JPH11171900A - Anti-fas antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new molecule for treatment and prevention, etc., of diseases caused by abnormality of Fas/Fas ligand system, comprising an anti-Fas antibody having antigen-binding region specific to epitope stored between Fas of primate and non-primate antmals. SOLUTION: This new molecule is a new anti-Fas antibody comprising a molecule having antigen-binding region specific to epitope stored between primate and non-primate animals and recognizes Fas antigen which is a cell membrane molecule which participates in apoptosis and is useful as an agent for preventing or treating diseases caused by abnormality of Fas/Fas ligand system, especially therapeutic agent for autoimmune diseases or therapeutic agent for rheumatism. The molecule is obtained by introducing an expression vector of fused protein of extracellular region of human Fas with extracellular region of mouse interleukin 3 receptor into cell strain COS-1 originating from monkey and immunizing the expressed antigen, fusing the spleen cell with myeloma cell, selecting the resultant hybridoma and cloning and culturing the hybridoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an anti-Fas antibody which recognizes a Fas antigen which is a cell membrane molecule involved in apoptosis, a DNA encoding a variable region thereof, and the like.

[0002] The present invention further relates to a prophylactic or therapeutic agent for a disease caused by abnormal Fas / Fas ligand system containing an anti-Fas antibody as an active ingredient, particularly a therapeutic agent for autoimmune disease or rheumatism.

[0003]

2. Description of the Related Art In vivo, physiological cell death caused by normal cell turnover is called apoptosis and is distinguished from necrosis (necrosis), which is a pathological cell death [Kerr, et al. (1972) Br. J. Cancer 26, 239]. So-called programmed cell death is the death of certain types of cells that have been programmed to die in vivo in advance, and apoptosis is one of them. In apoptosis, phenomena such as cell surface curvature, nuclear chromatin condensation, and chromosomal DNA fragmentation are characteristically observed.

[0004] Apoptosis plays a role in the differentiation of lymphocytes (T cells and B cells) by eliminating cells that recognize self antigens. In fact, it is known that more than 95% of cells, such as cells that react with self, die in the thymus during the maturation of T lymphocytes [see Nagata Shigeichi (1993) Protein Nucleic Acid Enzyme 38, 2208-2218]. The development of so-called autoimmune diseases is thought to be due to the generation of autoreactive lymphocytes due to apoptosis in lymphocyte differentiation [Nakayama et al., (1
995) Mebio Vol. 12, No. 10, 79-86].

As a molecule involved in such apoptosis, Fas [Yonehara, S. et al. (1989) J. Exp.
ed. 169, 1747-1756], Tumor Necrosis Factor Receptor [Loet
scher, H. et al. (1990) Cell 61, 351-359], C
D40 [Tsubata, T. et al. (1993) Nature 364, 645-
648] and Perforin / Granzyme A [Jenne,
DE et al. (1988) Immunol. Rev. 103, 53-71]. Fas is a transmembrane protein present on the cell surface. When a protein called Fas ligand binds to its extracellular region, it induces apoptosis in the cell. If the Fas / Fas ligand system is abnormal, cells that should be removed by apoptosis and that adversely affect the maintenance of homeostasis remain without being removed, or apoptosis occurs in cells that are essential for the maintenance of homeostasis. Various obstacles occur by being induced. In the present invention, they are collectively referred to as “F
Diseases caused by abnormalities in the as / Fas ligand system. "

[0006] The following common mechanism can be found in the onset or progression of disease caused by abnormal Fas / Fas ligand system. That is, Fas
Abnormal cells that express but are not removed (hereinafter referred to as “abnormal cells”) attack other normal tissues or cells or abnormally proliferate, causing damage to normal tissues or cells. May cause symptoms. In some cases, these disorders are caused or exacerbated by normal tissues or cells stimulated by abnormal cells that express Fas and undergo apoptosis. Specifically, examples of the diseases caused by abnormalities of the Fas / Fas ligand system include those described below.

Autoimmune diseases: Various human autoimmune diseases (Hashimoto's disease, systemic lupus erythematosus, Sjogren's syndrome, pernicious anemia, Addison's disease, insulin-dependent diabetes mellitus, scleroderma, Goodpasture's syndrome, Crohn's disease,
Many associations between the Fas / Fas ligand system and autoimmune hemolytic anemia, spontaneous infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, rheumatoid arthritis) have been reported. . As a genetic abnormality of the Fas / Fas ligand system, in mice, lpr
(Lymphoproliferation) mouse, lpr ^cg (lpr gene
complementing gld gene) gld (generalized lymphoproliferative) with abnormalities in mice and Fas ligand
disease) are known, and all of them have been shown to exhibit various autoimmune disease symptoms accompanied by specific systemic lymphadenopathy. MRLlpr mice, which are spontaneous models of human systemic lupus erythematosus, produce autoantibodies with marked lymphadenopathy and develop nephritis due to immune complexes. Since this mouse has an abnormality in the Fas gene, peripheral tolerance to self-antigen due to apoptosis via Fas did not occur in the periphery, and as a result, activated self-reactive T cells accumulated continuously. It is presumed to exhibit the above pathology [Stra
sser, A., Nature 373, 385 (1995)]. Lymphadenopathy, hypergammaglobulinemia, CD4 ^- C in humans
Two pediatric cases with significant D8 ^- T cell expansion [Snelle
r, MC. et al. (1992) J. Clin. Invest. 90, 334], and autoimmune disease-like cases were reported to be caused by abnormalities in the Fas gene [Fishe
r, GH. et al. (1995) Cell, 81, 935 and Rieux-Lau
cat, F. et al. (1995) Science, 268, 1347], autoimmune lymphoproliferative syndrome (Autoimmune Lymphoproli
ferative Syndrome (ALPS). Therefore, not only in mice but also in humans, the apoptosis-inducing system via Fas is greatly involved in the establishment and maintenance of immune tolerance to self-antigens, and it is considered that various autoimmune diseases are induced by the abnormalities.

[0008] Further, since most T cells involved in tissue destruction by infiltrating the affected area of rheumatoid arthritis patients express Fas, rheumatoid arthritis also has an aspect as an autoimmune disease. [Hoa,
TTM, et al. (1996) J. Rheumatol. 23, 1332-1337
reference].

[0009] Many insulin-dependent diabetes mellitus occurs because the destruction of pancreatic beta cells by autoreactive T cells leads to an absolute deficiency of insulin secretion.
Depletion of autoreactive T cells is important for the underlying treatment of the condition.

[0010] Furthermore, in graft-versus-host disease after bone marrow transplantation, the expression of Fas is increased in the target organ to be damaged, and the degree of the increased expression correlates with the degree of target organ damage [Chu, JL, et al. (1995) J. Exp. Med. 1
81, 393]. Therefore, in preventing or treating this disease, it is necessary to prevent apoptosis of cells of the target organ and reduce the number of cells that attack the target organ.

[0011] Allergic diseases: Inflammatory cells involved in allergic diseases are usually activated and infiltrate into lesions, apoptosis is suppressed, cell life is prolonged, and local populations are prolonged. To perform its function. In an experimental model of eosinophilic airway inflammation in mice, subcutaneous eosinophil infiltration seen after allergen inhalation can be abolished when an anti-Fas antibody with apoptosis-inducing activity is administered through the airway. Known [Tsuy
uki, S., et al. (1995) J. Clin. Invest. 96, 2924]. Therefore, it is possible to reduce symptoms by inducing apoptosis in inflammatory cells.

Rheumatoid arthritis: Apart from the aforementioned autoimmune disease aspects of rheumatoid arthritis, it is known that synovial cells that are abnormally proliferating in the affected area express Fas [Hoa, TTM, et al. (1996) J. Rheu
matol. 23, 1332]. When the synovial cells derived from the affected area are stimulated with an anti-Fas antibody having an apoptosis-inducing activity, apoptosis is induced [Nakajima, T. et al.
(1995) Arthritis Rheum. 38, 485]. That is,
Fas / Fas in the affected area of rheumatoid arthritis patients
Synovial cells in which the ligand system is not functioning properly and autoreactive T cells and abnormally proliferate are also in a state where they express Fas but are not removed.

Atherosclerosis: The final image of cell death at the center of atherosclerotic lesions is necrosis (necrosis), but it has been reported that apoptosis is involved in the progression and regression processes [Kenji Harada (1997) Modern Medicine , 29, 109]. Electron microscopic findings of atherosclerotic lesions show apoptosis of smooth muscle cells with nuclear enrichment [Isner,
JM, et al. (1995) Circulation 91, 2703].
It has also been reported that foam cells, which are a type of macrophage that gather in the intimal layer of the artery and phagocytose fat, express Fas in early lesions of arteriosclerosis, and that apoptosis is induced by an anti-Fas antibody having apoptosis-inducing activity. [Richardson, BC, et al. (1994) Eur.
J. Immunol. 24, 2640]. Atherosclerotic lesions are often accompanied by lymphocyte infiltration, suggesting that Fas ligand of T cells and Fas of macrophages may control atherosclerosis [Kenji Harada (1997) Modern Medicine
29, 109].

Myocarditis / cardiomyopathy: The Fas / Fas ligand system is also involved in the pathogenesis of ischemic heart disease, viral heart disease, and autoimmune heart diseases such as dilated cardiomyopathy and chronic cardiomyopathy. Likely to be. Myocarditis is inflammation of the heart muscle that is thought to be mainly caused by a virus such as Coxsackie virus, and typically develops from cold pain-like symptoms, followed by chest pain, arrhythmia, heart failure, shock, and the like. Cardiomyopathy is defined as "cardiomyopathy of unknown cause", and its cause is also considered to be viral infection. In an examination of a myocarditis model of a mouse with heart failure, apoptotic cells (nucleus enrichment, fragmentation) are observed in the mouse heart after virus inoculation. Also Fas in mouse heart
Increased expression was observed, suggesting that apoptosis was induced by Fas ligand derived from infiltrating inflammatory cells, mainly lymphocytes [Takehiko Yamada et al.
97) 29, 119]. It has been known that cultured rat cardiomyocytes undergo apoptosis due to ischemia, and at the same time, mRNA encoding Fas increases in the cells [Ta
naka M., et al. (1994) Circ. Res. 75, 426].

Renal disease: In many chronic kidney diseases,
Reconstruction of glomerular tissue results in accumulation of extracellular matrix in the glomerulus, which leads to progression of glomerular sclerosis, leading to abolition of the filtration function to the pathology of chronic renal failure. In the advanced glomerular sclerosis model, typical apoptotic images are observed in the hardened area at the electron microscopic level, and an increase in glomerular apoptosis is observed in accordance with the decrease in the number of glomerular cells as the hardening progresses [Sugiyama H., et al. (1996) K
idney Int. 49, 103]. In acute glomerulonephritis,
It is known that nephritis tends to recover by reducing apoptotic proliferation of mesangial cells [Shimizu A., et al. (1995) Kidney Int. 47, 114 and Baker AJ, et al. (1994) ) J. Clin. Invest. 9
4, 2105]. Furthermore, it has been reported that cells expressing Fas in the glomerulus are significantly increased in purpura nephritis, lupus nephritis and the like [Takemura T, et al.
(1995) Kidney Int. 48, 1886].

Aplastic anemia: In the surface of hematopoietic progenitor cells of patients with aplastic anemia, Fas is higher than that of normal humans.
Is remarkable, Fa decreases in hematopoietic stem cells.
s have been implicated [Maciejewski,
JP, et al. (1995) Br. J. Haematol. 91, 245].

Hepatitis: In fulminant hepatitis, it is known that apoptosis is induced in many hepatocytes, and when anti-Fas antibody Jo2 is intraperitoneally administered to mice, hepatic cells like fulminant hepatitis are observed. Fas because death is recognized
Apoptosis of hepatocytes mediated by E. coli is thought to be involved in its development [Kamogawa, Y., et al. (1996) Molecu
lar Medicine 33, 1284] [Ogasawara, J., et a
l. (1993) Nature 364,806]. Immunohistological studies showed that Fas expression was enhanced in the hepatocyte cytoplasm of hepatocyte necrosis in chronic hepatitis lesions and in the liver cell membranes of liver disease lesions such as fatty liver [Hiramatsu, N., et al. a
l. (1994) Hepatology 19, 1354 and Takatani, M., e.
t al. (1996) International Hepatology Commun. 4, 3
34]. In addition, Fas is expressed in foci of chronic persistent hepatitis C and chronic active hepatitis, all of which have hepatocytes stained sporadically and are accompanied by infiltrating lymphocytes that appear to be cytotoxic T cells. [Mita, E., et al. (1994) Bi
ochem. Biophys. Res. Commun. 204, 468]. Cytotoxic T cells have the function of inducing apoptosis in infected cells by Fas ligand on the cell surface, but also induce apoptosis in nearby normal cells. This phenomenon called neighbor injury is caused by the fact that many cells in the body express Fas when they or nearby cells are infected. In addition, in the case where RNA derived from hepatitis C virus disappears by administration of interferon for chronic hepatitis C, the expression of Fas in liver tissue is significantly reduced.

In hepatocytes of patients with acute liver failure, F on the cell surface
As is increased, the cells undergo apoptosis by an anti-Fas antibody having apoptosis-inducing activity. In addition, in alcoholic hepatitis, the hepatocytes inside the pseudolobules themselves have F
as ligand is expressed [Galle, PR, et al.
(1995) J. Exp. Med. 182, 1223]. In situ hybridization analysis of Fas ligand gene expression showed that expression was found in liver infiltrating lymphocytes in patients with acute liver failure and in hepatocytes themselves inside pseudolobules in patients with alcoholic cirrhosis. Apoptosis is thought to be induced by different mechanisms in cirrhosis, but in any case, it is speculated that the Fas / Fas ligand system is abnormal. In hepatitis model mice, it is known that liver damage is inhibited by administration of a substance having a property of inhibiting the binding of Fas ligand to Fas [Kondo, T., et a.
l. (1997) Nature Medicine 3, 409].

Acquired immunodeficiency syndrome: A cause of immunodeficiency in a patient infected with human immunodeficiency virus (hereinafter referred to as "HIV") is that a very large number of immune cells not infected with HIV die by apoptosis. Can be Helper T cells are killed by contact with HIV, but since growth factors derived from helper T cells are essential for suppressing apoptosis of cytotoxic T cells, when helper T cells disappear, cytotoxic T cells undergo apoptosis. Wake up. The expression of Fas in peripheral blood lymphocytes of HIV-infected persons is correlated with the progress of the disease state [Dhein, J., et al. (1
995) Behring Inst. Mitt. 96, 13, and McCloskey,
TW, et al. (1995) Cytometry 22, 111], Fas-positive peripheral blood lymphocytes do not readily undergo apoptosis by Fas stimulation in non-infected individuals, but Fas is expressed in peripheral blood lymphocytes of infected individuals. Induces apoptosis in a short period of time [Owen-Schaub, LB et al.
(1992) Cell Immunol. 140, 197].
Apoptosis of immune cells in V-infected persons is also Fas / F
It is thought to be due to an abnormality in the as ligand system.

Rejection after organ transplantation: The phenomenon of rejection after organ transplantation is similar to that of an autoimmune disease except that the transplanted organ targeted by cytotoxic T cells is derived from a donor. Therefore, improvement of symptoms can be expected by suppressing the function of cytotoxic T cells.

For each of these diseases, abnormal cells (eg, autoreactive T cells in autoimmune diseases, foam cells in arteriosclerosis, mesangial cells in acute glomerulonephritis, infected cells in viral infections, rheumatoid arthritis, etc.) Removal of synovial cells, etc.) to protect normal tissues or cells is an effective treatment.
A drug that only induces apoptosis through as is likely to cause damage to normal tissues even if it can remove abnormal cells, and a drug that only inhibits apoptosis through Fas can protect normal cells. Even abnormal cells cannot be removed. In fact, anti-mouse Fas monoclonal antibody Jo2 having apoptosis-inducing activity causes fulminant hepatitis in mice [Ogasawara, J., et a.
l. (1993) Nature 364, 806-809]. To date, an anti-Fas antibody that has been confirmed to have no apoptosis when administered to mice while having apoptosis-inducing activity has not been known.

Immunoglobulin G (hereinafter simply referred to as "IgG")
That. ) Is composed of two light polypeptide chains each having a molecular weight of about 23,000 (hereinafter referred to as “light chain”) and two heavy polypeptide chains each having a molecular weight of about 50,000 (hereinafter referred to as “heavy chain”). Both the heavy and light chains have a repeating structure of a region in which the amino acid sequence is conserved, consisting of about 110 residues, and these are the basic units of the three-dimensional structure of IgG (hereinafter, referred to as the basic unit).
It is called a "domain." ). The heavy and light chains are composed of four consecutive and two domains, respectively. In both heavy and light chains, the amino-terminal domain has a greater variation in amino acid sequence between antibody molecules than other domains, and this domain is a variable domain (hereinafter, referred to as “V domain”). Called. At the amino terminus of IgG, heavy and light chain V domains complementarily associate to form a variable region. In contrast, the remaining domains form a constant region as a whole. The constant region has a sequence characteristic of each animal species. For example, the constant region of mouse IgG is human I
Mouse IgG is recognized as foreign by the human immune system because it differs from the constant region of gG,
A human anti-mouse antibody (Human Anti Mouse Antibody: hereinafter referred to as “HAMA”) response occurs (Schloff et al.
Cancer Res., 45, 879-85 (1985)). Therefore, mouse antibodies cannot be repeatedly administered to humans. In order to administer such an antibody to humans, it is necessary to modify the antibody molecule so as not to cause a HAMA response while maintaining the specificity of the antibody.

According to the result of X-ray crystal structure analysis, such a domain generally has a long cylindrical structure in which two layers of antiparallel β sheets each having three to five β chains are overlapped. In the variable region, three loops for each of the heavy and light chain V domains assemble to form an antigen binding site. Each loop is a complementarity determining region.
determining region: Hereinafter, referred to as “CDR”. ), And the amino acid sequence variation is the most remarkable. The portion of the variable region other than the CDR generally has a role of maintaining the structure of the CDR, and is called a “framework”. Kabat et al. Collected a large number of primary sequences of the variable regions of heavy and light chains, and created a table in which each primary sequence was classified into CDRs and frameworks based on the conservation of the sequences (Kabato et al.,
SEQUENCES OFIMMUNOLOGICAL INTEREST, 5th edition, N
See IH publication, No. 91-3242, EAKabatt et al.). In addition, each framework was classified into a plurality of subgroups having common characteristics in amino acid sequences. In addition, it has been found that there is a corresponding framework between human and mouse.

From the study on the structural characteristics of IgG, the following method for producing a humanized antibody was devised.

Early in the research, a chimeric antibody was proposed in which the variable region of a mouse-derived antibody was conjugated to a human-derived constant region (Proc. Natl. Acad. Sci. USA 81 , 6851-6).
855, (1984)). However, such chimeric antibodies can still induce a HAMA response, especially when administered for long periods of time, as they contain many non-human amino acid residues (Begent et al. Br. J. Cancer, 62 , 487, ( 1990)).

As a method for further reducing the amino acid residues derived from non-human mammals which may express a HAMA response to humans, a method has been proposed in which only the CDR portion is incorporated into a human-derived antibody (Nature, 321, 522-525, (1
986)), but in general, CDR-only transplantation was insufficient to retain immunoglobulin activity against the antigen.

On the other hand, in 1987, using data from X-ray crystal structure analysis, Cottchia et al. (I) In the amino acid sequence of the CDR, there were a site directly binding to the antigen and a site maintaining the structure of the CDR itself. However, the possible three-dimensional structure of the CDR is
(Ii) that the class of the canonical structure is determined not only by the CDRs but also by the type of amino acid at a particular position in the framework portion ( J. Mol. Bio
l., 196, 901-917, (1987)).

Based on this finding, it has been suggested that when using the CDR grafting method, it is necessary to graft amino acid residues of some frameworks to human antibodies in addition to CDR sequences (see Japanese Patent Application Laid-Open No. 4-502408). ).

Generally, an antibody derived from a non-human mammal having a CDR to be transplanted is defined as a "donor", and a human antibody to which the CDR is transplanted is defined as an "acceptor". I do.

A point to be considered when performing the CDR transplantation method is to preserve the CDR structure as much as possible and to maintain the activity of the immunoglobulin molecule. To this end: (i) which subgroup should be selected for the acceptor; and (ii) which amino acid residues should be selected from the donor framework. is there.

Queen et al. Proposed a design method in which the amino acid residues of the framework of the donor are transplanted into the acceptor together with the CDR sequence when the amino acid residues meet at least one of the following criteria (see Japanese Patent Application Laid-Open No. 4-502408). ): (A) Amino acids in the acceptor framework region are rare at that position, and the corresponding amino acids of the donor are common at said position of the acceptor. (b) the amino acid is in the immediate vicinity of one of the CDRs;

(C) It is expected that the amino acid has a side chain atom within about 3% of the CDR in a three-dimensional immunoglobulin model and is capable of interacting with an antigen or with the CDR of a humanized antibody.

However, there has been no example of successfully obtaining an IgG-type humanized anti-Fas antibody having apoptosis-inducing activity. Further, the anti-Fas monoclonal antibody of the present invention is a monoclonal antibody having novel characteristics not known hitherto. Therefore, there is no known example of humanizing the monoclonal antibody.

[0034]

SUMMARY OF THE INVENTION The first object of the present invention is to provide a method for treating abnormal cells as described above with Fas on the surface thereof.
A novel anti-Fas having a function of inhibiting apoptosis induced by binding of Fas ligand to Fas on normal cells.
It is to provide an antibody or a molecule similar thereto.

Several monoclonal antibodies specifically binding to human Fas are known, including those having apoptosis-inducing activity, but none of them has been found to bind to mouse Fas. Similarly, mouse Fa
Although some monoclonal antibodies that bind to s are known, none of them bind human Fas. That is, in evaluating the efficacy of the existing anti-human Fas monoclonal antibody as a medicine, the above-mentioned disease state model mice cannot be used. A second object of the present invention is to provide an anti-Fas antibody that binds to human Fas and mouse Fas, or a molecule similar thereto.

A third object of the present invention is to provide a preventive or therapeutic agent for the above-mentioned various diseases, which contains the above-mentioned anti-Fas antibody or a molecule similar thereto as an active ingredient.

[0037]

The present invention relates to (1) a molecule comprising an antigen-binding region specific to an epitope conserved between primate and non-primate Fas, 2) The molecule according to (1), wherein the primate is a human; (3) the molecule according to (1) or (2), wherein the non-primate is a rodent The molecule, (4) the molecule according to (3), wherein the rodent is a mouse, (5) having an antigen-binding region specific to an epitope conserved among mammalian Fas. (6) a mouse-mouse hybridoma HFE7A (FERM BP-582)
8) a monoclonal antibody produced by (7) a mouse-mouse hybridoma HFE7A (FERM B
(8) mouse-mouse hybridoma HFE7A (FERM BP-5828), which has all the same CDRs as the six CDRs derived from the anti-human Fas monoclonal antibody produced by P-5828).
A molecule having an antigen-binding region specific to an epitope recognized by a monoclonal antibody produced by (9), (9) an antibody,
(1) a molecule according to any one of (5), (7) or (8), (10) a heavy chain polypeptide comprising an amino acid sequence represented by the following general formula (I): _{-FRH 1 -CDRH 1 -FRH 2 -CDRH 2} -FRH 3 -CDRH 3 -FRH 4 - (I) ( wherein, FRH ₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids, CDRH ₁ is represents the amino acid sequence shown in SEQ ID NO: 2, FRH ₂ represents an arbitrary amino acid sequence consisting of 14 amino acids, CDRH ₂ represents the amino acid sequence shown in SEQ ID NO: 3 of sequence Listing, FRH ₃ is represents the 32 arbitrary amino acid sequence consisting of amino acids, CDRH ₃ represents the amino acid sequence shown in SEQ ID NO: 4 in sequence Listing, FRH ₄ is any consisting of 11 amino acids acids Represents the column, each amino acid is linked at each peptide bond) and, following consists of an amino acid sequence represented by the general formula (II) a light chain polypeptide: -FRL ₁ -CDRL ₁ -FRL _{2 -CDRL 2 -FRL 3 -CDRL 3 -FRL} 4 - (II) ( wherein, FRL ₁ represents an arbitrary amino acid sequence consisting of 23 amino acids, CDRL ₁ SEQ ID NO: in the sequence Listing 5
And FRL ₂ represents an arbitrary amino acid sequence consisting of 15 amino acids, and
L ₂ represents the amino acid sequence shown in SEQ ID NO: 6 of Sequence Listing, FRL ₃ represents an arbitrary amino acid sequence consisting of 32 amino acids, CDRL ₃ represents the amino acid sequence shown in SEQ ID NO: 7, FRL ₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, and each amino acid is linked by a peptide bond.) (11) A molecule characterized by having at least the following a) to f) (1) to (5) or (7), having the biological property according to any one of (1) to (4).
Or a molecule according to any one of (10): a) having apoptosis-inducing activity on T cells expressing Fas; b) having an effect of ameliorating autoimmune disease symptoms of MRLgld / gld mice; c) Does not induce liver damage; d) has a therapeutic or preventive effect against fulminant hepatitis induced by anti-mouse Fas monoclonal antibody Jo2; e) has a preventive effect against collagen-induced arthritis; f) synovium from a rheumatoid arthritis patient (12) having all of the biological properties described in (12) a) to (f), which have an apoptosis-inducing activity on cells;
(1) the molecule described in (13), which is an antibody; (10) the molecule described in any one of (12) to (12), (14) being humanized,
(9) or (13), (15) an immunoglobulin G-type antibody, (9) or (13), (16) humanized The antibody according to (6), (17) Fas;
Any of (1) to (5) or (7) to (15), which is capable of inducing apoptosis in abnormal cells expressing, and preventing apoptosis of normal cells. The molecule described in one, (1
8) (1) to (5), (7) to (13), (1)
A method for evaluating the effect of the molecule according to any one of 5) or (17) or the antibody according to (6) as a medicament for a disease state affected by the interaction of Fas / Fas ligand. (A) administering the molecule or the antibody to a model animal of the disease state, and comparing the change in the disease state between an animal to which the molecule or the antibody is administered and an untreated animal; 19) at least one human immunoglobulin light chain or a fragment thereof;
And, for each at least one human immunoglobulin heavy chain or fragment thereof, an antibody that specifically binds to an epitope conserved between primate and non-primate Fas, (20) a human immunoglobulin, which has an antigen-binding region specific to an epitope conserved between primate and non-primate Fas, which can be obtained by transplantation; The light and human immunoglobulin heavy chains each have the highest similarity of the variable region to an antibody that specifically binds to an epitope conserved between primate and non-primate Fas. (19) the humanized antibody according to (19), which is conserved between primate and non-primate Fas. Amino acids that are important for maintaining the structure of the antigen-recognition site of the antibody in the heavy and / or light chain framework regions of the antibody from a human immunoglobulin light chain or (19) The humanized antibody according to (19) or (20), which is grafted on a fragment thereof and / or a human immunoglobulin heavy chain or a fragment thereof. (22) SEQ ID NO: 1 in the sequence listing. Characterized by binding to a peptide consisting of an amino acid sequence,
(1) to (5), (7) to (15), (17) or the molecule or antibody according to any one of (19) to (21), (23) the amino acid of SEQ ID NO: 50 in the sequence listing No. 1 to No. 218, Amino acid sequence No. 1 to No. 218 of Sequence No. 52 in the Sequence Listing, Amino acid sequence No. 1 to No. 218 of No. 54 in the Sequence No., Sequence of Sequence No. Number 10
7 or amino acid numbers 1 to 2 of SEQ ID NO: 109 in the sequence listing.
(1) to (5), (7) to (15), (17) or (19), having a light chain polypeptide comprising any one of the amino acid sequences shown in 18.
The molecule or antibody according to any one of (1) to (22),
(24) A heavy chain polypeptide comprising any one of the amino acid sequences of amino acid numbers 1 to 451 of SEQ ID NO: 89 in the sequence listing or the amino acid sequences of amino acid numbers 1 to 451 of SEQ ID NO: 117 of the sequence listing. (1) to (5), (7) to (1)
(5) the molecule or antibody according to any one of (17) or (19) to (23), (25) a light chain comprising the amino acid sequence of amino acids 1 to 218 of SEQ ID NO: 50 in the sequence listing The molecule or the antibody according to (23) or (24), comprising a polypeptide and a heavy chain polypeptide comprising an amino acid sequence represented by amino acid numbers 1 to 451 of SEQ ID NO: 89 in the sequence listing. (26) a light chain polypeptide comprising the amino acid sequence of amino acid Nos. 1 to 218 of SEQ ID NO: 107 in the sequence listing, and a heavy chain polypeptide comprising the amino acid sequence of amino acid Nos. 1 to 451 of SEQ ID NO: 117 of the sequence listing. (27) (1) The molecule or antibody according to (23) or (24), which is composed of a chain polypeptide.
To (5), (7) to (15), (17) or (1)
9) A DN encoding any one of the molecules or the polypeptide chains in an antibody according to any one of (26) to (26).
A, (28) DN encoding the light or heavy chain polypeptide of the antibody according to (6) or (16).
A, (29) Amino acid number 1 of SEQ ID NO: 11 in the sequence listing
(30) a DNA encoding a polypeptide comprising the amino acid sequence represented by amino acid numbers 1 to 445 of SEQ ID NO: 9 in the sequence listing, (31) an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 50 in the sequence listing; an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 52 in the sequence listing; From 2
Amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 107 in Sequence Listing
Amino acid numbers 1 to 218 of the amino acid sequence or amino acid numbers 1 to 21 of SEQ ID NO: 109 in the sequence listing
(32) a DNA encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 8; (32) an amino acid sequence of amino acid numbers 1 to 451 of SEQ ID NO: 89 in the sequence listing or SEQ ID NO: 117 of the sequence listing Encoding a polypeptide comprising any one of the amino acid sequences represented by amino acid numbers 1 to 451 of
NA, (33) the DNA according to (29), which comprises the nucleotide sequence of nucleotides 61 to 393 of SEQ ID NO: 10 in the sequence listing, (34) nucleotides 61 to 714 of SEQ ID NO: 10 in the sequence listing. (2) comprising the nucleotide sequence shown in
DNA according to (30), which comprises the nucleotide sequence represented by nucleotide numbers 58 to 420 of SEQ ID NO: 8 in the sequence listing, (35)
6) The DNA according to (30), comprising the nucleotide sequence of SEQ ID NO: 8 from nucleotide number 58 to 1392, (37) the nucleotide sequence of SEQ ID NO: 49 shown as nucleotide number 100 to 753. Nucleotide sequence, a nucleotide sequence represented by nucleotide numbers 100 to 753 of SEQ ID NO: 51 in the sequence listing,
Nucleotide numbers 100 to 7 of SEQ ID NO: 53 in the sequence listing
53, SEQ ID NO: 1 in the sequence listing
(31) comprising the nucleotide sequence of SEQ ID NO: 06 from nucleotide numbers 100 to 753 or the nucleotide sequence of SEQ ID NO: 108 from nucleotide numbers 100 to 753,
8) comprising the nucleotide sequence represented by nucleotide numbers 84 to 2042 of SEQ ID NO: 88 in the sequence listing or the nucleotide sequence represented by nucleotide numbers 78 to 2036 of SEQ ID NO: 116 in the sequence listing, (3)
2) The DNA described in (39), a recombinant DNA vector comprising the DNA described in any one of (27) to (38), (40) (29), (31), (3)
(3) a recombinant DNA vector comprising the DNA of any one of (34) or (37);
1) a recombinant DNA vector comprising the DNA of any one of (30), (32), (35), (36) or (38), (42) the recombination of (39) A host cell transformed with a DNA vector, (4
3) Recombinant D according to (40) and / or (41)
A host cell transformed with an NA vector, (44)
(40) The host cell according to any one of (41) to (43), wherein the host cell is transformed with the recombinant DNA vector according to (41) or (41), which is derived from a mammal. Host cells, (46) transformed E. coli
coli pME-L (FERM BP-5867),
(47) Transformed E. coli. coli pME-H
(FERM BP-5868), (48) Transformed E. coli. coli pHSGMM6 SANK 736
97 (FERM BP-6071), (49). coli pHSGHM17 SANK
73597 (FERM BP-6072), (50)
Transformed E. coli coli pHSGHH7 SAN
K 73497 (FERM BP-6073), (5
1) Transformed E. coli coli pHSHM2 S
ANK 70198 (FERM BP-6272),
(52) Transformed E. coli. coli pHSHH5
SANK 70398 (FERM BP-627
4), (53) Transformed E. coli. coli pgH
SL7A62 SANK73397 (FERM BP-
6074), (54). coli
pgHPDH3 SANK70298 (FERM B
(P-6273), (55) A method for producing an anti-Fas antibody, comprising culturing the host cell according to (44) or (45), and then recovering the anti-Fas antibody from the culture, (56) Mouse-mouse hybridoma HFE7
A (FERM BP-5828), (57) mouse-
Mouse hybridoma HFE7A (FERM BP-5
828) under conditions capable of producing immunoglobulin, and then recovering the immunoglobulin from the culture, the method for producing an antibody according to (6),
8) (1) to (5), (7) to (15), (1)
7) or a disease caused by an abnormality in the Fas / Fas ligand system, which comprises the molecule according to any one of (19) to (26) or the antibody according to (6) or (16) as an active ingredient. Prophylactic or therapeutic agent, (59) Fa
An autoimmune disease (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture syndrome) ,
Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus), allergies,
Atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis,
It consists of aplastic anemia, hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C, B, D) or alcoholic hepatitis), acquired immunodeficiency syndrome and rejection after organ transplantation The prophylactic or therapeutic agent according to (58), which is selected from the group; (60) Fa.
An autoimmune disease (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture syndrome) ,
(58) characterized by Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, and insulin-dependent diabetes mellitus. ) Or the prophylactic or therapeutic agent according to (59), (61) wherein the disease caused by an abnormality in the Fas / Fas ligand system is allergy,
8) or the prophylactic or therapeutic agent according to (59), (62)
(63) The prophylactic or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the Fas / Fas ligand system is rheumatoid arthritis.
The preventive or therapeutic agent according to (58) or (59), wherein the disease caused by an abnormality in the / Fas ligand system is atherosclerosis, (64) A disease caused by an abnormality in the Fas / Fas ligand system (58) or (59), wherein the disease caused by abnormality of the Fas / Fas ligand system is glomerulonephritis. The prophylactic or therapeutic agent according to (58) or (59),
(66) the disease caused by abnormal Fas / Fas ligand system is aplastic anemia, (5)
8) or the prophylactic or therapeutic agent according to (59), (67)
Diseases caused by abnormal Fas / Fas ligand system are hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C,
(58) or (59), wherein the disease caused by abnormality of the Fas / Fas ligand system is characterized by being hepatitis B, hepatitis D) or alcoholic hepatitis). The preventive or therapeutic agent according to (58) or (59), which is characterized by acquired immunodeficiency syndrome, (69) a disease caused by an abnormality in the Fas / Fas ligand system is rejection after organ transplantation. The prophylactic or therapeutic agent according to (58 or 59), which is characterized in that:

The portion where the amino acid sequence is conserved between human and mouse is defined as an epitope, and the human and mouse F
When a normal mouse such as a BALB / c mouse is immunized with human Fas in an attempt to obtain a mouse monoclonal antibody that binds to any of Are eliminated in the thymus as antibody-producing cells. It is presumed that the Fas-Fas ligand system is involved in the removal of self-reactive T cells in the thymus [Shin Yonehara (1994)
See Nikkei Science Supplement 110, 66-77] from mice in which the expression of the Fas-encoding gene has been disabled (hereinafter referred to as “Fas”).
When a mouse deficient in the Fas-Fas ligand system such as "s knockout mouse") is used as an immunized animal, an antibody that also binds to mouse Fas can be obtained. However, the immunized animal used in the present invention is not limited to the Fas knockout mouse.
Those deficient in the -Fas ligand system can be used as the immunized animal of the present invention.

The present inventors have produced a novel anti-Fas monoclonal antibody that binds to human and mouse Fas by immunizing a Fas knockout mouse with human Fas and fusing spleen cells of the mouse with mouse myeloma cells. A hybridoma was produced, and the monoclonal antibody was purified from the culture supernatant. This novel anti-Fas monoclonal antibody induced apoptosis in T cells of mouse and non-human primates expressing Fas. That is, according to the present invention, at least the primate F
As and rodent Fas, it was revealed that there is an epitope that can be commonly recognized by the antibody of the present invention and is capable of inducing apoptosis by binding to the antibody of the present invention.

The novel anti-Fas monoclonal antibody had an effect of alleviating the symptoms of an autoimmune disease model mouse. Even more surprisingly, this new anti-Fa
It has been found that the s monoclonal antibody has no side effect of inducing hepatic injury, which has been known for conventional anti-Fas antibodies having apoptosis-inducing activity. Further, the present inventors cloned the genes encoding the heavy and light chains of the novel anti-Fas monoclonal antibody from the above hybridomas, elucidated the nucleotide sequences thereof, and analyzed the nucleotide sequences encoded by those genes. The amino acid sequence of the CDRs in the chain and light chain amino acid sequences was determined. The present inventors have also prepared expression vectors comprising the genes encoding the heavy and light chains, respectively, and used them in culture supernatants obtained by co-transfection of cultured animal cells with these vectors. Produced a recombinant anti-Fas antibody that reacts with Fas. The thus-obtained anti-Fas antibody and its recombinant have an activity of protecting the liver from fulminant hepatitis caused by Fas-mediated apoptosis, and are also effective for the prevention and treatment of rheumatoid arthritis, respectively. From
The antibody has an activity of inducing apoptosis through Fas in abnormal cells and an activity of inhibiting apoptosis of normal cells through Fas in abnormal cells, and is effective in preventing or treating diseases caused by abnormal Fas / Fas ligand system. It has been found. The present inventors have determined that this mouse-derived anti-F
By transplanting the CDR amino acid sequence of the as monoclonal antibody into a human antibody, we succeeded in producing a recombinant antibody having no human immunogenicity while retaining the activity as an anti-Fas antibody, and completed the present invention. I let it.

When a monoclonal antibody commonly recognizing a homologous protein derived from a heterologous animal binds to a protein antigen, the amino acid sequence is conserved between the heterologous animals around the binding site (epitope) on the antigen side. It is thought that the amino acid sequence homology is often kept high.
The epitope in the s molecule is not limited to only the region where such primary structural homology is maintained. That is,
In the present invention, the "epitope conserved between primate and non-primate Fas" means that homology in higher-order structure is maintained between heterologous animals, and as a result, the homology is recognized by a single monoclonal antibody. And regions that can be used.

In the present invention, the term "antigen binding region" refers to an intramolecular region having the same function as an antigen binding region in an antibody molecule. This antigen binding region is not limited to those derived from a particular antibody or combination of antibodies.
Furthermore, the antigen-binding region and the binding region for an antigen in a specific antibody molecule need only be similar in that they can bind to the antigen. Is not a requirement of the antigen binding region. Therefore, for example, even when the antigen-binding region is designed based on the CDRs of a known antibody and transplanted into a human antibody, the resulting antigen-binding region is not necessarily similar to the antigen-binding region of the known antibody. However, from the viewpoint of maintaining the specific binding ability to the antigen, the similarity between the two is preferably high.

The “molecule characterized by having an antigen-binding region” in the present invention is preferably an antibody, but is not limited thereto. Any molecule whose antigen-binding region binds to an epitope in a Fas molecule can be used. I just need. Thus, for example, the antigen binding region may be added to the support of an affinity purification column. However, hereinafter, unless otherwise stated, "molecules of the invention" will generally refer to antibodies.

When the molecule of the present invention is an antibody, the antibody may be immunoglobulin G (IgG) or A (I
Any of gA), E (IgE) and M (IgM) can be preferably used, but IgG is usually more preferable.

In the present invention, the immunogenicity to humans while retaining the function as an antibody can be obtained by grafting at least the entire amino acid sequence of the CDR from an antibody derived from a non-human animal to a human antibody as described above. An artificially modified antibody in which is reduced is referred to as a “humanized antibody”.

In the present invention, "FR" indicates a framework region. For example, FRH ₁ refers to the framework region present in most N-terminal of the heavy chain subunit variable region, FRL ₄ refers to the fourth framework region from the N-terminus in the light chain subunit variable region. Similarly, for example, CDRH ₁ refers to the CDR present in the most N-terminal of the heavy chain subunit variable region, CDR
L ₃ refers to the third CDR from the N-terminus in the light chain subunit variable region.

[0047]

BEST MODE FOR CARRYING OUT THE INVENTION The anti-Fas monoclonal antibody of the present invention can
And culturing a hybridoma obtained by fusing spleen cells of the mouse with mouse myeloma cells.

In producing a monoclonal antibody,
At least the following work steps are required. That is, (a) purification of a biopolymer used as an antigen,
(B) a step of preparing an antibody-producing cell after immunizing an animal by injecting the antigen with an antigen, collecting blood and assaying the antibody titer to determine the timing of splenectomy, and (c) myeloma cells (Hereinafter referred to as "myeloma"), (d) cell fusion between antibody-producing cells and myeloma, (e) selection of hybridomas producing the desired antibody, and (f) division into single cell clones (cloning). ), (G) cultivation of hybridomas for producing monoclonal antibodies in large quantities, or breeding of animals transplanted with hybridomas, (h) biological activity of monoclonal antibodies thus produced, and recognition thereof Examination of specificity,
Alternatively, it is a test of characteristics as a labeling reagent.

Hereinafter, a method for preparing an anti-Fas monoclonal antibody will be described in detail along the above steps. However, the method for preparing the antibody is not limited thereto. it can.

(A) Purification of antigen As antigens, the extracellular region of human Fas and the extracellular region of mouse interleukin 3 receptor (hereinafter referred to as "IL3R") [Nishimura, Y. et al. (1995) J. Immunol. 15
4, 4395-4403], a recombinant protein obtained by introducing and expressing a fusion protein expression vector phFas-AIC2 into a monkey-derived cell line COS-1 and partially purifying the same (hereinafter referred to as “recombinant human Fas”). ) Is valid. phFas-AIC2 can be prepared by incorporating a DNA encoding a fusion protein of human Fas and mouse IL3R into an expression vector pME18S for animal cells. However, the DNA, vector, host and the like encoding Fas are not limited to these.

Specifically, CO is used in phFas-AIC2.
A transformant obtained by transforming S-1 cells is cultured, and human Fas and mouse I produced in the culture supernatant are cultured.
Recombinant Fas can be partially purified by performing ion exchange chromatography on the L3R fusion protein using a Resource Q column (trade name; manufactured by Pharmacia).

In addition, F present on the cell membrane of a human cell line
Purified as itself can also be used as the antigen. Furthermore, the primary structure of Fas is known [I
toh, N., et al. (1991) Cell 66, 233-243], a peptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing was chemically synthesized by a method well known to those skilled in the art. This can also be used as an antigen.

(B) Preparation of antibody-producing cells The antigen obtained in the step (a) is mixed with an adjuvant such as Freund's complete or incomplete adjuvant or alum, to immunize a laboratory animal as an immunogen. I do. Fas knockout mice are most preferably used as experimental animals, and such mice are described in Senju et al.
u, S., et al (1996) International Immunology 8, 42
3].

The method of immunogen administration for mouse immunization may be any of subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, and intramuscular injection, but subcutaneous injection or intraperitoneal injection is preferred.

The immunization can be performed once or multiple times at appropriate intervals (preferably at intervals of one to five weeks). Thereafter, the antibody titer against the antigen in the serum of the immunized animal is measured, and the effect of the subsequent operation can be enhanced by using the animal whose antibody titer is sufficiently high as a source of antibody-producing cells. Generally, 3 to 3 days after the last immunization
It is preferable to use antibody-producing cells derived from an animal 5 days later for cell fusion later.

The antibody titers used herein include radioisotope immunoassay (hereinafter referred to as "RIA"), enzyme-linked immunosorbent assay (hereinafter referred to as "ELISA"), fluorescent antibody assay, and passive immunoassay. Various known techniques such as a hemagglutination method can be mentioned, but from the viewpoints of detection sensitivity, speed, accuracy, and possibility of automation of the operation, the RIA method or the ELISA method is more preferable.

In the measurement of the antibody titer in the present invention, for example, E
According to the LISA method, it can be performed according to the procedure described below. First, purified or partially purified Fa
s is adsorbed on a solid surface such as a 96-well plate for ELISA, and a solid surface on which no antigen is adsorbed is exposed to a protein unrelated to the antigen, for example, bovine serum albumin (hereinafter referred to as “B
After washing the surface, the sample is contacted with a serially diluted sample (for example, mouse serum) as the first antibody, and the anti-Fas antibody in the sample is bound to the antigen. Further, an antibody against a mouse antibody that is enzyme-labeled as a second antibody is added and allowed to bind to the mouse antibody.After washing, a substrate of the enzyme is added, and the change in absorbance due to color development based on the decomposition of the substrate is measured to determine the antibody titer. calculate.

(C) Step of Preparing Myeloma Myeloma is generally used as a cell line obtained from a mouse, for example, an 8-azaguanine-resistant mouse (BALB)
/ C) Myeloma strain P3X63Ag8U.1 (P3-U1) [Yelton,
DE et al. Current Topics in Microbiology and Imm
unology, 81, 1-7 (1978)], P3 / NSI / 1-Ag4-1 (NS-1)
[Kohler, G. et al. European J. Immunology, 6, 511
-519 (1976)], Sp2 / O-Ag14 (SP-2) [Shulman, M.
et al. Nature, 276, 269-270 (1978)], P3X63Ag8.653
(653) [Kearney, JF et al. J. Immunology, 12
3, 1548-1550 (1979)], P3X63Ag8 (X63) [Horibata,
K. and Harris, AW Nature, 256, 495-497 (1975)]
It is preferable to use such as. These cell lines are grown in a suitable medium, for example, 8-azaguanine medium [RPMI-16
A medium in which 8-azaguanine is added to a medium in which glutamine, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter, referred to as “FCS”) are added to 40 media.
, Iscove's Modified Dulbecco's Medium
ulbecco's Medium (hereinafter referred to as "IMDM") or Dulbecco's Modified Eagle's medium
Eagle Medium (hereinafter referred to as "DMEM"), but 3-4 days before cell fusion, a normal medium [eg, 10
% FCS-containing ASF104 medium (manufactured by Ajinomoto Co., Inc.)], and secure 2 × 10 ⁷ or more cells on the day of fusion.

(D) Cell fusion The antibody-producing cells are plasma cells and lymphocytes which are precursor cells thereof, which may be obtained from any site of an individual. Alternatively, they can be obtained from a combination of these as appropriate, and splenocytes are most commonly used.

After the final immunization, a site where antibody-producing cells are present, for example, a spleen is removed from a mouse having a predetermined antibody titer, and spleen cells as antibody-producing cells are prepared. Currently, the most commonly used means for fusing the spleen cells with the myeloma obtained in step (c) is a method using polyethylene glycol, which has relatively low cytotoxicity and simple fusion operation. This method includes, for example, the following procedure.

The spleen cells and myeloma were thoroughly washed with a serum-free medium (for example, RPMI 1640) or a phosphate buffered saline (hereinafter referred to as “PBS”), and the ratio of spleen cells to myeloma was 5: 1 to 1: 1. Mix to about 10: 1 and centrifuge. The supernatant was removed, and the precipitated cell group was thoroughly loosened. Then, 1 ml of 50% (w
/ V) polyethylene glycol (molecular weight 1000 to 40)
00) is dropped. Then 10ml
And slowly centrifuge. The supernatant was discarded again, and the precipitated cells were placed in a normal medium (hereinafter referred to as "HAT") containing an appropriate amount of hypoxanthine / aminopterin / thymidine (hereinafter referred to as "HAT") solution and mouse interleukin-2 (hereinafter referred to as "IL-2"). Culture plate (hereinafter referred to as "plate")
And incubate at 37 ° C. for about 2 weeks in the presence of 5% carbon dioxide. HAT medium is supplemented as needed on the way.

(E) Selection of Hybridoma Group When the myeloma cells are 8-azaguanine-resistant strains, that is, when they are hypoxanthine / guanine / phosphoribosyltransferase (HGPRT) -deficient strains, the unfused myeloma cells , And fusion cells of myeloma cells cannot survive in a HAT-containing medium. On the other hand, a fusion cell between antibody-producing cells or a hybridoma of an antibody-producing cell and a myeloma cell can survive, but a fusion cell between antibody-producing cells has a life span. Therefore, by continuing the culture in the HAT-containing medium, only the hybridoma between the antibody-producing cell and the myeloma cell survives, and as a result, the hybridoma can be selected.

For the hybridoma that has grown in a colony, the medium is replaced with a medium (hereinafter, referred to as an “HT medium”) from which the aminopterin is removed from the HAT medium. Thereafter, a part of the culture supernatant is collected, and the anti-Fas antibody titer is measured, for example, by ELISA. However, when the above fusion protein is used as an antigen for ELISA, it is necessary to remove the clone so as not to select a clone that produces an antibody that specifically binds to the extracellular region of the mouse IL3 receptor. . The presence or absence of such a clone can be confirmed, for example, by ELISA using the mouse IL3 receptor or its extracellular region as an antigen.

Although the method using an 8-azaguanine-resistant cell line has been described above, other cell lines can be used according to the selection method of the hybridoma, and in that case, the composition of the medium used also changes.

(F) Cloning A hybridoma that has been found to produce a specific antibody by measuring the antibody titer in the same manner as described in step (b) is transferred to another plate and cloned. As the cloning method, a limiting dilution method in which one hybridoma is diluted and cultured so that one hybridoma is contained in one well of the plate, a soft agar method in which the colony is collected by culturing in a soft agar medium, and a micromanipulator one by one. Method of removing and culturing cells from a cell sorter
A "sort clone" for separating individual cells may be mentioned, but the limiting dilution method is simple and often used.

For the wells in which the antibody titer has been recognized, cloning by, for example, the limiting dilution method is repeated 2 to 4 times, and those having a stable antibody titer are selected as anti-Fas monoclonal antibody-producing hybridoma strains. further,
By selecting a hybridoma producing an antibody that binds to mouse Fas in the same manner, an anti-Fas monoclonal antibody-producing cell is obtained. The mouse Fas used at this time includes, for example, an expression plasmid vector pME18 for DNA encoding a fusion protein of the extracellular region of mouse Fas and the extracellular region of mouse IL3 receptor.
S-mFas-AIC [Nishimura, Y. et al. (1995)
J. Immunol. 154, 4395-4403] was introduced into cultured animal cells and expressed.
or cells expressing mouse Fas on the cell surface can be used.

The anti-Fas antibody suitable as a base antibody for preparing the humanized anti-Fas antibody of the present invention
Mouse-mouse hybridoma HFE7A, which is a cell producing a monoclonal antibody, has been internationally deposited on Feb. 19, 1997 with the Institute of Biotechnology, Industrial Science and Technology, and has the accession number FERM BP-5828. Therefore, for example, when preparing an antibody using the mouse-mouse hybridoma HFE7A, the preparation of the antibody can be performed from the step (g) described below, omitting the step (f).

(G) Preparation of Monoclonal Antibody by Hybridoma Culture After completion of cloning, the culture medium was HT
The medium is replaced with a normal medium and cultured. Large-scale culture is performed by rotary culture using a large culture bottle or spinner culture. The supernatant in this large-scale culture is purified using a method well known to those skilled in the art, such as gel filtration, so that the anti-Fas containing the prophylactic or therapeutic agent of the present invention as an active ingredient is obtained.
A monoclonal antibody can be obtained. In addition, mice of the same strain (for example, BALB / c described above) or N
By growing the hybridoma in the abdominal cavity of a u / Nu mouse, ascites containing a large amount of an anti-Fas monoclonal antibody contained as an active ingredient of the prophylactic or therapeutic agent of the present invention can be obtained. As a simple method of purification,
A commercially available monoclonal antibody purification kit (for example, MAb
Trap GII kit; manufactured by Pharmacia) and the like can also be used.

The monoclonal antibody thus obtained is
It has high antigen specificity for human and mouse Fas.

(H) Assay of Monoclonal Antibody The isotype and subclass of the monoclonal antibody thus obtained can be determined as follows. First, as an identification method, Ouchterlony (Ouchterlon
y) method, ELISA method, or RIA method.
The Otterlony method is simple, but requires a concentration operation when the concentration of the monoclonal antibody is low. on the other hand,
When the ELISA method or the RIA method is used, the culture supernatant is allowed to react with the antigen-adsorbed solid phase as it is, and various immunoglobulin isotypes and antibodies corresponding to subclasses are used as secondary antibodies, thereby isolating the monoclonal antibody isotype. It is possible to identify subclasses. Further, as a simpler method, a commercially available identification kit (for example, mouse typer kit; manufactured by Bio-Rad) can be used.

Further, protein quantification was performed by the Foreign Lowry method and the absorbance at 280 nm [1.4.
(OD280) = immunoglobulin 1 mg / ml].

The identification of the recognition epitope of the monoclonal antibody can be performed as follows. First, various partial structures of the molecule recognized by the monoclonal antibody are prepared. In preparing the partial structure, a method for preparing various partial peptides of the molecule using a known oligopeptide synthesis technique, a DNA sequence encoding the desired partial peptide using a gene recombination technique into a suitable expression plasmid Although there are methods for integration and production inside and outside the host such as Escherichia coli, it is common to use both in combination for the above purpose. For example, a series of polypeptides, which are sequentially shortened with an appropriate length from the C-terminus or N-terminus of an antigen protein, are prepared using a gene recombination technique well known to those skilled in the art, and the reactivity of the monoclonal antibody against them is examined. , Roughly determine the recognition site.

Thereafter, the oligopeptide of the corresponding portion, or a mutant of the peptide, etc., are further variously synthesized using oligopeptide synthesis techniques well known to those skilled in the art.
The epitope is limited by examining the binding of the monoclonal antibody contained as an active ingredient of the prophylactic or therapeutic agent of the present invention to those peptides, or examining the competitive inhibitory activity of the peptide on the binding between the monoclonal antibody and the antigen. As a simple method for obtaining various oligopeptides, commercially available kits (for example, SPOTs
A kit (manufactured by Genosis Biotechnologies), a series of multipin peptide synthesis kits using the multipin synthesis method (manufactured by Chiron), and the like can also be used.

Next, the DNA encoding the heavy or light chain of the anti-Fas monoclonal antibody of the present invention is
mRNA was prepared from the hybridoma cells producing the monoclonal antibody, and the mRNA was cDN
After conversion to A, the DNA can be obtained by isolating DNAs encoding the heavy chain or light chain of the antibody, respectively.

For the extraction of mRNA, a guanidine thiocyanate hot phenol method, a guanidine thiocyanate-guanidine hydrochloric acid method or the like can be employed, but a guanidine thiocyanate cesium chloride method is preferred. Preparation of mRNA from cells involves first
A is prepared and poly (A) ⁺ R such as oligo (dT) cellulose or oligo (dT) latex beads is prepared from the total RNA.
It can be carried out by a method of purifying using a carrier for NA purification or a method of directly purifying from a cell lysate using the carrier. As a method for preparing total RNA, alkaline sucrose density gradient centrifugation [Dougherty, WG and Hiebert,
E. (1980) Viology 101, 466-474], a guanidine thiocyanate / phenol method, a guanidine thiocyanate / trifluorocesium method, a phenol / SDS method and the like can be employed, but a method using guanidine thiocyanate and cesium chloride [Chirgwin, JM, et al. (197
9) Biochemistry 18, 5294-5299].

The poly (A) ⁺ R obtained as described above
Single-stranded cDN by reverse transcriptase reaction using NA as a template
After the synthesis of A, the double-stranded cDN
A can be synthesized. This method includes the S1 nuclease method [Efstratiadis, A., et al. (1976) Cel17,
279-288], the Gubler-Hoffman method [Gubler, U. a.
nd Hoffman, BJ (1983) Gene 25, 263-269],
The Okayama-Berg method [Okayama, H. and Berg, P. (198
2) Mol. Cell. Biol. 2, 161-170], but in the present invention, the polymerase chain reaction (hereinafter referred to as “PCR”) using single-stranded cDNA as a template [Saik
i, RK, et al. (1988) Science 239, 487-49]
The so-called RT-PCR method is preferred.

The double-stranded cDNA thus obtained is incorporated into a cloning vector, the resulting recombinant vector is introduced into a microorganism such as Escherichia coli and transformed, and transformation is performed using tetracycline resistance or ampicillin resistance as an index. You can choose the body. Transformation of E. coli is performed by the Hanahan method [Hanahan, D. (1983) J. Mol. Bio.
l. 166, 557-580], that is, by adding the recombinant DNA vector to competent cells prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. When a plasmid is used as a vector, it is necessary to have the above-mentioned drug resistance gene. It is also possible to use cloning vectors other than plasmids, for example, lambda phage and the like.

From the transformant obtained above, cD encoding each subunit of the desired anti-human Fas antibody was obtained.
As a method for selecting a strain having NA, for example, the following various methods can be adopted. When the target cDNA is specifically amplified by the RT-PCR method, these operations can be omitted.

(1) Method Using Polymerase Chain Reaction When all or a part of the amino acid sequence of the target protein has been elucidated, oligonucleotide primers of a sense strand and an antisense strand corresponding to a part of the amino acid sequence are synthesized. And combining them, the polymerase chain reaction [Saiki, RK, et al. (1988) Science
239, 487-49] to amplify a DNA fragment encoding the desired anti-human Fas antibody heavy or light chain subunit. As the template DNA used here, for example, mRN of a hybridoma (FERM BP-5828) producing an anti-human Fas monoclonal antibody HFE7A is used.
CDNA synthesized from A by reverse transcriptase reaction can be used.

The DNA fragment thus prepared can be directly incorporated into a plasmid vector using a commercially available kit or the like, or the fragment is labeled with ³² P, ³⁵ S, biotin, or the like, and this is used as a probe. The target clone can also be selected by performing colony hybridization or plaque hybridization using the DNA.

For example, the monoclonal antibody HFE7A suitable as the anti-Fas monoclonal antibody of the present invention is an immunoglobulin G1 (hereinafter referred to as “IgG1”) molecule, and each of the heavy chain (γ1 chain) and light chain (κ chain) It is a complex consisting of units. As a method for examining the partial amino acid sequence of each of these subunits, each subunit is isolated using a well-known method such as electrophoresis or column chromatography, and then an automatic protein sequencer (for example, manufactured by Shimadzu Corporation) A method of analyzing the N-terminal amino acid sequence of each subunit using PPSQ-10) or the like is preferable.

From the target transformant obtained as described above, a cDNA encoding each subunit of the anti-human Fas monoclonal antibody protein can be collected by a known method [Maniatis, T., et al. (1982) in "Mo
lecular Cloning A LaboratoryManual "Cold Spring Ha
rbor Laboratory, NY.]. For example, it can be carried out by separating a fraction corresponding to vector DNA from cells and cutting out a DNA region encoding a desired subunit from the plasmid DNA.

(2) Screening Method Using Synthetic Oligonucleotide Probes When the entire or partial amino acid sequence of the target protein has been elucidated (if the sequence is a plurality of consecutive specific sequences, Region), and synthesize an oligonucleotide corresponding to the amino acid sequence (in this case, any of a nucleotide sequence deduced with reference to codon usage or a plurality of nucleotide sequences obtained by combining possible nucleotide sequences) Can be used, and in the latter case, the type can be reduced by including inosine), which is used as a probe (labeled with ³² P, ³⁵ S or biotin, etc.) as
Hybridization is performed with a nitrocellulose filter in which NA is modified and fixed, and the obtained positive strain is selected.

The sequence of the DNA thus obtained can be determined, for example, by the Maxam-Gilbert chemical modification method [Maxa
m, AM and Gilbert, W. (1980) in "Methods in Enz
ymology "65, 499-576] and dideoxynucleotide chain termination [Messing, J. and Vieira, J. (1982) Gene 1
9, 269-276].

In recent years, an automatic base sequence determination system using a fluorescent dye has become widespread (for example, a sequence robot “CATALYST 800” manufactured by PerkinElmer Japan and a model 373A DNA sequencer).

By using such a system, DN
The A nucleotide sequencing operation can be performed efficiently and safely. From the nucleotide sequence of the DNA of the present invention thus determined and the N-terminal amino acid sequence data of the heavy and light chains, the entire amino acid sequence of the heavy and light chains of the monoclonal antibody of the present invention is determined. Can be.

Each of the heavy and light chains of the immunoglobulin is composed of a variable region and a constant region, and the variable region further comprises a complementarity determining region (hereinafter referred to as “CDR”; both heavy and light chains have three positions each). And framework regions adjacent to them (four at each of heavy and light chains).

Among them, the amino acid sequence of the constant region is common between antibodies having the same immunoglobulin subclass regardless of the type of antigen. On the other hand, the variable region, particularly the amino acid sequence of the CDR is unique to each antibody,
Studies comparing the amino acid sequence data of many antibodies have shown that CDR positions and framework sequence lengths are similar between antibody subunits belonging to the same subgroup [ Kabat, EA, et a
l. (1991) in "Sequence of Proteins of Immunologica
l Interest Vol.II ": US Department of Health and
See Human Services]. Therefore, for example, by comparing each amino acid sequence of the heavy chain and light chain of an antibody such as the anti-Fas monoclonal antibody HFE7A with known amino acid sequence data, the positions of CDRs, framework regions and constant regions in each amino acid sequence can be determined. Can be determined. The chain length of FRH ₁ , that is, the length of the framework region closest to the N-terminal side of the heavy chain is usually (30
Amino acids), for example, mouse IgG
As an example of the same subtype as HFE7A, there is known an example in which the framework region has a minimum of 18 amino acids [see Kabat et al., Supra]. From the above, in the antibody of the present invention, the chain length of the N-terminal framework region of the heavy chain should be 18 amino acids or more and 30 amino acids or less, as long as the function as an anti-Fas antibody is not impaired. It is. Note that amino acid number of the immunoglobulin heavy chain of the present invention, FRH ₁ unless otherwise stated it is assumed in the case of the 30 amino acids.

The peptide having the same amino acid sequence as the CDR of the light chain or heavy chain determined as described above, or a continuous partial amino acid sequence therein, is subjected to an artificial modification operation to obtain the peptide. By bringing the CDR closer to the three-dimensional structure formed in the anti-Fas antibody molecule, it is possible to impart binding activity to Fas alone [see, for example, US Pat. No. 5,331,573]. Accordingly, a peptide having the same amino acid sequence as that of the CDR or a continuous partial amino acid sequence therein so modified is also encompassed in the molecule of the present invention.
It can be used for prevention or treatment of diseases caused by abnormal Fas-Fas ligand system.

In preparing a variant in which one or more amino acids in the amino acid sequence have been deleted, a cassette mutation method [Toshimitsu Kishimoto, “Natural Chemistry Experiment Course 2, Nucleic Acid III, Recombinant DNA Technology” See 242-251].

[0091] Such various kinds of DNAs can be obtained, for example, by the phosphite triester method [Hunkapiller, M., et al.
984) See Nature 310, 105-111] and the like, and can be produced by chemical synthesis of nucleic acid. The codon for the desired amino acid is known per se, and its selection may be arbitrarily determined. For example, the codon for the desired amino acid may be determined according to a conventional method in consideration of the codon usage of the host to be used. These nucleotide sequence codons can be partially modified by a site-directed mutagenesis method (cytospecific mutagenesis) using a primer comprising a synthetic oligonucleotide encoding the desired modification [Mark, D. et al.
F., et al. (1984) Proc. Natl. Acad. Sci. USA 81, 5
662-5666].

Whether a certain DNA hybridizes with DNA encoding the heavy chain or light chain of the anti-Fas monoclonal antibody of the present invention can be determined, for example, by the random primer method [Feinberg, AP and Vogelstein,
B. (1983) Anal. Biochem. 132, 6-13] and the nick translation method [Maniatis, T., et al. (1982)
in "Molecular Cloning A laboratory Manual" Cold Sp
ring Harbor Laboratory, according NY. Browse etc. ^[alpha-32
[P] dCTP can be examined by performing an experiment as described below using a probe DNA labeled with dCTP or the like.

First, the DNA to be examined is adsorbed on, for example, a nitrocellulose membrane or a nylon membrane, and if necessary, treated with alkali modification or the like, and then solidified by heating or ultraviolet rays. This film was subjected to 6 × SSC
(1 × SSC is 0.15M sodium chloride, 0.01
5M Trisodium citrate solution), 5% Denhardt's solution, and 0.1% sodium dodecyl sulfate (SDS). Pre-hybridize solution, keep at 55 ° C for 4 hours or more. 1 × 1 final specific activity in prehybridization solution
It added to a 0 ⁶ cpm / ml, incubated overnight at 60 ° C.. Thereafter, the operation of washing the membrane with 6 × SSC at room temperature for 5 minutes is repeated several times, and further washed with 2 × SSC for 20 minutes, followed by autoradiography.

Using the above method, any cD
From an NA library or a genomic library, DNA that hybridizes to DNA encoding the heavy or light chain of the anti-Fas monoclonal antibody underlying the humanized anti-Fas antibody of the present invention can be isolated [Maniat
is, T., et al. (1982) in "Molecular Cloning A labo
ratory Manual "Cold Spring Harbor Laboratory, NY.
reference].

By incorporating each DNA obtained as described above into an expression vector, it is possible to introduce them into prokaryotic or eukaryotic host cells, and to express each gene in those host cells. .

Examples of prokaryotic host cells include Escherichia coli and Bacillus subtilis.
) And the like. In order to express the gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, i.e., a plasmid vector containing an origin of replication and a promoter sequence such as lacUV5. . The vector preferably has a sequence capable of imparting selectivity based on the phenotypic trait in the transformed cell. For example, Escherichia coli includes E. coli.
The JM109 strain derived from E. coli K12 strain and the like are often used, and as the vector, pBR322 and pUC-based plasmids are generally often used, but are not limited thereto, and any of various known strains and vectors can be used. As a promoter, in E. coli,
Tryptophan (trp) promoter, lactose (la
c) promoter, tryptophan lactose (tac
) Promoter, lipoprotein (lpp) promoter, bacteriophage-derived lambda (λ) PL promoter, polypeptide chain elongation factor Tu (tufB) promoter, and the like, but are not limited thereto.

As Bacillus subtilis, for example, strain 207-25 is preferable, and as a vector, pTUB228 [Ohmura,
K., et al. (1984) J. Biochem. 95, 87-93] and the like, but are not limited thereto. As the promoter, a regulatory sequence of the Bacillus subtilis α-amylase gene is often used, and if necessary, a DNA sequence encoding the signal peptide sequence of α-amylase can be ligated to allow extracellular secretion. .

Eukaryotic host cells include cells such as vertebrates and yeast. Examples of vertebrate cells include COS-1 [Gluzman, Y. (1981) C, a cell line derived from monkeys.
ell23, 175-182]. Yeasts include baker's yeast (Saccharomycescerevisiae), alcohol-assimilating yeast (Pichia pastoris), and fission yeast (Schizos
saccharomyces pombe) is used. Cells such as those exemplified here are commonly used as host cells, but are not limited thereto.

As a vertebrate cell expression vector, those having a promoter, an RNA splicing site, a polyadenylation site, a transcription termination sequence, etc. which are usually located upstream of the gene to be expressed can be used. Further, a replication origin may be provided if necessary. Examples of such expression vectors include pSV2dhfr with the SV40 early promoter [Subramani, S., et al. (198
1) Mol. Cell. Bio. 1, 854-864], but are not limited thereto.

Examples of yeast expression vectors include, for example, the promoter of alcohol dehydrogenase gene [Bennetzen,
JL and Hall, BD (1982) J. Biol. Chem. 257, 3
018-3025] and the promoter of a galactose metabolism enzyme gene (such as gal10) [Ichikawa, K.,
et al. (1993) Biosci. Biotech. Biochem. 57, 1686-1.
690] can be used, and if necessary, by linking a DNA sequence encoding the signal peptide sequence of the yeast gene, extracellular secretion becomes possible, but is not limited thereto.

As an example, when COS-1 is used as a host cell, the expression vector has an SV40 origin of replication, and is capable of autonomous growth in COS-1.
In addition, a transcription promoter, a transcription termination signal, and R
An expression vector having an NA splice site can be used. The expression vector is prepared by the DEAE-dextran method [Luthman, H. and Magnusson, G. (1983) Nucleic A
cids Res. 11, 1295-1308], calcium phosphate-
DNA coprecipitation method [Graham, FL and van der Eb, AJ
(1973) Virology 52, 456-457] and electroporation [see Neumann, E., et al. (1982) EMBO J. 1, 841-845].
Thus, a desired transformed cell can be obtained.

In particular, an expression vector comprising a DNA encoding the heavy chain and a DNA encoding the light chain
By co-transfection of COS-1 with an expression vector comprising, these DNAs can be simultaneously expressed, and a transformant producing a recombinant anti-Fas antibody can be obtained. However, recombinant anti-Fa
The method for producing the s antibody is not limited to the above. For example, a single expression vector capable of retaining both DNAs encoding each of the heavy chain and the light chain and simultaneously expressing them is constructed. A method of transforming COS-1 cells with a vector can also be used.

The desired transformant obtained above can be cultured according to a method well-known to those skilled in the art, and the culture allows the recombinant anti-Fas to be produced inside or outside the transformant cells.
Antibodies are produced. As a medium used for the culture,
Various conventional ones can be appropriately selected depending on the host cell employed. For example, in the case of COS-1, the RPMI16
A medium obtained by adding a serum component such as fetal calf serum (FCS) to a medium such as 40 medium or Dulbecco's modified Eagle medium (DMEM), if necessary, can be used.

The culturing temperature for culturing the transformant is not particularly limited as long as it does not remarkably reduce the ability to synthesize proteins in cells, but is preferably from 32 to 42 ° C, most preferably 37 ° C. Incubate with Also, if necessary, 1-1
The cells can be cultured in air containing 0% (v / v) carbon dioxide.

The E. coli strain E. coli transformed with a recombinant DNA vector expressing the DNA encoding the heavy or light chain of the anti-Fas monoclonal antibody of the present invention in animal cells. coli pME-H and E. coli. coli pM
ELs were internationally deposited with the Institute of Biotechnology and Industrial Technology, respectively, on March 12, 1997,
Accession numbers FERM BP-5868 and FERM B
P-5867 is attached. Therefore, a cultured animal cell such as COS-1 is transformed with the recombinant DNA vector isolated from the deposited strain, and the transformed cell is cultured to produce a recombinant anti-Fas antibody in the culture. be able to.

The fraction containing the anti-Fas antibody protein produced intracellularly or extracellularly in the transformant as described above can be obtained by various known protein separation procedures utilizing the physical properties, chemical properties, etc. of the protein. Can be separated and purified. Specific examples of such a method include, for example, treatment with a normal protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC). ), Various types of chromatography, dialysis methods, combinations thereof, and the like.

To humanize an anti-Fas mouse monoclonal antibody, the entire determined CDR sequence and FR
It is necessary to design the amino acid sequence of the variable region so that some amino acid residues of the sequence are transplanted into a human antibody. This design follows the following method. Conventionally, when designing humanization, as a guideline for selecting a subgroup of acceptors, a heavy chain of a known human antibody having a natural amino acid sequence,
The natural combination of light chains is used as is.The combination as a subgroup to which heavy and light chains belong is preserved, but the heavy and light chains are derived from different human antibodies, respectively, Either an amino acid sequence having high identity to the amino acid sequence of the chain or a consensus sequence is used. In the present invention, the above guidelines can be followed, but as a different method, without considering the combination of subgroups, the heavy chain and light chain FRs having the highest identity with the donor FR can be used as the human antibody. It is also possible to adopt a method of selecting from a library of primary sequences. With these selection methods,
The amino acid identity of the FR portion between the donor and the acceptor can be at least 70% or more. By adopting this method, the number of amino acid residues to be transplanted from the donor can be reduced, and the induction of the HAMA response can be reduced.

The operation of predicting the tertiary structure from the primary sequence of an antibody molecule (hereinafter, this operation is referred to as “molecular modeling”) is limited in its prediction accuracy, and rarely occurs in the subgroup to which the donor belongs. The role of amino acid residues cannot be fully specified. According to the method of Queen et al., It is generally difficult to determine which amino acid residue, donor or acceptor, should be selected at such a position. According to the selection method, the chance of making such a judgment can be significantly reduced.

We further extend this method of humanization by providing novel methods for identifying amino acids from the donor FRs that are important for maintaining the structure and function of the donor CDRs. Has improved.

After the selection of the human acceptor molecule for each of the light and heavy chains, the method of selecting the amino acid residue to be transplanted from the FR of the donor is as described below.

When the amino acid sequences of the donor and the acceptor are aligned, and the amino acid residues are different at the corresponding positions of the FRs, it is necessary to determine which residue should be selected. In this selection, , CD from donor
It is necessary to make a selection so as not to impair the three-dimensional structure of R.

Queen et al. Described in the above-mentioned National Publication of International Patent Application No. 4-50240.
In No. 8, a method was proposed in which the amino acid residue on the FR was grafted to the acceptor together with the CDR sequence if the amino acid residue met at least one of the following requirements: 1) Amino acids in the human FR region of the acceptor were rare at that position. And the corresponding amino acid of the donor is common at the said position of the acceptor; 2) the amino acid is in close proximity to one of the CDRs; 3) the amino acid is within about 3% of the CDR in a three-dimensional immunoglobulin model. It is expected that it will have a chain atom and be able to interact with the antigen or with the CDRs of the humanized antibody.

Here, the residue shown in 2) is often 3)
According to the present invention, the requirement 2) is deleted in the present invention, and two new requirements are separately provided. That is, in the present invention, for the amino acid residues on the donor FR to be grafted with the CDRs: a) whether the amino acids in the acceptor FR are rare at that position and the corresponding amino acids of the donor are common at that position B) whether the amino acid is expected to interact with a constituent amino acid atom of the CDR and an antigen or a CDR loop to be transplanted in a three-dimensional structural model; c) whether the position is a canonical class-determining residue; d. ) If the position constitutes the interface between the heavy and light chains, or, the amino acid residue will be grafted from the donor FR.

According to the requirement of a), amino acids found at a frequency of 90% or more at the relevant position for antibodies of the same subclass are “ordinary” and amino acids found at a frequency of less than 10% are “rare” according to the above-mentioned Kabat's table. Is defined.

In the requirement (c), whether or not “the position is a residue determined by a canonical class” can be uniquely determined in accordance with the above-mentioned table of Choccia.

For the requirements b) and d), molecular modeling of the antibody variable region is required in advance. As the software for molecular modeling, any commercially available software can be employed, but AbM (manufactured by Oxford Molecular Limited) can be preferably used.

Since the prediction accuracy of molecular modeling has a certain limit, the present invention refers to the experimental results of X-ray crystallography of the variable regions of various antibodies to determine the structure prediction obtained from molecular modeling. Probability is divided into two stages.

In the present invention, in the three-dimensional structure of the variable region constructed by molecular modeling software such as AbM, the distance between two atoms is obtained by adding 0.5 ° to the sum of each van der Waals radius. When the value is shorter than the calculated value, it is estimated that the two atoms are in van der Waals contact. When the distance between polar atoms such as amide nitrogen and carbonyl oxygen in the main chain and side chain is shorter than the average hydrogen bond distance of 2.9 ° plus 0.5 °, it is presumed that a hydrogen bond exists between them. Further, when the distance between atoms having opposite electric charges is shorter than the distance obtained by adding 0.5 ° to 2.85 °, it is estimated that an ion pair is formed between them.

On the other hand, from the experimental results of X-ray crystal structure analysis of the variable regions of various antibodies, regardless of the subgroup, the positions on the FRs where the contacts with the CDR are frequently found are: 2, 3, 4, 5, 23, 35, 3
Nos. 6, 46, 48, 49, 58, 69, 71, 88, heavy chains 2, 4, 27, 28, 29, 30, 3,
6, 38, 46, 47, 48, 49, 66, 67, 6
Positions 9, 71, 73, 78, 92, 93, 94 and 103 are specified (all numbers represent amino acid numbers defined in the above Kabat et al. Document; the same applies hereinafter). If you apply the same criteria as molecular modeling,
The amino acid residues at these positions are in contact with the amino acid residues of the CDR in two-thirds of the known antibody variable region. Based on these findings, b) "the amino acid is expected to interact with an antigen or a CDR loop to be transplanted in a three-dimensional structural model of the amino acid in the three-dimensional structural model" means the following requirements. .

In molecular modeling, the position of the FR where the possibility of contact between the FR and the CDR is predicted may be one of the positions where the contact between the FR and the CDR is frequently detected experimentally by X-ray crystallography. , The transplantation of donor amino acid residues is prioritized. Otherwise, this requirement b) is not taken into account.

The expression “d) constitutes the contact surface between the heavy chain and the light chain” in d) means the following requirements. From the experimental results of X-ray crystallography of the variable regions of various antibodies, 36, 38, 43, 44, 46, 49, 87, 98
The th amino acid residue, 37, 39, 4 in the heavy chain
It has been recognized that the amino acid residues at positions 5, 47, 91, 103 and 104 frequently make heavy chain-light chain contact. In molecular modeling, the potential for heavy-light chain contact is foreseen, and if the position matches any of the positions described above, transplantation of donor amino acid residues is preferred. Otherwise, this requirement d) is not taken into account.

[0122] DNAs encoding the variable regions of the heavy and light chains of the humanized anti-Fas antibody of the present invention can be produced by the method described below.

For example, a plurality of polynucleotide fragments of 60 to 70 nucleotides consisting of a partial nucleotide sequence of the DNA are chemically synthesized so as to be alternate on the sense side and the antisense side, and then each polynucleotide fragment is annealed. Ligated with DNA ligase to obtain a DNA having DNAs encoding the variable regions of the heavy and light chains of the desired humanized anti-Fas antibody.

As another method, DNA encoding the entire amino acid sequence of the variable region of the acceptor is isolated from human lymphocytes, and the region encoding the CDR is subjected to nucleotide substitution by a method well known to those skilled in the art, so that restriction enzyme cleavage is performed. Introduce an array. After cleavage of the region with the corresponding restriction enzyme, a nucleotide sequence encoding the CDR of the donor is synthesized and ligated with DNA ligase to encode the variable regions of the heavy and light chains of the desired humanized anti-Fas antibody. D
NA can be obtained.

Further, in the present invention, the overlap of the desired humanized anti-Fas antibody is preferably carried out according to the overlap extension PCR method described below (see Holton et al., Gene, 77 , 61-68, (1989)). DNA encoding the variable regions of the chains and light chains can be obtained.

That is, two kinds of DNAs respectively encoding two kinds of amino acid sequences desired to be connected are referred to as (A) and (B) for convenience. (A) a 20 to 40 nucleotide sense primer that anneals to the 5 ′ side (hereinafter, this primer is referred to as (C)) and (B) a 20 to 40 nucleotide antisense primer that anneals to the 3 ′ side (Hereinafter, this primer is referred to as (D)). Furthermore, 20 on the 3 ′ side of (A)
Chimera-type sense primer (hereinafter, this primer is referred to as (E)) and an antisense primer complementary thereto (hereinafter, this primer is referred to as (E)) Is defined as (F)). Using an appropriate vector DNA containing (A) as a substrate, P was prepared using sense primer (C) and chimeric antisense primer (F).
By performing CR, a DNA in which 20 to 30 nucleotides on the 5 ′ end side of (B) are added to the 3 ′ end of (A)
(This newly obtained DNA is referred to as (G)). Similarly, by using an appropriate vector DNA containing (B) as a substrate and performing PCR using an antisense primer (D) and a chimeric sense primer (E), (A) is added to the 5 ′ end of (B). ) Can be obtained with the addition of 20 to 30 nucleotides on the 3 ′ end side (this newly obtained DNA is referred to as (H)). (G) and (H) obtained in this way are:
Complementary nucleotide sequences are retained at the 3'-side 40 to 60 nucleotides of (G) and the 5'-side 40 to 60 nucleotides of (H). When PCR is performed by mixing the amplified (G) and (H), (G) and (H) become single-stranded in the first denaturation reaction, and most of the DNA is recovered in the subsequent annealing reaction. However, for some DNAs, a heteroDNA duplex is formed that anneals with the complementary nucleotide sequence region. In a subsequent extension reaction, the protruding single-stranded portion is repaired, and a chimeric DNA in which (A) and (B) are linked (hereinafter, this DNA is referred to as (I)
And ) Can be obtained. Further, by using this (I) as a substrate and performing PCR using a sense primer (C) and an antisense primer (D), (I) can be amplified. In the present invention, the DNA encoding the CDR regions of the heavy and light chains of the anti-human Fas mouse monoclonal antibody and the F of human immunoglobulin IgG are
The above ligation reaction can be carried out using DNA encoding the R region and further DNA encoding the secretion signal of human immunoglobulin IgG as (A) and (B), respectively, on a case-by-case basis.

[0127] The codon for the desired amino acid is known per se and may be selected arbitrarily. For example, it can be determined according to a conventional method in consideration of the frequency of codon usage of the host to be used. The partial modification of these nucleotide sequence codons is carried out according to a conventional method, using site-specific mutagenesis using a primer consisting of a synthetic oligonucleotide encoding the desired modification.
(Mark, DF, etal. (1984) Proc. Natl. Acad. Sc
i. USA 81, 5662-5666). Therefore, when chemically synthesizing each primer, by designing each primer so as to introduce a point mutation in advance, it is possible to obtain DNAs encoding the heavy and light chain variable regions of the desired anti-Fas antibody. it can.

Each DNA of the present invention thus obtained
Can be transformed into prokaryotic or eukaryotic host cells by incorporating them into expression vectors, respectively. Furthermore, each gene can be expressed in each host cell by introducing an appropriate promoter and a sequence involved in expression into these vectors.

[0129] Three types of transformants in which DNAs encoding the variable region of the light chain of the humanized anti-Fas antibody of the present invention were incorporated, E. coli. coli pHSGMM6 SANK 7
3697 strains, E. coli pHSGHM17 SAN
K 73597 strain and E. coli. coli pHSGHH7
A SANK 73497 strain, and a transformant into which DNA encoding the variable region of the heavy chain of the humanized anti-Fas antibody of the present invention has been incorporated. coli pgHSL7A6
2. The SANK73397 strain was established in 1997
On August 22, they were internationally deposited with the Institute of Biotechnology and Industrial Technology, and received the accession numbers FERM BP-607.
1, FERM BP-6072 and FERM BP-
6073 and FERM BP-6074. In addition, two transformants incorporating DNA encoding the variable region of the light chain of the humanized anti-Fas antibody of the present invention, coli pHSHM2 SANK 701
98 strains and E. coli. coli pHSHH5 SANK
70398 strain, and a transformant into which DNA encoding the variable region of the heavy chain of the humanized anti-Fas antibody of the present invention has been incorporated. coli pgHPDHV3 SANK
70298 strain was deposited on February 26, 1998 at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology,
Accession numbers FERM BP-6272 and FE respectively
RM BP-6274 and FERM BP-62
73 is attached. Therefore, it is possible to obtain a DNA encoding each subunit of the humanized anti-Fas antibody protein by, for example, isolating a plasmid from the deposited strain or performing PCR using the extract of the deposited strain as a template. it can.

According to the above method, a recombinant anti-Fas antibody can be easily produced with high yield and high purity. As a method for confirming that the thus-prepared recombinant anti-Fas antibody specifically binds to Fas, for example, the same ELISA method as in the case of evaluating the antibody titer at the time of mouse immunization is suitable.

The molecules of the invention (not limited to antibodies) have the following functional properties a) to f), each of which can be confirmed, for example, by the methods described in each section: a) Fas Apoptosis-inducing activity on T cells expressing thymus: The thymus is excised from a mouse to which the molecule of the present invention has been administered, and the obtained thymocytes are contacted with an anti-mouse Fas antibody and an antibody that specifically recognizes T cells. Investigate apoptosis-inducing activity on Fas-expressing T cells by measuring the percentage of cells bound by both antibodies by flow cytometry; b) Reducing autoimmune disease symptoms in MRLgld / gld mice: Fas MRLgld /, which has a mutation in the gene encoding the ligand and exhibits autoimmune disease-like symptoms
gld mouse [Shin Yonehara (1994) Nikkei Science separate volume 110
, 66-77] to determine whether the intraperitoneal administration of the molecule of the present invention reduces swelling of the limbs, which is an autoimmune disease-like symptom of the mouse; c) does not induce liver damage: Peripheral blood is collected from BALB / c mice to which the molecule of the present invention has been administered, and glutamic acid-oxaloacetate transaminase (hereinafter referred to as “GO”)
T ") and glutamate-pyruvate transaminase (hereinafter referred to as" GPT ") values using an automatic analyzer (for example, Model 7250; manufactured by Hitachi, Ltd.) and a reagent for the same (for example, transaminase-HRI).
I; manufactured by Wako Pure Chemical Industries, Ltd.). The molecule of the present invention does not increase blood GOT and GPT levels, which are indicators of liver damage, even when administered to a living body; d) therapeutic or preventive effect against fulminant hepatitis: anti-mouse Fas monoclonal antibody Jo2 is administered to mice In the experimental system which induces fulminant hepatitis by administration, simultaneous with Jo2 administration,
Alternatively, the effect of administering the molecule of the present invention after administration of Jo2 is examined; e) Effect of preventing the onset of collagen-induced arthritis; F) Investigation of the effect of administration of the molecule of the present invention; f) Apoptosis-inducing activity on synovial cells derived from a patient with rheumatoid arthritis: culturing synovial cells collected from the affected part of a patient with rheumatoid arthritis, and using the molecule of the present invention in a medium. The viability of the cells when they are included is examined.

The molecule of the present invention induces the activity described in a) and f) above, that is, the activity that induces apoptosis in abnormal cells, and the activity described in c) and d), that is, induces apoptosis in normal cells. Rather, it is a substance having a novel property, which has an activity of inhibiting apoptosis, and is useful as a component to be contained in a preventive or therapeutic agent for diseases caused by abnormal Fas / Fas ligand system.

Further, the fact that the molecule of the present invention has an apoptosis-inducing activity is based on the fact that cells (for example, the human lymphocyte cell line HPB-ALL (Morika
wa, S., et al. (1978) Int. J. Cancer 21, 166-170) or Jurkat (American Type Culture No.
TIB-152), etc.), and the viability thereof was measured by an MTT assay (Green, LM, et al. (1984) J. Immunological Met).
hods 70, 257-268).

The molecules of the present invention, in particular, anti-Fas antibodies having substantially the same immunogenicity to humans as human antibodies, can be obtained from Fas / F
diseases caused by the above as ligand system (autoimmune diseases (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia,
Addison's disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, spontaneous infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin dependence Diabetes, allergy, atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C, hepatitis B, hepatitis D) ) Or alcoholic hepatitis), acquired immunodeficiency syndrome or rejection after organ transplantation) can be used as a preventive or therapeutic agent containing this as an active ingredient. Such prophylactic or therapeutic agents can be administered in various forms, such as tablets, capsules, granules, powders,
Oral administration using a syrup or the like, or parenteral administration using an injection, a drip, a suppository or the like can be given.

The dose varies depending on symptoms, age, body weight and the like.
About 0.1 mg to 1000 mg per day.
It can be administered once or in several divided doses. Also,
For parenteral administration, a single dose of 0.1 mg to 1000 mg can be administered by subcutaneous injection, intramuscular injection or intravenous injection.

[0136]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 Preparation of Antigen To obtain soluble human Fas that does not contain a transmembrane region, the extracellular region of human Fas and the mouse interleukin 3 (IL3) receptor [Gorman, DM et al. (1990)
Natl. Acad. Sci. USA, 87, 5459-5463], a plasmid vector for expression of a fusion protein with the extracellular region (hereinafter referred to as "human Fas fusion protein") was prepared. The DNA encoding the human Fas fusion protein is
It was prepared by the PCR method.

A) Template In performing the PCR, the template used was an expression plasmid vector pME18S-mFas-AIC for a DNA encoding a fusion protein of the extracellular region of mouse Fas and the extracellular region of mouse IL3 receptor. [Nishim
ura, Y. et al. (1995) J. Immunol. 154, 4395-4403] and a plasmid vector pCEV4 carrying a cDNA encoding human Fas [Itoh, N., et al. (19)
91) Cell 66, 233-243].

B) Primers In performing PCR, the following oligonucleotide primers were synthesized: 5′-GGGGATATTCC AGTACGGAGTTGGGGAAGCTCTTT-3 ′ (N1: SEQ ID NO: 12 in Sequence Listing), 5′-GTTTCTTCTG CCTCTGTCAC CAAGTTAGAT CTGGA -3 '(C3N: SEQ ID NO: 13 in Sequence Listing), 5'-TCCAGATCTA ACTTGGTGAC
AGAGGCAGAA GAAAC-3 '(N3N: SEQ ID NO: 14 in Sequence Listing), 5'-CCCTCTAGAC GCGTCACGTG
GGCATCAC-3 '(CTN2: SEQ ID NO: 15 in Sequence Listing).

The synthesis of oligonucleotides such as primers for PCR was carried out entirely using an automatic DNA synthesizer (model 380B;
[Matteucci, MD and Caruthers, MH (1981)] using PerkinElmer Japan Applied Biosystems Division)
J. Am. Chem. Soc. 103, 3185-3191]. After completion of the synthesis, each oligonucleotide was cleaved from the support and deprotected, and the resulting solution was freeze-dried to make a powder. Dissolve this powder in distilled water, -20 ° C until use
And stored frozen.

C) First-step PCR DNA fragment encoding extracellular region of human Fas HF
AS was produced under the following conditions. When performing PCR, use the LA PCR kit (Takara Shuzo Co., Ltd.)
Was used.

Reaction solution composition: template pCEV4 DNA 20 ng Primer N1 0.5 μg C3N 0.5 μg 10-fold concentration LA PCR buffer (attached to kit) 25 μl dNTPs (attached to kit) 25 μl LA Taq polymerase (attached to kit) 5 units 250 μl with sterile distilled water (dNTPs are dAT
Represents an equimolar mixture of P, dCTP, dGTP and dTTP. The same applies in the following description); Temperature conditions: After heating at 94 ° C for 2 minutes, 1 hour at 94 ° C.
Temperature cycle for 1 minute at 55 ° C and 2 minutes at 72 ° C for 30 minutes.
After the repetition, the mixture was kept at 72 ° C. for 10 minutes (hereinafter, the reaction temperature of all PCRs in Examples was controlled by GeneAmp PCR System 9600; Perkin Elmer Co., Ltd.).
Japan was used).

On the other hand, the DNA fragment MAIC encoding the extracellular region of the mouse IL3 receptor was prepared under the following conditions.

Reaction solution composition: template pME18S-mFas-AIC DNA 20 ng Primer N3N 0.5 μg CTN2 0.5 μg 10-fold concentration LA PCR buffer 25 μl dNTPs 25 μl LA Taq polymerase 12.5 units Made up to 250 μl with sterile distilled water; Conditions: Same as above.

The amplified HFAS DNA and MAIC DNA after PCR were subjected to 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation, stained with 1 μg / ml ethidium bromide, and then exposed to ultraviolet light. Detected in. A gel piece of each detected band portion was cut off with a razor blade, and DNA was electrically eluted using a centriluter (manufactured by Amicon) equipped with a centrifugal tube type ultrafilter Centricon 10 (manufactured by Amicon). . Further, the Centricon 10 containing the eluate was taken out, and the DNA was concentrated by centrifugation at 7,500 × g for about 1 hour. This DNA was dissolved in 20 μl of distilled water after ethanol precipitation.

D) Second step PCR DNA fragment F encoding human Fas fusion protein
ASAIC was produced under the following conditions.

Reaction solution composition: template DNA solution HFAS 20 μl MAIC 20 μl Primer N1 0.5 μg CTN2 0.5 μg 10-fold concentration LA PCR buffer 25 μl dNTPs 25 μl LA Taq polymerase 12.5 units Made up to 250 μl with sterile distilled water; temperature conditions : Same as the first step.

Amplified FASAIC DN after PCR
A was subjected to 1% agarose gel electrophoresis after phenol extraction and ethanol precipitation, stained with 1 μg / ml ethidium bromide, and detected under ultraviolet irradiation. The gel piece of the detected band portion was cut off with a razor blade, DNA was eluted from this gel piece using a centuritor and a centricon, concentrated, and after ethanol precipitation, 50 μl
dissolved in 1 liter of distilled water.

E) Construction of vector The FASAIC DNA obtained in d) above was digested with the restriction enzyme E
digested with coRI and XbaI (FASAIC DNA
After the whole solution was used), phenol-chloroform extraction and ethanol precipitation were performed. This precipitate was suspended in 2 μl of sterile deionized water.

On the other hand, plasmid pME18S-mFas
2 μg of AIC was digested with EcoRI and XbaI, and then a dephosphorylated DNA fragment was prepared. This DNA fragment was combined with FASAIC D digested with EcoRI and XbaI.
After linking using NA and a ligation kit (Takara Shuzo Co., Ltd.), the method of Hanahan [Hanahan, D. (1983) J.
Mol. Biol. 166, 557-580] according to E. coli DH5α.
A strain (manufactured by Gibco BRL) was transformed. The alkaline SDS method [Mani
atis, T., et al. (1989) in Molecular Cloning: A La
boratory Manual (2nd Edition), Cold Spring Harbor
Laboratory, NY], to obtain the desired plasmid phFas-AIC2.

This plasmid was prepared using a plasmid mass preparation kit (Maxiprep DNA purification system: manufactured by Promega), and 20 μg of DNA was precipitated with ethanol, and the precipitate was sterilized with Dulbecco's PBS.
(-) (Hereinafter referred to as "PBS"; manufactured by Nissui Pharmaceutical Co., Ltd.)
Dissolved in 20 μl.

F) Expression COS-1 cells (American Type Culture Collection N)
o. CRL-1650) containing 10% fetal bovine serum (manufactured by Gibco) (hereinafter referred to as “FCS”) in modified Dulbecco's modified Eagle medium (hereinafter referred to as “DMEM”: Nissui Pharmaceutical Co., Ltd.)
Cell culture flask (culture area 225c)
m ² ; manufactured by Sumitomo Bakelite Co., Ltd.) at 37 ° C. under 5% carbon dioxide until it becomes semi-confluent. After removing the medium, 5 g / l was added to the flask.
A solution containing trypsin and 2 g / l disodium ethylenediaminetetraacetate (hereinafter "trypsin-EDT").
3 ml of “A solution”; Sigma) and kept at 37 ° C. for 3 minutes. The cells released from the flask were suspended and collected in PBS, washed twice with PBS, and then adjusted to 6 × 10 ⁷ cells / ml with PBS. 20 μl of this cell suspension (1.2 × 10 ⁶ cells) and 20 μl of the above-prepared plasmid solution were mixed, and the electrode spacing was 2 m.
m (manufactured by Shimadzu Corporation) and set in a gene transfer device (GTE-1; manufactured by Shimadzu Corporation), and then a pulse of 600 V-30 μsec was applied twice at 1 second intervals. . The cell-DNA mixture in the chamber was added to 10 ml of DMEM containing 10% FCS, and the mixture was placed in a cell culture flask (culture area: 75 cm ² ) at 37 ° C., 7.
The cells were cultured under 5% carbon dioxide for 24 hours. Next, the culture supernatant was removed, and the cells were washed with serum-free DMEM. Then, 10 ml of serum-free DMEM was added, and the mixture was further cultured at 37 ° C. under 7.5% carbon dioxide for 24 hours, and the culture supernatant was collected.

The obtained culture supernatant is placed in a dialysis tube (exclusion limit molecular weight: 12,000-14000; manufactured by Gibco BRL) and 10 mM Tris-hydrochloric acid (pH 8.
After dialysis against 0), crude purification of the human Fas fusion protein was performed using a Pharmacia FPLC device under the following conditions: Column: Resource Q column (trade name; φ6.4 × 30)
Solvent: 10 mM Tris-hydrochloric acid (pH 8.0) Flow rate: 5 ml / min; Elution: Sodium chloride 0.1 M-0.3 M, linear concentration gradient for 30 minutes.

The eluate was fractionated in 5 ml portions, and the Fas gene expression product was assayed by the ELISA method described below. First, 100 μl of each eluted fraction was placed in a well of a 96-well microplate (manufactured by Coaster) and kept at 37 ° C. for 1 hour. After removing the solution in each well, 0.1%
PBS containing Tween 20 (PB
(S-Tween) 3 times with 100 μl / well, 2
% Bovine serum albumin (hereinafter referred to as "BSA") in 100 μl / well of PBS was added and incubated at 37 ° C. for 1 hour. Add 100 μl of each well to PBS-Tween
Per well after washing three times, the anti-mouse IL-3 receptor β subunit monoclonal antibody HC (1 mg / m
l; manufactured by Institute of Medical Biology, Inc.)
100 μl / well of a solution diluted 1000-fold with n was added and incubated at 37 ° C. for 1 hour. In addition, PB
After washing three times with 100 μl of S-Tween, PB
100 μl / well of horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by Amersham) diluted 2000-fold with S-Tween was added thereto, and the mixture was incubated at 37 ° C. for 1 hour.
Washed three times with μl. Next, 100 μl / well of a horseradish peroxidase substrate (manufactured by Bio-Rad) was added and allowed to stand for 5 minutes, and then the absorbance at 415 nm was measured using a microplate reader (Model 450; manufactured by Bio-Rad). As a result, the 19th to 23rd fractions having high absorbance were collected and used as a sample of a crudely purified human Fas fusion protein.

Example 2 Immunization and Hybridization of Mice
Preparation (2-1) Immunization To 1 ml of the partially purified human Fas fusion protein solution obtained in Example 1 (total protein amount: 100 μg), 2
N hydrochloric acid 25 μl, 9% potassium alum 250 μl
(Final concentration 1.1%), 2N sodium hydroxide 25μ
After addition of about 1%, a pH 6.5 is obtained by adding about 120 μl of a 10% (w / v) aqueous sodium hydrogen carbonate solution.
To 7.0. After standing at room temperature for about 30 minutes, 200 μl of pertussis killed bacteria (manufactured by Wako Pure Chemical Industries, Ltd .; 1.2 × 10 ¹¹ bacteria / ml) was added for activation of T cells, and the cells were transferred to Fas knockout mice. Administered intraperitoneally. Fa
The s knockout mouse used was obtained by the method described in Senju et al. [Senju, S. et al.
(1996) International Immunology 8, 423]. 2
After a week, only the crude purified human Fas fusion protein (20 μl)
g protein / mouse) was intraperitoneally administered and boosted.

(2-2) Cell fusion The spleen was removed from the mouse three days after the booster immunization, and
mM HEPES buffer (pH 7.3), 350 mg /
ml sodium bicarbonate, 0.05 mM β-mercaptoethanol, 50 units / ml penicillin, 50μ
g / ml streptomycin, 300 μg / ml L
-Serum-free RPMI 1640 medium containing glutamic acid (1
0.4g / l RPMI1640 "Nissui"
: Nissui Pharmaceutical Co., Ltd.) (hereinafter referred to as "serum-free RPMI medium") and crushed on a mesh (Cell Strainer: Falcon) using a spatula. After the cell suspension passed through the mesh was centrifuged to precipitate spleen cells, the spleen cells were washed twice with a serum-free RPMI medium, and then suspended in a serum-free RPMI medium to measure the number of cells.

On the other hand, in an ASF104 medium (manufactured by Ajinomoto Co., Inc.) containing 10% FCS (manufactured by Gibco BRL) (hereinafter referred to as "ASF medium containing serum"), 37
Myeloma cells NS cultured at 5 ° C. in the presence of 5% carbon dioxide so that the cell concentration does not exceed 1 × 10 ⁸ cells / ml
1 (American Type Culture Collection TIB-18) was similarly washed with a serum-free RPMI medium, suspended in a serum-free RPMI medium, and the number of cells was measured.

NS1 cell suspension equivalent to 3 × 10 ⁷ cells,
After mixing 3 × 10 ⁸ spleen cell suspensions and centrifuging,
The supernatant was completely removed. The following cell fusion operation was performed while keeping the plastic centrifuge tube containing the pellet in a beaker containing warm water at 37 ° C. In this pellet, 50% (w / v) polyethylene glycol 15
After slowly adding 1 ml of 00 (manufactured by Boehringer Mannheim) while stirring the pellet with the tip of a pipette,
1 ml of serum-free RPMI medium preheated to 37 ° C
Was slowly added in two portions, followed by addition of 7 ml of serum-free RPMI medium. After centrifugation, the supernatant was removed and 10
10 ml of hypoxanthine-aminopterin-thymidine medium (hereinafter referred to as "HAT medium"; Boehringer Mannheim) containing% FCS was added while slowly stirring with a pipette tip. After addition of 20 ml of HAT medium containing 10% FCS,
Dispense 100 μl / well into a well microplate,
The cells were cultured at 37 ° C. under 5% carbon dioxide. After 7 to 8 days, 100 wells of fresh HAT medium was added to the wells with yellowish medium.
μl / well was added. The fusion cells thus obtained were subjected to screening by the limiting dilution method described below.

(2-3) Limiting dilution The thymus was isolated from a 4- to 10-week-old female BALB / c mouse (purchased from Japan SLC, Inc.), and was then subjected to a spatula on a mesh (Cell Strainer; Falcon). The cells that had been crushed and passed through the mesh were washed twice with a hypoxanthine / thymidine medium containing 10% FCS (hereinafter referred to as “HT medium”; Boehringer Mannheim).
Thymic cells for one mouse were suspended in 30 ml of HT medium containing 10% FCS, which was used as a feeder cell solution. The culture solution containing the fused cells obtained in the above (2-2) is mixed with a feeder cell solution for 10 to 10 times depending on the cell density.
0-fold dilution, and the density of fused cells was 5 cells / ml,
The cells were serially diluted with a feeder cell solution so as to be 1 cell / ml and 0.5 cell / ml. Each sample prepared in this manner was placed on a 96-well microplate for cell culture in an amount of 100 μl.
Each 1 / well was dispensed and cultured at 37 ° C. under 5% carbon dioxide for 5 days.

(2-4) Selection Mouse T lymphoma cells WR19L (American Type Cult)
WR19L12a cells expressing a gene encoding human Fas in ure collection TIB-52 [Itoh, N. et.
al. (1991) Cell 66, 233-243] with 10% FC
After culturing and growing in RPMI1640 medium containing S at 37 ° C. under 5% carbon dioxide, 1 × 10 ⁷ cells / ml
The prepared cell suspension was dispensed into a U-bottom 96-well microplate (manufactured by Nunc) at 50 μl / well and centrifuged (90 × g, 4 ° C., 10 minutes). Remove the supernatant,
50 μl of the culture supernatant of the fused cells cultured in the above (2-3)
After adding 1 / well and stirring, the mixture was allowed to stand on ice for 1 hour, and then centrifuged (90 × g, 4 ° C., 10 minutes) to remove the supernatant. The pellet was treated with 100 μl / well of flow cytometry buffer (5% FCS, 0.04% (w /
v) After washing twice with PBS containing sodium azide) and then diluting 500-fold, fluorescein-5-isothiocyanate;
50 µl of a labeled goat anti-mouse IgG antibody IgG fraction (manufactured by Organon Technica) was added as a secondary antibody, and the mixture was allowed to stand on ice for 1 hour. Centrifugation (90 ×
g, 4 ° C., 10 minutes), remove the supernatant, wash the pellet twice with 100 μl / well of a flow cytometry buffer, add 50 μl of a 3.7% formalin solution, and place on ice for 10 minutes. The cells were fixed by standing still. After centrifugation (90 × g, 4 ° C., 10 minutes) to remove the supernatant, the buffer for flow cytometry 100
After washing with μl / well, the pellet was suspended in 100 μl / well of a buffer for flow cytometry to obtain a sample for flow cytometry. FIT of cells in each sample
The C fluorescence intensity was measured with a flow cytometer (Epix Elite; manufactured by Coulter) (excitation wavelength: 488).
nm, detection wavelength: 530 nm). As a result, a clearly higher value (about 100-10) than WR19L12a (FITC fluorescence intensity about 0.3) to which no fusion cell culture supernatant was added.
The fused cells corresponding to the sample showing the FITC fluorescence intensity of (00) were selected.

(2-5) Cloning For the cell group selected in the above (2-4),
By repeating the series of steps from -3) to (2-4) five times, several clones producing hybridomas producing a single antibody that binds to WR19L12a but does not bind to WR19L were obtained. For the antibodies produced by these hybridomas, the binding ability to mouse Fas was examined using the same method as in (2-4). Cells expressing mouse Fas include cell line L5178 that hardly expresses Fas.
Y (American Type Culture Collection No. CRL-1722
L5178 into which a mouse Fas expression vector has been introduced
YA1 cells were used. As a result, a mouse-mouse hybridoma HFE7A that produces an antibody that binds to L5178YA1 cells and does not bind to L5178Y cells was selected. Mouse-mouse hybridoma HFE7A
Was deposited internationally with the Institute of Biotechnology and Industrial Technology on February 20, 2008, and was assigned the deposit number FERM BP-5828.

Mouse-mouse hybridoma HFE
As a result of examining the subclass of the antibody produced by 7A (hereinafter referred to as "HFE7A") using a monoclonal antibody isotyping kit (Pierce), IgG1, κ
Turned out to be.

Example 3 HFE7A monoclonal antibody
Mice were produced in the purification Example 2 - Mouse hybridoma H
FE7A (FERMBP-5828) with 10% FC
ASF medium containing S in 1 liter at 37 ° C., 5%
The cells were cultured under carbon dioxide and grown to 1 × 10 ⁶ cells / ml. The culture was centrifuged (1000 rpm, 2 minutes), the supernatant was discarded, and the precipitated cells were washed once with a serum-free ASF medium, resuspended in 1 liter of a serum-free ASF medium, and heated at 37 ° C., 5% CO 2 gas. The cells were cultured under the following conditions for 48 hours. This culture solution was centrifuged (1000 rpm, 2 minutes), the supernatant was recovered, and a dialysis tube (exclusion limit molecular weight 12000) was collected.
-14000; manufactured by Gibco BRL)
10 times the volume of 10 mM sodium phosphate buffer (pH
8.0). The crude purification of IgG from the solution in the dialysis tube was performed using a high performance liquid chromatography apparatus (FPLC system; manufactured by Pharmacia) under the following conditions: Column: DEAE-Sepharose CL-6B column (column size) 10 ml; manufactured by Pharmacia); Solvent: 10 mM sodium phosphate buffer (pH 8.
0) Flow rate: 1 ml / min; Elution: 1M linear concentration gradient of sodium chloride (0-5)
0%, 180 minutes).

The eluate was fractionated in 5 ml portions, and the anti-Fas antibody titer in each fraction was determined using E-Fus using human Fas fusion protein.
It was tested by the LISA method. First, the partially purified human Fas fusion protein solution prepared in Example 1 was used for ELISA.
100 μl / well in a 6-well microplate, 3
After incubating at 7 ° C. for 1 hour, this solution was discarded, and each well was washed three times with 100 μl / well of PBS-Tween. Next, PBS 10% containing 2% bovine serum albumin.
0 μl / well was added and incubated at 37 ° C. for 1 hour. PB
After washing three times with 100 μl / well of S-Tween, 100 μl of each eluted fraction was added and incubated at 37 ° C. for 1 hour. Further, after washing 3 times with 100 μl / well of PBS-Tween, 100 μl / well of horseradish peroxidase-labeled anti-mouse immunoglobulin antibody (manufactured by Amersham) diluted 2000-fold with PBS-Tween was added, and reacted at 37 ° C. for 1 hour. , PBS-Twe
Washed three times with 100 μl / well. Next, horseradish peroxidase substrate (manufactured by Bio-Rad) 100
After adding μl / well and allowed to stand for 5 minutes, the absorbance at 415 nm of each well was measured using a microplate reader.

As a result, it was found that the absorbance was 21-30.
The second fraction is collected, and the column for antibody affinity purification (Hitrap Protein G column, column volume 5
ml; Pharmacia). After the column was washed with 25 ml / column equilibration buffer (20 mM sodium phosphate buffer (pH 7.0)),
The antibody was eluted with 5 ml / column of an elution buffer (0.1 M glycine-hydrochloric acid (pH 2.7)). Each eluate contains 1.125 ml of 1M Tris-HCl (pH
9.0) in a test tube. Immediately after the elution is completed, the mixture is placed in the upper part of a centrifugal tube type ultrafilter (Centreprep 10; manufactured by Grace Japan Co., Ltd.), and the 3,000 × g,
Centrifuged at 2 ° C. for 2 hours. After removing the filtrate collected in the lower part of the filter, 15 ml of PBS was added to the upper part, and 3000 parts were removed again.
The operation of centrifugation at × g for 2 hours at 4 ° C. was repeated 5 times. However, the fifth centrifugation was performed until the liquid volume in the upper part of the filter became 0.5 ml, and the liquid remaining in the upper part of the filter was washed with HFE7.
Sample A was used.

Example 4 cDNA cloning (4-1) Preparation of poly (A) ⁺ RNA Mouse-mouse hybridoma HF prepared in Example 2
E7A (FERM BP-5828) with 10% FCS
After growing to 1 × 10 ⁶ cells / ml in 1 liter of ASF medium at 37 ° C. and 5% carbon dioxide,
The cells were collected by centrifugation, and a guanidine thiocyanate solution (4M guanidine thiocyanate, 1% sarkosyl, 20 mM ethylenediaminetetraacetic acid (EDT
A), 25 mM sodium citrate (pH 7.0),
The solution was dissolved in 100 mM 2-mercaptoethanol, 0.1% antiform A), and the solution was recovered. Isolation of poly (A) ⁺ RNA is essentially a “Molecular Cloning
A Laboratory Manual ”[Maniatis, T. et al. (1982) p
pp. 196-198]. Specifically, the procedure was as follows.

The collected cell lysate was repeatedly inhaled and discharged several times using a 10-ml syringe equipped with a 21-G injection needle. 3ml in a polyaromer centrifuge tube for RPS40-T rotor bucket manufactured by Hitachi, Ltd.
5.7M cesium chloride-0.1M EDTA (pH
7.5) was added in advance, and the cell lysate was overlaid thereon to fill a centrifuge tube. This is 30,000 rpm,
After centrifugation at 20 ° C. for 18 hours, the resulting pellet was
It was dissolved in 0 μl of distilled water and precipitated with ethanol. After dissolving the obtained pellet again in 400 μl of distilled water, an equal amount of chloroform / 1-butanol (4: 1, v
/ V) was added and stirred, and the aqueous layer was collected after centrifugation. This was again ethanol precipitated, and the precipitate dissolved in 600 μl of distilled water was used as a total RNA sample.

The thus obtained total RNA 600
Poly (A) ⁺ RNA was purified from μg by oligo (dT) cellulose column chromatography. That is, the total RNA was dissolved in an adsorption buffer (0.5 M sodium chloride, 20 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 0.1% sodium dodecyl sulfate (SD
S)), heated at 65 ° C. for 5 minutes, and then applied to an oligo (dT) cellulose column (type 7; manufactured by Pharmacia) packed with the same solution, and the elution solution (10 mM
Tris-HCl (pH 7.5), 1 mM EDTA,
0.05% SDS) to elute poly (A) ⁺ RNA,
Collected. By such an operation, 100 μg of a poly (A) ⁺ RNA fraction was obtained.

(4-2) Determination of N-terminal amino acid sequences of heavy and light chains of HFE7A A part of the solution containing the anti-human Fas antibody HFE7A purified in Example 3 was subjected to SDS-polyacrylamide gel electrophoresis (hereinafter referred to as " (SDS-PAGE) (gel concentration 1
2%, 100V constant voltage, 120 minutes). After the electrophoresis, the gel is transferred to a transfer buffer (25 mM Tris-HCl (pH 9.
5) After immersion in 20% methanol, 0.02% SDS), using a transfer device (KS-8451; manufactured by Marisol), a polyvinylidene difluoride film (hereinafter referred to as “polyvinylidene difluoride”) previously immersed in a transfer buffer. The protein in the gel was transferred to a PVDF membrane (pore size: 0.45 μm; manufactured by Millipore) (10 V constant voltage, 4 ° C., 14 hours). The PVDF membrane after the transfer is washed with a washing buffer (25 mM sodium chloride, 10 mM sodium borate buffer (pH 8.
0)), and then stained (50% methanol, 2%
0% acetic acid, 0.05% Coomassie brilliant blue)
For 5 minutes, and then destained with 90% methanol. The bands corresponding to the heavy chain (the band with the lower mobility) and the light chain (the band with the higher mobility) visualized on the PVDF membrane in this way are cut out,
After washing with deionized water, an automatic Edman method [Edman, P., et al. (1967) E] was performed using a gas phase protein sequencer (PPSQ-10; manufactured by Shimadzu Corporation).
ur. J. Biochem. 1, 80], the N-terminal amino acid sequence of each was determined.

As a result, the N-terminal amino acid sequence of the band corresponding to the heavy chain was Gln-Xaa-Gln-Leu-Gln-Gln-Pro-Gly-Ala-Glu-Leu (SEQ ID NO: 16 in the sequence listing). , The N-terminal amino acid sequence of the band corresponding to the light chain is Asp-Ile-Val-Leu-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ala-Va
1-Ser-Leu-Gly-Gln-Arg-Ala-Thr-Ile-Ser (SEQ ID NO: 17 in Sequence Listing).

These amino acid sequences were converted into the antibody amino acid sequence database [Kabat,
EA et al., (1991) in Sequences of Proteins of I
mmunological Interest Vol. II, US Department of
Health and Human Services]]
The heavy chain (γ1 chain) of HFE7A was found to be of subtype 2b, and the light chain (κ chain) was of subtype 3. Therefore, the following oligonucleotide primers were synthesized, which hybridize with a part of the 5′-terminal untranslated region and the most terminal part of the 3′-terminal translated region of the genes belonging to these mouse subtypes, respectively. Et al., Matti Kartinen et al. (1988) 25, 859-865.
And Heinrich, G. et al. (1984) J. Exp.Med. 159,
417-435]: 5'-GACCTCACCA TGGGATGGA-3 '(H1: SEQ ID NO: 18 in the sequence listing); 5'-TTTACCAGGA GAGTGGGAGA-3' (H2: SEQ ID NO: 19 in the sequence listing); 5'-AAGAAGCATC CTCTCATCTA- 3 '(L1: SEQ ID NO: 20 in Sequence Listing); 5'-ACACTCATTC CTGTTGAAGC -3' (L2: SEQ ID NO: 21 in Sequence Listing).

(4-3) cDNA Cloning The cDNAs encoding the heavy and light chains of the mouse anti-human Fas monoclonal antibody HFE7A were ligated with reverse transcriptase (R
T) reaction and PCR reaction in combination
From the poly (A) ⁺ RNA fraction derived from the HFE7A-producing hybridoma prepared in 1), an arbitrary sequence was cloned by a method of specifically amplifying (RT-PCR). RT
-PCR was performed using RNA PCR kit (AMV) version 2 (Takara Shuzo Co., Ltd.).

A) Reverse transcriptase reaction As a primer for RT-PCR, a set of oligonucleotide primers (5′-end and 3′-end primers) synthesized in the above (4-2) was used, RT-PCR reactions using each light chain primer set were individually performed.

Reaction solution composition: poly (A) ⁺ RNA 1 μg 3 ′ terminal side primer (H2 or L2) 0.3 μg Tris-HCl (pH 8.3) 10 mM potassium chloride 50 mM dNTPs 1 mM magnesium chloride 5 mM ribonuclease inhibitor (attached to the kit ) 0.5 units Reverse transcriptase (attached to kit) 0.25 units Make up to 20 μl with double distilled water; Temperature conditions: 55 ° C for 30 minutes, 99 ° C for 5 minutes, 5 ° C
For 5 minutes.

B) PCR reaction solution composition: Reverse transcriptase reaction solution 20 μl 10-fold concentration RNA PCR buffer (attached to kit) 10 μl magnesium chloride solution (attached to kit) 10 μl Taq polymerase (attached to kit) 2.5 units 5'-end primer (H1 or L1) Final concentration 0.2 μM The total volume was adjusted to 100 μl with sterile deionized water; temperature conditions: heated at 94 ° C. for 2 minutes, and then heated to 94 ° C. for 30 minutes.
A temperature cycle of 30 seconds at 60 ° C. and 1.5 minutes at 72 ° C. was repeated 28 times.

After the completion of the reaction, a part of the reaction solution was
% Agarose gel electrophoresis showed that a band of about 1.4 kbp was amplified in the reaction solution containing the primer for heavy chain, and a band of about 0.7 kbp was amplified in the reaction solution containing the primer for light chain. It had been.

The cDNA amplified by these PCRs was
Cloning was performed using an A cloning kit (Invitrogen). First, the PCR reaction solution and pCRII
50ng vector (attached to TA cloning kit)
With 1 μl of a 10-fold ligase reaction buffer (6 mM
Tris hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 5 mM sodium chloride, 7 mM β-mercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 m
M spermidine, 0.1 mg / ml bovine serum albumin) and 4 units of T4 DNA ligase (1 μl).
l) and make up to 10 μl with sterile deionized water.
It was kept at 14 ° C. for 15 hours. Then, competent Escherichia coli TOP10F '(attached to the TA cloning kit) 5 containing 2 μl of 0.5 M β-mercaptoethanol 5
Add 2 μl of the above ligase reaction solution to 0 μl, and add
After standing for 0 minutes, heated at 42 ° C. for 30 seconds,
Cooled again on ice for 5 minutes. Add SOC medium (2
% Tryptone, 0.5% yeast extract,
500 μl of 0.05% sodium chloride, 2.5 mM potassium chloride, 1 mM magnesium chloride, and 20 mM glucose) were added thereto, followed by shaking culture at 37 ° C. for 1 hour.
This culture solution was added to an L-broth agar medium containing 100 μg / ml ampicillin (1% tryptone, 0.5%
Yeast extract, 0.5% sodium chloride,
The cells were spread on a 0.1% glucose, 0.6% Bacto agar (manufactured by Difco) plate and incubated at 37 ° C. overnight. A single ampicillin-resistant colony that appeared on the plate was selected, and this colony was scraped off with a platinum loop to remove L-containing 100 μg / ml ampicillin.
After culturing in a broth medium, plasmid DNA was prepared from the cells collected by centrifugation by an alkali method.
Plasmids thus obtained were each cloned into pCR
-H (plasmid carrying the cDNA encoding the HFE7A heavy chain) and pCR-L (plasmid carrying the cDNA encoding the HFE7A light chain).

(4-4) Analysis of Nucleotide Sequence Plasmids pCR-H and pC obtained in (4-3)
The nucleotide sequences of the cDNA (1.4 kbp) encoding the HFA7A heavy chain and the cDNA (0.7 kbp) encoding the HFE7A light chain, respectively, held by RL were analyzed using a gene sequence analyzer (310 Genetic Analyzer; Inc.) Perkin Elmer Japan Co., Ltd.) [Sanger, F., et al. (1977) Proc.
Acad. Sci. USA 74, 5463-5467].

The nucleotide sequences of the cDNAs encoding the HFE7A heavy and light chains determined as a result are shown in SEQ ID NOs: 8 and 10 in the sequence listing, respectively. Also,
The entire amino acid sequences of the HFE7A heavy and light chains encoded by these cDNAs are shown in FIGS.
1 is shown. The N-terminal amino acid sequence of the HFE7A heavy chain found in (4-1) above (SEQ ID NO: 16 in the sequence listing) is the same as the sequence of amino acids 1 to 11 in SEQ ID NO: 9 except for an unknown residue. Matched. Also, the N of the HFE7A light chain
The terminal amino acid sequence (SEQ ID NO: 17 in the sequence listing) was identical to the sequence of amino acids 1 to 22 of SEQ ID NO: 11 in the sequence listing. That is, it was revealed that the N-terminals of the mature proteins of the HFE7A heavy and light chains are the amino acids of amino acid No. 1 of SEQ ID NOS: 9 and 11, respectively.

The amino acid sequences of the heavy and light chains were obtained from the database of the amino acid sequences of antibodies by Kabat et al. [Kabat, EA, et al. (1991) in "Sequence of Prot.
einsof Immunological Interest Vol.II ": US Depart
As a result of comparison with heavy chain, amino acid number 1 of SEQ ID NO: 9
To 121 are the variable regions, amino acids 122 to 445 are the constant regions, and for the light chain, amino acids 1 to 111 of SEQ ID NO: 11 are the variable regions, and amino acids 112 to 218 are the constant regions. It became clear that there was.

Further, the HFE7 determined as described above
The positions and sequences of the respective CDRs in the amino acid sequences of the heavy and light chain variable regions of A can be found in the database of antibody amino acid sequences according to Kabat et al. [See Kabat, EA, et al. (1991)]. Were determined by weighing the homology. According to the literature, the chain length of the framework region in the variable region is almost constant if the subtypes are the same even between different antibodies, and the amino acid sequences have commonality. On the other hand, CDR
It is a unique sequence that exists between these framework regions. Thus, the amino acid sequences of the heavy and light chains of HFE7A were compared with those of the same subtype, and as a result, the CDR of the heavy chain of HFE7A was found to be SEQ ID NO: 9 in the sequence listing.
Amino acid numbers 31~35 (CDRH _1), the amino acid number 50-66 (CDRH _2), and the amino acid numbers 9
9-110 was determined with an amino acid sequence represented by (CDRH _3). In addition, the CDR of the light chain of HFE7A corresponds to amino acids 24 to 38 (CDR
L _1), was determined with an amino acid sequence represented by the amino acid numbers 54 - 60 (CDRL _2), and the amino acid number 93~101 (CDRL _3).

Example 5 Preparation of Recombinant Antibody (5-1) Construction of Expression Plasmid cDNAs encoding the heavy and light chains of HFE7A cloned in Example 4 were expressed in an expression vector p for animal cells.
MS18S [Hara, T., et al. (1992) EMBO J. 11, 18
75] was constructed by the method described below. First, an oligonucleotide primer for adding a restriction enzyme EcoRI recognition site to the 5 ′ end of the cDNA held by the plasmid pCR-H, and a restriction enzyme XbaI recognition site and a termination codon at the 3 ′ end: 5′-GGGGATATTCG ACCTCACCAT GGGATGGA -3 '(H3: SEQ ID NO: 22 in the sequence listing) 5'-GGGGTCTAGAC TATTTACCAG GAGAGTGGGA GA -3' (H
4: SEQ ID NO: 23) in the sequence listing was synthesized. Further, the plasmid cCR-L carries c
Oligonucleotide primers for adding a restriction enzyme EcoRI recognition site to the 5 ′ end of the DNA and a restriction enzyme NotI recognition site and a termination codon at the 3 ′ end: 5′-GGGGGAATTCA AGAAGCATCC TCTCATCTA -3 ′ (L3:
SEQ ID NO: 24 in the sequence listing) 5'-GGGGCGGCCG CTTACTAACA CTCATTCCTG TTGAAGC -3 '
(L4: SEQ ID NO: 25 in Sequence Listing) was synthesized. Using each of the primers for the heavy and light chains, PCR was performed under the following conditions.

Reaction solution composition: Template (pCR-H or pCR-L) 1 μg Primer 5 ′ end (H3 or L3) 40 pmol 3 ′ end (H4 or L4) 40 pmol Tris-HCl (pH 8.0) 20 mM potassium chloride 10 mM ammonium sulfate 6 mM magnesium chloride 2 mM Triton X-100 0.1% bovine serum albumin, nuclease-free 10 μg / ml dNTPs 0.25 mM native Pfu DNA polymerase (manufactured by Stratagene) 5 units Sterile distilled water was added to make 100 μl. Temperature conditions: After heating at 94 ° C for 2 minutes, 30 ° C at 94 ° C.
Cycle for 30 seconds at 60 ° C. and 1.5 minutes at 75 ° C.
Repeated eight times.

The DNA amplified by the PCR described above was digested with restriction enzymes EcoRI and XbaI (heavy chain) or EcoR.
After digestion with I and NotI (light chain), the expression plasmid vector pME18S [Hara, T], which is also digested with the restriction enzymes EcoRI and XbaI (for heavy chain) or EcoRI and NotI (for light chain) and dephosphorylated, is used. ., et
al. (1992) EMBO J. 11, 1875] and a 10-fold concentration ligase reaction buffer (6 mM Tris-HCl (pH
7.5), 6 mM magnesium chloride, 5 mM sodium chloride, 7 mM β-mercaptoethanol, 0.1 m
MATP, 2 mM DTT, 1 mM spermidine,
In 0.1 mg / ml bovine serum albumin), 4 units of T4 DNA ligase were added and incubated at 14 ° C. for 15 hours. 2 μl of this ligase reaction solution was added to competent E. coli J.
M109 strain (manufactured by Takara Shuzo Co., Ltd.) was added to 50 μl, left on ice for 30 minutes, and then allowed to stand at 42 ° C. for 30 seconds and on ice for 5 minutes. Add SOC medium (2% tryptone, 0.5% yeast extract, 0.05%
500 μl of sodium chloride, 2.5 mM potassium chloride, 1 mM magnesium chloride, and 20 mM glucose) were added thereto, followed by shaking culture for 1 hour. Hereinafter, Example 4 (4-
A transformed E. coli strain was isolated by the method described in 3), and a plasmid DNA was prepared from the strain. The thus obtained plasmid was used as plasmid pME-H (HFE7
The plasmid was named as an expression plasmid vector carrying the cDNA encoding the A heavy chain and the plasmid pME-L (an expression plasmid vector carrying the cDNA encoding the HFE7A light chain). Transformed E. coli strains carrying these plasmids coli pME-H and E. coli. co
LipME-L was internationally deposited on March 12, 1997 at the Research Institute of Biotechnology and Industrial Technology, and given deposit numbers FERM BP-5868 and FERM BP-5867, respectively.

(5-2) Expression in COS-7 cells The plasmids pME-H and pME-L obtained in the above (5-1) were transformed into COS-7 cells using a gene transfer device (ECM600: (B.T.X Co., Ltd.) using an electroporation method. First, COS-7 cells (American Type Culture Collection No. CRL-1651)
) Was cultured in a cell culture flask (culture area: 225 cm ² ; manufactured by Sumitomo Bakelite Co., Ltd.) containing DMEM containing 10% FCS until the cells became semi-confluent. After removing the medium, 3 ml of trypsin was removed. −ED
A TA solution (manufactured by Sigma) was added and kept at 37 ° C. for 3 minutes. The released cells were collected, washed twice with PBS, and then adjusted to 5 × 10 ⁶ cells / ml with PBS. In addition, 20 μg of each of plasmids pME-H and pME-L prepared using a plasmid large-scale preparation kit (Maxiprep / DNA purification system: manufactured by Promega) was ethanol-precipitated, and then sterilized PBS 20 μg each.
l. When co-transfection was performed with two types of plasmids, each plasmid
Each μg was used. Cell suspension prepared as above
20 μl (1.2 × 10 ⁶ cells) and plasmid solution 20
The mixture was placed in a chamber (produced by Beatty X) with an electrode spacing of 2 mm, set in a gene transfer apparatus, and a pulse of 10 msec was applied once at a setting of 150 V and 900 μF. Remove the cell-DNA mixture in the chamber
It was added to 40 ml of DMEM containing 0% FCS, and cultured in a plastic dish for cell culture at 37 ° C. under 5% carbon dioxide for 24 hours. Next, the culture supernatant was removed, and the cells were washed with a serum-free DMEM medium.
After adding 40 ml, the mixture was further heated at 37 ° C. under 5% carbon dioxide gas for 72 hours.
After culturing for an hour, the culture supernatant was collected.

According to the above-mentioned method, COS-
Seven cells were transformed and the culture supernatant of the transformant was collected: [A] pME-H only; [B] pME-L only; [C] co-transfection of pME-H and pME-L .

(5-3) Anti-F in culture supernatant of transformant
Detection of As Antibody Whether the anti-Fas antibody was produced in the culture supernatant of the transformed COS-7 cell prepared in the above (5-2) was determined by using the human Fas fusion protein described in Example 3 as an antigen. It was examined by the ELISA method. As a result, it was confirmed that only in the case of co-transfection with pME-H and pME-L (above [C]), an antibody reactive with the human Fas fusion protein was produced in the culture supernatant.

Example 6 Restriction of Epitope (6-1) ELISA The peptide described below was subjected to Fmoc solid phase analysis using an automatic peptide synthesizer (Model 430A; PerkinElmer Japan Applied Biosystems Division). Synthesis method [Carpino, LA and Han, GY (1970) J. A
Mer. Chem. Soc. 92, 5748-5749] was synthesized as follows: Arg-Leu-Ser-Ser-Lys-Ser-Val-Asn-Ala-Gln-Val-Thr-As
p-Ile-Asn-Ser-Lys-Gly-Leu (P1: SEQ ID NO: 26 in Sequence Listing); Val-Thr-Asp-Ile-Asn-Ser-Lys-Gly-Leu-Glu-Leu-Arg-Ly
s-Thr-Val-Thr-Thr-Val-Glu (P2: SEQ ID NO: 27 in Sequence Listing); Glu-Leu-Arg-Lys-Thr-Val-Thr-Thr-Val-Glu-Thr-Gln-As
n-Leu-Glu-Gly-Leu-His-His-Asp (P3: SEQ ID NO: 28 in Sequence Listing); Thr-Gln-Asn-Leu-Glu-Gly-Leu-His-His-Asp-Gly-Gln- Ph
e-Cys-His-Lys-Pro-Cys-Pro-Pro (P4: SEQ ID NO: 29 in Sequence Listing); Gly-Gln-Phe-Cys-His-Lys-Pro-Cys-Pro-Pro-Gly-Glu- Ar
g-Lys-Ala-Arg-Asp-Cys-Thr-Val (P5: SEQ ID NO: 30 in the sequence listing); Gly-Glu-Arg-Lys-Ala-Arg-Asp-Cys-Thr-Val-Asn-Gly- As
p-Glu-Pro-Asp-Cys-Val-Pro-Cys-Gln (P6: SEQ ID NO: 31 in Sequence Listing); Asn-Gly-Asp-Glu-Pro-Asp-Cys-Val-Pro-Cys-Gln- Glu-Gl
y-Lys-Glu-Tyr-Thr-Asp-Lys-Ala (P7: SEQ ID NO: 32 in Sequence Listing); Glu-Gly-Lys-Glu-Tyr-Thr-Asp-Lys-Ala-His-Phe-Ser- Se
r-Lys-Cys-Arg-Arg-Cys-Arg (P8: SEQ ID NO: 33 in Sequence Listing); His-Phe-Ser-Ser-Lys-Cys-Arg-Arg-Cys-Arg-Leu-Cys-As
p-Glu-Gly-His-Gly-Leu-Glu-Val (P9: SEQ ID NO: 34 in Sequence Listing); Leu-Cys-Asp-Glu-Gly-His-Gly-Leu-Glu-Val-Glu-Ile- As
n-Cys-Thr-Arg-Thr-Gln-Asn-Thr (P10: SEQ ID NO: 35 in Sequence Listing); Glu-Ile-Asn-Cys-Thr-Arg-Thr-Gln-Asn-Thr-Lys-Cys- Ar
g-Cys-Lys-Pro-Asn-Phe-Phe-Cys (P11: SEQ ID NO: 36 in Sequence Listing); Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys-Asn-Ser- Th
r-Val-Cys-Glu-His-Cys-Asp-Pro (P12: SEQ ID NO: 37 in Sequence Listing); Asn-Ser-Thr-Val-Cys-Glu-His-Cys-Asp-Pro-Cys-Thr- Ly
s-Cys-Glu-His-Gly-Ile-Ile-Lys (P13: SEQ ID NO: 38 in Sequence Listing); Cys-Thr-Lys-Cys-Glu-His-Gly-Ile-Ile-Lys-Glu-Cys- Th
r-Leu-Thr-Ser-Asn-Thr-Lys-Cys (P14: SEQ ID NO: 39 in Sequence Listing); Glu-Cys-Thr-Leu-Thr-Ser-Asn-Thr-Lys-Cys-Lys-Glu- Gl
u-Gly-Ser-Arg-Ser-Asn (P15: SEQ ID NO: 4 in Sequence Listing
0); Ser-Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Asn-Ile-Tyr-Thr-Ly
s-Lys-Glu-Ala-Phe-Asn-Val-Glu (P16: SEQ ID NO: 41 in Sequence Listing).

P1 to P15 are partial sequences of the amino acid sequence from position 1 to position 157 of the extracellular region of human Fas, and the amino acid sequence of 9 to 11 residues overlaps with other peptides. P16 is a peptide for negative control and has no homology with human Fas.

Each of P1 to P16 was completely dissolved in 48 μl of dimethyl sulfoxide (hereinafter referred to as “DMSO”), and adjusted to a total volume of 0.8 ml by adding PBS containing 1 mM 2-mercaptoethanol. A peptide solution was used.

Human F having a carboxyl group at the C-terminus
10 mM peptide corresponding to the extracellular region of the as molecule
The solution was diluted to a concentration of 50 μg / ml with a 0.05 M carbonate-bicarbonate buffer (pH 9.6) containing 2-mercaptoethanol and placed in a 96-well ELISA plate (manufactured by Nunc).
0 μl / well was added. The plate was allowed to stand at 4 ° C. overnight to adsorb the peptide on the inner surface of the well. The solution in each well is discarded, and the well is replaced with PBS-Twee.
Washed 4 times with n. PBS containing 1% (w / v) bovine serum albumin (Sigma A3803) was added at 100 μl /
Each well was dispensed and kept at 37 ° C. for 1 hour. After washing each well four times with PBS-Tween, 5 μg /
50 μl of HFE7A and CH11 prepared in ml were added to each well. After incubating at 37 ° C. for 1 hour, each well was washed four times with PBS-Tween. A horseradish peroxidase-labeled goat anti-mouse immunoglobulin antibody (manufactured by Amersham) diluted 1000-fold with PBS was dispensed at 50 μl / well, incubated at 37 ° C. for 1 hour, and then each well was washed with PBS-Tween for 4 hours. Washed twice. A horseradish peroxidase substrate (manufactured by Bio-Rad) was dispensed at 100 μl / well into each well, allowed to stand at room temperature for 15 minutes, and then the absorbance at 415 nm of each well was measured with a microplate reader (manufactured by Corona). As a positive control, the human Fas fusion protein prepared in Example 1 was used instead of the synthetic peptide.

As a result, of the above synthetic peptides, P11
Since only the wells to which was adsorbed showed high absorbance, it was found that HFE7A specifically bound to the amino acid sequence contained in P11 (FIG. 3).

(6-2) Restriction of Epitope by Competition between Synthetic Peptide and Human Fas on Cell Surface (Competition Assay) First, the following peptide was synthesized: His-Gly-Leu-Glu-Val-Glu -Ile-Asn-Cys-Thr (P95: SEQ ID NO: 42 in Sequence Listing); Glu-Ile-Asn-Cys-Thr-Arg-Thr-Gln-Asn-Thr (P100: SEQ ID NO: 43 in Sequence Listing); Arg -Thr-Gln-Asn-Thr-Lys-Cys-Arg-Cys-Lys (P105: SEQ ID NO: 1 in the sequence listing); Lys-Cys-Arg-Cys-Lys-Pro-Asn-Phe-Phe-Cys (P110 : SEQ ID NO: 44 in the sequence listing); Pro-Asn-Phe-Phe-Cys-Asn-Ser-Thr-Val-Cys-Glu-His-Cy
s-Asp (P115L: SEQ ID NO: 45 in Sequence Listing); Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn-Asp-Asn (D355-364: SEQ ID NO: 46 in Sequence Listing).

P95, P100, P105 and P11
0 indicates the vicinity of the amino acid sequence corresponding to P11 in the extracellular region of human Fas (95% of the extracellular region of human Fas).
(Corresponding to position −128), which is a partial sequence consisting of 10 residues, each of which has an amino acid sequence overlapping with another peptide by 5 residues. P115L has an amino acid sequence of 10 residues overlapping with P110 by 5 residues: Pro-Asn-Phe-Phe-Cys-Asn-Ser-Thr-Val-Cys (P115: amino acid No. 1 to SEQ ID NO: 45 in the sequence listing)
It is obtained by elongating four residues at the C-terminal in anticipation of low solubility of 10). D355-364 is a peptide with no homology to human Fas and was used as a negative control. P
Each of the above peptides other than 115L had 16 μl of D
After dissolving in MSO, PBS containing 1 mM 2-mercaptoethanol was added to adjust the total volume to 0.8 ml, thereby preparing a peptide solution. P115L is 48 μl of DM
After dissolving in SO, PBS containing 1 mM 2-mercaptoethanol was added to adjust the total volume to 0.8 ml.

0.25 μg of HFE7A and each of the above peptide solutions (corresponding to 200 μg of peptide) were mixed in a micro test tube, and the total volume was adjusted to 100 μl with PBS containing 1 mM 2-mercaptoethanol. This is 37
After keeping the temperature for 2 hours while stirring at 10 to 20 rpm at room temperature, FCS was added to a final concentration of 5% to prepare a peptide-antibody mixed solution.

WR19L12a cells were grown by the method described in Example 2 and then collected by centrifugation.
The cell density was adjusted to 1 × 10 ⁷ cells / ml using MI medium. This cell suspension was dispensed at 100 μl / well into a U-shaped bottom 96-well plate, and the mixture was stirred at 4 ° C. and 1000 rpm using a microplate swing rotor.
After centrifugation at pm for 3 minutes, the supernatant was removed. 100 μl of each peptide-antibody mixture was added to the pellet,
Pipette ~ 2 times. After standing at 4 ° C. for 30 minutes, the mixture was centrifuged and the supernatant was removed. The pellet was washed three times with a flow cytometry buffer, and then a 50 μl / well FITC-labeled goat anti-mouse IgG antibody (manufactured by Kappel) diluted 250-fold with the flow cytometry buffer was added. Pipette once. The plate was allowed to stand at 4 ° C. for 30 minutes in the dark, and then centrifuged to remove the supernatant. The pellet was washed three times with a flow cytometry buffer, and a 10% neutral buffered formalin solution for tissue fixation (manufactured by Wako Pure Chemical Industries, Ltd.) diluted 10-fold with PBS was used.
0 μl / well was added to each well, pipetting was performed once or twice, and the plate was fixed at 4 ° C. for 12 hours or more while shielding the plate from light. Buffer for flow cytometry 100
After suspending by adding μl / well, the mixture was centrifuged and the supernatant was removed.
After the pellet was washed three times with a flow cytometry buffer, the suspension suspended in 500 μl / well of a flow cytometry buffer was analyzed with a flow cytometer (Cytoace-150; manufactured by JASCO Corporation). (Excitation wavelength: 488 nm, detection wavelength: 530 nm), and the average value of FITC fluorescence intensity per cell was calculated. further,
0% when no peptide-antibody mixture was added,
The average value of the fluorescence intensity of each sample was calculated with the value of D355-364 as 100%.

As a result, P105 was HFE7A and WR1
It was found that P100 and P110, which strongly inhibit the binding to 9L12a cells and half overlap with the amino acid sequence of P105, respectively, inhibit the binding between HFE7A and WR19L12a cells by about 50% and 60%, respectively. P9 that does not completely overlap the amino acid sequence of P105
No binding inhibition was observed with 5 and P115L (FIG. 4). From the above results, P105 was composed of HFE7A and human F
an amino acid sequence sufficient to inhibit as binding;
The HFE7A epitope was shown to be contained in the amino acid sequence corresponding to P105. The portion corresponding to P105 is a region where the amino acid sequence is conserved between human Fas and mouse Fas.

Example 7 HFE7A against monkey Fas
That binding capacity chimpanzee peripheral blood 40ml of (Sanwa Kagaku Kenkyusho Kumamoto primates Park), peripheral blood 20ml of Japanese macaques, of peripheral blood 20ml or marmoset of cynomolgus monkey peripheral blood 3m
Using l, the tests described below were performed. First, peripheral blood to which heparin (Novoheparin: manufactured by Novo) was added was adjusted to an equal volume of Ficoll-Pak solution (specific gravity: 1.077, except that for cynomolgus monkeys, specific gravity: 1.072:
(Pharmacia) and centrifuged at 1700 rpm for 30 minutes to collect a peripheral blood mononuclear cell fraction. The peripheral blood mononuclear cell fraction was washed twice with Hanks' balanced salt solution,
1 × 10 5 in RPMI 1640 medium containing 10% FCS
^The cells were suspended at ⁶ cells / ml. Phytohemagglutinin-P (Sigma) was added to this suspension at a final concentration of 5 μg.
/ Ml, and cultured at 37 ° C under 5% carbon dioxide for 24 hours. The cells were collected by centrifugation, washed, and then resuspended in RPMI1640 medium containing 10% FCS. Interleukin 2 (manufactured by Amersham) was added to this suspension to a final concentration of 10 units / ml, and 3
By culturing at 7 ° C. under 5% carbon dioxide for 72 hours,
Activated lymphocytes were obtained.

Next, 1 × 10 ^{6 of the} activated lymphocytes were placed in a test tube, and 50 μl of 20 μg / ml HFE7A was added.
Alternatively, the cells were suspended in 50 μl of PBS, left on ice for 1 hour, and washed three times with 500 μl of PBS. The cells were suspended in 50 μl of a 20 μg / ml FITC-labeled anti-mouse IgG antibody (manufactured by BioResource) and incubated on ice for 30 minutes.
After leaving still for minutes, it was washed three times with 500 μl of PBS.
Using the cells resuspended in 500 μl of PBS as a sample, the fluorescence intensity was measured using a flow cytometer (Cytoace: manufactured by JASCO Corporation). The frequency distribution of the number of cells based on the fluorescence intensity was obtained, and the ratio of the number of stained cells to the total number of cells was calculated. As a result, in the sample without HFE7A, less than 3% of the cells were stained in any of the above species, whereas in the sample with HFE7A, at least 17% and at most 82% of the cells were stained. It had been. Therefore, HFE7A was shown to bind to a wide range of primates, including humans.

Example 8 For T Cells in Mice
HFE7A apoptosis-inducing activity 0.05 mg or 0.1 mg / individual HFE7A monoclonal antibody (500 μl each) against 6-week-old female C3H / HeJ mice (purchased from CLEA Japan)
Dissolved in PBS) or 500 μl of PB
Only S was intraperitoneally administered (3 mice per group), and 42 hours after administration, the mice were anesthetized with ether and the thymus was removed. The extracted thymus was washed with an RPMI medium containing 10% FCS, and then crushed using a spatula on a mesh (Cell Strainer; manufactured by Falcon), and the cells that passed through the mesh were twice washed with an RPMI1640 medium containing 10% FCS. Washed. After measuring the cell number, 10% FCS
1 × 10 ⁶ cells / 50μ in RPMI1640 medium containing
The thymus cells suspended to 1 l were dispensed at 50 μl / well into a U-shaped bottom 96-well microplate (manufactured by Nunc) and centrifuged (90 × g, 4 ° C., 10 minutes).

The supernatant was removed, and the following fluorescently labeled antibody solution (a) or (b) was added to each well: (a) 0.5 mg / ml FITC-labeled anti-mouse CD9
5 (Fas) antibody (Jo2: manufactured by Pharmingen) 1
0 μl / well and 10 μl / well of 0.5 mg / ml phycoerythrin (hereinafter referred to as “PE”) labeled anti-mouse CD90 antibody (Thy-1.2: manufactured by Cedarane) (CD90 is specifically expressed on T cells) (B) 0.5 mg / ml FITC-labeled anti-mouse CD4
Antibody (L3T4: Pharmingen) 10 μl / well and 0.2 mg / ml PE-labeled anti-mouse CD8
Antibody (Ly-2: manufactured by Pharmingen) 10 μl / well.

The plate was shaken to stir the inside of the well, allowed to stand on ice for 1 hour, and then centrifuged (90 × g, 4 ° C., 1 ° C.).
0 minutes). After removing the supernatant and washing twice with 100 μl / well of a flow cytometry buffer, 3.7%
Add 50 μl / well of formalin solution and place on ice for 10
The cells were fixed by standing still. Centrifugation (90
× g, 4 ° C., 10 minutes) to remove the supernatant. The precipitated cells were washed again with 100 μl / well of a flow cytometry buffer, and then washed with a flow cytometry buffer 100.
Resuspended in μl / well. Using the cell suspension in each well thus obtained as a sample, using a flow cytometer (Epix Elite: manufactured by Coulter),
The fluorescence of 1 × 10 ⁴ cells / sample was measured under the conditions described below: excitation wavelength: 488 nm; detection wavelength: 530 nm (FITC), 600 nm (P
E).

From the measurement data, the fluorescence distribution of FITC and PE of the cell population of each sample was prepared, and for the sample to which the fluorescently labeled antibody of (a) was added, Fas positive and CD90 positive cells (hereinafter referred to as “ The ratio of “Fas ⁺ CD90 ⁺ ” to the total cell number was calculated. Similarly,
For the sample to which the fluorescently labeled antibody of (b) above was added, C
D4-positive CD8-positive cells (hereinafter referred to as “CD4 ⁺ CD8 ⁺ ”) or CD4-positive CD8-negative cells (hereinafter “CD4 ⁺
The ratio of the number of “CD8 ⁻ ” to the total number of cells was calculated.

The results are shown in Table 1.

[0205]

[Table 1]

(Numerical value is%) In the thymocytes of the HFE7A-administered group mice, the Fas-expressing T cells (F
as ⁺ CD90 ⁺ ) was significantly reduced at all doses. In addition, the CD4 ⁺ CD8 ⁺ cell group and the CD4 ⁺ C
The D8 ^- cell group also had a higher HF compared to the group receiving only PBS.
E7A administration significantly reduced the number.

From the above results, it was shown that the anti-Fas monoclonal antibody HFE7A has an apoptosis-inducing activity on T cells expressing Fas molecules in vivo.

Example 9 H against an autoimmune disease model
Effect of FE7A MR, a mutant of the Fas ligand gene, a model mouse for systemic lupus erythematosus-like autoimmune disease
Using Lgld / gld mice, the effect of the administration of anti-Fas monoclonal antibody HFE7A on autoimmune disease symptoms was examined according to the method described below.

Example 3 Intraperitoneally of 18-week-old MRL gld / gld mice (purchased from Japan SLC, Inc.)
0.2 mg or 0.5 mg / HFE7A prepared in
An individual (dissolved in 500 μl of PBS) or a single dose of 500 μl of PBS was administered.

Swelling of the wrists and ankles of each test mouse caused by autoimmune disease symptoms was observed, the degree of swelling was evaluated, and changes over time in each group were examined [Yonehara Shin (1994) Nikkei Science Supplement 110, 66-77]. As a result, H
It was clarified that the administration of FE7A markedly reduced the degree of swelling of the wrists and ankles.

The thymus was excised from these test mice, and the proportion of T cells expressing Fas in the thymus was examined by the method described in Test Example 1 above. As a result, the number of Fas-expressing T cells in the thymus was significantly reduced.

Example 10 Hepatotoxicity test BALB / c mice were intraperitoneally administered any of the following: i) HFE7A 0.2 mg (dissolved in 500 μl PBS); ii) HFE7A 0.5 mg (500 μl PBS Iii) 0.1 mg of Jo2 (Pharmingen)
Iv) 500 μl PBS only.

Of the above, Jo2 is a known anti-mouse Fas antibody having apoptosis-inducing activity. Eight hours, 24 hours and 72 hours after administration (only mice administered Jo2 3 hours after administration), whole blood was collected from the posterior vena cava under light ether anesthesia, and glutamate-oxaloacetate transaminase in each sample blood was collected. (Hereinafter "GO
T ") and glutamate-pyruvate transaminase (hereinafter referred to as" GPT ") values were measured using an automatic analyzer (Model 7250; manufactured by Hitachi, Ltd.) and a reagent for the same (Transaminase-HRII; Wako Pure Chemical Industries, Ltd.). (Manufactured by K.K.). As a result, the GOT value and GPT value in the group administered with Jo2 rapidly increased after 3 hours, whereas the GOT value and GPT value in the group administered with HFE7A increased.
The PT value hardly changed as in the group to which only PBS was administered (FIG. 5). From the above results, it was shown that HFE7A did not induce liver damage.

Example 11 Effect on Fulminant Hepatitis Model It is known that when the anti-mouse Fas antibody Jo2 is administered intraperitoneally to mice, the mice develop fulminant hepatitis within a few hours and die [Ogasawara, J., et al. (1993) N
ature 364, 806]. Therefore, in order to examine the effect of HFE7A on liver injury caused by Jo2, the survival rate of mice when HFE7A was administered simultaneously with or after Jo2 was examined. Intraperitoneally in 6 week-old BALB / c mouse females (3 per group: purchased from Japan SLC, Inc.)
Antibodies were administered under the following conditions and observed over time: (A) Jo2 0.1 mg / 0.5 ml; (B) Jo2 0.01 mg / 0.5 ml; (C) 0.1 mg of Jo2 and 0.1 mg of HFE7A. 5mg
/0.5 ml (simultaneous administration); (D) Jo2 0.1 mg and HFE7A 0.05 m
g / 0.5 ml (simultaneous administration); (E) 2 mg after administration of Jo2 0.01 mg / 0.2 ml
0 minutes later, HFE7A was administered at 0.1 mg / 0.2 ml.

The results are shown in FIG. In the case of single administration of Jo2, all mice died within 9 hours in both cases of 0.1 mg / individual and 0.01 mg / individual ((A) and (B)). However, simultaneous administration of HFE7A (0.5 mg / individual, 0.05 m
g / individual) ((C) and (D)), the mice showed no abnormality even after several weeks of administration.
It has been found that the administration of E7A can prevent the onset of fulminant hepatitis. Furthermore, even when HFE7A was administered 20 minutes after the administration of Jo2, the mouse was normal and no external symptoms were observed. HFE7A has been shown to have prophylactic and therapeutic effects on various diseases.

Example 12 Effect on Rheumatoid Arthritis
Result 1) Preventive effect against collagen-induced arthritis CD1F1 mouse (6), which is an F1 mouse obtained by crossing a female BALB / c mouse and a male DBA / 1J mouse
1 week-old female (purchased from Japan Charles River Co., Ltd.)
Acclimated for a week. [Phadke, K. (198
5) Refer to Immunopharmacol. 10, 51-60] and dilute a 0.3% solution of bovine type II collagen (50 mM acetic acid solution, manufactured by Collagen Technology Training Institute) to 0.2% (2 mg / ml) with 50 mM acetic acid. After adding an equal dose of Complete Freund's Adjuvant (manufactured by Difco) and emulsifying,
Using a plastic 1 ml syringe equipped with a tuberculin needle, 100 μl (equivalent to 100 μg of bovine type II collagen) was administered intradermally to the base of the ridge of a mouse held in an intravenous incubator. One week after the first sensitization, booster immunization was performed under the same conditions as above. At the time of boosting, 100 μg of HFE7A or mouse IgG for control was intraperitoneally administered (6 per group).
One). From 5 weeks after the first sensitization, swelling of the extremities began to be observed. The joints of the limbs were observed macroscopically, and the method described in the literature [Wood, FD, et al. (1969) Int. Arch.
Appl. Immunol. 35, 456-467], and the degree of swelling was scored. The score was determined for each limb according to the following criteria: 0: No symptoms; 1: Only one small joint such as a finger of the limb swollen and red; 2: Two or more small joints or relatively large such as a wrist or ankle 3: swelling and redness of joints; 3: swelling and redness of one hand or entire foot.

That is, when the limbs are swollen to the maximum, 1
The score of the individual is 12. An individual having a total limb score of 1 or more was defined as an “onset mouse”.

The results are shown in FIG. Non-specific mouse I
In the control group to which gG was administered, all mice developed by 7 weeks after the first sensitization, while half of the mice in the HFE7A administration group did not develop any disease by 8 weeks (FIG. 7).
A). The average score was lower in the HFE7A-administered group than in the control group (FIG. 7B).

2) Apoptosis-inducing ability for synovial cells derived from rheumatoid patients The effect of HFE7A on the survival rate of synovial cells derived from patients with rheumatoid arthritis was examined by the following method using mitochondrial reducing ability as an index. . First, synovial tissue collected from the affected part of a human rheumatoid arthritis patient was minced with scissors in Dulbecco's modified Eagle medium (manufactured by Gibco) containing 10% fetal bovine serum (manufactured by Summit), and fat was removed. Collagenase (manufactured by Sigma) was added at 5 μg / ml, and the mixture was incubated at 37 ° C. for 90 minutes. Cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 ° C. under 5% carbon dioxide gas was used as rheumatoid patient-derived synovial cells.

The thus-obtained synovial cells derived from a rheumatic patient were converted into single cells by trypsinization, and then 1 × 10 5 in Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
^After suspending the cells at ⁵ cells / ml, the cells were seeded on a 96-well plate at 2 × 10 ⁴ cells / 200 μl / well, and cultured at 37 ° C. for 5 days under 5% CO 2 gas conditions. After removing the culture supernatant, Hanks buffer (Gibco)
And washed 3 times with HFE7A from 10 ng / ml to 100
200 μl of Dulbecco's modified Eagle's medium containing 0 ng / ml (10-fold dilution series) and 10% fetal bovine serum was added. After culturing at 37 ° C. and 5% carbon dioxide for 20 hours, 1 mg / ml XTT (2,3-bis [2-methoxy-4-nitro-5-sulfophenyl] -2H-tetrazolium-5-carboxanilide inner salt: Sigma Corporation 50 μl (final concentration: 250 μg /) of 25 μM PMS (phenazine methosulfate: Sigma)
(ml XTT; 5 μM PMS) and further cultured for 4 hours, and the absorbance at 450 nm of each well was measured.

The viability of the cells in each well was calculated by the following formula: Viability (%) = 100 × (ab) / (c−b) (where a is the measured value of the test well, b represents the measured value of the cell-free well, and c represents the measured value of the well without the antibody.) The results are shown in Table 2. HFE7A inhibited the survival of synovial cells from rheumatic patients in a concentration-dependent manner.

[0222]

[Table 2]

Example 13 Design of HFE7A Humanized Antibody (1) Molecular Modeling of Variable Region of HFE7A Molecular modeling of the variable region of HFE7A is generally known as homology modeling [Methods in Enz.
ymology 203, 121-153 (1991)]. That is, the Protein Data Bank (Protein Data Bank)
Data Bank: Chemistry Department, Building 555, Br
rokheaven National Laboratory, PO Box 5000, Upto
n, NY 11973-5000, USA: hereinafter referred to as “PDB”). The primary sequence of the variable region of human immunoglobulin subjected to X-ray crystal structure analysis was HFE7.
The three-dimensional structure of the framework with the highest homology compared to the framework part of A is 1 G for the light chain.
GI, 2HFL was selected as the heavy chain. By combining both, a three-dimensional structure of the framework part (hereinafter referred to as “framework model”) was constructed.

According to the classification of Choccia et al., HFE7A
Of the CDR, CDRL ₂ is canonical class 1, C
DRL ₃ was classified into canonical class 1 and CDRH ₁ was classified into canonical class 1. CDRL ₁ , CDRH ₂ , CDR
H ₃ did not correspond to a particular canonical class. C
The CDR loops of DRL ₂ , CDRL ₃ and CDRH ₁ were anchored in a conformation specific to each canonical class and incorporated into a framework model. CDRL ₁ is Soronton et al classification of [J. Mol. Biol. 263, 800-815 (19
96), the conformation of cluster 15B was used. For CDRH ₂ and CDRH _3, conformations of highly homologous sequences were retrieved from the PDB,
By using energy calculations together, the most likely CDR loop conformation was constructed and incorporated into the framework model. Finally, energy calculation was performed so as to eliminate the contact between atoms that are disadvantageous in terms of energy, and a molecular model of HFE7A was obtained. The above operation can be performed using a commercially available general molecular modeling system. In the present invention, AbM (manufactured by Oxford Molecular Limited) was used.

Regarding the obtained molecular model, a software procheck (PROCHECK: J. Appl. Cryst. (199)
3) Refer to 26, 283-291), and after evaluating the accuracy of the structure, the surface exposure of each residue was calculated and the presence or absence of interatomic contact was searched.

(2) Selection of Acceptor The HFE7A light chain and heavy chain subgroups were compared with the consensus sequence of each human antibody subgroup.
The light chain showed 79% identity with subgroup κIV and the heavy chain showed 79% identity with subgroup I. However, no human antibody with this combination was present. 8E10'CL whose light chain is subgroup κIII and whose heavy chain is subgroup I was selected as a human antibody in which the light chain and heavy chain were derived from the same antibody and both showed 70% or more identity. .

(3) Selection of Donor Residues to be Transplanted into Acceptor Using a chameleon software (manufactured by Oxford Molecular Limited), the light chain and heavy chain of HFE7A were aligned with the amino acid sequence of the acceptor. According to the requirements of a) to d), the humanized variable region sequence shown in the following example was designed to
The following plasmids were constructed as recombinant vectors containing the nucleotide sequence of the DNA encoding the humanized antibody.

Example 14 DN Encoding Humanized Light Chain
Preparation of A (1) Cloning of cDNA Encoding the Full Length of Human Light Chain (κ Chain) Prior to humanizing the light chain amino acid sequence of the mouse anti-human Fas antibody HFE7A, human immunoglobulin containing a constant region (hereinafter referred to as “Ig ) Was cloned.

1) Synthesis of primers The cDNA encoding the human light chain was separated by PCR. In performing PCR, the following 2
The following primers were synthesized: 5'-GCGAATTCTG CCTTGACTGA TCAGAGTTTC CTCA-3 '(HVKII5-4: SEQ ID NO: 47 in Sequence Listing); 5'-GCTCTAGATG AGGTGAAAGA TGAGCTGGAG GA-3' (HKCL3-3: SEQ ID NO in Sequence Listing) 48).

2) Construction of Plasmid Containing Human Ig Light Chain cDNA A cDNA containing the full length human Ig light chain was prepared by PCR, inserted into the plasmid, and cloned in E. coli.

HL-D encoding full length human Ig light chain
The NA fragment was prepared under the following conditions.

Reaction solution composition: Human lymphocyte cDNA library (manufactured by Life Technology) 25 ng oligonucleotide primer HVKII5-4 50 pmol oligonucleotide primer HKCL3-3 50 pmol 25 mM dNTP mixture 10 μl 100 mM Tris-HCl buffer (pH 8.5) 10 μl 1 M potassium chloride 5 μl 25 mM magnesium chloride 10 μl Taq DNA polymerase (manufactured by PerkinElmer) 1 unit The reaction solution having the above composition was mixed with double distilled water to a final volume of 100 μl.
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times,
Warmed for minutes.

The HL-DNA fragment thus prepared was inserted into a plasmid pCR3 DNA using a TA cloning kit for eukaryotes (manufactured by Invitrogen) according to the protocol specified by the manufacturer, and was previously attached to the kit. Competent E. coli TOP10F '
Introduced into the strain. Thus, the plasmid pHL15 carrying the HL-DNA fragment, which is the cDNA of the human Ig light chain,
-27 was obtained.

(2) Construction of Humanized Antibody Light Chain Expression Vector for HFE7A 1) Construction of Humanized HFE7A-Light Chain Expression Vector Plasmid The 47th amino acid (proline) and the 49th amino acid (lysine) from the N-terminal side of the amino acid sequence (hereinafter referred to as "region I") are replaced with alanine and arginine, respectively, which are conserved in a human light chain (κ chain). 80th amino acid (histidine), 81st amino acid (proline),
82nd amino acid (valine), 84th amino acid (glutamic acid), 85th amino acid (glutamic acid), 87th amino acid (alanine) and 89th amino acid (threonine) (hereinafter referred to as "region II")
Is a serine conserved in the human light chain (κ chain),
What was replaced with arginine, leucine, proline, alanine, phenylalanine, and valine was designated as "HH type".

Further, those obtained by replacing the region I with human amino acid residues and leaving the region II as mouse amino acid residues were designated as "HM type".

Furthermore, those in which the amino acid residues of the mouse in both the region I and the region II were left as the "MM type" were designated.

In the following, expression plasmids having these three types of humanized anti-human Fas antibody HFE7A-light chain amino acid sequences were constructed.

2) Synthesis of primers for preparing light chain variable and constant regions of humanized HFE7A HFE obtained by fusing the variable region of each humanized anti-Fas antibody HFE7A-light chain with the constant region of human Ig light chain (κ chain) DNA encoding the type polypeptide chain (SEQ ID NO: 50 in the sequence listing) (SEQ ID NO: 49 in the sequence listing), DNA encoding the HM type polypeptide chain (SEQ ID NO: 52 in the sequence listing)
(SEQ ID NO: 51 in the sequence listing) and DNA (SEQ ID NO: 53 in the sequence listing) encoding the MM type polypeptide chain (SEQ ID NO: 54 in the sequence listing) were each synthesized by PCR.

In performing PCR, the following 1
Three oligonucleotide primers were synthesized: 5'-CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCT-3 '(7AL1P: SEQ ID NO: 55 in the sequence listing); 5'-GAGAGGGTGG CCCTCTCCCC TGGAGACAGA GACAAAGTAC CTGG-3' (7AL1N: SEQ ID NO in the sequence listing) 5'-CCAGGTACTT TGTCTCTGTC TCCAGGGGAG AGGGCCACCC TCTC-3 '(7AL2P: SEQ ID NO: 57 in Sequence Listing); 5'-GATTCGAGAT TGGATGCAGC ATAGATGAGG AGTCTGGGTG CCTG-3' (7AL2N: SEQ ID NO: 58 in Sequence Listing); 5'- GCTGCATCCA ATCTCGAATC TGGGATCCCCA
GACAGGTTTA GTGGC -3 '(7AL3PA: SEQ ID NO: 59 in Sequence Listing); 5'-AAAATCGCCC GGCTCCAGAC GAGAGATGGT
GAGGGTGAAG TCTGTCCCAG AC-3 '(7AL3N: SEQ ID NO: 60 in Sequence Listing); 5'-CTCGTCTGGA GCCGGCGGAT TTTGCAGTCT ATTACTGTCA GCAAAGTAAT GAGGATCC -3' (7AL4P: SEQ ID NO: 61 in Sequence Listing); 5'-GGTCAAGGCA CCAGGCTGGA AATCAAACGG ACTGTGGCTG CACCATCTGT CTTCA -3 '(7ALCP: SEQ ID NO: 63 of sequence listing); 5'-CCCGAATTCT TACTAACACT CTCCCCTGTT GAAGCTCTTT GTGAC -3'5'-TCTGTCCCAG ACCCACTGCC ACTAAACCTG TCTGGGATCC CAGATTCGAG ATTGG -3 '(M7AL2N: SEQ ID NO: 65 in the sequence listing); 5'-GTTTAGTGGC AGTGGGTCTC GGACAGACTT CACCTCTACC ATCCATCCTG TGGAG -3' sequence No. 66); 5′-ATGGTGCAGC CACAGTCCGT TTGATTTCCA GCCTGGTGCC TTGACCGAAC GTCCG -3 ′ (7AL4NA: Sequence listing) SEQ ID NO: 67).

3) Construction of Plasmid p7AL-HH (Humanized HH Type HFE7A-Light Chain Expression Plasmid) It was made by performing a step-wise PCR, inserted into a plasmid vector, and cloned in E. coli.

A) First-step PCR FIG. 8 shows an outline of the first-step PCR for preparing VHH-DNA.

[0243] 5 'ends were added HindIII restriction enzyme cleavage site, L7A1-DNA fragment encoding a portion of the secretion signal sequence and FRL ₁ region was produced under the following conditions.

Reaction solution composition: plasmid pME-L DNA 200 ng oligonucleotide primer 7AL1P 80 pmol oligonucleotide primer 7AL1N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase (manufactured by Stratagene) 10 units 200 µl final volume with double distilled water
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times,
Warmed for minutes.

[0246] Some of FRL _1, L7A2-DNA encoding a portion of CDRL _1, FRL ₂ and CDRL ₂ region
The fragment was prepared under the following conditions.

Reaction solution composition: plasmid pME-L DNA 200 ng oligonucleotide primer 7AL2P 80 pmol oligonucleotide primer 7AL2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

[0249] L7A3-DNA fragment encoding a portion of the CDRL ₂ and FRL ₃ was prepared under the following conditions.

Reaction solution composition: plasmid pME-L DNA 200 ng oligonucleotide primer 7AL3PA 80 pmol oligonucleotide primer 7AL3N 80 pmol dNTP mixed solution 20 μl 10 × Pfu buffer solution 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was finalized in double distilled water. 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

[0252] Some of the FRL _3, L7A4-DNA fragment encoding a portion of CDRL _3, FRL ₄ and the constant region was prepared under the following conditions.

Reaction solution composition: plasmid pME-L DNA 200 ng oligonucleotide primer 7AL4P 80 pmol oligonucleotide primer 7AL4N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

[0255] encoding part and the constant region of the FRL _4, 3 'L7A5-DNA fragment obtained by adding an EcoRI restriction enzyme cleavage sites at the ends was produced under the following conditions.

Reaction solution composition: plasmid pHL15-27 200 ng oligonucleotide primer 7ALCP 80 pmol oligonucleotide primer 7ALCN 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of the reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

To 200 μl of each product after PCR, add an equal volume of phenol / chloroform (50% water-saturated phenol,
48% chloroform, 2% isoamyl alcohol)
Was added and stirred vigorously for 1 minute. Then 10,000
An xg centrifugation was performed to collect an aqueous layer. An equal amount of chloroform / isoamyl alcohol (96
% Chloroform, 4% isoamyl alcohol), vigorously stirred again for 1 minute, and centrifuged at 10,000 × g to collect an aqueous layer (hereinafter, this series of operations is referred to as “phenol extraction”).

The recovered supernatant was added to a 1 / 10-fold amount of 3M
After adding sodium acetate (pH 5.2) and 2.5 times the volume of ethanol, and freezing on dry ice, 10,000 ×
g was centrifuged to collect the DNA as a precipitate (hereinafter, this series of operations is referred to as "ethanol precipitation").

The obtained DNA precipitate was dried in vacuum, dissolved in double-distilled water, and separated by 5% polyacrylamide gel electrophoresis. The gel after electrophoresis is 1 μg / m
The product D was stained with ethidium bromide at a concentration of 1 l.
NA was detected under UV irradiation. L7A1-D detected
NA, L7A2-DNA, L7A3-DNA, L7A4
-DNA and the DNA band corresponding to L7A5-DNA were excised with a razor and eluted from the gel using Centricon-10 and Centriluter. Eluted D
NA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

B) Second-step PCR The outline of the second-step PCR for preparing VHH-DNA is shown in FIG.

The above L7A1-DNA fragment and L7A2-D
The L7A1.2-DNA fragment fused with the NA fragment was prepared under the following conditions.

Reaction solution composition: L7A1-DNA solution prepared by first-stage PCR 10 μl L7A2-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer 7AL2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

The aforementioned L7A4-DNA fragment and L7A5-D
The L7A4.5-DNA fragment fused with the NA fragment was prepared under the following conditions.

Reaction solution composition: L7A4-DNA solution prepared by first-stage PCR 10 μl L7A5-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL4P 80 pmol Oligonucleotide primer 7ALCN 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times, and then at 72 ° C. for 10 minutes.
Warmed for minutes.

Amplified L7A1.2-DN after PCR
A and L7A4.5-DNA fragments were separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. Gel after electrophoresis
Stained with ethidium bromide at a concentration of g / ml and the product DNA was detected under UV irradiation. Each detected fusion DNA band was cut out with a razor and centricon-10.
And eluted from the gel using a centuritor.
The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

C) Third Step PCR The outline of the third step PCR for preparing VHH-DNA is shown in FIG.

L7A1.2-DNA fragment and L7A
A 4.5-DNA fragment and a VHH-DNA fragment fused with L7A3-DNA were prepared under the following conditions.

Reaction solution composition: L7A1.2-DNA solution prepared by second-stage PCR 10 μl L7A4.5-DNA solution prepared by second-stage PCR 10 μl L7A3-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80pmol Oligonucleotide primer 7ALCN 80pmol dNTP mixture 20µl 10x Pfu buffer 20µl Pfu DNA polymerase 10 units Final volume of reaction solution of the above composition in double distilled water 200µl
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

After the PCR, the amplified VHH-DNA fragment was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VHH-DNA band was excised with a razor and eluted from the gel using Centricon-10 and Centriluter. 7500xg of eluted DNA
After centrifugation, and ethanol precipitation.
dissolved in 1 liter of distilled water.

FIG. 11 shows an outline of a method for preparing an expression plasmid holding a VHH-DNA fragment.

The obtained VHH-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with the restriction enzymes HindIII and EcoRI. Cloning plasmid pHSG399 DNA
1 g of Takara Shuzo Co., Ltd.
It was digested with III and EcoRI, and dephosphorylated with alkaline phosphatase (bovine intestinal origin: hereinafter referred to as “CIP”). The thus obtained dephosphorylated plasmid p
HSG399 DNA and VH previously digested with restriction enzymes
The H-DNA fragment was ligated using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.) according to the protocol specified by the manufacturer.

The ligated DNA was recovered by ethanol precipitation, dissolved in 5 μl of double-distilled water. coli JM1
09 Electro-Cell (manufactured by Takara Shuzo Co., Ltd.). The mixture was mixed with Gene Pulser / E. coli pulsar cuvette 0.1 cm (Bio-Rad)
According to the protocol specified by the manufacturer, Gene Pulser I
I (manufactured by Bio-Rad) was introduced into E. coli JM109 strain (hereinafter, this series of operations is referred to as "transformation").
After the transformation, the cells were treated with a final concentration of 1 mM IPT.
G (isopropylthio-β-D-galactoside, manufactured by Takara Shuzo Co., Ltd.), 0.1% X-Gal (5-bromo-4)
-Chloro-3-indolyl-β-D-galactoside, Takara Shuzo Co., Ltd.) and LB agar medium containing 50 μg / ml of chloramphenicol [Bactotripton (Difco) 10 g, Bacto yeast extract ( 5 g, sodium chloride 10 g, and Bacto agar (Difco) 15 g. Dissolved in distilled water to make 1 liter], and cultured at 37 ° C. overnight to obtain an E. coli transformant.

The obtained white transformants were 50
LB liquid medium containing 10 μg / ml of chloramphenicol [10 g of bactotripton (manufactured by Difco), 5 g of bactoeast extract (manufactured by Difco), and 10 g of sodium chloride. Dissolve in distilled water to make up 1 liter] in 2 ml at 37 ° C. overnight, and use the alkaline SDS method [Sambrook, J. et al., (19)
89) in "Molecular Cloning A Laboratory Manual Sec
ond edition. "Cold Spring Harbor Laboratory Pres
s.], to extract plasmid DNA.

The plasmid DNA thus extracted was digested with the restriction enzyme Hi.
After digestion with ndIII and EcoRI, a clone holding the VHH-DNA fragment was selected by 1% agarose gel electrophoresis analysis.

By the above operation, humanized HH type HF
E7A-plasmid pH holding a fusion fragment of the light chain variable region and the DNA encoding the human Igκ chain constant region
SGHH7 was obtained. In addition, transformed Escherichia coli carrying the plasmid pHSGHH7. coli pHSGHH
7 SANK 73497 was deposited internationally on August 22, 1997 with the Institute of Biotechnology and Industrial Technology, and was assigned accession number FERM BP-6073.

DNA encoding humanized HH type HFE7A-light chain polypeptide (SEQ ID NO: 50 in Sequence Listing)
The expression vector plasmid p7AL-HH carrying (SEQ ID NO: 49 in the sequence listing) was prepared using the above-described plasmid pHSGHH.
7 was produced.

Expression plasmid vector p for mammalian cells
EE. 1 μg of 12.1 (Lonza) DNA was digested with restriction enzymes HindIII and EcoRI, and dephosphorylated with CIP. This dephosphorylated pEE. 12.1 DNA
100 ng and 10 μg of the pHSGHH7 DNA fragment digested with HindIII and EcoRI
Ligation kit version 2.0 (Takara Shuzo Co., Ltd.)
After transformation, E. coli JM109 strain was transformed and spread on LB agar medium containing 50 μg / ml ampicillin.

The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin,
Plasmid DNA was extracted according to the SDS method. The extracted plasmid DNA was digested with HindIII and EcoRI, and subjected to 1% agarose gel electrophoresis to confirm the presence of the target insert. Thus, humanized HH
A plasmid p7AL-HH was obtained in which a fusion fragment of the variable region of type HFE7A-light chain and DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the cytomegalovirus (hereinafter referred to as CMV) promoter.

4) Construction of plasmid p7AL-HM (Humanized HM type HFE7A-light chain expression plasmid) The VHM-DNA fragment shown in SEQ ID NO: 51 encoding the amino acid shown in SEQ ID NO: 52 in the Sequence Listing is It was made by performing a step-wise PCR, inserted into a plasmid vector, and cloned in E. coli.

A) First Step PCR The outline of the first step PCR for preparing VHM-DNA is shown in FIG.

[0285] 5 'ends were added HindIII restriction enzyme cleavage site, L7A1-DNA fragment encoding a portion of the secretion signal sequence and FRL _1, a portion of FRL _1,
CDRL _1, FRL ₂ and L7A2-DNA fragment encoding a portion of the CDRL _2, and encodes a portion and the constant region of the FRL _4, L7A5-DNA fragment obtained by adding an EcoRI restriction enzyme cleavage site in the 3 'end, VHH-D
Using the DNA obtained in the preparation of the NA fragment [“(2) Construction of plasmid p7AL-HH (humanized HH type HFE7
A-Light chain expression plasmid) "and" a) First stage PC
R "].

CDRL ₂ , FRL ₃ , CDRL ₃ , FRL ₄
And ML7A3-DNA encoding part of the constant region
The fragment was prepared under the following conditions.

Reaction solution composition: plasmid pME-L DNA 200 ng oligonucleotide primer 7AL3PA 80 pmol oligonucleotide primer 7AL4NA 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times, and then at 72 ° C. for 10 minutes.
Warmed for minutes.

After PCR, each product was separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. 1 μg of gel after electrophoresis
The DNA was stained with ethidium bromide at a concentration of / ml and the product DNA was detected under UV irradiation. ML7A detected
The DNA band corresponding to 3-DNA was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. 7500xg of eluted DNA
After centrifugation, and ethanol precipitation.
dissolved in 1 liter of distilled water.

B) Second-step PCR The outline of the second-step PCR for preparing VHM-DNA is shown in FIG.

L7A1.2-DNA fragment and ML7A
The VHM-DNA fragment obtained by fusing the 3-DNA fragment and the L7A5-DNA fragment described above was prepared under the following conditions.

Reaction solution composition: L7A1.2-DNA solution prepared by second-stage PCR 10 μl ML7A3-DNA solution prepared by first-stage PCR 10 μl L7A5-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer 7ALCN 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times,
Warmed for minutes.

After the PCR, the amplified VHM-DNA fragment was separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VHM-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. 7500 xg of eluted DNA
After centrifugation, and ethanol precipitation.
dissolved in 1 liter of distilled water.

FIG. 14 shows an outline of a method for preparing an expression plasmid having a VHM-DNA fragment.

The resulting VHM-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and EcoRI.

Cloning plasmid pHSG399
1 μg of DNA (Takara Shuzo Co., Ltd.) was added to restriction enzyme Hin.
It was digested with dIII and EcoRI and dephosphorylated with CIP. This dephosphorylated pHSG399 DNA and Hin
The VHM-DNA fragment digested with dIII and EcoRI was combined with DNA ligation kit version 2.0.
(Takara Shuzo Co., Ltd.) and then Escherichia coli JM
109 strains were transformed to a final concentration of 1 mM IPTG,
It was spread on LB agar medium containing 0.1% X-Gal and 50 μg / ml chloramphenicol. The resulting white transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and plasmid DNA was extracted by the alkaline SDS method. After digestion of the extracted plasmid DNA with HindIII and EcoRI, 1% agarose gel electrophoresis analysis was performed to select a clone holding the VHM-DNA fragment.

By the above operation, humanized HM type HF
E7A-plasmid pH holding a fusion fragment of the light chain variable region and the DNA encoding the human Igκ chain constant region
SGHM17 was obtained. In addition, plasmid pHSGHM1
Transformed E. coli harboring E.7 coli pHSGH
M17 SANK 73597 was internationally deposited on August 22, 1997, with the Research Institute of Biotechnology, Industrial Science and Technology, under the accession number FERMBP-6072.

Humanized H exemplified in SEQ ID NO: 52 in the sequence listing
The expression vector plasmid p7AL-HM carrying the DNA exemplified in SEQ ID NO: 51 in the sequence listing encoding the M type HFE7A-light chain polypeptide is a plasmid p
It was prepared using HSGHM17.

Expression plasmid vector p for mammalian cells
EE. 12.1 1 μg of DNA was added to HindI restriction enzyme.
Digested with II and EcoRI and dephosphorylated with CIP.
This dephosphorylated pEE. 12.1 DNA 100ng
And pHSGH digested with HindIII and EcoRI
After ligation of 10 µg of the M17-DNA fragment using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), Escherichia coli JM109 strain was transformed, and
It was spread on LB agar medium containing g / ml ampicillin. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with EcoRI and HindIII, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, a plasmid p7AL-HM in which a fusion fragment of the variable region of the humanized HM type HFE7A-light chain and the DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the CMV promoter was obtained. .

5) Construction of Plasmid p7AL-MM (Humanized MM Type HFE7A-Light Chain Expression Plasmid) The VMM-DNA fragment shown in SEQ ID NO: 53, which encodes the amino acid shown in SEQ ID NO: 54 of the Sequence Listing, It was made by performing a three-step PCR, inserted into a plasmid vector, and cloned in E. coli.

A) First Step PCR The outline of the first step PCR for preparing VMM-DNA is shown in FIG.

[0303] 5 'were added to the ends of the HindIII cleavage site, L7A1-DNA fragment encoding a portion of the secretion signal sequence and FRL _1, and encodes a portion and the constant region of the FRL _4, 3' EcoRI terminated The L7A5-DNA fragment to which the cleavage site has been added is VHH-DN
A fragment obtained by preparing the A fragment was used [“(2) Construction of plasmid p7AL-HH (humanized HH type HFE7A
-Light chain expression plasmid) "and" a) First stage PC
R "].

[0304] part of the _{FRL 1, CDRL 1, FRL 2} , C
ML7A2M encoding part of DRL ₂ and FRL ₃
-The DNA fragment was prepared under the following conditions.

Reaction solution composition: plasmid pME-L DNA 200 ng oligonucleotide primer 7AL2P 80 pmol oligonucleotide primer M7AL2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was finally distilled redistilled water. 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times,
Warmed for minutes.

[0307] Some of the FRL _3, ML7A3M-DNA fragment encoding a portion of CDRL _3, FRL ₄ and the constant region was prepared under the following conditions.

Reaction solution composition: plasmid pME-L DNA 200 ng oligonucleotide primer M7AL3PA 80 pmol oligonucleotide primer 7AL4NA 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was finalized in double distilled water. 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

After PCR, the products were separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. 1 μg of gel after electrophoresis
The DNA was stained with ethidium bromide at a concentration of / ml and the product DNA was detected under UV irradiation. ML7A detected
D corresponding to 2M-DNA and ML7A3M-DNA
The NA band was excised with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7500 × g,
After ethanol precipitation, it was dissolved in 50 μl of distilled water.

B) Second Step PCR The outline of the second step PCR for preparing VMM-DNA is shown in FIG.

The above L7A1-DNA fragment and ML7A2M
The ML7A1.2-DNA fragment obtained by fusing the DNA fragment was prepared under the following conditions.

Reaction solution composition: L7A1-DNA solution prepared by first-stage PCR 10 μl ML7A2M-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer M7AL2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

Amplified ML7A1.2-D after PCR
The NA fragment was obtained after phenol extraction and ethanol precipitation.
% Polyacrylamide gel electrophoresis. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected fusion DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. 7500 eluted DNA
And concentrated by centrifugation at × g.
It was dissolved in 0 μl of distilled water.

C) Third Step PCR The outline of the third step PCR for preparing VMM-DNA is shown in FIG.

The ML7A1.2 DNA fragment and ML7
A VMM-DNA fragment obtained by fusing the A3M-DNA fragment and the L7A5-DNA fragment was prepared under the following conditions.

Reaction solution composition: ML7A1.2-DNA solution prepared by second-stage PCR 10 μl ML7A3M-DNA solution prepared by first-stage PCR 10 μl L7A5-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer 7ALCN 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution of the above composition was mixed with double distilled water to a final volume of 200 μl
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times,
Warmed for minutes.

The amplified VMM-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VMM-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. 7500 xg of eluted DNA
After centrifugation, and ethanol precipitation.
dissolved in 1 liter of distilled water.

FIG. 18 shows an outline of a method for preparing a plasmid holding a VMM-DNA fragment. The obtained VMM-D
The NA fragment was further purified by phenol extraction and ethanol precipitation, followed by restriction enzymes HindIII and EcoRI.
Digested.

Cloning plasmid pHSG399
(Takara Shuzo Co., Ltd.) 1 μg of DNA was added to HindIII
And EcoRI, and dephosphorylated with CIP. This dephosphorylated pHSG399 DNA, EcoRI and H
The indIII digested VMM-DNA fragment and DNA
Ligation kit version 2.0 (Takara Shuzo Co., Ltd.)
After the ligation using E. coli JM109 strain, the final concentration was 1 mM IPTG and 0.1% X-G.
It was spread on LB agar medium containing al and 50 μg / ml chloramphenicol. The resulting white transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and plasmid DNA was extracted by the alkaline SDS method. Extracted plasmid DN
A was digested with HindIII and EcoRI, and then 1%
A clone holding the VMM-DNA fragment was selected by agarose gel electrophoresis analysis.

By the above operation, humanized MM type HF
E7A-plasmid pH holding a fusion fragment of the light chain variable region and the DNA encoding the human Igκ chain constant region
SGMM6 was obtained. In addition, transformed Escherichia coli carrying the plasmid pHSGMM6. coli pHSGMM
6 SANK 73697 was deposited on August 22, 1997, with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology, and assigned the accession number FERM BP-6071.

The humanized M exemplified in SEQ ID NO: 54 in the sequence listing
The expression vector plasmid p7AL-MM carrying the DNA exemplified in SEQ ID NO: 53 of the sequence listing encoding the M-type HFE7A-light chain polypeptide is prepared by using the plasmid p.
It was produced using HSGMM6.

Expression plasmid vector p for mammalian cells
EE. 12.1 1 μg of DNA was added to HindI restriction enzyme.
Digested with II and EcoRI and dephosphorylated with CIP.
This dephosphorylated pEE. 12.1 DNA 100ng
And pHSGM digested with HindIII and EcoRI
After ligation of 10 μg of M6 DNA fragment with DNA ligation kit version 2.0 (Takara Shuzo Co., Ltd.), E. coli JM109 strain was transformed and 50 μg was transformed.
/ Ml of LB agar medium containing ampicillin. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with EcoRI and HindIII, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. In this way, a plasmid p7AL-MM was obtained in which a fusion fragment of the humanized HFE7A-light chain MM type variable region and the DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the CMV promoter. .

6) Confirmation of Nucleotide Sequence Plasmids p7AL-HH, p7AL-HM and p7
The nucleotide sequence was determined in order to confirm that the inserted DNA of AL-MM retained the nucleotide sequence of interest. Oligonucleotide primers generated in determining the nucleotide sequence were as described below: 5'-CCCAAGCTTA AGAAGCATCC-3 '(SP1: SEQ ID NO: 68 in Sequence Listing); 5'-ATCTATGCTG CATCCAATCT-3' ( 5'-GTTGTGTGCC TGCTGAATAA-3 '(SP3: SEQ ID NO: 70 in the sequence listing); 5'-CCCGAATTCT TACTAACACT-3' (SP4: SEQ ID NO: 71 in the sequence listing); 5 ' -TTATTCAGCA GGCACACAAC-3 '(SP5: SEQ ID NO: 72 in the sequence listing); 5'-AGATTGGATG CAGCATAGAT-3' (SP6: SEQ ID NO: 73 in the sequence listing).

FIG. 19 shows the positions where the primers bind. The nucleotide sequence is determined by alkaline SDS
Method and the cesium chloride method [both in Sambrook, J. et al. (1
989) in "Molecular Cloning, A Laboratory Manual.
Second edition. "Cold Spring Harbor Laboratory Pre
ss.], and using the plasmid DNA purified as a template, the dideoxynucleotide chain termination method [Sanger, FS
et al. (1977) Pro. Natl. Acad. Sci. USA. 74, 5463]. That is, 3 μg of the purified plasmid DNA was dissolved in 13 μl of double distilled water, and 2 μl of 2 m
M EDTA and 2 μl of 2N NaOH were added, and the mixture was allowed to stand at room temperature for 5 minutes. Thereafter, 4 μl of a 10 M ammonium acetate solution and 100 μl of ethanol were added, and after stirring, the mixture was allowed to stand on dry ice for 10 minutes. After centrifugation at 15000 × g, the obtained precipitate was washed with 80% ethanol and dried under vacuum. Vacuum dried DNA was dissolved in 7 μl of double distilled water and used for nucleotide sequencing reactions.
For the nucleotide sequencing reaction, 7-deaza-sequenase version 2.0 kit (for dCTP: manufactured by Amersham) was used. To 7 μl of the above plasmid solution, 1 pmol of a primer synthesized in advance and 1 μl of a reaction buffer (buffer supplied in advance with the kit) were added, and the mixture was added at 65 ° C.
For 2 minutes. Thereafter, the mixture was gradually cooled to room temperature, annealed with the primer, and labeled with [α- ³² P] dCTP (Amersham). The reaction product was 1 × TBE (100
mM Tris, 100 mM boric acid, 1 mM EDT
A, pH 8.3) 5% polyacrylamide gel electrophoresis containing 8 M urea in buffer was performed. After drying the gel, autoradiography was performed to decode the nucleotide sequence (hereinafter, the nucleotide sequence was determined by the above method).

As a result, p7AL-HH was obtained by converting the nucleotide sequence shown in SEQ ID NO: 49 which encodes the polypeptide shown in SEQ ID NO: 50 of the Sequence Listing to p7AL-H
M is the nucleotide sequence shown in SEQ ID NO: 51 of the sequence listing encoding the polypeptide shown in SEQ ID NO: 52 in the sequence listing, and p7AL-MM is the sequence listing encoding the polypeptide shown in SEQ ID NO: 54 in the sequence listing. Has been confirmed to have the nucleotide sequence shown in SEQ ID NO: 53, respectively.

Example 15 Humanized antibody heavy chain of HFE7A
Construction of expression vector (1) Construction of plasmid holding humanized HFE7A heavy chain variable region DNA 1) Synthesis of primer for preparing humanized heavy chain variable region Humanized anti-Fas antibody HFE7A-variable region of heavy chain and I
D encoding a polypeptide chain (SEQ ID NO: 75 in the sequence listing) containing 5 amino acids at the N-terminal side of the gG-CH1 region
The synthesis of NA (SEQ ID NO: 74 in the sequence listing) was performed by a combination of PCR methods.

In performing the PCR, the following eight primers were prepared: 5′-GGGAAGCTTG GCTTGACCTC ACCATGGGAT GGAGCTGTAT -3 ′ (7AH1P: SEQ ID NO: 76 in Sequence Listing); 5′-TGAAGCCCCA GGCTTCTTGA CCTCAGCCCC AGACTGCACC AGTTGGAC − 5'-TCCACTCAAG CCTCTGTCCA GGGGCCTGTT TTACCC-3 '(7AH2N: SEQ ID NO: 78 of sequence listing); 5'-GTCTGGGGCT GAGGTCAAGA AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA AG-3'E 5'-CAGGCCCCTG GACAGAGGCT TGAGTGGATG GGAGAGATT-3 '(7AH3P: SEQ ID NO: 80 in the sequence listing); 5'-TCAGATCTCA GGCTGCTGAG CTCCATGTAG GCTGTGCTAG CGGATGTGTC-3' (7AH3N: SEQ ID NO: 81 in the sequence listing); 5'-TGGAGCTCAG CAGCCTGAGA TCTGAGGACA CGGCGGTCTA TTAC-3 '(7AH4P: SEQ ID NO: 82 in the sequence listing); 5'-GATGGGCCCT TGGTGGAGGC TGAGGAGACG GTGACCAGGG TCCCTTCGCC C CAGT-3 '(7AH4N: SEQ ID NO: 83 in Sequence Listing).

2) Construction of plasmid pBL7A27 The VD-DNA fragment shown in SEQ ID NO: 74 encoding the amino acid shown in SEQ ID NO: 75 in the sequence listing was prepared by performing three-step PCR, and inserted into the plasmid. And cloned in E. coli.

A) First-Step PCR FIG. 2 shows an outline of the first-step PCR for preparing VD-DNA.
0.

[0333] the 5 'ends were added HindIII restriction enzyme cleavage site, H7A1-DNA fragment encoding the N-terminal secretion signal sequence and FRH ₁ were produced under the following conditions.

Reaction solution composition: plasmid pME-H DNA 200 ng oligonucleotide primer 7AH1P 80 pmol oligonucleotide primer 7AH1NNEW 80 pmol dNTP mixed solution 20 μl 10 × Pfu buffer solution 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was finalized in double distilled water. 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

[0336] A portion of FRH _1, CDRH ₂ and FRH ₂
The H7A2-DNA fragment encoding a part of was prepared under the following conditions.

Reaction solution composition: plasmid pME-H DNA 200 ng oligonucleotide primer 7AH2N 80 pmol oligonucleotide primer 7AH2PNEW 80 pmol dNTP mixed solution 20 μl 10 × Pfu buffer solution 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was finalized in double distilled water. 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times, and then at 72 ° C. for 10 minutes.
Warmed for minutes.

[0339] part of the FRH _2, CDRH ₂ and FRH ₃
The H7A3-DNA fragment encoding a part of was prepared under the following conditions.

Reaction solution composition: plasmid pME-H DNA 200 ng oligonucleotide primer 7AH3P 80 pmol oligonucleotide primer 7AH3N 80 pmol dNTP mixed solution 20 μl 10 × Pfu buffer solution 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was finalized in double distilled water. 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times, and then at 72 ° C. for 10 minutes.
Warmed for minutes.

[0342] Some of FRH _3, H encoding the N-terminal 5 amino acid residues of CDRH _3, FRH ₄ and CH1 regions
The 7A4-DNA fragment was prepared under the following conditions.

Reaction solution composition: Plasmid pME-H DNA 200 ng Oligonucleotide primer 7AH4P 80 pmol Oligonucleotide primer 7AH4N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was finalized in double distilled water. 200 μl volume
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times, and then at 72 ° C. for 10 minutes.
Warmed for minutes.

Each product after PCR was separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. 1 μg of gel after electrophoresis
The DNA was stained with ethidium bromide at a concentration of / ml and the product DNA was detected under UV irradiation. H7A1 detected
-DNA bands corresponding to DNA, H7A2-DNA, H7A3-DNA and H7A4-DNA were cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. 7500 eluted DNA
And concentrated by centrifugation at × g.
It was dissolved in 0 μl of distilled water.

B) Second-Step PCR FIG. 2 shows an outline of the second-step PCR for preparing VD-DNA.
1 is shown.

The above H7A1-DNA fragment and H7A2-D
The H7A1.2-DNA fragment fused with the NA fragment was prepared under the following conditions.

Reaction solution composition: H7A1-DNA solution prepared by first-stage PCR 10 μl H7A2-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AH1P 80 pmol Oligonucleotide primer 7AH2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

The above H7A3-DNA fragment and H7A4-D
The H7A3.4-DNA fragment fused with the NA fragment was prepared under the following conditions.

Reaction solution composition: H7A3-DNA solution prepared by first-stage PCR 10 μl H7A4-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AH3P 80 pmol Oligonucleotide primer 7AH4N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

Amplified H7A1.2-DN after PCR
A and H7A3.4-DNA fragments were separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. Gel after electrophoresis
Stained with ethidium bromide at a concentration of g / ml and the product DNA was detected under UV irradiation. Each detected fusion DNA band was cut out with a razor and centricon-10.
And eluted from the gel using a centuritor.
The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

C) Third-Step PCR FIG. 2 shows an outline of the third-step PCR for preparing VD-DNA.
2 is shown.

H7A1.2-DNA fragment and H7A
The VD-DNA fragment obtained by fusing the 3.4-DNA fragment was prepared under the following conditions.

Reaction solution composition: H7A1.2-DNA solution prepared by second-stage PCR 10 μl H7A3.4-DNA solution prepared by second-stage PCR 10 μl Oligonucleotide primer 7AH1P 80 pmol Oligonucleotide primer 7AH4N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times, and then at 72 ° C. for 10 minutes.
Warmed for minutes.

The amplified VD-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected VD-DNA band is cut out with a razor,
It was eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

FIG. 23 shows an outline of a method for preparing a plasmid holding a VD-DNA fragment.

The obtained VD-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and ApaI.

The plasmid vector pBLUESCRI
PT-II SK + DNA (Stratagene) 1
μg was digested with HindIII and ApaI and dephosphorylated with CIP. This dephosphorylated pBLUESCRIPT
-II SK + DNA, HindIII and ApaI
Was ligated to the VD-DNA fragment digested with, using DNA Ligation Kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.). Escherichia coli JM109 was transformed, and IPTG (isopropylthio-β-isopropyl) having a final concentration of 1 mM was used. D-galactoside, manufactured by Takara Shuzo Co., Ltd.), 0.1% X-Gal
(5-Bromo-4-chloro-3-indolyl-β-D-
Galactoside, manufactured by Takara Shuzo Co., Ltd.) and 50 μg / m
Plated on LB agar containing 1 ampicillin. The resulting white transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and alkali-S
Plasmid DNA was extracted by the DS method. This plasmid was digested with ApaI and HindIII and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, a plasmid pBL7A27 into which the VD-DNA fragment was inserted was obtained.

(2) Human IgG1 constant region genomic DNA
1) Synthesis of Primer for Preparation of Human IgG1 Genome 5 ′ DNA Fragment The synthesis of human IgG1 genomic 5 ′ DNA fragment was prepared by PCR. In performing the PCR, the following two oligonucleotide primers were prepared: 5′-GGGAAGCTTC CGCGGTCACA TGGCACCACC TCTCTTGCA -3 ′ (5 ′ Hind: SEQ ID NO: 84 in the sequence listing); 5′-GCTCTGCAGA GAGAAGATTG GGAGTTACTG GAATC − 3 '(IGGCPSTN: SEQ ID NO: 85 in Sequence Listing).

2) Construction of plasmid pIG5'03 Following the HindIII cleavage sequence, the CH1 of human IgG1
Genomic DNA containing regions and introns is the human genome D
The target DNA fragment was separated and amplified by the PCR method using NA as a template, inserted into a plasmid pHSG399 (manufactured by Takara Shuzo Co., Ltd.), and cloned in E. coli. FIG. 24 shows an outline of a method for preparing a DNA fragment (hereinafter, referred to as “IG5′-DNA”) containing the desired human IgG1 CH1 region and intron.

The IG5'-DNA fragment was prepared under the following conditions.

Reaction solution composition: Human genomic DNA (manufactured by Clontech) 2 μg Oligonucleotide primer 5 ′ Hind 80 pmol Oligonucleotide primer IGGCPSTN 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units 200 µl final volume with double distilled water
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times, and then at 72 ° C. for 10 minutes.
Warmed for minutes.

[0367] The amplified IG5'-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected IG5'-DNA band was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. 7500 xg of eluted DNA
After centrifugation, and ethanol precipitation.
dissolved in 1 liter of distilled water.

The IG5'-DNA fragment obtained was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and PstI.

Plasmid pHSG399 DNA (manufactured by Takara Shuzo Co., Ltd.) (1 μg) was digested with restriction enzymes HindIII and Ps.
Digested with tI and dephosphorylated with CIP. The dephosphorylated plasmid pHSG399 DNA and HidIII
And IG5'-DNA fragment digested with PstI
Ligation kit version 2.0 (Takara Shuzo Co., Ltd.)
After the ligation using E. coli JM109 strain, the final concentration was 1 mM IPTG and 0.1% X-G.
It was spread on LB agar medium containing al and 50 μg / ml chloramphenicol. The resulting white transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and the plasmid DNA was extracted by the alkaline SDS method. This plasmid is called Pst
After digestion with I and HindIII, agarose gel electrophoresis was performed to confirm the presence or absence of the target insert. Thus, the plasmid p into which the IG5'-DNA fragment has been inserted
IG5'03 was obtained.

(3) Genomic DNA of human IgG1 constant region
1) Synthesis of Primer for Preparation of Human IgG1 Genome 3 ′ DNA Fragment The synthesis of human IgG1 genomic 3 ′ DNA fragment was prepared by PCR. In carrying out the PCR, the following two primers were prepared: 5′-TCTCTGCAGA GCCCAAATCT TGTGACAAAA CTCAC-3 ′ (IGGCPSTP: SEQ ID NO: 86 in the sequence listing); ': SEQ ID NO: 87 in Sequence Listing).

2) Construction of plasmid pIG3'08 Intron, hinge region, intron of human IgG1
CH2 region, intron, CH3 region and EcoRI
DNA containing the cleavage sequence was isolated and amplified by PCR using a human genomic DNA as a template, and then inserted into a plasmid pHSG399 (Takara Shuzo) and cloned in E. coli. Genomic DNA containing the hinge region, CH2 region, CH3 region and intron of the target human IgG1 (hereinafter referred to as “IG3′-
FIG. 25 shows an outline of a method for preparing a fragment (referred to as “DNA”).

The IG3'-DNA fragment was prepared under the following conditions.

Reaction solution composition: Human genomic DNA (manufactured by Clontech) 2 μg Oligonucleotide primer IGGCPSTP 80 pmol Oligonucleotide primer Eco3 ′ 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units 200 µl final volume with distilled water
And used for PCR.

PCR temperature conditions: After heating at 94 ° C. for 2 minutes, a temperature cycle of 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes was repeated 30 times.
Warmed for minutes.

The amplified IG3'-DNA fragment after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. Each detected DNA fragment was cut out with a razor and eluted from the gel using Centricon-10 and Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

The obtained IG3'-DNA fragment was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes EcoRI and PstI.

[0377] 1 µg of plasmid pHSG399 DNA (manufactured by Takara Shuzo Co., Ltd.) was digested with EcoRI and PstI, and dephosphorylated with CIP. This dephosphorylated pHSG
After ligating 399 DNA with an IG3'-DNA fragment digested with EcoRI and PstI using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), E. coli JM109 strain was transformed and the final concentration was 1 mM.
IPTG, 0.1% X-Gal and 50 μg / m
It was spread on LB agar medium containing 1 chloramphenicol. White colonies were selected to obtain a plasmid pIG3'08 into which the IG3'-DNA fragment was inserted.

(4) Construction of Humanized HFE7A-Heavy Chain Expression Vector Plasmid The DNA encoding the humanized HFE7A-heavy chain polypeptide shown in SEQ ID NO: 89 in the Sequence Listing and having the DNA shown in SEQ ID NO: 88 in the Sequence Listing is retained. Expression vector plasmid pEg7
AH-H was obtained from plasmid pBL7A27, pIG
It was prepared using 5'03 and pIG3'08. FIG. 26 shows an outline of the manufacturing procedure.

Human IgG1-pIG5'03 plasmid DNA containing the CH1 region of heavy chain and intron 10 μg
Was digested with restriction enzymes ApaI and KpnI.
On the other hand, 1 μg of the aforementioned plasmid pBL7A27 DNA
Was digested with restriction enzymes ApaI and KpnI and dephosphorylated with CIP. This dephosphorylated pBL7A27 DNA
100 ng of p digested with ApaI and KpnI
After ligation with 10 μg of IG5'03 DNA using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), the clone was cloned into Escherichia coli JM109 strain. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was transformed with ApaI and KpnI, or HindIII and Pst
After digestion with I, agarose gel electrophoresis was performed to confirm the presence of the target insert. Thus, the VD-DNA fragment of humanized HFE7A and IG5'-DNA
The plasmid pBL7AF1 to which the fragments were connected and inserted
84 was obtained.

Next, the obtained plasmid pBL7AF1
10 μg of 84 DNA was digested with restriction enzymes HindIII and PstI. On the other hand, the plasmid pIG3 '
08 DNA was mixed with the restriction enzymes HindIII and Ps
Digested with tI and dephosphorylated with CIP. 100 ng of the dephosphorylated pIG3'08 DNA and HindI
PBL7AF184D digested with II and PstI
After ligation of 10 μg of NA using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.),
It was cloned in E. coli JM109 strain. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid is called PstI
And HindIII or HindIII and EcoRI
And agarose gel electrophoresis was performed to confirm the presence of the desired insert. Thus, humanized HFE
The VD-DNA fragment of 7A was connected to a genomic DNA fragment encoding a human IgG1 constant region to obtain an inserted plasmid pgHSL7A62. In addition, transformed Escherichia coli carrying the plasmid pgHSL7A62.
coli pgHSL7A62 SANK73397
Was deposited internationally on August 22, 1997 with the Institute of Biotechnology and Industrial Technology, and received the accession number FERM BP-6.
074 was added.

The resulting plasmid pgHSL7A62D
10 μg of NA was added to the restriction enzymes HindIII and EcoR.
I cut. On the other hand, the expression plasmid pEE. 6.1 μg of DNA was digested with HindIII and EcoRI and dephosphorylated with CIP. This dephosphorylated pEE. 6.1 100 ng of DNA and Hi
pgHSL7A digested with ndIII and EcoRI
After 10 μg of 62 DNA was ligated using a ligation kit, it was cloned into E. coli JM109 strain. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted by an alkaline SDS method. This plasmid was digested with HindIII and EcoRI, and subjected to agarose gel electrophoresis to confirm the presence or absence of the target insert. Thus, the VD-DNA fragment of humanized HFE7A and the genomic DN encoding the human IgG1 constant region
A plasmid pEg7AH-H in which the fusion fragment with A was inserted in the forward direction downstream of the CMV promoter was obtained.

(5) Confirmation of nucleotide sequence The nucleotide sequence was determined to confirm that the inserted DNA of pEg7AH-H retained the sequence of the nucleotide of interest. Oligonucleotide primers prepared in determining the nucleotide sequence were as described below: 5'-ACAGCCGGGA AGGTGTGCAC-3 '(IG01: SEQ ID NO: 90 in the sequence listing); 5'-AGACACCCTC CCTCCCTGTG-3' ( 5′-GTGCAGGGCC TGGGTTAGGG-3 ′ (IG03: SEQ ID NO: 92 in the sequence listing); 5′-GCACGGTGGG CATGTGTGAG-3 ′ (IG04: SEQ ID NO: 93 in the sequence listing); 5 ′ -GTTTTGGGGG GAAGAGGAAG-3 '(IG05: SEQ ID NO: 94 in the sequence listing); 5'-CCAGTCCTGG TGCAGGACGG-3' (IG06: SEQ ID NO: 95 in the sequence listing); 5'-CCTGTGGTTC TCGGGGCTGC-3 '(IG07: SEQ ID NO: 5'-CGTGGTCTTG TAGTTGTTCT-3 '(IG08: SEQ ID NO: 97 in Sequence Listing); 5'-CTTCCTCTTC CCCCCAAAAC-3' (IGP5: SEQ ID NO: 98 in Sequence Listing); 5'-CCGTCCTGCA CCAGGACTGG -3 '(IGP6: SEQ ID NO: 99 in Sequence Listing) 5'-GCAGCCCCGA GAACCACAGG-3 '(IGP7: SEQ ID NO: 100 in the sequence listing); 5'-AGAACAACTA CAAGACCACG-3' (IGP8: SEQ ID NO: 101 in the sequence listing); 5'-GCCTGACATC TGAGGACTC-3 '(H5 +: 5'-GAGTCCTCAG ATGTCAGGC-3 '(H5-: SEQ ID NO: 103 in the sequence listing); 5'-GAGCAGTACT CGTTGCTGCC GCGCGCGCCA CCAG-3' (PEEF: SEQ ID NO: 104 in the sequence listing); 5 '-GGTATGGCTG ATTAATGATC AATG-3' (PEEB: SEQ ID NO: 105 in Sequence Listing).

FIG. 27 shows the positions where the respective primers bind. The nucleotide sequence is determined by alkaline SDS
Plasmid DNA purified by the method and cesium chloride method
Was used as a template by the dideoxynucleotide chain termination method. As a result, pEg7AH-H was sequence number 8 in the sequence listing.
It was confirmed that the polypeptide retained the nucleotide sequence shown in SEQ ID NO: 88 in the sequence listing, encoding the polypeptide shown in FIG.

Example 16 Expression in COS-1 Cells Introduction of the humanized HFE7A-heavy chain expression plasmid and each humanized HFE7A-light chain expression plasmid obtained above into monkey kidney-derived cell line COS-1 cells. , 2m between electrodes
Electrophoresis was performed using a gene transfer apparatus GTE-1 (manufactured by Shimadzu Corporation) equipped with a m / chamber / FCT-13 (manufactured by Shimadzu Corporation). First, COS-1 cells (American Type Culture Collecti
on No. CRL-1650) is a minimum essential alpha medium (hereinafter "α (+) MEM") containing 10% fetal bovine serum (hereinafter "FCS" manufactured by Moregate): manufactured by Gibco BRL ) In a cell culture flask (culture area: 225 cm ² : manufactured by Sumitomo Bakelite Co., Ltd.) until the cells became semi-confluent. Thereafter, the medium was removed, and the cells were treated in 3 ml of a trypsin-EDTA solution (manufactured by Sigma) at 37 ° C. for 3 minutes to release COS-1 cells from the flask. The released cells were collected by centrifugation at 800 rpm for 2 minutes.
Phosphate buffer [0.02% potassium chloride, 0.02%
Potassium dihydrogen phosphate, 0.8% sodium chloride,
1.15% disodium hydrogen phosphate. See “PBS
(−) ”; Manufactured by Nissui Pharmaceutical Co., Ltd.]. Washed COS-1 cells with PBS (-) at 1 × 10
^{It was} prepared to be ⁸ cells / ml to prepare a COS-1 cell suspension.

On the other hand, humanized HFE7A prepared by the alkali-SDS method and the cesium chloride density gradient centrifugation method
4 μg of heavy chain expression plasmid DNA and humanized HF
After mixing 4 μg of E7A-light chain expression plasmid DNA, the mixture was precipitated with ethanol in the same tube, and PBS (−)
Suspended in 20 μl. Obtained plasmid suspension 20
μl to 20 μl of the previously prepared COS-1 cell suspension
(2 × 10 ⁶ cells), transferred to a chamber / FCT-13 (manufactured by Shimadzu Corporation) with an electrode spacing of 2 mm, and set in a gene transfer apparatus GTE-1 (manufactured by Shimadzu Corporation). Thereafter, a pulse of 600 V and 50 μF is applied twice at 1-second intervals, whereby the desired plasmid D
NA was introduced into COS-1 cells. The cell-DNA mixture in the chamber after the pulse was applied to 10% FC
The suspension was suspended in 5 ml of α (+) MEM containing S and transferred to a cell culture flask (culture area: 25 cm ² : manufactured by Sumitomo Bakelite Co., Ltd.). 37 ° C, 72% under 5% carbon dioxide
After culturing for a period of time, the culture supernatant was collected, and the expression product contained in the main culture supernatant was analyzed.

According to the above method, COS-1 cells were transformed with the following combinations of plasmids, respectively, and the culture supernatant was recovered: [A]: conditions without plasmid DNA; [B]: pEg7AH- H and p7AL-MM
Transfection; [C]: pEg7AH-H and p7AL-HM
Transfection; [D]: pEg7AH-H and p7AL-HH
Transfection. Example 17 Quantification of Expression Product by ELISA The expression of the humanized antibody in the culture supernatant prepared in Example 16 and the quantitative assay of the expression product were determined by the enzyme-linked antibody method using an antibody against anti-human IgG ( The following "ELISA
Method). That is, in each well of a 96-well plate (Maxisorp: Nunc), a solid phase buffer (0.05 M sodium bicarbonate, 0.02% sodium azide, pH 9.0) was added at a final concentration of 1 μg / ml.
6) 100 μl of a goat anti-human IgG Fc-specific polyclonal antibody (manufactured by Capel) dissolved in 6) was added thereto.
After incubation for 2 hours, the antibody was immobilized on a plate. afterwards,
PBS (-) containing 0.05% Tween-20 (manufactured by Bio-Rad) (hereinafter referred to as "PBS-T") 350
Washed four times with μl. 10 wells after each wash
The culture supernatant diluted with α (+) MEM containing% FCS was added, and the mixture was kept at 37 ° C. for 2 hours. After washing with PBS-T again, 100 μl of an alkaline phosphatase-labeled goat anti-human IgG Fc-specific polyclonal antibody (manufactured by Caltag Lab) diluted 5000-fold with PBS-T
Was added to each well and incubated at 37 ° C. for 2 hours. again,
After washing with PBS-T, p-nitrophenylphosphoric acid was added with 10% diethanolamine (pH 9.8) at 1 mg / p.
ml of the prepared substrate solution was added. After keeping the temperature at 37 ° C. for 30 minutes to 1 hour, the absorbance at 405 nm was measured. In this experiment, humanized HF contained in the culture supernatant was used.
As a concentration control for the E7A antibody, human plasma immunoglobulin G / subclass 1 (IgG1) (manufactured by BioPure AG) diluted to a certain concentration with α (+) MEM containing 10% FCS was used.

As a result, of the expression products in the culture supernatant prepared in Example 16, those under the conditions of [B], [C] and [D] were specifically detected by the anti-human IgG antibody. Was.

Example 18 Measurement of binding activity to Fas
According to the method described in constant below, ELIS binding activity to Fas transformed cell culture supernatant prepared in Example 16
The test by the method A was performed. First, the culture supernatant of the COS-1 cell expressing the human Fas fusion protein prepared in Example 1 was diluted 5-fold with the immobilization buffer, and 50 μl was added to each well of a 96-well plate (Maxisorp: Nunc). Get in,
By incubating overnight at 4 ° C, human Fa was added to the well surface.
The s fusion protein was adsorbed. After that, each well is
Washed 4 times with 350 μl of BS-T. 5% after washing
PB containing bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.)
50 μl of ST was added to each well, and blocking was performed by incubating at 37 ° C. for 1 hour. Thereafter, each well was washed again with PBS-T. The culture supernatant prepared in Example 16 was previously prepared by the method described in Example 17.
The concentration of the target expression product to be contained was calculated, and 10% F was adjusted so that the final target product concentration was 100 ng / ml.
It was prepared in an α (+) MEM medium containing CS. 10
Each expression product solution adjusted to a concentration of 0 ng / ml was added to 1
A 2-fold dilution was repeated with an α (+) MEM medium containing 0% FCS to prepare a dilution series. 50 μl of each of the thus prepared expression product dilution series solutions was added to each well, and the mixture was added at 37 ° C.
The reaction was performed for 2 hours. Then, after washing again with PBS-T, 50 μl of an alkaline phosphatase-labeled goat anti-human IgG Fc-specific polyclonal antibody (manufactured by Caltag Lab) diluted 10000-fold with PBS-T was dispensed into each well, and the temperature was lowered at 37 ° C. And reacted for 2 hours. The mouse hybridoma HFE7 used as a control
Detection of HFE7A (IgG1) purified from A was performed using PBS-T instead of alkaline phosphatase-labeled goat anti-human IgG Fc-specific polyclonal antibody.
Alkaline phosphatase-labeled goat anti-mouse IgG + IgA + IgM (manufactured by Gibco BRL) diluted 000-fold was used. After washing with PBS-T, 50μ
A substrate solution prepared by adjusting 1 l of p-nitrophenyl phosphoric acid to 1 mg / ml with 10% diethanolamine (pH 9.8) was added, and the mixture was kept at 37 ° C. for 30 minutes to 1 hour. By measuring the absorbance at 405 nm of each well, the binding activity of the expression product contained in each culture supernatant to the human Fas fusion protein was examined.

As a result, among the expression products in the culture supernatant prepared in Example 16, those expressed under the conditions of [B], [C] and [D] have the binding property to the human Fas fusion protein. It was confirmed that it was a product (FIG. 28).

Example 19 Binding of HFE7A to Fas
Competition Inhibition Against Fas Binding of the humanized anti-Fas antibody obtained in the present invention should inhibit the binding of the anti-Fas mouse monoclonal antibody, HFE7A, on which the humanization design was based. Thus, the competitive inhibitory activity of HFE7A and the expression product prepared in Example 16 on human Fas fusion protein was measured.

[0391] 1 mg of the purified HFE7A monoclonal antibody obtained in Example 3 was used in a commercially available alkaline phosphatase labeling kit (Immuno-Link AP and AP).
Labeling was carried out using an L labeling kit (manufactured by Genosis) according to the attached protocol (hereinafter, this labeled antibody is referred to as “AP-HFE7A”).

COS containing human Fas fusion protein
1) The culture supernatant is diluted 5-fold with the immobilization buffer, and 50 μl is added to each well of a 96-well plate for luminescence detection (luminescent solid assay plate, high binding property: manufactured by Coaster) at 4 ° C. overnight. By keeping warm,
The human Fas fusion protein was adsorbed on the well surface.
After the adsorption, each well was washed four times with 350 μl of PBS-T. After washing, 100 μl of PBS-T containing 5% bovine serum albumin (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well.
One block at a time was added and blocking was performed by incubating at 37 ° C. for 1 hour. Thereafter, each well was washed again with PBS-T. The culture supernatant prepared in Example 16 was prepared in advance in Example 1
The concentration of the target expression product contained was calculated by the method shown in 7, and the final target product concentration was adjusted to 1 μg / ml with α (+) MEM containing 10% FCS. Each expression product solution adjusted to a concentration of 1 μg / ml was repeatedly double-fold diluted with α (+) MEM containing 10% FCS to prepare a dilution series. On the other hand, AP-HFE
7A was converted to 5% by α (+) MEM containing 10% FCS.
It was diluted to 0 ng / ml and mixed with the prepared dilution series in an equal amount. Expression product dilution series and AP-HFE7A mixture
50 μl was added to each well after washing, and the plate was allowed to stand at room temperature overnight. Thereafter, each well was washed again with PBS-T, and 100 μl of CDP-star buffer (9.58 ml diethanolamine, 0.2 g magnesium chloride, 0.25 g sodium azide, pH
8.5) was added and left at room temperature for 10 minutes. afterwards,
After removing the CDP-star buffer, the CDP-star substrate (1.2 ml sapphire II (manufactured by Tropics)),
200 μl CDP-star (manufactured by Tropics), CD
50 μl / well was added thereto, and the mixture was allowed to stand at room temperature for 40 minutes. The competitive inhibitory activity of the expression product contained in each culture supernatant with HFE7A against the human Fas fusion protein was determined by measuring the luminescence intensity using a luminoscan (manufactured by Titertec).

As a result, it was confirmed that the expression product prepared in Example 16 specifically inhibited the binding of HFE7A prepared from a mouse hybridoma to a human Fas fusion protein (FIG. 29).

Example 20 Apoptosis Inducing Activity W in which human Fas molecule was expressed in mouse T lymphoma cells
R19L12a cells [Itoh, N. et. Al. (1991) Cell 6
6, 233-243], the apoptosis-inducing activity of COS cell culture supernatant expressing humanized anti-Fas antibody was examined. First, at 37 ° C., 5% in an RPMI1640 medium containing 10% FCS (manufactured by Gibco BRL).
WR19L12a cells (1 × 10 ⁵ cells / 50 μl) cultured for 3 days in the presence of carbon dioxide were added to each well of a 96-well microplate (Sumitomo Bakelite Co., Ltd.).
Dispensed in μl. The culture supernatant prepared in Example 16 was
The concentration of the target expression product contained was calculated by the method described in Example 17 in advance, and the final target product concentration was 1%.
RP containing 10% FCS to be 00 ng / ml
It had been prepared in MI1640 medium. 100 ng / ml
Each expression product solution adjusted to a concentration of 10% FCS
Was repeated twice in an RPMI1640 medium containing, to prepare a dilution series. Add 50 μl of each expression product dilution,
The solution was added to each well and kept at 37 ° C. for 1 hour. afterwards,
After washing the cells with RPMI 1640 medium containing 10% FCS, the cells in each well were added to 75 μl of 10% FCS.
Was suspended in an RPMI1640 medium containing. Then, RPMI1 containing 10% FCS was used as a secondary antibody in each well.
Goat anti-human IgG Fc-specific polyclonal antibody (Capel) prepared at 1.25 μg / ml in 640 medium
Was added. After reacting at 37 ° C for 12 hours,
1 mg / ml of XTT (2,3-bis-2-methoxy-4
-Nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide inner salt: manufactured by Sigma)
50 μl (final concentration 250 μg / ml) of 25 μM PMS (phenazine methosulfate: Sigma) containing
XTT, 5 μM PMS) was added. After culturing for 3 hours, the absorbance at 450 nm of each well was measured, and the cell viability was calculated using the mitochondrial reducing ability as an index.

The viability of the cells in each well was calculated by the following formula: Viability (%) = 100 × (ab) / (c−b) (where a is the measured value of the test well) , B represents the measured value of the cell-free well, and c represents the measured value of the well without the antibody).

As a result, it was shown that the expression product prepared in Example 16 induces apoptosis in a T lymphoma cell line expressing human Fas antigen (FIG. 3).
0).

Example 21 Humanized Development of the Light Chain of HFE7A
Construction of Current Vector (1) Construction of Humanized HFE7A-Light Chain Expression Vector Plasmid In order to make the HH type humanized light chain prepared in Example 14 closer to humans, the first amino acid from the N-terminus of the amino acid of the HH type light chain was used. Amino acids (aspartic acid), 85th (alanine) and 107th (arginine) were designed by replacing glutamic acid, glutamic acid and lysine conserved in the human light chain (κ chain), respectively, and this was designated as “PDHH type”. And Similarly, an HM-type light chain amino acid in which the first amino acid (aspartic acid) and the 107th amino acid (arginine) from the N-terminus were respectively replaced with glutamic acid and lysine conserved in the human light chain, and this was designated as " PDHM type ".
Hereinafter, expression plasmids carrying DNAs encoding the amino acid sequences of these two types of humanized anti-human Fas antibody light chains were respectively constructed.

1) Synthesis of primer PDHH type polypeptide chain (SEQ ID NO: 10 in the sequence listing)
7) and a DNA encoding a PDHM type polypeptide chain (SEQ ID NO: 109 in the sequence listing) (SEQ ID NO: 10 in the sequence listing).
The synthesis of 8) was performed by combining the PCR method.

In performing the PCR, in addition to the above-mentioned two oligonucleotide primers 7AL1P (SEQ ID NO: 55 in the sequence listing) and 7ALCN (SEQ ID NO: 64),
Six more primers in the sequence listing: 5'-GGTGAGATTG TGCTCACCCA ATCTCCAGG-3 '(LPD1P: SEQ ID NO: 110); 5'-CCTGGAGATT GGGTGAGCAC AATCTCACC-3' (LPD1N: SEQ ID NO: 111); 5'-CCATCTCTCG TCTGGAGCCG GAGGATTTTG C 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) was prepared.

2) Construction of PDDHH-type humanized light chain expression plasmid pLPDHH75 [0400] LPDHH-DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 107 in the sequence listing (SEQ ID NO: 10 in the sequence listing)
6) is made by repeating the PCR method three times,
After insertion into a plasmid, it was cloned in E. coli.

A) First Step PCR The outline of the first step PCR for producing LPDHH-DNA is shown in FIG.

[0402] 5 'ends were added HindIII restriction enzyme cleavage site, LPD1-DNA fragment encoding a portion of the secretion signal sequence and FRL ₁ region was produced under the following conditions.

Reaction solution composition: plasmid pHSGHH7 DNA 200 ng oligonucleotide primer 7AL1P 80 pmol oligonucleotide primer LPD1N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of the reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C for 2 minutes, 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

Part of FRL ₁ region, CDRL ₁ region, FR
L ₂ region, LPDHH1-DNA fragment encoding a portion of the CDRL ₂ region and FRL ₃ region, was prepared under the following conditions.

Reaction solution composition: plasmid pHSGHH7 DNA 200 ng Oligonucleotide primer LPD1P 80 pmol Oligonucleotide primer LPD2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of the reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C for 2 minutes, 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The LPDHH2-DNA fragment encoding a part of the FRL ₃ region, the CDRL ₃ region and the FRL ₄ region was prepared under the following conditions.

Reaction solution composition: plasmid pHSGHH7 DNA 200 ng Oligonucleotide primer LPD2P 80 pmol Oligonucleotide primer LPD3N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The final volume of the reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C for 2 minutes, 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

An LPDC-DNA fragment encoding a part of the FRL ₄ region and the constant region and having an EcoRI restriction enzyme cleavage site added to the 3 ′ end was prepared under the following conditions.

Reaction solution composition: plasmid pHSGHH7 DNA 200 ng oligonucleotide primer LPD3P 80 pmol oligonucleotide primer 7ALCN 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The final volume of the reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

Each product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. LPD1-DNA, LPDHH1-DNA, LP detected
D corresponding to DHH2-DNA and LPDC-DNA
The NA band was cut out with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter, respectively. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

B) Second Step PCR The outline of the second step PCR for the preparation of LPDHH-DNA is shown in FIG.

The above LPD1-DNA fragment, LPDHH1
-L fused with DNA and LPDHH2-DNA fragment
The PDHH1.2-DNA fragment was prepared under the following conditions. Reaction solution composition: LPD1-DNA solution prepared by first-stage PCR 10 μl LPDHH1-DNA solution prepared by first-stage PCR 10 μl LPDHH2-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer LPD3N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C for 2 minutes, the mixture was heated at 94 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The products after PCR were separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected band of DNA corresponding to LPDHH1.2-DNA was cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

C) Third Step PCR FIG. 33 shows an outline of the third step PCR for producing LPDHH-DNA.

The LPDHH1.2-DNA fragment and LP
The LPDHH-DNA fragment fused with the DC-DNA fragment was prepared under the following conditions.

Reaction solution composition: LPDHH1.2-DNA solution prepared by second-stage PCR 10 μl LPDC-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer 7ALCN 80 pmol dNTP mixture 20 μl 10 × Pfu Buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C for 2 minutes, 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The product after PCR was separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The band corresponding to the detected LPDHH-DNA was cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. 7 eluted DNA
It was concentrated by centrifugation at 500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

FIG. 34 shows an outline of a method for preparing an expression plasmid carrying the LPDHH-DNA fragment.

The obtained LPDHH-DNA fragment was further purified by phenol extraction and ethanol precipitation.
It was digested with restriction enzymes HindIII and EcoRI.

Cloning plasmid pHSG399
1 µg of DNA (manufactured by Takara Shuzo Co., Ltd.)
Digested with III and EcoRI and dephosphorylated with CIP. Dephosphorylated plasmid pH thus obtained
SG399 DNA and LPDH previously digested with restriction enzymes
The H-DNA fragment was ligated using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.) according to the protocol specified by the manufacturer, followed by transformation of Escherichia coli JM109 strain, to a final concentration of 1 mM IPTG, 0.1% X- G
It was spread on LB agar medium containing al and 50 μg / ml chloramphenicol. After culturing this at 37 ° C,
A white colony was isolated, cultured in an LB liquid medium containing 50 μg / ml of chloramphenicol, and plasmid DNA was isolated from this culture by the alkaline SDS method.
Was extracted. The extracted plasmid DNA was digested with the restriction enzyme Hi.
After digestion with ndIII and EcoRI, analysis by 1% agarose gel electrophoresis revealed LPDHH-DN.
A clone retaining the A fragment was selected.

By the above operation, a plasmid pHSHH5 carrying a fusion fragment of the DNA encoding the variable region of the humanized light chain of the PDHH type and the DNA encoding the constant region of the human Igκ chain was obtained. In addition, plasmid pHSH
Transformed E. coli carrying H5 coli pHS
HH5 SANK 70398 has been established on February 26, 1998.
It was deposited internationally with the National Institute of Biotechnology and Industrial Technology at the National Institute of Advanced Industrial Science and Technology under the accession number FERMBP-6274.

Next, using the plasmid pHSHH5,
An expression vector plasmid pLPDHH75 carrying a DNA encoding a PDHH type humanized light chain polypeptide was prepared by the method described below.

The expression plasmid pEE. 1
2.1 (Lonza)
It was digested with indIII and EcoRI and dephosphorylated with CIP. This dephosphorylated plasmid pEE. 12.1D
NA 100 ng, restriction enzymes HindIII and Ec
After 10 μg of pHSHH5 DNA fragment digested with oRI was ligated using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), E. coli JM109 strain was transformed and spread on LB agar medium containing 50 μg / ml ampicillin.

The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and alkaline
Plasmid DNA was extracted according to the SDS method. This plasmid DNA was digested with the restriction enzymes HindIII and EcoRI.
And the presence or absence of the inserted fragment was confirmed by 1% agarose gel electrophoresis. By the above operation, humanized PDHH
A plasmid pLP in which a fusion fragment of a variable region of type HFE7A-light chain and DNA encoding a human Igκ chain constant region was inserted in a forward direction downstream of a CMV promoter.
DHH75 was obtained.

(3) Construction of PDHM-Type Humanized Light Chain Expression Plasmid pLPDHM32 An LPDHM-DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 109 in the sequence listing (SEQ ID NO: 10 in the sequence listing)
8) is made by repeating the PCR method three times,
After insertion into a plasmid, it was cloned in E. coli.

A) First Step PCR The outline of the first step PCR for preparation of LPDHM-DNA is shown in FIG.

[0433] 5 'ends were added HindIII restriction enzyme cleavage site, LPD1-DNA fragment encoding a portion of the secretion signal sequence, and FRL ₁ region, and, F
RL ₄ encoding part and a constant region domain, the 3 'end E
LPDC-DNA added with a coRI restriction enzyme cleavage site
All of the fragments are LPDHH-DNA as described in (2) above.
The one obtained in the process of preparing the fragment was used.

Part of CDRL ₁ region, FRL ₂ region, CD
RL ₂ region, FRL ₃ region, CDRL ₃ region, and FR
LPDHM1-DNA fragment encoding the L ₄ region was prepared under the following conditions.

Reaction solution composition: plasmid pHSGHM17 DNA 200 ng Oligonucleotide primer LPD1P 80 pmol Oligonucleotide primer LPD3N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of the reaction solution with double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

Each product after the PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. LPD1-DNA, LPDHM1-DNA and L
A DNA band corresponding to PDC-DNA was cut out with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter. 75 eluted DNA
It was concentrated by centrifugation at 00 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

B) Second-step PCR The outline of the second-step PCR for LPDHM-DNA production is shown in FIG.

The LPD1-DNA fragment and LPDHM1
The LPDHM1.2-DNA fragment fused with the DNA fragment was prepared under the following conditions.

Reaction solution composition: LPD1-DNA solution prepared by first-stage PCR 10 μl LPDHM1-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer LPD3N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C for 2 minutes, 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The band corresponding to the detected LPDHM1.2-DNA was excised with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter. Eluted DN
A was concentrated by centrifugation at 7500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

C) Third Step PCR FIG. 37 shows an outline of the third step PCR for producing LPDHM-DNA.

The LPDHM1.2-DNA fragment and LP
The LPDHM-DNA fragment fused with the DC-DNA fragment was prepared under the following conditions.

Reaction solution composition: LPDHM1.2-DNA solution prepared by second-stage PCR 10 μl LPDC-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AL1P 80 pmol Oligonucleotide primer 7ALCN 80 pmol dNTP mixture 20 μl 10 × Pfu Buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The product after PCR was separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. A band corresponding to the detected LPDHM-DNA was cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. 7 eluted DNA
It was concentrated by centrifugation at 500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

FIG. 38 shows an outline of a method for preparing an expression plasmid carrying the LPDHM-DNA fragment.

First, the LPDHM-DNA obtained above
After further purification of the fragment by phenol extraction and ethanol precipitation, the restriction enzymes HindIII and Eco
Digested by RI.

Next, the cloning plasmid pHSG3 was used.
99 (manufactured by Takara Shuzo) DNA (1 μg) and restriction enzyme HindI
Digested with II and EcoRI and dephosphorylated with CIP.
This dephosphorylated plasmid pHSG399 DNA;
LPDHM previously digested with HindIII and EcoRI
-The DNA fragment was ligated using a DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.) according to the protocol specified by the manufacturer, followed by transformation of Escherichia coli JM109 strain to a final concentration of 1 mM IPTG, 0.1% X −
It was spread on LB agar medium containing Gal and 50 μg / ml chloramphenicol. After culturing this at 37 ° C., a white colony was isolated, and 50 μg / ml
Cultured in LB liquid medium containing chloramphenicol,
From this culture, plasmid D
NA was extracted. This plasmid DNA is digested with the restriction enzyme Hi.
After digestion with ndIII and EcoRI, analysis by 1% agarose gel electrophoresis revealed LPDHM-DN.
A clone retaining the A fragment was selected.

By the above operation, the plasmid pHSHM2 containing the fusion fragment of the variable region of the PDHM-type humanized light chain and the DNA encoding the human Igκ chain constant region was prepared.
I got In addition, transformed Escherichia coli carrying the plasmid pHSHM2. colipHSHM2 SANK 7
No. 0198 was internationally deposited on February 26, 1998 with the Institute of Biotechnology and Industrial Technology, and received the accession number FERM.
BP-6272 was attached.

Next, using the plasmid pHSHM2,
An expression vector plasmid pLPDHM32 carrying a DNA encoding a PDHM-type humanized light chain polypeptide was prepared by the method described below.

Expression plasmid pEE. 1
2.1 (Lonza) DNA (1 μg) was digested with restriction enzymes HindIII and EcoRI in advance and dephosphorylated with CIP. This dephosphorylated plasmid pEE. 12.1
DNA (100 ng) and restriction enzyme HindII
I and pHSHM2-DNA fragment (10 μg) digested with EcoRI were ligated using DNA ligation kit version 2.0 (Takara Shuzo Co., Ltd.).
The M109 strain was transformed and spread on an LB agar medium containing 50 μg / ml ampicillin. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes EcoRI and HindIII, and the presence or absence of the inserted fragment was confirmed by agarose gel electrophoresis. By the above operation, a plasmid pLPDHM32 in which a fusion fragment of the DNA encoding the variable region of the PDHM type humanized light chain and the DNA encoding the human Igκ chain constant region was inserted in the forward direction downstream of the CMV promoter was obtained. .

(4) Confirmation of nucleotide sequence Plasmid pLP prepared in (2) and (3) above
Nucleotide sequences were determined to confirm that DHH75 and pLPDHM32 each had a DNA having the target nucleotide sequence. The primers used here were SP1 (SEQ ID NO: 68 in the sequence listing), SP2 (SEQ ID NO: 69 in the sequence listing),
They were SP3 (SEQ ID NO: 70 in the Sequence Listing), SP4 (SEQ ID NO: 71 in the Sequence Listing), SP5 (SEQ ID NO: 72 in the Sequence Listing) and SP6 (SEQ ID NO: 73 in the Sequence Listing). The positions to which each primer binds are shown in FIG. The nucleotide sequence was determined by dideoxynucleotide chain termination using each plasmid DNA purified by the alkali-SDS method and the cesium chloride method as a template. As a result, pLPD
HH75 is the nucleotide sequence shown in SEQ ID NO: 106 in the sequence listing, and pLPDH32 is SEQ ID NO: 10 in the sequence listing.
It was confirmed that each of them retains the nucleotide sequence shown in FIG.

Example 22 Construction of Humanized Heavy Chain Expression Vector
To make the humanized HFE7A-heavy chain produced in Construction Example 15 closer to humans, the amino acid sequence of the humanized heavy chain
The 44th amino acid from the end (arginine) and 76
Those in which the third amino acid (alanine) was replaced with glycine and threonine conserved in the human heavy chain, respectively, were designed and designated as “HV type”. An expression plasmid carrying the DNA encoding the HV type humanized heavy chain amino acid sequence was constructed by the method described below.

(1) DNA encoding the synthesized HV type humanized heavy chain polypeptide chain (SEQ ID NO: 117 in the sequence listing)
(SEQ ID NO: 116 in the sequence listing) was synthesized by a PCR method.

In performing the PCR, in addition to the primer 7AH1P (SEQ ID NO: 76 in the sequence listing), three more primers: 5′-CAGGCCCCTG GACAGGGCCT TGAGTGGATG -3 ′ (HPD1P: SEQ ID NO: 118 in the sequence listing); 5′-CATCCACTCA AGGCCCTGTC CAGGGGCCTG-3 ′ (HPD1N: SEQ ID NO: 119 in Sequence Listing); and 5′-GCTGAGCTCC ATGTAGGCTG TGCTAGTGGA TGTGTCTAC-3 ′ (HPD2N: SEQ ID NO: 120 in Sequence Listing) were prepared.

(2) Construction of plasmid pgHPDHV3 Amino acid numbers -19 to 84 of SEQ ID NO: 117 in the sequence listing
HPD1.2-D encoding the amino acid sequence shown in
The NA fragment was prepared by repeating the PCR method twice, inserted into a plasmid, and cloned in E. coli.

A) First Step PCR The outline of the first step PCR for producing HPD1.2-DNA is shown in FIG. An HPD1-DNA fragment encoding a secretory signal sequence, a part of the FRH ₁ region, a CDRH ₁ region, and a part of the FRH ₂ region to which a HindIII restriction enzyme cleavage site was added at the 5 ′ end was prepared under the following conditions.

Reaction solution composition: plasmid pgHSL7A62 DNA 200 ng oligonucleotide primer 7AH1P 80 pmol oligonucleotide primer HPD1N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The final volume of the reaction solution with double distilled water was 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 1 minute at 94 ° C., 1 minute at 55 ° C., 2 minutes at 72 ° C.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

Next, HPD2-DN encoding part of the FRH ₂ region, CDRH ₃ region and part of the FRH ₃ region
The A fragment was prepared under the following conditions.

Reaction solution composition: Plasmid pgHSL7A62 DNA 200 ng Oligonucleotide primer HPD1P 80 pmol Oligonucleotide primer HPD2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of the reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

After PCR, each product was separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. DNA bands corresponding to the detected HPD1-DNA and HPD2-DNA were cut out with a razor, and eluted from an acrylamide gel using a Centricon and a Centriluter. The eluted DNA was concentrated by centrifugation at 7,500 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

B) Second-step PCR The outline of the second-step PCR for preparing HPD1.2-DNA is shown in FIG. HPD1-DNA fragment and HP
The HPD1.2-DNA fragment fused with the D2-DNA fragment was prepared under the following conditions.

Reaction solution composition: HPD1-DNA solution prepared by first-stage PCR 10 μl HPD2-DNA solution prepared by first-stage PCR 10 μl Oligonucleotide primer 7AH1P 80 pmol Oligonucleotide primer HPD2N 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units The reaction solution having the above composition was mixed with double distilled water to a final volume of 200 μl.
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. A band corresponding to the detected fusion DNA was cut out with a razor, and eluted from an acrylamide gel using a Centricon and a Centriluter. 7500x eluted DNA
g by centrifugation and, after ethanol precipitation, 50 g.
Dissolved in μl of distilled water.

FIG. 41 shows an outline of a method for preparing a plasmid holding the HPD1.2-DNA fragment.

The HPD1.2-DNA fragment obtained above was further purified by phenol extraction and ethanol precipitation, and then digested with restriction enzymes HindIII and SacI.

The above plasmid pgHSL7A62 DNA
1 μg was previously digested with restriction enzymes HindIII and SacI and dephosphorylated with CIP. This dephosphorylated plasmid pgHSL7A62 DNA and HindI
After ligation of the HPD1.2-DNA fragment digested with II and SacI using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), E. coli JM109 strain was transformed and chloramphenicol at a final concentration of 50 μg / ml. Was spread on an LB agar medium containing. The obtained transformant was transformed into L containing 50 μg / ml chloramphenicol.
B liquid culture medium, and alkali-SDS
Plasmid DNA was extracted by the method. This plasmid DNA was digested with restriction enzymes SacI and HindIII,
Perform agarose gel electrophoresis and determine HPD1.2-DN
A clone retaining the A fragment was selected.

[0473] By the above operation, a plasmid pgHPDHV3 carrying a fusion fragment of the DNA encoding the variable region of the HV type humanized heavy chain and the genomic DNA fragment encoding the human IgG1 constant region was obtained. In addition, transformed Escherichia coli carrying the plasmid pgHPDHV3
E. FIG. E. coli pgHPDHV3 SANK 7029
8 was deposited on February 26, 1998 at the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, under the accession number FERM BP.
-6273.

Next, pgHPDHV3 DNA 10 μm
g was digested with the restriction enzymes HindIII and EcoRI. On the other hand, the expression plasmid p for mammalian cells
EE. 6.1 (Lonza Co., Ltd.) of 1 μg
It was digested with indIII and EcoRI and dephosphorylated with CIP. This dephosphorylated plasmid pEE. 6.1 DN
A (100 ng) and pgHPDHV3 DNA (10 μg) digested with HindIII and EcoRI were added to D
After ligation was performed using NA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), the clone was cloned into E. coli JM109 strain. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes HindIII and EcoRI, and subjected to agarose gel electrophoresis to confirm the presence or absence of the inserted fragment. By the above operation, the plasmid pEgPDH in which the DNA encoding the HV type humanized heavy chain was inserted in the forward direction downstream of the CMV promoter.
V3-21 was obtained.

(3) Confirmation of nucleotide sequence The plasmid pEgPDHV3-2 obtained in the above (2)
The nucleotide sequence was determined to confirm that Sample No. 1 had a DNA having the target nucleotide sequence. The primer used here was PE
EF (SEQ ID NO: 104 of Sequence Listing), HPD1P (SEQ ID NO: 118 of Sequence Listing), HPD1N (SEQ ID NO: 1 of Sequence Listing)
19) and HPD2N (SEQ ID NO: 120 in the sequence listing). The position where each primer binds is shown in FIG. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using plasmid DNA purified by the alkali-SDS method and the cesium chloride method as a template. As a result, pEgPDHV3-21 was obtained from SEQ ID NO: 1 in the sequence listing.
It was confirmed that the nucleotide sequence was retained as shown in FIG.

Example 23 Expression Vector for COS-1 Cells
Various humanized H made in the construction Example 14 and Example 21 over
FE7A-light chain expression vector (p7AL-HH, p7A
L-HM, p7AL-MM, pLPDHH75 and p
LPHM32) and various humanized HFE7A-heavy chain expression vectors (p
Using Eg7AH-H and pEgPDHV3-21), an expression vector optimized for gene expression using COS-1 cells was prepared by the method described below.

[0477] 1. Construction of Light Chain Expression Vector for COS-1 Cells FIG. 43 shows an outline of the construction method of the expression vector for COS-1 cells of various humanized HFE-7A light chains.

(1) Synthesis of Promoter Amplification Primer (for Light Chain) In preparing a DNA fragment containing the SRα promoter region by PCR, the following two oligonucleotide primers: 5′-TGCACGCGTG GCTGTGGAAT GTGTGTCAGT TAG − 3 ′ (MLUA: SEQ ID NO: 121 in Sequence Listing); and 5′-TCCGAAGCTT TTAGAGCAGA AGTAACACTT C-3 ′ (HINDB: SEQ ID NO: 122 in Sequence Listing) were prepared.

(2) Construction of Expression Vector (for Light Chain) The LSRα-DNA fragment containing the SRα promoter region and having a MulI restriction enzyme cleavage site at the 5 ′ end and a HindIII restriction enzyme cleavage site at the 3 ′ end, respectively, is ,
It was prepared under the following conditions using the plasmid pME18S as a template.

Reaction solution composition: plasmid pME18S DNA 200 ng oligonucleotide primer MLUA 80 pmol oligonucleotide primer HINDB 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. for 2 minutes
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The product after PCR was separated by phenol extraction and ethanol precipitation by 5% polyacrylamide gel electrophoresis. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. A band corresponding to the detected LSRα-DNA was cut out with a razor and eluted from an acrylamide gel using a centricon and a centriluter. 75 eluted DNA
It was concentrated by centrifugation at 00 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

Next, the plasmids p7AL-HH, p7AL-HM, p7 prepared in Examples 14 and 21 were prepared.
7AL-MM, pLPDHH75 or pLPDHM3
21 μg was digested with restriction enzymes MulI and HindIII and dephosphorylated with CIP. After ligating the dephosphorylated plasmid DNA with an LSRα-DNA fragment previously digested with MulI and HindIII using DNA ligation kit version 2.0 (Takara Shuzo),
E. coli JM109 strain was transformed and the final concentration was 50 μg /
It was spread on LB agar medium containing ml of ampicillin.
The obtained transformant is cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and an alkaline
Plasmid DNA was extracted by the SDS method. This plasmid DNA was digested with restriction enzymes MulI and HindIII and subjected to agarose gel electrophoresis to obtain LSRα-D
A clone retaining the NA fragment was selected.

By the above operation, the expression vector pSRHH (H
H type, derived from p7A-HH), pSRHM (HM type, derived from p7A-HM), pSRMM (MM type, derived from p7A-HM)
7A-MM), pSRPDHH (PDHH type,
pLPDHH75) and pSRPDHM (PDM
M type, derived from pLPDH32).

[0485] 2. Construction of heavy chain expression vector for COS-1 cells The outline of the construction method of the expression vector for various humanized heavy chains for COS-1 cells is shown in FIG.

(1) Synthesis of Promoter Amplification Primer (for Heavy Chain) In preparing a DNA fragment containing the SRα promoter region by PCR, one more primer was added to the above-mentioned primer HINDB (SEQ ID NO: 122 in the sequence listing). Primer: 5'-AAAGCGGCCG CTGCTAGCTT GGCTGTGGAA TGTGTG-3 '(NOTA: SEQ ID NO: 123 in Sequence Listing) was prepared.

(2) Construction of Expression Vector (for Heavy Chain) The HSRα-DNA fragment containing the SRα promoter region and having a NotI restriction enzyme cleavage site at the 5 ′ end and a HindIII restriction enzyme cleavage site at the 3 ′ end was added. ,
It was prepared under the following conditions using the plasmid pME18S as a template.

Reaction solution composition: Plasmid pME18S DNA 200 ng Oligonucleotide primer NOTA 80 pmol Oligonucleotide primer HINDB 80 pmol dNTP mixture 20 μl 10 × Pfu buffer 20 μl Pfu DNA polymerase 10 units Final volume of reaction solution of the above composition in double distilled water 200 μl
And used for PCR.

PCR reaction temperature conditions: After heating at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 2 minutes.
After repeating the temperature cycle for 30 minutes 30 times, the mixture was heated at 72 ° C. for 10 minutes.

The product after PCR was separated by 5% polyacrylamide gel electrophoresis after phenol extraction and ethanol precipitation. The acrylamide gel after electrophoresis was stained with ethidium bromide at a concentration of 1 μg / ml, and the product DNA was detected under ultraviolet irradiation. The detected band corresponding to HSRa-DNA was cut out with a razor and eluted from an acrylamide gel using a Centricon and a Centriluter. 75 eluted DNA
It was concentrated by centrifugation at 00 × g, and after ethanol precipitation, dissolved in 50 μl of distilled water.

Next, the plasmid pEg7AH-H or pEgPDH prepared in Example 15 and Example 22 was used.
1 μg of V3-21 was added to NotI and HindI restriction enzymes.
Digested with II and dephosphorylated with CIP. This dephosphorylated plasmid DNA was combined with NotI and HindIII in advance.
The DNA fragment was ligated to the HSRα-DNA fragment digested with the above using DNA ligation kit version 2.0 (manufactured by Takara Shuzo Co., Ltd.), and then transformed into Escherichia coli JM109 strain, and placed on an LB agar medium containing ampicillin at a final concentration of 50 μg / ml. Applied. The obtained transformant was cultured in an LB liquid medium containing 50 μg / ml of ampicillin, and a plasmid DNA was extracted from the culture by an alkaline SDS method. This plasmid DNA was digested with restriction enzymes NotI and Hin.
Digest with dIII and perform agarose gel electrophoresis,
A clone retaining the HSRa-DNA fragment was selected.

By the above operation, the expression vector pSRg7AH for COS-1 cells encoding various humanized heavy chains
(From pEg7AH-H) and pSRgPDH (pEg7AH-H)
gPDHV3-21).

Example 24 Each subunit of humanized HFE7A
Expression of knit-encoding gene in COS-1 cells COS-1 cells were transformed with the expression vector for COS-1 cells obtained in Example 23 by the method described in Example 16. First, COS-1 cells were
Cell culture flask (culture area 225) containing α (+) MEM (manufactured by Gibco BRL) containing% FCS
cm ² : manufactured by Sumitomo Bakelite Co., Ltd.) until the cells became semi-confluent. Then remove the medium,
In 3 ml of trypsin-EDTA solution (manufactured by Sigma),
COS-1 cells were released from the flask by incubating at 37 ° C. for 3 minutes. Release the cells at 800 rpm
m, collected by centrifugation for 2 minutes, washed twice with PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd.), and then washed with PBS (-).
The COS-1 cell suspension was prepared so as to have a cell count of 1 × 10 ⁸ cells / ml.

On the other hand, a humanized heavy chain expression vector (pSRg7AH or pSRgPDH) DNA prepared using the alkali-SDS method and the cesium chloride density gradient centrifugation method
4 μg, and a humanized light chain expression vector (pSRH
H, pSRHM, pSRMM, pSRPDHH or p
After mixing 4 μg of (SRPDHM) DNA in a combination described below, ethanol precipitation was performed, and the obtained precipitate was dissolved in 20 μl of PBS (−). A mixture of 20 μl of the obtained plasmid solution and 20 μl (2 × 10 ⁶ cells) of the previously prepared COS-1 cell suspension was mixed with a chamber having an electrode spacing of 2 mm (FCT-13: manufactured by Shimadzu Corporation). And set in a gene transfer device GTE-1 (manufactured by Shimadzu Corporation). After that, 600V
The target plasmid DNA was introduced into COS-1 cells by applying a pulse of 50 μF twice at 1 second intervals. Cells in chamber after pulse-
The DNA mixture was prepared using α (+) M containing 10% fetal bovine serum.
The cells were suspended in 5 ml of EM and transferred to a cell culture flask (culture area: 25 cm ² : manufactured by Sumitomo Bakelite). This was cultured at 37 ° C. for 72 hours under 5% CO ₂ , and the culture supernatant was recovered.

According to the above method, COS-1 cells were transformed under the following conditions [A] to [F], and the culture supernatant was recovered.

[A]: Conditions without plasmid DNA; [B]: Co-transfection of pSRgPDH and pSRPDHH; [C]: Co-transfection of pSRgPDH and pSRPDHM; [D]: Co-transfection of pSRg7AH and pSRHH [E]: co-transfection of pSRg7AH and pSRHM; [F]: co-transfection of pSRg7AH and pSRMM.

Example 25 Assay of Binding Ability to Fas For each culture supernatant sample prepared in Example 24, the concentration of the target expression product contained was calculated by the method described in Example 17, and the final concentration was calculated. Target product concentration is 1
Α containing 10% FCS to be 00 ng / ml
Prepared with (+) MEM. When the target product concentration in the culture supernatant sample was lower than 100 ng / ml, the obtained culture supernatant was first concentrated using Centriprep-10 (manufactured by Amicon). That is, 5 ml of culture supernatant
Was transferred to Centriprep-10, concentrated by centrifugation at 3000 × g, and the concentration of the target product was calculated again by the method described in Example 17.

Next, the operation of diluting each sample solution prepared at a concentration of 100 ng / ml twice with α (+) MEM containing 10% FCS was repeated to prepare a dilution series. For these samples, the binding ability to the human Fas fusion protein was measured by the ELISA method described in Example 18.

As a result, of the culture supernatant samples prepared in Example 24, those under the conditions of [B], [C], [D], [E] and [F] were different from the human Fas fusion protein. (FIG. 45).

Example 26 Binding of HFE7A to Fas
For each culture supernatant samples prepared in competitive inhibition Example 24 against, by the method shown in advance Example 17, to calculate the concentration of the target expression product, each of which contains, alpha containing 10% FCS
(+) 1 μg / ml final target product concentration in MEM
It was prepared so that When the concentration of the target product in the culture supernatant sample is lower than 1 μg / ml, the culture supernatant is concentrated using Centriprep-10 and then concentrated to 1 μg / ml.
g / ml.

Next, the operation of diluting the sample prepared at a concentration of 1 μg / ml twice with α (+) MEM containing 10% FCS was repeated to prepare a dilution series. Meanwhile, AP
Α-(+) MEM containing 10% FCS
Was adjusted to a concentration of 50 ng / ml, and mixed with the diluted series sample in an equal amount. Thereafter, the competitive inhibitory activity of each sample on HFE7A was measured by the method described in Example 19.

As a result, of the culture supernatant samples prepared in Example 24, those under the conditions of [B], [C], [D], [E] and [F] were prepared from the hybridoma culture. It was confirmed that HFE7A specifically inhibited the binding to the human Fas fusion protein (FIG. 46).

Example 27 Apoptosis Inducing Activity The culture supernatant sample prepared in Example 24 was subjected to the method described in Example 22 to give a final target product concentration of 100 ng / ml.
(However, the medium for adjustment was 1
RPMI 1640 medium containing 0% FCS was used).
This 100 ng / ml sample was treated with R containing 10% FCS.
The operation of diluting twice with PMI1640 medium was repeated to prepare a dilution series. Thereafter, the cytotoxic activity of each sample on WR19L12a cells was measured by the method described in Example 20.

As a result, of the culture supernatant samples prepared in Example 24, those under the conditions of [B], [C], [D], [E] and [F] were prepared from the hybridoma culture. Similarly to HFE7A, it was shown to induce apoptosis in a T lymphoma cell line that expressed the human Fas antigen (FIG. 47).

Formulation Examples The molecules of the present invention are provided for use as ampules of sterile solutions or suspensions in water or other pharmacologically acceptable solutions. Alternatively, a sterile powder preparation (preferably freeze-dried of the molecule of the present invention) may be filled in an ampoule and diluted with a pharmacologically acceptable solution before use.

[0506]

According to the present invention, various diseases caused by the Fas / Fas ligand system (autoimmune diseases (systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft versus host disease, Sjogren's syndrome, pernicious anemia, Addison) disease,
Scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, natural infertility, myasthenia gravis, multiple sclerosis, Basedow's disease, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus), allergies , Atopy, arteriosclerosis, myocarditis, cardiomyopathy, glomerulonephritis, aplastic anemia, hepatitis (fulminant hepatitis, chronic hepatitis, viral hepatitis (hepatitis C,
Conserved between primate and non-primate Fas, useful as prophylactic or therapeutic agents against hepatitis B, hepatitis D) or alcoholic hepatitis), acquired immunodeficiency syndrome or rejection after organ transplantation. Molecules having an antigen-binding region specific for the epitope in question have been provided.

[Brief description of the drawings]

FIG. 1 is a construction diagram of phFas-AIC2.

FIG. 2 is a construction diagram of pME-H and pME-L.

FIG. 3. ELI for epitope definition of HFE7A.
The figure showing the result of SA.

FIG. 4 shows the results of a competition assay for defining HFE7A epitopes.

FIG. 5 shows the results of a toxicity test of HFE7A.

FIG. 6 is a diagram showing the results of a test using a fulminant hepatitis model.

FIG. 7 is a diagram showing the results of a test for preventing the onset of collagen-induced arthritis.

FIG. 8: First step PCR for VHH-DNA production
Overview.

FIG. 9: Second step PCR for VHH-DNA production
Overview.

FIG. 10: Third step PC for VHH-DNA production
Outline of R.

FIG. 11 shows an outline of a method for preparing an expression plasmid holding a VHH-DNA fragment.

FIG. 12: First step PC for VHM-DNA production
Outline of R.

FIG. 13: Second step PC for VHM-DNA production
Outline of R.

FIG. 14 shows an outline of a method for preparing an expression plasmid having a VHM-DNA fragment.

FIG. 15: First step PC for VMM-DNA production
Outline of R.

FIG. 16: Second stage PC for VMM-DNA production
Outline of R.

FIG. 17: Third step PC for VMM-DNA production
Outline of R.

FIG. 18 shows an outline of a method for preparing a plasmid holding a VMM-DNA fragment.

FIG. 19 is a diagram showing binding positions of humanized light chain sequencing primers.

FIG. 20. First-step PCR for VD-DNA production
Overview.

FIG. 21. Second step PCR for VD-DNA production
Overview.

FIG. 22. Third step PCR for VD-DNA production
Overview.

FIG. 23 shows an outline of a method for preparing a plasmid holding a VD-DNA fragment.

FIG. 24 shows an outline of a method for producing a DNA (IG5′-DNA) fragment containing the CH1 region and intron of human IgG1.

FIG. 25: Hinge region, CH2 region of human IgG1,
Genomic DNA (IG) containing CH3 region and intron
Outline of a method for producing a 3′-DNA) fragment.

FIG. 26. Expression vector plasmid pEg7AH-H
Outline of preparation procedure.

FIG. 27 shows the binding positions of humanized heavy chain sequencing primers.

FIG. 28 shows the binding activity of a humanized anti-Fas antibody to a human Fas fusion protein.

FIG. 29. HFE for human Fas fusion protein
The figure which shows the competitive inhibition of 7A and a humanized anti-Fas antibody.

FIG. 30 shows the cytotoxic activity of humanized HFE7A on WR19L12a.

FIG. 31. Overview of first step PCR for LPDHH-DNA production.

FIG. 32. Overview of the second step PCR for LPDHH-DNA production.

FIG. 33. Summary of the third step PCR for LPDHH-DNA production.

FIG. 34 shows an outline of a method for preparing an expression plasmid retaining an LPDHH-DNA fragment.

FIG. 35. Summary of first step PCR for LPDHM-DNA production.

FIG. 36. Summary of the second step PCR for LPDHM-DNA production.

FIG. 37. Summary of the third step PCR for LPDHM-DNA production.

FIG. 38 shows an outline of a method for preparing an expression plasmid holding an LPDHM-DNA fragment.

FIG. 39. Summary of first step PCR for HPD1.2-DNA production.

FIG. 40. Summary of the second step PCR for HPD1.2-DNA production.

FIG. 41 shows an outline of a method for preparing a plasmid holding an HPD1.2-DNA fragment.

FIG. 42. DNA encoding an HV type humanized heavy chain
FIG. 5 is a diagram showing the binding position of the sequencing primer in FIG.

FIG. 43 is a construction diagram of an expression vector optimized for expressing various humanized light chains in COS-1 cells.

FIG. 44 is a construction diagram of an expression vector optimized for expressing various humanized heavy chains in COS-1 cells.

FIG. 45 shows the binding activity to human Fas fusion protein in the culture supernatant of various transformed COS-1 cells.

FIG. 46 is a graph showing competitive inhibitory activity on the binding of human Fas fusion protein to HFE7A in the culture supernatant of various transformed COS-1 cells.

FIG. 47 is a view showing cytotoxicity to WR19L12a in the culture supernatant of various transformed COS-1 cells.

[Sequence list]

SEQ ID NO: 1 Sequence length: 10 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: peptide sequence Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys 1 5 10 SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Protein Fragment type: Middle fragment Sequence Ser Tyr Trp Met Gln 15 SEQ ID NO: 3 Sequence length : 17 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein Fragment type: middle fragment sequence Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly SEQ ID NO: 4 Sequence length: 12 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein Fragment type: middle fragment Sequence Asn Arg Asp Tyr Ser Asn Asn Trp Tyr Phe Asp Val 1 5 10 SEQ ID NO: 5 Sequence Length: 15 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein Fragment type: middle fragment sequence Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn 15 10 15 SEQ ID NO: 6 Sequence length: 7 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: protein Fragment type: middle fragment sequence Ala Ala Ser Asn Leu Glu S
er 15 SEQ ID NO: 7 Sequence length: 9 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein Fragment type: middle fragment sequence Gln Gln Ser Asn Glu Asp Pro Arg Thr 15 SEQ ID NO: 8 Sequence length: 1392 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Hypothetical: No Antisense: No Origin Organism name: Mouse Cell type: Hybridoma Cell line: HFE7A Sequence characteristics Characteristic code: CDS Location: 1.1392 Method for determining characteristics: S Character code: mat peptide Location: 58.1392 Characteristic determination Method: S Symbol indicating characteristics: sig peptide Location: 1..57 Method for determining characteristics: S sequence ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCA ACA GCT ACA GGT 48 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala 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 Ala 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 Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 ACC AGC TAC TGG ATG CAG TGG GTA AAA CAG AGG CCT GGA CAG GGC CTT 192 Thr Ser Tyr Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 GAG TGG ATC GGA GAG ATT GAT CCT TCT GAT AGC TAT ACT AAC TAC AAT 240 Glu Trp Ile Gly Glu Ile Asp 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 Ala Thr Leu Thr Val Asp Thr Ser Ser Ser 65 70 75 ACA GCC TAC ATG CAG CTC AGC AGC CTG ACA TCT GAG GAC TCT GCG GTC 336 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 80 85 90 TAT TAC TGT GCA AGA AAT AGG GAC TAT AGT AAC AAC TGG TAC TTC GAT 384 Tyr Tyr Cys Ala Arg Asn Arg Asp Tyr Ser Asn Asn Trp Tyr Phe Asp 95 100 105 GTC TGG GGC ACA GG G ACC ACG GTC ACC GTC TCC TCA GCC AAA ACG ACA 432 Val Trp Gly Thr Gly Thr Thr Val Val Val Ser Ser Ala Lys Thr Thr 110 115 120 125 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 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 GTG ACA GTG ACC TGG AAC TCT GGA TCC CTG TCC AGC GGT GTG CAC ACC 576 Val Thr Val Thr Trp 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 Ala 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 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 210 215 220 GA T TGT GGT TGT AAG CCT TGC ATA TGT ACA GTC CCA GAA GTA TCA TCT 768 Asp Cys Gly Cys Lys Pro Cys Ile 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 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu 240 245 250 ACT CCT AAG GTC ACG TGT GTT GTG GTA GAC ATC AGC AAG GAT GAT CCC 864 Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys 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 Asp Asp Val Glu Val His Thr Ala 270 275 275 280 285 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 Ile Met His Gln Asn Trp 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 Ile Glu Lys Thr 320 325 330 ATC TCC AAA ACC AAA GGC AGA CCG AAG GCT CCA CAG GTG TAC ACC ATT 1104 Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile 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 Ala Lys Asp Lys 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 Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp 370 375 380 AAT GGG CAG CCA GCG GAG AAC TAC AAG AAC ACT CAG CCC ATC ATG AAC 1248 Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn 385 390 395 395 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 GGA AAT ACT TTC ACC TGC TCT GTG TTA CAT GAG GGC 1344 Asn Trp Glu Ala 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 H is Ser Pro Gly Lys 430 435 440 445 SEQ ID NO: 9 Sequence length: 464 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys 1 5 10 Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Ser Tyr Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Ile Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 50 55 60 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser 65 70 75 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Asn Arg Asp Tyr Ser Asn Asn Trp Tyr Phe Asp 95 100 105 Val Trp 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 Ala Pro Gly Ser Ala Ala Gln Thr Asn 130 135 140 Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro 145 150 155 Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr 160 165 170 Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val 175 180 185 Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val 190 195 200 205 Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg 210 215 220 Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser 225 230 235 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu 240 245 250 Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro 255 260 265 Glu Val Gln Phe Ser Trp 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 290 295 300 300 Ser Glu Leu Pro Ile Met His Gln Asn Trp Leu Asn Gly Lys Glu Phe 305 310 315 Lys Cys Arg Val Asn Ser Ala Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr 320 325 330 Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile 335 340 345 Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys 350 355 360 365 Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp 370 375 380 Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile 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 Trp Glu Ala 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 SEQ ID NO: 10 Length: 714 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical: No antisense: No Origin Organism name: Mouse Cell type: Hybridoma Cell line: Characteristic of HFE7A sequence Symbol indicating the characteristic: CDS Location: 1..714 Method for determining the characteristic: S Symbol indicating the characteristic: mat peptide Location: 61..714 Method for determining the characteristic: Symbol indicating the S characteristic: sig peptide Location: 1..60 Method for determining characteristics: S sequence ATG GAG ACA GAC ACA ATC CTG CTA TGG GT G ATG ATG CTC TGG ATT CCA 48 Met Glu Thr Asp Thr Ile Leu Leu Trp Val Met Met Leu Trp Ile Pro -20 -15 -10 -10 -5 GGC TCC ACT GGT GAC ATT GTG CTG ACC CAA TCT CCA GCT TCT TTG GCT 96 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala 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 Ala Thr Ile Ser Cys Lys Ala 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 Trp Tyr Gln Gln Lys Pro 30 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 Ile Tyr Ala Ala 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 Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 CTC AAC ATC CAT CCT GTG GAG GAG GAG GAT GCT GCA ACC TAT TAC TGT 336 Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys 80 85 90 CAG CAA AGT AAT GAG GAT CCT CGG ACG TTC GGT GG A 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 Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile 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 Ile Asn Val Lys Trp Lys Ile 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 Asn Ser Trp Thr Asp 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 Ala 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 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 205 210 215 SEQ ID NO: 11 Sequence length: 238 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Glu Thr Asp Thr Ile Leu Leu Trp Val Met Met Leu Trp Ile Pro -20 -15 -10 -5 Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala 1 5 10 Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser 15 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 Ile Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Asn Ile 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 Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe 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 Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 145 150 155 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp 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 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 205 210 215 SEQ ID NO: 12 Sequence length: 34 Sequence type : Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GGGGAATTCC AGTACGGAGT TGGGGAAGCT CTTT 34 SEQ ID NO: 13 Sequence length: 35 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GTTTCTTCTG CCTCTGTCAC CAAGTTAGAT CTGGA 35 SEQ ID NO: 14 Sequence length Length: 35 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of sequence: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence TCCAGATCTA ACTTGGTGAC AGAGGCAGAA GAAAC 35 SEQ ID NO: 15 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence CCCTCTAGAC GCGTCACGTG GGCATCAC 28 SEQ ID NO: 16 Sequence length: 11 Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Protein Fragment type: N-terminal fragment Sequence Gln Xaa Gln Leu Gln Gln Pro Gly Ala
Glu Leu 15 1
0 SEQ ID NO: 17 Sequence length: 22 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: protein Fragment type: N-terminal fragment Sequence Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser 20 SEQ ID NO: 18 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid (synthetic DNA) Hypothetical: No antisense: No sequence GACCTCACCA TGGGATGGA 19 SEQ ID NO: 19 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid (synthetic DNA) Hypothetical: No Antisense: No Sequence AAGAAGCATC CTCTCATCTA 20 SEQ ID NO: 20 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypocete Cal: No Antisense: No Sequence TTTACCAGGA GAGTGGGAGA 20 SEQ ID NO: 21 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypo Setical: No Antisense: No Sequence ACACTCATTC CTGTTGAAGC 20 SEQ ID NO: 22 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GGGGAATTCG ACCTCACCAT GGGATGGA 28 SEQ ID NO: 23 Sequence length: 32 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthesis DNA) Hypothetical: No Antisense: No Sequence GGGTCTAGAC TATTTACCAG GAGAGTGGGA GA
32 SEQ ID NO: 24 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GGGGAATTCA AGAAGCATCC TCTCATCTA 29 SEQ ID NO: 25 Sequence length: 37 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GGGGCGGCCG CTTACTAACA CTCATTCCTG TTGAAGC 37 SEQ ID NO: 26 Sequence length: 19 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser 1 5 10 15 Lys Gly Leu SEQ ID NO: 27 Sequence length: 19 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg Ly s Thr Val Thr 1 5 10 15 Thr Val Glu SEQ ID NO: 28 Sequence length: 20 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence Glu Leu Arg Lys Thr Val Thr Thr Val
Glu Thr Gln Asn Leu Glu Gly 15
10 15 Leu His His Asp 20 SEQ ID NO: 29 Sequence length: 20 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence 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 SEQ ID NO: 30 Sequence length: 20 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence 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 SEQ ID NO: 31 Sequence length: 21 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp 1 5 10 15 Cys Val Pro Cys Gln 20 SEQ ID NO: 32 Sequence length: 20 Sequence type: Amino acid Number of chains : Single chain Topology: Linear Sequence type: Peptide sequence Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr 1 5 10 15 Thr Asp Lys Ala 20 SEQ ID NO: 33 Sequence length: 19 Sequence type: Amino acid Number of chains: Single chain Topology: Linear sequence Type: Peptide Sequence Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg 1 5 10 15 Arg Cys Arg SEQ ID NO: 34 Sequence length: 20 Sequence type: Amino acid Number of chains: Single chain Topology : Linear Sequence type: Peptide sequence 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 SEQ ID NO: 35 Sequence length: 20 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn Cys Thr Arg 1 5 10 15 Thr Gln Asn Thr 20 SEQ ID NO: 36 Sequence length Length: 20 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence Glu le Asn Cys Thr Arg Thr Gln Asn
Thr Lys Cys Arg Cys Lys Pro 15
10 15 Asn Phe Phe Cys 20 SEQ ID NO: 37 Sequence length: 20 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu 1 5 10 15 His Cys Asp Pro 20 SEQ ID NO: 38 Sequence length: 20 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu His 1 5 10 15 Gly Ile Ile Lys 20 SEQ ID NO: 39 Sequence length: 20 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser 1 5 10 15 Asn Thr Lys Cys 20 SEQ ID NO: 40 Sequence length: 18 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg 1 5 10 15 Ser Asn SEQ ID NO: 41 Sequence length: 20 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence Ser Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala 1 5 10 15 Phe Asn Val Glu 20 SEQ ID NO: 42 Sequence length: 10 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Type Sequence type: Peptide sequence His Gly Leu Glu Val Glu Ile Asn Cys Thr 1510 SEQ ID NO: 43 Sequence length: 10 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type : Peptide sequence Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr 1510 SEQ ID NO: 44 Sequence length: 10 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys 1 5 10 SEQ ID NO: 45 Sequence length: 14 Sequence type: amino acid Number: single chain Topology: linear Sequence type: peptide sequence Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 1 5 10 SEQ ID NO: 46 Sequence length: 10 Sequence type: amino acid chain Number: single-chain Topology: linear Sequence type: peptide sequence Gly Lys Ile Ala Ser Cys Leu Asn Asp Asn 1 5 10 SEQ ID NO: 47 Sequence length: 34 Sequence type: Nucleic acid Number of strands: One Main strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GCGAATTCTG CCTTGACTGA TCAGAGTTTC CTCA 34 SEQ ID NO: 48 Sequence length: 32 Sequence type: Nucleic acid Number: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GCTCTAGATG AGGTGAAAGA TGAGCTGGAG GA 32 SEQ ID NO: 49 Sequence length: 768 Sequence type: Number of nucleic acid strands: double strand Topology: Type of linear sequence: cDNA to mRNA Hypothetical: NO Antisense: NO Sequence characteristics Characteristic code: CDS location: 40..753 Character code: mat_peptide Location: 100..753 Characteristic Symbol: sig_peptide Location: 40..99 Sequence 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 Ile Leu Leu Leu Leu Leu Trp 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 Ile 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 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 35 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro 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 Ile Ser Arg 70 75 80 CTG GAG CCG GCG GAT TTT GCA GTC TAT TAC TGT CAG CAA AGT AAT GAG 390 Leu Glu Pro Ala Asp Phe Ala 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 Pro Arg Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 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 Ile 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 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 150 155 160 AAC TCC CAG GAG AGT GTC ACA GAG CAG GAC AGC AA G 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 GCA GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 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 SEQ ID NO: 50 Sequence length: 238 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20 -15 -10 -5 Gly Ser Thr Gly Asp Ile 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 15 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 30 35 40 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly I le Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Thr Ile Ser Arg Leu Glu Pro Ala Asp Phe Ala 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 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 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 Ala Lys Val Gln Trp 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 Ala 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 SEQ ID NO: 51 Sequence length: 768 Sequence length Type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical: NO Antisense: NO Sequence characteristics Characteristic symbol: CDS present Location: 40..753 Symbol indicating characteristics: mat_peptide Location: 100..753 Symbol indicating characteristics: sig_peptide Location: 40..99 Sequence 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 Ile Leu Leu Trp Val Leu Leu Leu Trp 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 Ile 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 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 35 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile 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 Ile 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 Ile 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 Ile 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 Trp 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 GCA GAC TA C GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 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 SEQ ID NO: 52 Sequence length: 238 Sequence type: 238 Sequence type: Amino acid Topology: Type of linear sequence: Protein sequence Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20 -15 -10 -5 Gly Ser Thr Gly Asp Ile 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 15 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 30 35 40 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Thr Ile 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 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 110 115 120 Ser Asp Glu 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 Ala Lys Val Gln Trp 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 Ala 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 Sequence No .: 53 Sequence length: 768 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Hypothetical: NO Antisense: NO Sequence characteristics Characteristic symbols: CDS location: 40..753 Characteristic code: mat_peptide Location: 100..753 Character code: sig_peptide Place: 40..99 Sequence 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 Ile Leu Leu Trp Val Leu Leu Leu 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 Ile 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 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 35 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile 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 Ile His Pro 70 75 80 GTG GAG GAG GAG GAT GCT GCA ACC TAT T AC 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 Ile 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 Ile 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 Trp 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 GCA GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 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 SEQ ID NO: 54 Sequence length: 238 Sequence type: amino acid Topology: linear Sequence type: protein sequence Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20 -15 -10 -5 Gly Ser Thr Gly Asp Ile 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 15 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 Ile Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Thr Ile 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 Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Ph e 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 Ala Lys Val Gln Trp 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 Ala 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 SEQ ID NO: 55 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence CCCAAGCTTA AGAAGCATCC TCTCATCTAG TTCT 34 SEQ ID NO: 56 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence Type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GAGAGGGTGG CCCTCTCCCC TGGAGAC AGA GACAAAGTAC CTGG 44 SEQ ID NO: 57 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence CCAGGTACTT TGTCTCTGTC TCCAGGGGAG AGGGCCACCC TCTC 44 SEQ ID NO: 58 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GATTCGAGAT TGGATGCAGC ATAGATGAGG AGTCTGGGTG CCTG 44 SEQ ID NO: 59 Sequence length: 45 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GCTGCATCCA ATCTCGAATC TGGGATCCCA GACAGGTTTA GTGGC 45 SEQ ID NO: 60 Sequence length: 52 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Hypothetical: No Antisense: No sequence AAAATCCGCC GGCTCCAGAC GAGAGATGGT GAGGGTGAAG TCTGTCCCAG AC 52 SEQ ID NO: 61 Sequence length: 58 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of sequence: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No Sequence CTCGTCTGGA GCCGGCGGAT TTTGCAGTCT ATT
ACGTTCA GCAAAGTAAT GAGGATCC 58 SEQ ID NO: 62 Sequence length: 55 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence TGAAGACAGA TGGTGCAGCC ACAGTCCGTT TGATTTCCAG CCTGGTGCCT TGACC 55 SEQ ID NO: 63 Sequence length: 55 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical : No Antisense: No Sequence GGTCAAGGCA CCAGGCTGGA AATCAAACGG ACTGTGGCTG CACCATCTGT CTTCA 55 SEQ ID NO: 64 Sequence length: 45 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthesis) DNA) Hypothetical: No Antisense: No sequence CCCGAATTCT TACTAACACT CTCCCCTGTT GAAGCTCTTT GTGAC 45 SEQ ID NO: 65 Sequence length: 55 sequences Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence TCTGTCCCAG ACCCACTGCC ACTAAACCTG TCTGGGATCC CAGATTCGAG ATTGG 55 SEQ ID NO: 66 Sequence length Length: 55 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GTTTAGTGGC AGTGGGTCTG GGACAGACTT CACCTCTACC ATCCATCCTG TGGAG 55 SEQ ID NO: : 67 Sequence length: 55 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (Synthetic DNA) Hypothetical: No Antisense: No Sequence ATGGTGCAGC CACAGTCCGT TTGATTTCCA GCCTGGTGCC TTGACCGAAC GTCCG 55 SEQ ID NO: 68 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) High Cetical: No Antisense: No Sequence CCCAAGCTTA AGAAGCATCC 20 SEQ ID NO: 69 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypo Setical: No antisense: No sequence ATCTATGCTG CATCCAATCT 20 SEQ ID NO: 70 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GTTGTGTGCC TGCTGAATAA 20 SEQ ID NO: 71 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence CCCGAATTCT TACTAACACT 20 SEQ ID NO: 72 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic) DNA Hypothetical: No Antisense: No Sequence TTATTCAGCA GGCACACAAC 20 SEQ ID NO: 73 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence AGATTGGATG CAGCATAGAT 20 SEQ ID NO: 74 Sequence length: 457 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Tikal: NO Antisense: Characteristics of NO sequence Symbol indicating characteristics: CDS Location: 21. . 455 Characteristic symbol: mat_peptide Location: 78..455 Characteristic signature: sig_peptide Location: 21..77 Sequence AAGCTTGGCT TGACCTCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG 50 Met Gly Trp Ser Cys Ile Ile 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 15 GGG GCT GAG GTC AAG AAG CCT GGG GCT TCA GTG AAG GTG TCC TGC AAG 146 Gly Ala Glu Val Lys Lys Pro Gly Ala 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 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Trp Val Lys Gln 25 30 35 GCC CCT GGA CAG AGG CTT GAG TGG ATG GGA GAG ATT GAT CCT TCT GAT 242 Ala Pro Gly Gln Arg Leu Glu Trp Met Gly Glu Ile 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 Ala Thr Leu Thr 60 65 70 GTA GAC ACA TCC GCT AGC ACA GCC TAC ATG GA G 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 Ala Val Tyr Tyr Cys Ala 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 Trp Tyr Phe Asp Val Trp Gly Glu Gly Thr Leu Val Thr Val 105 110 115 TCC TCA GCC TCC ACC AAG GGC CC 457 Ser Ser Ala Ser Thr Lys Gly 120 125 SEQ ID NO: 75 Sequence length: 145 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 -5 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Ser Tyr Trp Met Gln Trp Val Lys Gln Ala Pro Gly Gln Arg Leu 30 35 40 45 Glu Trp Met Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 50 55 60 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ala Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Asn Arg Asp Tyr Ser Asn Asn Trp Tyr Phe Asp 95 100 105 Val Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 110 115 120 125 Gly SEQ ID NO: 76 Sequence length: 40 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GGGAAGCTTG GCTTGACCTC ACCATGGGAT GGAGCTGTAT 40 SEQ ID NO: 77 Sequence length: 48 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence Type: Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence TGAAGCCCCA GGCTTCTTGA CCTCAGCCCC AGACTGCACC AGTTGGAC 48 SEQ ID NO: 78 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Single strand Topology: Type of linear sequence: Other nucleic acids (synthetic DNA) Posetical: No Antisense: No Sequence TCCACTCAAG CCTCTGTCCA GGGGCCTGTT TTACCC 36 SEQ ID NO: 79 Sequence length: 51 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GTCTGGGGCT GAGGTCAAGA AGCCTGGGGC TTCAGTGAAG GTGTCCTGCA A 51 SEQ ID NO: 80 Sequence length: 39 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence CAGGCCCCTG GACAGAGGCT
GAGATT 39 SEQ ID NO: 81 Sequence length: 50 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence TCAGATCTCA GGCTGCTGAG CTCCATGTAG GCTGTGCTAG CGGATGTGTC 50 SEQ ID NO: 82 Sequence length: 44 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Anti Sense: No sequence TGGAGCTCAG CAGCCTGAGA TCTGAGGACA CGGCGGTCTA TTAC 44 SEQ ID NO: 83 Sequence length: 55 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Tikal: No Antisense: No Sequence GATGGGCCCT TGGTGGAGGC TGAGGAGACG GTGACCAGGG TCCCTTCGCC CCAGT 55 SEQ ID NO: 84 Sequence length: 39 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of row: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GGGAAGCTTC CGCGGTCACA TGGCACCACC TCTCTTGCA 39 SEQ ID NO: 85 Sequence length: 35 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GCTCTGCAGA GAGAAGATTG GGAGTTACTG GAATC 35 SEQ ID NO: 86 Sequence length: 35 Sequence type: Nucleic acid Number of strands: One Chain Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence TCTCTGCAGA GCCCAAATCT TGTGACAAAA CTCAC 35 SEQ ID NO: 87 Sequence length: 39 Sequence type: Nucleic acid Number of strands : Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GGGGAATTCG GGAGCGGGGC TTGCCGGCCG TCGCACTCA 39 SEQ ID NO: 88 Sequence Length: 2077 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: DNA (genomic) Hypothetical: NO antisense: NO Sequence characteristics Characteristic symbol: sig_peptide Location : 27..83 Symbol indicating feature: intron Location: 741..1131 Symbol indicating feature: intron Location: 1177..1294 Symbol indicating feature: intron Location: 1625..1721 Symbol indicating feature: exon Location: 27..740 Symbol indicating feature: exon Location: 1132..1176 Symbol indicating feature: exon Location: 1295.1624 Symbol indicating feature: exon Location: 1722..2077 Symbol indicating feature : Mat_peptide Location: join (84..740, 1132..1176, 1295..1624, 17
22..2042) Characteristic symbol: CDS Location: join (27..740, 1132..1176, 1295..1624, 17
22..2042) Sequence GGGCGAAAGC TTGGCTTGAC CTCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC 53 Met Gly Trp Ser Cys Ile Ile Leu Phe -19 -15 TTG GTA GCA ACA GCT ACA GGT GTC CAC TCT CAG GTC CAA CTG GTG CAG Leu Val Ala Thr Ala Thr Gly Val His 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 Ala Glu Val Lys Lys Pro Gly Ala 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 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Trp 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 Trp Met Gly Glu Ile 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 Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala 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 Ala Ser Thr Ala 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 Arg 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 Trp Tyr Phe Asp Val Trp 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 Ser 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 Ala 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 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 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 Ala Val Leu Gln Ser Ser Gly 170 175 180 CTC TAC TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC 677 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 185 190 195 ACC CAG AC C TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG 725 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 200 205 210 GTG GAC AAG AGA GTT GGTGAGAGGC CAGCACAGGG AGGGAGGGTG TCTGCTGGAA 780 Val Asp Lys Arg GCCAGGCTCA GCGCTCCTGC CTGGACGCAT CCCGGCTATG CAGTCCCAGT CCAGGGCAGC 840 AAGGCAGGCC CCGTCTGCCT CTTCACCCGG AGGCCTCTGC CCGCCCCACT CATGCTCAGG 900 GAGAGGGTCT TCTGGCTTTT TCCCCAGGCT CTGGGCAGGC ACAGGCTAGG TGCCCCTAAC 960 CCAGGCCCTG CACACAAAGG GGCAGGTGCT GGGCTCAGAC CTGCCAAGAG CCATATCCGG 1020 GAGGACCCTG CCCCTGACCT AAGCCCACCC CAAAGGCCAA ACTCTCCACT CCCTCAGCTC 1080 GGACACCTTC TCTCCTCCCA GATTCCAGTA ACTCCCAATC TTCTCTCTGC A GAG CCC 1137 Glu Pro 220 AAA TCT TGT GAC AAA ACT CAC ACA TGC CCA CCG TGC CCA GGTAAGCCAG 1186 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 CCCAGGCCTC GCCCTCCAGC TCAAGGCGGG ACAGGTGCCC TAGAGTAGCC TGCATCCAGG 1246 GACAGGCCCC AGCCGGGTGC TGACACGTCC ACCTCCATCT CTTCCTCA GCA CCT GAA 1303 Ala 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 TGC GTG GTG GTG GAC 1399 Thr Leu Met Ile 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 Trp Tyr Val Asp Gly 270 275 275 280 285 GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC 1495 Val Glu Val His Asn Ala 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 Trp 305 310 315 CTG AAT GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA 1591 Leu Asn Gly Lys Glu Tyr Lys Cys 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 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 335 340 AGGGCCACAT GGACAGAGGC CGGCTCGGCC CACCCTCTGC CCTGAGAGTG ACC GCTGTAC 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 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 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 Ile Ala Val Glu Trp 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 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 Trp 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 2042 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 4 40 445 450 TGAGTGCGAC GGCCGGCAAG CCCCGCTCCC GAATT 2077 Sequence length: 89 Sequence length: 470 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly -19 -15 -10 -5 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Ser Tyr Trp Met Gln Trp Val Lys Gln Ala Pro Gly Gln Arg Leu 30 35 40 45 Glu Trp Met Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 50 55 60 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ala Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Asn Arg Asp Tyr Ser Asn Asn Trp Tyr Phe Asp 95 100 105 Val Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 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 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 160 165 170 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 175 180 185 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 190 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 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 Ile 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 Trp Tyr Val Asp Gly 270 275 280 285 Val Glu Val His 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 His Gln Asp Trp 305 310 315 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 320 325 330 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 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 Ile 370 375 380 Ala Val Glu Trp 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 Trp 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 SEQ ID NO: 90 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence ACAGCCGGGA AGGTGTGCAC 20 SEQ ID NO: 91 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence AGACACCCTC CCTCCCTGTG 20 SEQ ID NO: 92 Sequence length: 20 Sequence type: Nucleic acid Number: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GTGCAGGGCC TGGGTTAGGG 20 SEQ ID NO: 93 Sequence length: 20 Sequence type: Nucleic acid Number of chains: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GCACGGTGGG CATGTGTGAG 20 SEQ ID NO: 94 Sequence length: 20 Sequence type: Nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GTTTTGGGGG GAAGAGGAAG 20 SEQ ID NO: 95 Sequence length: 20 Sequence type : Nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No antisense: No sequence CCAGTCCTGG TGCAGGACGG 20 SEQ ID NO: 96 Sequence length: 20 Sequence Type Nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence CCTGTGGTTC TCGGGGCTGC 20 SEQ ID NO: 97 Sequence length: 20 Sequence type : Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence CGTGGTCTTG TAGTTGTTCT 20 SEQ ID NO: 98 Sequence length: 20 Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence CTTCCTCTTC CCCCCAAAAC 20 SEQ ID NO: 99 Sequence length: 20 sequences Type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence CCGTCCTGCA CCAGGACTGG 20 SEQ ID NO: 100 Sequence length: 2 0 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence GCAGCCCCGA GAACCACAGGG
20 SEQ ID NO: 101 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence AGAACAACTA CAAGACCACG 20 SEQ ID NO: 102 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GCCTGACATC TGAGGACTC 19 SEQ ID NO: 103 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GAGTCCTCAG ATGTCAGGC 19 SEQ ID NO: 104 Sequence length: 34 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GAGCAGTACT CGTTGCTGCC GCGCGCGCCA CCAG 34 SEQ ID NO: 105 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Anti Sense: No sequence GGTATGGCTG ATTAATGATC AATG 24 SEQ ID NO: 106 Sequence length: 768 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical: NO Antisense: Characteristic of NO sequence Characteristic code: CDS Location: 40..753 Characteristic code: mat_peptide Location: 100..753 Characteristic code: sig_peptide Location: 40..99 Sequence 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 Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Glu -15 -5 -5 1 ATT GTG CTC ACC CAA TCT CCA GGT ACT TTG TCT CTG TCT CCA GGG GAG 150 Ile 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 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 35 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro 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 Ile 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 Ala Val 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 Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 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 Ile 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 Trp 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 GCA GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 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 SEQ ID NO: 107 Sequence length: 238 Sequence type: A Mino acid Topology: Linear Sequence type: Protein sequence Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20 -15 -10 -5 Gly Ser Thr Gly Glu Glu Ile 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 15 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 30 35 40 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 80 85 90 Gln Gln Ser Asn Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu 95 100 105 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 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 Ala Lys Val Gln Trp 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 Le u Ser Ser Thr Leu Thr Leu Ser Lys Ala 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 Sequence No .: 108 Sequence length: 768 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Hypothetical: NO Antisense: NO Sequence characteristics Characteristic symbols: CDS location: 40..753 Symbol indicating characteristics: mat_peptide Location: 100..753 Symbol indicating characteristics: sig_peptide Location: 40..99 Sequence 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 Ile Leu Leu Trp Val Leu Leu Leu Trp 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 Ile 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 G CC 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 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 35 40 45 CTC CTC ATC TAT GCT GCA TCC AAT CTC GAA TCT GGG ATC CCA GAC AGG 294 Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile 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 Ile 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 AAG CTG GAA ATC AAA CGG ACT 438 Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 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 Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 AAA TCT GGA ACT GCC TCT GTT GTG T GC 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 Trp 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 GCA GAC TAC GAG AAA CAC 678 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 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 SEQ ID NO: 109 Sequence length: 238 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro -20- 15 -10 -5 Gly Ser Thr Gly Glu Ile 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 15 20 25 Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro 30 35 40 Gly Gln Ala Pro Arg Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser 45 50 55 60 Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 65 70 75 Leu Thr Ile 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 Lys Leu 95 100 105 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile 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 Ala Lys Val Gln Trp 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 Ala 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 SEQ ID NO: 110 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence GGTGAGATTG TGCTCACCCA ATCTCCAGG 29 SEQ ID NO: 111 Sequence length: 29 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids ( Synthetic DNA) Hypothetical: No Antisense: No Sequence CCTGGAGATT GGGTGAGCAC AATCTCACC 29 SEQ ID NO: 112 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid (synthetic DNA) Hypothetical: No Antisense: No sequence CCATCTCTCG TCTGGAGCCG GAGGATTTTTG C
31 SEQ ID NO: 113 Sequence length: 31 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence GCAAAATCCT CCGGCTCCAG ACGAGAGATG G 31 SEQ ID NO: 114 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Sequence CAAGGCACCA AGCTGGAAAT CAAACGGACT G 31 SEQ ID NO: 115 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence CAGTCCGTTT GATTTCCAGC TTGGTGCCTT G 31 SEQ ID NO: 116 Sequence length: 2071 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: DNA (genomic) Hypothetical: NO Characteristic of NO sequence: Characteristic of NO sequence Symbol indicating characteristics: sig_peptide Location: 21..77 Symbol indicating characteristics: intron Location: 741..1131 Symbol indicating characteristics: intron Location: 1177..1294 Symbol indicating characteristics: intron Location: 1619..1715 Symbol indicating feature: exon Location: 21..734 Symbol indicating feature: exon Location: 1126..1170 Symbol indicating feature: exon Location: 1289. . 1618 Symbol indicating feature: exon Location: 1716..2071 Symbol indicating feature: mat_peptide Location: join (78..734, 1126..1170, 1289..1618, 17
16..2036) Symbol indicating feature: CDS Location: join (21..734, 1126..1170, 1289..1618, 17
16..2036) Sequence AAGCTTGGCT TGACCTCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG 50 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu -19 -15 -10 -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 Ala Glu Val Lys Lys Pro Gly Ala 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 Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Gln Trp Val Lys Gln 25 30 35 GCC CCT GGA CAG GGC CTT GAG TGG ATG GGA GAG ATT GAT CCT TCT GAT 242 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Glu Ile 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 Ala 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 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 Ala Val Tyr Tyr Cys Ala 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 Trp Tyr Phe Asp Val Trp 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 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 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 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 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 Trp 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 Ala 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 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 185 190 195 CAG ACC TA C ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG GTG 722 Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 200 205 210 215 GAC AAG AGA GTT GGTGAGAGGC CAGCACAGGG AGGGAGGGTG TCTGCTGGAA GC Asp LysGCGCGC 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 A 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 ACAGGTGCCC TAGAGTAGCC TGCATCCAGG 1240 GACAGGCCCC AGCCGGGTGC GTC TGACACCTTCTC GCC TGACACCTTCTC GCC GG 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 Ile 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 Trp Tyr Val Asp Gly 270 275 280 280 285 GTG GAG GTG CAT AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC 1489 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 AGC ACG 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 Trp 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 Ala Leu Pro 320 325 330 GCC CCC ATC GAG AAA ACC ATC TCC AAA GCC AAA GGTGGGACCC GTGGGGTGCG 1638 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 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 Serg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 360 365 370 CTG GTC AAA GGC TTC TAT CCC AGC GAC ATC GCC GTG GAG TGG GAG AGC 1844 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 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 TCC 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 Trp 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 4 50 TGAGTGCGAC GGCCGGCAAG CCCCGCTCCC GAATT 2071 SEQ ID NO: 117 Sequence length: 470 Sequence type: Amino acid Topology: Linear Sequence type: Protein Sequence Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly-19 -15 -10 -5 Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 1 5 10 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 20 25 Thr Ser Tyr Trp Met Gln Trp Val Lys Gln Ala Pro Gly Gln Gly Leu 30 35 40 45 Glu Trp Met Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn 50 55 60 Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Thr Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 80 85 90 Tyr Tyr Cys Ala Arg Asn Arg Asp Tyr Ser Asn Asn Trp Tyr Phe Asp 95 100 105 Val Trp Gly Glu Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 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 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 1 45 150 155 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 160 165 170 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 175 180 185 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 190 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 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 Ile 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 Trp Tyr Val Asp Gly 270 275 280 285 Val Glu Val His 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 His Gln Asp Trp 305 310 315 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 320 325 330 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 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 Three 55 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp 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 Trp 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 SEQ ID NO: 118 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Anti Sense: No sequence CAGGCCCCTG GACAGGGCCT TGAGTGGATG 30 SEQ ID NO: 119 Sequence length: 30 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No sequence CATCCACTCA AGGCCCTGTC CAGGGGCCTG 30 SEQ ID NO: 120 Sequence length: 39 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (Synthetic DNA) Hypothetical: No Antisense: No sequence GCTGAGCTCC ATGTAGGCTG TGCTAGTGGA TGTGTCTAC 39 SEQ ID NO: 121 Length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (Synthetic DNA) Hypothetical: No Antisense: No sequence TGCACGCGTG GCTGTGGAAT GTGTGTCAGT TAG 33 SEQ ID NO: 122 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (Synthetic DNA) Hypothetical: No Antisense: No Sequence TCCGAAGCTT TTAGAGCAGA AGTAACACTT C 31 SEQ ID NO: 123 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical: No Antisense: No Column AAAGCGGCCG CTGCTAGCTT GGCTGTGGAA TGT
GTG 36

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 1/21 C12N 1/21 5/10 C12P 21/02 C 15/02 C12Q 1/68 A C12P 21/02 C12P 21/08 C12Q 1/68 C12N 5/00 B // A61K 38/00 AED 15/00 C C12P 21/08 A61K 37/02 AED (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) ( C12P 21/02 C12R 1:19) (C12P 21/08 C12R 1:91) (72) Inventor Hiroko Yoshida 1-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Ichikawa Kunihisa 1-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Inventor Jun Osumi 1-2-58, Hiromachi, Shinagawa-ku, Tokyo Sanko Co., Ltd. (72) Inventor Masahiko Otsuki Tokyo 1-2-5 Hiromachi, Shinagawa-ku, Tokyo No. 8 Sankyo Co., Ltd. (72) Inventor Akio Shiraishi 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd.

Claims (58)

[Claims] 1. A molecule comprising an antigen-binding region specific for an epitope conserved between primate and non-primate Fas. 2. The primate is a human,
The molecule of claim 1. 3. The molecule according to claim 1, wherein the non-primate animal is a rodent. 4. The molecule according to claim 3, wherein the rodent is a mouse. 5. A molecule having an antigen-binding region specific for an epitope conserved among mammalian Fas. 6. A mouse-mouse hybridoma HFE7.
A (FERM BP-5828) produced monoclonal antibody. 7. A mouse-mouse hybridoma HFE7
A (FERM BP-5828) produced anti-F
C identical to the six CDRs from the as monoclonal antibody
A molecule characterized by having all of DR. 8. A mouse-mouse hybridoma HFE7
A. A molecule having an antigen-binding region specific to an epitope recognized by a monoclonal antibody produced by A (FERM BP-5828). 9. The method according to claim 1, wherein the antibody is an antibody.
9. The molecule according to any one of 5, 7 and 8. 10. The following consists of an amino acid sequence represented by the general formula (I) a heavy chain _{polypeptide: -FRH 1 -CDRH 1 -FRH 2 -CDRH} 2 -FRH 3 -CDRH 3 -FRH 4 - ( I) (wherein, FRH ₁ represents an arbitrary amino acid sequence consisting of 18 to 30 amino acids, CDRH ₁ represents the amino acid sequence shown in SEQ ID NO: 2, FRH ₂ consists of 14 amino acids represents any amino acid sequence, CDRH ₂ represents the amino acid sequence shown in SEQ ID NO: 3 of sequence Listing, FRH ₃ represents an arbitrary amino acid sequence consisting of 32 amino acids, CDRH ₃ in SEQ ID NO: 4 represents the amino acid sequence shown, FRH ₄ represents an arbitrary amino acid sequence consisting of 11 amino acids, are linked with each respective amino acid peptide bonds .) And the following consists of an amino acid sequence represented by the general formula (II) a light chain _{polypeptide: -FRL 1 -CDRL 1 -FRL 2 -CDRL} 2 -FRL 3 -CDRL 3 -FRL 4 - (II (Wherein, FRL ₁ represents an arbitrary amino acid sequence consisting of 23 amino acids, and CDRL ₁ represents SEQ ID NO: 5 in the sequence listing.)
And FRL ₂ represents an arbitrary amino acid sequence consisting of 15 amino acids, and
L ₂ represents the amino acid sequence shown in SEQ ID NO: 6 of Sequence Listing, FRL ₃ represents an arbitrary amino acid sequence consisting of 32 amino acids, CDRL ₃ represents the amino acid sequence shown in SEQ ID NO: 7, FRL ₄ represents an arbitrary amino acid sequence consisting of 10 amino acids, and each amino acid is connected by a peptide bond. ). 11. The method according to claim 1, which has at least one of the following biological properties a) to f): Molecules: a) has apoptosis-inducing activity on T cells expressing Fas; b) has an effect of ameliorating autoimmune disease symptoms of MRLgld / gld mice; c) does not induce liver damage; d) anti-mouse Fas monoclonal It has a therapeutic or preventive effect on fulminant hepatitis induced by antibody Jo2; e) has a preventive effect on collagen-induced arthritis; and f) has an apoptosis-inducing activity on rheumatoid arthritis patient-derived synovial cells. 12. The molecule according to claim 11, which has all of the biological properties according to a) to f). 13. The molecule according to claim 10, which is an antibody. 14. It is humanized,
The antibody according to claim 9 or 13. 15. The molecule according to claim 9 or 13, which is an immunoglobulin type G antibody. 16. It is humanized,
The antibody according to claim 6. 17. The method according to claim 1, wherein apoptosis can be induced in abnormal cells expressing Fas and apoptosis of normal cells can be prevented. The molecule according to one. 18. A pathological condition of a molecule according to any one of claims 1 to 5, 7 to 13, 15 or 17, or an antibody according to claim 6, which is affected by a Fas / Fas ligand interaction. A method for evaluating the effect of a drug on the disease model animal, comprising administering the molecule or the antibody to the model animal of the disease state, and then comparing the change in the disease state between the animal to which the molecule or the antibody is administered and the non-administered animal. A method characterized by comparing between. 19. The at least one human immunoglobulin light chain or fragment thereof, and the at least one human immunoglobulin heavy chain or fragment thereof, respectively, are conserved between primate and non-primate Fas. An antigen-binding region specific for an epitope that is conserved between primate and non-primate Fas, which can be obtained by grafting the respective CDRs of the antibody from an antibody that specifically binds to the epitope. A humanized antibody comprising: 20. Variable regions between the human immunoglobulin light chain and the human immunoglobulin heavy chain, respectively, with antibodies that specifically bind to an epitope conserved between primate and non-primate Fas. 20. The one having the highest similarity is selected.
The humanized antibody according to any of the preceding claims. 21. An antibody that specifically binds to an epitope that is conserved between primate and non-primate Fas from the antigen of the antibody in the framework regions of the heavy and / or light chains of the antibody. The amino acid important for maintaining the structure of the recognition site is grafted on a human immunoglobulin light chain or a fragment thereof and / or a human immunoglobulin heavy chain or a fragment thereof. 21. The humanized antibody according to 20. 22. A binding to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing,
Claims 1 to 5, 7 to 15, 17, or 19 to 21
The molecule or antibody according to any one of the above. 23. An amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 50 in the sequence list, an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 52 in the sequence list, and an amino acid number represented by SEQ ID NO: 54 of the sequence list 1 to 21
8, the amino acid sequence of amino acid numbers 1 to 218 of SEQ ID NO: 107 in the sequence listing or the amino acid sequence of amino acid numbers 1 to 218 of SEQ ID NO: 109 in the sequence listing
Characterized in that it has a light chain polypeptide comprising any one of the amino acid sequences shown in claims 1 to 5,
23. The molecule or antibody according to any one of 7 to 15, 17 or 19 to 22. 24. A heavy chain polyprotein comprising any one of the amino acid sequences represented by amino acid numbers 1 to 451 of SEQ ID NO: 89 in the sequence listing or the amino acid sequence represented by amino acid numbers 1 to 451 of SEQ ID NO: 117 of the sequence listing Claims 1 to 5, 7 to 15, characterized by having a peptide.
24. The molecule or antibody according to any one of 17 or 19 to 23. 25. A light chain polypeptide comprising the amino acid sequence of amino acid Nos. 1 to 218 of SEQ ID No. 50 in the sequence listing, and the amino acid sequence of amino acid No. 1 to 451 of SEQ ID No. 89 of the sequence listing The molecule or antibody according to claim 23 or 24, which is composed of a heavy chain polypeptide. 26. A light chain polypeptide comprising the amino acid sequence of amino acids 1 to 218 of SEQ ID NO: 107 in the sequence listing, and the amino acid sequence of amino acids 1 to 451 of SEQ ID NO: 117 of the sequence listing. 3. It is composed of a heavy chain polypeptide.
25. The molecule or antibody according to 3 or 24. 27. DN encoding a molecule according to any one of claims 1 to 5, 7 to 15, 17 or 19 to 26 or any one of the polypeptide chains in an antibody.
A. 28. A DN encoding the light or heavy chain polypeptide of the antibody according to claim 6 or 16.
A. 29. A DNA encoding a polypeptide comprising an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 11 in the sequence listing. 30. Amino acid No. 1 of SEQ ID No. 9 in the sequence listing
A DNA encoding a polypeptide comprising the amino acid sequence of SEQ ID NOs: to 445. 31. An amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 50 in the sequence list, an amino acid sequence represented by amino acid numbers 1 to 218 of SEQ ID NO: 52 in the sequence list, and an amino acid number represented by SEQ ID NO: 54 of the sequence list 1 to 21
8, the amino acid sequence of amino acid numbers 1 to 218 of SEQ ID NO: 107 in the sequence listing or the amino acid sequence of amino acid numbers 1 to 218 of SEQ ID NO: 109 in the sequence listing
A DNA encoding a polypeptide comprising any one of the amino acid sequences of 32. It comprises any one of the amino acid sequences represented by amino acid numbers 1 to 451 of SEQ ID NO: 89 in the sequence listing or the amino acid sequences represented by amino acid numbers 1 to 451 of SEQ ID NO: 117 of sequence listing. DNA encoding a polypeptide. 33. The DNA according to claim 29, comprising a nucleotide sequence represented by nucleotide numbers 61 to 393 of SEQ ID NO: 10 in the sequence listing. 34. The DNA according to claim 29, comprising the nucleotide sequence represented by nucleotide numbers 61 to 714 of SEQ ID NO: 10 in the sequence listing. 35. The DNA according to claim 30, which comprises a nucleotide sequence represented by nucleotide numbers 58 to 420 of SEQ ID NO: 8 in the sequence listing. 36. The DNA according to claim 30, which comprises a nucleotide sequence represented by nucleotide numbers 58 to 1392 of SEQ ID NO: 8 in the sequence listing. 37. A nucleotide sequence represented by nucleotide numbers 100 to 753 of SEQ ID NO: 49 in the sequence listing, and a nucleotide sequence represented by nucleotide numbers 100 to 75 of SEQ ID NO: 51 in the sequence listing.
SEQ ID NO: 53 in the nucleotide sequence shown in SEQ ID NO: 3
The nucleotide sequence represented by nucleotide numbers 100 to 753 of the sequence listing, the nucleotide sequence represented by nucleotide numbers 100 to 753 of the sequence listing SEQ ID NO: 106, or the nucleotide sequence represented by the nucleotide numbers 100 to 753 of the sequence listing SEQ ID NO: 108 32. The DNA of claim 31, consisting of: 38. The method according to claim 32, comprising the nucleotide sequence represented by nucleotide numbers 84 to 2042 of SEQ ID NO: 88 in the sequence listing or the nucleotide sequence represented by nucleotide numbers 78 to 2036 of SEQ ID NO: 116 in the sequence listing. DNA. 39. A recombinant DNA vector comprising the DNA according to any one of claims 27 to 38. 40. A recombinant DNA vector comprising the DNA according to any one of claims 29, 31, 33, 34 or 37. 41. A recombinant DNA vector comprising the DNA according to any one of claims 30, 32, 35, 36 and 38. 42. A host cell transformed with the recombinant DNA vector according to claim 39. 43. The claim 40 and / or 41.
A host cell transformed with the recombinant DNA vector according to the above. (44) A host cell transformed with the recombinant DNA vector according to (40) or (41). 45. The host cell according to any one of claims 41 to 43, wherein the host cell is derived from a mammal. 46. A transformed E. coli E. coli. coli pME
-L (FERM BP-5867). 47. A transformed E. coli E. coli. coli pME
-H (FERM BP-5868). 48. A transformed E. coli E. coli. coli pHS
GMM6 SANK73697 (FERM BP-60
71). 49. A transformed E. coli E. coli. coli pHS
GHM17 SANK73597 (FERM BP-6
072). 50. A transformed E. coli E. coli. coli pHS
GHH7 SANK73497 (FERM BP-60
73). 51. A transformed E. coli E. coli. coli pHS
HM2 SANK 70198 (FERM BP-62
72). 52. A transformed E. coli E. coli. coli pHS
HH5 SANK 70398 (FERM BP-62
74). 53. A transformed E. coli E. coli. coli pgH
SL7A62 SANK73397 (FERM BP-
6074). 54. A transformed E. coli E. coli. coli pgH
PDHV3 SANK70298 (FERM BP-6
273). 55. A method for producing an anti-Fas antibody, comprising culturing the host cell according to claim 44 or 45, and then recovering the anti-Fas antibody from the culture. 56. Mouse-mouse hybridoma HFE
7A (FERM BP-5828). 57. Mouse-mouse hybridoma HFE
The method for producing an antibody according to claim 6, wherein 7A (FERM BP-5828) is cultured under conditions capable of producing immunoglobulin, and then the immunoglobulin is recovered from the culture. 58. A medicament comprising the molecule according to any one of claims 1 to 5, 7 to 15, 17 or 19 to 26, or the antibody according to claim 6 or 16 as an active ingredient.