JPWO2005019268A1 - Antibodies against novel cancer antigens - Google Patents

Abstract

By detecting DHCR24 that is highly expressed in cancer cells, highly accurate cancer diagnosis can be performed. In addition, an antibody that recognizes DHCR24 is effective for simple and highly accurate cancer diagnosis and treatment.

Description

  The present invention relates to an antibody that recognizes DHCR24. Furthermore, the present invention relates to a method for diagnosing cancer, which comprises detecting DHCR24.

Hepatitis C virus (HCV) is the main causative virus of non-A non-B hepatitis after blood transfusion, and hepatitis caused by this virus has a high chronicity rate. The cDNA of this virus was cloned by Choo et al. in 1989 (Choo, QL et al., Science, 244, 359-362, 1989) and is a single-stranded RNA virus belonging to the Flaviviridae family. Known (Kato, N. et al., Proc. Natl. Acad. Sci. USA, 87, 9524-9528, 1990). Further, the entire base sequence and amino acid sequence have been elucidated by some groups.
The present inventors have previously produced a vector expressing a full-length gene of HCV (WO99/67394).
There is an urgent need to find a therapeutic means for hepatitis C, and one of the reasons for this is that there is a high probability of transition from hepatitis C to liver cancer/cirrhosis. In particular, for liver cancer that shifts from hepatitis C, there is a need for immediate establishment of therapeutic means.
In recent years, a cancer antigen protein has been attracting attention as a target for efficiently attacking cancer cells. Cancer antigen protein is a generic term for antigens that are present in cancer cells and are rarely expressed in normal cells. As cancer antigen proteins, CEA (J. Natl. Cancer. Inst., 87:982, 1995) is used. ), gp100 (J. Exp. Med., 179: 1005, 1994), MART-1 (Proc. Natl. Acad. Sci. USA, 91: 3515, 1994), tyrosinase (J. Exp. Med., 178: 489, 1993), HER2 (J. Exp. Med., 181:2109, 1995), PSA (J. Natl. Cancer. Inst., 89: 293, 1997) and the like.
Proteins such as cancer antigen proteins that are highly expressed specifically in cancer cells are used as tumor markers for cancer diagnosis, and antibodies that specifically recognize cancer antigen proteins are used. Like other anti-cancer drugs, it can be a target for cancer drugs. For example, a humanized anti-HER2 monoclonal antibody that specifically recognizes HER2 highly expressed in breast cancer cells is used as a therapeutic agent for breast cancer (WO89/06692). Thus, a protein highly expressed in cancer cells and an antibody that binds to the protein are considered to be particularly useful for cancer diagnosis and treatment.

An object of the present invention is to provide an antibody that enables simple and highly accurate cancer diagnosis. Another object of the present invention is to provide a method for diagnosing cancer by detecting an antigen specifically expressed in cancer cells.
The present inventors first immunized mice with cells into which a gene encoding an HCV protein was introduced as an immunogen to obtain antibodies. After that, an antibody specifically recognizing liver cancer cells was selected from the obtained antibodies. Subsequent analysis revealed that the antibody was an antibody that recognizes DHCR24. Furthermore, the present inventors have found that DHCR24 is highly expressed in the cancerous part of the liver cancer patient and the liver cancer cell line as compared to the normal cells and the non-cancerous part of the liver cancer patient. It was speculated that DHCR24 was particularly highly expressed in HCV-positive liver cancer patients.
Therefore, it has been found that highly accurate cancer diagnosis can be performed by detecting DHCR24 highly expressed in cancer cells. Further, they have found that an antibody that recognizes DHCR24 is effective for cancer diagnosis and treatment. The present invention has been completed based on these findings.
That is, the present invention provides an antibody that recognizes DHCR24.
The present invention also provides an antibody that specifically recognizes DHCR24.
Furthermore, the present invention provides an antibody that recognizes DHRC24, which can diagnose cancer.
Furthermore, the present invention provides an antibody that recognizes DHCR24, which can be obtained by a method for producing an antibody, which comprises introducing a gene encoding a hepatitis C virus protein into a cell and immunizing an animal with the cell.
Furthermore, the present invention provides an antibody that recognizes DHCR24, which can be obtained by a method for producing an antibody that includes immunizing an animal with liver cancer cells.
The present invention also provides a diagnostic agent for cancer, which comprises the above antibody.
Further, the present invention provides an anticancer agent containing the above antibody and a pharmaceutically acceptable carrier.
Furthermore, the present invention provides a method for diagnosing cancer, which comprises detecting DHCR24.
Furthermore, the present invention provides a method for detecting DHCR24 for diagnosing cancer, which comprises reacting the above antibody with living cells or cell lysates.
Furthermore, the present invention provides a method for treating cancer, which comprises administering the above antibody.
Furthermore, the present invention provides a method of killing a cancer cell expressing DHCR24, a method of causing a cytotoxicity to the cancer cell, and a method of suppressing the growth of the cancer cell using an anti-DHCR24 antibody. To do.

FIG. 1A is a diagram showing an outline of an HCV protein expression system using the Cre/loxP system.
FIG. 1B is a diagram showing the expression of HCV protein in a cell line incorporating the HCV protein expression system shown in FIG. 1A.
FIG. 2 is a diagram showing the results of producing a monoclonal antibody against Rz-HepM2-18-648d cells and screening using a live cell ELISA system.
FIG. 3 is a diagram showing the results of analyzing the expression of antibody-recognizing antigen molecules in various cell lines using the monoclonal antibody clones 2-152, 2-243 and 2-433.
FIG. 4 is a diagram showing the results of Western blotting analysis of the expression levels of the recognition antigens in the cancerous and non-cancerous tissues of liver cancer patients.
FIG. 5: is a figure which shows the result of having analyzed the intracellular localization of P55 by the fluorescent antibody method using the monoclonal antibody clone 2-152.
FIG. 6 shows that (A) HCR6-Rz gene was transfected into WRL68 and HepG2 cells, and (B) HCV was expressed in Rz-HepM2-18-6 cells (M6 Tm+) and HCV in the same cells. It is a result of having analyzed about the expression of P55 in the case of expressing and subculturing (M6-48d).
FIG. 7 shows (A) HepG2 cells transfected with the HCV gene and double-stained with 2-152 and anti-HCV-core (rabbit RR8 serum). (B) HepG2 cells were transfected with a CAG control plasmid, and were similarly stained with multiple stains. (C) WRL68 cells were transfected with the HCV gene and double stained with 2-152 and anti-HCV-core (rabbit RR8 serum). (D) WRL68 cells were transfected with the CAG control plasmid, and multiple staining was similarly performed.
FIG. 8 is a diagram in which HCV was expressed in Rz-HepM2-18-6 cells and double stained with 2-152, an anti-core antibody (4 days after the expression).
FIG. 9 is a diagram in which HCV was expressed and passaged (48 days or more) in Rz-HepM2-18-6 cells, and double stained with 2-152 and anti-core antibody.
FIG. 10 shows the results of recognizing DHCR24 using the monoclonal antibody clone 2-152. (A) In vitro translation of pGEM-T-DHCR24 (Exp.1), (B) Reaction of in vitro translated protein of pGEM-T-DHCR24 with 2-152 (western blotting), (C) pCAG-DHCR24 (Exp) 2) shows the reaction (Western blotting) with 2-152 after transfection of WRL68 cells.
FIG. 11 is a graph showing the CDC activity of monoclonal antibody 2-152 or normal mouse IgG against cell lines WRL68, HepG2 or HuH-7.
FIG. 12 is a micrograph (600 times) of immunostaining the cancerous tissue (A) or non-cancerous tissue (B) of the liver of an HCV-infected HCC patient. The left panel shows the monoclonal antibody 2-152 staining, TO-PRO-3 staining, double staining (Merge), and normal IgG (Normal) staining, respectively. The right panel is a photomicrograph stained with HE to observe the cell morphology.

It is considered that DHCR24 is hardly expressed on the cell membrane in normal hepatocytes, but its distribution is disrupted and the cells are expressed on the cell membrane due to the carcinogenesis of hepatocytes. This indicates that the anti-DHCR24 antibody can easily reach hepatoma cells, but it is difficult to reach normal hepatocytes. Therefore, the anti-DHCR24 antibody is considered to be useful for cancer diagnosis/treatment.
The antibody that recognizes DHCR24 of the present invention may be any antibody as long as it has the ability to bind to DHCR, and is not limited by its origin or shape. The ability to bind to DHCR preferably binds specifically to DHCR.
The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but when used for treatment or diagnosis, it is preferably a monoclonal antibody.
The antibody of the present invention may be any animal-derived antibody such as mouse antibody, human antibody, rat antibody, rabbit antibody, goat antibody and camel antibody.
Furthermore, for example, a modified antibody in which the amino acid sequence is substituted, such as a chimeric antibody or a humanized antibody, may be used, or an antibody modified product to which various molecules are bound, an antibody fragment, a low molecular weight antibody, a sugar chain modified antibody, etc. It may be an antibody.
1. Anti-DHCR24 antibody
The anti-DHCR24 antibody used in the present invention can be obtained as a polyclonal antibody or a monoclonal antibody using known means. As the anti-DHCR24 antibody of the present invention, a mammal-derived monoclonal antibody is particularly preferable. Mammalian-derived monoclonal antibodies include those produced by hybridomas, those produced by a host transformed with an expression vector containing the antibody gene by a genetic engineering technique, and the like.
Such an antibody can be obtained by a method known to those skilled in the art, for example, as follows.
2. Antibody-producing hybridoma
A hybridoma producing a monoclonal antibody can be basically prepared using a known technique, for example, as follows. That is, DHCR24 protein or DHCR24-expressing cells (for example, hepatocytes into which a gene encoding HCV protein has been introduced, hepatoma cells, etc.) are used as a sensitizing antigen, and are immunized according to a usual immunization method to obtain It can be prepared by fusing an immune cell with a known parent cell by a usual cell fusion method and screening a monoclonal antibody-producing cell by a usual screening method.
Specifically, a monoclonal antibody may be prepared as follows.
First, the DHCR24 protein used as a sensitizing antigen for antibody acquisition is obtained by expressing the DHCR24 gene/amino acid sequence disclosed in GenBank: NM_014762 (or Am. J. Hum. Genet. 69(4), 685). -694 (2001); J. Neurosci. 20 (19), 7345-7352 (2000)). That is, after inserting a gene sequence encoding DHCR24 into a known expression vector system to transform an appropriate host cell, the desired human DHCR24 protein is purified from the host cell or the culture supernatant by a known method. To do.
Next, this purified DHCR24 protein is used as a sensitizing antigen. Alternatively, a partial peptide of DHCR24 can be used as a sensitizing antigen. At this time, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human DHCR24.
The epitope on the DHCR24 molecule that the anti-DHCR24 antibody of the present invention recognizes is not limited to a particular one, and any epitope existing on the DHCR24 molecule may be recognized. Therefore, as the antigen for producing the anti-DHCR24 antibody of the present invention, any fragment can be used as long as it is a fragment containing an epitope present on the DHCR24 molecule.
The mammal to be immunized with the sensitizing antigen is not particularly limited, but it is preferable to select it in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, rabbits, monkeys, etc. are used.
Immunization of animals with a sensitizing antigen is performed according to a known method. For example, as a general method, it is carried out by injecting the sensitizing antigen into the mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted with PBS (Phosphate-Buffered Saline), physiological saline or the like to an appropriate amount, and suspended in a usual adjuvant, for example, Freund's complete adjuvant in an appropriate amount, and after emulsification, The mammal is dosed several times every 4 to 21 days. In addition, a suitable carrier can be used during immunization with the sensitizing antigen.
As described above, after immunizing a mammal and confirming that the desired antibody level in serum is increased, immune cells are collected from the mammal and subjected to cell fusion. Cells.
Mammalian myeloma cells are used as the other parent cells to be fused with the immune cells. The myeloma cells are various known cell lines such as P3 (P3x63Ag8.653) (J. Immunol. 123, 1548-1550, 1979), P3x63Ag8U. 1 (Current Topics in Microbiology and Immunology, 81, 1-7, 1978), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol., 6, 511-519, 1976), MPC-11. (Margulies. DH et al., Cell, 8, 405-415, 1976), SP2/0 (Shulman, M. et al., Nature, 276, 269-270, 1978), FO (deSt. Groth). , SF et al., J. Immunol. Methods, 35, 1-21, 1980), S194 (Trobridge, I, SJ, Exp. Med., 148, 313-323, 1978), R210 (S. Galfre, G. et al., Nature, 277, 131-133, 1979) and the like are preferable.
The cell fusion between the immune cell and the myeloma cell is basically a known method, for example, the method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol., 73, 3-46, 1981) and the like.
More specifically, the cell fusion is carried out in an ordinary nutrient medium in the presence of, for example, a cell fusion promoter. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like are used, and if desired, an auxiliary agent such as dimethyl sulfoxide can be added and used to enhance the fusion efficiency.
The use ratio of immune cells and myeloma cells can be set arbitrarily. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture medium used for the cell fusion, for example, RPMI 1640 culture medium suitable for the growth of the myeloma cell line, MEM culture medium, and the like, a normal culture medium used for this type of cell culture can be used, Furthermore, a serum supplement such as fetal calf serum (FCS) can be used in combination.
For cell fusion, a predetermined amount of the immune cells and myeloma cells are well mixed in the culture medium, and a PEG solution (eg, average molecular weight of about 1,000 to 6,000) preheated to about 37° C. is usually used for 30 times. The desired fused cells (hybridomas) are formed by adding at a concentration of -60% (w/v) and mixing. Then, an appropriate culture solution is sequentially added, and the procedure of centrifuging and removing the supernatant is repeated to remove the cell fusion agent and the like which are not preferable for the growth of the hybridoma.
The hybridoma thus obtained is selected by culturing in a usual selective culture medium, for example, HAT culture medium (culture medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium is continued for a sufficient time (usually several days to several weeks) to kill cells (non-fused cells) other than the target hybridoma. Then, an ordinary limiting dilution method is carried out to screen and single clone the hybridoma producing the desired antibody.
In addition to immunizing a non-human animal with an antigen to obtain the above hybridoma, human lymphocytes are sensitized to DHCR24 in vitro, and the sensitized lymphocytes are fused with human-derived myeloma cells having permanent division ability, It is also possible to obtain a desired human antibody having a binding activity to DHCR24 (see Japanese Patent Publication No. 1-59878). Furthermore, DHCR24 serving as an antigen may be administered to a transgenic animal having a full repertoire of human antibody genes to obtain anti-DHCR24 antibody-producing cells, and human antibodies against DHCR24 may be obtained from cells immortalized. (See WO94/25585, WO93/12227, WO92/03918, and WO94/02602).
The hybridoma producing the monoclonal antibody thus produced can be subcultured in an ordinary culture medium and can be stored in liquid nitrogen for a long period of time.
To obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant thereof, or the hybridoma is administered to a mammal compatible with this to proliferate the ascites fluid. The method of obtaining as. The former method is suitable for obtaining a high-purity antibody, while the latter method is suitable for mass production of antibodies.
3. Recombinant antibody
In the present invention, as a monoclonal antibody, a recombinant antibody produced by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing this into a host, and using a gene recombination technique can be used. (See, for example, Vandamme, AM et al., Eur. J. Biochem., 192, 767-775, 1990).
Specifically, mRNA encoding the variable (V) region of the anti-DHCR24 antibody is isolated from the hybridoma producing the anti-DHCR24 antibody. Isolation of mRNA is known in the art, for example, guanidine ultracentrifugation method (Chirgwin, J, M. et al., Biochemistry, 18, 5294-5299, 1979), AGPC method (Chomczynski, P. et al., Anal. Biochem., 162, 156-159, 1987) etc. to prepare total RNA, and the mRNA of interest is prepared using mRNA Purification Kit (manufactured by Pharmacia). Alternatively, mRNA can be directly prepared by using the QuickPrep mRNA Purification Kit (manufactured by Pharmacia).
The cDNA of the antibody V region is synthesized from the obtained mRNA using a reverse transcriptase.
DNA is synthesized using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (manufactured by Seikagaku Corporation). In order to synthesize and amplify cDNA, 5'-Ampli FINDER RACE Kit (manufactured by Clontech) and 5'-RACE method using PCR (Frohman, MA et al., Proc. Natl. Acad. Sci. USA, 85, 8998-9002, 1988; Belyavsky, A. et al., Nucleic Acids Res., 17, 2919-2932, 1989) and the like can be used.
The desired DNA fragment is purified from the obtained PCR product and ligated with the vector DNA. Furthermore, a recombinant vector is prepared from this and introduced into Escherichia coli or the like to select colonies to prepare a desired recombinant vector. Then, the nucleotide sequence of the desired DNA is confirmed by a known method such as the dideoxynucleotide chain termination method.
After obtaining the DNA encoding the V region of the anti-DHCR24 antibody of interest, this is incorporated into an expression vector containing the DNA encoding the desired antibody constant region (C region).
To produce the anti-DHCR24 antibody used in the present invention, the antibody gene is usually incorporated into an expression vector so that it is expressed under the control of an expression control region, for example, an enhancer or a promoter. Next, a host cell is transformed with this expression vector to express an antibody.
For the expression of the antibody gene, the DNA encoding the antibody heavy chain (H chain) or the light chain (L chain) may be separately incorporated into an expression vector to cotransform the host cell, or alternatively, the H chain and the L chain may be transformed. The host cell may be transformed by incorporating the DNA encoding the above into a single expression vector (see WO94/11523).
In addition to the above host cells, transgenic animals can be used for the production of recombinant antibodies. For example, an antibody gene is inserted in the middle of a gene encoding a protein (eg, goat β casein) produced uniquely in milk to prepare a fusion gene. A DNA fragment containing the fusion gene having the antibody gene inserted therein is injected into a goat embryo, and this embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its offspring. In addition, hormones may optionally be used in transgenic goats to increase the amount of milk containing the desired antibody produced by the transgenic goats (Ebert, KM et al., Bio/Technology, 12). , 699-702, 1994).
4. Modified antibody
In addition to the above-mentioned antibodies, the antibodies of the present invention include modified antibodies that have been artificially modified for the purpose of reducing xenoantigenicity to humans, such as chimeric antibodies and humanized antibodies. These modified antibodies can be produced using known methods.
The chimeric antibody can be obtained by ligating the DNA encoding the antibody V region obtained as described above with the DNA encoding the human antibody C region, incorporating this into an expression vector and introducing it into a host to produce. Chimeric antibodies can be obtained using this known method.
A humanized antibody is also called a reshaped human antibody, which is a non-human mammal, for example, a complementarity determining region (CDR) of a mouse antibody transplanted to a complementarity determining region of a human antibody. The general gene recombination technique is also known (see EP125023 and WO96/02576).
Specifically, a DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody is designed to have a portion overlapping both terminal regions of CDR and FR. The prepared oligonucleotides are used as primers to synthesize by PCR (see the method described in WO98/13388).
The human antibody framework regions linked via CDRs are selected such that the complementarity determining regions form a favorable antigen-binding site. If desired, amino acids in the framework regions of the variable region of the antibody may be substituted so that the complementarity determining regions of the reshaped human antibody form appropriate antigen binding sites (Sato, K. et al., Cancer Res., 53, 851-856, 1993).
For the C regions of the chimeric antibody and humanized antibody, those of human antibodies are used. For example, Cγ1, Cγ2, Cγ3, and Cγ4 can be used for the H chain, and Cκ and Cλ can be used for the L chain. In addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production.
Generally, a chimeric antibody consists of a variable region of a non-human mammal-derived antibody and a human antibody-derived constant region. On the other hand, a humanized antibody is composed of a complementarity determining region of a non-human mammal-derived antibody, a human antibody-derived framework region and a C region. Since the chimeric antibody and humanized antibody have reduced antigenicity in the human body, they are considered to be useful when administered to humans for therapeutic purposes.
5. Modified antibody
The antibody used in the present invention is not limited to the whole antibody molecule, and may be a fragment of the antibody or a modified product thereof as long as it binds to DHCR24, and includes a bivalent antibody and a monovalent antibody. For example, antibody fragments include Fab and F(ab'). _Two , Fv, Fab/c having one Fab and complete Fc, or single chain Fv (scFv) in which Fv of H chain or L chain is linked with an appropriate linker, diabody and the like. Specifically, an antibody is treated with an enzyme such as papain or pepsin to produce an antibody fragment, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, and then in an appropriate host cell. Express (eg, Co, MS et al., J. Immunol., 152, 2968-2976, 1994; Better, M. & Horwitz, AH, Methods in Enzymology, 178, 476-496, 1989). Academic Press, Inc., Pleckthun, A. & Skerra, A. Methods in Enzymology, 178, 476-496, 1989; Academic Press, Inc., Lamoyi, E., 65, verses. Rousseaux, J. et al., Methods in Enzymology, 121, 663-669, 1989; Bird, RE et al., TIBTECH, 9, 132-137, 1991).
scFv can be obtained by linking the H chain V region and the L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, JS et al., Proc. Natl. Acad. Sci. US. A., 85, 5879-5883, 1988). The H chain V region and the L chain V region in scFv may be derived from any of the antibodies described herein. As the peptide linker connecting the V regions, for example, an arbitrary single-chain peptide having about 5 to 20 amino acid residues is used.
The scFv-encoding DNA may be the entire H-chain or H-chain V-region encoding DNA or the L-chain or L-chain V-region encoding all or desired amino acid sequences of those sequences. A DNA portion encoding the template is used as a template and amplified by PCR using a pair of primers defining both ends thereof, and then DNA encoding a peptide linker portion and both ends thereof are linked to an H chain and an L chain, respectively. It can be obtained by combining and amplifying the primer pairs defined in 1.
Further, once the DNA encoding the scFv is produced, an expression vector containing them and a host transformed with the expression vector can be obtained by a conventional method. ScFv can be obtained according to the method.
Fragments of these antibodies can be produced by the host by obtaining and expressing the gene in the same manner as described above. The “antibody” in the present invention also includes fragments of these antibodies.
Examples of modified antibodies include anti-DHCR24 antibody bound to various molecules such as cytotoxic substances and polyethylene glycol (PEG). Examples of cytotoxic substances include radioisotopes, chemotherapeutic agents, and bacterial toxins. The “antibody” in the present invention also includes modified antibodies bound to such other substances. Such a modified antibody can be obtained by chemically modifying the obtained antibody. It should be noted that antibody modification methods have already been established in this field.
Further, the antibody used in the present invention may be a bispecific antibody. The bispecific antibody may be a bispecific antibody having an antigen binding site that recognizes a different epitope on the DHCR24 molecule, or one antigen binding site recognizes DHCR24 and the other antigen binding site is chemically bound. It may also recognize cytotoxic substances such as therapeutic agents, cell-derived toxins and radioactive substances. In this case, it is possible to suppress the proliferation of tumor cells by directly acting a cytotoxic substance on the cells expressing DHCR24 to specifically damage the tumor cells. The bispecific antibody can be prepared by binding HL pairs of two kinds of antibodies, or can be prepared by fusing hybridomas producing different monoclonal antibodies to prepare bispecific antibody-producing fused cells. it can. Furthermore, it is also possible to produce bispecific antibodies by genetic engineering techniques.
Further, the sugar chain of the antibody may be modified for the purpose of enhancing the cytotoxic activity of the antibody. Techniques for modifying sugar chains of antibodies are already known (for example, WO/0061739, WO02/31140, etc.).
The anti-DHCR24 antibody may have ADCC (antibody-dependent cellular cytotoxity) activity or CDC (complement-dependent cytotoxity) activity as cytotoxic activity.
The ADCC activity can be measured, for example, by mixing the anti-DHCR antibody and effector cells with the target cells and examining the degree of ADCC. As the effector cells, for example, mouse spleen cells, mononuclear cells isolated from human peripheral blood or bone marrow, and the like can be used. As the target cells, human cell lines such as human hepatocyte cell line HuH-7 can be used. Target cells in advance ⁵¹ After labeling with Cr, anti-DHCR24 antibody is added thereto for incubation, and then an effector cell at an appropriate ratio to the target cell is added for incubation. ADCC activity can be measured by collecting the supernatant after the incubation and counting the radioactivity in the supernatant.
Further, the CDC activity can be measured by mixing the above-mentioned labeled target cells with the anti-DHCR24 antibody, then adding complement and incubating, and counting the radioactivity in the supernatant after culturing.
The Fc portion is usually required for antibodies to exert cytotoxic activity. Therefore, when the anticancer agent of the present invention utilizes the cytotoxic activity of the antibody, the anti-DHCR24 antibody used in the present invention preferably contains an FC portion.
By contacting an anti-DHCR24 antibody with a cancer cell expressing DHCR24, particularly a liver cancer cell, it is possible to cause a cytotoxicity to the cancer cell and further to kill the cancer cell. Is. Furthermore, by using an anti-DHCR24 antibody, it is possible to suppress the growth of cancer cells expressing DHCR24. Therefore, the present invention provides a method of killing a cancer cell expressing DHCR24, a method of causing cytotoxicity to the cancer cell, or a method of suppressing the growth of the cancer cell, using an anti-DHCR24 antibody. provide.
6. Expression and production of recombinant or modified antibodies
The antibody gene constructed as described above can be expressed and obtained by a known method. In the case of mammalian cells, a commonly used useful promoter, an antibody gene to be expressed, and a poly A signal can be functionally linked downstream of the 3'side of the gene for expression. For example, the promoter/enhancer can include human cytomegalovirus immediate early promoter/enhancer.
In addition, other promoters/enhancers that can be used for antibody expression in the present invention include viral promoters/enhancers such as retrovirus, polyoma virus, adenovirus, simian virus 40 (SV40), or human elongation factor 1α (HEF1α). ) And other mammalian cell-derived promoters/enhancers.
When the SV40 promoter/enhancer is used, the method of Mulligan et al. (Nature, 277, 108, 1979), and when the HEF1α promoter/enhancer is used, the method of Mizushima et al. (Nucleic Acids Res., 18, 5322, 1990). Thus, gene expression can be easily performed.
In the case of Escherichia coli, a commonly used useful promoter, a signal sequence for antibody secretion and an antibody gene to be expressed can be functionally linked to express the gene. Examples of the promoter include lacZ promoter and araB promoter. When using the lacZ promoter, the method of Ward et al. (Nature, 341, 544-546, 1998; FASEB J., 6, 2422-2427, 1992), or when using the araB promoter, the method of Better et al. (Science). , 240, 1041-1043, 1988).
As a signal sequence for antibody secretion, a pelB signal sequence (Lei, SP et al J. Bacteriol., 169, 4379, 1987) may be used when it is produced in the periplasm of Escherichia coli. Then, after separating the antibody produced in the periplasm, the structure of the antibody is appropriately refolded and used.
As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. Furthermore, since the gene copy number is amplified in the host cell system, the expression vector serves as a selectable marker. An aminoglycoside transferase (APH) gene, a thymidine kinase (TK) gene, an Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc. can be included.
Any expression system, such as eukaryotic or prokaryotic cell lines, can be used for the production of the antibodies used in the present invention. Examples of eukaryotic cells include animal cells such as established mammalian cell lines, insect cell lines, filamentous fungal cells and yeast cells, and examples of prokaryotic cells include bacterial cells such as Escherichia coli cells.
Preferably, the antibody used in the present invention is expressed in mammalian cells such as CHO, COS, myeloma, BHK, Vero, HeLa cells.
Next, the transformed host cell is cultured in vitro or in vivo to produce the desired antibody. Culturing of host cells is performed according to a known method. For example, DMEM, MEM, RPMI 1640, IMDM can be used as the culture medium, and serum supplement such as fetal calf serum (FCS) can also be used together.
7. Separation and purification of antibody
The antibody expressed and produced as described above can be isolated from cells or host animals and purified to homogeneity. The antibody used in the present invention can be separated and purified using an affinity column. For example, as a column using a protein A column, Hyper D, POROS, Sepharose F. F. (Pharmacia) and the like. In addition, the separation and purification methods used for ordinary proteins may be used without any limitation. For example, the antibody can be separated and purified by appropriately selecting and combining a chromatography column other than the affinity column, a filter, ultrafiltration, salting out, dialysis, etc. (Antibodies A Laboratory Manual. Ed Harlow, David Lane). , Cold Spring Harbor Laboratory, 1988).
8. Confirmation of antibody activity
Known methods can be used to measure the antigen-binding activity of the antibody (Antibodies A Laboratory Manual, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988).
As a method of measuring the antigen-binding activity of the anti-DHCR24 antibody used in the present invention, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked immunosorbent assay), RIA (radio-immunoassay) or fluorescent antibody method is used. be able to. For example, when the enzyme immunoassay is used, a sample containing anti-DHCR24 antibody, for example, a culture supernatant of anti-DHCR24 antibody-producing cells or a purified antibody is added to a plate coated with DHCR24. To evaluate the antigen-binding activity by adding a secondary antibody labeled with an enzyme such as alkaline phosphatase, incubating the plate, washing, and then adding an enzyme substrate such as p-nitrophenyl phosphate and measuring the absorbance. You can
9. Administration method and formulation
When the antibody of the present invention is used as an anticancer agent, the effective dose can be selected within the range of 0.001 to 1,000 mg per 1 kg of body weight. Alternatively, a dose of 0.01-100,000 mg/body per patient can be selected. However, the anti-cancer agent containing the anti-DHCR24 antibody of the present invention is not limited to these doses.
The anticancer agent of the present invention can be administered before or after the clinical symptoms of the disease occur.
The anticancer agent of the present invention can be administered 1 to 3 times a day for 1 to 7 days a week.
The anticancer agent of the present invention is usually administered parenterally, for example, by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), but is not particularly limited, and is transdermal or transmucosal. , Nasal, pulmonary, orally.
However, the anticancer agent of the present invention is not limited to the above dose and administration method.
The anticancer agent containing the anti-DHCR24 antibody of the present invention as an active ingredient can be formulated according to a conventional method (Remington's Pharmaceutical Sciences, latest edition, Mark Publishing Company, Easton, USA), and is pharmaceutically acceptable. The carrier and the additive to be used may be included together.
Examples of such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran. , Sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol , Sorbitol, lactose, and surfactants acceptable as pharmaceutical additives.
The actual additives are selected from the above alone or in an appropriate combination according to the dosage form of the anticancer agent of the present invention, but are not of course limited thereto. For example, when used as an injectable preparation, the purified anti-DHCR24 antibody is dissolved in a solvent such as physiological saline, buffer solution, glucose solution and the like, and an anti-adsorption agent such as Tween 80, Tween 20, gelatin, human serum albumin And the like can be used. Alternatively, it may be lyophilized to obtain a dosage form that is reconstituted before use, and as an excipient for lyophilization, for example, mannitol, sugar alcohol such as glucose, or sugar is used. be able to.
10. Cancer diagnosis
The present invention relates to a method for diagnosing cancer by detecting DHCR24.
The DHCR24 gene may be detected by detecting the DHCR24 gene (for example, mRNA) or the DHCR24 protein.
The method for diagnosing cancer of the present invention is particularly useful for diagnosing liver cancer.
In the present invention, detection includes quantitative or non-quantitative detection. For example, non-quantitative detection simply measures whether or not DHCR24 protein exists, whether DHCR24 protein exists in a certain amount or more. Examples of the quantitative detection include measurement of whether or not DHCR24 protein is compared with other samples (eg, control sample), and quantitative detection includes measurement of DHCR24 protein expression level. it can.
The test sample is not particularly limited as long as it is a sample that may contain the DHCR24 protein or the DHCR24 gene, and specific examples of the test sample include, for example, patients who require cancer diagnosis. Alternatively, cells (hepatocytes, etc.) collected from a healthy person, a sample obtained by disrupting the cells, and the like can be mentioned.
When the DHCR24 protein is detected, the DHCR24 protein may be detected by any method, but for example, it can be detected by an immunological method using the anti-DHCR24 antibody of the present invention. Examples of the immunological method include radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescent immunoassay, immunoprecipitation method, immunoturbidimetric method, western blot, immunostaining, immunodiffusion method and the like.
Western blotting is a method of detecting proteins separated by electrophoresis using an antigen-antibody reaction. The DHCR24 protein is solubilized with a denaturing agent such as urea, SDS, 2-mercaptoethanol or a reducing agent, and separated on an SDS polyacrylamide gel. The separated bands are electrophoretically transferred to a solid support such as a nitrocellulose membrane and reacted with an antigen-specific antibody. Antibodies bound to peroxidase, alkaline phosphatase, etc. ¹²⁵ Detection is performed using I-labeled protein A or the like.
Further, as another detection method using the anti-DHCR24 antibody, for example, the anti-DHCR24 antibody is immobilized on a support, a test sample is added thereto, incubation is performed, and the anti-DHCR24 antibody and DHCR24 are bound and then washed. Then, a method of detecting DHCR24 in a test sample by detecting DHCR24 bound to a support through an anti-DHCR24 antibody can be mentioned. Examples of the support include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resin, polystyrene resin, polyacrylamide resin, nylon resin and polycarbonate resin, and insoluble supports such as glass. .. These supports can be used in the form of beads or plates. In the case of beads, a column packed with these can be used. In the case of a plate, a multiwell plate (96-well multiwell plate or the like), a biosensor chip, or the like can be used. The anti-DHCR24 antibody and the support can be bound by a commonly used method such as chemical binding or physical adsorption. All of these supports may be commercially available ones.
The binding between the anti-DHCR24 antibody and DHCR24 is usually performed in a buffer. As the buffer solution, for example, a phosphate buffer solution, a Tris buffer solution, a citrate buffer solution, a borate buffer solution, a carbonate buffer solution, or the like is used. As the incubation conditions, the conditions often used, for example, incubation at 4° C. to room temperature for 1 to 24 hours are performed. The washing after the incubation may be any as long as it does not prevent the binding between the DHCR24 and the anti-DHCR24 antibody, and for example, a buffer solution containing a surfactant such as Tween 20 is used.
In the DHCR24 detection method of the present invention, a control sample may be installed in addition to the test sample for which DHCR24 is desired to be detected. The control sample includes a negative control sample containing no DHCR24 and a positive control sample containing DHCR24. In this case, it is possible to detect DHCR24 in the test sample by comparing the result obtained with the negative control sample containing no DHCR24 with the result obtained with the positive control sample containing DHCR24. In addition, a series of control samples with varying concentrations were prepared, the detection results for each control sample were obtained as numerical values, a standard curve was created, and the standard curve was constructed from the numerical values of the test sample. It is also possible to quantitatively detect DHCR24 contained in the sample.
A preferred embodiment of detecting DHCR24 bound to a support through an anti-DHCR24 antibody is a method using an anti-DHCR24 antibody labeled with a labeling substance.
For example, a test sample is brought into contact with an anti-DHCR24 antibody immobilized on a support, and after washing, detection is performed using a labeled antibody that specifically recognizes DHCR24.
At this time, the anti-DHCR24 antibody immobilized on the support and the anti-DHCR24 antibody labeled with a labeling substance may recognize the same epitope of the DHCR24 molecule, but preferably recognize different epitopes.
Labeling of the anti-DHCR24 antibody can be carried out by a generally known method. As the labeling substance, labeling substances known to those skilled in the art such as fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, and radioactive substances can be used, and specific examples include radioisotopes ( ³² P, ¹⁴ C, ¹²⁵ I, ^Three H, ¹³¹ I, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, microperoxidase, and biotin. You can When biotin is used as the labeling substance, it is preferable to further add avidin to which an enzyme such as alkaline phosphatase is bound after adding the biotin-labeled antibody. A known method such as a glutaraldehyde method, a maleimide method, a pyridyl disulfide method, or a periodic acid method can be used for binding the labeling substance and the anti-DHCR24 antibody.
Specifically, a solution containing the anti-DHCR24 antibody is added to a support such as a plate to fix the anti-DHCR24 antibody. After washing the plate, it is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add test sample to plate. After incubation, wash and add labeled anti-DHC24 antibody. After a suitable incubation, the plate is washed and the labeled anti-DHCR24 antibody remaining on the plate is detected. The detection can be performed by a method known to those skilled in the art. For example, in the case of labeling with a radioactive substance, it can be detected by liquid scintillation or RIA method. In the case of labeling with an enzyme, a substrate can be added, and an enzymatic change of the substrate, for example, color development can be detected by an absorptiometer. Specific examples of the substrate include 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,2-phenylenediamine (ortho-phenylenediamine), 3,3′. , 5,5′-tetramethylbenzidine (TME) and the like. In the case of a fluorescent substance, it can be detected by a fluorometer.
Another embodiment of the DHCR24 detection method of the present invention is a method using an anti-DHCR24 antibody labeled with biotin and avidin.
Specifically, a solution containing the anti-DHCR24 antibody is added to a support such as a plate to fix the anti-DHCR24 antibody. After washing the plate, it is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add test sample to plate. After incubation, wash and add biotin labeled anti-DHCR24 antibody. After proper incubation, the plates are washed and avidin coupled with enzymes such as alkaline phosphatase, peroxidase, etc. is added. After the incubation, the plate is washed, a substrate corresponding to the enzyme bound to avidin is added, and DHCR24 is detected using the enzymatic change of the substrate as an index.
Another aspect of the method for detecting DHCR24 of the present invention is a method using a primary antibody that specifically recognizes DHCR24 and a secondary antibody that specifically recognizes the primary antibody.
For example, a test sample is brought into contact with an anti-DHCR24 antibody immobilized on a support, incubated, washed, and then bound DHCR24 specifically recognizes the primary anti-DHCR24 antibody and the primary antibody. It is detected by the secondary antibody. In this case, the secondary antibody is preferably labeled with a labeling substance.
Specifically, a solution containing the anti-DHCR24 antibody is added to a support such as a plate to fix the anti-DHCR24 antibody. After washing the plate, it is blocked with, for example, BSA to prevent non-specific binding of proteins. Wash again and add test sample to plate. After incubation, wash and add primary anti-DHCR24 antibody. After appropriate incubation, the plate is washed and then a secondary antibody that specifically recognizes the primary antibody is added. After appropriate incubation, wash to detect secondary antibody left on the plate. The detection of the secondary antibody can be performed by the method described above.
As another aspect of the DHCR24 detection method of the present invention, a detection method utilizing an agglutination reaction can be mentioned. In the method, DHCR24 can be detected using a carrier sensitized with an anti-DHCR24 antibody. As a carrier for sensitizing an antibody, any carrier may be used as long as it is insoluble, does not cause a non-specific reaction, and is stable. For example, latex particles, bentonite, collodion, kaolin, fixed sheep red blood cells and the like can be used, but latex particles are preferably used. As the latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyltoluene latex particles and the like can be used, but polystyrene latex particles are preferably used. After mixing the sensitized particles with the sample and stirring for a certain period of time, the higher the concentration of the anti-DHCR24 antibody in the sample, the greater the degree of particle aggregation. You can Further, it is possible to detect the turbidity due to aggregation by measuring with a spectrophotometer or the like.
As another embodiment of the DHCR24 detection method of the present invention, for example, a method using a biosensor utilizing the surface plasmon resonance phenomenon can be mentioned. A biosensor utilizing the surface plasmon resonance phenomenon can observe a protein-protein interaction in real time as a surface plasmon resonance signal using a trace amount of protein and without labeling. For example, it is possible to detect the binding between DHCR24 and anti-DHCR24 antibody by using a biosensor such as BIAcore (manufactured by Pharmacia). Specifically, a test sample is brought into contact with a sensor chip on which anti-DHCR24 antibody is immobilized, and DHCR24 binding to the anti-DHCR24 antibody can be detected as a change in resonance signal.
The detection method of the present invention can be automated using various automatic inspection devices, and it is also possible to inspect a large number of samples at once.
In addition, the test cells are contacted with an anti-DHCR24 antibody labeled with a fluorescent substance or the like to remove the anti-DHCR24 antibody that is not bound to DHCR24, and then the labeling substance such as a fluorescent substance is detected to allow the cells to detect the DHCR24. It is also possible to detect whether or not it is expressed. Detection of labeling substances such as fluorescence can be performed by a method known to those skilled in the art.
Furthermore, in the present invention, the DHCR24 protein may be detected using a method that does not use an anti-DHCR24 antibody, such as SDS polyacrylamide gel electrophoresis.
In the present invention, not only DHCR24 protein detection but also DHCR24 gene detection may be performed to diagnose cancer.
For example, as one aspect of the diagnostic method of the present invention, first, an RNA sample is prepared from a subject. Then, the amount of RNA encoding the DHCR24 protein contained in the RNA sample is measured. The measured amount of RNA is then compared to the control.
In another embodiment, first, a cDNA sample is prepared from a subject. Then, the amount of cDNA encoding the DHCR24 protein contained in the cDNA sample is measured. The measured amount of cDNA is then compared to a control.
Examples of such methods include methods well known to those skilled in the art, such as Northern blotting method, RT-PCR method, and DNA array method.
For example, in the DNA array method, a cDNA sample is prepared using RNA prepared from a subject as a template, and is brought into contact with a substrate on which an oligonucleotide or a polynucleotide capable of hybridizing with the DHCR24 gene is fixed, and the cDNA sample and the The expression level of the gene contained in the cDNA sample is measured by detecting the intensity of hybridization with the nucleotide probe immobilized on the substrate. Then, the expression level of the measured gene is compared with the control. Hybridization means that DNA or its corresponding RNA binds to another DNA or RNA molecule in solution or on a solid support by hydrogen bonding interactions. The strength of such interaction can be evaluated by changing the stringency of the hybridization conditions. Depending on the desired specificity and selectivity, different stringency hybridization conditions can be used, and stringency can be adjusted by varying the salt concentration or denaturant concentration. Such methods of controlling stringency are well known in the art, and are described, for example, in “Molecular Cloning: A Laboratory Manual”, 2nd Edition, Cold Spring Harbor Laboratory, Sambrook, Fritsch & Maniatis, ed. , 1989.
An example of stringent hybridization conditions is 50% formamide, 5×SSC, 50 mM NaH. _Two PO _Four , PH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and hybridization in 5x Denhardt's solution at 42°C overnight; 2xSSC, 45% in 0.1% SDS. Washing with 0.2×SSC, 0.1% SDS at 45° C.
The nucleotide probe used in the present invention can be produced by a method known to those skilled in the art. For example, it can be synthesized by a commercially available DNA synthesizer (for example, 394 synthesizer, manufactured by Applied Biosystems) using a protocol known in the art. Alternatively, it can be produced by a PCR amplification technique well known in the art using a combination of an appropriate template and a primer based on the sequence information of DHCR24.
A nucleotide probe usually has a contiguous base sequence of at least 12 bases, 20, 30, 50 or 100 bases or more of a DHCR24 base sequence or a base sequence complementary thereto, and is specific to a specific region of the DHCR24 gene. Selected to hybridize with each other.
The probe may be immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastic, agarose, sepharose, polyacrylamide, latex beads, nitrocellulose, and the like. Techniques for attaching probes to such solid supports are well known in the art. The probe can be visualized by labeling using a standard labeling technique such as radioactive labeling, enzyme labeling (horseradish peroxidase, alkaline phosphatase), fluorescent labeling, biotin-avidin labeling, chemiluminescence and the like.
Preparation of a cDNA sample from a subject can be performed by a method well known to those skilled in the art. In a preferred embodiment of preparing a cDNA sample, first, total RNA is extracted from a cell or tissue (for example, liver) of a subject. Extraction of total RNA can be performed by a method known to those skilled in the art, for example, as follows. For extraction of total RNA, existing methods and kits can be used, as long as high-purity total RNA can be prepared. For example, pretreatment is performed using RNA later manufactured by Ambion, and then total RNA is extracted using Isogen manufactured by Nippon Gene. The specific method may follow those attached protocols.
Then, using the extracted total RNA as a template, cDNA is synthesized using reverse transcriptase to prepare a cDNA sample. Synthesis of cDNA from total RNA can be performed by a method well known to those skilled in the art. If necessary, the prepared cDNA sample is labeled for detection. The labeling substance is not particularly limited as long as it can be detected, and examples thereof include a fluorescent substance and a radioactive element. The labeling can be carried out by a method generally performed by those skilled in the art (L Luo et al., Gene expression profiles of laser-captured adjuvant neural subtypes. Nat Med. 1999, 117-122).
The detection of the intensity of hybridization between the nucleotide probe and the cDNA can be appropriately performed by those skilled in the art depending on the type of the substance labeling the cDNA sample. For example, when the cDNA is labeled with a fluorescent substance, it can be detected by reading a fluorescent signal with a scanner.
In the above method, when the expression level of the gene or protein is significantly increased as compared with the control, it is determined that the subject has or is likely to develop cancer. .
The present invention also provides a test agent for use in a method for testing cancer. Examples of such a test agent include a test agent containing a nucleotide probe capable of hybridizing with DHCR24 (including a substrate on which the nucleotide probe is immobilized), a test agent containing the antibody of the present invention, and the like. The above-mentioned antibody is not particularly limited as long as it can be used for the test. The probe or antibody is labeled if necessary.
In the above test agents, in addition to the nucleotide probe and the antibody which are active ingredients, for example, sterile water, physiological saline, vegetable oil, surfactants, lipids, solubilizing agents, buffers, protein stabilizers (BSA, gelatin, etc.) ), a preservative and the like may be mixed if necessary. Further, it may appropriately contain a blocking solution, a reaction solution, a reaction termination solution, a reagent for treating the sample, and the like.
The expression of DHCR24 is enhanced in specific human cancer tissues. Therefore, when inhibiting the expression of the DHCR24 gene for therapeutic purposes, antisense oligonucleotides, ribozymes, siRNAs and the like can be used.
When using antisense, ribozyme, siRNA, etc., the polynucleotide or oligonucleotide preferably has a chain length of at least 12 nucleotides or more, more preferably 12 to 50 nucleotides, particularly preferably 12 to 25 nucleotides. Is. These polynucleotides or oligonucleotides are mutants in which one or several bases have been deleted, substituted or added from the complementary sequence of the target site, as long as they have the desired antisense, ribozyme or siRNA activity. Good. Such variants preferably have a nucleotide sequence with at least 70%, preferably 90% or more, more preferably 95% or more identity with the complementary sequence of the target site.
The identity of the nucleotide sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993) by Karlin and Altschul. Programs called BLASTN and BLASTX have been developed based on this algorithm (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When a base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, score=100 and word length=12. When using BLAST and Gapped BLAST programs, the default parameters for each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm, nih.gov.).
Alternatively, such polynucleotides or oligonucleotides can hybridize under stringent conditions to DNA or RNA having the gene sequence encoding DHCR24 or its complementary sequence.
Methods of controlling gene expression using antisense, ribozyme and siRNA technologies are well known in the art. For example, antisense, ribozyme, siRNA may be administered with a suitable carrier, or a vector encoding antisense, ribozyme or siRNA may be administered to induce their expression in vivo.
The term "ribozyme" refers to a nucleic acid molecule that has catalytic activity for cleaving mRNA. Ribozymes generally exhibit endonuclease, ligase or polymerase activity. Various types of trans-acting ribozymes are known, such as hammerhead and hairpin type ribozymes.
“Antisense” refers to a nucleic acid molecule or derivative thereof that specifically hybridizes to genomic DNA and/or mRNA and thereby inhibits the expression of the protein by inhibiting its transcription and/or translation. Binding may be by general base pair complementarity or, for example in the case of binding to a DNA duplex, a specific interaction in the major groove of the double helix. The target site of the antisense nucleic acid is preferably the 5'end of the mRNA, for example, the 5'untranslated sequence up to and including the AUG start codon, but the 3'untranslated sequence of the mRNA or the sequence of the coding region can also be used for translation of the mRNA. It is known to be effective in inhibition.
By "siRNA" is meant a double-stranded nucleic acid capable of RNA interference (RNAi) (eg, Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494). -498). siRNA decomposes mRNA in a sequence-specific manner, which can suppress gene expression. siRNAs are typically double stranded RNAs 20-25 base pairs in length that contain sequences complementary to the target sequence. siRNA molecules may include chemically modified nucleotides and non-nucleotides.

これらの細胞株はＡｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）から購入した。ＨｕＨ−７、２９３、ＨｅｐＧ２細胞はＤｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ ｍｅｄｉｕｍ（ＤＭＥＭ；ＳＩＧＭＡ）に１０％ＦＣＳ（ＳＩＧＭＡ）を加えて培養した。ＷＲＬ６８細胞は１０％ＦＣＳ−ＤＭＥＭに１ｍＭ Ｎａ Ｐｙｒｕｖａｔｅ（ＧＩＢＣＯ−ＢＲＬ）、０．１ｍＭ Ｎｏｎ ｅｓｓｅｎｔｉａｌ ａｍｉｎｏ ａｃｉｄ（ＧＩＢＣＯ−ＢＲＬ）を加えて培養した。ＮＩＨ３Ｔ３細胞は、ＤＭＥＭに５％ｎｅｏｎａｔａｌ ｃａｌｆ ｓｅｒｕｍ（ＳＩＧＭＡ）を加えて培養した。
ハイブリドーマやミエローマのＰＡＩ細胞は、１０％ＦＣＳを加えたＲＰＭＩ１６４０（ＳＩＧＭＡ）で培養した。
全長ＨＣＶ ｃＤＮＡクローン（ＨＣＲ６−Ｒｚ）をＣｒｅ／ｌｏｘＰのスイッチング発現システムの下流に組み込んだユニット（図１Ａ、上段）を、ｎｅｏ遺伝子を利用してＨｅｐＧ２細胞に組み込み、細胞株を樹立した（Ｒｚ−Ｈｅｐ２−１８細胞）。さらに、これにＭｅｒ−Ｃｒｅ−Ｍｅｒシステム（図１Ａ、下段）を組み込んだ細胞株も樹立した（Ｒｚ−ＨｅｐＭ２−１８−６細胞）。Ｒｚ−ＨｅｐＭ２−１８−６細胞株をタモキシフェン（Ｔｍ）で処理するとＣｒｅ酵素が産生され、全長ＨＣＶがスイッチング発現する（図１Ｂ）。
１．２ 患者材料
東京都立駒込病院・肝臓内科を訪れた１５人の肝癌患者検体を用いた（表２）。癌部、非癌部の検体は外科的に切除した組織から得た。これらの患者から本研究利用へのインフォームドコンセントは得ている。
１．３ ＥＬＩＳＡ系
（１）生細胞ＥＬＩＳＡ系
丸底の９６ウェルプレート（Ｎｕｎｃ）をＰｏｌｙ Ｌ ｌｉｓｉｎｅ １０μｇ／ｍｌ−ＰＢＳ（−）で室温３０分インキュベートした。ＰＢＳ（−）で３回洗浄し、細胞をウェル当たり１〜５×１０^５でまき、１５００ｒｐｍ、４℃、１分遠心後、上清を吸引除去し、細胞ペレットをボルテックスミキサーで撹拌した。細胞を遠心しながら（１５００ｒｐｍ、１分、４℃）冷ＰＢＳ（−）で４回洗浄した。細胞を１％ＢＳＡ−ＰＢＳで室温１〜２時間反応させた。１％ＢＳＡ−ＰＢＳで適当に希釈した抗体液を１００μｌずつ加え、室温で１５分間に１回緩やかに撹拌しながら２時間反応させた。細胞を同様に２回洗浄後、ＨＲＰ標識２次抗体Ｆ（ａｂ’）_２−ｅｎｚｙｍｅ（Ｂｅｃｔｏｎ−Ｄｉｃｋｉｎｓｏｎ）（１００ｎｇ／ｍｌ）１００μｌを加えた。４℃で１時間半、１５分間隔で振盪しながら反応させ、細胞を３回洗浄して基質を加え、吸光度をＯＤ_６５５で測定した。
（２）細胞溶解物ＥＬＩＳＡ系
１０^６個の細胞をライセートバッファー（１％ＳＤＳ／０．５％ＮＰ−４０／０．１５Ｍ ＮａＣｌ／１０ｍＭ Ｔｒｉｓ ｐＨ７．４／５ｍＭ ＥＤＴＡ／１ｍＭ ＤＴＴ）１００μｌで溶解し、これを抗原として（２μｇ／ウェル）常法に従いＥＬＩＳＡを行った。
１．４ モノクロナール抗体の作製
Ｒｚ−ＨｅｐＭ２−１８−６細胞をタモキシフェン処理してＨＣＶを発現させた後、４８日以上継代した細胞Ｒｚ−ＨｅｐＭ２−１８−６ ４８ｄ（５×１０^６個）を、ＢＡＬＢ／ｃマウスの腹腔内に８回以上免疫し、脾臓を取りだし、ＰＥＧ １５００（Ｂｏｈｒｉｎｇｅｒ）を用いてＰＡＩミエローマ細胞と融合させた（Ｋｏｈｌｅｒ ａｎｄ Ｍｉｌｓｔｅｉｎ，１９７５）。ハイブリドーマ細胞はＨＡＴ（ＧＩＢＣＯ−ＢＲＬ）で選択し、培養上清を生細胞ＥＬＩＳＡ系で解析した。
１．５ イムノブロット解析
１０^６個の細胞を１００μｌのＲＩＰＡ溶液（１％ＳＤＳ／０．５％ＮＰ−４０／０．１５Ｍ ＮａＣｌ／１０ｍＭ Ｔｒｉｓ ｐＨ７．４／５ｍＭ ＥＤＴＡ／１ｍＭ ＤＴＴ）に溶かし、ＳＤＳ−ＰＡＧＥを行った。ゲルはナイロン膜（Ｉｍｍｏｂｉｌｏｎ Ｐ）に転写してモノクロナール抗体とＨＲＰ標識抗マウス２次抗体（ＤＡＫＯ）を反応させた。凍結した患者組織はダンスホモジェナイザー（Ｗｈｅａｔｏｎ ｓｃｉｅｎｃｅ ｐｒｏｄｕｃｔ）を用いてＲＩＰＡに氷中で溶解した。抗アクチンポリクロナール抗体（Ｃ−１１、Ｓａｎｔａ−Ｃｒｕｚ）を内部標準として使用した。
１．６ 蛍光抗体法（ＩＦＡ）
細胞はアセトン−メタノール（１：１）溶液を用いて−２０℃で１０分間固定するか、ＰＢＳでバッファライズしたホルマリン（ＷＡＫＯ）で固定した。これらを抗体と室温で６０分間反応させ、ＰＢＳで洗浄後、ＦＩＴＣ標識抗マウス２次抗体（ＤＡＫＯ）と室温で３０分間反応させた。
１．７ 抗原精製とＭＡＬＤＩ−ＴＯＦＦ ＭＳによる抗原決定
モノクロナール抗体クローン２−１５２ ０．７ｍｇをＨｉ ＴｒａｐＮＨＳ−ａｃｔｉｖａｔｅｄ ＨＰカラム（ファルマシア）に結合させた抗体カラムを作製し、１０^７個のＨｕＨ−７細胞の溶解液（ライセートバッファー：１％Ｔｒｉｔｏｎ Ｘ１００／２０ｍＭ Ｈｅｐｅｓ−ＮａＯＨ ｐＨ７．５／１ｍＭ ＥＤＴＡ／１ｍＭ ＤＴＴ／１ｍＭ ＤＦＰ）１ｍｌを抗体カラムに流速１滴／２秒で流し、室温で３０分通し続けた。ライセートバッファーで未吸着画分を洗浄し、結合画分を溶出バッファー（０．２Ｍグリシン−塩酸緩衝液、ｐＨ３．０）５ｍｌで溶出した。ピーク画分を凍結乾燥機にかけて濃縮し、さらにＳＤＳ−ＰＡＧＥで泳動後クマシー染色を行い、染色されたバンドを切り出し、ＭＡＬＤＩ−ＴＯＦＦＭＳによってアミノ酸配列を決定した。
１．８ ＤＨＣＲ２４タンパク質の発現
ＤＨＣＲ２４の遺伝子配列（Ｇｅｎｂａｎｋ ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ ＮＭ＿０１４７６２）に基づいてプライマー（センス；５’−ＧＴＴＣＴＣＧＡＧＣＡＧＴＧＡＣＡＧＧＡＧＧＣＧＡＡＣ−３’、アンチセンス；５’ −ＧＴＴＣＴＣＧＡＧＴＣＣＡＧＧＣＧＧＧＣＴＣＣＡＧＣＴＣＡ−３’）を合成し、ＨｕＨ−７細胞のｍＲＮＡからｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｂｅｄ ＰＣＲ法によってｃＤＮＡを得た。これをｐＧＥＭ−Ｔベクター（プロメガ）に組み込み、ＴＮＴ ｃｏｕｐｌｅｄ ｒｅｔｉｃｕｌｏｃｙｔｅ ｌｙｓａｔｅ ｓｙｓｔｅｍｓ（プロメガ）、^３５Ｓメチオニン（ＮＥＮ）を用いてＤＨＣＲ２４タンパク質を合成した。また、^３５Ｓメチオニンを加えずに合成した細胞溶解物を抗原とし、ウェスタンブロッティング法によりモノクロナール抗体クローン２−１５２との反応を検討した。さらに、ＤＨＣＲ２４遺伝子をｐＣＡＧ−ＰＵＲＯベクターに組み込み、ＷＲＬ６８細胞にリン酸カルシウム法か、Ｌｉｐｏｆｅｃｔｉｎ ２０００でトランスフェクション後、ＤＨＣＲ２４タンパク質の発現をウェスタンブロッティング法で解析した。
２． 結果
Ｒｚ−Ｈｅｐ細胞を用いた生細胞ＥＬＩＳＡ系の開発とモノクローナル抗体の 樹立
発明者らは、先に全長ＨＣＶ遺伝子：ＨＣＲ６−Ｒｚを発現するスイッチング発現系を確立し（図１Ａ）、これをＨｅｐＧ２細胞に組み込んで細胞株を樹立している（Ｒｚ−Ｈｅｐ２−１８−６細胞；Ｋｏｈａｒａ ｅｔ ａｌ．，ｓｕｂｍｉｔｔｅｄ）。ＨＣＶ発現細胞にがんに関連するような細胞表面抗原が表出するかを解析するため、生細胞の表面抗原を認識するようなＥＬＩＳＡ系を確立した。この系は免疫したマウスの血清に特異的に反応することが確認された（図２）。同時に、タモキシフェン（Ｔｍ）処理によりＨＣＶを発現させ、その後４８日以上継代することによって腫瘍原性が亢進したＲｚ−Ｈｅｐ細胞（Ｒｚ−ＨｅｐＭ２−１８−６ ４８ｄ細胞；Ｋｏｈａｒａ ｅｔ ａｌ．，Ｊｏｕｒｎａｌ ｏｆ Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ，ｉｎ ｐｒｅｓｓ）をマウスに免疫し、モノクロナール抗体を樹立した。免疫したマウスの血清は、３２，０００倍まで細胞に反応した（図２）。
次に、Ｒｚ−ＨｅｐＭ２−１８−６ ４８ｄ細胞を８回以上マウスに免疫し、脾臓細胞をミエローマ細胞と融合してハイブリドーマを作製した。１，０００クローン以上から上清を回収し、生細胞ＥＬＩＳＡ系で解析した。このうち、Ｒｚ−ＨｅｐＭ２−１８−６ ４８ｄ細胞への反応性とＲｚ−ＨｅｐＭ２−１８−６未発現細胞への反応性が異なるクローンを抽出し、さらにその反応性を蛍光抗体法（ＦＡ）とＷＢ法で解析した。その結果、Ｒｚ−ＨｅｐＭ２−１８−６ ４８ｄ細胞に反応する３つのクローンを得た。１つはクローン２−１５２で５５ｋＤａの分子（Ｐ５５）を認識し、クローン２−２４３は７０ｋＤａの分子（Ｐ７０）、クローン２−４３３は３０ｋＤａの分子（Ｐ３０）を認識した（図３）。これら分子の発現レベルを各種細胞株：ヒト肝芽腫細胞ＨｅｐＧ２；ヒト肝癌細胞ＨｕＨ−７；ヒト胎児肝細胞ＷＲＬ６８；ヒト胎児腎細胞２９３細胞；及びマウス線維芽細胞ＮＩＨ３Ｔ３（図３）で解析した。これら細胞の腫瘍原性を表１に示す。Ｐ５５、Ｐ７０、Ｐ３０はＨｕＨ−７細胞での発現が最も高かった。Ｐ５５、Ｐ３０のこれら細胞群での発現は腫瘍原性に応じて変化していた。腫瘍原性の低いＮＩＨ３Ｔ３細胞におけるＰ５５、Ｐ７０、Ｐ３０の発現レベルは低かった。
以上より、肝臓由来細胞株では腫瘍原性が高い程、各分子の発現が上昇している傾向がみられた。また上皮細胞（２９３細胞）やＮＩＨ３Ｔ３細胞では発現が低かった。
さらに、これら分子の発現レベルが細胞の腫瘍原性と関連する可能性が考えられたので、肝癌患者（ＨＣＣ）の癌部、非癌部における発現を解析した（図４）。５例がＨＣＶ陽性（ＨＣＶ（＋））患者群（Ｎｏ．１、２、３、９及び１０）、５例がＨＣＶ、ＨＢＶ感染陰性（ＮＢＮＣ）患者群（Ｎｏ．４、５、６、７及び８）、５例がＨＢＶ陽性（ＨＢＶ（＋））患者群（Ｎｏ．１１、１２、１３、１４及び１５）であった（表２）。

これら患者の凍結した各組織の細胞溶解物をＷＢ法で解析した結果、Ｐ５５の発現は、ＨＣＶ（＋）患者５例全例の癌部で上昇していた（図４）。ＨＢＶ（＋）では５例中３例が、ＮＢＮＣ患者群では５例中２例が癌部での発現が上昇していた。Ｐ７０の発現は、一部の患者群（Ｎｏ．１、３、６、７、１０、１１、１２及び１４）の癌部では上昇していたが、他の患者群では癌部での発現上昇は見られなかった（Ｎｏ．２、４、５、９、１３及び１５）。Ｐ３０の発現は、患者Ｎｏ．１、２、３、８、１０、１１、１２及び１４の癌部で発現上昇が見られたが、患者Ｎｏ．５、６、９、１３及び１５では見られなかった。このように、Ｐ５５はＨＣＶ（＋）患者群において最も高い割合で発現上昇が見られた（表３）。

以上より、モノクローナル抗体クローン２−２４３や２−４３３の認識する７０ｋＤａ、３０ｋＤａ分子は、２０〜６０％の患者で癌部での発現上昇が見られた。これに対しクローン２−１５２の認識する５５ｋＤａ分子はＨＣＶ（＋）の全ての患者で発現が上昇していた。またＨＢＶ（＋）やＮＢＮＣ肝癌患者での発現上昇率は４０〜６０％だった。
さらに、Ｐ５５に関して解析した。まず、Ｐ５５の細胞内局在を明らかにするため、蛍光抗体法を行った（図５）。細胞を１％パラフォルムアルデヒドで固定し、１％Ｔｒｉｔｏｎ Ｘ−１００で処理（Ｆｉｘ）あるいは未処理（ｎｏｎ−ｆｉｘｅｄ）後、モノクローナル抗体２−１５２と反応させた。また、小胞体のマーカーであるＰＤＩに対する抗体（Ｓｔｒｅｓｓｇｅｎ社製）とも同時に反応させた。その結果、小胞体のマーカーであるＰＤＩは細胞を固定（透過）しないと検出されないのに対し、Ｐ５５は細胞を固定（透過）しなくとも検出されるため、細胞表面膜に存在すると考えられた。
次にＨＣＶの発現と、ｐ５５の発現の関連性を検討した。
まず、ＨＣＶ遺伝子（ＨＣＲ６−Ｒｚ）の発現とｐ５５の発現に関連性があるか検討した。ＷＲＬ６８細胞やＨｅｐＧ２細胞にＨＣＶ遺伝子をトランスフェクションしたところ、ＷＲＬ６８細胞では発現は見られなかったが、ＨｅｐＧ２細胞でＰ５５の発現が上昇した（図６Ａ）。同様な結果が蛍光抗体法でも観察された（図７）。ＨＣＶ発現細胞株Ｒｚ−ＨｅｐＭ２−１８−６細胞、あるいは発現継代細胞（Ｒｚ−ＨｅｐＭ２−１８−６ ４８ｄ）でのｐ５５の発現変化も観察したが、ＨＣＶの発現に伴う発現上昇が見られた（図６Ｂ）。蛍光抗体法の観察によるとＲｚ−ＨｅｐＭ２−１８−６細胞でＨＣＶを発現しているもののうち５０％程度でｐ５５の発現が上昇していた（図８及び図９）。
そこで、ｐ５５を同定するため、モノクローナル抗体クローン２−１５２の抗体カラムを作製し、ＨｕＨ−７細胞溶解物を流してカラムに結合させた。結合分画を凍結乾燥後、ＳＤＳ−ＰＡＧＥを行いＣＢＢ染色した。染色されたバンドを切り出してアミノ酸配列を決定するため、ＭＡＬＤＩ−ＴＯＦ型質量分析計（ミリポア）でアミノ酸配列を決定した。その結果、ｐ５５はＤＨＣＲ２４と呼ばれる分子である確率が最も高いと考えられた。
次に、ＤＨＣＲ２４遺伝子を上述の方法でクローニングし、ｐＧＥＭ−Ｔ ｅａｓｙベクターのＴ７プロモーター下流に組み込んで（ｐＧＥＭ−Ｔ−ＤＨＣＲ２４）試験管内で合成した。その結果、分子量約５５ｋＤａのタンパク質が合成され（図１０Ａ）、これがクローン２−１５２抗体と反応した（図１０Ｂ）。また、ＣＡＧプロモーター下流にＤＨＣＲ２４を組み込んだｐＣＡＧ−ＤＨＣＲをＷＲＬ６８細胞にトランスフェクションしたところ、発現が見られた（図１０Ｃ）。従って、モノクロナール抗体クローン２−１５２はＤＨＣＲ２４分子を認識していると考えられた。
［実施例２］
ＣＤＣ活性の測定
標的細胞（１０^６／ｍｌ、０．１ｍｌ）に抗体（０．１ｍｌ）を加えて３０℃で３０分間インキュベーションした。次に細胞をＧＶＢバッファーで洗浄し、希釈したモルモット血清（ＳＩＧＭＡ）０．１ｍｌを加えて３７℃で６０分間インキュベーションした。細胞を０．０５％トリパンブルー０．１ｍｌで染色することにより、生存率を測定した。用いた標的細胞、抗体および補体を以下に示す。
標的細胞：ＷＲＬ６８、ＨｅｐＧ２、ＨｕＨ−７
抗体：２−１５２（１、１０μｇ／ｍｌ）
正常マウスＩｇＧ（１、１０μｇ／ｍｌ）
補体：モルモット由来（ＧＶＢバッファーで１：８に希釈）
本抗体２−１５２は、補体存在下で容量依存的かつ選択的にＨｕＨ−７細胞を障害することが明らかとなった（図１１）。
蛍光抗体法（ＩＦＡ）
Ｔｉｓｓｕｅ−Ｔｅｋ（登録商標）ＯＣＴ化合物（ＴＥＤ ＰＥＬＬＡ Ｉｎｃ．）中のＨＣＣ患者肺のがん組織及び非がん組織の凍結切片を室温で融解し、ＰＢＳ（−）で洗浄後、ＰＢＳ（−）中の１％パラホルムアルデヒドを用いて室温で１０分間固定した。組織切片を１ｍＭ ＥＤＴＡ／１％ＢＳＡ／ＰＢＳ（−）を用いて室温で１時間ブロッキングし、ＰＢＳ（−）で洗浄後、モノクローナル抗体２−１５２（５μｇ／ｍｌ）を加えて４℃で一晩反応させた。さらに、切片をＰＢＳ（−）で３回洗浄後、０．０５％Ｔｗｅｅｎ２０／ＰＢＳ（−）中の抗マウスＩｇＧ Ａｌｅｘａ ４８８（１：１０００；ヤギ抗マウスＩｇＧ（Ｆａｂ’）_２フラグメントＦｌｕｏｒ４８８、Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を加えて室温で３０分間反応させた。切片をＰＢＳ（−）で３回洗浄後、ＴＯ−ＰＲＯ−３（１０μｇ／ｍｌ；Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ）を含有するベクターシールド（Ｖｅｃｔｏｒ Ｉｎｃ．）を用いてマウントした。
本抗体２−１５２は、ＨＣＣ患者の肝臓組織のうち、非癌部よりも癌部を強く染色し、かつ癌部組織の細胞膜を染色することが明らかとなった（図１２）。[Example 1]
1. Materials and methods
1.1 Cultured cell line The properties of the cell line used in this study are shown in Table 1.

These cell lines were purchased from the American Type Culture Collection (ATCC). HuH-7, 293 and HepG2 cells were cultured by adding 10% FCS (SIGMA) to Dulbecco's modified Eagle's medium (DMEM; SIGMA). WRL68 cells were cultured by adding 1 mM Na Pyruvate (GIBCO-BRL) and 0.1 mM Non essential amino acid (GIBCO-BRL) to 10% FCS-DMEM. NIH3T3 cells were cultured by adding 5% neonatal calf serum (SIGMA) to DMEM.
Hybridoma and myeloma PAI cells were cultured in RPMI1640 (SIGMA) supplemented with 10% FCS.
A unit in which a full-length HCV cDNA clone (HCR6-Rz) was incorporated downstream of the Cre/loxP switching expression system (FIG. 1A, upper row) was incorporated into HepG2 cells using the neo gene to establish a cell line (Rz-. Hep2-18 cells). Furthermore, the cell line which integrated the Mer-Cre-Mer system (FIG. 1A, lower part) into this was also established (Rz-HepM2-18-6 cell). When the Rz-HepM2-18-6 cell line is treated with tamoxifen (Tm), Cre enzyme is produced and full-length HCV is switch-expressed (FIG. 1B).
1.2 Patient Material We used 15 liver cancer patient specimens who visited the Department of Liver Internal Medicine, Tokyo Komagome Hospital (Table 2). The cancerous and non-cancerous specimens were obtained from surgically resected tissue. Informed consent for the use of this study was obtained from these patients.
1.3 ELISA system (1) Live cell ELISA system A 96-well plate (Nunc) with a round bottom was incubated with Poly L lisine 10 µg/ml-PBS(-) at room temperature for 30 minutes. The cells were washed 3 times with PBS(-), the cells were seeded at 1 to 5 x 10 ⁵ per well, centrifuged at 1500 rpm, 4°C for 1 minute, the supernatant was removed by suction, and the cell pellet was stirred with a vortex mixer. The cells were washed 4 times with cold PBS(−) while centrifuging (1500 rpm, 1 minute, 4° C.). The cells were reacted with 1% BSA-PBS at room temperature for 1-2 hours. 100 μl of an antibody solution appropriately diluted with 1% BSA-PBS was added to each, and the mixture was allowed to react for 2 hours at room temperature with gentle stirring once every 15 minutes. After washing the cells twice in the same manner, 100 μl of HRP-labeled secondary antibody F(ab′) ₂ -enzyme (Becton-Dickinson) (100 ng/ml) was added. The reaction was carried out at 4°C for 1 and a half hours with shaking at 15-minute intervals, the cells were washed 3 times, the substrate was added, and the absorbance was measured at OD ₆₅₅ .
(2) Cell lysate ELISA system 10 ⁶ cells were lysed with 100 μl of lysate buffer (1% SDS/0.5% NP-40/0.15M NaCl/10 mM Tris pH 7.4/5 mM EDTA/1 mM DTT). Using this as an antigen (2 μg/well), an ELISA was performed according to a conventional method.
1.4 Preparation of Monoclonal Antibody Rz-HepM2-18-6 cells were treated with tamoxifen to express HCV, and then cells were passaged for 48 days or more Rz-HepM2-18-6 48d (5×10 ⁶ cells) Was immunized into the abdominal cavity of BALB/c mice 8 times or more, and the spleen was taken out and fused with PAI myeloma cells using PEG 1500 (Bohringer) (Kohler and Milstein, 1975). Hybridoma cells were selected by HAT (GIBCO-BRL), and the culture supernatant was analyzed by a live cell ELISA system.
1.5 Immunoblot analysis 10 ⁶ cells were dissolved in 100 μl of RIPA solution (1% SDS/0.5% NP-40/0.15M NaCl/10 mM Tris pH 7.4/5 mM EDTA/1 mM DTT) to obtain SDS. -Performed PAGE. The gel was transferred onto a nylon membrane (Immobilon P) and reacted with a monoclonal antibody and an HRP-labeled anti-mouse secondary antibody (DAKO). The frozen patient tissues were thawed in RIPA on ice using a dance homogenizer (Wheaton science product). Anti-actin polyclonal antibody (C-11, Santa-Cruz) was used as an internal standard.
1.6 Fluorescent antibody method (IFA)
The cells were fixed with an acetone-methanol (1:1) solution at -20°C for 10 minutes or with PBS-buffered formalin (WAKO). These were reacted with the antibody for 60 minutes at room temperature, washed with PBS, and then reacted with FITC-labeled anti-mouse secondary antibody (DAKO) for 30 minutes at room temperature.
1.7 Antigen purification and antigen determination by MALDI-TOFF MS 0.7 mg of the monoclonal antibody clone 2-152 was bound to a Hi Trap NHS-activated HP column (Pharmacia) to prepare an antibody column, and 10 ⁷ HuH-7 were prepared. 1 ml of cell lysate (lysate buffer: 1% Triton X100/20 mM Hepes-NaOH pH 7.5/1 mM EDTA/1 mM DTT/1 mM DFP) was flowed through the antibody column at a flow rate of 1 drop/2 seconds, and continued to be passed at room temperature for 30 minutes. It was The unadsorbed fraction was washed with the lysate buffer, and the bound fraction was eluted with 5 ml of the elution buffer (0.2 M glycine-hydrochloric acid buffer, pH 3.0). The peak fraction was concentrated in a freeze-dryer, further subjected to SDS-PAGE and subjected to Coomassie staining, the stained band was cut out, and the amino acid sequence was determined by MALDI-TOFFMS.
1.8 Expression of DHCR24 protein Based on the gene sequence of DHCR24 (Genbank accession number NM_014762), a primer (sense; 5'-GTTCTCCGAGCAGTGACAGGAGGCGAAC-3', antisense; CDNA was obtained from the cell mRNA by reverse transcribed PCR. This was incorporated into a pGEM-T vector (Promega), and TNT coupled reticulocyte lysate systems (Promega) and ³⁵ S methionine (NEN) were used to synthesize a DHCR24 protein. In addition, the reaction with monoclonal antibody clone 2-152 was examined by Western blotting using a cell lysate synthesized without adding ³⁵ S methionine as an antigen. Further, the DHCR24 gene was incorporated into the pCAG-PURO vector, and WRL68 cells were transfected with the calcium phosphate method or Lipofectin 2000, and then the expression of the DHCR24 protein was analyzed by the Western blotting method.
2. result
Development of live cell ELISA system using Rz-Hep cells and establishment of monoclonal antibody The inventors previously established a switching expression system for expressing the full-length HCV gene: HCR6-Rz (Fig. 1A), which was used for HepG2 cells. To establish a cell line (Rz-Hep2-18-6 cells; Kohara et al., submitted). To analyze whether cell surface antigens related to cancer are expressed in HCV expressing cells, an ELISA system that recognizes surface antigens of living cells was established. This system was confirmed to react specifically with the serum of immunized mice (Fig. 2). At the same time, Rz-Hep cells (Rz-HepM2-18-6 48d cells, whose tumorigenicity was enhanced by expressing HCV by tamoxifen (Tm) treatment and then passaged for 48 days or longer; Kohara et al., Journal of. The mouse was immunized with Biological Chemistry, in press) to establish a monoclonal antibody. The serum of immunized mice reacted with cells up to 32,000 times (Fig. 2).
Next, mice were immunized with Rz-HepM2-18-648d cells 8 times or more, and spleen cells were fused with myeloma cells to prepare hybridomas. Supernatants were collected from over 1,000 clones and analyzed by a live cell ELISA system. Of these, clones having different reactivity to Rz-HepM2-18-6 48d cells and reactivity to Rz-HepM2-18-6 non-expressing cells were extracted, and the reactivity was determined by the fluorescent antibody method (FA). It was analyzed by the WB method. As a result, three clones reactive with Rz-HepM2-18-648d cells were obtained. One was clone 2-152 which recognized a 55 kDa molecule (P55), clone 2-243 recognized a 70 kDa molecule (P70) and clones 2-433 recognized a 30 kDa molecule (P30) (FIG. 3). The expression levels of these molecules were analyzed in various cell lines: human hepatoblastoma cells HepG2; human hepatoma cells HuH-7; human fetal liver cells WRL68; human fetal kidney cells 293 cells; and mouse fibroblasts NIH3T3 (FIG. 3). .. The tumorigenicity of these cells is shown in Table 1. P55, P70, and P30 had the highest expression in HuH-7 cells. The expression of P55 and P30 in these cell groups was changed depending on tumorigenicity. The expression levels of P55, P70, and P30 were low in NIH3T3 cells with low tumorigenicity.
From the above, in the liver-derived cell line, the higher the tumorigenicity, the higher the expression of each molecule. The expression was low in epithelial cells (293 cells) and NIH3T3 cells.
Furthermore, since it was considered that the expression levels of these molecules might be related to the tumorigenicity of cells, the expression in the cancerous part and non-cancerous part of the liver cancer patient (HCC) was analyzed (FIG. 4). 5 cases are HCV positive (HCV(+)) patient group (No. 1, 2, 3, 9 and 10), 5 cases are HCV, HBV infection negative (NBNC) patient group (No. 4, 5, 6, 7) And 8), 5 cases were HBV positive (HBV(+)) patient groups (No. 11, 12, 13, 14 and 15) (Table 2).

As a result of analyzing the cell lysates of frozen tissues of these patients by the WB method, the expression of P55 was elevated in the cancerous part of all 5 HCV(+) patients (FIG. 4). In HBV(+), 3 out of 5 cases and 2 out of 5 cases in the NBNC patient group had increased expression in the cancerous part. The expression of P70 was increased in the cancerous part of some patient groups (No. 1, 3, 6, 7, 10, 11, 12 and 14), but increased in the cancerous part of other patient groups. Was not found (No. 2, 4, 5, 9, 13 and 15). Expression of P30 is shown in Patient No. Although increased expression was observed in the cancerous areas of 1, 2, 3, 8, 10, 11, 12, and 14, patient No. It was not found in 5, 6, 9, 13 and 15. Thus, P55 showed the highest expression increase in the HCV(+) patient group (Table 3).

From the above, the 70 kDa and 30 kDa molecules recognized by the monoclonal antibody clones 2-243 and 2-433 showed increased expression in the cancerous part in 20 to 60% of patients. On the other hand, the 55 kDa molecule recognized by clone 2-152 had elevated expression in all HCV(+) patients. The expression increase rate in HBV(+) and NBNC liver cancer patients was 40 to 60%.
Furthermore, P55 was analyzed. First, a fluorescent antibody method was performed to clarify the intracellular localization of P55 (FIG. 5). The cells were fixed with 1% paraformaldehyde, treated with 1% Triton X-100 (Fix) or untreated (non-fixed), and then reacted with the monoclonal antibody 2-152. Further, they were also reacted with an antibody against PDI which is a marker of endoplasmic reticulum (manufactured by Stressgen). As a result, PDI, which is an endoplasmic reticulum marker, was not detected without fixing (permeating) cells, whereas P55 was detected without fixing (permeating) cells, and was therefore considered to be present on the cell surface membrane. ..
Next, the relationship between the expression of HCV and the expression of p55 was examined.
First, it was examined whether there is a relationship between the expression of the HCV gene (HCR6-Rz) and the expression of p55. When WRL68 cells or HepG2 cells were transfected with the HCV gene, expression was not seen in WRL68 cells, but expression of P55 was increased in HepG2 cells (FIG. 6A). Similar results were observed with the fluorescent antibody method (Fig. 7). Although an expression change of p55 in the HCV-expressing cell line Rz-HepM2-18-6 cells or expression-passaged cells (Rz-HepM2-18-648d) was also observed, an increase in expression due to the expression of HCV was observed. (FIG. 6B). According to the observation by the fluorescent antibody method, the expression of p55 was elevated in about 50% of those expressing HCV in Rz-HepM2-18-6 cells (FIGS. 8 and 9).
Therefore, in order to identify p55, an antibody column of monoclonal antibody clone 2-152 was prepared, and HuH-7 cell lysate was flown and bound to the column. The bound fraction was freeze-dried and then subjected to SDS-PAGE and CBB staining. The amino acid sequence was determined with a MALDI-TOF mass spectrometer (Millipore) in order to cut out the stained band and determine the amino acid sequence. As a result, p55 was considered to have the highest probability of being a molecule called DHCR24.
Next, the DHCR24 gene was cloned by the method described above, integrated into the downstream of the T7 promoter of the pGEM-T easy vector (pGEM-T-DHCR24), and synthesized in vitro. As a result, a protein having a molecular weight of about 55 kDa was synthesized (Fig. 10A), which reacted with the clone 2-152 antibody (Fig. 10B). Expression was observed when pCAG-DHCR incorporating DHCR24 downstream of the CAG promoter was transfected into WRL68 cells (Fig. 10C). Therefore, the monoclonal antibody clone 2-152 was considered to recognize the DHCR24 molecule.
[Example 2]
Measurement of CDC activity An antibody (0.1 ml) was added to target cells (10 ⁶ /ml, 0.1 ml) and incubated at 30°C for 30 minutes. Next, the cells were washed with GVB buffer, 0.1 ml of diluted guinea pig serum (SIGMA) was added, and the mixture was incubated at 37° C. for 60 minutes. Viability was measured by staining the cells with 0.1 ml of 0.05% trypan blue. The target cells, antibodies and complements used are shown below.
Target cells: WRL68, HepG2, HuH-7
Antibody: 2-152 (1, 10 μg/ml)
Normal mouse IgG (1, 10 μg/ml)
Complement: Derived from guinea pig (diluted 1:8 with GVB buffer)
It was revealed that this antibody 2-152 damages HuH-7 cells in a dose-dependent and selective manner in the presence of complement (FIG. 11).
Fluorescent antibody method (IFA)
Frozen sections of cancerous and non-cancerous tissues of HCC patient lungs in Tissue-Tek® OCT compound (TED PELLA Inc.) were thawed at room temperature, washed with PBS(-), and then PBS(-). Fixed with 1% paraformaldehyde in 10 minutes at room temperature. The tissue section was blocked with 1 mM EDTA/1% BSA/PBS(−) at room temperature for 1 hour, washed with PBS(−), and then monoclonal antibody 2-152 (5 μg/ml) was added to it at 4° C. overnight. It was made to react. Furthermore, after washing the section 3 times with PBS(-), anti-mouse IgG Alexa 488 (1:1000; goat anti-mouse IgG(Fab') ₂ fragment Fluor488, Molecular Probes in 0.05% Tween20/PBS(-) was used. ) Was added and reacted at room temperature for 30 minutes. The section was washed three times with PBS(-) and then mounted using a vector shield (Vector Inc.) containing TO-PRO-3 (10 µg/ml; Molecular Probes).
It was revealed that the present antibody 2-152 more strongly stains the cancerous part than the non-cancerous part in the liver tissue of the HCC patient and also stains the cell membrane of the cancerous part tissue (FIG. 12).

  By detecting DHCR24, highly accurate cancer diagnosis is possible. Further, by using the DHCR24-recognizing antibody of the present invention, it is possible to diagnose or treat cancers that have not been detected up to now.

Claims (16)

An antibody that recognizes DHCR24. An antibody that specifically recognizes DHCR24. An antibody that recognizes DHRC24 and can diagnose cancer. The antibody according to claim 3, which can diagnose liver cancer. An antibody that recognizes DHCR24, which can be obtained by a method for producing an antibody, which comprises introducing a gene encoding a hepatitis C virus protein into a cell and immunizing an animal with the cell. An antibody that recognizes DHCR24, which can be obtained by a method for producing an antibody that includes immunizing an animal with liver cancer cells. A diagnostic agent for cancer, comprising the antibody according to any one of claims 1 to 6. The diagnostic agent for cancer according to claim 7, which diagnoses liver cancer. An anticancer agent comprising the antibody according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. The anticancer agent according to claim 9, which is effective for liver cancer. A method for diagnosing cancer, which comprises detecting DHCR24. The diagnostic method according to claim 11, wherein the DHCR24 is a protein. A method for detecting DHCR24 for diagnosing cancer, which comprises reacting the antibody according to any one of claims 1 to 6 with live cells or cell lysates. The detection method according to claim 13, wherein the cells are liver cancer cells. A method for treating cancer, which comprises administering the antibody according to any one of claims 1 to 6. The treatment method according to claim 15, which is effective for liver cancer.

Images