in line with the steps described above. However, it will be appreciated that the method described below only represents one way of preparing an adequate antiquake, and other methods can be followed, as in eeg, such as for example using other antifungal producing cells different from spleen cells and other cell lines different from myeloma. a) Preparation of the antigen A recombinant protein (hereinafter referred to as a "recombinant human Fas"), effective as the Fas antigen, can be obtained by transfecting the monkey cell line C0S-1 with the expression vector pME18S-mFas-AIC, which encodes a fusion protein comprising the extracellular domain of human Fae and the extracellular domain of the mouse interleukin 3 receptor [IL3R - see Niehi ura, Y., and other, (1995), J. Immunol, 154, 4395- 4403], and collecting and partially purifying the expulsion product. The plamidido phFae-AIC2 was constructed by inserting DNA encoding human Fas and a mouse IL3R fusion protein into pMEISS, which is an expression vector for animal cells. As indicated above, the materials used, such as the DNA encoding Fas, the vector and the host, are not restricted to those mentioned. The resulting human Fas and IL3R fusion protein, referred to herein as recombinant human Fas, harvested from the culture supernatant of the transformed C0S-1 cells, can be partially purified by means of a suitable method such as ion exchange chromatography. using a Q Resource column (Pharmacia brand). As a suitable alternative, it can be used as a purified Fas antigen, obtained from cell membranes of human cellulare lines. In addition, since the primary structure of Fae is known [see Itoh, N, and other, (1991), Cell, 6_6, 233-243], a peptide comprising a suitable portion of the amino acid sequence of Fas can be chemically synthesized. human, such as SEQ ID No. 1 of the sequence listing, by any suitable method, and used as the antigen. b) Preparation of antishock producing cells An experimental animal is immunized with the immunogen produced in step a), suitably mixed with an adjuvant, such as complete and incomplete Freund's medium, adjuvant and alum. In this case, a suitable experimental animal is a destroyed Fas mouse, which can be produced by the method of Senju et al., [Senju, S., et al., (1996), International Immunology, 8, 423]. Appropriate adieme routing routes to immunize the mouse include the subcutaneous, intraperitoneal, intravenous, intradermal and intramuscular injection routes, with subcutaneous and intraperitoneal injections being preferred. The immunization can be by means of a single dose or, preferably, by several repeated doses at appropriate intervals (preferably 1 to 5 weeks). The immunized mice are monitored for their anti-Fas anti-drug activity in their sera, and an animal with a high anti-ring titre is selected as the antibody-producing cell agent. The selection of an animal with a high degree makes the process more efficient. Cells for subsequent fusion are usually harvested from the animal three to five days after the final immunization. Methods for analyzing anti-coterie titer include several well known techniques such as radio immunoassay (RIA), solid-based enzyme immunoassay (ELISA), fluorescent antibody test and passive hemagglutination test, RIA and ELISA being preferred for sensibility detection reasons. , speed, accuracy and automation potential. The determination of the anti-cough label can be effected, for example, by means of ELISA as follows. First, purified or partially purified Fae is absorbed over the surface of an eolian fae, such as a 96-well ELISA plate, followed by blocking any remaining surface, to which Fas was not bound, with a protein not related to the antigen, such as bovine serum albumin (BSA). After washing, the surfaces of the well are contacted with serially diluted samples of the antifog preparations to be tested (for example, mouse serum) to allow the anti-Fas antifouling in the samples to the antigen. An anti-mouse anti-enzyme-labeled anti-cough is added, such as the secondary anti-cough, to bind to the mouse anti-cuspid. After washing, the substrate for the enzyme is added, and the anti-Fas binding activity can then be analyzed by determining an appropriate change, such as change in absorption due to color development. c) Preparation of myeloma cells In general, cells from established mouse cell lines serve as the source of myeloma cells. Cell lines include: mouse myeloma resistant to 8-azaguanine (BALB / c derivatives), strain P3X63Ag8LFl (P3-U1) [Treiton, DE and other, Current Topice in Microbiology and Immunology, 81, 1-7, (1978 )], P3 / NSI / l-Ag4-l (NS-1) [Kohier, G., and other, European J. I munology 6, 511-519 (1976)], Sp2 / 0-Agl4 (SP-2) ) [Shulman, M., et al., Nature, 276, 269-270 (1978)], P3X63Ag8.653 (653) [Kearney, JF, et al., J.

Immunology, 123, 1548-1550 (1979)] and P3X63Ag8 (X63) [Horibata, K. and Harris, A.W., Nature, 256, 495-497 (1975)]. The selected cell line is serially transferred in an appropriate medium, such as 8-asaguanine medium [RPI1640 medium supplemented with glutamine, 2-mercaptoethanol, gentamicin, fetal calf serum (FCS), and 8-azaguanine], modified Iscove Medium from Dulbecco (IMDM) or modified Dulbecco's Eagle Medium (DMEM). The cells are then transferred to a normal medium, such as ASF104 medium (Ajinomoto, KK), containing 10% FCS w / v, 3 to 4 days before fusion, to ensure that at least 2 x 10 7 cells are available on the cell. day of the merger. d) Cell fusion The antifungal producing cells used in fusion are plae cells and their precursor cells, lymphocytes, which can be obtained from any suitable part of the animal. Typical areas are the spleen, lymph nodes, peripheral blood, or any suitable combination thereof, most commonly using spleen cells. After the last boost of reinforcement, tissue is extracted in which antisense producing cells, such as the spleen, of the mouse having the predetermined anti-cotuex title are prepared for preparing antisense producing cells. The currently favored technique for fusion of spleen cells with myeloma cells prepared in case c), employs polyethylene glycol, which has relatively low cytotoxicity and the fusion procedure that uses it in simple. An example of this technique is as follows. Spleen and myeloma cells are well washed with serum-free medium (such as RPMI 1640) or phosphate buffered saline (PBS), and then mixed, so that the numerical ratio of spleen cells to myeloma cells is approximately between 5: 1 and 10: 1, and then centrifuged. After the supernatant has been discarded and the pelleted cells have been sufficiently loosened, a suitable amount, usually 1 ml, of serum-free medium containing 50% polyethylene glycol (p / v) (1,000 ppm) is added dropwise. 4,000) with agitation. Subsequently, 10 ml of serum-free medium is added slowly and then the mixture is centrifuged. The supernatant is discarded again and the pelleted cells are suspended in an appropriate amount of HAT medium [a solution of hypoxanthine, aminopterin and thymidine medium (these three compounds, together, are also known as "HAT") and mouse interleukin 2 ( IL-2)]. Afterwards, the suspension is dispensed into the wells of the culture plates (also referred to herein simply as "plates") and incubated in the presence of 5% CO2 v / v at 57 ° C for about 2 weeks, with the addition supplemental medium HAT as appropriate. e) Selection of hybridomas When the myeloma strain used is resistant to 8-azaguanine, that is, it is deficient in the enzyme guanine hypoxanthine foeforibosyl transferase (HGPRT), any unfused myeloma cell and any myeloma / myeloma fusion are unable to survive in the middle HAT. On the other hand, fusions of anti-coterminous producing cells with each other, as well as antigens producing cell hybridomas with myeloma cells, can survive, the latter having only a limited life. Therefore, continuous incubation in HAT medium results in the selection of only the desired hybridomas. Then, the resulting hybridomas develop in colonies in the HAT medium lacking aminopterin (HT medium). Then, aliquots of the culture supernatant are removed to determine anti-Fas antishock titer by means of, for example, ELISA. When the above recombinant human Fas fusion protein is used as the antigen for ELISA, it is also necessary to eliminate clones that produce an antiquase that binds specifically to the extracellular domain of the mouse IL3 receptor. The presence or presence of said clone can be verified, for example, by means of ELISA ueando the mouse IL3 receptor, or eu extracellular domain, as the antigen. Although the above selection procedure is exemplified using an 8-azaguanine resistant cell line, it will be appreciated that other cell lines may be used with appropriate selection mark and with appropriate modifications to the media used. f) Cloning Hybridomas that have been seen to produce specific anti-Fas anti-cues, using a method similar to that described in step b) to determine the anti-cough caption, are then transferred to another plate for cloning. Suitable cloning methods include: the limiting dilution method, in which the hybridomas are diluted to contain one cell per well of a plate and then grown; the soft agar method, in which the colonies are recovered after culturing the soft agar medium; use a micro anipulator to separate a single cell per culture; and "selecting a clone", in which individual cells are separated by means of a cell sorter. The limiting dilution is usually the simplest and the most commonly used. Any selected cloning procedure is repeated 2 to 4 times for each well demonstrating an anti-fasting titer, and clones having stable anti-clumping titers are selected as hybridomas producing anti-Fas monoclonal antibodies. Hybridomas producing a mouse anti-Fas antiquase are selected by a similar method to obtain a monoclonal anti-Fas anti-cell producing cell line. A suitable mouse Fas useful for this purpose, for example, is the fusion protein expressed by cultured animal cells transfected with the expression vector pME18S-mFas-AIC. This plamidmid has DNA encoding a fusion protein comprising the extracellular domain of mouse Fas and the extracellular domain of the mouse IL3 receptor [see Nishimura, Y, et al., (1995), J. Immunol, 154, 4395-4403 , incorporated herein by reference]. Other sources of Fas of urid include mouse Fae and cells expressing mouse Fas on its surface. The mouse-mouse hybridoma HFE7A was selected by means of the above methodology. Its specific preparation is described in the attached examples. HFE7A is a cell line that produces an anti-fas monoclonal anti-fastener suitable as the baee in the preparation of a humanized anti-Fas antiquague of the present invention. Therefore, when an anti-cotue is prepared using the mouse-mouse hybridoma HFE7A, the Preparation can be carried out following a procedure starting from step g) below, with steps a) to f) above omitted. g) Hybridoma culture to prepare monoclonal anti-cough. The hybridoma obtained by the preceding steps was then cultured in normal medium, instead of HT medium. Large-scale culture can be done by cylinder-bottle culture, using large, rotating culture bottles. The large-scale culture supernatant is then harvested and purified by a suitable method, such as gel filtration, which is well known to the person skilled in the art, to obtain a anti-Fas anti-clump.

The hybridoma can also be developed intraperitoneally in a non-genomic mouse, such as a BALB / c mouse or Nu / Nu mouse, to obtain ascitic fluid containing anti-Fas anti-fasting monoclonal in large quantities. Conventionally available monoclonal anti-cough purification kits (for example, MabTrap Gil Kit, Pharmacia) can be conveniently used to purify the harvested anti-cues. The monoclonal antibodies prepared as explained above, and which have been selected for their specificity for human and mouse Fas, have high specificity for human and mouse Fas. h) Monoclonal anti-cell test. The determination of the isotype and the subclause of the monoclonal antiquake obtained in this way can be carried out as follows: suitable identification methods include the Ouchterlony method, ELISA and RIA. The Ouchterlony method is simple, but requires concentration of the solutions used when the monoclonal anti-cell concentration is low. In contrast, when ELISA or RIA is used, the culture supernatant can be reacted directly with an absoFido antigen on an eolid phase and with secondary antibodies that have specificity for immunoglobulin varioe and prototypes to identify the type and subclass of the monoclonal antichode. . However, in general, it is preferred to use a commercial equipment for identification, such as a Mouse Typer equipment (commercial name; BioRad). The quantification of protein can be carried out by means of the Folin-Lowry method, for example, or by calculation based on absorption at 280 nm [1.4 (OD 280) = 1 mg / ml of immunoglobulin]. The identification of the epitope of the Fas antigen that recognizes the monoclonal antifog can be carried out as follows. In the first place, the partial structure of Fas is varied. The partial structures may be prepared synthetically, for example by means of oligopeptide synthesis, or in vivo, using a suitable host such as E. coli. which has been transformed by means of a suitable vector that uncovers the DNA encoding the desired fragments. Both methods are frequently used in combination to identify the epitope recognized by the antigen binding region. For example, a series of polypeptides having appropriately reduced lengths can be prepared by working from the C- or N- terminus of the antigen protein, by means of genetic engineering techniques well known to those skilled in the art. By establishing which fragments react with the antiquake, a rough idea of the epitopic site can be obtained. The epitope can be identified more closely by synthesizing a variety of smaller oligopeptides corresponding to portions or mutants of the peptide or peptides, recognized by the antiquake. Synthesis of oligopeptides is generally used for the preparation of these smaller fragments. Identification of the epitope can then be established by binding studies or by means of competitive inhibition study with the fusion protein of recombinant human Fas in ELISA, for example. They can conveniently use commercially available equipment, such as the SPOTs equipment (Genosys Biotechnologies, Inc.) and a series of multiple spike peptide kits based on the multi-pin synthesis method (Chiron Corp.), to obtain a large variety of oligopeptides. DNA encoding the heavy and light chains of the monoclonal anti-Fas antibody prepared above can be obtained by preparing mRNA from hybridoma cells that produce the anti-Fas monoclonal antibody, converting the mRNA into cDNA by reverse transcription and then isolating the DNA encoding the antibodies. heavy and / or light chains of the antiquake, respectively. This DNA can then be used to generate the humanized anti-Fas antiquague of the present invention. MRNA extraction can be performed with the hot guanidinium-thiocyanate-phenol method, or with the guanidinium thiocyanate-guanidinium HCl method, for example, however, the guanidinium chloride-thiocyanate method is preferred. The mRNA preparation of cells is generally carried out by first preparing total RNA, and then, by purifying mRNA from total RNA using a poly (A) + RNA purification matrix, such as globules of oligo (dT) cellulose and oligo (dT) latex . Alternatively, mRNA can be prepared directly from a cell lysate using said matrix. Methods for preparing total RNA include: alkaline sucrose density gradient centrifugation [see Dougherty, W.G and Hiebert, E. (1980), Virology, 101, 466-474]; the guanidine thiocyanate-phenol method; the guanidine-trifluorocesium thiocyanate method and the phenol-SDS method. The presently preferred method uses guanidinium thiocyanate and cesium chloride [see Chirgwin, J.M. et al. (1979), Biochemistry, 18, 5294-5299]. The poly (A) + RNA thus obtained can be used as the template in a reverse tranecriptase reaction to prepare single-stranded cDNA [cDNA (ss)]. The cDNA (ss) obtained by the use of transcriptaea inverea, as described above, can then be converted to double-stranded cDNA (ds). Suitable methods for obtaining the cDNA include the SJ nuclease method (see Eifstradiatis, A. et al. (1976), Cell, 7, 279-288] and the Gubler-Hoff method [see Gubler, U. and Hoffman, BJ (1983), Gene, 25, 263-269], and the Okaya a-Berg method [see Okayama, H. and Berg, P. (1982), Mol Cell Biol. 2, 161-170]. However, the currently preferred method includes the polymerase chain reaction [RCP- see Saiki, RK et al. (1988), Science, 239, 487-491, hereby incorporated by reference] using single-stranded cDNA as the template In this way, the preferred method is labeled "TI-PCR", since it includes reverse transcription and PCR The ds cDNA obtained above can then be integrated into a cloning vector and the resulting recombinant vector can be used to transform a suitable microorganism, such as E. coli The ransformant can be selected using a standard method, for example, by selecting Resistance to tetracycline or re-competition to ampicillin, encoded by the recombinant vector. If E.coli is used, then the transformation can be effected by the Hanshan method [véaee Hanehan, D (1983), J. Mol. Biol. 166, 557-580]. Alternatively, the recombinant vector can be introduced into competent cells, prepared by co-exposure to calcium chloride and either magnesium chloride or rubidium chloride. If a plasmid is used as a vector, then it is highly desirable that the plamidid host a drug-responsive gene, as mentioned above, in order to facilitate selection. Selection by brute force is possible, but it is not preferred. While the plasmids have been discussed, it will be appreciated that other cloning vehicles, such as lambda phages, can be used. In order to select for transformants carrying cDNA encoding a subunit of a human anti-Fas antiquase of interest, various methods can be used, such as those described below. When the cDNA of interest is specifically amplified by means of TI-PCR, this may be omitted. (1) Selection by Polymerase Chain Reaction If all or a portion of the amino acid sequence of the desired protein has been elucidated, then sense and antisense oligonucleotide primers corresponding to separate non-contiguous portions of the amino acid sequence can be synthesized. These initiators can then be used in the polymerase chain reaction technique [see Saiki, RK et al. (1988), Science, 239, 487-491] to amplify the desired DNA fragment encoding the monoclonal anti-cell subunit of human anti-Fas mouse. The DNA template used in PCR can be, for example, cDNA synthesized by reverse transcription from mRNA of the hybridoma that produces the anti-human Fae monoclonal antibody HFE7A (FERM BP-5828). The DNA fragment synthesized in this way can be integrated either directly into a plasmid vector, for example using commercial equipment, or labeled for example with 32P, 35S, or biotin, and then used as a probe for hybridization of colony or plate hybridization to obtain the desired clone. The DNA harvesting that encodes each subunit of the anti-human anti-Fas anti-clump, from the transformants obtained above, can be performed by well-known techniques, such as those described by Maniatis, T. et al. [In "Molecular Cloning a Laboratory Manual, "Cold Spring Ha or Laboratory, NY (1982), hereby incorporated by reference]. For example, the region of DNA encoding the desired subunit can be excised from the plasmid DNA after separating the fraction corresponding to the vector DNA from a transformant that had been determined to be plamido neceae. (2) Selection using a synthetic oligonucleotide probe. If all or a portion of the amino acid sequence of the desired protein has been elucidated, then a contiguous short sequence, which is also representative of the desired protein, can be used to construct an oligonucleotide probe. The probe encodes the amino acid sequence but, due to the degeneracy of the genetic code, there may be a large number of probes that can be prepared. In this manner, an amino acid sequence will normally be selected from an amino acid sequence that can be encoded only by a limited number of oligonucleotides. The number of oligonucleotides that need to be produced can be further reduced by the substitution of inosine wherein any of the four normal bases can be used. The probe is then labeled suitably, for example with 32P or 35S or biotin and then hybridized with denatured DNA transformed from the transformant that has been immobilized on a cellulose filter. Strains poeitivae appear by detection of the mark on the probe. Whenever appropriate, DNA sequences can be determined according to various methods well known in the art including, for example, the Maxam-Gilbert chemical modification technique [see Maxam, A.M. and Gilbert, W. (1980) in "Methods in Enzymology" 65, 499-276] and the dideoxy chain termination method, using the M13 phage [see Meesing, J. and Vieira, J. (1982), Gene, 19, 269-276]. In recent years, an additional method for the determination of DNA sequence has gained wide acceptance and includes the use of a fluorogenic dye in place of the conventional radioisotope in the dideoxy method. The entire process is done by computation, including the reading of the nucleotide sequence from the elect roforeey. The suitable machinery for the procedure, for example, is the sequence determination robot of Perkin-Elmer "CATALYST 800" and the DNA sequencer of Perkin-Elmer model 373A. The use of this technique makes the determination of nucleotide sequence of DNA efficient and safe. Using techniques such as those described above, the determination of the DNA sequence can be carried out, efficiently and safely. Based on the data of the nucleotide sequences reepectivae determined from the DNA of the present invention, and the respective N-terminal amino acid sequences of the heavy and light chains, all the amino acid sequences of the heavy and light chains of a monoclonal antibody of the present invention. For example, the HFE7A monoclonal antiquague of the present invention, which is suitable for providing RDC for grafting in a humanized antiquague of the present invention, is an immunoglobulin molecule Gl (IgGl) and, thus, is a complex composed of subunit chain peeada tl and light chain K. Loe preferred methods for determining the partial amino acid sequences of these respective subunits include, for example, isolating the respective subunits by means of a suitable technique, such as electrophoresis or column chromatography, and then analyzing the N-terminal amino acid sequences of the respective subunits using, for example, an automatic protein sequencer (eg, PPSQ-10, Shimadzu Seisakusyo, KK). The light and heavy chains of an immunoglobulin each consist of a variable region and a constant region; The variable region of each chain also consists of three RDCs and four structure regions that flank the RDCs. The amino acid sequence of the constant region is constant within any given subclass, regardless of the recognized antigen. On the other hand, the amino acid sequence of the variable region, at least for the RDCs, is specific for each antiquake. However, it has been established by comparison studies, using data on amino acid sequences of numerous anticueds, that both the location of the RDC and the lengths of the structure sequences are approximately similar among the antiquase subunits belonging to the same subgroup [ see Kabat, EA et al. (1991), in "Sequences of proteins of imological interest Vol. II", Department of Health and Human Services of the U.S.A., hereby incorporated by reference]. Therefore, by comparing the amino acid sequences of the heavy and light chains of the anti-Fas monoclonal antibody HFE7A with the known data of amino acid sequences, for example, the RDCs and the structure regions, as well as the location of the constant region, in each of the amino acid sequences determined above. Occasionally it has been found that the length of FRHi, that is, the region of the most N-terminal structure of the heavy chains, is shorter than the normal length of 30 amino acids. For example, the shortest known FRHI in mouse IgGl, of the same subtype as HFE7A, is only 18 amino acids [see Kabat et al., Ibid.]. Therefore, in the antiquase of the present invention, it will be appreciated that the length of that part of the total molecule corresponding to FRHi may be of appropriate length, typically between 18 and 30 amino acids, but preferably about 30 amino acids, provided that no the necessary Fae binding activity is lost. The three-dimensional structure of the binding region of Fae is determined mainly by the frequency in the variable regions, the support being provided by the constant regions. The structure regions provide structure to the RDCs that are configured chemically and structurally to interact with the antigen. Therefore, an exogenous antibody, or a portion of the mRNA, which recognizes an antigen other than Fas, can be selected and modified to recognize Fae by an appropriate alteration of the RDCs, according to the above guidelines (see, for example, U.S. Patent Publication No. 5,331,573). To preserve as much binding activity as possible, it is generally preferred to select receptor chains that have the greatest similarity to the donor chains. Said peptides modified in this manner are useful in the present invention, for example in the prevention or treatment of diseases attributable to Abnormalities of the Fas / Fas ligand system. Construction of a mutant in which one or more amino acids in an amino acid sequence may be suppressed, for example, by cassette mutagenesis (see Toshimitsu Kishi oto, "Shin-Seikagaku Jikken Kouza 2: Kakusan III Ku ikae DNA Gijutsu" , 242-251). DNA sequences can be prepared by any appropriate method, and many are known. A suitable method, especially for shorter sequences, is chemical synthesis using a conventional method, such as the pheephite triester method [see Hunkapiller, M. et al. (1984), Nature, 310, 105-111]. The selection of codons for any amino acid can be from any of the recognized codons corresponding to the desired amino acid, and such selection can be aFitrary, or take into account the frequency of a given codon in a host, or because it is possible to create a site of restriction by appropriate selection, without changing the amino acid sequence, for example. Partial modification of the nucleotide sequence can be effected by means of site-specific utosis using synthetic oligonucleotide primers coding for the desired modifications [Mark, D.F. and other (1984) Proc. Nati Acad. Sci. USA, 81, 5662-5666], by means of conventional techniques. Hybridization of DNA with DNA encoding the heavy or light chain of an anti-Fae monoclonal antiquague of the present invention can be determined, for example, by using an appropriate fragment of DNA of the invention labeled with (-32P) dCTP, for example, as a probe with a method such as the random primer method [see Feinberg, AP and Vogelstein B. (1983), Anal. Biochem., 132, 6-13] or with the critical point translation method [see Maniatis, T. et al. (1982) in "Molecular Cloning a Laboratory Manual", Cold Spring HaFor Laboratory, NY]. A suitable technique is as follows. First, the potentially hybridizing DNA is adsorbed onto a nitrocellulose or nylon membrane, for example, being subjected to alkaline treatment if necessary, and after being fixed by heating or UV radiation. In a preferred method, the membrane is then immersed in prehybridization solution containing 6 X SSC (1 x SSC is an aqueous solution of 0.15 M NaCl and 0.015 M citric acid trisodium), 5% v / v of Denhart's solution and 0.1% v / v sodium dodecyl sulfate (SDS), and incubate at 55 ° C for 4 hours or more. Then, the probe prepared above is dissolved in similar prehybridization solution to a final specific activity of 1 x 106 cpm / ml, followed by incubation at 60 ° C overnight. Subsequently, the membrane is washed at room temperature by repeated washing with 6 x SSC for 5 minutes and then with 2 x SSC for 20 minutes, and then autoradiographed. Using such a method, DNA can be isolated with the DNA encoding the heavy or light chain of a monoclonal anti-Fas anti-clump which can serve as the basis for a humanized anti-Fas antiquague of the present invention., from any genomic library or cDNA library [see Maniatis, T. et al. (1982) in "Molecular Cloning to Laboratory Manual", Cold Spring Ha or Laboratory, NY]. Said DNA is within the scope of the present invention, the essential characteristics of the hybridization being 6x SSC and 55 ° C, preferably 60 ° C, and preferably 70 ° C. The integration of the DNA of the present invention thus obtained, into an expression vector, allows the transformation of prokaryotic or eukaryotic host cells. Such expression vectors will typically contain suitable promoters, sites of replication and sequence involved in the expression of genes, which allow the DNA to be expressed in the host cell. Suitable prokaryotic host cells include, for example, E. coli Escherichia coli) and Bacillus eubtilis. In order to express the gene of interest in those host cells, said host cells can be transformed with a plasmid vector containing a replicon derived from a species compatible with the host, typically having an origin of replication and a promoter sequence, such as lac UV5. Preferably these vectors have sequences capable of conferring a selection phenotype to the transformed cell. A suitable strain of E. coli is strain JM109 derived from E. coli K12. Suitable vectors include the eeries of plasmids pBR322 and pCU. Suitable promoters include the lactose promoter (lac) and the tryptophan-lactose (tre) promoter, the tryptophan (tf) promoter, the lipoprotein promoter (Ipp), the PL lambda promoter derived from bacteriophage lambda, and the promoter of the elongation factor of the Tu (tufB) polypeptide chain. In general, it will be appreciated that the present invention is not limited to the use of those hosts, vectors, promoters, etc., which are exemplified herein, and that any suitable system may be used, as desired.

A suitable preferred strain of Bacillue subtilis is strain 207-25, and a preferred vector is pTUB228 [see Obmura, K. et al. (1984), J. Biochem., 95, 87-93]. A suitable promoter is the regulatory sequence of the α-amylase gene of Baciilus eubtilis. If desired, the DNA sequence encoding the signal peptide sequence of an a-a -lase can be linked to the DNA of the present invention to allow extracellular secretion. Eukaryotic hosts include huésoedes cells of vertebrates and yeast species. An example of vertebrate cells used is the monkey C0S-1 cell line [see Gluz an, Y. (1981), Cell, 25, 175-182]. Suitable hosts of yeast cells include baker's yeast (Saccharomyces cerevisiae). methyl yeast (Pichia pastoris) and fission yeast (Schieosaccharomyces pombe). It will be appreciated that other guests may also be used. In general, what is required for the expression vectors suitable for vertebrate cells is that they comprise: a promoter, usually located towards the 5 'end of the gene to be expressed; an RNA splice site, a polyadenylation site; and a transcription termination sequence, as well as any other required functionality, such as an origin of replication. A suitable plasmid is pS ~ V2dhfr, which contains the SV40 early promoter [see Subremani, S. et al. (1981), Mol. Cell. Biol. 1, 854-884], but many others are known to those skilled in the art. Suitable eukaryotic microorganisms are yeasts, such as S. cerevisiae and suitable expression vectors for yeasts include pAH301, pAH82 and YEp51. Suitable vectors contain, for example, the promoter of the alcohol dehydrogenase gene [see Bennetzen, Y.L. and Hali, B.D. (1982) J. Biol. Chem. 257, 3018-3025] or of the caFoxypeptidase Y GALIO promoter [see Ichikawa, -K. And others (1993), Biosci. Biotech, Biochem, 57, 1686-1690]. If desired, the DNA sequence encoding the signal peptide sequence of caFoxypeptidase Y can be linked to the DNA to be expressed, in order to allow extacellular secretion. When COS cells are used as a host, suitable vectors comprise the SV40 reproduction origin, which allows autonomous replication; a transcription promoter, a transcription termination signal and an RNA splice site. Expression vectors can be used to transform the cells by any suitable method, such as the DEAE-dextran method [see Luthman, H. and Magnusson, G. (1983), Nucleic Acids Res. 11, 1295-1308], calcium phosphate-DNA coprecipitation method [see Graham, FL and van der Eb, A.J. (1973), Virology, 52, 456-457] and the electroporation method with electric pulse [see Neumann, E. et al. (1982), EMBO J. 1, 841-845]. In a preferred embodiment COS cells are co-transfected with two separate expression vectors - one containing the DNA encoding a protein comprising at least the variable region of the heavy chain of the anti-HFE7A antibody, preferably as part of a humanized heavy chain complete, and one containing the DNA encoding a protein comprising at least the variable region of the anti-HFE7A anti-chain light, preferably as part of a complete humanized light chain; these vectors being simultaneously expressed to generate a recombinant, humanized anti-Fas antishock. . The tranexicants of the present invention may be cultured using conventional method, the proteins being expressed as desired intracellularly or extracellularly. Suitable culture media include various commonly used media, and will generally be selected according to the selected host. For example, suitable media for COS cells include RPMI-1640 and Dulbecco's Modified Eagle Minimum Essential Medium (DMEM), which can be supplemented, when desired, with fetal bovine serum (FBS). The culture temperature can be any suitable temperature that does not significantly depress the protein synthesis capacity of the cells and, preferably, will be in the range of 32 to 42 ° C, most preferably, 37 ° C, especially for cells of mammals If desired, cultivation can be carried out in an atmosphere containing 1 to 10% (v / v) of carbon dioxide. The E. coli transformants pME-H and E. coli pME-L, each transformed with a recombinant DNA vector for the expression in animal cells of DNA encoding the heavy and light chains, respectively, of an anti-monoclonal anti-convolver. Useful fas to prepare humanized anti-Fas antiquands of the present invention, were deposited according to the Budapest Treaty in Kogyo Gijutsuin Seimei-Kogaku Kogyo Gijuteu Kenkyujo on March 12, 1997, and were assigned the numbers FERM BP-5868 and BP-5867, respectively. Therefore, by transforming cultured animal cells such as C0S-1, with the activated recombinant vectors of the depoeitadae strains and culturing the transforming cells, a recombinant anti-Fas anti-cough can be produced in culture. The protein expressed by the transformants of the present invention can be isolated and purified by various well-known methods for separation, according to whether the protein is expressed inside or outside the cells, and depending on considerations such as the properties Physical and chemical proteins. Specific methods suitable for separation include: treatment with precipitating agents commonly used for proteins; various chromatography methods, such as ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography (HPLC); dialysis and combinations thereof. By using those methods described above, the protein can be easily obtained in high yields and high purity. To optimally humanise, in this case, a monoclonal anti-Fas mouse, it is preferred to graft the variable regions in a human anti-cough, at least so that all CDRs are incubated in the human anti-cough, and preferably also for residues. Eignificants of the FR sequences are grafted onto the human anti-cotuefo in order to maintain as much of the structure of the binding site as possible. This can be accomplished by any of the following three methods: 1) using heavy and light chains of the same concocted human antiquake; or 2) uear peeada and light chains derived from different human anticuefos that have high homology of sequences, or that share sequence of connection with the chains of the donor, maintaining at the same time the combination of the subgroups of the receptor chains; or 3) select the FRs of peeda and light chains that have the highest homologies with the FRs of the donor of a library of the primary sequences of human anticueves, regardless of the combination of the subgroups. Said selection method based only on sequence homology, without other restrictions, makes it possible for the donor and the recipient to share at least 7C% of amino acid identity in the FR portions. Adopting this approach, it is possible to reduce the number of grafted amino acids of the donor, with respect to known methods, and thereby minimize the HAMA response induction. The term "amino acid sequence homology", as used herein, refers to the sequence similarity of amino acids between two different polypeptides or proteins. The amino acid sequence homology can be determined by means of one of several methods, which generally include the computerized search of baeee of sequence data. These methods are well known to the person skilled in the art. The authors of the present prefer that the homology be determined on the length of the regions of structure. It will be appreciated that the function of the amino acid residues that rarely occur in the donor subgroup can not be fully defined, since the techniques for predicting the three-dimensional structure of an antiquake molecule from its primary sequence (hereinafter referred to as " molecular model elaboration ") have limited accuracy. Known methods, such as the method of Queen et al. (Queen et al., cited above), do not indicate whether the amino acid residue of the donor or receptor should be selected at that position. The selection of a receptor molecule based only on sequence homology can significantly reduce the need to make this type of selection. Additionally, the present inventors have discovered an additional refinement for this method, through the provision of an additional selection procedure, designed to identify the amino acids from donor FRs, which are important in the maintenance of structure and function. from the DRC regions of the donor. Once the human receptor molecule has been selected for a given chain, the selection of the amino acid residues to be grafted, coming from an FR of a donor, is carried out in the following manner: amino acids of the donor and the recipient. If the aligned amino acid residues of the FRs differ in any position, it is necessary to decide which residues should be selected. The selected waste must interfere with, or only have a minimal effect on, the three-dimensional structure of the CDRs derived from the donor. Queen et al. (International Patent Publication No. W090 / 07861, hereby incorporated herein by reference) proposed a method for deciding whether an amino acid residue from the donor FR should be grafted together with the RDC sequence. According to that method, an amino acid residue from an FR region is grafted into the receptor along with the CDR sequence if the residue satieves at least one of the following criteria: (1) The amino acid in the region of human structure The receptor is rarely found in that position in the receptor, whereas the corresponding amino acid in the donor is commonly found in that position in the recipient. (2) The amino acid is close to one of the RDC; and (3) The amino acid has a side chain atom within about 3oA of a CDR, when judged by the three-dimensional model of immunoglobulin, and is potentially capable of interacting with an antigen or CDR of a humanized antiquake. A residue identified by criterion (2) above frequently exhibits the characteristic of criterion (3). Thus, in the present invention, criterion (2) is omitted and two new criteria are introduced. Accordingly, in the present invention, an amino acid residue from a donor FR is grafted together with the RDC, if the residue satieves at least one of the following criteria: (a) The amino acid in the human structure region of the receptor is rarely found in that position in the recipient, whereas the corresponding amino acid in the donor is commonly found in that position in the recipient; (b) The amino acid has a side chain atom within about 3 ° A of a CDR, when judged by a three-dimensional model of the immunoglobulin, and is potentially capable of interacting with an antigen and a CDR of a humanized anti-cue; (c) The amino acid is found in a position that is involved in the determination of the structure of the canonical class of the RDC; (d) The position of the amino acid is found at the contact surface of the light and heavy chains. With respect to criterion (a), the amino acid is defined as "common" when it is in that position in 90% or more of the antiquands of the same subclass (Kabat et al., Above). An amino acid is defined as "rare" when it is found in less than 10% of the antiquands of the same subclass. With respect to criterion (c), the position of a canonical class of determinant residues can be determined unambiguously, according to the information provided by Chothia et al. [Chothia et al., Cited above]. With respect to criteria (b) and (d) it is necessary to carry out the elaboration of the molecular model of the variable regions of the antiquake, in advance. While any application program commercially available for the formation of the molecular model can be used, the authors hereby prefer that AbM software be used [Oxford Molecular Limited, Inc.]. The predictions made through the elaboration of the molecular model have limited precision. Therefore, in the present invention, the structure prediction obtained by the molecular model formation was determined by comparing it with X-ray crietalography data from variable regions of various anticuefos. When a structural model generated by molecular model elaboration or molecular model determination (such as AbM software) is used, it is presumed that two atoms will be in contact with each other through the Van der Waals forces, when the distance between the two atoms is lower. than the sum of its Van der Waals radios, plus 0.5 °. It is presumed that a hydrogen bridge is present when the distance between the polar atoms, such as an amide nitrogen and a cafonon oxygen of the main and side chains, is less than 2.9oA, that is, the average bond length of hydrogen plus 0.5 ° A. Additionally, when the distance between the two atoms of opposite charge is shorter than 2-85 ° A plus 0.5 ° A, they are presumed to form an ion pair. Thus, in this case, an amino acid residue from the donor is conserved if: i) the side chain atom is within a distance of a second atom of less than the sum of its Van der Waals radii, plus 0.5 TO; ii) the side chain atom is polar and is less than 3.4 A of a second polar atom, or iii) the side chain atom is charged and is less than 3.35 A of an atom with opposite charge. The positions of the amino acids in the FRs, which make frequent contact with an RDC, were identified, based on X-ray crystallography data from the variable regions of various antibodies. Those poeicionee were determined independently of the subgroups. For light chains, these positions are: 1, 2, 3, 4, 5, 23, 35, 36, 46, 48, 49, 58, 69, 71 and 88 and, for heavy chains, the positions are: 2, 4, 27, 28, 29, 30 , 36, 38, 46, 47, 48, 49, 66, 67, 69, 71, 73, 78, 92, 93, 94 and 103. The above amino acid numbering is defined in relation to the N-terminus of the heavy chain or mature light chain (see Kabat et al., above), which is called position "1". The numbering is applied as if the FRHi region were 30 amino acid residues long, even if it is shorter, for example 18 residues. This numbering system is eagerly pursued. When the same data is analyzed by molecular model elaboration, the amino acid residues in this demoetre were found to be in contact with the RDC amino acid residue in two thirds of the anti-cough variable regions that were examined. These discoveries were used to define criterion (b) above. Specifically, if it is predicted that an amino acid position in a FR will be both in contact with an RDC by molecular model elaboration, and is also frequently found experimentally that makes contact with an RDC by X-ray crystallographic analysis, then it becomes a priority is the grafting of the amino acid residue of the donor. In any other case, criterion (b) is not considered. Similarly, with respect to criterion (d), X-ray crystallography data from the variable regions of many anti-convolutions indicate that the amino acid residues at positions 36, 38, 43, 44, 46, 49, 87 and 98 in light chains, and those in positions 37, 39, 45, 47, 91, 103 and 104 in heavy chains, are frequently involved in contact between heavy and light chains. If either of these amino acids is predicted to be involved in light and heavy chain contact by molecular model making, then the graft of the amino acid residue of the donor is given priority. In any other case, criterion (d) is not considered. The DNA encoding the variable regions of the H and L chains of a humanized anti-Fas humanized antiquague of the present invention can be prepared in several ways. In one method, polynucleotide fragments of between 60 and 70 nucleotides long, which represent partial nucleotide sequences of the desired DNA, can be synthesized. This synthesis procedure is arranged so that the ends of the fragments of the sense chain alternate with those of the antisense chain. The resulting polynucleotide fragments can be fixed to one another and ligated by DNA ligase. In this way, the desired DNA fragment coding for the variable regions of the H and L chains of the humanized anti-Fas antiscan can be obtained. Alternatively, the DNA encoding the entire variable region of the receptor can be isolated from human lymphocytes. Site-directed mutagenesis can be used to introduce restriction sites in the regions encoding the RDCs of the donor. The CDRs can then be effected by the receptor using the relevant restriction enzyme. The DNA encoding the RDC of the donor can then be synthesized, and ligated into the receptor molecule, using DNA ligase. The present authors prefer that the DNA encoding the variable regions of the H and L chains of a humanized anti-Fas human antibody is obtained by the overlapping extension PCR technique [Horton et al. (1989), Gene, 77, 61-68, hereby incorporated by reference]. The overlapping extension PCR allows two DNA fragments, each encoding a desired amino acid sequence, to join. By way of example, the two fragments are designated here as (A) and (B). A sense primer (C) of 20 to 40 nucleotides, which is fixed with a 5 'region of (A) ee synthesized, together with a counter-sense initiator, of 20 to 40 nucleotides (D), which is fixed with a 3 'region of (B). Other initiating doe are required: first a chimeric (E) sense primer, comprising from 20 to 30 nucleotides of a 3 'region of (A) linked to 20 to 30 nucleotides of a 5' region of (B). Secondly, a counter-sense initiator (F) is required, complementary to the sense initiator. A PCR reaction can be carried out using primers (C) and (F) in combination with a DNA template containing fragment A. This allows a DNA product comprising 20 to 30 nucleotides of the DNA to be produced. 5 'region of (B), attached to the 3' end of (A). This fragment is called the fragment (G). Similarly, PCR can be carried out using primers (D) and (E), in combination with a DNA template containing fragment B. This allows a DNA product to be produced comprising 20 to 30 nucleotides of DNA. the 3 'region of (A), attached to the 5' end of (B). This fragment is called the fragment (H). Fragments (G) and (H) carry complementary sequences of 40 to 60 nucleotides in the 3 'region of (G) and 40 to 60 nucleotides in the 5' region of (H), respectively. A PCR reaction can be carried out using a mixture of the fragments (G) and (H) as a template. In the first step of denaturing, the DNA becomes a single chain. Most of the DNA returns to the original form in the subsequent binding step. However, a part of the DNA forms a heterologous DNA duplex, due to the binding of the fragments (G) and (H) in the region of overlap of the sequence. In the subsequent extension step, the portions of a single protruding chain are repaired, to result in the chimeric DNA representing a ligation of (A) and (B). This DNA fragment is referred to below as fragment (I). The fragment (I) can be amplified using the initiator (C) and the initiator (D). In the embodiments of the present invention, the fragments (A) and (B) can represent DNA encoding the RDC regions and the H and L chains of a humanized monoclonal anti-mouse mouse anti-human Fae; DNA encoding the FR regions of human IgG or DNA encoding the secretion signal of human IgG. The codon or codons that correspond to a specific amino acid are known. When designing a DNA sequence from which a protein is produced, any suitable codon can be selected. For example, a codon can be selected based on the codon order of the host. The partial modification of a nucleotide sequence can be obtained by means of the standard technique of site-directed mutagenesis, using synthetic oligonucleotide primers that encode the desired modifications [Mark, D.F. and others (1984) Proc. Nati Acad. Sci. USA, 81, 5662-5666]. Using the primers selected to introduce one or more point-specific mutations, the DNA encoding the variable regions of the H and L chains can be obtained from any desired anti-human humanized anti-Fas. The integration of the DNA of the present invention, thus obtained in an expression vector, allows the transformation of prokaryotic or eukaryotic host cells. Said expression vectors will typically contain suitable vectors, sites of reproduction and sequence involved in the expression of genes, which allow the DNA to be expressed in the host cell. The five transformant strains carrying DNA coding for the variable regions of the light chains of a humanized anti-Fae antiquake of the present invention, to eaber, E. coli pHSGMM6 SANK73697, E. coli pHSGHM17 SANK73597, E. coli pHSGHH7 SANK73497, E coli pHSHM2 SANK 70198 and E. coli pHSHH5 SANK 70398, as well as two transformant strains carrying DNA encoding the variable region of the peadal chain of a humanized anti-Fas antiquase of the present invention, namely E. coli pgHSL7A62 SANK 73397 and E. coli pgHPDHV3 SANK 70298, were deposited in accordance with the Budapest Treaty in Kogyo Gijutsuin Seimei-Kogaku Kogyo Gijutsu Kenkyujo on August 22, 1997, and were assigned the numbers FERM BP-6071, FERM BP-6072, FERM BP-6073, FERM-6272 and FERM-6274 (light chains) and FERM Bp-6074 and FERM BP-6273 (heavy chains), respectively, in accordance therewith. Therefore, DNA encoding each subunit of the humanized anti-Fas antiquase protein can be obtained, for example, by isolating a plasmid from staged cepae, or by performing PCR using as template an extract of the deposited strains. A high purity anti-Fas recombinant antibody, in high yield, can easily be produced by the methodology described above. To check that a recombinant anti-Fas antifungal prepared as indicated above binds specifically to Fas, ELISA can be performed in a manner similar to that described above for the evaluation of anti-cotus titers in immunized mice. The HFE7A antibody, and the humanized anti-Fas antibodies of the present invention, have functional properties a) to f) below, each of which can be verified, for example, by a method described. Induction of apoptosis in T cells expressing Fas. The apoptosis-inducing activity in Fas expressing T cells can be analyzed by extracting the thymus from a mouse which has been administered a anti-Fas humanised antiquase of the present invention (also referred to hereafter as "the antiquase"), fragmenting the and by contacting the cells obtained with T cells and a specific anti-CFC for mouse Fas, and measuring the proportion of the cells to which both anti-buds are bound, by means of flow cytomety. Decrease in autoimmune symptoms of MRL gld / gld mice.

The antiquase is administered intraperitoneally to an MRL gld / gld mouse. These mice carry a mutation in the gene encoding the Fas ligand and exhibit symptoms that resemble autoimmune diseases [see Shin Yonehara (1994), Nikkei Science Beseateu, 110, 66-77]. The anti-convulsant is capable, in many cases, of preventing or at least reducing, swelling of the limbs, which is one of the symptoms similar to the autoimmune disease. Failure to induce liver disorders. Peripheral blood is extracted from a BALB / c mouse to which the antibody was administered, and the blood levels of glutamic-oxalacetic enzymes transaase inase (GOT) and glutamic-pyruvic transaminase (GPT) are measured using an automatic analyzer ( for example, Model 7250; Hitachi Seisakueyo, KK) together with the reagent for the analyzer (eg, trane-aminase-HRII; Wako Puré Chemical Industries, Ltd.). Failure to cause high blood levels of GOT and GPT indicate that the anti-convulsant does not induce liver disorders by in vivo administration. Therapeutic or prophylactic effect on fulminating hepatitis. In an experimental system in which fulminant hepatitis is induced in mice by administering the anti-Fas mouse monoclonal anti-Fas Jo2, the effects of the anterior antifog adinjection can be examined simultaneously with Jo2 or after the administration of Jo2. The anti-foe of the invention can prevent, to a large extent, all the effects of Jo2 in mice, thereby demonstrating a protective effect on the liver. Preventive effect on the appearance of collagen-induced arthritis. The effects of administering the antibody in a rheumatoid arthritis model are examined, caused by administering to an mouse an emulsion comprising collagen and complete Freund's adjuvant. The antiquake has prophylactic properties. Induction of apoptosis in synovialee cells of a patient with rheumatoid arthritis. Synovial cells obtained from an affected region of a patient with rheumatoid arthritis are cultured, and the viability of the cells is examined when the previous antiquake is contained in the culture medium.

Surprisingly, the proliferation of synovial cells is inhibited. In this manner, the anti-convolutions of the present invention, unlike the known anti-Fas anti-Fas anti-cues, not only protect the normal cells, but also kill the abnormal cells. Therefore, they are useful as prophylactic and therapeutic agents for diseases attributable to abnormality of the eietema.

Fas / Fas ligand. The ability of the proteins of the present invention to induce apoptosis can be established, for example, by culturing cells such as the human lymphocyte cell line HPB-ALL [Morikawa, S et al. (1978), Int. J. Cancer 21, 166- 170] or Jurkat (American Cultivation Type No. TIB-1520) in the middle in which the test sample has been, or will be, added. The survival rate can then be determined by, for example, an MTT test [Green, L.M. and others (1984), J. Immunological Methods, 70, 257-268]. The antiquands of the present invention can be used in several respective pharmaceutical preparations for the different pathological conditions related to abnormalities of the seventh Fae / Fas ligand, such as those indicated above. Said prophylactic or therapeutic agent can be administered in any form of a variety. Suitable forms of administration include oral administration, for example by tablets, capsules, granules, powder and syrup, or parenteral administration, for example by any form of injection, including intravenous, intramuscular and intradermal, as well as infusions and suppositories. In this manner, the present invention also provides methods and therapeutic compositions for treating the conditions referred to above. Said compositions typically comprise a therapeutically effective amount of the protein of the present invention in admixture with a pharmaceutically acceptable carrier thereof., and can be administered in any suitable manner, for example by parenteral, intravenous, subcutaneous or topical administration. In particular, in case the condition to be treated is local, it is preferred to administer the protein as close to the site as possible. For example, serious rheumatic pain may be experienced in the major joints, and the protein may be administered at such sites. The proteins of the present invention to be administered in general form, are preferably administered in the form of a therapeutically and parenterally acceptable, pyrogen-free aqueous solution. The preparation of such pharmaceutically acceptable protein solutions with respect to products such as pH, isotonicity, stability and the like is within the skill of the skilled artisan. In addition, the compositions of the present invention may comprise additional ingredients, as deemed appropriate, such as cell growth retardants and other medicaments. It will be appreciated that the dosage may vary, depending on factors such as the condition, age, and coforal weight of the patient, but usually the dosage for oral administration for an adult varies between about 0.1 mg and 1,000 mg per day, which can be administered in a single dose or in several divided dosies. The dosage for parenteral administration typically varies between 0.1 and 1,000 mg, which can be administered by injection (or injections) subcutaneously, intramuscularly or intravenously. A suitable oral administration form of the humanized anti-Fas antifungal of the present invention is as a vial of a sterile solution or suspension in water or a pharmaceutically acceptable solution. Alternatively, a sterile powder (preferably prepared by lyophilization of the anti-humanized anti-Fas antifoulant) can be filled into an ampule, which can be diluted for later use with a pharmaceutically acceptable solution. Due to the fact that the antiquaves of the present invention used for human treatment have been humanized, their toxicity is very low. The invention will now be illustrated in greater detail with reference to the following examples, these being illustrative but not mandatory of the present invention. The examples show specific embodiments of the present invention. Any method, preparation, solution and similar that are not specifically defined can be found in "Molecular Cloning -A Laboratory Handbook" (above, hereby incorporated by reference). All solutions are aqueous and made in sterile deionized water, unless otherwise specified.

REFERENCE EXAMPLE 1 Preparation of Fas antigen A vector was constructed to obtain a soluble version of human Fas that lacks the transmembrane domain. This vector was designed to encode a fusion protein (the "Fas fusion protein") comprising the extracellular domain of human Fas fused to the extracellular domain of the mouse interleukin 3 (IL3) receptor [see Gorman, DM and others , (1990), Proc. Nati Acad. Sci. U.S.A., 87. 5459-5463]. DNA encoding the human Fas fusion protein was prepared from this vector by PCR. The construction of the vector and the preparation of the DNA was as follows. a) Mold The molds used for the CPR to construct the insert that codifies the protein of fire, were doe plaemidoe. The first plaemid, [see Nishimura, Y. and others, (1995), J. Immunol. 154, 4395-4403], was the plasmid vector of DNA expression encoding a fusion protein, comprising the extracellular domain of mouse Fas and the extracellular domain of the mouse IL3 receptor. The second plasmid, pCEV4 [see Itoh, N., et al., (1991), Cell, 66, 233-243], carries cDNA encoding human Fas. b) PCR primers The following oligonucleotide primers were synthesized: 5'-GGGGAATTCC AGTACGGAGT TGGGGAAGCT CTTT-3 '(NI: SEQ ID No. 12 of sequence list); 5'-GTTTCTTCTG CCTCTGTCAC CAAGTTAGAT CTGGA-3 '(C3N; SEQ ID No. 13 of the sequence listing); 5'-TCCAGATCTA ACTTGGTGAC AGAGGCAGAA GAAAC-3 '(N3N: SEQ ID No. 14 of the sequence listing); and 5'-CCCTCTAGAC GCGTCACGTG GGCATCAC-3 '(CTN2: SEQ ID No. 15 of the sequence listing).

Unless otherwise specified, all oligonucleotides in these examples were synthesized using an automated DNA synthesizer (model 380B, Perkin Elmer Japan, Applied Biosyeteme Division) following the instructions in the manual [see Matteucci, MD and Caruthers , M: H:, (1981), J. Am. Chem. Soc., 103, 3185-3191]. After the synthesis, each oligonucleotide (initiator) was removed from the support, unprotected, and the resulting solution was lyophilized to obtain a powder. This powder was then dissolved in distilled water and stored at -20 ° C until required. c) First step of PCR i) A DNA fragment, designated as HFAS and encoding the extracellular domain of human Fas, was prepared as follows. CPR was performed using the RCP LA (Long and Accurate) equipment (Takara Shuzo Co., Ltd., Japan).

Composition of the PCR reaction solution: DNA template pCEV4, 20 ng; NI primer, 0.5 μg; C3N initiator, 0.5 μg; Concentrated 10X RCP pH regulator (supplied with the equipment), 25 μl; dNTP's (provided with the equipment), 25 μl; and Taq LA polymerase (provided with the equipment), 12.5 units. Sterile distilled water was added to the solution to a total volume of 250 μl. Unless otherwise specified, dNTP's were supplied as an equimolar mixture of dATP, dCTP, dGTP and dTTP. The PCR reaction was carried out as follows. First, the solution was heated to 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After completion of this procedure, the reaction solution was heated at 72 ° C for 10 minutes (in all the PCR reactions described in the reference examples, the temperature was regulated using the seven RPM GeneAmp 9600, Perkin Elmer, Japan). ii) A DNA fragment, denoted as MAIC and encoding the extracellular domain of the mouse IL3 receptor, was prepared as follows: Composition of the PCR reaction solution: DNA template pME18S-mFas-AIC, 20 ng; initiator N3N, 0.5 μg; CTN2 primer, 0.5 μg; RCP LA pH regulator concentrated 10 times (provided with the equipment), 25 μl; dNTP's, 25 μl; Taq LA polymerase, 12.5 units; and sterile distilled water up to a total volume of 250 μl. The PCR reaction was carried out as before. The fragment of DNA HFAS and amplified MAIC aei obtainedthey were first removed first with phenol extraction, after ethanol precipitation [these two procedures are defined in example 2 (2) 3) a) below], after which the purified fragments were subjected to electrophoresis on a gel of polyacrylamide at 5% w / v. The gel was stained with 1 μg / ml of ethidium bromide to show DNA under UV light. Bands in which it was determined to contain the desired DNA fragments were cut using a safety knife blade and the DNA was electroeluted from it using an Amicon Centriruter apparatus equipped with Centricon-10 Centrifugal type ultrafiltration device ( Amicon). After electroelution, the Centricon-10 unit containing the eluted product was discarded and centrifuged at 7,500 x g for about 1 hour to concentrate the DNA. The DNA was precipitated with ethanol and then dissolved in 20 μl of distilled water. d) Second PCR step The FASAIC fragment of DNA encoding the fusion protein of human Fas (human Fas / murine IL3 receptor) was prepared as follows: Composition of the PCR reaction solution: HFAS DNA template solution 20 μl; MAIC DNA template solution, 20 μl; NI primer, 0.5 μg; CTN2 primer, 0.5 μg; RCP LA pH regulator concentrated 10 times, 25 μl; dNTP's, 25 μl; Taq LA polymerase, 12.5 units; and sterile distilled water up to a total volume of 250 μl. The PCR reaction was carried out as in c) above. The amplified FASAIC DNA fragment thus obtained was first extracted with phenol, then precipitated with ethanol, after which it was subjected to electrophoresis on a 1% w / v polyacrylamide gel. The gel was stained with 1 μg / ml ethidium bromide to show the DNA under UV light. The band that was determined to contain the used DNA fragment was cut using a safety razor blade and the DNA was electroeluted from it by using an Amicon Centriruter equipped with a Centricon-10 device, as described above. After electroelution, the Centricon-10 unit containing the eluted product was removed and centrifuged at 7,500 xg for about 1 hour to concentrate the DNA, and the DNA was then precipitated with ethanol and finally dissolved in 50 μl of distilled water. e) Construction of Vectoree All the DNA FASAIC, obtained in b) above, was digested with the restriction enzymes EcoRI and Xbal, after which it was extracted with a mixture of phenol / cyoroform (50% v / v of phenol saturated with water, 48% v / v chloroform, 2% v / v of isoamyl alcohol), then precipitated with ethanol. The resulting precipitate was suspended in 2 μl of sterile deionized water. Two micrograms of plasmid pME18S-mFae-AIC were subjected to digestion with the EcoRI and Xbal reentrant enzyme and X-phosphorylation (the phosphorylation procedure is defined in procedure 2 (2) 3) a) below]. The resulting DNA fragment was then ligated with the FASAIC of restriction digested DNA obtained above, using a ligation kit ((Takara Shuzo Co., Ltd.), then the ligation product was used in the transformation of the E. coli DH5a (Gibco BRL) as described by Hanahan [Hanahan, D., (1983), J. Mol. Biol., 166, 557-580.] The transformed E. coli plasmid was then obtained by the method of alkaline SDS [see Maniatis, T., et al., (1989), in Molecular Cloning: A Laboratory Manual (23 Edition), Cold Spring HaFor Laboratory, NY] The plasmid thus obtained was designated as phFas-AIC2. This plasmid was purified using a large-scale plasmid preparation kit (MaxiPrep DNA purification system, Promega) 20 μg of purified plasmid DNA was precipitated with ethanol and the precipitate was dissolved in 20 μl of Dulbecco's PBS medium (- ) (hereinafter referred to as PBS; Nissui Pharmaceutical Co., Ltd.). f) Expression COS-1 cells (American Type Culture Collection - American Type Culture Collection - No. CRL-1650) were developed up to semi-inflow in a culture flask (culture area: 225 cm2; Sumitomo Bakelite, KK) containing medium Modified Dulbecco Eagle (DMEM; Nissui Pharmaceutical Co., Ltd., Japan) supplemented with 10% v / v fetal calf serum (FCS; Gibco) at 37 ° C under a 5% v / v gaeeoeo CO2 atmosphere. The growth medium was then distilled, and 3 ml of an aqueous solution of 5 g / l trypsin and 2 g / l of diethyldiaminotetraacetic acid (trypsin-EDTA solution, Sigma Chemicals, Col.) were added to the flask, which was then incubated at room temperature. 37 ° C for 3 minutes to separate the cell from the flask. Lae harvested cells were suspended in PBS. They were washed twice with PBS and adjusted to 6 x 10 7 cells / ml with PBS. 20 μl of the resulting cell suspension (1.2 x 10 6 cells) was mixed with 20 μl of the plasmid solution prepared above, and the mixture was placed in a chamber with electrodes fixed with 2 mm separation (Shimadzu Seisakusyo, K.K.). Then, the chamber was loaded into a gene transfection apparatus (GTE-1; Shimadzu Seisakusyo, K.K.) and puleacionee of 600 V, duration of 30 μec, 2 times, with one second of separation were applied. The cell-DNA mixture in the chamber was then introduced in 10 ml of DMEM supplemented with 10% FCS v / v and incubated in a culture flask (culture area: 55 cm2) under 7.5% CO2 v / v at 37 ° C C for 24 hours. After this time, the supernatant culture was discarded and the cells were washed with serum free DMEM. Subsequently, 10 ml of the serum-free DMEM was added to the washed cells and the mixture was then incubated under 7.5% v / v CO2 at 37 ° C for 24 hours, after which the supernatant was recovered. The recovered supernatant was dialyzed against 10 M Tris-HCl (pH 8.0), in a dialysis tube (PM exclusion 12,000-14,000, Gibco BRL) and the human Fas fusion protein after partial purification using the FPLC apparatus from Pharmacia under The following conditions: Column: Reeource Q (brand: 06.4 x 30 mm, Pharmacia); Eluent: 10 mM Tris-HCl (pH 8.0); Flow rate: 5 ml / min; Elution: 0.1-0.3 M linear gradient of NaCl, in 30 minutes. The eluted product was collected in 5 ml fractions and these were analyzed to determine the Fas gene expression product by ELISA (enzyme-linked immunosorbent assay), as described below. First, 100 μl of each separate fraction was placed in wells of 1 96-well microplate (Costar) and incubated at 37 ° C for 1 hour. After this time, the solution in the wells was tilted, and the plate was washed 3 times with 100 μl / well of PBS containing 0.1% v / v Tween 20 (PBS-Tween). After washing, PBS containing 10% w / v bovine serum albumin ("BSA") was added in amounts of 100 μl / well, and the plate was then incubated at 37 ° C for 1 hour. After this time, the wells were washed three more times with 100 μl / well of PBS-Tween, after which 100 μl / well of a solution of the monoclonal anti-cough HC of the beta subunit of the IL-3 receptor was added. anti-mouse (1 mg / ml; Igaku Seibutsugaku Kenkyujo, KK) diluted 100 vecee with PBS-Tween to each well, and the plate was incubated once again at 37 ° C for 1 hour. The wells were then washed 3 times with 100 μl / well in PBS-Tween, and then 100 μl / well anti-mouse anti-mouse immunoglobulin labeled anti-horseradish peroxidase (Amersham) diluted 2000-fold with PBS- was added to each well. Tween, and the plate was incubated at 3 ° C for another hour, after which each well was again washed 3 times with 100 μl PBS-Tween. Then added horseradish peroxidase substrate (BioRad) in an amount of 100 μl / well was left for 5 minutes. After this time, the absorption at 415 nm was measured with a microplate reader (model 450, BioRad). Fractions 19 to 23, inclusive, which had high wavelength rating at high wavelength, were collected to prepare the raw fusion sample of human Fas.

REFERENCE EXAMPLE 2 Immunization of mice and hybridoma preparation (2-1) Immunization A 1 ml sample of the crude human fusion fusion protein solution obtained in Reference Example 1 above (total protein: 100 μg) was taken, and 25 μl of 2N HCl was added, 250 μl of potassium alum 9% w / v (final concentration: 1.1% w / v) and 25 μl of 2N NaOH. The resulting mixture was adjusted to a pH between about 6.5 and 7.0 by the addition of about 120 μl of a 10% (w / v) aqueous sodium caFonate solution and allowed to stand at room temperature for about 30 minutes. After this time, 200 μl of Bordetella pertuseie muertae (Wako Pure Chemical Induetriee, Ltd, 1.2 x 1011 cells / ml) was added to the mixture to activate the T cells, and the mixture was int ritely administered to a mouse Fas -destroyed. The mouse used was prepared according to the method described by Senju et al [see Senju, s. and others, (1996), International Immunology, S. 423]. The mouse was given an intrap ritoneal booster injection after only two weeks of crude fusion protein from human Fas (20 μg protein / mouse). (2-2) Fusion of Cells On the third day after the booster injection, the spleen was removed from the mouse and placed in 10 ml of serum-free RPMI 1640 medium (10.4 g / l RPMI1640"Nussiu" 1; Nieeui Pharmaceutical Co., Ltd.) containing 20 mM pH buffer HEPES (pH 7.3), 350 mg / ml of sodium acid caFonate, 0.05 mM of β-mercaptoethanol, 50 units / ml of penicillin, 50 μg / ml of streptomycin and 300 μg / ml of L-glutamic acid, and was broken by passing the organ on a mesh (Cell Strainer; Falcon) using a eepule. The re-emerging cellular euepeneion was centrifuged to form pellets of the spleen cells which were subsequently washed with RPMI medium free of serum. The washed cells were deferred in RPMI medium free of serum and counted. Meanwhile, NS1 myeloma cells (American Type Culture Collection TIB-18) were developed to a cell density no greater than 1 x 108 cells / ml in ASF104 medium (Ajinomoto, KK) containing FCS at 10% v / v (Gibco BRL) ("ASF medium with serum") at 37 ° C under 5% v / v CO2, and these were also shredded, washed, suspended and they told An amount of the NS1 cell suspension that was calculated to contain 3 x 10 7 cells, was mixed with an amount of the spleen cell suspension that was calculated to contain 3 x 10 8 cells. The resulting mixture was centrifuged and the supernatant was discarded. In the meantime, the following cellular heat was carried out, maintaining the plastic tube containing the pellet in a flask of warm water at 37 ° C all the time. Deepuée slowly added one ml of 50% (w / v) polyethylene glycol 1500 (Boehringer Manheim) to the tube while stirring the pellet with the tip of a pipette. Subsequently, one ml of serum-free RPMI medium, preheated to 37 ° C, was added slowly in two portions, followed by the addition of 7 ml of serum-free RPMI medium. The resulting mixture was then centrifuged, the supernatant was discarded and 10 ml of hypoxanthine-aminopterin-thymidine medium ("HAT medium") was added.; Boehringer Manheim) containing FCS at 10% v / v while gently shaking with a pipette tip. An additional 20 ml of HAT medium containing 10% v / v FCS was added, and the suspension was dispensed in 96-well cell culture microplates at 100 μl / well and incubated at 37 ° C under 5% CO2 v / v . After 7 or 8 days, 100 μl / well of fresh HAT medium was used to replace the medium if any of the wells exhibited a yellowish tint. The fusion cells of these wells were screened by limiting the dilution as described below. (2-3) Dilution of limitation Thymuses were removed from female BALB / c mice from 4 to 10 weeks of age (from Japan SLC, Inc.) they were shredded on a mesh (Cell Strainer; Falcon) as described above, and Fragmented cells were washed twice with hypoxanthine-ti-idine medium ("HT medium", Boehringer Manheim) containing 10% FCS v / v. An amount of thymus cells corresponding to those of a mouse was suspended in 30 ml of HT medium containing 10% v / v FCS to produce a supply cell suspension. The fusion cell preparation obtained before (2-2) was diluted with this abatement cell suspension from 10 to 100 vecee, and then serially diluted with supplying cell suspension to make the suspensions having melting cell densities. of 5, 1 and 0.5 cells / ml. The samples thus prepared were dispensed in wells of 95-well cell culture microplates at 100 μl / well and incubated for 5 days at 37 ° C under 5% v / v CO2. (2-4) Selection WR19L12a cells were propagated [see Itoh, N. and others, (1991), Cell. 66, 233-243] by incubation in RPMI1640 medium containing 10% v / v FSC at 37 ° C under 5% v / v CO2. WR19L12a cells are derived from WR19L cells of mouse T lymphoma (American Type Culture Collections TIB-52) and have been modified to express a gene encoding human Fas. The suspension of WR19L12a propagated cells was adjusted to a cellular deficiency of 1 x 107 cells / ml and dieed aliquots of 50 μl / well in the well of a 96-well microplate, the wells having U-shaped bottoms (Nunc) and the plate was centrifuged (90 xg, 4 ° C, 10 minutes). The supernatant was discarded, and 50 μl / well of culture supernatant obtained from the fusion cells cultured in 2-3 above were added to the wells with shaking. The resulting mixtures were allowed to stand on ice for 1 hour and after centrifugation (90 x g, 4 ° C, 10 minutes), and the supernatant was removed. Each of the pellets was washed twice with 100 μl / well flow cytometry regulator [PBS containing 5% v / v FCS and 0.4% (w / v) sodium azide]. A secondary antibody [50 μl of a goat anti-cough IgG fraction against mouse IgG, labeled with fluoroscein-5-isothiocyanate (FITC) ((Organon Technika) diluted 500-fold] was added to the washed cells, and the mixture it was left to stand on ice for 1 hour.After further centrifugation (90 xg, 4 ° C, 10 minutes), and removal of the supernatant, the pellet was washed 2 times with 100 μl / well of flow cytometry pH regulator, and the cells were fixed by adding 50 μl of formaldehyde solution at 3.7% v / v and repoeating on ice for 10 minutes.After centrifugation (90 xg, 4 ° C, 10 minutes) and removal of the copolymer, the pellets were again washed with 100 μl / well of flow cytometry pH regulator, and were suspended in 100 μl / well of flow cytometry pH regulator to produce the flow cytometry sample.The FITC fluorescence intensity of the cells was measured in each shows with a citometer or flow (Epics Elite; Coulter; Exitation wavelength: 488 mm; detection wavelength: 530 nm) and sample fusion cells were selected that had clearly higher FITC fluorescence intensities (FITC fluorescence intensities of approximately 100 to 1000) than those of the control WR19L12a cells to which they did not supernatant of fusion cells had been added (FITC fluorescence intensity of approximately 0.3). (2-5) Cloning The steps described in (2-3) and (2-4) above were repeated 5 times for the cells selected in (2-4), thus allowing the selection of several hybridization clones that produced, each, a single antiquake that joins WR19L12a but not WR19L. The binding of these anticuefos with mouse Fas was examined using a test similar to that described in (2-4), but using L5178YA1 cells. The cell line L5178YA1 expresses murine Fas. L5178YA1 is a cell line produced by transfection of L5178Y cells with a mouse Fas expression vector. L5178Y cells (American Type Culture Collection No. CRL-1722) almost do not express Fas. As a result of this selection procedure, a mouse-mouse hybridoma, designated HFE7A, was obtained and produces an antiquase that binds to L5178YA1 cells, but not to L5178Y cells. This hybridoma, HFE7A, was deposited with Kogyo Gijutsuin Seimei Kogaku Kogyo Gijuteu Kenkyujo on February 20, 1997, in accordance with the treaty of Budapeet for the deposit of microorganism, and has an assigned access No. FERM BP-5828. The anti-cough subclass produced by the mouse-mouse hybridoma HFE7A (hereinafter referred to simply as "HFE7A") was shown to be IgGl, K, after testing with a monoclonal anti-cell styling kit (Pierce).

EXAMPLE PE REFERENCE 3 Purification of Monoclonal Antifluid HFE7A The mouse-mouse hybridoma HFE7A obtained in Reference Example 2 (FERM BP-5828), a cell deficiency of 1 x 106 cells / ml was developed haeta by incubation in 1 1 of ASF medium, containing 10% FCS v / v , at 37 ° C under CO2 at 5% v / v. Later, the culture was centrifuged (1,000 rpm, 2 minutes) and the erobanadane reagent. The cell pellet was washed once with serum free ASF medium, was swapped in 1 liter of serum free ASF medium and incubated for 48 hours at 37 ° C under 5% v / v CO2. After this time, the culture was centrifuged (1,000 r.p.m. for 2 minutes) to recover the supernatant. This supernatant was then placed in a dialysis tube (exclusion v: 12,000-14,000, Gibco BRL), and dialyzed against 10 volumes of 10 M sodium phosphate pH buffer (pH 8.0). Partial purification of IgG was achieved from the internal solution using a high resolution liquid chromatography apparatus (FPMA FPMA, Pharmacia) under the following conditions: column: DEAE-Sepharose CL-6B (column size 10 ml, Pharmacia) . eluent: regulator (pH 8.0) of 10 mM epoxide solution; flow rate: 1 ml / min; elution: linear gradient of 1 M NaCl (0 to 50%, 180 min). The eluted product was collected in 5 ml fractions and each fraction was analyzed for anti-Fas anti-cough titre by ELISA using the human Fas fusion protein prepared above. First, 100 μl / well of the crude fusion protein solution of human Fas prepared in the Reference Example 1, in the wells of a 96-well ELISA microplate. After incubation at 37 ° C for 1 hour, the solution was discarded and the wells were each washed 3 times with 100 μl / well of PBS-Tween. Afterwards, 100 μl / well of PBS containing 2% BSA was added and incubated at 37 ° C for 1 hour. After this time, the cells were washed 3 times with 100 μl / well of PBS-Tween, and then samples of 100 μl of the fractions to be analyzed were added to the wells, and the plate was incubated at 37 ° C for 1 hour. . Then, after washing each of the wells 3 times with 100 μl / well of PBS-Tween, we added 100 μl / well anti-mouse anti-mouse immunoglobulin labeled with horseradish peroxidase (Amersham), diluted 2,000 times with PBS -Tween, and let it react at 37 ° C for 1 hour. After this time, each well was washed 3 times with 100 μl / well of PBS-Tween. Radish peroxidase substrate (BioRad) was added in an amount of 100 μl / well and left for 5 minutes before reading the absorption of each well at 415 nm with a microplate reader. Fractions 21 to 30, inclusive, which had high absorption values, were concentrated and applied to 2 anti-affinity affinity purification columns (HighTrap Protein G Column, 5 ml column volume, Pharmacia). After washing the column with equilibrium pH regulator [20 mM sodium phosphate pH regulator (pH 7.0), 25 ml / column], the antiquake was eluted with 15 ml per elution pH regulator column [0.1 M glycine-HCl (pH 2.7)]. The eluted product was collected in tubes containing each 1-125 ml of 1 M Trie-HCl (pH 9.0) and centrifuged at 3,000 xg at 4 ° C for 2 hours at the top of a tube-type ultrafiltration dispoeitive. centrifuge (CentriPrep 10; Grace Japan, KK) immediately after finishing the elution. The filtrate recovered at the bottom of the device was discarded, and 15 ml of PBS was added to the top, and the preparation was once again centrifuged at 3,000 x g at 4 ° C for 2 hours. These same steps were repeated 5 times in everything. The fifth centrifugation was stopped when the volume of the solution remaining at the top reached 0.5 ml, and this was retained as the mueetra HFE7A.

REFERENCE EXAMPLE 4 Cloning of cDNA (4-1) Preparation of poly (A) - RNA Mouse-mouse hybridoma cells were developed HFE7A (FERM BP-5828), obtained in Reference Example 2, to a cell density of 1 x 106 cells / ml in 1 liter of ASF medium supplemented with 10% FCS v / v at 37 ° C under 5% v / v CO2. Eetae cells were cultured by centrifugation and they were in the presence of the guanidine thiocyanate solution [4 M guanidine thiocyanate, 1% v / v Sarcosil, 20 mM EDTA, 25 mM eodium citrate (pH 7.0), 2-mercaptoethanol 100 M, antifoaming A at 0.1% v / v] and the lysate recovered. Isolation of poly (A) + RNA was performed, essentially as described in "Molecular Cloning A Laboratory Manual" [see Maniati, T., et al., (1982), p. 196-198]. More specifically, the procedure was as follows: The recovered cell lysate was suctioned and discharged from a 10 ml syringe equipped with a 21-gauge needle several times. The cell lysate was applied in a layer on 3 ml of an aqueous solution of 5.7 M cesium chloride, 0.1 M EDTA solution (pH 7.5) in a polysail centrifuge tube for the hub of a RPS-40T rotor (Hitachi Seisakusyo , KK). The lysate was centrifuged after 30,000 r.p.m. at 20 ° C for 18 hours, and the resulting pellet was dissolved in 400 μl of distilled water and subjected to ethanol precipitation. The resulting precipitate was re-dissolved in 400 μl of distilled water, mixed with an equal volume of a mixture of chloroform and 1-butanol (4: 1, v / v), the aqueous layer was recovered after centrifuging to 5,000 rpm for 10 minutes. This aqueous layer was again precipitated with ethanol and the precipitate was dissolved in 600 μl of distilled water. The resulting solution was retained as the total RNA sample. Poly (A) + RNA was purified from 600 μg (dry weight) of the total RNA sample obtained above, by oligo (dT) cellulose chromatography. More specifically, the total RNA was dissolved in 200 μl of adsorption pH buffer [0.5 M NaCl, 20 M Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% v / v sodium dodecyl sulfate (SDS)], Then it was heated to 65 ° C for 5 minutes, and then applied to an oligo (dT) cellulose column (type 7).; Pharmacia) that had been loaded with an adsorption pH regulator. Poly (A) + RNA was eluted and recovered from the column using elution pH regulator [10 M Tris-HCl (pH 7.5), EDTA ImM, 0.05% SDS v / v]. A total of 100 μg of poly (A) + RNA fraction was obtained by this procedure. (4-2) Determination of the amino acid sequences of the N- terminus of heavy and light chains of HFE7A. 8 microliters of the solution containing the anti-human anti-Fas anti-Fas HFE7A, obtained in reference example 3, using a 12% gel concentration, was subjected to elect roforesie on SDS polyacrylamide gel ("SDS-PAGE"). p / v, constant voltage of 100 v, for 120 minutes. After the electrophorea, the gel was immersed in a transfer pH regulator [25 mM Tris-HCl (pH 9.5), 20% methanol, SDS 0.02% v / v] for 5 minutes. After this time, the protein content of the gel was transferred to a polyvinylidene difluoride membrane ("PVDF membrane", pore size 0.45 μm, Millipore, Japan), previously rinsed in a pH transfer buffer, using a blotting (KS-8451; Marysol) under conditions of 10 v constant voltage, 4 ° C, for 14 hours. After this time, the PVDF membrane was washed with washing pH regulator [25 mM NaCl, pH buffer of 10 mM sodium borate (pH 8.0)], then stained in a dyeing solution (methanol 50 % v / v, acetic acid 20% v / v and Coomassie brilliant blue 0.05% w / v) for 5 minutes to locate the protein band. The PVDF membrane was then decolorized with 90% v / v aqueous methanol, and the bands corresponding to the heavy chain (the band with the least mobility) and the light chain (the band with the greatest mobility) previously located on the PVDF membrane, and washed with de-ionized water. Then the sequences of the N-terminus of the heavy and light chains could be determined, by means of the automatic method of Edman [see Edman, P. et al. (1967), Eur. J. Biochem., 1, 80] using a sequencer of Gaseoea phase proteins (PPSQ-10; Shimadzu Seieakueyo, KK). It was determined that the amino acid sequence of the N-terminus of the band corresponding to the heavy chain is: Gln-Xaa-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu (SEQ ID No. 16 of the listing of sequences); and it was determined that the amino acid sequence of the N-terminus of the band corresponding to the light chain is: Asp-Ile-Val-Leu-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ala-Val-Ser-Leu -Gly-Gln-Arg-Ala-Thr-Ile-Ser (SEQ ID No.17 of the sequence listing). Comparison of these amino acid sequences with the database of amino acid sequences of the anti-convolutions produced by Kabat et al. [See Kabat E.A., et al., (1991), in "Sequences of protein of immunological interest Vol. II" U.S. Department of Health and Human Services - Department of Health and Human Services of the U.S., revealed that the heavy chain (tl chain) and the light chain (chain k) of HFE7A belong to subtypes 2b and 3, respectively. Based on these findings, oligonucleotide primers were synthesized, which could be expected to be hybridized with portions of the 5 'untranslated regions and the true ends of the 3'-traducidae regions of the genes belonging to these subtypes. of mouse [see Kabat et al., ibid; Matti Kartinen and other (1988), 25, 859-865; and Heinrich, G. and others (1984), J. Exp.

Med. 159, 417-435]: 5'-GACCTCACCA TGGGATGGA-3 '(Hl: SEQ ID No. 18 of the sequence listing); 5'-TTTACCAGGA GAGTGGGAGA-3 '(H2: SEQ ID No. 19 of the sequence listing); 5'-AAGAAGCATC CTCTCATCTA-3 '(Ll: SEQ ID No. 20 of the sequence listing); and 5'-ACACTCATTC CTGTTGAAGC-3 '(L2: SEQ ID No. 21 of the sequence listing). (4-3) Cloning of cDNA cDNA encoding the heavy and light chains of the anti-human anti-Fas anti-Fas antibody HFE7A was cloned by means of a combination of reverse transcription and PCR ("TI-RCP"). Amplification was performed on the poly (A) + RNA fraction obtained from hybridoma cells producing HFE7A as described above in (4-1). The TI / RCP reaction was performed using an RNA-PCR (AMV) version 2 kit (Takara Shuzo Co., Ltd). a) The reverse riptaea transection reaction. Lae eeriee of oligonucleotide initiator 5 (5 'end and 3' end primers), synthesized before in (4-2), were used as initiator pairs for the TI / RCP reaction for the heavy and light chains. _. Composition of the reaction solution: poly (A) + RNA (heavy or light chain, as required), lμg; initiator 3 '(H2 or L2), 0.3 μg; Tris-HCl (pH 8.3), 10 mM; Potassium chloride, 50 M; dNTP's, 1 mM; 5 Magnesium chloride, 5 mM; RNase inhibitor (provided with the equipment), 0.5 units; Reverse transcriptase (provided with the equipment), 0.25 units; and 0 Re-distilled water to a total volume of 20 μl. The reaction solution was incubated at 55 ° C for 30 minutes, at 99 ° C for 5 minutes, and then at 5 ° C for 5 minutes. The IT solution treated was developed in the next CPR stage. 5 b) RCP. Composition of the reaction solution: Reaction solution of transeriptaea inverea, 20 μl; PH regulator for 10-fold concentrated RNA RCP (provieto con el equipo), 10 μl; Magneeium chloride solution (provieta con el equipo), 10 μl; Polimeraea Taq (provieta con el equipo), 2.5 unid; Primer 5 '(Hl or Ll), final concentration 0-2 mM; Y Water deionized eetéril haeta a total volume of 100 μl. The solution of the PCR reaction was heated at 94 ° C for 2 minutes, followed after a cycle of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 1-5 minutes, repeated 28 times. After the PCR reaction, aliquots of the reaction solutions were electrophoresed on 1.5% w / v agarose gels. It was found that bands of approximately 1.4 kbp and approximately 0.7 kbp were amplified in the reaction solutions, using the primers for the heavy chain and light chain initiators, respectively. This confirmed that the cDNAs encoding the heavy and light chains were amplified, as intended. Therefore, the amplified PCR reaction solutions can be used in the next cloning step of the amplified cDNAs using the TA Cloning kit (Invitrogen). This was done as follows.

The relevant CPR reaction solution, together with 50 ng of the pCRII vector (provid with the TA Cloning kit), were mixed in 1 μl of the lOx ligand reaction regulator [6 mM Tris-HCl, (pH 7.5) , 6 mM magnesium chloride, 5 mM eodium chloride, 7 mM β-mercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 mM spermidine, and 0.1 mg / ml bovine serum albumin] to which 4 units of DNA ligase had been added T4 (1 μl). The total volume of the mixture was adjusted to 10 μl with sterile deionized water, and the resulting ligase solution was incubated at 14 ° C for 15 hours. After this time, 2 μl of the ligase reaction solution was added to 50 μl of the competent E. coli strain T0P10F '(provided with the TA Cloning kit and brought to competition according to the instruction manual of the team ) to which 2 μl of 0.5 M β-mercaptoethanol had been added, and the resulting mixture was kept on ice for 30 minutes, then at 42 ° C for 30 seconds, and again on ice for 5 minutes. they added to the culture 500 μl of SOC medium (tryptone 2% v / v, yeast extract 0.5% w / v, sodium chloride 0.05% w / v, potassium chloride 2.5 mM, magnesium chloride 1 mM and glucose, 20 mM), and the mixture was incubated for 1 hour at 37 [deg.] C. with shaking.After this time, the culture was spread on a L broth agar plate [tryptone 1% v / v, yeast extract 0.5% w / v , 0.5% w / v eodium chloride, 0.1% w / v glucoea and 0.6% w / v bacto-agar (Difco)], which contained 100 μg / ml ampicillin, and incubated at 37 ° C, overnight. The individual ampicillin-resistant colonies that appeared on the plate were selected, scraped off with a platinum transfer loop and cultured in L-broth medium containing 100 μg / ml ampicillin at 37 ° C overnight. with stirring at 200 rpm After incubation, the cells were harvested by centrifugation, of which plasmid DNA was prepared by the alkali method. The plamidoids thus obtained were assigned as plamide RCP-H (the plamidid carries cDNA encoding the peeled chain of HFE7A) or RCP-L (the plasmid carrying the cDNA encoding the light chain of HFE7A). (4-4) Analysis of nucleotide sequences The nucleotide sequences of the cDNAs encoding both the heavy chain of HFE7A (1.4 kbp) and the light chain of HFE7A (0.7 kbp) carried by the plasmids RCP-H and RCP-L , obtained earlier in (4-3), were determined by the dideoxy method [see Sanger, FS and others (1977), Proc. Nati Acad. Sci. USA 74: 5463-5467] using a gene sequence analyzer (Model 310 Genetic Analyzer, Perkin Elmer, Japan). The nucleotide sequences of the cDNA of the peeled and light chains of HFE7A, determined in this way, are given as SEQ ID Noe. 8 and 10, respectively, in the statement of sequence. The complete amino acid sequence sequences of the heavy and light chains of HFE7A, encoded by the cDNAs, are given as SEQ ID Nos. 9 and 11, respectively, of the sequence listing. The amino acid sequence of the N-terminus of the HFE7A heavy chain set forth above in (4-1) (SEQ ID No. 16 of the sequence listing) is perfectly matched to amino acid sequence Nos. 1 to 11 of SEQ ID No .9, except for the only uncertain residue. The amino acid sequence of the N-terminus of the light chain of HFE7A (SEQ ID No. 17 of the sequence listing) exactly matches the amino acid sequences Nos. 1 to 22 of SEQ ID No. 11. Thus, it was shown that the N-terminus of the heavy and light mature proteins of HFE7A are amino acids Nos. 1 to 11 and Nos. 1 to 22 in SEQ ID Nos. 9 and 11, respectively. In addition, when the amino acid sequences of the heavy and light chains were compared with the database of anticuefoe amino acid sequences [Kabat E.A. and others (1991), in "Sequences of Proteins of immunological interest Vol. II" U.S. Department of Health and Human Services (US Department of Health and Human Services), it was established that, for the heavy chain, amino acids Nos. 1 to 121 of SEQ ID No. 9 constituted the variable region, while amino acids Nos. 122 to 445 constituted the constant region. For the light chain, amino acids Nos. 1 to 111 of SEQ ID No. 11 constituted the variable region, while amino acids Nos. 112 to 218 constituted the receptor region. The localization and sequence of the RDC in the amino acid sequences of the variable regione of the heavy and light chain of HFE7A, as determined above, were also elucidated by comparing the homologies with the same database of amino acid sequences of anticuefos [see Kabat EA, et al. (1991), ibid.]. From this publication, you can establish that the lengths of the structure regions in the variable regions are subetantially lae miemae, and that the amino acid sequences share common characteristics, between different anti-buds of the subtype. The RDCs are unique sequences located between the regione of the structure. Therefore, by comparing the amino acid sequence of the heavy and light chains of HFE7A with those of the same subtypes in the Kabat work, it was possible to identify the RDCs of HFE7A. Therefore, it was established that, in the heavy chain of HFE7A (SEQ ID No. 9 in the sequence listing), amino acids Nos. 31 to 35 form CDRHi, amino acids Nos. 50 to 66 form CDRH2 and amino acids Nos. 99 to 110 form CDRH3. The RDCs in the light chain of HFE7A (SEQ ID No. 11 in the sequence listing) were identified as amino acids Nos. 24 to 38 (CDRLi), amino acids Nos. 54 to 60 (CDRL2) and amino acids Nos. 93 to 101 (CDRL3).

REFERENCE EXAMPLE 5 Preparation of Recombinant Antibody (5-1) Construction of expression plasmid Recombinant expression vectors were constructed for animal cells by inserting the cDNAs encoding the heavy and light chains of HFE7A (cloned in reference example 4) into the expression vector pMS18S [Hara, T ., and others, (1992), EMBO J., 11, 1875]. This was done as follows. First, oligonucleotide primers were synthesized: 5'-GGGGAATTCG ACCTCACCAT GGGATGGA-3 '(H3: SEQ ID No. 22 of the sequence listing) and 5'GGGTCTAGAC TATTTACCAG GAGAGTGGGA GA-3' (H4: SEQ ID No-23 of the listing of sequences) These primers serve for the introduction of a recognition site for the restriction enzyme EcoRI, for a recognition site for the restriction enzyme Xbal, as well as a stop codon, at the 5 'end and the 3' end , respectively, of the last strand cDNA carried by the plasmid RCP-H. Oligonucleotide primers were also synthesized: 5'-GGGGAATTCA AGAAGCATCC TCTCATCTA-3 '(L3: SEQ ID No. 24 of sequence listing) and 5'-GGGGCGGCCG CTTACTAACA CTCATTCCTG TTGAAGC-3' (L4: SEQ ID No.25 of the listing These sequences are initiated for the introduction of a recognition site for the EcoRI reentrant enzyme, for a recognition site for the restriction enzyme NotI, as well as for a stop codon, at the 5 'end and at the end 3 ', respectively, of the light chain cDNA carried by the plasmid RCP-L. Using respective primers for the heavy and light chains, PCR was performed as follows. Composition of the reaction solution: Mold (RCP-H or RCP-L), 1 μg; Initiated r 5 '(H3 or L3), 40 pmol; Initiated r 3 '(H4 or L4), 40 pmol; T ris-HCl (pH 8.0), 20 M; Potassium chloride, 10 mM; Ammonium sulfate, 6 mM; Magnesium chloride, 2 mM; Triton X-100, 0.1%; Nuclease-free bovine serum albumin, 10 μg / ml; dNTP's 0.25 mM; Native Pfu DNA polymerase (Stratagene), 5 units; Y Sterile distilled water up to a total volume of 100 μl. Thermal conditions of PCR: The initial heating of the reaction solution was at 94 ° C for 2 minutes, after which a thermal cycle of 94 ° C was repeated 28 times for 30 seconds, 60 ° C for 30 seconds and 75 ° C for 1.5 minutes. The resulting amplified DNA was digested with the restriction enzymes EcoRI and Xbal (for the heavy chain) or EcoRI and NotI (for the light chain) and then mixed with the animal cell expression plasmid pME18S [see, Hara. T., et al., (1995), EMBo J., 11, 1875] which had been digested with the restriction enzymes EcoRI and Xbal (for the heavy chain) or EcoRI and NotI (for the light chain) and dephosphorylated using CIP [as described in example 2 (2) 3) c) below]. One microliter of 4 units of T4 DNA ligase was added to 8 microliters of the resulting mixture, and then one microliter of lOx ligase reaction pH regulator [6 mM Tris-HCl (pH 7.5), 6 mM magnesium chloride was added. , 5 mM eodium chloride, 7 M β-mercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 mM spermidine, and 0.1 mg / ml bovine serum albumin] were added to the mixture, which was then incubated at 14 ° C for 15 hours. After this time, 2 μl of the incubated ligand reaction solution was mixed with 50 μl of competent E. coli strain JM109 at a cell density of 1-2 x 109 cells / ml (Takara Shuzo Co., Ltd), and the mixture was kept on ice for 30 minutes, then at 42 ° C for 30 seconds, and again on ice for 5 minutes. Then 500 μl of SOC medium (tryptone 2% v / v, yeast extract 0.5% w / v, sodium chloride 0.05% w / v, potassium chloride 2.5 mM w / v, magnesium chloride 1 was added to the mixture. mM, and 20 mM glucoea), which was incubated for a further hour with shaking. Transformant strains were then isolated, and plasmid DNA was prepared from the strains, following the methods described in reference example 4 (4-3). The resulting plasmids were designated pME-H (the expression plasmid vector bearing cDNA encoding the heavy chain HFE7A) and pME-L (the expression plasmid vector bearing cDNA encoding the light chain of HFE7A). The transformant E. coli strains that host these plasmids, designated as E. coli pME-H and E, coli pME-L, were deposited with Kogyo Gijuteuin Seimei-kogaku Gijutdu Kenkyujo, on March 12, 1997, in accordance with the Budapest Treaty for the deposit of microorganisms, and ee lee granted the access numbers FERM BP-5868 and FERM BP-5867, respectively. (5-2) Expression in COS-7 cells Transfection of C0S-7 cells was performed with the expresion plasmids pME-H and pME-L obtained in (5-1) above by means of electroporation using a gene tranefection apparatus (ECM600; BTX), C0S-7 cells were developed (Ametican Type Culture Collection No, CRL-1651) up to eemiconfluence in a culture flask (culture area: 225 cm2, Sumitomo Bakelite, K.K.) containing DMEM supplemented with 10% FCS v / v.

Subsequently, the medium was added and 3 ml trypsin solution EDTA (Sigma Chemicals Co.) was added to the cells, followed by incubation at 37 ° C for 3 minutes. The cells separated by this procedure were harvested, washed with doe vecee with PBS and after adjusted to a cell density of 5 x 106 cells / ml with PBS. In the meantime, 20 μg of each of the plasmids pME-H and pME-L, prepared using a large-scale plasmid preparation kit (MaxiPrep DNA purification system, Promega) with ethanol, were separately precipitated and dissolved in 20 μg of each of the plasmids pME-H and pME-L. μl each of sterile PBS. Where C0S-1 cells were cotransfected with both plasmids; 20 μg of each of the plasmids were used and dissolved together in 20 μl of sterile PBS. Twenty μl of the prepared cell suspension (1.2 x 10 6 cells) and 20 μl of the relevant plasmid solution were mixed and transferred to a chamber with electrodes fixed at a separation distance of 2 mM (BTX). The chamber was then loaded into the gene transfection apparatus and read to give a 10 meec puleation at 150 V, to provide a total charge of 900 μF. The cell-DNA mixture in the chamber was added to 40 ml of DMEM supplemented with 10% v / v FCS and incubated in plastic cell culture discs under 5% v / v CO2 at 37 ° C for 24 hours. After this time, the culture extract was deepened and the cells were washed with serum free DMEM medium. After that, 40 ml of serum-free DMEM was added to each of the plastic discs and the supernatant was recovered after culturing the cells under 5% v / v CO2 at 37 ° C for a further 72 hours. Using the above method, COS-7 cells were obtained that were transfected with some or both plasmids (as shown below) and recovered the supernatant of each of the transformants: (A): only pME-H; (B): only pME-L; and (C): cotnefection of pME-H and pME-L. (5-3) Detection of anti-Fae antibody in eobrenatant of transforming culture. The expression of anti-Fas antibody in the culture supernatants obtained earlier in (5-2) was determined by ELISA, in a manner similar to that described in reference example 3, and by flushing the human Fae protein as the antigen . It was established that the production of an antibody that reacts with the human Fae antigen fusion protein in the culture supernatant only occurred when both pME-H and pME-L were used to cotransfect COS-I cells [5-2].

(C)].

REFERENCE EXAMPLE 6 Determination of epitope (6-1) ELISA The following peptides were synthesized by Fmoc solid phase synthesis (see Carpino, LA and Han, GY, (1970), J. Am. Chem. Soc., 92, 5748-5749), using a synthesizer. of automatic peptides (model 430A; Perkin Elmer, Japan, Applied Biosystems Division). Arg-Leu-Ser-Ser-Lys-Ser-Val-Aen-Ala-Gln-Val-Thr-Aep-Ile-Asn-Ser-Lys-Gly-Leu (Pl: SEQ ID No. 26 of the list of secuncias); Val-Thr-Asp-Ile-Asn-Ser-Lys-Gly-Leu-Glu-Leu-Arg-Lys-Thr-Val-Thr-Thr-Val-Glu (P2: SEQ ID No. 27 of the sequence listing); Glu-Leu-Arg-Lys-Thr-Val-Thr-Thr-Val-Glu-Thr-Gln-Asn-Leu-Glu-Gly-Leu-His-Hie-Aep (P3: SEQ ID No. 28 of the eecuenciae); Thr-Gln-Asn-Leu-Glu-Gly-Leu-His-His-His-Asp-Gly-Gln-Phe-Cys-His-Lys-Pro-Cys-Pro-Pro (P4: SEQ ID No. 29 of the listing of sequence); Gly-Gln-Phe-Cye-His-Lys-Pro-Cys-Pro-Pro-Gly-Glu-Arg-Lys-Ala-Arg-Asp-Cys-Thr-Val (P5: SEQ ID No. 30 of the sequences); Gly-Glu-Arg-Lys-Ala-Arg-Asp-Cys-Thr-Val-Asn-Gly-Asp-Glu-Pro-Aep-Cye-Val-Pro-Cye-Gln (P6: SEQ ID No. 31 of lietado de eecuenciae); Aen-Gly-Aep-Glu-Pro-Aep-Cye-Val-Pro-Cye-Gln-Glu-Gly-Lys-Glu-Tyr-Thr-Asp-Lys-Ala (P7: SEQ ID NO. eecuenciae); Glu-Gly-Lys-Glu-Tyr-Thr-Asp-Lye-Ala-Hie-Phe-Ser-Ser-Lye-Cye-Arg-Rg-Cye-Arg (P8: SEQ ID No. 33 of the sequence of sequences); His-Phe-Ser-Ser-Lye-Cye-Arg-Arg-Cys-Arg-Leu-Cys-Asp-Glu-Gly-Hie-Gly-Leu-Glu-Val (P9: SEQ ID No. 34 of the eecuenciae); Leu-Cye-Asp-Glu-Gly-His-Gly-Leu-Glu-Val-Glu-Ile-Asn-Cys-Thr-Arg-Thr-Gln-Aen-Thr (PlO: SEQ ID No. 35 of the sequences); Glu-Ile-Asn-Cys-Thr-Arg-Thr-Gln-Asn-Thr-Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys (Pll: SEQ ID No. 36 of the eecuenciae); Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys-Asn-Ser-Thr-Val-Cys-Glu-His-Cys-Asp-Pro (P12: SEQ ID No. 37 of the listing of sequence); Aen-Ser-Thr-Val-Cye-Glu-Hie-Cye-Asp-Pro-Cys-Thr-Lys-Cys-Glu-Hie-Gly-Ile-Ile-Lye (P13: SEQ ID No. 38 of the eecuenciae); Cys-Thr-Lys-Cys-Glu-His-Gly-Ile-Ile-Lys-Glu-Cys-Thr-Leu-Thr-Ser-Asn-Thr-Lys-Cys (P14: SEQ ID No. 39 of the listing of sequences); Glu-Cys-Thr-Leu-Thr-Ser-Asn-Thr-Lys-Cys-Lys-Glu-Glu-Gly-Ser-Arg-Ser-Asn (P15: SEQ ID No. 40 of the sequence listing); and Ser-Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Asn-Ile-T r-Thr-Lys-Lys-Glu-Ala-Phe-Asn-Val-Glu (P16: SEQ ID No. 41 of sequence listing).

Pl to P15 are partial sequences of the amino acid sequence of numbers 1 to 157 of the human Fas extracellular domain, translapping between 9 and 11 amino acid residues. P16 is a negative control that has no homology with human Fas. Pl to P16 were respectively completely dissolved in 48 μl of dimethyl sulfoxide (DMSO) and each was adjusted after a final volume of 0.8 ml by the addition of 752 μl of PBS containing 1 mM li-mercaptoethanol. The above peptides each correspond to a portion of the extracellular domain of the human Fas molecule, but with a C -oxoyl group added to the C-terminus. Each peptide was diluted to 50 μg / ml with 0.05 M carbonate-bicarbonate pH buffer (pH 9.6 ), containing 10 mM 2-mercaptoethanol and 50 μl of each were introduced into a well of a 96-well ELISA microplate (Nunc). The plate was left at 4 ° C overnight to allow adsorption of the peptide to the well surface. After this time, the solution in the wells was discarded and each well was washed 4 times with PBS-Tween. Then, 100 μl of PBS containing 1% (w / v) bovine eeroalbumin (A3803, Sigma Chemicale Co.) was added to each well and the plate was incubated at 37 ° C for 1 hour. Then, the wells were washed 4 more times with PBS-Tween, and then 50 μl of HFE7A or CH11 adjusted to 5 μg / ml in PBS was added to each well. The plate was then incubated at 37 ° C for 1 hour, and the wells were washed again 4 times with PBS-Tween. After washing, 50 μl per well of goat anti-cough was added to mouse immunoglobulin labeled with horseradish peroxidase (Amersham), diluted 1000 times with PBS, and plate ee incubated again at 37 ° C for 1 hour, after which which wells were washed 4 times with PBS-Tween. Added after horseradish peroxidase (BioRad) substrate in an amount of 100 μl / well and the plate was left at room temperature for 15 minutes before reading the absorption of each well at 415 nm using a microplate reader (Corona). As a positive control, the human Fas fusion protein prepared in Reference Example 1 was used in place of the synthetic peptides. Using the previous methodology, it was established that only wells with Pll adsoFido showed high aberation values, demonstrating that HFE7A binds specifically to an amino acid sequence contained in Pll (figure 3). (6-2) Identification of the epitope recognized by HFA7A in Pll by proficiency test The following peptides were synthesized: His-Gly-Leu-Glu-Val-Glu-Ile-Asn-Cys-Thr (P95: SEQ ID No. 42 from the list of sequences); Glu-Ile-Asn-Cye-Thr-Arg-Thr-Gln-Aen-Thr (P100: SEQ ID No. 43 of the sequence listing); Arg-Thr-Gln-Asn-Thr-Lys-Cys-Arg-Cys-Lys (P105: SEQ ID No. 1 of the sequence listing); Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys (PllO: SEQ ID No. 44 of the sequence listing); Pro-Asn-Phe-Phe-Cys-Asn-Ser-Thr-Val-Cys-Glu-His-Cye-Asp (P115: SEQ ID No. 45 of the sequence listing); and Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn-Asp-Aen (D355-364: SEQ ID No. 46 of the sequence listing.

P95, P100, P105 and PllO are each partial sequences of 10 residues of the flanking region (corresponding to amino acids 95 to 128 of the extracellular domain of human Fas) of the amino acid sequence corresponding to Pll in the extracellular domain of human Fas, each with 5 overlapping amino acid residues with the following. Desired peptide P115, Pro-Asn-Phe-Phe-Cye-Asn-Ser-Thr-Val-Cye (P115: amino acids Nos. 1 to 10 of SEQ ID No. 45 of the sequence listing) has an overlap of 5 reeiduoe with a peptide of 10 P10, but it was expected to have low solubility, so that 4 extra residues were added at the C-terminus of P115 to produce P115L. D355-364 was used as a negative control, this peptide had no homology with human Fas.

Each of the peptides, except P115L, was completely dissolved in 16 μl of DMSO, and then each was adjusted to a final volume of 0.8 ml by the addition of 784 μl of PBS containing 1 M 2-mercaptoethanol. P115L was completely dissolved in 48 μl of DMSO and then adjusted to a final volume of 0.8 ml by addition of 752 μl of PBS containing 1 mM 2-mercaptoethanol. Each of the above peptide solutions (corresponding to 200 μg of peptides) were mixed with 0.25 μg of HFE7A in a microtube and adjusted to a total volume of 100 μl with PBS containing 1 mM 2-mercaptoethanol. The mixture was incubated at 37 ° C for 2 h with shaking from 10 to 20 fm., followed by the addition of FCS to a final concentration of 5%, to thereby produce the peptide-antiquake mixture. WR19L12a cells were developed by a method similar to that described in reference example 2. Then, the cells were recovered by centrifugation and adjusted to a cell density of 1 x 10 7 cells / ml with serum-free RPMI medium. The cell suspension was dispensed into a 96-well plate, the wells having U-bottoms, at 100 μl / well and centrifuged at 4 ° C, 1,000 fm for 3 minutes using an oscillating rotor for the microplates, and the supernatant was discarded later. Then, 100 μl of the peptide-antibody mixture was added to each pellet and mixed by pipetting a few times, as described above. The plate was left after 4 ° C for 30 minutes, and then it was centrifuged and the supernatant discarded. The pellet was washed 3 times with flow cytometry pH regulator, and then 50 μl of goat anti-cough anti-mouse IgG labeled with FITC (Kappel), diluted 250 times with flow cytometry pH regulator, was added per well. followed by light pipetting to mix the contents of the wells. The plate was kept in the dark at 4 ° C for 30 minutes, after which it was centrifuged and the epenatant was deepened. The pellet was washed 3 times with flow cytometry pH regulator, which contained formaldehyde neutral solution regulated at 10% v / v pH (Wako Puré Chemical Industries, Ltd) for tissue fixation, this solution was diluted 10 times with PBS and 50 μl / well was added and mixed with light pipetting. Deepuée, the plate was kept in the dark at 4 ° C for at least 12 hours to fix the cells. After this time, the cells were collected in 100 μl / well of flow cytometry pH regulator and centrifuged to remove the supernatant. The pellet was washed 3 times with flow cytometry pH regulator and suspended in 500 μl / well of flow cytometry pH regulator, and the resulting suspension was analyzed with a flow cytometer (Cytoace-150; Nippon Bunko, K.K, -exitation wavelength: 488 nm; detection wavelength: 530 nm) to calculate average FITC fluorescence intensities per cell. The mean FITC fluorescence intensities for each sample were calculated by taking the value without peptide-anti-cough mixture as 0% and the value of the sample containing 355-364 as 100%. By the above procedure, it was established that P105 is capable of strongly inhibiting the binding between HFE7A and WR19L12a cells, and P100 and PllO, the amino acid sequence of each of which overlaps 50% with P105, each inhibiting the binding between HFE7A and WR19L12a cells in approximately 50% and 60%, respectively. No inhibition was observed with P95 or with P115L, which also did not have shared compartments with P105 (figure 4). From this, it was established that P105 represents an amino acid sequence capable of inhibiting the binding between human HFE7A and Fas and that, consequently, the epitope for HFE7A must be located within the amino acid sequence reproduced in P105. This epitope amino acid sequence is a region that is conserved between human FAS and mouse Fas.

REFERENCE EXAMPLE 7 Union of HFE7A to simian Fas The following test was carried out, to establish if HFE7A was able to bind to the Fas antigen of different primate species. First, samples of peripheral blood were taken from a chimpanzee (Sanwa Kagaku Kumamoto Primates Park, 40 ml), 20 ml of either a Japanese monkey (Macaca fuscata), or a crab monkey (Macaca irue), and 3 ml of a marmoset (of the genus Hapalida). The blood samples had 1 ml of heparin (Novoheparin; Novo) added, and the samples were slowly layered over an equal volume of Ficol Pague solution (specific gravity: 1.092 for monkey crab, Pharmacia) and centrifuged at 1700 rpm. for 30 minutes to obtain a fraction of peripheral blood mononuclear cells. This fraction of mononuclear cells was washed twice with Hanke's balanced salt solution and then suspended in RPMI 1640 medium with 10% FCS v / v to a cell density of 1 x 106 cells / ml. Phytohemagglutinin-P ( Sigma Chemicals, Co.) to the resulting suspension to a concentration of 5 μg / ml and the sample was incubated at 37 ° C under 5% v / v CO2 for 24 hours. After this time, the cells were recovered by centrifugation, washed and resuspended in RPMI 1640 medium with 10% v / v FCS. Then, to activate the recovered cells, interleukin-2 (Amersham) was added to the suspension to a final concentration of 10 units / ml, and this was incubated at 37 ° C under 5% v / v CO2 for 72 hours. An amount of the activated preparation that was calculated contained 1 x 10 6 activated lymphocyte cells, was placed in a test tube and suspended in 50 μl of 20 μg / ml HFE7A in PBS, or in 50 μl of PBS alone. The resulting melting was left on ice for 1 hour, after which the cells were washed 3 times with aliquot of 500 μl of PBS and then swapped in 50 μl of anti-mouse IgG antibody labeled with FITC, 20 μg / ml. ml (Bioresource) in PBS. This suepeneion was then placed on ice for 30 minutes, and washed 3 times with aliquots of 500 μl of PBS. Using the cells suspended in 500 μl of PBS as control, the fluorescence intensities were measured using a flow cytometer (Cytoace, Nippo Bunko, K.K.). Distributions of the number of cells were obtained by the fluorescence intensity and the proportions of the number of cells stained to the total cell number were calculated. As a result, in the samples without HFE7A, the stained cells constituted less than 3% of all the species. However, in the samples treated with HFE7A, at least 17% of the cells were stained, the maximum being 82%. Therefore, HFE7A is able to bind to a broad range of primate Fas, including humans, against which HFE7A was originally prepared.

REFERENCE EXAMPLE 8 Apoptosis inducing activity of HFE7A on murine T cells in vivo 500 μl of PBS alone, or 0.05 or 0.1 mg of HFE7A monoclonal anti-cough (in 500 μl of PBS) was administered intraperitoneally to the member of the groups of three six-week-old female C3H / HeJ mice (from Japan Clea ). The mice were anesthetized with ether, 42 hours after administration, and their thymuses were removed. These thymuses were washed with RPMI medium containing 10% FCS v / v, and were subsequently fragmented, using a eepule on a mesh (Cell Strainer; Falcon). The fragmented cells (which had passed through the mesh) were washed twice with RPMI 1640 medium containing 10% v / v FCS. Where washing is concerned more than once in any of the present examples, it will be understood that the medium with which the washing is performed is replaced with said fresh medium for each washing, unless otherwise required. The washed cells obtained above were counted and adjusted to 1 x 10e cells in 50 μl of RPMI 1640 medium containing 10% v / v FCS. Each of the resultant suspensions was disperse in a well of a 96 well microplate, the well having U-shaped bottoms (Nunc) and the plate then centrifuged (90 x g, 4 ° C, 10 minutes). The supernatants were decanted and then one of the two anti-cough solutions labeled for fluorescence in PBS was added to each well, (a) or (b): (a) 10 μl anti-CD95 (Fas) anti-mouse mouse labeled with FITC at 0.5 mg / ml (Jo2; PharMingen), and 10 μl of anti-mouse anti-CD90 antibody labeled with phycoerythrin (PE) of 0.5 mg / ml (Thy-1.2, Cedarlane, CD90 is a cell surface antigen expressed only on T cells); (b) 10 μl of anti-CD4 anti-CD4 labeled with FITC, 0.5 mg / ml (L3T4 PharMingen), and 10 μl of anti-CD8 anti-CD8 labeled with PE, 0.2 mg / ml (Ly-2; PharMingen) . After the addition of the antibody mixtures, the plate was shaken to mix the contents of the wells and then kept on ice for one hour before centrifugation (90 x g, 4 ° C, 10 minutes). After discarding the supernatant and washing the wells twice with 100 μl / well of flow cytometry pH regulator, the cells were fixed by adding 50 μl / well of 3.7% v / v formaldehyde solution and then remaining on ice for 10 hours. minutes After further centrifugation (90 xg, 4 ° C, 10 minutes) to remove the supernatant, the cell pellets were washed again with 100 μl / well of flow cytometry pH regulator and euependieron in 100 μl / well regulator Flow cytometry pH - Using the cells obtained from each well as samples, fluorescence was measured from samples of 1 x 104 cells, using a flow cytometer (Epios Elite; Coulter) under the following conditions: wavelength excitation: 488 nm; Detection wavelength: 530 nm (FITC) or 600 nm (PE). Then FITC and PE fluorescence distributions could be prepared for the cell populations of each sample. For the samples to which the anti-cough mixture (a) was added, the proportion of the number of cells that were positive for Fas and CD90 (hereinafter referred to as "Fas + CD90 +") in relation to the total number of cells was calculated. . Similarly, for the patients to whom the anti-cough sample (b) was added, the proportion of the number of cells that were positive for CD4 and CD8 (hereinafter referred to as "CD4 + CD8 +") or those that were positive for CD4 but negative for CD8 (hereinafter referred to as "CD4 + CD8-") in relation to the total number of cells. The results are shown as a percentage in Table 1 below.

TABLE 1 Compared with the group to which only PBS was administered, the proportions of T cells expressing Fae (Fae + CD90 +) in the mouse thymus cells of the groups to which HFE7A was administered were markedly reduced at both doses. In addition, the CD4 + CD8 + and CD4 + CD8- cell populations, known by substantial expression of Fas, were also markedly reduced in number after administration of HFE7A, as compared to the PBS group alone. Therefore, it was deduced that anti-Fas antifungal monoclonal, HFE7A, has apoptosis-inducing activity, in vivo on T cells expressing Fas.

EXAMPLE OF REFERENCE 9 Effects of HFE7A on a model of autoimmune disease The effect of administration of monoclonal anti-Fas anti-Fas, HFE7A, on autoimmune disease symptom using MRL gld / gld mice was examined. These mice carry a mutant of the Fas ligand gene and serve as an animal model of general autoimmune disease lupus erythematosus. MRL mice gld / gld of 18 week old were treated (from Japan SLC, K.K.), intraperitoneally with an eola doeie, already at 0.2 or 0.5 mg, of monoclonal anti-HFE7A prepared in reference example 3 (in 500 μl of PBS) or with 500 μl of Polsol. Each test mouse was monitored to observe swelling of the ankles as a symptom of autoimmune disease. The degree of swelling was evaluated and recorded with time for each group [see Shin Yonehara, (1984), Nikkei Science Beseateu, 110, 66-77). It was observed that the degree of swelling of the ankle decreased markedly with the administration of HFE7A.

The tymuses of the test mice were extirpated and the proportions of T cells expressing Fas in the thymus were determined by the method described in reference example 8 above. The results showed that the number of T cells expressing Fae was lowered significantly after the administration of HFE7A, in accordance with the results of reference example 8.

REFERENCE EXAMPLE 10 Hepato Oxicity Tests BALB / c mice were inoculated intraperitoneally with an eola doe of one of the following: i) 0.2 mg of HFE7A in 500 μl of PBS; ii) 0.5 mg of HFE7A in 500 μl of PBS; iii 0.1 mg of Jo2 (PharMingen) in 500 μl of PBS; and iv) 500 μl of PBS alone. Of the foregoing, Jo2 is a known anti-Fas anti-mouse anti-fastener that has apoptosis-inducing activity. Blood was extracted from the posterior aorta of the mice at 8 hours, 24 hours, or 72 hours after administration. Blood was drawn at 3 hours after administration for mice treated with Jo2, while they were still alive. All the blood was taken under light anesthesia with ether. Blood levels of gluta-ico-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) were measured for each blood sample, using an automatic analyzer (Model 7250, Hitachi Seisakusyo, KK) together with the appropriate reagent for the analyzer (Transa inasa-HRII; Wako Puré Chemical Industries, Ltd). As a result, the group treated with Jo2 showed rapid elevation of the GOT and GPT values after 3 hours, while the corresponding values for the groups treated with HFE7A showed little change, as with the group treated with PBS alone (figure 5) . From these results, it could be established that HFE7A does not induce acute hepatic disorders.

REFERENCE EXAMPLE 11 Effects on the fulminant hepatitis model It is known that, by intraperitoneal administration of the mouse anti-Fas anti-Fas Jo2, a mouse develops fulminant hepatitis and dies within a few hours [see Owasawara, J. and otroe (1993), Nature, 364, 806]. Therefore, to evaluate the effect of HFE7A on the hepatic traetornoe induced by Jo2, the viability of the mice was tested by administering HFE7A simultaneously with, or subsequent to, the administration of Jo2. BALB / c female mice 6 weeks of age (three mice per group, from Japan SLC) received intraperitoneal administration of an anti-cough preparation as follows: A) 0.1 mg of Jo2 in 0.5 ml of PBS; B) 0.01 mg of Jo2 in 0.5 ml of PBS; C) 0.1 mg of Jo2 and 0.5 mg of HFE7A together in 0.5 ml of PBS (simultaneous administration); D) 0.1 mg of Jo2 and 0.05 mg of HFE7A in 0.5 ml of PBS (simultaneous administration); and E) 0.01 mg of Jo2 in 0.2 ml of PBS, followed by 0.1 mg of HFE7A in 0.2 ml of PBS after 20 minutes; and the mice were then observed over time. The results are shown in Figure 6. When Jo2 was administered alone, all mice died within 9 hours, regardless of which were administered with 0.1 mg or with 0.01 mg of Jo2 per mouse, ie, all mice groups A) and B) above died in the transcureo of 9 hours after administration. In contrast, when HFE7A was administered simultaneously with Jo2 (both 0.5 mg / mouse and 0.05 mg / mouse), that is, in groups C) and D) above, the mice did not exhibit disturbances even for several weeks after administration, demonstrating that the administration of HFE7A can block the dementrollo de hepatitie fulminante. In addition, the mice remained normal, without developing evident symptoms, even when HFE7A was administered 20 minutes after the administration of Jo2. Thus, HFE7A has preventive and therapeutic effects on several diseases involving disorders of normal tissues mediated by the Fae / Fas ligand system, both in the liver and in other organs.

REFERENCE EXAMPLE 12 Effects on Rheumatoid Arthritis 1) Preventive effect on the development of collagen-induced arthritis. Fl mice obtained from the pairing of a female BALB / c mouse and a male DBA / 1J mouse (female CD1F1 mice, 6 weeks of age, from Japan Charles River, K.K.) were tamed for 1 week. After this time, the mice were treated with collagen to induce arthritis. In more detail, the method was based on one described in the literature [see Phadke, K. (1985), Immunopharmacol. , 10, 51-60]. In this method, a 0.3% w / v solution of type II bovine collagen (Collagen Gijutsu Kensyukai, supplied in a 50 mM acetic acid solution) was diluted to 0.2% (2 mg / ml) with additional 50 M acetic acid and it was then emulsified with an equal volume of Freund's complete adjuvant (Difco). This emulsion was then administered, in an amount of 100 μl (corresponding to 100 μg of type II bovine collagen), intradermally in the proximal position of the tail, which was held in a fixator for intravenous injection, using a syringe plastic 1 ml equipped with a tuberculin needle. An identical booster dose was administered under similar conditions, 1 week after the original exposure. At the same time as the booster injection, an injection of 100 μg of intraperitoneally was administered HFE7A or mouse IgG as control in 0.5 ml of PBS (6 mice per group). Beginning five weeks after the original exposure, limb swelling was monitored visually. The degree of swelling in the joints of the limbs was described in Wood's method, F.D., and other [Int. Arch, Alergy Appl. Im unol., (1969), 35, 456-467]. Therefore, the following criterion was used in the calculation of scores for each of the members: Score 0: there is no symptom; 1: swelling and redness of only one of the small joints, e.g. of the hoof; 2: swelling and redness of 2 or more small joints, or of a relatively large joint, such as an ankle; and 3: swelling and redness of a limb in its entirety. Therefore, the maximum score for an animal is when all four limbs swell, and it is 12. The animal with a score of at least 1 for all four limbs was designated as an "affected mouse." The results are shown in Figure 7. In the control group, to which non-specific mouse IgG was administered, all mice were affected in the seventh week after the initial exposure, while in the group to which HFE7A was administered. , half of the mice did not show any redness of any of the joints until the eighth week (Figure 7A). In addition, the group treated with HFE7A had a lower average score compared to the control group (Figure 7B). 2) Induction of apoptosis in synovial cells of rheumatic patients The effects of HFE7A on the viability of synovial cells of patients with rheumatoid arthritis were evaluated. The method was like the one described below, using the reducing power of the itchondria as the index. Silyvial tissue obtained from an affected region of a rheumatoid arthritis patient was cut with scissors, in small pieces, in Dulbecco's modified Eagle's medium (Gibco), supplemented with FCS 10% v / v (Summit). The fat was removed and then collagenase (Sigma Chemical Co.) was added to a final concentration of 5 μg / ml and the mixture was incubated at 37 ° C for 90 minutes under 5% v / v CO2. The resulting incubated cells then served as the synovial cells for the replacement of the experiment. The synovial cells thus obtained were separated into individual cells by treatment with an aqueous solution of trypsin 0.05% w / v at 37 ° C for 2 minutes; after which, the Dulbecco modified Eagle medium containing FCS at 10% v / v was diluted to a cell density of I X 10 5 cells / ml. This cell suspension was then dispensed into wells of a 96-well plate at 2 X 10 4 cells / 200 μl per well, and incubated at 37 ° C under 5% v / v CO2. The culture supernatant was discarded and the cells were washed 3 times with Hank's pH regulator (Gibco). After washing, 200 μl of Dulbecco's modified Eagle's medium containing FCS 10% v / v and between 10 and 1,000 ng / ml of HFE7A (dilute in 10 times) was added to each well, and the plate was further incubated. 37 ° C under CO2 5% v / v for 20 hours. Then, 50 μl of an aqueous solution of 1 mg / ml of XTT (internal salt of 2,3-bis [2-methoxy-4-nitro-5-eulfophenyl] -2H-tetrazolium-5) was added to each well. -caFoxanilide; Sigma Chemical Co.) and 25 μM PMS (phenazine methosulfate; Sigma Chemical Co.) (final concentrations: 250 μg / ml XTT and 5 μM PMS). After a further 4 hours of incubation at 37 ° C under CO2 5% v / v, the absorption of each well was read at 450 nm. The viability of the cells in each well was calculated according to the following formula: Viability% = 100 x (ab) / (cb), in which "a" is the absorption of a test well, "b" is the absorption of a well without cells, and "c" is the absorption of a well without an antiquake added. The results are shown in Table 2. HFE7A inhibited, in a dose-dependent manner, the survival of the synovial cells of patients with rheumatism, TABLE 2 -0 EXAMPLE 1 Design of a Humanized Version of the Antifluid HFE7A (1) Molecular model of the variable regione of HFE7A 5 A molecular model of the variable regione of HFE7A was made by means of the method generally known as the homology model elaboration [see Methods in Enzymology, 203. 121-153, (1991) ] The primary sequences of the regione variable of 0 immunoglobulin regietradae in the protein data bank (hereinafter referred to as the "PDB", Chemistry Department, Building 555, Brookhaven National Laboratory, PO Box 5000, Upton, New York 11973-5000, USA), for which crietalography of. X ray, ee compared with the regione of HFE7A structure determined above. As a result, IGGI and 2HFL were selected, since they have the highest homologies of the three-dimensional structures of the structure regions for the light and peaked chains, respectively. The three-dimensional structures of the structure regions were generated by combining the properties of 1GGI and 2HFL and calculating the properties of the HFE7A regione, as described below, to obtain the "structure model". Using the classification described by Chothia and others, the RDCs of HFE7A could be classified as follows: CDRL2, CDRL3 and CDRHi, all, belong to the canonical class 1, while CDRLi, CDRH2 and CDRH3 do not currently seem to belong to any specific canonical class. . Lae buFujae CDR CDRL2, CDRL3 and CDRHi were attributed to the confirmations inherent to sue respective canonical classes, and then they were integrated into the structure model- CDRLi was attributed to the conformation of cluster 15B, in accordance with the classification of Thornton et al. [See J. Mol. Biol., 263, 800-815, (1996)]. For CDRH2 and CDRH3, sequence conformations were selected with high homology of the PDB, and then these were combined with the energy calculation results. Then they took the conformation of the buDujae RDC with the highest probabilities, and they were integrated into the structure model. Finally, energy calculations were carried out to eliminate undesirable contact between inappropriate atoms, in terms of energy, to obtain a global molecular model of HFE7A. The above procedure was carried out using the commercially available, common molecular model system, AbM (Oxford Molecular Limited, Inc.), although any other appropriate form may be used. For the molecular model obtained, the accuracy of the structure was further evaluated using the software PROCHECK, [J. Appl. Cryst., (1993), 26, 283-291], and ee calculated the degree of surface exposure of each residue to determine which atoms and surface groups interacted. (2) Selection of the receptors The subgroups of the light and heavy chains of HFE7A share identities of 79% with the subgroup klV and also 79% with the subgroup I, respectively, in comparison with the sequence sequences of the subgroups reepectivoe of anticuefoe humanoe However, there is no human antiquake that has a combination of a light chain klV and a peedal chain of subgroup I. In this way, SEIO'CL, which has a light chain of subgroup KIII and a heavy chain of subgroup I, which has 72% and 77% sequence identity with the light chain and heavy chain of HFE7A, respectively, was selected as the individual human antibody having light and heavy chains that have an identity of more than 70% with light and heavy chains of HFE7A. (3) Selection of donor residues to be grafted onto the receptors Using the Cameleon software (Oxford Molecular Limited, Inc.), the amino acid sequence of each of the light and heavy chains of HFE7A was aligned with that of the relevant SEIO chain. CL, and humanized variables of the variable region were made as described in the following examples, in accordance with the general guidelines indicated in a) to e) above. Plasmids were constructed that could serve as recombinant vectors comprising DNA nucleotide sequences encoding humanized anti-Fas human anti-bud.

EXAMPLE 2 Preparation of DNA encoding humanized light chain (1) Cloning of cDNA encoding a full-length human light chain (k-chain) Before the humanization of the amino acid sequence of the light chain of the anti-human mouse anti-Fas anti-Fas, HFE7A, first cDNA cloning was carried out. a light chain of human immunoglobulin comprising the constant region. 1) Synthesis of initiation The carrying out of cDNA encoding a human light chain by PCR was carried out. For PCR, the following two primers were synthesized: 5'-GCGAATTCTG CCTTGACTGA TCAGAGTTTC CTCA-3 '(HVKII5-4: SEQ ID No.47 of sequence listing); and 5'-GCTCTAGATG AGGTGAAAGA TGAGCTGGAG GA-3 '(HKCL3-3: SEQ ID No. 48 of the sequence sequence). 2) Construction of a plasmid containing human immunoglobulin light chain cDNA. CDNA encoding a full-length human immunoglobulin light chain was prepared by PCR, inserted into a plasmid and cloned into E. coli. The HL-DNA fragment encoding a full-length human immunoglobulin light chain was prepared under the following conditions: Compounding the PCR reaction solution: human lymphocyte cDNA library (Life Technologies), 25 ng; oligonucleotide primer HVKII5-4, 50 pmoles; oligonucleotide primer HKCL3-3, 50 pmoles; 25 mM dNTP cocktail, 10 μl; pH buffer Tris-HCl 100 mM (pH 8.5), 10 μl; 1M potassium chloride [KCl], 5 μl; 25 mM magnesium chloride [MgCl2], 10 μl; DNA polymerase Taq (Perkin Elmer Japan), 1 unit; Water redeetilated haeta a total volume of 100 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 74 ° C for 10 minutes. The thus-prepared DNA-HL fragment (human light chain DNA) was inserted into plasmid pCR3DNA using a TA Cloning kit of Eukaryote (Invitrogen), following the manufacturer's protocol and introduced into competent E. coli T0P10F ' the equipment, and following the instructions on the equipment. Plasmid pHL15-27 carrying the DNA-HL fragment, ie, cDNA for a human immunoglobulin light chain, was obtained therewith. (2) Construction of expression vectors for the light chain of the humanized version of the HFE7A antibody 1) Construction of expression vectors for the light chain of HFE7A The humanization of the amino acid sequence of the light chain of anti-Fas anti-Fas mouse human, HFE7A encompassed the replacement of amino acid 47 (proline) and amino acid 49 (lysine) from the N-terminus of the amino acid sequence of the light chain (hereinafter referred to as "region 15") with alanine and arginine, respectively . Alanine (47) and arginine (49) are conserved in the human light chain (K chain). Additional humanization was also performed, and included the replacement of amino acid 80 (hietidine), amino acid 81 (proline), amino acid 82 (valine), amino acid 84 (glutamic acid), amino acid 85 (glutamic acid), amino acid 87 (alanine) and amino acid 89 (threonine) (hereinafter referred to as "region II") with serine, arginine, leucine, proline, alanine, phenylalanine, and valine, respectively, since these are also conserved in the light chain human (chain K). Where regions I and II were humanized, the sequence was designated as "HH type". Where region I was only humanized, the sequence was designated as "HM type". Where no region was humanized, the sequence was designated as "MM type". Expression plasmids were constructed as follows, which respectively carry these three types of humanized light chain amino acid sequences of the anti-human Fae anti-HFE7A. 2) Sinteeis of initiators to prepare the variable regione and the light chain chain of humanized HFE7A. PCR was used to construct the following DNA sequence, each of which comprises one of the sequences HH, HM or MM described above, together with the constant region of the human immunoglobulin light chain (K chain): DNA (SEQ ID. No. 49 of the sequence listing) encoding the HH-type polypeptide chain (SEQ ID No, 50 of the sequence listing); DNA (SEQ ID No. 51 of the sequence listing) encoding the HM type polypeptide chain (SEQ ID No. 52 of the sequence listing); and DNA (SEQ ID No. 53 of the sequence listing) encoding the MM-type polypeptide chain (SEQ ID No. 54 of the sequence of sequences). The following oligonucleotide PCR primers were synthesized: 5'-CCCAAGCTTAA GAAGCATCCT CTCATCTAGT TCT-3 '(7ALIP: SEQ ID No. 55); 5'-GAGAGGGTGG CCCTCTCCCC TGGAGACAGA GACAAAGTAC CTGG-3 '(7ALIN: SEQ ID No. 56); 5'-CCAGGTACTT TGTCTCTGTC TCCAGGGGAG AGGGCCACCC TCTC-3 '(7AL2P: SEQ ID No. 57); 5'-GATTCGAGAT TGGATGCAGC ATAGATGAGG AGTCTGGGTG CCTG-3 '(7AL2N; SEQ ID No. 58); 5'-GCTGCATCCA ATCTCGAATC TGGGATCCCA GACAGGTTTA GTGGC-3 '(7AL3PA; SEQ ID No. 59); 5 '-AAAATCCGCC GGCTCCAGAC GAGAGATGGT GAGGGTGAAG TCTGTCCCAG AC-3' (7AL3N; SEQ ID No. 60); 5'-CTCGTCTGGA GCCGGCGGAT TTTGCAGTCT ATTACTGTCA GCAAAGTAAT GAGGATCC-3 '(7AL4P: SEQ ID No. 61); 5'-TGAAGACAGA TGGTGCAGCC ACAGTCCGTT TGATTTCCAG CCTGGTGCCT TGACC-3 '(7AL4N, SEQ ID No. 62); 5'-GGTCAAGGCA CCAGGCTGGA AATCAAACGG ACTGTGGCTG CACCATCTGT CTTCA-3 '(7ALCP; SEQ ID No. 63); 5'-CCCGAATTCT TACTAACACT CTCCCCTGTT GAAGCTCTTT GTGAC-3 '(7ALCN; SEQ ID No. 64); 5 '-TCTGTCCCAG ACCCACTGCC ACTAAACCTG TCTGGGATCC CAGATTCGAG ATTGG-3 '(M7AL2N; SEQ ID No. 65); 5'-GTTTAGTGGC AGTGGGTCTG GGACAGACTT CACCTCTACC ATCCATCCTG TGGAG-3 '(M7AL3PA: SEQ ID No. 66); and 5'-ATGGTGCAGC CACAGTCCGT TTGATTTCCA GCCTGGTGCC TTGACCGAAC GTCCG-3 '(7AL4NA; SEQ ID No. 67). 3) Construction of the plaemid P7AL-HH (expiry plaemid for the humanized HFE7A light chain type HH) The VHH fragment of DNA (SEQ ID No. 49 of the sequence) which encodes the amino acid sequence of SEQ ID No. 50 of the sequence list, was prepared by performing PCR of 3 steps, and then inserted into a plasmid vector and cloned into E. coli. a) First step of CPR In figure 8 the boequejo of the first step of CPR for the preparation of DNA-VHH is shown.

The DNA fragment L7A1, which encodes a secretion signal sequence and a portion of the altered FRLi region to contain a cleavage site of the Hind III restriction enzyme at the 5 'end, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pME-L, 200 ng; Oligonucleotide initiator 7AL1P, 80 pmoles; Oligonucleotide initiator 7AL1N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and redispersed water haeta a final volume of 200 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The L7A2 fragment of DNA encoding a portion of the FRLi, CDRLi, FRL_ and a portion of the CDRL2 region, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pME-L, 200 ng; Oligonucleotide primer 7AL2P, 80 pmolee; Oligonucleotide primer 7AL2N, 80 pmolee; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; DNA poli eraea Pfu (Stratagene), 10 unidadee; and Water redeetilated haeta a final volume of 200 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The L7A3 fragment of DNA, which encodes CDRL2 and a portion of FRL3, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pME-L, 200 ng; Oligonucleotide initiator 7AL3PA, 80 pmol; Oligonucleotide initiator 7AL3N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The DNA fragment L7A4, which encodes a portion of FRL3, CDRL3, FRL4, and a portion of the constant region, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pME-L, 200 ng; Oligonucleotide initiator 7AL4P, 80 pmoles; Oligonucleotide initiator 7AL4N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 C for 10 minutes. The L7A5 fragment of DNA, which encodes a portion of FRL4 and the altered constant region to have an EcoRI restriction enzyme cleavage site at the 3 'end, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pHL15-27, 200 ng; Oligonucleotide initiator 7ALCP, 80 pmol; Oligonucleotide initiator 7ALCN, 80 pmol; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. An equal volume of phenol-chloroform (50% v / v of phenol saturated with water, 48% v / v of chloroform, 2% v / v of isoamyl alcohol) was added to 200 μl of each of the PCR products , and mixed vigorously for 1 minute.

After this time, the mixture was centrifuged at 10,000 xg, and the aqueous layer was recovered and mixed with an equal volume of chloroform-isoamyl alcohol (96% v / v chloroform and 4% v / v isyl alcohol), which was stirred again vigorously for 1 minute. The resulting mixture was centrifuged at ,000 x g and the aqueous layer was recovered (the series of step cited in this paragraph is referred to herein as "phenol extraction"). Then, ethanol precipitation was performed on the recovered aqueous layer. As used and referred to herein, "ethanol precipitation" consists of the addition, with mixing, of one-tenth volume of 3M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol, to the solution to be treated. , and freezing the mixture using dry ice. The resulting mixture is then centrifuged at 10,000 x g to recover DNA as a precipitate. After extraction with phenol and precipitation with ethanol, the resulting DNA precipitate was dried under vacuum, dissolved in a minimum amount of redistilled water and separated by polyacrylamide gel electrophoresis at 5% w / v.

After electrophoreation, the gel was stained with an aqueous solution of 1 μg / ml ethidium bromide to allow detection of DNA under UV light. The DNA bands corresponding to DNA-L7A1, DNA-L7A2, DNA-L7A3, DNA-L7A4 and DNA-L7A5, were extracted using a safety razor blade and eluted from the gel using Centriruter and Centricon-10, as described above. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation and finally dissolved in 50 μl of distilled water. b) Second step of PCR In figure 9 the sketch of the second step of PCR for the production of DNA-VHH is shown. ADN-L7A1.2, in which the fragments were merged DNA-L7A1 and DNA-L7A2, described above, was prepared as follows. Composition of the PCR reaction solution: DNA-L7A1 solution prepared in the first PCR step, 10 μl; DNA-L7A2 solution prepared in the first PCR step, 10 μl; Oligonucleotide initiator 7AL1P, 80 pmoles; Oligonucleotide initiator 7AL2N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 days at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. DNA-L7A4.5, in which the DNA-L7A4 and DNA-L7A5 fragments described above were fused, was prepared as follows. Composition of the reaction solution: DNA-L7A4 solution prepared in the first PCR step, 10 μl; DNA-L7A5 solution prepared in the first step of RCP, 10 μl; Oligonucleotide initiator 7AL4P, 80 pmoles; Oligonucleotide initiator 7ALCN, 80 pmol; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle at 94 ° C was repeated 30 times for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. First, phenol extraction was performed and then ethanol precipitation of the amplified fragments with PCR, DNA-L7A1.2 and DNA-L7A4.5, and these fragments were separated by putative electrophoresis in 5% polyacrylamide gel p / v. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide, and the bands detected under UV light were extracted by using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as described above. The eluted DNA was first concentrated by centrifugation at 7,500 x g, then by precipitation with ethanol, and then dissolved in 50 μl of distilled water. c) third step of PCR In figure 10 the sketch of the third step of PCR for the production of DNA-VHH is shown. The VHH DNA fragment amplified by PCR was first subjected to extraction with phenol and then to ethanol precipitation, before separation by electrophoreation in 5% w / v polyacrylamide gel. Deepuée of electrophoresis, the gel was stained with 1 μg / ml of ethidium bromide and the VHH band of DNA, detected under UV light, was cut using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10 , as described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. In figure 11 the connection of an expression plasmid carrying a VHH fragment of DNA is boequeja. The DNA fragment VHH obtained above was further purified by extraction with phenol, followed by ethanol precipitation, and was digested with the restriction enzymes Hind III and EcoR1. One μg of cloning plasmid pHSG399 DNA (Takara Shuzo Co., Ltd) was digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated with alkaline phosphataea (derived from intenetine of calf, hereinafter abbreviated as CIP). The pHSG399 DNA from the reemic deefoephorylated plamidid, and the digested VHH DNA fragment, were ligated using a DNA Ligation Kit, version 2.0 (Takara Shuzo Co., Ltd.) using the manufacturer's protocol. The ligated DNA was recovered by ethanol precipitation, dissolved in 5 μl of redistilled water, and then mixed with E.coli JM109 Electro-Cell (Takara Shuzo Co., Ltd.). The mixture was transferred to a pulsator cell Gene Pulser / E. coli, 0.1 cm (BioRad) and the ligated mixture was then used to transform E. coli JM 109 using Gene Pulser II (BioRad) by the manufacturer's protocol (the series of steps in this paragraph is referred to herein as "transformation"). "). After tracification, the cells were plated on LB agar [Bacto-tryptone (Difco) 10 g, Bacto-yeast extract (Difco) 5 g, 10 g NaCl, Bacto-agar (Difco) 15 g; dissolved in deethylated water, c.e.p. 1 1] containing final concentrations of 1 mM of IPTG (isopropylthio-β-D-galactose, Takara Shuzo Co., Ltd.), X-Gal (5-bromo-4-chloro-3-indolyl- &-D- galactoside, Takara Shuzo Co., Ltd.) 0.1% w / v) and 50 μg / ml chloramphenicol, and plaques were incubated at 37 ° C overnight to obtain E. coli transformants. Any obtained white transformant was cultured in 2 ml of liquid LB medium at 37 ° C overnight, and plasmid DNA was extracted from the resulting culture by the alkaline SDS method [Sambrook, J. et al. (1989) in "Molecular Cloning: A Laboratory Manual (23 Edition) ", Cold Spring HaFor Laboratory Press]. The resulting extracted plasmid DNA was digested with the restriction enzymes Hind III and EcoRI and a clone carrying the VHH DNA fragment was then selected by means of 1% w / v agarose gel electrophoresis. Therefore, plasmid pHSGHH7 carrying a fusion fragment of the light chain variable region of HFE7A type HH and DNA encoding the constant region of the human immunoglobulin k chain was obtained. The E. coli pHSGHH7 SANK 73497 that hosts the plasmid pHSGHH7 was depoeited with the Kogyo Gijuteuin Sei ei-Kogaku Kogyo Gijutdu Kenkyujo, on Aug. 22, 1997, in accordance with the Budapeet Treaty, and was granted the accession number FERM BP-6073. Using the plasmid pHSGHH7 described above, it was then possible to construct the plasmid expression vector P7AL-HH, which carries the DNA of SEQ ID No. 49 of the sequence of eequencee and which encodes the HFE7A light chain polypeptide type HH of SEQ ID No. 50 of the sequence of sequences. One μg of DNA pEE.12.1 (Lonza), an expression vector for mammalian cells, was digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated using CIP. The resulting digested dephosphorylated plasmid DNA (100 ng) was ligated with 10 μg of the DNA fragment pHSGHH7 which had also been digested with Hind III and EcoRI, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.) . The ligation mixture was then used to transform E, coli JM109 (as described above), which was then plated onto LB agar plates containing 50 μg / ml ampicillin. The transformants obtained by this method were cultured in 2 ml of liquid LB medium containing 50 μg / ml of ampicillin at 37 ° C overnight, and the plasmid DNA was extracted subecreasingly from the re-culture culture by the alkaline SDS method. The extracted plasmid DNA was digested with Hind III and EcoRI and subjected to electrophoresis in 1% w / v agarose gel to confirm the presence or absence of the insert of interest. This allowed the isolation of the plasmid p7AL-HH, which contains a fusion fragment having the variable region of the humanized HH-type HH-type light chain, together with DNA encoding the constant region of the human immunoglobulin k chain. It was found that the fusion fragment is located towards the 3 'end of the cytomegalovirus (CMV) promoter in the correct orientation. 4) Construction of the plasmid p7AL-HM (expression plasmid for the humanized HM type HFE7A light chain) The VHM DNA fragment of SEQ ID No. 51 of the sequence listing coding for the amino acid sequence of SEQ ID No. 52 of the sequence listing, was produced by performing a 3-step CPR; it was inserted into a plasmid vector and then cloned into E. coli. a) First step of PCR In figure 12 the sketch of the first step of PCR for the preparation of the DNA VHM fragment is shown. In this procedure, the L7A1 fragment of DNA, which encodes a secretion signal sequence and a portion of FRLi, which has a Hind III restriction enzyme cleavage site added at the 5 'end, the L7A2 fragment of DNA, which encodes a portion of FRL, CDRLi, FRL2 and a portion of CDRL2, and the L7A5 fragment of DNA, which encodes a portion of FRL4 and the constant region having an EcoRI site added at the 3 'end, which are those obtained in the preparation of the DNA fragment VHH in 2) previous. A ML7A3 fragment of DNA encoding CDRL2, FRL3, CDRL3, FRL4 and a portion of the constant region was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pME-L, 200 ng; Oligonucleotide initiator 7AL3PA, 80 pmol; Oligonucleotide initiator 7AL4NA, 80 pmoles; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 days at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 * C for 10 minutes. The PCR products were first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophorea, the gel was stained with 1 μg / ml ethidium bromide and the DNA band detected under UV light, corresponding to DNA-ML7A3, was cut using a safety knife and eluted from the gel using a Centriruter and Centricon-10, as described above. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. b) Second step of PCR In figure 13 the sketch of the second step of PCR for the production of DNA-VHM is shown. A DNA-VHM fusion fragment, comprising DNA-L7A1.2, DNA-ML7A3, and the above DNA-L7A5 fragment, was prepared as follows. Composition of the PCR reaction solution: DNA-L7A1.2 solution prepared in the second PCR step, 10 μl; DNA-ML7A3 solution prepared in the first PCR step, 10 μl; DNA-L7A5 eolution prepared in the first step of PCR, 10 μl Oligonucleotide primer 7AL1P, 80 pmol; Oligonucleotide initiator 7ALCN, 80 pmol; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The VHM fragment of the re-amplified amplified DNA was first subjected to phenol extraction and then to ethanol precipitation, and was separated by 5% w / v polyacrylamide gel electrophoresis. After electrophoreation, the gel was stained with 1 μg / ml ethidium bromide and the VHH band of detected DNA was cut using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and dissolved in 50 μl of distilled water. Figure 14 depicts the construction of an expulsion plasmid carrying a DNA VHM fragment. The VHM fragment of DNA obtained antee ee was further purified by extraction with phenol, followed by ethanol precipitation, and was then digested with the restriction enzymes Hind III and EcoR1. One μg of plasmid DNA from cloning pHSG399 (Takara Shuzo Co., Ltd) was digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated using CIP. The resulting dephosphorylated DNA PHSG399 was then ligated with DNA-VHM, which had also been digested with Hind III and EcoRI, using a DNA Ligation Kit, version 2.0 (Takara Shuzo Co., Ltd.). Afterwards, E. coli JM109 was transformed with the ligated DNA and spread on LB agar medium containing final concentrations of 1 mM IPTG, 0.1% w / v X-gal and 50 μg / ml chloramphenicol. Any obtained white transformant was cultured in 2 ml of liquid LB medium containing 50 μg / ml of chloramphenicol, at 37 ° C overnight, and the plasmid DNA was extracted from the resulting culture by the alkaline SDS method. The extracted plasmid DNA was then digested with Hind III and EcoRI, a clone carrying the VHM DNA fragment was selected using 1% w / v agarose gel electrophoresis. Therefore, plasmid pHSGHM17 was obtained, which carries a fusion fragment of the variable region of the light chain of HFE7A type humanized and DNA encoding the constant region of the human Ig chain k. The transforming E. coli PHSGHM17 SANK 73597 harboring the plasmid pHSGHM17 was deposited with the Kogyo Gijutsuin Seimei-Kogaku Kogyo Gijutdu Kenkyujo, on Aug. 22, 1997, in accordance with the Budapest Treaty, and was granted the FERM accession number. BP-6072. Using the plasmid pHSGHM17 described above, a plasmid p7AL-HM expression vector was constructed, which carries the DNA of SEQ ID No. 51 of the sequence of eequencee, which encodes the humanized HM-type HMP7A light chain polypeptide of SEQ ID No. 52 of the sequence of events. One μg of pEE.12.1 DNA (Lonza), an expression vector for mammalian cells, was digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated using CIP. The resulting digested dephosphorylated plasmid DNA (100 ng) was ligated with 10 μg of the DNA fragment pHSGHM17 which had also been digested with Hind III and EcoRI, using a Versiói 2.0 DNA ligation kit (Takara Shuzo Co., Ltd.) . The ligation mixture was then used to transform E, coli JM109 (as described above), which was plated on then LB agar plates containing 50 μg / ml ampycillin. The transformants obtained by this method were cultured in 2 ml of liquid LB medium containing 50 μg / ml of ampicillin at 37 ° C overnight, and the plasmid DNA was extracted from the resulting culture by the SDS alkaline method. The extracted plasmid DNA was digested with Hind III and EcoRI and subjected to electrophoresis in 1% w / v agaroea gel to confirm the presence or absence of the ineree of interest. This allowed the isolation of the plasmid p7AL-HM, which contains a fusion fragment having the variable region of the light chain of HFE7A humanized type, together with DNA encoding the constant region of the human immunoglobulin k chain. It was found that the fusion fragment is located towards the 3 'end of the cytomegalovirus (CMV) promoter in the correct orientation.

) Construction of plasmid p7AL-MM (expression plasmid for humanized type HFE7A light chain MM) The VMM DNA fragment of SEQ ID No. 53 of the sequence listing coding for the amino acid sequence of SEQ ID No. 54 of Sequence listing, was produced by performing a 3-step CPR; it was inserted into a plasmidic vector and deepuée was cloned into E. coli. a) First step of PCR In figure 15 ee shows the sketch of the first step of PCR for the preparation of the DNA VMM fragment. The L7A1 DNA fragment, which encodes a secretion signal sequence and a portion of FRLi, and has a Hind III restriction enzyme cleavage site added at the 5 'end, and the L7A5 DNA fragment, which encodes a portion of FRL4 and the constant region having an EcoRI restriction site added at the 3 'end, are those obed in the preparation of the VHH fragment of DNA in 2) above. These fragments were used in the first step of PCR for the construction of DNA-VMM. The DNA fragment ML7A2M, which encodes a portion of FRLi, CDRLi, FRL2, CDRL2 and a portion of FRL3, was prepared as follows: Composition of the PCR reaction solution: plasmid DNA pME-L, 200 ng; Oligonucleotide primer 7AL2P, 80 pmoles; Oligonucleotide primer M7AL2N, 80 pmoles; dNTP cock, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The DNA fragment ML7A3M, which encodes a portion of FRL3, CDRL3, FRL4 and a portion of the constant region, was prepared as follows: Composition of the PCR reaction eXolution: pEM-L plaemid DNA, 200 ng; Oligonucleotide primer M7AL3PA, 80 pmoles; Oligonucleotide initiator 7AL4NA, 80 pmoles; dNTP cock, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After the procedure, the reaction solution was heated at 72 ° C for 10 minutes.The PCR products were first subjected to phenol extraction and then to ethanol precipitation, and were subsequently removed by 5% w / v polyacrylamide gel electrophoresis. After electrophoresis, the gel was sed with 1 μg / ml ethidium bromide and the DNA bands corresponding to DNA-ML7A2M, and DNA-ML7A3M, detected under UV light, were cut using a safety razor blade and eluted of the gel using a Centriruter and Centricon-10, as described above.The eluted DNAs were concentrated by centrifugation at 7,500 xg, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. Figure 16 shows the trace of the second step of PCR for the preparation of DNA-VMM. The ML7A1.2 fragment of DNA, comprising a fusion of the above DNA-ML7A1 and DNA-ML7A2M fragments, was prepared as follows. Composition of the PCR reaction eolium: DNA-L7A1 solution prepared in the first step of RCO, 10 μl; DNA-ML7A2M solution prepared in the first step of RCO, 10 μl; oligonucleotide primer 7AL1P, 80 pmoles; Oligonucleotide primer M7AL2N, 80 pmoles; dNTP cock, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was heated first at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The resulting amplified DNA ML7A1.2 fragment was first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophoreation, the gel was stained with 1 μg / ml ethidium bromide and the band of DNA thus detected was cut using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as described. described earlier. The eluted DNA was concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and dissolved in 50 μl of distilled water. c) Third step of PCR: Figure 17 shows the sketch of the third step of PCR for the preparation of DNA-VMM. The VMM fragment of DNA, comprising a fusion of the above DNA-ML7A1.2, DNA-ML7A3M and the DNA fragment-L7A5, was prepared as follows: Composition of the reaction solution PCR: DNA-ML7A1.2 solution prepared in the second step of CPR, 10 μl; DNA-ML7A3M solution prepared in the first step of RCP, 10 μl; DNA-L7A5 solution prepared in the first PCR step, 10 μl; oligonucleotide primer 7AL1P, 80 pmoles; Oligonucleotide initiator 7ALCN, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The resulting amplified DNA-VMM fragment was first subjected to extraction with phenol and after precipitation with ethanol, and was separated by polyacrylamide gel electrophoresis 5% w / v. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA-VMM band thus detected was extracted using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as shown in FIG. described earlier. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and dissolved in 50 μl of distilled water. The construction of a plasmid carrying the VMM fragment of DNA is outlined in Figure 18. The DNA-VMM fragment obtained above was further purified by extraction with phenol, followed by precipitation with ethanol, and then subjected to digestion with the restriction enzymes Hind III and EcoR1. One μg of DNA from the cloning plasmid pHSG399 (Takara Shuzo Co., Ltd) was digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated using CIP. The resulting dephosphorylated DNA pHSG399 was then ligated with DNA-VMM, which had also been digested with Hind III and EcoRI, using a DNA Ligation Kit, version 2.0 (Takara Shuzo Co., Ltd.). Then, E. coli JM109 was transformed with the ligated DNA and spread on LB agar medium containing final concentrations of 1 mM IPTG, 0.1% w / v X-gal and 50 μg / ml chloramphenicol. The obtained white transformants were grown in 2 ml of liquid LB medium containing 50 μg / ml of chloramphenicol, at 37 ° C overnight, and the plasmid DNA was extracted from the resulting culture by means of the alkaline SDS method. extracted was then digested with Hind III and EcoRI, and a clone carrying the VMM DNA fragment was selected using electrophoresis in 1% w / v agaroea gel, therefore, plasmid pHSGMM6, which carries a fusion fragment, was obtained. of the variable region of the light chain of HFE7A type MM and DNA encoding the constant region of the human immunoglobulin k chain The transforming E. coli pHSGMM6 SANK 73697 harboring the plasmid pHSGMMó was deposited with the Kogyo Gijuteuin Seimei-Kogaku Kogyo Gijutdu Kenkyujo, on August 22, 1997, in accordance with the Budapest Treaty, and was granted accession number FERM BP-6071. Plasmid expression vector p7AL-MM was constructed using the plasmid pHSG MMo described above, and carries the DNA of SEQ ID No. 53 of the sequence listing, which encodes the light chain polypeptide of HFE7A type MM of SEQ ID No. 54 of the sequence listing. One μg of pEE.12.1 DNA (Lonza), an expression vector for mammalian cells, was digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated using CIP. The resulting digested dephosphorylated plasmid DNA (100 ng) was ligated with 10 μg of the DNA fragment pHSGMM6 which had also been digested with Hind III and EcoRI, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.) . The ligation mixture was then used to transform E. coli JM109 (as described above), which was then plated onto LB agar plates containing 50 μg / ml ampicillin.

The trainees obtained by this method were cultured in 2 ml of liquid LB medium containing 50 μg / ml of ampicillin at 37 ° C overnight, and the plaemid DNA was subsequently extracted from the resulting culture by means of the alkaline SDS method. . The extracted plasmid DNA was digested with Hind III and EcoRI and subjected to electrophoresis in 1% w / v agarose gel to confirm the presence or absence of the insert of interest. This allowed the isolation of plasmid p7AL-MM, which contains a fusion fragment having the variable region of the light chain of HFE7A type MM, together with DNA coding for the constant region of the human immunoglobulin k chain. It was found that the fusion fragment is located towards the 3 'end of the cytomegalovirus (CMV) promoter in the correct orientation. 6) Verification of the nucleotide sequence To verify that the DNA inserts of the plasmids p7AL-HH, p7AL-Hm and P7AL-MM have the desired nucleotide sequences, their DNA inserts were analyzed to determine the nucleotide sequences. The oligonucleotide primers prepared for nucleotide eequencing are as follows: '-CCCAAGCTTA AGAAGCATCC-3 '(SP1, SEQ ID No. 68); 5'-ATCTATGCTG CATCCAATCT-3 '(SP2, SEQ ID No. 69); 5'-GTTGTGTGCC TGCTGAATAA-3 '(SP3, SEQ ID No. 70); '-CCCGAATTCT TACTAACACT-3 '(SP4, SEQ ID No. 71); 5'-TTATTCAGCA GGCACACAAC-3 '(SP5, SEQ ID No. 72); and 5'-AGATTGGATG CAGCATAGAT 3 '(SP6, SEQ ID No. 73); Fi 19 shows the positions in which each initiator joins. The determination of the nucleotide sequences was carried out by the chain termination method of dideoxynucleotide [Sanger, F.S. and others (1977), Proc. Nati Acad. Sci. USA 74, 5463]. The templates used were the respective plasmid DNAs purified by the alkaline SDS method and by the cesium chloride method [see Sambrook, J. et al. (1989) in "Molecular Cloning: A Laboratory Manual, Second Edition", Cold Spring HaFor Laboratory Press, for both methods] More specifically, 3 μg of the purified plasmid DNA was dissolved in 13 μl of redistilled water, followed by the addition of 2 μl of 2mM EDTA and 2 μl of 2N NaOH, and the mixture was allowed to stand at room temperature for 5 minutes. Then, 4 μl of 10 M ammonium acetate solution and 100 μl of 100% ethanol were added and mixed, and the mixture was placed on dry ice for 10 minutes. At the end of this time, the mixture was centrifuged at 15,000 x g, and the obtained pellet was washed with 80% v / v aqueous ethanol and then dried under vacuum. The resulting dried DNA was dissolved in 7 μl of redistilled water and used for nucleotide sequencing. The nucleotide sequencing reaction was carried out by a 7-Deaza-Sequanaee team, version 2.0 (for dCTP, Amersham). A mixture of 7 μl of the plasmid solution described above, 1 pmol of an initiator, which had been synthesized in advance, and 1 μl of reaction pH buffer (supplied with the equipment) was made, and this mixture was incubated after 65 'C for 2 minutes. Subsequently, the DNA was fixed with the initiator by gradually cooling to room temperature, followed by labeling with [α-32P] dCTP (Amersham). Then, the reaction product was subjected to gel electrophoresis on a 5% w / v polyacrylamide gel containing 8M urea in pH regulator TBE lx (100 mM Tris, 100MM boric acid, 1mM EDTA, pH 8.3). After drying, the sequences on the gel were read by autoradiography. As used herein, any sequence determination was made as indicated above, unless otherwise specified. As a result, it was established that the DNA inserts of the plasmids p7AL-HH, P7AL-HM, and p7AL-MM, had the expected nucleotide sequences, ie, SEQ ID No. 49 encoding the SEQ ID polypeptide sequence. No. 50; SEQ ID No. 51, which encodes the polypeptide sequence SEQ ID No. 52; and SEQ ID No. 53, which encodes the polypeptide sequence SEQ ID No. 54; respectively, of the sequence listing.

EXAMPLE 3 Construction of an Expression Vector for the Heavy Chain of the Humanized Version of the Antifog HFE7A (1) Construction of a plasmid carrying the variable region of the humanized HFE7A DNA heavy chain 1) Synthesis of primers to prepare the variable region of the humanized heavy chain DNA synthesis (SEQ ID No. 74 of the sequence listing ) encoding a polypeptide chain comprising the variable region of the heavy chain of the anti-HFE7A anti-Fas human antifungal and the 5 amino acid residues at the N-terminus of the IgG-CH1 region (SEQ ID No. 75 of the sequence listing) , was performed using a combination of CPR. The following 8 CPR primers were synthesized as described above: '-GGGAAGCTTG GCTTGACCTC ACCATGGGAT GGAGCTGTAT-3 '(7AH1P; SEQ ID No. 76); 5'-TGAAGCCCCA GGCTTCTTGA CCTCAGCCCC AGACTGCACC AGTTGGAC-3 ' (7AH1NNEW; SEQ ID No. 77); 5'-TCCACTCAAG CCTCTGTCCA GGGGCCTGTT TTACCC-3 '(7AH2N; SEQ ID No. 78); 5'-GTCTGGGGCT GAGGTCAAGA AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA AG-3 '(7AH2PNEW; SEQ ID No. 79); 5'-CAGGCCCCTG GACAGAGGCT TGAGTGGATG GGAGAGATT-3 '(7AH3P; SEQ ID No. 80); 5'-TCAGATCTCA GGCTGCTGAG CTCCATGTAG GCTGTGCTAG CGGATGTGTC-3 '(7AH3N; SEQ ID No. 81); 5'-TGGAGCTCAG CAGCCTGAGA TCTGAGGACA CGGCGGTCTA TTAC-3 '(7AH4P; SEQ ID No, 82); 5'-GATGGGCCCT TGGTGGAGGC TGAGGAGACG GTGACCAGGG TCCCTTCGCC CCAGT-3 '(7AH4N; SEQ ID No. 83); 2) Construction of plamidmid PBL7A27 The DNA fragment VD of SEQ ID No. 74 of the sequence listing that encodes the amino acid sequence of the SEQ ID No. 75 of the sequence listing was prepared by performing a 3-step PCR, then inserted into a plamidid and cloned in E. coli. a) First step of CPR Figure 20 shows the sketch of the first step of PCR for the preparation of the DNA-RV. The DNA H7A1 fragment, which encodes a secretion signal sequence and an N-terminal portion of FRHi and has a Hind III restriction enzyme cleavage site added at the 5 'end, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pME-H, 200 ng; Oligonucleotide primer 7AH1P, 80 pmoles; oligonucleotide primer 7AH1NNEW, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. In this procedure, the reaction solution was heated to 72 [deg.] C. for 10 minutes.The DNA fragment H7A2, which encodes a portion of FRHi, CDRHi, and a portion of FRH2, was prepared as follows: Compounding the PCR reaction Plasmid DNA pME-H, 200 ng, oligonucleotide primer 7AH2N, 80 pmoles, oligonucleotide primer 7AH2PNEW, 80 pmoles, cocktail dNTP, 20 μl, pH buffer Pfu lOx, 20 μl, Pfu DNA polymerase, 10 units; redistilled water to a final volume of 200 μl The PCR reaction was carried out as follows The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle at 94 ° C was repeated 30 times for 1 hour. minute, 55 ° C for 1 minute and 72 ° C du After 2 minutes, after finishing this procedure, the reaction solution was heated at 72 [deg.] C. for 10 minutes.

The DNA H7A3 fragment, which encodes a portion of FRH2, CDRH2, and a portion of FRH3, was prepared as follows: Composition of the PCR reaction solution: plasmid DNA pME-H, 200 ng; oligonucleotide primer 7AH3P, 80 pmolee; Oligonucleotide initiator 7AH3N, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was heated first at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The DNA H7A4 fragment, which encodes a portion of FRH3, CDRH3, FRH4, and the 5 amino acid residues of the N-terminus of the CH1 region, was prepared as follows: Composition of the PCR reaction solution: Plasmid DNA pME- H, 200 ng; Oligonucleotide primer 7AH4P, 80 pmoles; oligonucleotide initiator 7AH4N, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was heated first at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 minutes at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The respective PCR products were first subjected to extraction with phenol and then to ethanol precipitation, and then they were separated by elect roforeey in 5% w / v polyacrylamide gel. Deepuée de electroforeeie, the gel was stained with 1 μg / ml of ethidium bromide and the corresponding lae of DNA-H7A1, DNA-H7A2, DNA-H7A3 and DNA-H7A4, detected under UV light, were cut using a razor blade of safety and eluted from the gel using a Centriruter and Centricon-10, as described above. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. b) Second CPR pae In Figure 21 the sketch of the second step of PCR for the preparation of DV-DNA is shown. The H7A1.2 fragment of DNA, in which the DNA-H7A1 and DNA-H7A2 fragments described above were fused, was prepared as follows: Compoeición of the reaction solution RCP: DNA-H7A1 solution prepared in the first phase of PCR , 10 μl; DNA-H7A2 solution prepared in the first PCR step, 10 μl; Oligonucleotide primer 7AH1P, 80 pmoles; 7AH2N oligonucleotide primer, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The DNA fragment H7A3.4, in which the fragment DNA-H7A3 and DNA-H7A4 described above were extracted, was prepared as follows: Compoeición of the reaction solution RCP: solution of DNA-H7A3 prepared in the first step of PCR , 10 μl; DNA-H7A4 solution prepared in the first PCR step, 10 μl; oligonucleotide primer 7AH3P, 80 pmolee; Oligonucleotide primer 7AH4N, 80 pmolee; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was first heated to 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes.The resulting amplified DNA-H7A1.2 and DNA-H7A3.4 fragments were first subjected to extraction with phenol and then to ethanol precipitation, and then eepa raron by electrophoresis in 5% w / v polyacrylamide gel After electrophoreation, the gel was stained with 1 μg / ml of ethidium bromide and the relevant band was detected using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as described above.The eluted DNA was then concentrated by centrifugation at 7,500 xg, followed by ethanol precipitation, and dissolved in 50 μl of distilled water c) Third step of PCR: the figu Ra 22 shows the sketch of the third step of PCR for the preparation of DNA-VD. The DNA fragment VD, in which the DNA-H7A1.2 and DNA-H7A3.4 fragments described above were fused, was prepared as follows: Composition of the PCR reaction solution: DNA-H7A1.2 solution prepared in the second step CPR, 10 μl; DNA-H7A3.4 solution prepared in the second PCR step, 10 μl; Oligonucleotide primer 7AH1P, 80 pmoles; oligonucleotide initiator 7AH4N, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was performed as follows. The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes.The resulting amplified DNA-VD fragment was first subjected to extraction with phenol and then to ethanol precipitation, and then separated by gel electrophoresis. 5% w / v polyacrylamide After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA-VD band thus detected was extracted using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and dissolved in 50 μl of distilled water. In Figure 23 the construction of a plasmid carrying the DNA-VD fragment is outlined. The DNA-VD fragment obtained above was further purified by extraction with phenol, followed by ethanol precipitation, and then subjected to digestion with the restriction enzymes Hind III and Apal. One μg of plasmid vector DNA pBLUESCRIPT-II SK + (Strategene), was digested with Hind III and Apal, and then dephosphorylated using CIP. The resulting dephosphorylated plasmid DNA and 100 ng of the DNA fragment VD, which had also been digested with Hind III and Apal, were ligated using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). The resulting ligation mixture was then used to transform E. coli JM109, which was then plated onto LB agar plates containing final concentrations of 1 mM IPTG, 0.1% w / v X-gal and 50 μg / ml ampicillin Any resulting white transformant was cultured in 2 ml of liquid LB medium containing 50 μg / ml ampicillin at 37 ° C overnight, and the plasmid DNA was extracted from the culture by the alkaline SDS method. The resulting plasmid was digested with Hind III and Apal and subjected to agarose gel electrophoresis to confirm the presence or absence of the enzyme. Therefore, plasmid pBL7A27 was obtained with a DNA-VD insert. (2) Construction of a plasmid carrying genomic DNA from the constant region of human IgGl 1) Synthesis of primers to prepare a genomic DNA fragment of human IgGl from the 5 'end. A genomic DNA fragment of human IgGl from the 5 'end was synthesized by means of PCR. For this, the following 2 oligonucleotide primers were prepared: 5'-GGGAAGCTTC CGCGGTCACA TGGCACCACC TCTCTTGCA-3 '(5'Hind: SEQ ID No. 84 of the sequence listing); and 5'-GCTCTGCAGA GAGAAGATTG GGAGTTACTG GAATC-3 '(IGGCPSTN: SEQ ID No. 85 of the sequence listing). 2) Construction of plamidmid pIG5'03 Separated and amplified by PCR, genomic DNA comprising the CH1 region of human IgGl together with an intron close to a Hind III cleavage sequence, using human genomic DNA as a template, and then inserted into the plaster PHSG399 (Takara Shuzo Co., Ltd.) and cloned in E. coli. The preparation of this DNA (hereinafter referred to as "DNA-IG5" ') is outlined in Figure 24. A DNA-IG5' fragment was prepared as follows. Composition of the RCP reaction solution: human genomic DNA (Clonetech), 2 μg; oligonucleotide primer 5'Hind, 80 pmoles; Oligonucleotide primer IGGCPSTN, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and redispersed water haeta a final volume of 200 μl. The CPR reaction was performed as follows. The solution was heated first at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 minutes at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The resulting amplified DNA-IG5 'fragment was first subjected to phenol extraction and then to ethanol precipitation, and then quenched by 5% w / v polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA-IG5 'band thus detected was extracted using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as It was described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and dissolved in 50 μl of distilled water. The DNA-IG5 'fragment thus obtained was further purified by extraction with phenol, followed by ethanol precipitation, and then digested with the restriction enzymes Hind III and PstI. One μg of plasmid DNA pHSG399 (Takara Shuzo Co., Ltd.) was digested with the restriction enzyme Hind III and Petl, and was dephosphorylated using CIP. The resulting dephosphorylated plasmid DNA and 100 ng of the IG5 'fragment of DNA, which had also been digested with Hind III and PstI, were ligated by Vereión 2.0 DNA ligation kit (Takara Shuzo Co., Ltd.). The resulting ligation mixture was then transformed into E. coli JM109, which was then plated on LB agar medium containing final concentrations of 1 mM IPTG., 0.1% w / v of X-gal and 50 μg / ml of chloramphenicol. Any white trainer obtained was cultured in 2 ml of liquid LB medium containing 50 μg / ml chloramphenicol at 37 ° C overnight, and the plasmid DNA was extracted from the resulting culture by the alkaline SDS method. The extracted plasmid DNA was then digested with Hind III and PstI and subjected to 1% w / v agarose gel electrophoresis to confirm the presence or absence of the insert of interest. Therefore, plasmid pIG5'03 containing a fragment insert DNA-IG5 'was obtained. (3) Construction of a plasmid carrying genomic DNA from the constant region of human IgGl 1) Synthesis of initiate to prepare a genomic DNA fragment of human IgGl from the 3 'end. A genomic DNA fragment of human IgGl from the 3 'end was synthesized by means of PCR. For this, the following 2 oligonucleotide primers were prepared: 5'-TCTCTGCAGA GCCCAAATCT TGTGACAAAA CTCAC-3 '(IGGCPSTP: SEQ ID No. 86 of the sequence of sequences); and 5'-GGGGAATTCG GGAGCGGGGC TTGCCGGCCG TCGCACTCA-3 '(Eco3': SEQ ID No. 87 of the sequence listing). 2) Construction of plaemid pIG3'08 DNA was prepared and amplified by PCR, comprising the sequence: intron of human IgGl; hinge region; human IgGl intron; CH2 region; human IgGl intron; CH3 region; and an EcoRI breaking sequence, using human genomic DNA as a template, and then inserted into the plasmid pHSG399 (Takara Shuzo Co., Ltd.) and cloned into E. coli. The preparation of the above DNA (hereinafter referred to as "DNA-IG3" ') is boequeja in Figure 25. DNA-IG3' was prepared as follows. Composition of the PCR reaction solution: human genomic DNA (Clonetech), 2 μg; Oligonucleotide primer IGGCPSTP, 80 pmoles; Oligonucleotide primer Eco3 ', 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase, 10 units; and water redeetilated haeta a final volume of 200 μl. The CPR reaction was performed as follows. The solution was first heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 days at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The resulting amplified DNA-IG3 'fragment was first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA-IG3 'band thus detected was extracted using a safety razor blade and eluted from the gel using a Centriruter and Centricon-10, as It was described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and dissolved in 50 μl of distilled water. The DNA-IG3 'fragment thus obtained was further purified by extraction with phenol, and then by precipitation with ethanol, and then digested with the EcoRI and PstI reentrant enzyme. One μg of plasmid DNA pHSG399 (Takara Shuzo Co., Ltd.) was digested with EcoRI and PstI, and was dephosphorylated after CIP. The resulting dephosphorylated plasmid DNA was then ligated with 100 ng of IG3 'fragment of DNA, which also had been digested with EcoRI and PstI, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). The resulting ligation mixture was then used to transform E. coli JM109, which was then seeded onto LB agar medium containing final concentrations of 1 mM IPTG, 0.1% w / v X-gal and 50 μg / ml Chloramphenicol Any white colony was selected, and plasmid pIG3'08 containing an insert of DNA-IG3 'was obtained. (4) Construction of a plasmid expression vector for the humanized HFE7A heavy chain The expression vector plasmid pEg7AH-H, which carries the DNA of SEQ ID No. 88 of the sequence listing and encoding the chain polypeptide, was constructed. Humanized HFE7A of SEQ ID No. 89 of the sequence listing using the plasmids pBL7A27, pIG5'03 and pIG3'08 described above. The procedure is outlined in Figure 26. Ten μg of plasmid DNA pIG5'03, comprising the CH1 region of the human IgG1 heavy chain and an intron, were digested with the restriction enzymes Apal and Kpnl. In addition, 1 μg of the above pBL7A27 DNA was also digested with the restriction enzymes Apa I and Kpn I, and then dephosphorylated using CIP. The rheumatoid rhemoeff pBL7A27 DNA (100 ng) was ligated with 10 μg of the pIG5'03 digested and deefoephorylated DNA, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). The resulting ligation mixture was then used to transform E. coli JM109, which was then plated onto LB medium containing 50 μg / ml ampicillin. The resulting transformants were cultured in 2 ml of liquid LB medium containing 50 μg / ml of ampicillin, at 37 C during the night, and the plasmid DNA was extracted from the culture by the alkaline SDS method. The plasmid DNA was digested with Apa I and Kpnl, or with Hind III and Pst I, to confirm the presence or absence of the insert of interest by means of electrophoresis in 1% w / v agarose gel. In this manner, plasmid pBL7AF184 containing a VD fragment of humanized HFE7A DNA connected to the DNA-IG5 'fragment was obtained. Next, 10 μg of the plamidid pBL7AF184 thus obtained was digested with the restriction enzymes Hind III and Pst I, and also 1 μg of the plasmid DNA pIG3'08 above was digested with Hind III and Pst I, and dephosphorylated using CIP. The resulting dephosphorylated pIG3'08 DNA (100 ng) was ligated with 10 μg of the digested pBL7AF184 DNA, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). With the ligation mixture, E. coli JM109 was transformed and plated on LB medium containing 50 μg / ml of ampicillin. Any resulting transformant was cultured in 2 ml of liquid LB medium containing 50 μg / ml of chloramphenicol, at 37 ° C overnight, and the plasmid DNA was extracted from the culture by the alkaline SDS method. The plasmid was digested with Hind III and Pst I, or with Hind III and EcoRI, to confirm the presence or absence of the insert of interest by means of electrophoresis in 1% w / v agarose gel. Plasmid pgHSL7A62 was obtained, which contains a DNA-VD fragment of HFE7A connected to a genomic DNA fragment encoding the constant region of human IgGl. The transformant E. coli pgHSL7A62 SANK 73397 harboring the plasmid pgHSL7A62 was depoeited with the Kogyo Gijuteuin Seimei-Kogaku Kogyo Gijutdu Kenkyujo, on Aug. 22, 1997, in accordance with the Treaty of Budapeet, and was granted the FERM accession number. BP-6074. Ten micrograms of the plasmid DNA pgHSL7A62 obtained was digested with the restriction enzymes Hind III and Pst I, and likewise, 1 μg of the expression plasmid DNA pEE6.1 was digested with Hind III and EcoRI, and defoefo rilated. CIP. The resulting dephosphorylated DNA (100 ng) DNA was ligated with 10 μg of the digested pgHSL7A62 DNA, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). The E. coli JM109 was transformed with the ligation mixture and plated on LB medium containing 50 μg / ml of ampicillin. Any resulting ransformant was grown in 2 ml of liquid LB medium containing 50 μg / ml ampicillin at 37 ° C overnight, and the plasmid DNA was extracted from the culture by the alkaline SDS method The plasmid was digested with Hind III and EcoRI, to confirm the presence or absence of the insert of interest by means of electrophoresis in 1% w / v agarose gel The resulting plasmid, pEg7AH-H, contains a fusion fragment comprising a DNA-VD fragment of HFE7A humanized and a genomic DNA fragment encoding the constant region of human IgGl in connection and inserted towards the 3 'end of the CMV promoter in the correct orientation. (5) Verification of the nucleotide sequence To verify that the DNA insert of pEg7AH-H has the expected nucleotide sequence, the DNA insert was analyzed to determine the nucleotide sequence. For this, the following primers were synthesized: 5 '-ACAGCCGGGA AGGTGTGCAC-3' (1601: SEQ ID No. 90); 5'-AGACACCCTC CCTCCCTGTG-3 '(1G02: SEQ ID No. 91); 5'-GTGCAGGGCC TGGGTTAGGG-3 '(1G03: SEQ ID No. 92); 5'-GCACGGTGGG CATGTGTGAG-3 '(1G04: SEQ ID No. 93); 5'-GTTTTGGGGG GAAGAGGAAG-3 '(1G05: SEQ ID No. 94); 5'-CCAGTCCTGG TGCAGGACGG-3 '(1G06: SEQ ID No. 95); 5'-CCTGTGGTTC TCGGGGCTGC-3 '(1G07: SEQ ID No. 96); 5'-CGTGGTCTTG TAGTTGTTCT-3 '(1G08: SEQ ID No. 97); 5'-CTTCCTCTTC CCCCCAAAAC-3 '(1GP5: SEQ ID No. 98); 5'-CCGTCCTGCA CCAGGACTGG-3 '(1GP6: SEQ ID No. 99); 5 '-GCAGCCCCGA GAACCACAGG-3' (1GP7: SEQ ID No. 100); 5'-AGAACAACTA CAAGACCACG-3 '(1GP8: SEQ ID No. 101); 5'-GCCTGACATC TGAGGACTC-3 '(H5 +: SEQ ID No. 102); 5'-GAGTCCTCAG ATGTCAGGC-3 '(H5-: SEQ ID No. 103); 5'-GAGCAGTACT CGTTGCTGCC GCGCGCGCCA CCAG-3 '(PEEF: SEQ ID No. 104); and 5'-GGTATGGCTG ATTAATGATC AATG-3 '(PEEB: SEQ ID No. 105); Figure 27 shows the positions in which each initiator is linked. The determination of the nucleotide sequence was carried out by means of the chain termination method of dideoxynucleotide (Ibide), with the respective plasmids purified by means of the alkaline SDS method and the cesium chloride method (ibidem). It was confirmed that pEg7AH-H has the nucleotide sequence of SEQ ID No. 88 of the sequence listing, which encodes the polypeptide of SEQ ID No. 89 of the sequence listing.

EXAMPLE 4 Expression in COS-1 Cells C0S-1 cells (derived from the kidney of a monkey) were transfected with the expression plasmids for the heavy chain of humanized HFE7A, and with the expresion plasmids for each of the light chains of humanized HFE7A obtained above. Transfection was effected by electroporation using the gene transfection apparatus GTE-1 (Shimadzu Seisakusyo, K.K.), equipped with a FCT-13 chamber having electrodes separated by 2 mm (Shimadzu Seieakueyo, K.K.). C0S-1 cells were developed (American Type Culture Collection No. CRL-1650) haeta semiconfluence in a culture flask (culture area: 225 cm2; Sumitomo Bakelite) containing Minimum Essential Medium a ["oc (+) MEM"; Gibco BRL] supplemented with FCS at 10% v / v (Mo dribble). Subsequently, the medium was discarded and the C0S-1 cells were removed from the flask by means of treatment with 3 ml of tripeine-EDTA solution (Sigma Chemicale, Col.) at 37 ° C. for 3 minutes, and the separated cells were harvested afterwards. by means of centrifugation at 800 rpm for 2 minutes, discarding the supernatant and washing twice with pH buffer of phosphate (potassium chloride [KCl] 0.02% w / v, potassium dihydrogen phosphate [KH2PO4] 0.02% w / v, Eodium chloride [NaCl] 0-8% w / v, disodium acid phosphate [Na2HP04] 1.15% w / v, hereinafter referred to as "pH buffer of PBS (-)", Niesui Pharmaceutical Co. Ltd.). Washed COS-1 cells were then suspended to a cell density of 1 x 10 8 cells / ml in pH buffer PBS (-) - In parallel, 4 μg of humanized HFE7A heavy chain expression plasmid was mixed with 4 μg of humanized HFE7A light chain expulsion plasmid DNA, each purified by the alkaline SDS method and gradient centrifugation of ceeium chloride. The resulting mixture was subjected to ethanol precipitation and then suspended in 20 μl pH buffer PBS (-). These steps of mixing, precipitation and resuspension were all carried out in the same tube. All the resulting plasmid suspension (20 μl) was mixed with 20 μl of the cell eosinase C0S-1 (2 x 106 cells) prepared above, and the mixture was transferred to an FCT-13 electroporation chamber Shimadzu Seisakusyo, KK) which has a set of electrodes with 2 mm separation, which was loaded in the transfection apparatus of genes GTE-1 (Shimadzu Seisakueyo, KK). Pulsations of 600 V, each of 50 μF, were applied twice, with an interval of 1 second, to transform the C0S-1 cells with the plamid DNA. After electroporation, the cell-DNA mixture in the chamber was suspended in 20 μl of a (+) MEM supplemented with 10% v / v FCS, and transferred to a culture flask (culture area: 75 cm2; Sumitomo; Bakelite). After incubating under 5% v / v CO2 at 37 ° C at 37 ° C for 72 hours, the culture supernatant was recovered and an analysis of the supernatants was carried out to determine which expression products were present. Using the above method, but modifying it as appropriate, C0S-1 cells were transfected in a varied manner with each of the following plasmids or plasmid combinations: (A): non-plasmid DNA; (B): cotransfection with pEg7AH-H and p7AL-MM; (C): cotransfection with pEg7AH-H and p7AL-HM; (D): cotransfection with pEg7AH-H and p7AL-HH.

EXAMPLE 5 Quantification of Expression Products by means of ELISA It was carried out by means of ELISA, verification and quantitative analysis of the expression of humanized anti-convolutions as expression products in the culture supernatant fluids prepared in example 4, using a anti-human anti-IgG anti-trap. Goat Fc specific polyclonal antibody was dissolved against human IgG (Kappel) to a final concentration of 1 μg / ml in adsorption pH buffer (0.05M sodium acid caFonate, 0.02% w / v sodium azide, pH 9.6) and 100 μl aliquots were added to each well of a 96 well plate (MaxiSoF, Nunc), and the plate was incubated at 37 ° C for 2 hours to stimulate the antiquase adsorption. Next, each well was washed four times with 350 μl of PBS-T [PBS (-) with 0.05% w / v of Tween-20 (BioRad)]. After washing, culture supernatant diluted with oc (+) MEM containing 10% v / v FCS was added to the wells and the plate was further incubated at 37 ° C for 2 hours. After this time, the wells were again washed 4 times with PBS-T, and then 100 μl of goat anti-human IgG labeled polyclonal antibody labeled with alkaline phosphataea (Caltag Lab) diluted 5,000 was added to each well. vecee with PBS-T, and the plate was incubated at 37 ° C for 2 hours. Each well was then again washed 4 times with PBS-T, and 100 μl of a 1 mg / ml p-nitrophenyl phosphate substrate solution, prepared in 10% v / v diethanolamine (pH 9.8), was added to each well. ). After an incubating incubation at 37 ° C for 0.5 to 1 hour, the absorption at 405 nm was measured. In these experiments, human plasma IgG subclass 1 (IgG1; Biopure AG) diluted with a (+) MEM containing 10% FCS v / v was used up to certain desired concentrations to provide reference concentration samples of humanized HFE7A antibodies in Fluids of culture supernatant. As expected, it was determined that each supernatant of the transformants (B), (C) and (D), expressed human anti-cotuefo, as detected by anti-anti-human IgG. The negative control, (A), showed no expression of human antiquake.

EXAMPLE 6 Test for Fas binding activity The Fas binding activity test was performed on the cell culture supernatants prepared in Example 4, by means of ELISA, as follows. The culture supernatant of the C0S-1 cells expressing the human Fae fusion protein, as obtained in reference example 1 above, diluted 5 times with adsorption pH regulator, was dispeneled into wells of a 96-well plate. wells (MaxisoF; Nunc) at 50 μl per well and the plate was incubated at 4 ° C overnight to allow adsorption of the human Fas fusion protein to the surface of the wells. Then, each of the wells was washed 4 times with 350 μl of PBS-T. After washing, PBS-T containing 5% v / v BSA (bovine serum albumin; Wako Puré Chemical Industriee, Ltd) was added to the wells at 50 μl per well and the plate was incubated at 37 ° C for 1 hour to block the rest of the surface of each well. Afterwards, the wells were again washed 4 times with PBS-T. The culture eobrenants obtained in the example 4 were adjusted to have a final concentration of product of interest of 100 ng / ml in cx (+) MEM containing 10% FCS v / v. The concentrations were estimated by the method described in Example 5. Each of the resulting solutions of 100 ng / ml was then used to produce serial dilutions by serial two-fold dilution with oc (+) MEM containing 10% FCS. % v / v. Then, 50 μl of each of the resulting serial dilutions of each expression product was added to a well prepared as above, and the plate was incubated at 37 ° C for two hours to allow the reaction. After this time, the wells were again washed 4 times with PBS-T and then 50 μl of goat polyclonal antibody specific for Fc anti-human IgG labeled with alkaline phosphatase (Caltag Lab) was dispensed in each well., diluted 10,000 times with PBS-T, and the reaction was allowed to proceed at 37 ° C for 2 hours. HFE7A purified from mouse HFE7A hybridoma was used as control (IgGl), and was detected using goat anti-IgG + IgA + IgM from goat mice, labeled with alkaline phosphatase (Gibco BRL), diluted 5,000 times with PBS-T, instead of goat polyclonal antibody specific for Fc anti-human IgG labeled with alkaline phosphatase. The wells were again washed 4 times with PBS-T, and then 50 μl of substrate solution [1 mg / ml of p-nitrophenyl phosphate in diethanolamine 20% v / v (pH 9.8)] was dispenerated in each well and the plate was incubated at 37 ° C for 0.5 to 1 hour. The binding activity of the expression product contained in each culture supernatant fluid was evaluated with the human Fas fusion protein, reading the absorption of each well at 405 nm. As expected, binding activity was demonstrated for the human Fas fusion protein for the supernatants (B), (C) and (D) above (Figure 2B).

EXAMPLE 7 Competitive inhibition of binding of HFE7A to Fas Humanized anti-Fas anti-cues of the examples should inhibit the binding of HFE7A to Fas, since the anti-copies of the examples were derived from HFE7A. Therefore, the ability of the expression products obtained in Example 4 to competitively inhibit the binding of HFE7A to the human Fas fusion protein was measured. One milligram of the purified HFE7A monoclonal anti-cell obtained in Reference Example 3 was labeled using commercially available alkaline phosphatase labeling equipment (muno-Link AP and APL labeling equipment; Genosis), using the protocol provided with the equipment. The resulting labeled antiquake is also referred to herein as "AP-HFE7A". The culture supernatant of C0S-1 cells containing the human Fas fusion protein, as obtained in Reference example 1, was diluted 5-fold with adsorption pH buffer, and dispensed into wells of a 96-well plate. Wells for luminescence detection (Luminescent Test Plate in solid, high binding property, Costar) with 50 μl per well. Then, the plate was incubated at 4 ° C overnight to allow adsorption of the human Fas fusion protein to the surface of the wells. After this time, each well was washed 4 times with 350 μl of PBS-T, and then 100 μl of PBS-T containing 5% v / v BSA was added to each well, and the plate was incubated at 37 ° C for 1 hour to block the rest of the surface of each well. The wells were washed again 4 times with PBS-T. The culture supernatants obtained in Example 4 were adjusted to final anti-cotuex concentrations of 1 μg / ml in a (+) MEM containing 10% FCS v / v by means of the method of Example 5. Each of the resulting solutions of the expression products were used to produce serial dilutions by serial dilution of 2 times with a (+) MEM containing 10% v / v FCS. AP-HFE7A was diluted to 50 ng / ml with a (+) MEM containing 10% v / v FCS, and 25 μl of the resulting solution was mixed with an equal volume of each of the diluted in prepared series. Each of the wells was again washed 4 times with PBS-T, and then 50 μl of each of the resulting antiquake mixtures was added to the individual wells, and the plate was left at room temperature overnight. Subsequently, after washing each well with PBS-T again 4 times, 100 μl of CDP-star pH buffer (9.58 ml of diethanolamine, 0.2 g of magnesium chloride, 0.25 g of sodium azide, pH were dispensed in each well). 8.5), and the plate was left at room temperature for 10 minutes. After this time, the CDP-star pH regulator was discarded and CDP-star substrate was added [1.2 ml sapphire II (Tropix), 200 μl CDP-star (Tropix), q.s. for 12 μl with CDP-star pH regulator) at 50 μl per well, and then the plate was allowed to stand at room temperature for an additional 40 minutes. The competitive inhibition of the expreation products of Example 4 of the binding of HFE7A to the human Fae-firing protein was evaluated by measuring the inteneity of the luminescence with Lu inoscan (Titertech). As re-examined, it was verified that the expression products prepared in Example 4 specifically inhibited the binding of HFE7A to the human Fas fire protein (Figure 29).

EXAMPLE 8 Apoptosis-inducing activity WR19L12a cells (see Itoh, N. et al., Ibid.) Were used to examine the apoptosis-inducing activity of the culture supernatant of C0S-1 cells of the Example. 4. WR19L12a cells were cultured in RPMI1640 medium with 10% v / v FCS (Gibco BRL) at 37 ° C for 3 days under 5% v / v CO2, and then 50 μl (1x10 * cells) were dispensed into each well. ) of the resulting culture, from a 96-well microplate (Sumitomo Bakelite). The culture supernatants obtained in Example 4 were adjusted to a final anti-cotuene concentration of 100 ng / ml in RPMI1640 medium containing 10% v / v FCS. Concentrations were calculated by the method of Example 5. Each of the adjusted solutions of the expression products was used to produce serial dilutions by serial dilution of two times with RPMI1640 containing 10% FCS v / v. Each of the diluted reagents of each expression product solution was added to individual wells, at 50 μl per well.; and the plate was incubated at 37 ° C for 1 hour. After this time, the cells in each well were washed 4 times with RPMI1640 containing 10% FCS v / v and then the washed cells were suspended in 75 μl per well of RPMI1640 containing 10% v / v FCS. Subsequently, 75 μl of goat anti-polyclonal goat anti specific human IgG, 1.25 μg / ml in RPMI1640 medium containing 10% v / v FCS, (Kappel) as a secondary antibody was added to each well. The plate was left at 37 ° C for 12 hours and then 50 μl of 25 μM PMS (phenazine methosulfonate, Sigma Chemical Co.), containing 1 mg / ml of internal salt XTT [2,3-bis] was added to each well. (2-methoxy-4-nitro-5-eulfophenyl) -2H-tetrazolium-5-carboxanilide; Sigma Chemical Co.] at final concentrations of 250 μg / ml for XTT and 5 μM for PMS. The plate was then incubated for 3 hours at 37 ° C, and the absorption at 450 nm of each well was measured to calculate the viability of the cells, using the reducing power of the mitochondria as an index. The viability of the cells in each well was calculated according to the following formula: viability (%) = 100 x (a-b) / (c-b) in which "a" is the absorption of a test well, "b" is the absorption of a well without cells, and "c" is the absorption of a well without added antibody. As expected, it was shown that each of the expression products (B), (C) and (D) of Example 4 induce apoptosis in T cells expressing the human Fas antigen (Figure 30).

EXAMPLE 9 Preparation of DNA encoding the humanized light chain (1) Vector construction for the light chains of the humanized versions of the HFE7A antibody. To humanize the amino acid sequence of the anti-Fas mouse anti-Fas light chain, HFE7A, the first amino acid (aspartic acid), the amino acid 85 (alanine) and the amino acid 107 (arginine) of the N-terminus of the amino acid sequence of the light type strain HH, were replaced with glutamic acid, glutamic acid and lysine, respectively. These replacement items are stored in the human light chain (K chain). The referenced sequence was designated as "PDHH type". For the light chain HM, the first amino acid (aepartic acid) and amino acid 107 (arginine) of the N-terminus of the amino acid sequence were replaced with the conserved residues of glutamic acid and glycine, respectively. The consecutive sequence was designated as "PDHM type". Expreenement plaemidoe were constructed with eetoe doe types of humanized light chain amino acid sequencing (PDHH and PFHM), as eigue. 1) Synthesis of initiation to prepare the variable region or conetantee of the humanized HFE7A light chain By means of PCR, DNA (SEQ ID No. 106 of sequence listing) coding for the PDHH type polypeptide chain (SEQ ID No. 107 of the sequence listing) and DNA (SEQ ID No. 108 of the sequence listing) which encodes the PDHM type polypeptide chain (SEQ ID No. 109 of the sequence listing). Each of these sequences is a fusion of one of the humanized versions of the light chain variable region HFE7A with the constant region of the human Ig light chain (K chain). 7AL1P (SEQ ID No. 55) and 7ALCN (SEQ ID No. 64) [example 2 (2) 2) above] had already been synthesized, and the following oligonucleotide initiators were also synthesized by PCR: 5 '- GGTGAGATTG TGCTCACCCA ATCTCCAGG - 3 '(L PDIF; SEQ ID No: 110); 5 '- CCTGGAGATT GGGTGAGCAC AATCTCACC - 3' (LPD1N; SEQ ID No: 111); 5 '- CCATCTCTCG TCTGGAGCCG GAGGATTTTG C-3' (LPD2P; SEQ ID No. 112); 5'-GCAAAATCCT CCGGCTCCAG ACGAGAGATG G-3 '(LPD2N; SEQ ID No. 113) 5'-CAAGGCACCA AGCTGGAAAT CAAACGGACT G-3' (LPD3P; SEQ ID No. 114; and 5 '- CAGTCCGTTT GATTTCCAGC TTGGTGCCTT G-3' ( LPD3N; SEQ ID No: 115). 2) Construction of plasmid pLPDHH75 (expression plasmid for humanized PDHH type HFEF7A light chain) DNA-LPDHH (light chain receptor region fused with DNA-PDHH) was prepared, as defined in SEQ ID No. 106 of listing of sequences, and which encodes the amino acid sequence of SEQ ID No. 107 of the sequence listing, performing three-step PCR, was inserted into a plasmid vector and cloned into E. coli. a) First step of PCR In figure 31 the sketch of the first step of PCR for the preparation of DNA-LPDHH is shown. The DNA LPD1 fragment, which encodes a secretion signal sequence and a portion of FRLi, but which has a HindIII restriction enzyme cleavage site added at the 5 'end, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pHSGHH7, 200 ng; Oligonucleotide initiator 7AL1P, 80 pmoles; Oligonucleotide primer LPD1N, 80 pmolee; DNTP cocktail, 20 μl; PH regulator lOxPfu, 20 μl; DNA polymerase Pfu (Stratagene), 10 unidadee; and 'Water redeetilated haeta a final volume of 200 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes.

The DNA fragment LPDHH1 encoding a portion of FRLi, CDRLi, FRL2, CDRL2 and a portion of the FRL3 region, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pHSGHH7, 200 ng; Oligonucleotide primer LPD1P, 80 pmol; Oligonucleotide primer LPD2N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator lOxPfu, 20 μl; DNA polymerase Pfu (Stratagene), 10 unidadee; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes- after After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The DNA fragment LPDHH2, which encodes a portion of FRL3, CDRL3 and FRL4, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pHSGHH7, 200 ng; Oligonucleotide primer LPD2P, 80 pmoles; Oligonucleotide primer LPD3N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator lOxPfu, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The LPDC fragment of DNA encoding a portion of FRL4 and the constant region of the light chain of HEF7A, but having an EcoRI restriction enzyme cleavage site added at the 3 'end, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pHSGHH7, 200 ng; Oligonucleotide primer LPD3P, 80 pmoles; Oligonucleotide initiator 7ALCN, 80 pmol; DNTP cocktail, 20 μl; PH regulator lOxPfu, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction was heated to 72 ° C for 10 minutes. After PCR, the fragments of amplified DNA were first subjected to phenol extraction and then to ethanol precipitation, and then passed through electrophoresis in 5% w / v polyacrylamide gel. After elect roforeeie, the gel was stained with 1 μg / ml of ethidium bromide and the DNA band detected in UV light, extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon- 10, as described above. The eluted DNA was concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. b) Second step of PCR In figure 32 the sketch of the second step of PCR for the production of DNA-LPDHH is shown. DNA-LPDHH1.2, in which the DNA-LPD1, DNA-LPDHH1, and DNA-LPDHH2 fragments described above are fused, is prepared as follows. Composition of the PCR reaction solution: DNA-LPD1 solution (from the first step of PCR), 10 μl; DNA-LPDHH1 solution (from the first step of PCR), 10 μl: DNA-LPDHH2 solution (from the first step of PCR), 10 μl; oligonucleotide primer 7AL1P, 80 pmoles; Oligonucleotide primer LPD3N, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and redistilled water to a final volume of 200 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle at 94 ° C was repeated 30 times for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. After PCR, the amplified LPDHH1.2 fragment was first subjected to extraction with phenol and after precipitation with ethanol, and was then separated by polyacrylamide gel electrophoresis 5% w / v. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the fusion DNA band thus detected, under UV light, was extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon -10, as described above. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. c) Third step of PCR In figure 33 the sketch of the third step of PCR for the production of DNA-LPDHH is shown. The DNA fragment LPDHH, in which the fragment DNA-LPDHH1.2 and DNA-LPDC described above, were prepared as eigue. Composition of the PCR reaction eolution: DNA-LPDHH1.2 (second PCR) eXolution, 10 μl; DNA-LPDC solution (from the first step of PCR), 10 μl; oligonucleotide primer 7AL1P, 80 pmoles; 5 oligonucleotide primer 7ALCN, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows: First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute. minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. After PCR, the amplified DNA-LPDHH fragment ee was first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA band thus detected, under UV light, was extracted with a safety razor blade and eluted from the gel by a Centriruter and Centricon- 10, as described above. The DNA eluted was concentrated by centrifugation at 7,500 25 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water.

The construction of an expression plasmid carrying the DNA fragment LPDHH is sketched in Figure 34. The DNA fragment LPDHH, obtained above, was further purified by means of extraction with phenol, followed by ethanol precipitation, and then subjected to digestion with the enzymes of HindIII and EcoRl. A microgram of cloning plasmid DNA pHSG399 was subjected to digethion with the restriction enzymes HindIII and EcoRI and then dephosphorylated with CIP. The resulting dephosphorylated pHSG399 DNA was then ligated with the DNA LPDHH fragment, which had also been previously digested with HindIII and EcoRI, using a DNA ligation kit version 2.0 (Takara Shuzo, Co. Ltd). E. coli JM109 was then transformed with the ligation mixture and seeded onto plates and onto LB agar medium containing final concentrations of 1 mM IPTG, 0.1% w / v X-Gal and 50 μg / ml chloramphenicol. Any obtained white transformant was cultured in 2 ml of LB liquid medium containing 50 μg / ml of chloramphenicol at 37 ° C overnight, and the plasmid DNA was extracted from the resulting culture by the alkaline SDS method. The extracted plasmid DNA was digested with HindIII and EcoRI, and then a clone carrying the LPDHH DNA fragment was selected by means of 1% w / v agarose gel electrophoresis. Therefore, the plasmid pHSHHS, which carries a fusion insert comprising the variable region of humanized DNA-LPDHH and DNA encoding the constant region of the human immunoglobulin K chain, was isolated. The transforming E. coli pHSHHS SANK 70398, which hosts the plasmid pHSHH5, was deposited with the Kogyo Gijutsuin Seimei-Kogaku Gijutsu Kenkyujo, on February 26, 1998, in accordance with the Budapest Treaty on the deposit of microorganisms, and was granted access number FERM BP-6274. The expression vector plasmid pLPDHH75, which carries the DNA fragment SEQ ID No. 106 from the sequence listing, which encodes the humanized PDHH type HFE7A light chain polypeptide of SEQ ID No, 107 of the sequence listing, was prepared after using the Plasmid pHSHHS. One μg of pEE.12.1 DNA (Lonza), an expression vector for mammalian cells, was digested with the restriction enzymes Hind III and EcoRI, and then deepened using CIP. The resulting digested dephosphorylated plasmid DNA (100 ng) was ligated with 10 μg of the DNA pHSHHS fragment that had also been digested with Hind III and EcoRI, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.) . The ligation mixture was then used to transform E. coli JM109 (as described above), which was seeded in plate depuped onto LB agar plates containing 50 μg / ml ampicillin. The transformants obtained by this method were cultured in 2 ml of liquid medium containing 50 μg / ml of ampicillin at 37 ° C overnight, and plasmid DNA was subsequently extracted from the resulting culture by means of the alkaline SDS method. The extracted plasmid DNA was digested with HindIII and EcoRI, and subjected to electrophoresis in 1% w / v agarose gel to confirm the presence or absence of the insert of interest. This allowed for the isolation of the plasmid pLPDHH75, which contains a fusion fragment having the variable region of the humanized PDHH type HFE7A light chain together with DNA encoding the human immunoglobulin K chain congering region. It was found that the fusion fragment was located towards the 3 'end of the cytomegalovirue (CMV) promoter in the correct orientation. 3) Construction of plasmid PLPDHM32 (expression plasmid for the humanized PDHM type HFE7A light chain) The DNA LPDHM fragment (SEQ ID No. 108 of the sequence listing, which encodes the amino acid sequence of SEQ ID No. 109 thereof) ) was performed by performing three-step PCR, the fragment then being inserted into a plasmid vector and cloned into E. coli. a) First step of PCR In figure 35 the sketch of the first step of PCR for the preparation of DNA-LPDHM is shown. The DNA fragment LPD1, which encodes a sequence of secretion signal and a portion of FRLi having an eitio of cleavage of HindIII-restriction enzyme added at the 5 'end, and the LPDC fragment of DNA, which encodes a portion of FRL4 and the region that attracts has an EcoRI re-entry enzyme cleavage site added at the 3 'end, were those obtained in the preparation of the DNA LPDHH fragment [see "2) Construction of plasmid PLPDHH75 (expression plasmid for humanized PDHH type HFEF7A light chain) "and" a) First step of CPR "]. The DNA fragment LPDHM1, which encodes a portion of CDRLi, FRL2, CDRL2, FRL3, CDRL3 and FRL, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pHSGHM17, 200 ng; Oligonucleotide primer LPD1P, 80 pmolee; Oligonucleotide primer LPD3N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator lOxPfu, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes- After After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. After CPR, the LPD1, LPDHM1 and LPDC fragments of Amplified DNAs were first subjected to phenol extraction and then to ethanol precipitation, and then removed by electrophoreation on 5% w / v polyacrylamide gel. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the fusion DNA bands thus detected, under UV light, were extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon -10, as described above. The eluted DNA was concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. b) Second step of CPR. The sketch of the second step of PCR for the production of DNA-PDHM is shown in Figure 36. The DNA-LPDHM1.2, in which the above DNA-LPD1 and DNA-LPDHM1 fragments were fused, was prepared as follows. Composition of the PCR reaction solution: DNA-LPD1 solution (from the first step of PCR), 10 μl; DNA-LPDHM1 solution (from the first step of PCR), 10 μl; oligonucleotide primer 7AL1P, 80 pmoles; Oligonucleotide primer LPD3N, 80 pmoles; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and water redeetilated haeta a final volume of 200 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. After PCR, the amplified DNA-LPDHM1.2 fragment was first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophorea, the gel was stained with 1 μg / ml ethidium bromide and the DNA band thus detected, under UV light, was extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon-10. , as described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. c) Third step of CPR. The sketch of the third step of PCR for the preparation of DNA-LPDHM is shown in Figure 37. The DNA LPDHM fragment, comprising a fusion of the above DNA-LPDHM1.2 fragments and DNA-LPDC fragments, was prepared as follows. Composition of the PCR reaction solution: DNA-LPDHM1.2 solution (from the second PCR step), 10 μl; DNA-LPDC solution (from the first step of PCR), 10 μl; Oligonucleotide primer 7AL1P, 80 pmolee; Oligonucleotide primer 7ALCN, 80 pmolee; dNTP cocktail, 20 μl; pH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. After PCR, the amplified DNA-LPDHM fragment was first subjected to phenol extraction and then to ethanol precipitation, and then separated by electrophoresis in 5% w / v polyacrylamide gel. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA band thus detected, under UV light, was extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon- 10, as described above. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. The construction of a plasmid carrying the DNA LPDHM fragment is outlined in Figure 38. The DNA-LPDHM obtained before was further purified by extraction with phenol followed by precipitation with ethanol, and then digethion was carried out with the restriction enzymes HindIII and EcoRl. One μg of plasmid DNA from cloning pHSG399 was digested with the restriction enzymes HindIII and EcoRI, and then dephosphorylated with CIP. The resulting dephosphorylated pHSG399 DNA was ligated with the DNA LPDHM fragment, which had also previously been digested with Hind III and EcoRI, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). E.coli JM109 was then transformed with the ligation mixture and then plated on LB agar medium containing final concentrations of 1 mM IPTG, 0.1% w / v X-Gal and 50 μg / ml chloramphenicol. Any obtained white transformant was grown in 2 ml of liquid LB medium containing 50 μg / ml of chloramphenicol at 37 ° C overnight, and the plasmid DNA was extracted from the resulting culture by means of the alkaline SDS method. The extract was digested with Hind III and EcoRI, and a clone carrying the DNA-LPDHM fragment was selected by means of elect roforeey in 1% w / v agaroea gel. As a result of the previous procedure, the plasmid pHSHM2 carrying a fusion insert comprising the variable region of the light chain of HFE7A type humanized PDMM and DNA encoding the constant region of the chain K of human Ig. The transformant E. coli pHSHM2 SANK 70198, which hosts the plasmid pHSHM2, was deposited with Kogyo Gijutsuin Seimei-Kogaku Kogyo Gijutsu Kenkyujo on February 26, 1998, in accordance with the Budapest Treaty on the deposit of microorganisms, and was granted Accession number FERM BP-6272. Plasmid expression vector was constructed PLPDHM32, which carries the DNA of SEQ ID No. 108 of the sequence of eequencee, which encodes the light chain polypeptide of HFE7A type humanized PDHM of SEQ ID No. 109, using the plamidido pHSHM2 obtained above. One μg of pEE.12.1 (Lonza) DNA, an expiation vector for mammalian cells, was digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated using CIP. The resulting digested dephosphorylated plasmid DNA (100 ng) was ligated with 10 μg of the DNA pHSHM2 fragment, which had also been digested with Hind III and EcoRI, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd. ). The ligation mixture was then used to transform E. coli JM109 (as described above), which was seeded on plate after LB agar plate containing 50 μg / ml ampicillin. The transformants obtained by this method were cultured in 2 ml of liquid medium containing 50 μg / ml of ampicillin at 37 ° C overnight, and plaemidic DNA was extracted from the resulting culture by means of the alkaline SDS method. The extracted plasmid DNA was digested with HindIII and EcoRI, and subjected to electrophoresis in 1% w / v agaroea gel to confirm the presence or absence of the insert of interest.

This allowed the isolation of plasmid pLPDHM32, which contains a fusion fragment having the variable region of the humanized PDHM type HFE7A light chain together with DNA encoding the human immunoglobulin K chain congering region. It was found that the fusion fragment was located towards the 3 'end of the cytomegalovirus (CMV) promoter in the correct orientation. (4) Verification of nucleotide sequences To verify that the plasmid DNA inserts pLPDHH75 and pLPDHM32 have the desired nucleotide sequences, the DNA inserts were analyzed to determine their nucleotide sequences. The nucleotide primers used for nucleotide sequencing were SP1 (SEQ ID No. 68, SP2 (SEQ ID No. 69), SP3 (SEQ ID No. 70), SP4 (SEQ ID No. 71), SPS (SEQ ID No 72) and SP6 (SEQ ID No. 73) The position in which each primer is bound is shown in Figure 19. The determination of the nucleotide sequence was carried out by means of the chain termination method of diseoxynucleotide as mold the plamidid containing the sequence to be confirmed, having purified the plasmid by means of the alkaline SDS method and the cesium chloride method As expected, it was confirmed that pLPDHH75 has the nucleotide sequence of SEQ ID No. 106 of the listing of sequence, which encodes the polypeptide SEQ ID No. 107, and that pLPDHM32 has the nucleotide sequence of SEQ ID No. 108 of the sequence of sequences, which encodes the polypeptide of SEQ ID No. 109.

EXAMPLE 10 Preparation of APN encoding the humanized heavy chain (1) Construction of a vector for the heavy chain of the humanized version of the HFE7A antibody To further humanize the amino acid sequence of SEQ ID No. 75 of the sequence listing (the humanized heavy chain of the anti-human mouse anti-Fas antifungal, HFE7A ), amino acid 44 (arginine), and amino acid 76 (alanine) in the amino acid sequence of SEQ ID No. 75, were replaced with glycine and threonine, respectively; These residues are conserved in the human peedal chain. The reecuing sequence was designated as "the HV type". Plasmids of expression carrying the amino acid sequences of the humanized heavy chain type HV of the anti-human anti-Fas anti-Fas, HFE7A, were constructed as follows. (1) Synthesis of primers for preparing the variable region of the humanized heavy chain The DNA synthesis (SEQ ID No. 116 of the sequence listing) encoding the anti-humanized anti-Fas anti-chain heavy chain, HFE7A (SEQ ID No. 117) of the sequence listing), was performed using a combination of PCR step. In addition to 7AH1P (SEQ ID No: 76 above), the following 3 initiators were etintized for CPR.

'- CAGGCCCCTG GACAGGGCCT TGAGTGGATG-3' (HPD1P; SEQ ID No. 118); 5 '- CATCCACTCA AGGCCCTGTC CAGGGGCCTG -3' (HPD1N; SEQ ID No. 119); and 5 '- GCTGAGCTCC ATGTAGGCTG TGCTAGTGGA TGTGTCTAC -3' (HPD2N; SEQ ID No. 120). 2) Construction of the plamidido pgHPDHV3 The fragment HPD1.2 of DNA, which encodes amino acids Nos. -19 to +84 of SEQ ID No. 117, of the sequence listing, was prepared by performing two-step PCR, was inserted into a plasmid and then it was cloned in E. coli. a) First step of PCR In figure 39 the sketch of the first step of PCR for the preparation of DNA-HPD1.2 is shown. The DNA fragment HPD1, which encodes a secretion signal sequence and FRHi, CDRHi and a portion of FRH2 with a HindIII restriction enzyme cleavage site added at the 5 'end, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pgHSL7A62, 200 ng; Oligonucleotide initiator 7AH1P, 80 pmoles; Oligonucleotide primer HPD1N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator lOxPfu, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Water redeetilated haeta a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. The DNA fragment HPD2, which encodes a portion of FRH, CDRH3, and a portion of FRH3, was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pgHSL7A62, 200 ng; Oligonucleotide primer HPD1P, 80 pmoles; Oligonucleotide primer HPD2N, 80 pmoles; DNTP cocktail, 20 μl; PH regulator lOxPfu, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and Redistilled water to a final volume of 200 μl. The PCR reaction was carried out as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle at 94 ° C was repeated 30 times for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. After PCR, the amplified DNA fragments HPD1 and HPD2 were first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with 1 μg / ml of ethidium bromide and the DNA bands thus detected, under UV light, were extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon- 10, as described above. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. _ b) Second CPR pae "10 In figure 40 ee shows the sketch of the second step of PCR for the preparation of DNA-HPD1.2 The HPD1.2 fragment of DNA, in which DNA fragments HPD1 were fused and DNA HPD2, described above, was prepared as follows: Composition of the PCR reaction solution: DNA-HPD1 solution (from the first PCR step), 10 μl; DNA-HPD2 solution (from the first step of PCR) ), 10 μl, 7AH1P oligonucleotide primer, 80 pmoles, HPD2N oligonucleotide primer, 80 pmoles, 20 dNTP cocktail, 20 μl, Pfu lOx pH regulator, 20 μl, Pfu DNA polymerase (Stratagene), 10 units, and water redistilled to a final volume of 200 μl The PCR reaction was carried out as follows: First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle at 94 ° C was repeated 30 times during 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the The reaction was heated at 72 ° C for 10 minutes. Deepuée PCR, the amplified DNA-HPD1.2 fragment was first extracted with phenol and then subjected to ethanol precipitation, and then separated by polyacrylamide gel electrophoresis 5% w / v. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA band thus detected, under UV light, was extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon-10 , as described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. The construction of a plasmid carrying the fragment DNA HPD1.2, is sketched in Figure 41. The HPD1.2 fragment of DNA obtained above was further purified by phenol extraction followed by ethanol precipitation, and then digested with the restriction enzyme HindIII and Sacl. Next, 10 μg of the plasmid DNA pgHSL7A62 was digested with the restriction enzymes HindIII and Sacl, and dephosphorylated with CIP. The resulting dephosphorylated pgHSL7A62 DNA (100 ng) was ligated with 10 μg of DNA-HPD1.2, which had previously been digested with HindIII and Sacl, using a DNA ligation kit Version 2.0 (Takara Shuzo, Co. Ltd.) and the ligation mixture was cloned into E. coli JM109. Any resulting transformant was cultured in 2 ml of LB liquid medium, containing 50 μg / ml of chloramphenicol at 37 ° C overnight, and plasmid DNA was extracted from the culture by the alkaline SDS method. The extracted plasmid was digested with Hind III and Sacl, to confirm the presence or absence of the insert of interest, by means of electrophoresis in agarose gel 1% w / v. In this manner, the plasmid pgHPDHV3 was obtained, which carries a fusion insert comprising the DNA fragment encoding the variable region of the humanized Hv type Hv H7 heavy chain, and a genomic DNA fragment encoding the constant region of the heavy chain of human IgGl. The transforming E. coli pgHPDHV3 SANK 70298, which hosts the plasmid pgHPDH3, was depoeited with the Kogyo Gijuteuin Seimei-Kogaku Gijutsu Kenkyujo, on February 26, 1998, in accordance with the Budapest Treaty on the deposit of microorganisms, and was granted the access number FERM BP-6273. Ten micrograms of the plasmid DNA pgHPDHV3 thus obtained were digested with the restriction enzymes Hind III and EcoRl. In parallel, 1 μg of mammalian expression plasmid DNA pEE6.1 was digested with Hind III and EcoRI, and dephosphorylated with CIP. The resulting dephosphorylated pEE6.1 DNA (100 ng) was ligated with 10 μg of the digested pgHPDHV3 DNA, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.), and cloned into E. coli JM109.

Any resulting transformant was cultured in 2 ml of LB liquid medium containing 50 μg / ml ampicillin, at 37 ° C overnight, and the plasmid DNA was extracted from the culture by the alkaline SDS method. The plasmid was digested with Hind III and EcoRI, to confirm the presence or absence of the insert of interest by means of electrophoresis in 1% w / v agarose gel. In this manner, the plasmid pEgPDHV3-21 is obtained, which contains a fusion insert comprising the DNA fragment encoding the variable region of the heavy chain HFE7A humanized type HV and a genomic DNA fragment encoding the constant region of the heavy chain of human IgGl towards the 3 'end of the CMV promoter, and in the correct orientation. (3) Verification of the nucleotide sequence To verify that the DNA insert of the plasmid pEqPDHV3-21 had the desired nucleotide sequence, the DNA sample was analyzed to determine the nucleotide sequence. For this sequencing, the PEEF primers (SEQ ID No. 104), HPD1P (SEQ ID No. 1118), HPDIN (SEQ ID No. 119) and HPD2N (SEQ ID No. 120) were used. The positions in which the initiators are bound are shown in Figure 42. The determination of the nucleotide sequences was carried out by means of the dideoxynucleotide chain termination method, using the plasmids purified by the alkaline SDS method as templates. and the cesium chloride method, which contain the sequences for confirmation.

As expected, it was verified that pEgPDHV3-21 has the nucleotide sequence of SEQ ID No. 116 of the sequence listing, which encodes the polypeptide of SEQ ID No. 117.

EXAMPLE 11 Construction of high level expression vectors optimized for C0S-1 cells High level expression vectors, optimized for C0S-1 cells, were constructed using the vectors described above p7AL-HH, p7AL-HM, p7AL-MM, pLPDHH75, PLPDHM32, pEg7AH-H and pEgPDHV3-21. (1) High-level expression vectors for humanized light chains In Figure 43, the construction of high-level vectors for humanized light chains is outlined. 1) Synthesis of primers for preparing the SR alpha promoter DNA fragment The SR a promoter DNA fragment was synthesized for insertion into the expression vectors for humanized light chains using PCR. The following two oligonucleotide primers were synthesized by PCR: 5'-TGCACGCGTG GCTGTGGAAT GTGTGTCAGT TAG -3 '(MLUA: SEQ ID No. 121); and 5 '- TCCGAAGCTT TTAGAGCAGA AGTAACACTT C -3' (HINDB: SEQ ID No. 122). 2) Construction of plasmids After synthesis, the DNA fragment of the SR a promoter was inserted into the vectors p7AL-HH, p7AL-HM, P7AL-MM, pLPDHH75 or pLPDHM32, and then cloned in E. coli. A DNA LSR fragment was prepared, comprising the SR a promoter with a MulI restriction enzyme cleavage site added at the 5 'end, and a HindIII restriction enzyme cleavage site added at the 3' end, as follow. Composition of the PCR reaction solution: plasmid DNA pME18S, 200 ng; Oligonucleotide initiator MLUA, 80 pmoles; Oligonucleotide primer HINDB, 80 pmolee; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; DNA polymerase Pfu (Stratagene), 10 unidadee; and Water redeetilated haeta a final volume of 200 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated to 72 ° C for 10 minutes.

After PCR, the LSR fragment of amplified DNA was first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA band thus detected, under UV light, was extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon-10 , as described before. The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. One μg of plasmid DNA p7AL-HH, p7AL-HM, p7AL-MM, PLPDHH75 or pLPDHM32 was digested with restriction enzymes MulI and HindIII, and then dephosphorylated with CIP. The resulting dephosphorylated plasmid DNA (100 ng) was ligated with 10 μl of the solution containing the DNA LSR fragment, which had previously been digested with MulI and Hind III, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). E. coli JM109 was then transformed with the ligation mixture and seeded on plate after LB agar medium containing a final concentration of 50 μg / ml ampicillin. The traneformants obtained were cultured in 2 ml of liquid LB medium containing 50 μg / ml of ampicillin at 37 ° C overnight, and the plamidido DNA was extracted from the resulting culture by means of the alkaline SDS method. The extracted plasmid DNA was digested with MulI and Hind III, and a clone carrying the DNA-LSRoc fragment was selected by means of 1% w / v agarose gel electrophoresis. Following the above procedure, the plasmids and high level expression vectors pSRHH (type HH), pSRHM (type HM), pSRMM (type MM), pSRPDHH (type PDHH) and pSRPDHM (type PDHM) were obtained. (2) High-level expression vectors for humanized heavy chains Figure 44 outlines the construction of high-level expulsion vectors for humanized heavy chains. 1) Synthesis of initiation to prepare the DNA fragment of the SR promoter The DNA fragment of the SR ct promoter, for in vitro injection of expresion for humanized heavy chains, was synthesized using PCR. In addition to HINDB (SEQ ID No. 122), it was synthesized by RCP the next oligonucleotide primer. 5'- AAAGCGGCCG CTGCTAGCTT GGCTGTGGAA TGTGTG -3 '(NOTE: SEQ ID No. 123). 2) Plasmid construct The DNA fragment of the pR rom a SR was synthesized using PCR, inserted in the vector before the degradation, pEg7AH-H or pEgPDHV3-21, and then cloned into E. coli The HSR or DNA fragment, which comprises the p romote r SR ce with a Notl restriction enzyme cleavage site added at the 5 'end, and a HindIII restriction enzyme cleavage site added at the 3' end , it was prepared as follows. Composition of the PCR reaction solution: plasmid DNA pME18S, 200 ng; Oligonucleotide primer NOTE, 80 pmol; HINDB oligonucleotide primer, 80 pmoles; DNTP cocktail, 20 μl; PH regulator Pfu lOx, 20 μl; Pfu DNA polymerase (Stratagene), 10 units; and redispersed water haeta a final volume of 200 μl. The CPR reaction was performed as follows. First, the solution was heated at 94 ° C for 2 minutes, after which a heating cycle was repeated 30 times at 94 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 2 minutes. After finishing this procedure, the reaction solution was heated at 72 ° C for 10 minutes. After PCR, the HSR a fragment of amplified DNA was first subjected to phenol extraction and then to ethanol precipitation, and then separated by 5% w / v polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained with 1 μg / ml ethidium bromide and the DNA band thus detected, under UV light, was extracted with a safety razor blade and eluted from the gel using a Centriruter and Centricon-10 , as described before.

The eluted DNA was then concentrated by centrifugation at 7,500 x g, followed by ethanol precipitation, and finally dissolved in 50 μl of distilled water. One μg of plasmid DNA pEg7AH-H or pEgPDHV3-21 was then digested with the restriction enzymes Notl and HindIII, and then dephosphorylated with CIP. The resulting dephosphorylated plasmid DNA (100 ng) was ligated with 10 μl of the solution containing the HSR a fragment of DNA, which had also been previously digested with Notl and Hind III, using a DNA ligation kit Version 2.0 (Takara Shuzo Co., Ltd.). E. coli JM109 was then transformed with the ligation mixture and plated on LB agar medium containing final concentrations of 0.1 μM IPTG, 0.1% w / v X-Gal and 50 μg / ml ampicillin. The obtained white transplants were cultured in 2 ml of liquid LB medium containing 50 μg / ml of ampicillin at 37 ° C overnight, and the plasmid DNA was extracted from the resulting culture by the alkaline SDS method. The extracted plasmid DNA was digested with Notl and Hind III, and a clone carrying the DNA-HSRoc fragment was selected by means of 1% w / v agarose gel electrophoresis. Following the above procedure, high level expression vector plasmids pSRg7AH and pSRgPDH (type HV) were obtained.

EXAMPLE 12 Expression in C0S-1 cells Transfection of C0S-1 cells was performed with the high level expulsion plasmids for each of the humanized HEA7A heavye chains and for each of the humanized HFE7A light chains obtained above, by means of electroporation, in a manner similar to that described in Example 4. Cell COSe-1 haeta semiconfluence was de-coiled in a culture flask (culture area: 225 cm2) containing medium to (+) MEM supplemented with 10% FCS v / v. Afterwards, the medium was discarded and the C0S-1 cells were separated from the flask by means of treatment with 3 ml of trypsin-EDTA solution (Sigma Chemicals, Col.) at 37 βC for 3 minutes. The eeparadae cells were harvested after centrifugation at 800 r.p.m. for 2 minutes, and then washed twice with pH buffer PBS (-). The washed C0S-1 cells were then adjusted to a cell density of 1 x 10 8 cells / ml in pH buffer PBS (-) to produce a suspension of C0S-1 cells. In parallel, 4 μg of humanized HFE7A heavy chain expression plasmid DNA was mixed with 4 μg of humanized HFE7A light chain expression plasmid DNA, prepared by the alkaline SDS method and density gradient centrifugation. cesium chloride, and then precipitated with ethanol. before being suspended in 20 μl of pH regulator PBS (-), performing the whole operation in the same tube. All of the resulting plasmid suspension (20 μl) was mixed with 20 μl of the suspension of C0S-1 cells (2 x 106 cells) prepared above, and the mixture was transferred to a FCT-13 chamber (Shimadzu Seisakusyo, KK) that has a set of electrodes with 2 mm separation. This chamber was then loaded into the gene transfection apparatus GTE-1 (Shimadzu Seisakusyo, KK), and two pulses were applied, each of 600 V, 50 μF, applied with a second interval to transform the C0S-1 cells with the plasmid DNA of interest. After electroporation, the cell-DNA mixture in the chamber was suspended in 5 ml of a (+) MEM, supplemented with 10% FCS v / v, in a culture flask (culture area 25 cm2).; Sumitomo Bakelite) and incubated under 5% CO2 at 37 ° C for 72 hours. After this time, the culture supernatant was taken and analyzed for the expression products. Following the above method, transfected C0S-1 cells were obtained with each of the following plasmid combinations: (A) non-plasmid DNA; (B) cotransfection of pSRgPDH and pSRPDHH; (C) cotransfection of pSRgPDH and pSRPDHM; (D) cotransfection of pSRg7AH and pSRHH; (E) cotransfection of pSRg7AH and pSRHM; and (F) cotransfection of pSRg7AH and pSRMM.

EXAMPLE 13 Test for Fas binding activity The test for Fas binding activity in the cell culture supernatant fluids prepared in Example 12 was carried out by ELISA as follows. The culture supernatant of the C0S-1 cells expressing the human Fas fusion protein, as obtained in reference example 1 above, diluted 5 times with adsorption pH buffer, was dispensed into wells of a 96-well plate. wells (MaxisoF; Nunc) at 50 μl per well and the plate was incubated at 4 ° C overnight to allow adsorption of the human Fae fusion protein to the surface of the pozoe. Deepuée, each of the wells was washed 4 times with 350 μl of PBS-T. After washing, PBS-T containing 5% v / v BSA (bovine serum albumin; Wako Pur Chemical Industry, Ltd) was added to the wells at 50 μl per well and the plate was incubated at 37 ° C for 1 hour block the rest of the surface of each well. Afterwards, the wells were again washed four times with PBS-T. The culture supernatants obtained in Example 4 were adjusted to have a final product concentration of interest of 100 ng / ml in a (+) MEM containing 10% v / v FCS. The concentrations were estimated by the method described in Example 5. Each of the 100 ng / ml reagent solutions was then used to produce serial dilutions by serial two-fold dilution with oc (+) MEM containing 10% FCS. % v / v. Afterwards, 50 μl of each of the diluted samples was prepared in a prepared well as antecedent, and the plate was incubated at 37 ° C for two hours to allow the reaction. After this time, the wells were again washed 4 times with PBS-T and then 50 μl of goat anti-human polyclonal anti-IgG, labeled with alkaline phosphatase (Caltag Lab) diluted 10,000 times, was dispensed into each well. with PBS-T, and the reaction was allowed to proceed at 37 ° C for 2 hours. HFE7A purified from mouse HFE7A hybridoma was used as control (IgGl), and was detected using goat anti-IgG + IgA + IgM from goat mice, labeled with alkaline phosphatase, (Gibco BRL), diluted 5,000 times with PBS-T, in place of the goat polyclonal antiquase specific for human anti-IgG Fc, labeled with alkaline phosphatase. The wells were again washed with PBS-T, and then 50 μl of the substrate solution [p-nitrophenyl phosphate 1 mg / ml in 10% v / v diethanolamine (pH 9.8)] was dispensed into each well, and the plate was incubated at 37 ° C for 0.5 to 1 hour. The binding activity of the expression product contained in each culture supernatant fluid was evaluated with the human Fas fusion protein, reading the absorption of each well at 405 nm. As expected, binding activity was demonstrated for the human Fas fusion protein for the supernatants of categories (B), (C), (D), (E) and (F) above of Example 12, and is shown in figure 45.

EXAMPLE 14 Competitive inhibition of Fas binding activity of HFE7A Humanized anti-Fas antibodies obtained in the Example 12, they must inhibit the binding of HFE7A to Fas, since the anticuefos of this example were derived from HFE7A. Therefore, the ability of the expression products obtained in Example 12 to competitively inhibit the binding of HFE7A to the human Fas fusion protein was measured. The culture supernatant of C0S-1 cells containing the human Fas fusion protein, as obtained in Reference Example 1, was diluted 5 times with an adsorption pH regulator, and dispeneled in a 96 well plate. Wells for luminescence detection (Solid Luminescent Test Plate, high binding property, Costar) at 50 μl per well. Then, the plate was incubated at 4 ° C overnight to allow adsorption of the human Fas fusion protein to the surface of the wells. After this time, each well was washed 4 times with 350 μl of PBS-T, and then 100 μl of PBS-T containing 5% v / v BSA was added to each well, and the plate was incubated at 37 ° C. C for 1 hour to block the rest of the surface of each well. The wells were again washed four times with PBS-T. The culture supernatants obtained in Example 12 were adjusted to final anti-cotuex concentrations of 1 μg / ml in a (+) MEM containing 10% v / v FCS by the method of Example 5. Each of the resulting solutions of the expression products was used to produce serial dilutions by 2-fold dilution in eceie with oc (+) MEM containing 10% FCS v / v. AP-HFE7A was diluted to 50 ng / ml with a (+) MEM containing 10% FCS v / v, and 25 μl of the resulting solution was mixed with an equal volume of each of the prepared serial dilutions. Each of the wells was again washed four times with PBS-T, and then 50 μl of each of the resulting antiquake mixtures were added to the individual wells, and the plate was left at room temperature overnight. Subsequently, after washing each well with PBS-T again four times, 100 μl of CDP-star pH buffer (9.58 ml of diethanolamine, 0.2 g of magnesium chloride, 0.25 g of sodium azide, pH were dispensed into each well). 8.5), and the plate was left at room temperature for 10 minutes. After this time, the CDP-star pH regulator was discarded and CDP-star substrate [1.2 ml sapphire II (Tropix), 200 μl CDP-star (Tropix), c.e. for 12 μl with CDP-star pH regulator) at 50 μl per well, and after the plate was left at room temperature for an additional 40 minutes. The competitive inhibition of the expression products of Example 12 of the binding of HFE7A to the fusion protein of human Fas was evaluated, measuring the intensity of the luminescence with Luminoecan (Titertech). As expected, it was verified that each of the expression products of the supernatants (B), (C), (D), (E) and (F) obtained in example 12 above, specifically inhibited the binding of the anti-HFE7A antifluid to the fusion protein of human Fas. The results are shown in the figure 46.

EXAMPLE 15 Apoptosis Inducing Activity The apoptosis-inducing activity of the expression products in the supernatant fluids of the cultures obtained in example 12, was examined in a manner similar to that described in example 8. WR19L12a cells were cultured in RPMI1640 medium with 10% FCS. / v (Gibco BRL) at 37 ° C for 3 days under 5% v / v CO2, and 50 μl (lxlO5 cells) of the resulting culture were dispensed into each well from a 96-well microplate (Sumitomo Bakelite). The culture supernatants obtained in Example 12 were adjusted to a final anti-cotuene concentration of 100 ng / ml in RPMI1640 medium containing 10% v / v FCS. Concentrations were calculated by the method of Example 5. Each of the adjusted solutions of the expression products was used to produce serial dilutions by serial dilution two times with RPMI1640 containing 10% v / v FCS. Each of the resulting dilutions of each expression product solution was added to individual wells, at 50 μl per well; and the plate was incubated at 37 ° C for 1 hour. After this time, the cells in each well were washed once with RPMI1640 containing 10% v / v FCS, and then the washed cells were suspended in 100 μl per goat anti-human IgG-specific IgG polyclonal well, (Kappel), 0.5 μg / ml, in RPMI1640 containing FCS at 10% v / v. The plate was left at 37 ° C for 12 hours and then 50 μl of 25 μM PMS (phenazine methosulfonate, Sigma Chemical Co.), containing 1 mg / ml of internal salt XTT [2,3-bis] was added to each well. (2-methoxy-4-nitro-5-eulpho-phenyl) -2H-tetrazolium-5-caFoxianilide; Sigma Chemical Co,] a finale concentration of 333 μg / ml for XTT and 8.3 μM for PMS. The plate was incubated for 3 hours at 37 ° C, and the absorption at 450 nm of each well was measured to calculate the viability of the cells, using the reducing power of the mitochondria as an index. The viability of the cells in each well was calculated according to the following formula: viability (%) = 100 x (ab) / (cb) in which "a" is the absorption of a test well, "b" is the absorption of a well without cells, and "c" is the absorption of a well without an antiquake added. As expected, it was demonstrated that each of the expression products of the culture supernatant fluids (B), (C), (D) (E) and (F) obtained in Example 12 above, induce apoptosis in cells. T of eeta cell line of lymphoma expressing the human Fae antigen (figure 47).

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Sankyo Company, Limited (B) ADDRESS: 5-1, Nihonbashi Honcho 3-chome, Chuo-ku (C) CITY: Tokyo (E) COUNTRY: Japan (F) POSTAL CODE (ZIP): 103-8426 (G) TELEPHONE: 81-3-5255-7111 (ii) TITLE OF THE INVENTION: Anti-Fasc Antibody (iii) SEQUENCE NUMBER: 123 (iv) COMPUTER FORMAT : (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Rel ase # 1.0, Version # 1.30 (EPO) (vi) ) PREVIOUS APPLICATION INFORMATION: (A) APPLICATION NUMBER: JP Hei 9-82953 (B) SUBMISSION DATE: Ol-Apr-1997 (vi) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: JP Hei 9-169088 (B) DATE OF SUBMISSION: JUNE 25, 1997 (vi) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: JP Hei 9-276064 (B) DATE OF SUBMISSION: 08-0CT-1997 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1: Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys 1 .5 10 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) CHAIN CARTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Ser Tyr Trp Met Gln 1 5 (2) INF0RMACION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Glu lie Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly (2) INF0RMACI0N FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: Asn Arg Asp Tyr Ser Asn Asn Tf Tyr Phe Asp Val 1 5 10 (2) INF0RMACION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Lys Wing Ser Gln Ser Val Asp Tyr Asp Gly Aep Ser Tyr Met Asn 1 5 10 15 (2) INF0RMACION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Ala Ala Ser Asn Leu Glu Ser 1 5 (2) INF0RMACION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acid (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: eencilla (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: internal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Gln Gln Ser Asn Glu Asp Pro Arg Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1392 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA to mRNA (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (G) TYPE OF CELL: Hybridoma (H) CELLULAR LINE: HFE7A (ix) FEATURE: (A) NAME / KEY: CDS (B) L0CALIZACI0N-.1.3992 (ix) CHARACTERISTICS: (A) NAME / KEY: peptide mat (B) L0CALIZACI0N: 58..1392 (ix) FEATURE : (A) NAME / KEY: peptide sig__ (B) L0CALIZACI0N: 1..57 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCT ACA GGT 48 Met Gly Tf Ser Cye lie lie Leu Phe Leu Val Wing Thr Wing Thr Gly -19- 15 -10 -5 GTC CAT TCT CAG GTC CAA CTG CAG CAG CCT GGG GCT GAG CTT GTG AAG 96 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Wing Glu Leu Val Lys 1 5 10 CCT GGG GCT TCA GTG AAG CTG TCC TGC AAG GCT TCT GGC TAC ACC TTC 144 Pro Gly Wing Ser Val Lys Leu Ser Cys Lys Wing Ser Gly Tyr Thr Phe 15 20 25 10 ACC AGC TAC TGG ATG CAG TGG GTA AAA CAG AGG CCT GGA CAG GGC CTT 192 Thr Ser Tyr Tf Met Gln Tf Val Lys Gln Arg Pro Gly Gln Gly Leu 35 40 45"15 GAG TGG ATC GGA GAG ATT GAT CCT TCT GAT AGC TAT ACT AAC TAC AAT 240 Glu Tf lie Gly Glu lie Aep Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 50 55 60 CAA AAG TTC AAG GGC AAG GCC ACA TTG ACT GTA GAC ACA TCC TCC AGC 288 Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr Val Asp Thr Ser Ser Ser 65 70 75 25 ACA GCC TAC ATG CAG CTC AGC AGC CTG ACA TCT GAG GAC TCT GCG GTC 336 Thr Wing Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Wing Val 80 85 90 30 TAT TAC TGT GCA AGA AAT AGG GAC TAT AGT AAC AAC TGG TAC TTC GAT 384 Tyr Tyr Cys Wing Arg Asn Arg Asp Tyr Ser Asn Asn Tf Tyr Phe Asp 95 100 105 35 GTC TGG GGC ACA GGG ACC ACG GTC ACC GTC TCC TCA GCC AAA ACG ACA 432 Val Tf Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr 110 115 120 125 40 CCC CCA TCT GTC TAT CCA CTG GCC CCT GGA TCT GCT GCC CAA ACT AAC 480 Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn 130 135 140 45 TCC ATG GTG ACC CTG GGA TGC CTG GTC AAG GGC TAT TTC CCT GAG CCA 528 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 145 150 155 50 GTG ACA GTG ACC TGG AAC TCT GGA TCC CTG TCC AGC GGT GTG CAC ACC 576 Val Thr Val Thr Tf Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 160 165 170 TTC CCA GCT GTC CTG CAG TCT GAC CTC TAC ACT CTG AGC AGC TCA GTG 624 Phe Pro Wing Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 175 180 185 ACT GTC CCC TCC AGC ACC TGG CCC AGC CAG ACC GTC ACC TGC AAC GTT 672 Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val 190 195 200 205 GCC CAC CCG GCC AGC AGC ACC AAG GTG GAC AAG AAA ATT GTG CCC AGG 720 Wing His Pro Wing Ser Ser Thr Lys Val Asp Lys Lys lie Val Pro Arg 210 215 220 GAT TGT GGT TGT AAG CCT TGC ATA TGT ACA GTC CCA GAA GTA TCA TCT 768 Asp Cys Gly Cys Lys Pro Cys lie Cys Thr Val Pro Glu Val Ser Ser 225 230 235 GTC TTC ATC TTC CCC CCA AAG CCC AAG GAT GTG CTC ACC ATT ACT CTG 816 Val Phe lie Phe Pro Pro Lys Pro Lys Asp Val Leu Thr lie Thu Leu 240 245 250 ACT CCT AAG GTC ACG TGT GTT GTG GTA GAC ATC AGC AAG GAT GAT CCC 864 Thr Pro Lye Val Thr Cys Val Val Val Asp lie Ser Lye Asp Asp Pro 255 260 265 GAG GTC CAG TTC AGC TGG TTT GTA GAT GAT GTG GAG GTG CAC ACA GCT 912 Glu Val Gln Phe Ser Trp Phe Val Aep Aep Val Glu Val His Thr Ala 270 275 280 285 CAG ACG CAA CCC CGG GAG GAG CAG TTC AAC AGC ACT TTC CGC TCA GTC 960 Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val 290 295 300 AGT GAA CTT CCC ATC ATG CAC CAG AAC TGG CTC AAT GGC AAG GAG TTC 1008 Ser Glu Leu Pro lie Met His Gln Asn Tf Leu Asn Gly Lys Glu Phe 305 310 315 AAA TGC AGG GTC AAC AGT GCA GCT TTC CCT GCC CCC ATC GAG AAA ACC 1056 Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro lie Glu Lys Thr 320 325 330 ATC TCC AAA ACC AAA GGC AGA CCG AAG GCT CCA CAG GTG TAC ACC ATT 1104 lie Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr lie 335 340 345 CCA CCT CCC AAG GAG CAG ATG GCC AAG GAT AAA GTC AGT CTG ACC TGC 1152 Pro Pro Pro Lys Glu Gln Met Wing Lys Asp Lye Val Ser Leu Thr Cys 350 355 360 365 ATG ATA ACA GAC TTC TTC CCT GAA GAC ATT ACT GTG GAG TGG CAG TGG 1200 Met lie Thr Asp Phe Phe Pro Glu Asp He Thr Val Glu Trp Gln Tf 370 375 380 AAT GGG CAG CCA GCG GAG AAC TAC AAG AAC ACT CAG CCC ATC ATG AAC 1248 Asn Gly Gln Pro Wing Glu Asn Tyr Lys Asn Thr Gln Pro He Met Asn 385 390 395 ACG AAT GGC TCT TAC TTC GTC TAC AGC AAG CTC AAT GTG CAG AAG AGC 1296 Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser 400 405 410 AAC TGG GAG GCA GAT AAT ACT TTC ACC TGC TCT GTG TTA CAT GAG GGC 1344 Asn Tf Glu Wing Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly 415 420 425 CTG CAC AAC CAC CAT ACT GAG AAG AGC CTC TCC CAC TCT CCT GGT AAA 1392 Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 430 435 440 445 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 464 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Met Gly Tf Ser Cys He He Leu Phe Leu Val Wing Thr Wing Thr Gly -19 -15 -10 -5 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Wing Glu Leu Val Lys 1 5 10 Pro Gly Wing Ser Val Lys Leu Ser Cys Lys Wing Ser Gly Tyr Thr Phe 15 20 25 Thr Ser Tyr Tf Met Gln Tf Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 Glu Tf He Gly Glu He Asp Pro Being Asp Being Tyr Thr Asn Tyr Asn 50 55 60 Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr Val Asp Thr Ser Ser Ser 65 70 75 Thr Wing Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Wing Val 80 85 90 Tyr Tyr Cys Wing Arg Asn Arg Asp Tyr Ser Asn Asn Tf Tyr Phe Asp 95 100 105 Val Tf Gly Thr Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr 110 115 120 125 Pro Pro Ser Val Tyr Pro Leu Pro Wing Gly Wing Ala Wing Gln Thr Asn 130 135 140 Ser Met Val Thr Leu Gly Cys Leu Val Lye Gly Tyr Phe Pro Glu Pro 145 150 155 Val Thr Val Thr Tf Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 160 165 170 Phe Pro Wing Val Leu Gln Ser Asp Leu Tyr Thr Leu Being Ser Val 175 180 185 Thr Val Pro Ser Ser Thr Tf Pro Ser Gln Thr Val Thr Cys Asn Val 190 195 200 205 Ala His Pro Wing Being Ser Thr Lys Val Asp Lys Lys He Val Pro Arg 210 215 220 Asp Cys Gly Cys Lys Pro Cys He Cry Thr Val Pro Glu Val Ser Ser 225 230 235 Val Phe He Phe Pro Pro Lys Pro Lys Asp Val Leu Thr He Thr Leu 240 245 250 Thr Pro Lys Val Thr Cys Val Val Val Asp He Ser Lys Asp Asp Pro 255 260 265 Glu Val Gln Phe Ser Tf Phe Val Asp Asp Val Glu Val His Thr Ala 270 275 280 285 Gln Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val 290 295 300 Ser Glu Leu Pro He Met His Gln Asn Tf Leu Asn Gly Lys Glu Phe 305 310 315 Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro He Glu Lys Thr 320 325 330 He Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr He 335 340 345 Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys 350 355 360 365 Met He Thr Asp Phe Phe Pro Glu Asp He Thr Val Glu Tf Gln Tf 370 375 380 Asn Gly Gln Pro Wing Glu Asn Tyr Lys Asn Thr Gln Pro He Met Asn 385 390 395 Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser 400 405 410 Asn Tf Glu Wing Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly 415 420 425 Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 430 435 440 445 (2) INF0RMACION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 714 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (vi) ORIGINAL SOURCE: (A) ORGANISM: Mus musculus (G) TYPE OF CELL: Hybridoma (H) CELLULAR LINE: HFE7A (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION.l..714 (ix) FEATURE: (A) NAME / KEY: peptide mat (B) L0CALIZACI0N: 61..714 (ix) FEATURE: (A) NAME / KEY: peptide sig_ (B) LOCATION:! .. 60 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: ATG GAG ACA GAC ACA ATC CTG CTA TGG GTG ATG ATG CTC TGG ATT CCA 48 Met Glu Th r Asp Th r He Leu Leu T f Val Met Met Leu T f He P ro -20 -15 -10 -5 GGC TCC ACT GGT GAC ATT GTG CTG ACC CAA TCT CCA GCT TCT TTG GCT 96 Gly Ser Thr Gly Asp He Val Leu Thr Gln Ser Pro Wing Ser Leu Wing 1 5 10 GTG TCT CTA GGG CAG AGG GCC ACC ATC TCC TGC AAG GCC AGC CAA AGT 144 Val Ser Leu Gly Gln Arg Wing Thr He Ser Cys Lys Wing Ser Gln Ser 15 20 25 GTT GAT TAT GAT GGT GAT AGT TAT ATG AAC TGG TAC CAA CAG AAA CCA 192 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Tf Tyr Gln Gln Lys Pro 35 40 GGA CAG CCA CCC AAA CTC CTC ATC TAT GCT GCA TCC AAT CTA GAA TCT 240 Gly Gln Pro Pro Lys Leu Leu He Tyr Ala Wing Ser Asn Leu Glu Ser 45 50 55 60 GGG ATC CCA GCC AGG TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC 288 Gly He Pro Wing Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 CTC AAC ATC CAT CCT GTG GAG GAG GAT GCT GCA ACC TAT TAC TGT 336 Leu Asn He His Pro Val Glu Glu Glu Aep Wing Wing Thr Tyr Tyr Cye 80 85 90 CAG CAA AGT AAT GAG GAT CCT CGG ACG TTC GGT GGA GGC ACC AAG CTG 384 Gln Gln Ser Asn Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu 95 100 105 GAA ATC AAA CGG GCT GAT GCT GCA CCA ACT GTA TCC ATC TTC CCA CCA 432 Glu He Lys Arg Ala Asp Ala Wing Pro Thr Val Ser He Phe Pro Pro 110 115 120 TCC AGT GAG CAG TTA ACA TCT GGA GGT GCC TCA GTC GTG TGC TTC TTG 480 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 125 130 135 140 AAC AAC TTC TAC CCC AAA GAC ATC AAT GTC AAG TGG AAG ATT GAT GGC 528 Asn Asn Phe Tyr Pro Lys Asp He Asn Val Lys Tf Lys He Asp Gly 145 150 155 AGT GAA CGA CAA AAT GGC GTC CTG AAC AGT TGG ACT GAT CAG GAC AGC 576 Ser Glu Arg Gln Asn Gly Val Leu Aen Ser Tf Thr Aep Gln Asp Ser 160 165 170 AAA GAC AGC ACC TAC AGC ATG AGC AGC ACC CTC ACG TTG ACC AAG GAC 624 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 175 180 185 GAG TAT GAA CGA CAT AAC AGC TAT ACC TGT GAG GCC ACT CAC AAG ACA 672 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Wing Thr His Lys Thr 190 195 200 TCA ACT TCA CCC ATT GTC AAG AGC TTC AAC AGG AAT GAG TGT 714 Ser Thr Ser Pro He Val Lys Ser Phe Asn Arg Asn Glu Cys 205 210 215 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION FROM THE SEQUENCE: SEQ ID NO: 11: Met Glu Th r Asp Th r He Leu Leu T rp Val Met Met Leu T f He P ro -20 -15 -10 -5 Gly Se r Th r Gly Asp He Val Leu Th r Gln Se r P ro Ala Se r Leu Ala 1 5 10 Val Se r Leu Gly Gln Arg Ala Th r He Se r Cys Lys Ala Se r Gln Se r 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 30 35 40 Gly Gln Pro Pro Lys Leu Leu He Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly He Pro Wing Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Asn He His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 80 85 90 Gln Gln Ser Asn Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu 95 100 105 Glu He Lys Arg Ala Asp Ala Ala Pro Thr Val Ser He Phe Pro Pro 110 115 120 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 125 130 135 140 Asn Aen Phe Tyr Pro Lye Aep He Aen Val Lye Tf Lye He Aep Gly 145 150 155 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Tf Thr Asp Gln Asp Ser 160 165 170 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 175 180 185 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 190 195 200 Ser Thr Ser Pro He Val Lys Ser Phe Aen Arg Aen Glu Cye 205 210 215 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 paree de baeee (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA eintético" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: GGGGAATTCC AGTACGGAGT TGGGGAAGCT CTTT 34 (2) INF0RMACI0N FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iü) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: GTTTCTTCTG CCTCTGTCAC CAAGTTAGAT CTGGA 35 (2) INF0RMACION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 pairs of baees (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: TCCAGATCTA ACTTGGTGAC AGAGGCAGAA GAAAC 35 (2) INF0RMACION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: CCCTCTAGAC GCGTCACGTG GGCATCAC 28 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) CHAIN CARTER: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: Gln Xaa Gln Leu Gln Gln Pro Gly Ala Glu Leu 1 5 10 (2) INF0RMACION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (v) TYPE OF FRAGMENT: N-terminal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: Asp He Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr He Ser 20 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GACCTCACCA TGGGATGGA 19 (2) INF0RMACION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: TTTACCAGGA GAGTGGGAGA 20 (2) INF0RMACION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: AAGAAGCATC CTCTCATCTA 20 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CARTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: ACACTCATTC CTGTTGAAGC 20 (2) INF0RMACION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid 10 (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" • 15 (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No 20 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: GGGGAATTCG ACCTCACCAT GGGATGGA 28 25 (2) INF0RMACION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: 30 (A) LENGTH: 32 pairs of base (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: eincilla (D) TOPOLOGY: linear 35 (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec = "DNA eintético" (iii) HYPOTHETICAL: No 40 (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: 45 GGGTCTAGAC TATTTACCAG GAGAGTGGGA GA 32 (2) INFORMATION FOR SEQ ID NO: 24: 50 (ij SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: GGGGAATTCA AGAAGCATCC TCTCATCTA 29 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: GGGGCGGCCG CTTACTAACA CTCATTCCTG TTGAAGC 37 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp He Aen Ser 1 5 10 15 Lys Gly Leu (2) INF0RMACION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27: Val Thr Asp He Asn Ser Lys Gly Leu Glu Leu Arg Lys Thr Val Thr 1 5 10 15 Thr Val Glu (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28: Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly 1 5 10 15 Leu His His Asp 20 (2) INF0RMACION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHAIN CARTER: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29: Thr Gln Asn Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys 1 5 10 15 Pro Cys Pro Pro 20 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHAIN CARTER: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30: Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala Arg 1 5 10 15 Asp Cys Thr Val 20 (2) INF0RMACI0N FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31: Gly Glu A rg Lys Wing A rg Asp Cys Th r Val Asn Gly Asp Glu P ro Asp 1 5 10 15 Cye Val Pro Cys Gln 20 (2) INF0RMACION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32: Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr 1 5 10 15 Thr Asp Lys Wing 20 (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (Xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 33: Glu Gly Lys Glu Tyr Thr Asp Lys Wing His Phe Ser Ser Lys Cys Arg 1 5 10 15 Arg Cys Arg (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (Ü) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34: His Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His 1 5 10 15 Gly Leu Glu Val 20 (2) INFORMATION FOR SEQ ID NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35: Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu He Asn Cys Thr Arg 1 5 10 15 Thr Gln Asn Thr 20 (2) INF0RMACI0N FOR SEQ ID NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 36: Glu He Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro 1 5 10 15 Asn Phe Phe Cys 20 (2) INFORMATION FOR SEQ ID NO: 37: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 37: Lye Cye Arg Cye Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu 1 5 10 15 His Cys Asp Pro 20 (2) INFORMATION FOR SEQ ID NO: 38: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 38: Aen Ser Thr Val Cye Glu His Cys Asp Pro Cys Thr Lys Cys Glu His 1 5 10 15 Gly He He Lys 20 (2) INFORMATION FOR SEQ ID NO: 39: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39: Cys Thr Lys Cys Glu His Gly He He Lys Glu Cys Thr Leu Thr Ser 1 5 10 15 Asn Thr Lys Cys 20 (2) INF0RMACION FOR SEQ ID NO: 40: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 40: Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg 1 5 10 15 Ser Asn (2) INFORMATION FOR SEQ ID NO: 41: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 41: Ser Gly Lys Tyr Glu Gly Gly Asn He Tyr Thr Lys Lys Glu Ala 1 5 10 15 Phe Asn Val Glu 20 (2) INFORMATION FOR SEQ ID NO: 42: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 42: His Gly Leu Glu Val Glu He Aen Cye Thr 1 5 10 (2) INF0RMACION FOR SEQ ID NO: 43: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 43: Glu He Asn Cys Thr Arg Thr Gln Asn Thr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 44: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 44: Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 45: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 45: Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 1 5 10 (2) INF0RMACION FOR SEQ ID NO: 46: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 46: Gly Lys He Ala Ser Cys Leu Asn Asp Aen 1 5 10 (2) INF0RMACI0N FOR SEQ ID NO: 47: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 paree de baeee (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 47: GCGAATTCTG CCTTGACTGA TCAGAGTTTC CTCA 34 (2) INF0RMACION FOR SEQ ID NO: 48: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc "Synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 48: GCTCTAGATG AGGTGAAAGA TGAGCTGGAG GA 32 (2) INF0RMACION FOR SEQ ID NO: 49: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 768 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: CDS (B) L0CALIZACION: 40..753 (ix) CHARACTERISTIC: (A) NAME / KEY: peptide mat (B) L0CALIZACI0N: 100..753 (ix) FEATURE: (A) NAME / KEY: sig peptide (B) L0CALIZACI0N: 40..99 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 49: CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCTCAGAG ATG GAG ACA GAC ACA 54 Met Glu Thr Asp Thr -20 ATC CTG CTA TGG GTG CTG CTG CTC TGG GTT CCA GGC TCC ACT GGT GAC 102 He Leu Leu Tf Val Leu Leu Leu Tf Val Pro Gly Ser Thr Gly Asp -15 -10 -5 1 ATT GTG CTC ACC CAA TCT CCA GGT ACT TTG TCT CTG TCT CCA GGG GAG 150 He Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 5 10 15 AGG GCC ACC CTC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT 198 rg Wing Thr Leu Ser Cys Lys Wing Ser Gln Ser Val Asp Tyr Asp Gly 20 25 30 GAT AGT TAT ATG AAC TGG TAC CAA CAG AAA CCA GGA CAG GCA CCC AGA 246 Asp Se r Ty r Met Asn Tf Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu He Tyr Ala Wing As As Leu Glu Se r Gly He P ro Asp A rg 50 55 60 65 TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC ACC ATC TCT CGT 342 Phe S r Gly Ser Gly Ser Gly Th r Asp Phe Th r Leu Th r He Se r A rg 70 75 80 CTG GAG CCG GCG GAT TTT GCA GTC TAT TAC TGT CAG CAA AGT AAT GAG 390 Leu Glu P ro Wing Asp Phe Wing Val Tyr Tyr Cys Gln Gln Ser Asn Glu 85 90 95 GAT CCT CGG ACG TTC GGT CAA GGC ACC AGG CTG GAA ATC AAA CGG ACT 438 Asp P ro A rg Th r Phe Gly Gln Gly Th r A rg Leu Glu He Lys A r g Th r 100 105 110 GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG 486 Val Ala Ala P ro Se Val Phe He Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC 534 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 145 AGA GAG GCC AAA GTA CAG TGG AAA GTG GAT AAC GCC CTC CAA TCG GGT Arg Glu Ala Lys Val Gln Tf Lys Val Asp Asn Ala Leu Gln Ser Gly 150 155 160 AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC 630 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GAC GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His 180 185 190 AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC 726 Lys Val Ty.r Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 ACA AAG AGC TTC AAC AGG GGA TGT TAGTAAGAAT TCGGG 768 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 (2) INF0RMACION FOR SEQ ID NO: 50: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 50: Met Glu Thr Asp Thr He Leu Leu Trp Val Leu Leu Leu Tf Val Pro -20 -15 -10 -5 -5 Gly Ser Th r Gly Asp He Val Leu Th r Gln Se r P ro Gly Th r Leu Se r 1 5 10 Leu Se r P ro Gly Glu A rg Ala Th r Leu Se r. Cys Lys Ala Se r Gln Se r 20 25 Val Asp Ty r Asp Gly Aep Ser Ty r Met Asn T f Ty r Gln Gln Lys Pro 30 35 40 Gly Gln Wing P ro A rg Leu Leu He Tyr Wing Ala Se r Asn Leu Glu Se r 45 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Thr He Ser Arg Leu Glu Pro Wing Asp Phe Wing Val Tyr Tyr Cys 80 85 90 Gln Gln Ser Asn Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Arg Leu 95 100 105 Glu He Lys Arg Thr Val Wing Ala Pro Ser Val Phe He Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 125 130 135 140 Asn Asn Phe Tyr Pro Arg Glu Wing Lys Val Gln Trp Lys Val Asp Asn 145 150 155 Wing Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 160 165 170 Lye Aep Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lye Wing 175 180 185 Asp Tyr Glu Lys His Lys Val Tyr Wing Cys Glu Val Thr His Gln Gly 190 195 200 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 215 (2) INFORMATION FOR SEQ ID NO: 51: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 768 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) L0CALIZACI0N: 40..753 (ix) CHARACTERISTICS: (A) NAME / KEY: peptide mat.

(B) L0CALIZACI0N: 100..753 (ix) CHARACTERISTICS: (A) NAME / KEY: sig peptide "_ (B) L0CALIZACI0N: 40..99 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 51: CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCTCAGAG ATG GAG ACA GAC ACA 54 Met Glu Thr Asp Thr -20 ATC CTG CTA TGG GTG CTG CTG CTC TGG GTT CCA GGC TCC ACT GGT GAC 102 He Leu Leu Tf Val Leu Leu Leu Tf Val Pro Gly Ser Thr Gly Asp -15 -10 -5 1 ATT GTG CTC ACC CAA TCT CCA GGT ACT TTG TCT CTG TCT CCA GGG GAG 150 He Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 5 10 15 AGG GCC ACC CTC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT 198 Arg Wing Thr Leu Ser Cys Lys Wing Ser Gln Ser Val Asp Tyr Asp Gly 20 25 30 GAT AGT TAT ATG AAC TGG TAC CAA CAG AAA CCA GGA CAG GCA CCC AGA 246 Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu He Tyr Ala Wing Ser Asn Leu Glu Ser Gly He Pro Asp Arg 50 55 60 65 TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC ACC ATC CAT CCT 342 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He His Pro 70 75 80 GTG GAG GAG GAG GAT GCT GCA ACC TAT TAC TGT CAG CAA AGT AAT GAG 390 Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu 85 90 95 GAT CCT CGG ACG TTC GGT CAA GGC ACC AGG CTG GAA ATC AAA CGG ACT 438 Asp Pro Arg Thr Phe Gly Gln Gly Thr Arg Leu Glu He Lys Arg Thr 100 105 110 GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG 486 Val Ala Wing Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC 534 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 145 AGA GAG GCC AAA GTA CAG TGG AAA GTG GAT AAC GCC CTC CAA TCG GGT 582 Arg Glu Ala Lys Val Gln Tf Lys Val Asp Asn Ala Leu Gln Ser Gly 150 155 160 AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC 630 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GAC GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His 180 185 190 AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC 726 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 ACA AAG AGC TTC AAC AGG GGA GAG TGT TAGTAAGAAT TCGGG 768 thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 (2) INFORMATION FOR SEQ ID NO: 52: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 52: Met Glu Thr Asp Thr He Leu Leu Tf Val Leu Leu Leu Tf Val Pro -20 -15 -10 -5 Gly Ser Thr Gly Asp He Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 1 5 10 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Tf Tyr Gln Gln Lys Pro 35 40 Gly Gln Wing Pro Arg Leu Leu He Tyr Wing Wing Being Asn Leu Glu Being 45 50 55 60 Gly He Pro Asp rg Phe Ser Gly Ser Gly Be Gly Thr Asp Phe Thr 65 70 75 Leu Thr He His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 80 85 90 Gln Gln Ser Asn Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Arg Leu 95 100 105 Glu He Lys Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 125 130 135 140 Asn Asn Phe Tyr Pro Arg Glu Wing Lys Val Gln Tf Lys Val Asp Asn 145 150 155 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 160 165 170 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing 175 180 185 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 190 195 200 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cye 205 210 215 (2) INFORMATION FOR SEQ ID NO: 53: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 768 paree of bases (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) L0CALIZACION: 40..753 (ix) CHARACTERISTICS: (A) NAME / KEY: peptide mat. "(B) L0CALIZACI0N: 100..753 (ix) CHARACTERISTICS: (A) NAME / KEY: peptide eig "" (B) L0CALIZACI0N: 40..99 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 53: CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCTCAGAG ATG GAG ACA GAC ACA 54 Met Glu Thr Aep Thr -20 ATC CTG CTA TGG GTG CTG CTG CTC TGG GTT CCA GGC TCC ACT GGT GAC 102 He Leu Leu Tf Val Leu Leu Leu Tf Val Pro Gly Ser Thr Gly Asp -15 -10 -5 1 ATT GTG CTC ACC CAA TCT CCA GGT ACT TTG TCT CTG TCT CCA GGG GAG 150 He Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 5 10 15 AGG GCC ACC CTC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT 198 Arg Wing Thr Leu Ser Cys Lys Wing Ser Gln Ser Val Asp Tyr Asp Gly 20 25 30 GAT AGT TAT ATG AAC TGG TAC CAA CAG AAA CCA GGA CAG CCA CCC AAA 246 Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu He Tyr Ala Ala Ser Asn Leu Glu Ser Gly He Pro Asp Arg 50 55 60 65 TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC ACC ATC CAT CCT 342 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He His Pro 70 75 80 GTG GAG GAG GAT GAT GCT GCA ACC TAT TAC TGT CAG CAA AGT AAT GAG 390 Val Glu Glu Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Gln Ser Asn Glu 85 90 95 GAT CCT CGG ACG TTC GGT CAA GGC ACC AGG CTG GAA ATC AAA CGG ACT 438 Asp Pro Arg Thr Phe Gly Gln Gly Thr Arg Leu Glu He Lys Arg Thr 100 105 110 GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG 486 Val Ala Wing Pro Ser Val Phe He Phe Pro Pro Be Asp Glu Gln Leu 115 120 125 AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC 534 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 145 AGA GAG GCC AAA GTA CAG TGG AAA GTG GAT AAC GCC CTC CAA TCG GGT 582 Arg Glu Ala Lys Val Gln Tf Lys Val Asp Asn Ala Leu Gln Ser Gly 150 155 160 AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC 630 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GAC GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His 180 185 190 AAA GTC TAC GCC TGC GAA GTC ACC CAG GGC CTG AGC TCG CCC GTC 726 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 ACA AAG AGC TTC AAC AGG GGA GAG TGT TAGTAAGAAT TCGGG 768 Thr Lys Ser Phe Aan Arg Gly Glu Cye 210 215 (2) INFOMACITY FOR SEQ ID NO: 54: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 54: Met Glu Thr Asp Thr He Leu Leu Trp Val Leu Leu Leu Tf Val Pro -20 -15 -10 -5 Gly Ser Thr Gly Asp He Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 1 5 10 Leu Ser Pro Gly Glu Arg Wing Thr Leu Ser Cys Lys Wing Being Gln Ser 15 20 25 Val Aep Tyr Aep Gly Asp Being Tyr Met Asn Trp Tyr Gln Gln Lye Pro 30 35 40 Gly Gln Pro Pro Lys Leu Leu He Tyr Wing Wing Being Asn Leu Glu Being 45 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 '70 75 Leu Thr He His Pro Val Glu Glu Glu Asp Wing Wing Thr Tyr Tyr Cys 80 85 90 Gln Gln Ser Asn Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Arg Leu 95 100 105 Glu He Lys Arg Thr Val Wing Ala Pro Ser Val Phe He Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys Ser Gly Thr Wing Ser Val Val Cys Leu Leu 125 130 135 140 Asn Aen Phe Tyr Pro Arg Glu Wing Lye Val Gln Tf Lye Val Asp Asn 145 150 155 Wing Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 160 165 170 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing 175 180 185 Asp Tyr Glu Lys His Lys Val Tyr Wing Cys Glu Val Thr His Gln Gly 190 195 200 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 215 (2) INF0RMACION FOR SEQ ID NO: 55: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 pairs of base (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: eingle (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec = "Synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 55: CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCT 34 (2) INFORMATION FOR SEQ ID NO: 56: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 pairs of base (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 56: GAGAGGGTGG CCCTCTCCCC TGGAGACAGA GACAAAGTAC CTGG 44 (2) INF0RMACION FOR SEQ ID NO: 57: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 57: CCAGGTACTT TGTCTCTGTC TCCAGGGGAG AGGGCCACCC TCTC 44 (2) INFORMATION FOR SEQ ID NO: 58: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: eincilla (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec = "Synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 58: GATTCGAGAT TGGATGCAGC ATAGATGAGG AGTCTGGGTG CCTG 44 (2) INFORMATION FOR SEQ ID NO: 59: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 59: GCTGCATCCA ATCTCGAATC TGGGATCCCA GACAGGTTTA GTGGC 45 (2) INF0RMACION FOR SEQ ID NO: 60: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 60: AAAATCCGCC GGCTCCAGAC GAGAGATGGT GAGGGTGAAG TCTGTCCCAG AC 52 (2) INF0RMACION FOR SEQ ID NO: 61: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 58 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA eintético" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 61: CTCGTCTGGA GCCGGCGGAT TTTGCAGTCT ATTACTGTCA GCAAAGTAAT GAGGATCC 58 (2) INFORMATION FOR SEQ ID NO: 62: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 62: TGAAGACAGA TGGTGCAGCC ACAGTCCGTT TGATTTCCAG CCTGGTGCCT TGACC 55 (2) INF0RMACION FOR SEQ ID NO: 63: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 63: GGTCAAGGCA CCAGGCTGGA AATCAAACGG ACTGTGGCTG CACCATCTGT CTTCA 55 (2) INF0RMACION FOR SEQ ID NO: 64: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) CHAIN CARTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 64: CCCGAATTCT TACTAACACT CTCCCCTGTT GAAGCTCTTT GTGAC 45 (2) INFORMATION FOR SEQ ID NO: 65: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 65: TCTGTCCCAG ACCCACTGCC ACTAAACCTG TCTGGGATCC CAGATTCGAG ATTGG 55 (2) INF0RMACION FOR SEQ ID NO: 66: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 66: GTTTAGTGGC AGTGGGTCTG GGACAGACTT CACCTCTACC ATCCATCCTG TGGAG 55 (2) INF0RMACION FOR SEQ ID NO: 67: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 67: ATGGTGCAGC CACAGTCCGT TTGATTTCCA GCCTGGTGCC TTGACCGAAC GTCCG 55 (2) INFORMATION FOR SEQ ID NO: 68: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CARTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 68: CCCAAGCTTA AGAAGCATCC 20 (2) INF0RMACI0N FOR SEQ ID NO: 69: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: eingle (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec = "DNA eintético" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 69: ATCTATGCTG CATCCAATCT 20 (2) INF0RMACION FOR SEQ ID NO: 70: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 70: GTTGTGTGCC TGCTGAATAA 20 (2) INF0RMACION FOR SEQ ID NO: 71: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 71: CCCGAATTCT TACTAACACT 20 (2) INF0RMACION FOR SEQ ID NO: 72: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 72: TTATTCAGCA GGCACACAAC 20 (2) INF0RMACION FOR SEQ ID NO: 73: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 73: AGATTGGATG CAGCATAGAT 20 (2) INF0RMACION FOR SEQ ID NO: 74: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 457 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: CDS (B) L0CALIZACION: 21..455 (ix) CHARACTERISTICS: (A) NAME / KEY: mat peptide "(B) L0CALIZACI0N: 78..455 (ix) FEATURE : (A) NAME / KEY: sig_ peptide (B) L0CALIZACI0N: 21..77 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 74: AAGCTTGGCT TGACCTCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG 50 Met Gly Tf Ser Cys He He Leu Phe Leu -19 -15 -10 GTA GCA ACA GCT ACA GGT GTC CAC TCT CAG GTC CAA CTG GTG CAG TCT 98 Val Ala Thr Ala Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser -5 1 5 GGG GCT GAG GTC AAG AAG CCT GGG GCT TCA GTG AAG GTG TCC TGC AAG 146 Gly Wing Glu Val Lys Lys Pro Gly Wing Ser Val Lys Val Ser Cys Lys 10 15 20 GCT TCT GGC TAC ACC TTC ACC AGC TAC TGG ATG CAG TGG GTA AAA CAG 194 Wing Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Tf Val Lys Gln 30 35 GCC CCT GGA CAG AGG CTT GAG TGG ATG GGA GAT ATT GAT CCT TCT GAT 242 Wing Pro Gly Gln Arg Leu Glu Tf Met Gly Glu He Asp Pro As Asp 40 45 50 55 AGC TAT ACT AAC TAC AAT CAA AAG TTC AAG GGC AAG GCC ACA TTG ACT 290 Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr 60 65 70 GTA GAC ACA TCC GCT AGC ACA GCC TAC ATG GAG CTC AGC AGC CTG AGA 338 Val Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg 75 80 85 TCT GAG GAC ACG GCG GTC TAT TAC TGT GCA AGA AAT AGG GAC TAT AGT 386 Ser Glu Asp Thr Wing Val Tyr Tyr Cys Wing Arg Asn Arg Asp Tyr Ser 90 95 100 AAC AAC TGG TAC TTC GAT GTC TGG GGC GAA GGG ACC CTG GTC ACC GTC 434 Asn Asn Tf Tyr Phe Asp Val Tf Gly Glu Gly Thr Leu Val Thr Val 105 110 115 TCC TCA GCC TCC ACC AAG GGC CC 457 Ser Ser Wing Ser Thr Lys Gly 120 125 (2) INFORMATION FOR SEQ ID NO: 75: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 145 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION FROM THE SEQUENCE: SEQ ID NO: 75: Met Gly Tf Ser Cys He He Leu Phe Leu Val Wing Thr Wing Thr Gly -19 -15 -10 -5 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys 1 5 10 Pro Gly Wing Ser Val Lys Val Ser Cye Lys Wing Ser Gly Tyr Thr Phe 15 20 25 Thr Ser Tyr Tf Met Gln Tf Val Lys Gln Wing Pro Gly Gln Arg Leu 30 35 40 45 Glu Tf Met Gly Glu He Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 50 55 60 Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr Val Asp Thr Ser Wing Ser 65 70 75 Thr Wing Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Wing Val 80 85 90 Tyr Tyr Cys Wing Arg Asn Arg Asp Tyr Ser Asn Asn Tf Tyr Phe Asp 95 100 105 Val Tf Gly Glu Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys .0 115 120 125 Gly (2) INF0RMACI0N FOR SEQ ID NO: 76: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 76: GGGAAGCTTG GCTTGACCTC ACCATGGGAT GGAGCTGTAT 40 (2) INFORMATION FOR SEQ ID NO: 77: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 77: TGAAGCCCCA GGCTTCTTGA CCTCAGCCCC AGACTGCACC AGTTGGAC 48 (2) INF0RMACION FOR SEQ ID NO: 78: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 78: TCCACTCAAG CCTCTGTCCA GGGGCCTGTT TTACCC 36 (2) INFORMATION FOR SEQ ID NO: 79: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 52 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 79: GTCTGGGGCT GAGGTCAAGA AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA AG 52 (2) INFORMATION FOR SEQ ID NO: 80: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 80: CAGGCCCCTG GACAGAGGCT TGAGTGGATG GGAGAGATT 39 (2) INF0RMACI0N FOR SEQ ID NO: 81: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA eintético" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 81: TCAGATCTCA GGCTGCTGAG CTCCATGTAG GCTGTGCTAG CGGATGTGTC 50 (2) INF0RMACI0N FOR SEQ ID NO: 82: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 82: TGGAGCTCAG CAGCCTGAGA TCTGAGGACA CGGCGGTCTA TTAC 44 (2) INF0RMACION FOR SEQ ID NO: 83: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 83: GATGGGCCCT TGGTGGAGGC TGAGGAGACG GTGACCAGGG TCCCTTCGCC CCAGT 55 (2) INF0RMACION FOR SEQ ID NO: 84: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs ( B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 84: GGGAAGCTTC CGCGGTCACA TGGCACCACC TCTCTTGCA 39 (2) INFORMATION FOR SEQ ID NO: 85: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iü) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 85: GCTCTGCAGA GAGAAGATTG GGAGTTACTG GAATC 35 (2) INF0RMACI0N FOR SEQ ID NO: 86: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 86: TCTCTGCAGA GCCCAAATCT TGTGACAAAA CTCAC 35 (2) INFORMATION FOR SEQ ID NO: 87: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 87: GGGGAATTCG GGAGCGGGGC TTGCCGGCCG TCGCACTCA 39 (2) INF0RMACI0N FOR SEQ ID NO: 88: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2077 pairs of baeee (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTICS: (A) NAME / KEY: sig peptide "_ (B) L0CALIZACI0N: 27..83 (ix) ) CHARACTERISTICS: (A) NAME / KEY: int ron (B) LOCATION: 741..1131 (ix) CHARACTERISTICS: (A) NAME / KEY: int ron (B) LOCATION: 1177..1294 (ix) FEATURE: (A) NAME / KEY: intron (B) LOCATION: 1625..1721 (ix) FEATURE: (A) NAME / KEY: exon (B) L0CALIZACI0N: 27..740 (ix) CHARACTERISTIC: (A) NAME / KEY: exon (B) L0CALIZACI0N.-1132..1176 (ix) FEATURE: (A) NAME / KEY: exon (B) LOCATION: 1295..1624 (ix) FEATURE: (A) NAME / KEY: exon (B) LOCATION: 1722..2077 (ix) CHARACTERISTICS: (A) NAME / KEY: peptide mat (B) L0CALIZACI0N: union (84..740, 1132..1176, 1295..1624, 1722..2042) (ix) FEATURE: (A) NAME / KEY: CDS (B) L0CALIZACI0N: union (27..740, 1132..1176, 1295..1624 , 1722..2042) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 88: GGGCGAAAGC TTGGCTTGAC CTCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC 53 Met Gly Tf Ser Cys He lie Leu Phe -19-15 TTG GTA GCA ACA GCT ACA GGT GTC CAC TCT CAG GTC CAA CTG GTG CAG 101 Leu Val Ala Thr Ala Thr Gly Val Hie Ser Gln Val Gln Leu Val Gln -10 -5 1 5 TCT GGG GCT GAG GTC AAG AAG CCT GGG GCT TCA GTG AAG GTG TCC TGC 149 Ser Gly Wing Glu Val Lye Lys Pro Gly Wing Ser Val Lys Val Ser Cys 10 15 20 AAG GCT TCT GGC TAC ACC TTC ACC AGC TAC TGG ATG CAG TGG GTA AAA 197 Lye Wing S r Gly Tyr Thr Phe Thr Ser Tyr Tf Met Gln Tf Val Lys 25 30 35 CAG GCC CCT GGA CAG AGG CTT GAG TGG ATG GGA GAG ATT GAT CCT TCT 245 Gln Ala Pro Gly Gln Arg Leu Glu Tf Met Gly Glu He Asp Pro Ser 40 45 50 GAT AGC TAT ACT AAC TAC AAT CAA AAG TTC AAG GGC AAG GCC ACA TTG 293 Asp Ser Tyr Thr Aen Tyr Asn Gln Lye Phe Lys Gly Lys Wing Thr Leu 55 60 65 70 ACT GTA GAC ACA TCC GCT AGC ACA GCC TAC ATG GAG CTC AGC AGC CTG 341 Thr Val Asp Thr Ser Wing Ser Thr Wing Tyr Met Glu Leu Ser Ser Leu 75 80 85 AGA TCT GAG GAC ACG GCG GTC TAT TAC TGT GCA AGA AAT AGG GAC TAT 389 Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn rg Asp Tyr 90 95 100 AGT AAC AAC TGG TAC TTC GAT GTC TGG GGC GAA GGG ACC CTG GTC ACC 437 Ser Asn Asn Tf Tyr Phe Asp Val Tf Gly Glu Gly Thr Leu Val Thr 105 110 115 GTC TCC TCA GCC TCC ACC AAG GGC CCA TCG GTC TTC CCC CTG GCA CCC 485 Val Ser Ser Ala Be Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 120 125 130 TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG GTC 533 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Wing Leu Gly Cys Leu Val 135 140 145 150 AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC 581 Lys Asp Ty r Phe P ro Glu P ro Val Th r Val Se r T rp Asn Ser Gly Wing 155 160 165 CTG ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA 629 Leu Thr Ser Gly Val His Thr Phe Pro Wing Val Leu Gln Ser Ser Gly 170 175 180 10 CTC TAC TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGG TTG GGC 677 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Leu Gly 185 190 195? 5 ACC CAG ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG 725 Thr Gln Thr Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 200 205 210 20 GTG GAC AAG AGA GTT GGTGAGAGGC CAGCACAGGG AGGGAGGGTG TCTGCTGGAA 780 Val Asp Lys Arg Val 215 25 GCCAGGCTCA GCGCTCCTGC CTGGACGCAT CCCGGCTATG CAGTCCCAGT CCAGGGCAGC 840 AAGGCAGGCC CCGTCTGCCT CTTCACCCGG AGGCCTCTGC CCGCCCCACT 30 CATGCTCAGG 900 GAGAGGGTCT TCTGGCTTTT TCCCCAGGCT CTGGGCAGGC ACAGGCTAGG TGCCCCTAAC 960 35 CCAGGCCCTG CACACAAAGG GGCAGGTGCT GGGCTCAGAC CTGCCAAGAG CCATATCCGG 1020 GAGGACCCTG CCCCTGACCT AAGCCCACCC CAAAGGCCAA ACTCTCCACT CCCTCAGCTC 1080 0 GGACACCTTC TCTCCTCCCA GATTCCAGTA ACTCCCAATC TTCTCTCTGC for GAG CCC 1137 Glu Pro 5 220 AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GGTAAGCCAG 1186 Lys Ser Cys Asp Lye Thr Hie Thr Cye Pro Pro Cye Pro 0 225 230 CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG TAGAGTAGCC TAGAGTAGCC TGCATCCAGG 1246 GACAGGCCCC AGCCGGGTGC TGACACGTCC ACCTCCATCT CTTCCTCA GCA CCT GAA 1303 Wing Pro Glu 235 CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC 1351 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 240 245 250 ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA GTG GTG GTG GAC 1399 Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 255 260 265 GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC 1447 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Tf Tyr Val Asp Gly 270 275 280 285 GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC 1495 Val Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG 1543 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Tf 305 310 315 CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA 1591 Leu Asn Gly Lye Glu Tyr Lye Cye Lys Val Ser Asn Lys Ala Leu Pro 320 325 330 GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGTGGGACCC GTGGGGTGCG 1644 Wing Pro He Glu Lye Thr He Ser Lye Wing Lys 335 340 AGGGCCACAT GGACAGAGGC CGGCTCGGCC CACCCTCTGC CCTGAGAGTG ACCGCTGTAC 1704 CAACCTCTGT CCCTACA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG 1754 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 345 350 355 CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC 1802 Pro Pro Ser Arg Glu Glu Met Thr Lye Aen Gln Val Ser Leu Thr Cye 360 365 370 CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC 1850 Leu Val Lys Gly Phe Tyr Pro Ser Asp He Wing Val Glu Tf Glu Ser 375 380 385 AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC 1898 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Pro Pro Val Leu Asp 390 395 400 TCC GAC GGC TCC TTC TTC CTC TAT AGC AAG CTC ACC GTG GAC AAG AGC 1946 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT 1994 Arg Tf Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Wing 420 425 430 435 CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCC CCG GGT AAA 2042 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 440 445 450 TGAGTGCGAC GGCCGGCAAG CCCCGCTCCC GAATT 2077 (2) INF0RMACION FOR SEQ ID NO: 89: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 470 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 89: Met Gly Tf Ser Cys He He Leu Phe Leu Val Wing Thr Wing Thr Gly -19 -15 -10 -5 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Wing Glu Val Lye Lye 1 5 10 Pro Gly Wing Ser Val Lye Val Ser Cye Lys Wing Ser Gly Tyr Thr Phe 20 25 Thr Ser Tyr Tf Met Gln Tf Val Lys Gln Wing Pro Gly Gln Arg Leu 35 40 45 Glu Trp Met Gly Glu He Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 50 55 60 Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr Val Asp Thr Ser Wing Ser 65 70 75 Thr Wing T r Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Wing Val 80 85 90 Ty r Ty r Cys Wing A rg Asn A rg Asp Ty r Asn Asn T f Ty r Phe Asp 95 100 105 Val T f Gly Glu Gly Th r Leu Val Th r Val Se r Se r Ala Se r Th r Lys 110 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Wing Wing Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 Val Thr Val Ser Tf Asn Ser Gly Wing Leu Thr Ser Gly Val His Thr 160 165 170 Phe Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 175 180 185 Val Thr Val Pro Being Ser Leu Gly Thr Gln Thr Tyr He Cys Asn 190 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lye Val Aep Lye Arg Val Glu Pro 210 215 220 Lye Ser Cye Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Glu 225 230 235 Leu Leu Gly Pro Ser Val Phe Pro Pro Lys Pro Lys Asp 240 245 250 Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 255 260 265 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Tf Tyr Val Asp Gly 270 275 280 285 Val Glu Val Hie Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Hie Gln Aep Tf 305 310 315 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 320 325 330 Wing Pro He Glu Lys Thr He Ser Lys Wing Lys Gly Gln Pro Arg Glu 335 340 345 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 350 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He 370 375 380 Wing Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 Thr Pro Pro Val Leu Aep Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 400 405 410 Leu Thr Val Asp Lys Ser Arg Tf Gln Gln Gly Asn Val Phe Ser Cys 415 420 425 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 430 435 440 445 Ser Leu Ser Pro Gly Lys 450 (2) INFORMATION FOR SEQ ID NO: 90: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 pairs of bases (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 90: ACAGCCGGGA AGGTGTGCAC 20 (2) INF0RMACION FOR SEQ ID NO: 91: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 91: AGACACCCTC CCTCCCTGTG 20 (2) INF0RMACION FOR SEQ ID NO: 92: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 92: GTGCAGGGCC TGGGTTAGGG 20 (2) INFORMATION FOR SEQ ID NO: 93: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 93: GCACGGTGGG CATGTGTGAG 20 (2) INFORMATION FOR SEQ ID NO: 94: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 94: GTTTTGGGGG GAAGAGGAAG 20 (2) INF0RMACION FOR SEQ ID NO: 95: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 95: CCAGTCCTGG TGCAGGACGG 20 (2) INFORMATION FOR SEQ ID NO: 96: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 pairs of baeee (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 96: CCTGTGGTTC TCGGGGCTGC 20 (2) INFORMATION FOR SEQ ID NO: 97: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 97: CGTGGTCTTG TAGTTGTTCT 20 (2) INF0RMACI0N FOR SEQ ID NO: 98: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 98: CTTCCTCTTC CCCCCAAAAC 20 (2) INF0RMACION FOR SEQ ID NO: 99: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 pairs of baeee (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: eincilla (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec = "DNA eintético" (üi) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 99: CCGTCCTGCA CCAGGACTGG 20 (2) INFORMATION FOR SEQ ID NO: 100: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 pairs of bases (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 100: GCAGCCCCGA GAACCACAGG 20 (2) INFORMATION FOR SEQ ID NO: 101: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 101: AGAACAACTA CAAGACCACG 20 (2) INFORMATION FOR SEQ ID NO: 102: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 102: GCCTGACATC TGAGGACTC 19 (2) INF0RMACION FOR SEQ ID NO: 103: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: eingle (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec =. "Synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 103: GAGTCCTCAG ATGTCAGGC 19 (2) INFORMATION FOR SEQ ID NO: 104: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA eintético" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 104: GAGCAGTACT CGTTGCTGCC GCGCGCGCCA CCAG 34 (2) INF0RMACION FOR SEQ ID NO: 105: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 105: GGTATGGCTG ATTAATGATC AATG 24 (2) INFORMATION FOR SEQ ID NO: 106: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 768 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME / KEY: CDS (B) L0CALIZACION: 40..753 (ix) CHARACTERISTIC: (A) NAME / KEY: peptide mat_ (B) L0CALIZACI0N: 100..753 (ix) FEATURE: (A) NAME / KEY: sig peptide "(B) L0CALIZACI0N: 40..99 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 106: CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCTCAGAG ATG GAG ACA GAC ACA 54 11et (31u "Thr (sp Thr -20 ATC CTG CTA TGG GTG CTG CTG CTC TGG GTT CCA GGC TCC ACT GGT GAG 102 He Leu Leu Tf Val Leu Leu Leu Tf Val Pro Gly Ser Thr Gly Glu -15 -10 -5 1 ATT GTG CTC ACC CAA TCT CCA GGT ACT TTG TCT CTG TCT CCA GGG GAG 150 He Val Leu Thr Gln S r Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 5 10 15 AGG GCC ACC CTC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT 198 Arg Ala Thr Leu Ser Cys Lys Wing Ser Gln Ser Val Asp Tyr Asp Gly 20 25 30 GAT AGT TAT ATG AAC TGG TAC CAA CAG MM CCA GGA CAG GCA CCC AGA 246 Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg 35 40 45 CTC CTC ATC TAT GCT GCC TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu He Tyr Wing Wing Being Asn Leu Glu Being Gly He ro Asp Arg 50 55 60 65 TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC ACC ATC TCT CGT 342 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Arg 70 75 80 CTG GAG CCG GAG GAT TTT GCA GTC TAT TAC TGT CAG CAA AGT AAT GAG 390 Leu Glu Pro Glu Asp Phe Wing Val T r T r Cys Gln Gln Ser Asn Glu 85 90 95 GAT CCT CGG ACG TTC GGT CAA GGC ACC AAG CTG GAA ATC AAA CGG ACT 438 Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu He Lys Arg Thr 100 105 110 GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG 486 Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC 534 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 145 AGA GAG GCC AAA GTA CAG TGG AAA GTG GAT AAC GCC CTC CAA TCG GGT 582 Arg Glu Ala Lys Val Gln Tf Lys Val Asp Asn Ala Leu Gln Ser Gly 150 155 160 AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC 630 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GAC GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His 180 185 190 AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC 726 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 ACA AAG AGC TTC AAC AGG GGA GAG TGT TAGTAAGAAT TCGGG 768 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 (2) INFORMATION FOR SEQ ID NO: 107: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 107: Met Glu Thr Asp Thr He Leu Leu Tf Val Leu Leu Leu Tf Val Pro -20 -15 -10 -5 -5 Gly Ser Thr Gly Glu He Val Leu Thr Gln Ser Pro Gly Thr Leu Ser 1 5 10 Leu Ser P ro Gly Glu A rg Ala Th r Leu Se r Cys Lys Ala Se r Gln Se r 15 20 25 Val Asp Ty r Asp Gly Asp Ser Tyr Met Asn T f Ty Gln Gln Lys P ro 30 35 40 Gly Gln Wing P ro A rg Leu Leu He Ty r Wing Ala Se r Asn Leu Glu Se r 45 50 55 60 Gly He P ro Aep A rg Phe It is Gly Se r Gly Se r Gly Th r Aep Phe Th r 65 70 75 Leu Thr He Ser Arg Leu Glu Pro Glu Aep Phe Wing Val Tyr Tyr Cye 80 85 90 Gln Gln Ser Aen Glu Aep Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu 95 100 105 Glu He Lys Arg Thr Val Wing Ala Pro Ser Val Phe He Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 125 130 135 140 Aen Aen Phe Tyr Pro Arg Glu Wing Lye Val Gln Tf Lye Val Asp Asn 145 150 155 Wing Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 160 165 170 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing 175 180 185 Asp Tyr Glu Lys His Lye Val Tyr Ala Cys Glu Val Thr His Gln Gly 190 195 200 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 215 (2) INFORMATION FOR SEQ ID NO: 108: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 768 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) L0CALIZACI0N: 40..753 (ix) CHARACTERISTICS: (A) NAME / KEY: peptide mat.

(B) L0CALIZACION: 100..753 (ix) CHARACTERISTIC: (A) NAME / KEY: sig peptide (B) L0CALIZACI0N: 40..99 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 108: CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCTCAGAG ATG GAG ACA GAC ACA 54 Met Glu Thr Asp Thr -20 ATC CTG CTA TGG GTG CTG CTG CTC TGG GTT CCA GGC TCC ACT GGT GAG 102 He Leu Leu Tf Val Leu Leu Leu Tf Val Pro Gly Ser Thr Gly Glu -15 -10 -5 1 ATT GTG CTC ACC CAA TCT CCA GGT ACT TTG TCT CTG TCT CCA GGG GAG 150 He Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu 5 10 15 AGG GCC ACC CTC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT 198 Arg Ala Thr Leu Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly 25 30 GAT AGT TAT ATG AAC TGG TAC CAA CAG AAA CCA GGA CAG GCA CCC AGA 246 Asp Ser Tyr Met Asn Tf Tyr Gln Gln Lys Pro Gly Gln Wing Pro Arg 35 40 45 CTC CTC ATC TAT GCT GCC TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu He Tyr Ala Wing Ser Aen Leu Glu Ser Gly He Pro Asp Arg 50 55 60 65 TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC ACC ATC CAT CCT 342 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He His Pro 70 75 80 GTG GAG GAG GAG GAT GCT GCA ACC TAT TAC TGT CAG CAA AGT AAT GAG 390 Val Glu Glu Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Gln Ser Asn Glu 85 90 95 GAT CCT CGG ACG TTC GGT CAA GGC ACC AAG CTG GAA ATC AAA CGG ACT 438 Asp P ro A rg Th r Phe Gly Gln Gly Th r Lys Leu Glu He Lys Arg Th r 100 105 110 GTG GCT GCA CCA TCT GTC TTC ATC TTC CCG CCA TCT GAT GAG CAG TTG 486 Val Ala Wing Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 AAA TCT GGA ACT GCC TCT GTT GTG TGC CTG CTG AAT AAC TTC TAT CCC Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 145 AGA GAG GCC AAA GTA CAG TGG AAA GTG GAT AAC GCC CTC CAA TCG GGT 582 Arg Glu Ala Lys Val Gln Tf Lys Val Asp Asn Ala Leu Gln Ser Gly 150 155 160 AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AAG GAC AGC ACC TAC 630 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 AGC CTC AGC AGC ACC CTG ACG CTG AGC AAA GAC GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing Asp Tyr Glu Lys His 180 185 190 AAA GTC TAC GCC TGC GAA GTC ACC CAT CAG GGC CTG AGC TCG CCC GTC 726 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 ACA AAG AGC TTC AAC AGG GGA GAG TGT TAGTAAGAAT TCGGG 768 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 (2) INFORMATION FOR SEQ ID NO: 109: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 109: Met Glu Thr Asp Thr He Leu Leu Tf Val Leu Leu Leu Tf Val Pro -20 -15 -10 -5 Gly Se r Th r Gly Glu He Val Leu Th r Gln Se r P ro Gly Th r Leu Se r 1 5 10 Leu Ser Pro Gly Glu Arg Wing Thr Leu Ser Cys Lys Wing Ser Gln Ser 15 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Tf Tyr Gln Gln Lys Pro 30 35 40 Gly Gln Ala Pro Arg Leu Leu He Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly He Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Thr He His Pro Val Glu Glu Glu Asp Wing Wing Thr Tyr Tyr Cys 80 85 90 Gln Gln Ser Aen Glu Aep Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu 95 100 105 Glu He Lys Arg Thr Val Wing Wing Pro Ser Val Phe He Phe Pro Pro 110 115 120 Ser Asp Glu Gln Leu Lys Ser Gly Thr Wing Ser Val Val Cys Leu Leu 125 130 135 140 Asn Asn Phe Tyr Pro Arg Glu Wing Lys Val Gln Tf Lys Val Asp Asn 145 150 155 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 160 165 170 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Wing 175 180 185 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 190 195 200 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 205 210 215 (2) INFORMATION FOR SEQ ID NO: 110: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 110: GGTGAGATTG TGCTCACCCA ATCTCCAGG 29 (2) INFORMATION FOR SEQ ID NO: 111: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs. (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 111: CCTGGAGATT GGGTGAGCAC AATCTCACC 29 (2) INFORMATION FOR SEQ ID NO: 112: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec = "DNA eintético" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 112: CCATCTCTCG TCTGGAGCCG GAGGATTTTG C 31 (2) INFORMATION FOR SEQ ID NO: 113: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 113: GCAAAATCCT CCGGCTCCAG ACGAGAGATG G 31 (2) INF0RMACION FOR SEQ ID NO: 114: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 pairs of baees (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 114: CAAGGCACCA AGCTGGAAAT CAAACGGACT G 31 (2) INF0RMACION FOR SEQ ID NO: 115: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 pairs of baees (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 115: CAGTCCGTTT GATTTCCAGC TTGGTGCCTT G 31 (2) INFORMATION FOR SEQ ID NO: 116: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2071 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (üi) HYPOTHETICAL: NO (iv) ANTICIPATION: NO (ix) CHARACTERISTICS: (A) NAME / KEY: sig peptide (B) L0CALIZACI0N: 21..77 (ix) FEATURE: (A) NAME / KEY: int ron (B) LOCATION: 741..1131 (ix) FEATURE : (A) NAME / KEY: intron (B) LOCATION: 1177..1294 (ix) FEATURE: (A) NAME / KEY: intron (B) L0CALIZACI0N: 1619..1715 (ix) FEATURE: (A) NAME / KEY: exon (B) L0CALIZACI0N: 21..734 (ix) CHARACTERISTICS: (A) NAME / KEY: exon (B) LOCATION: 1126..1170 (ix) FEATURE: (A) NAME / KEY: exon (B) LOCATION: 1289..1618 (ix) FEATURE: (A) NAME / KEY: exon (B) LOCATION: 1716..2071 (ix) CHARACTERISTICS: (A) NAME / KEY: peptide mat (B) L0CALIZACI0N: union (78) ., 734, 1126..1170, 1289..1618, 1716..2036) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) L0CALIZACI0N: union (21..734, 1126..1170, 1289 ..1618, 1716..2036) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 116: AAGCTTGGCT TGACCTCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG 50 Met Gly Trp Ser Cys He He Leu Phe Leu -19 -15 -10 GTA GCA ACA GCT ACA GGT GTC CAC TCT CAG GTC CAA CTG GTG CAG TCT 98 Val Ala Thr Ala Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser J * J) GGG GCT GAG GTC AAG AAG CCT GGG GCT TCA GTG AAG GTG TCC TGC AAG 146 Gly Wing Glu Val Lys Lys Pro Gly Wing Ser Val Lys Val Ser Cys Lys 10 15 20 GCT TCT GGC TAC ACC TTC ACC AGC TAC TGG ATG CAG TGG GTA AAA CAG 194 Wing Ser Gly Tyr Thr Phe Thr Ser Tyr Tf Met Gln Tf Val Lys Gln 25 30 35 GCC CCT GGA CAG GGC CTT GAG TGG ATG GGA GAG ATT GAT CCT TCT GAT 242 Pro Wing Gly Gln Gly Leu Glu Tf Met Gly Glu He Asp Pro Ser Asp 40 45 50 55 AGC TAT ACT AAC TAC AAT CAA AAG TTC AAG GGC AAG GCC ACA TTG ACT 290 Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr 60 65 70 GTA GAC ACA TCC ACT AGC ACA GCC TAC ATG GAG CTC AGC AGC CTG AGA 338 Val Asp Thr Ser Thr Ser Thr Wing Tyr Met Glu Leu Ser Ser Leu Arg 75 80 85 TCT GAG GAC ACG GCG GTC TAT TAC TGT GCA AGA AAT AGG GAC TAT AGT 386 Ser Glu Asp Thr Wing Val Tyr Tyr Cys Wing Arg Aen Arg Asp Tyr Ser 90 95 100 AAC AAC TGG TAC TTC GAT GTC TGG GGC GAA GGG ACC CTG GTC ACC GTC 434 Asn Asn Tf Tyr Phe Asp Val Tf Gly Glu Gly Thr Leu Val Thr Val 105 110 115 TCC TCA GCC TCC ACC AAG GGC CCA TCG GTC TTC CCC CTG GCA CCC TCC 482 Ser Ser Wing Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Wing Pro Ser 120 125 130 135 TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG GTC AAG 530 Ser Lys Be Thr Ser Gly Gly Thr Wing Wing Leu Gly Cye Leu Val Lye 140 145 150 GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC CTG 578 Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Tf Asn Ser Gly Ala Leu 155 160 165 ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA CTC 626 Thr Ser Gly Val His Thr Phe Pro Wing Val Leu Gln Ser Ser Gly Leu 170 175 180 TAC TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC ACC 674 Tyr Ser Leu Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 185 190 195 CAG ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTG 722 Gln Thr Tyr He Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 200 205 210 215 GAC AAG AGA GTT GGTGAGAGGC CAGCACAGGG AGGGAGGGTG TCTGCTGGAA 774 Asp Lys Arg Val GCCAGGCTCA GCGCTCCTGC CTGGACGCAT CCCGGCTATG CAGTCCCAGT CCAGGGCAGC 834 AAGGCAGGCC CCGTCTGCCT CTTCACCCGG AGGCCTCTGC CCGCCCCACT CATGCTCAGG 894 GAGAGGGTCT TCTGGCTTTT TCCCCAGGCT CTGGGCAGGC ACAGGCTAGG TGCCCCTAAC 954 CCAGGCCCTG CACACAAAGG GGCAGGTGCT GGGCTCAGAC CTGCCAAGAG CCATATCCGG 1014 GAGGACCCTG CCCCTGACCT AAGCCCACCC CAAAGGCCAA ACTCTCCACT CCCTCAGCTC 1074 GGACACCTTC TCTCCTCCCA GATTCCAGTA ACTCCCAATC TTCTCTCTGC for GAG CCC 1131 Glu Pro 220 AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GGTAAGCCAG 1180 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG TAGAGTAGCC TGCATCCAGG 1240 GACAGGCCCC AGCCGGGTGC TGACACGTCC ACCTCCATCT CTTCCTCA GCA CCT GAA 1297 Wing Pro Glu 235 CTC CTG GGG GGA CCG TCA GTC TTC CTC TTC CCC CCA AAA CCC AAG GAC 1345 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 240 245 250 ACC CTC ATG ATC TCC CGG ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC 1393 Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 255 260 265 GTG AGC CAC GAA GAC CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC 1441 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Tf Tyr Val Asp Gly 270 275 280 285 GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC 1489 Val Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 AGC ACG TAC CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG 1537 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Tf 305 310 315 CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA 1585 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Wing Leu Pro 320 325 330 GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGTGGGACCC GTGGGGTGCG 1638 Wing Pro He Glu Lys Thr He Ser Lys Wing Lye 335 340 AGGGCCACAT GGACAGAGGC CGGCTCGGCC CACCCTCTGC CCTGAGAGTG ACCGCTGTAC 1698 CAACCTCTGT CCCTACA GGG CAG CCC CGA GAA CCA CAG GTG TAC ACC CTG 1748 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 345 350 355 CCC CCA TCC CGG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG ACC TGC 1796 Pro Pro Ser Arg Glu Glu Met Thr Lye Aen Gln Val Ser Leu Thr Cye 360 365 370 CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC 1844 Leu Val Lye Gly Phe Tyr Pro Ser Asp He Wing Val Glu Tf Glu Ser 375 380 385 AAT GGG CAG CCG GAG AAC AAC TAC AAG ACC ACG CCT CCC GTG CTG GAC 1892 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 390 395 400 TCC GAC GGC TTC TTC TTC CTC TAT AGC AAG CTC ACC GTG GAC AAG AGC 1940 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 AGG TGG CAG CAG GGG AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT 1988 Arg Tf Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 435 CTG CAC AAC CAC TAC ACG CAG AAG AGC CTC TCC CTG TCC CCG GGT AAA 2036 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 440 445 450 TGAGTGCGAC GGCCGGCAAG CCCCGCTCCC GAATT 2071 (2) INFORMATION FOR SEQ ID NO: 117: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 470 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION FROM THE SEQUENCE: SEQ ID NO: 117: Met Gly Tf Ser Cys He He Leu Phe Leu Val Wing Thr Wing Thr Gly -19 -15 -10 -5 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Wing Glu Val Lys Lys 1 5 10 P ro Gly Ala Se r Val Lys Val Se r Cye Lye Wing Se r Gly Ty r Th r Phe 15 20 25 Th r Se r Ty r T f Met Gln T f Val Lys Gln Wing P ro Gly Gln Gly Leu 30 35 40 45 Glu T f Met Gly Glu He Asp P ro Se r Asp Se r Ty r Th r Asn Ty r Asn 50 55 60 Gln Lys Phe Lys Gly Lys Wing Thr Leu Thr Val Asp Thr Ser Thr Ser 65 70 75 Thr Wing Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Wing Val 80 85 90 Tyr Tyr Cys Wing Arg Asn Arg Asp Tyr Ser Aen Tf Tyr Phe Aep 95 100 105 Val Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser Ala Be Thr Lys 110 115 120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lye Ser Thr Ser Gly 130 135 140 Gly Thr Wing Wing Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 Val Thr Val Ser Tf Aen Ser Gly Wing Leu Thr Ser Gly Val Hie Thr 160 165 170 Phe Pro Wing Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 175 180 185 Val Thr Val Pro Ser Ser Leu Gly Thr Gln Thr Tyr He Cys Asn 190 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Aep Lye Arg Val Glu Pro 210 215 220 Lye Ser Cye Aep Lye Thr Hie Thr Cys Pro Pro Cys Pro Pro Glu 225 230 235 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 240 245 250 Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Aep 255 260 265 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Tf Tyr Val Asp Gly 270 275 280 285 Val Glu Val His Asn Wing Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Be Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Tf 305 310 315 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 320 325 330 Wing Pro He Glu Lys Thr He Ser Lys Wing Lye Gly Gln Pro Arg Glu 335 340 345 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Aen 350 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He 370 375 380 Wing Val Glu Tf Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 400 405 410 Leu Thr Val Asp Lys Ser Arg Tf Gln Gln Gly Asn Val Phe Ser Cys 415 420 425 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 445 Ser Leu Ser Pro Gly Lys 450 (2) INFORMATION FOR SEQ ID NO: 118: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 118: CAGGCCCCTG GACAGGGCCT TGAGTGGATG 30 (2) INFORMATION FOR SEQ ID NO: 119: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 pairs of baeee (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: eingle (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / deec = "Synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 119: CATCCACTCA AGGCCCTGTC CAGGGGCCTG 30 (2) INF0RMACION FOR SEQ ID NO: 120: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No 15 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 120; GCTGAGCTCC ATGTAGGCTG TGCTAGTGGA TGTGTCTAC 39 * 20 (2) INFORMATION FOR SEQ ID NO: 121: (i) CHARACTERISTICS OF THE SEQUENCE: 25 (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) CHARACTER OF THE CHAIN : simple (D) TOPOLOGY: linear 30 (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "DNA eintético" (iii) HYPOTHETICAL: No 35 (iv) ANTICIPATION: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 121: 40 TGCACGCGTG GCTGTGGAAT GTGTGTCAGT TAG_33_(2) INFORMATION FOR SEQ ID NO: 122: 45 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple 50 (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 122: TCCGAÁGCTT TTAGAGCAGA AGTAACACTT C 31 (2) INFORMATION FOR SEQ ID NO: 123: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN CHARACTER: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA" (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 123: AAAGCGGCCG CTGCTAGCTT GGCTGTGGAA TGTGTG 36

Claims (40)

NOVELTY OF THE INVENTION CLAIMS

1. - A molecule having a specific antigen binding region for an epitope of the Fas antigen, said epitope being conserved between a primate and a non-primate animal.

2 - The molecule according to claim 1, characterized in that the primate is human.

3. The molecule in accordance with the claim 1, characterized because the non-primate animal is a rodent.

4. The molecule according to claim 3, characterized in that the rodent is a mouse.

5. The molecule according to claim 1, characterized in that the primate is the human being and the non-primate animal is a mouse.

6. A molecule that has a specific antigen binding region for a conserved epitope of the mammalian Fas antigen.

7. An antibody produced by hybridoma HFE7A having accession number FERM BP-5828.

8, - A molecule having at least one anti-CDR signal, said specific anti-virus for human Fae, wherein said RDCs have identity with the CDRs of the anti-virus produced by the hybridoma HFE7A having the access number FERM BP-5828.

9. - A molecule having an antigen binding region, said binding region having specificity for the antigenic determinant recognized by the anti-primer produced by the hybridoma HFE7A, which has the accession number FERM BP-5828.

10. The molecule according to claim 1, characterized in that it is an antiquake.

11. The molecule according to claim 6, characterized in that it is an antibody.

12. The molecule in accordance with the claim 8, characterized because it is an antiquake.

13.- The molecule in accordance with the claim 9, characterized because it is an antiquake.

14. A molecule comprising a polypeptide light chain and a polypeptide heavy chain, the heavy chain has the following general formula (I): -FRHi-CDRH-FRH2 -CDRH2 -FRH3 -CDRH3 -FRH «- (I) , in which FRHi represents any amino acid sequence consisting of 18 to 30 amino acids, CDRHi represents the sequence defined in SEQ ID No. 2 of the sequence listing, FRH2 represents any amino acid sequence consisting of 14 amino acids, CDRH2 represents the sequence defined in SEQ ID No. 3 of the sequencing listing, FRH3 repreeenta any amino acid sequence consisting of 32 amino acids, CDRH3 represents the sequence defined in SEQ ID No. 4 of the sequence listing, FRH4 repreeenta any amino acid sequence that connects 11 amino acid, and each amino acid is bound to the other by a peptide bond; the light chain has the following general formula (II): -FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4- (II), in which FRLi represents any amino acid sequence consisting of 23 amino acids, CDRLi represents the sequence defined in SEQ ID No. 5 of the sequence listing, FRL2 represents any amino acid sequence consisting of 15 amino acids, CDRL2 represents the sequence defined in SEQ ID No. 6 of the sequence listing, FRL3 represents any amino acid sequence consisting of 32 amino acids, CDRL3 represents the sequence defined in SEQ ID No. 7 of the sequence listing, FRL4 represents any amino acid sequence consisting of 10 amino acids, and each amino acid is linked to the other by a peptide bond.

15.- The molecule in accordance with the claim 14, characterized because it is an antibody.

16. The antibody according to any of claims 10, 11, 12, 13 and 15, which is of the type of immunoglobulin G.

17. The molecule according to any of claims 1 to 9 and 14, characterized in that It is humanized.

18. The anti-cable according to any of claims 10, 11, 12, 13 and 15, characterized in that it is humanized.

19. The molecule according to claim 1, characterized in that it is capable of inducing apoptosis in abnormal cells expressing Fas, and that is able to inhibit apoptosis in normale cells.

20. The molecule according to claim 8, characterized in that it is capable of inducing apoptosis in abnormal cells expressing Fas, and that it is capable of inhibiting apoptosis in normal cells.

21 - The use of the molecule according to claim 8, to evaluate therapies for conditions in humans affected by the Fas / Fas ligand interaction, said evaluation being in animals that are models for such conditions.

22. A humanized molecule having an antigen-binding region specific for an epitope of the Fas antigen conserved between a primate and a non-primate animal; said molecule is obtainable by grafting the respective CDRs of a specific antiquake for said epitope of the Fas antigen, into at least one human light chain, or fragment thereof, and into at least one human heavy chain, or fragment thereof.

23. The molecule according to claim 22, the antiquase from which the RDC is obtainable has variable regions comprising said RDC, and wherein said human light and heavy chains are selected based on the closest similarity between the variable regione understood in the same and the variable regions of said anticuefo.

24. - The molecule according to claim 22, characterized in that there are also grafted into said heavy and light chains, significant amino acids of the structure regions comprised in an epitope recognition site of the antiquake from which the RDCs are obtainable, in order to maintain the structure at the epitope recognition site.

25. The molecule according to any of claims 1, 6, 8 and 9, which binds to a peptide comprising the amino acid sequence of SEQ ID No. 1 of the sequence of eequence.

26. The molecule according to any of claim 1, 6, 8 and 9, characterized in that it comprises a light chain polypeptide protein selected individually from the group consisting of amino acid sequence 1 to 218 of SEQ ID No. 50 , amino acid sequence 1 to 218 of SEQ ID No. 52, amino acid sequence 1 to 218 of SEQ ID No. 54, amino acid sequence 1 to 218 of SEQ ID No. 107, and amino acid sequence 1 to 218 of SEQ ID No. 109 of the sequence listing.

27. The molecule according to any of claims 1, 6, 8 and 9, characterized in that it comprises a heavy chain polypeptide protein selected individually from the group consisting of amino acid sequence 1 to 451 of SEQ ID No. 89 and amino acid sequence 1 to 451 of SEQ ID No. 117 of the sequence listing.

28. The molecule according to any of claims 1, 6, 8 and 9, comprising a light chain polypeptide protein having the sequence of amino acids 1 to 218 of SEQ ID No. 50 of the sequence listing, and a heavy chain polypeptide protein having the amino acid sequence 1 to 451 of SEQ ID No. 89 of the sequence of sequences.

29. The molecule according to any of claims 1, 6, 8 and 9, comprising a light chain polypeptide protein having the sequence of amino acids 1 to 218 of SEQ ID No. 107 of the sequence listing, and a heavy chain polypeptide protein having amino acid sequence 1 to 451 of SEQ ID No. 117 of the sequence listing.

30.- DNA encoding any individual portion of polypeptide of a molecule according to any of claims 1, 6, 8 and 9.

31.- DNA comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence 100 at 753 of SEQ ID No. 49, the nucleotide sequence 100 to 753 of SEQ ID No. 51, the nucleotide sequence 100 to 753 of SEQ ID No. 53, the nucleotide sequence 100 to 753 of SEQ ID No. 106 , and the nucleotide sequence 100 to 753 of SEQ ID No. 108.

32. - DNA comprising a nucleotide sequence selected from the group consisting of nucleotide sequence 84 to 2042 of SEQ ID No. 88 and nucleotide sequence 84 to 2042 of SEQ ID No. 116 of the sequence listing.

33.- A recombinant DNA vector comprising DNA encoding a light chain polypeptide protein selected individually from the group consisting of amino acid sequence 1 to 218 of SEQ ID No. 50, amino acid sequence 1 to 218 of SEQ ID No. 52, amino acid sequence 1 to 218 of SEQ ID No. 54, amino acid sequence 1 to 218 of SEQ ID No. 107 and amino acid sequence 1 to 218 of SEQ ID No. 109 of the sequence listing .

34.- A recombinant DNA vector comprising DNA encoding a peeled chain polypeptide protein individually selected from the group that you connected from amino acid sequence 1 to 451 of SEQ ID No. 89 and the amino acid sequence 1 to 451 of SEQ ID No. 117 of the sequence listing.

35.- A host cell transformed with a recombinant DNA vector comprising DNA encoding a light chain polypeptide protein selected individually from the group consisting of amino acid sequence 1 to 218 of SEQ ID No. 50, the amino acid sequence 1 to 218 of SEQ ID No. 52, amino acid sequence 1 to 218 of SEQ ID No. 54, amino acid sequence 1 to 218 of SEQ ID No. 107 and amino acid sequence 1 to 218 of SEQ ID No. 109 of sequence listing.

36.- A huéeped cell traded with a recombinant DNA vector comprising DNA encoding a heavy chain polypeptide protein selected individually from the group consisting of amino acid sequence 1 to 451 of SEQ ID No. 89 and the amino acid sequence 1 to 451 of SEQ ID No. 117 of the sequence listing.

37.- A host cell transformed with at least one recombinant DNA vector comprising DNA encoding a light chain polypeptide protein and DNA encoding a heavy chain polypeptide; said light chain polypeptide comprises a sequence selected individually from the group that you connected from amino acid sequence 1 to 218 of SEQ ID No. 50, amino acid sequence 1 to 218 of SEQ ID No. 52, amino acid sequence 1 to 218 of SEQ ID No. 54, amino acid sequence 1 to 218 of SEQ ID No. 107 and amino acid sequence 1 to 218 of SEQ ID No. 109 of the sequence listing; said heavy chain polypeptide protein comprises an individually selected sequence from the group consisting of amino acid sequence 1 to 451 of SEQ ID No. 89 and amino acid sequence 1 to 451 of SEQ ID No. 117 of sequence listing.

38.- The huéeped cell according to claim 35, 36 or 37, characterized in that it is a mammalian ee.

39.- E. coli selected from the group consisting of E. coli pHSGMMÓ SANK73697 (FERM BP-6071), E.coli PHSGHM17 SANK73597 (FERM BP-6072), E.coli pHSGHH7 SANK73497 (FERM BP-6073), E. coli PHSHM2 SANK 70198 and E. coli pHSHHS SANK 70398 (FERM BP-6272), E, coli pgHSL7A62 (FERM BP-6274) SANK73397 (FERM BP-6074) and E. coli pgHPDHV3 SANK 70298 (FERM BP-6273).

40. A method for producing a humanized anti-Fas antibody comprising culturing the host cell according to claim 37, and then recovering the anti-humanized anti-Fas anti-Fas from the culture. 41.- An agent for the treatment or prophylaxis of conditions attributable to abnormality of the Fae / Fas ligand system, which comprises, as an active ingredient, the molecule according to claim 1, 6, 8 or 9. 42.- An agent for the treatment or prophylaxis of conditions attributable to abnormalities of the Fas Fas / Fas ligand, which comprises as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is selected from the group consisting of diseases autoimmune, allergy, atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia, hepatitis, acquired immunodeficiency syndrome and rejection after organ transplantation. 43.- An agent for the treatment or prophylaxis of conditions attributable to abnormality of the Fae / Fas ligand system, which comprises, as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is a autoimmune disease selected from the group consisting of general lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjögren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, sterility, myasthenia gravis, multiple sclerosis, Baeedow's disease, thrombocytopenia, and diabetee mellitue dependent on ineulin. 44.- An agent for the treatment or prophylaxis of conditions attributable to abnormalities of the Fas / Fas ligand system, which comprises, as an active ingredient, the molecule according to claim 1, 6, 8 or 9, characterized in that said condition is a allergy. 45.- An agent for the treatment or prophylaxis of conditions attributable to abnormality of the Fae / Fae ligand system, which comprises, as an active ingredient, the molecule according to claim 1, 6, 8 or 9, characterized in that said condition is a rheumatoid 46.- An agent for the treatment or prophylaxis of conditions attributable to abnormalities of the seventh Fae / Fae ligand, which comprises as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is arteriosclerosis .

47. An agent for the treatment or prophylaxis of conditions attributable to Fas systemic Fas / ligand abnormalities, comprising as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is selected from the group which consists of myocarditis and cardiomyopathy. 48.- An agent for the treatment or prophylaxis of conditions attributable to Abnormalities of the Fas / Fas ligand system, comprising as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is nephritis Glomerular 49.- An agent for the treatment or prophylaxis of conditions attributable to Abnormalities of the Fas / Fas ligand system, comprising as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is anemia hypoplastic 50.- An agent for the treatment or prophylaxis of conditions attributable to abnormality of Fas system / Fas ligand, which comprises as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is hepatitis . 51.- An agent for the treatment or prophylaxis of conditions attributable to abnormalities of the Fas / Fae ligand system, which comprises as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is selected of the group consisting of fulminating hepatitis, chronic hepatitis, viral hepatitis, hepatitis C, hepatitis B, hepatitis D, and alcoholic hepatitis. 52.- An agent for the treatment or prophylaxis of conditions attributable to Abnormalities of the Fas / Fas ligand system, comprising as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is rejection after organ transplantation. 53.- An agent for the treatment or prophylaxis of conditions attributable to abnormalities of the Fas / Fae ligand system, which comprises as an active ingredient, the molecule according to claims 1, 6, 8 or 9, characterized in that said condition is the acquired immunodeficiency syndrome. 54.- The molecule according to claim 1, 6, 8 or 9, characterized in that it has a property selected from the group consisting of: inducing apoptosis in T cells expressing Fae; decrease autoimmune symptoms in MRL mice gld / gld; not induce liver disorders; a therapeutic or prophylactic effect on fulminating hepatitis; a preventive effect of the onset of collagen-induced arthritis; induce apoptosis in synovial cells of a patient with rheumatoid arthritis. 55.- The molecule according to claims 1, 6, 8 or 9, characterized in that it has the following properties: induces apoptosis in T cells expressing Fas; decreases autoimmune symptoms in MRL mice gld / gld; does not induce liver disorders; a therapeutic or prophylactic effect on fulminating hepatitis; a preventive effect of the onset of collagen-induced arthritis; induces apoptosis in synovial cells of a patient with rheumatoid arthritis. 56.- The use of a molecule as claimed in claims 1, 6, 8 or 9, in the preparation of compositions for the treatment or prophylaxis of a condition attributable to an abnormality of the Fas / Fae ligand system. 57.- The use of the molecule according to claims 1, 6, 8 or 9, wherein said condition is selected from the group consisting of autoimmune diseases, allergy, atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia , hepatitis, acquired immunodeficiency syndrome and rejection after organ transplantation.