HK1079568B - A diagnostic reagent for cancer and a diagnostic kit for cancer, comprising an anti-gpc3 antibody - Google Patents

Description

Cancer diagnostic reagent and cancer diagnostic kit comprising anti-GPC 3 antibody

Technical Field

The present invention relates to a cancer marker soluble in blood, and in particular to a method for diagnosing cancer by detecting soluble glypican 3(GPC3) in clinical samples.

Background

The phosphatidylinositolglycan family has been reported to be a new family of heparan sulfate proteoglycans present on the cell surface. To date, five members of the glypican family have been reported: glypicans (glypican 1, glypican 2, glypican 3, glypican 4, and glypican 5). These family members have the same size core protein (approximately 60kDa), share specific and more conserved cysteine sequences, and bind to the cell membrane via Glycosylphosphatidylinositol (GPI) anchors.

Glypican 3(GPC3) is known to be involved in cell division, or regulation of cell division pattern during development, and GPC3 gene is highly expressed in liver cancer cells. Thus, the GPC3 gene can be used as a hepatoma marker.

We have previously found that an anti-GPC 3 antibody has ADCC activity and CDC activity and is useful for treating liver tumors, and have applied for a patent (Japanese patent application No. 2001-189443).

GPC3 is a membrane-bound protein. There has been no report that secretory GPC3 exists in blood and GPC3 protein can be used as a cancer marker.

Summary of The Invention

We found that glypican 3(GPC3) is cleaved between 358 th arginine and 359 th serine, and thus it was assumed that soluble GPC3 is secreted in the blood of liver cancer patients. Then we established a sandwich ELISA system of GPC3 and revealed that GPC3 was present in the culture supernatant of HepG2 (human hepatoma cells) highly expressing the GPC3 gene. Furthermore, we also successfully determined soluble GPC3 in the plasma of mice transplanted with HepG2 and in the serum of human liver cancer patients. Since the gene expression of GPC3 in liver cancer is observed at an earlier stage than the gene expression of AFP as a cancer marker, the detection of GPC3 protein is considered to be useful for the diagnosis of cancer. Furthermore, detection of soluble GPC3 using anti-GPC 3 antibodies that recognize C-terminal fragments is somewhat difficult. It is inferred that the secretory GPC3 protein mainly contains an N-terminal fragment. Therefore, we speculated that the GPC3 antibody recognizing the N-terminus can be used for detecting solubility, thereby completing the present invention.

The expression of GPC3 protein was also detected in cancer cell lines other than liver cancer cell lines, such as lung cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer, and lymphoid cancer. Therefore, GPC3 may be used for the diagnosis of liver cancer as well as many other cancers.

The present invention is as follows.

(1) A method of diagnosing cancer comprising detecting soluble GPC3 protein in a test sample.

(2) The method for diagnosing cancer as described in (1), wherein the soluble GPC3 protein is an N-terminal peptide of GPC 3.

(3) The method for diagnosing cancer according to (2), wherein the N-terminal peptide of GPC3 is a peptide fragment contained in the amino acid sequence of amino acids from amino acid 1 to amino acid 374 of GPC3, or in the amino acid sequence of amino acids from amino acid 1 to amino acid 358 of GPC 3.

(4) The diagnostic method according to any one of (1) to (3), wherein the test sample is selected from the group consisting of blood, serum and plasma.

(5) The diagnostic method according to any one of (1) to (4), wherein the cancer is liver cancer.

(6) The method according to any one of (1) to (5), which comprises using an anti-GPC 3 antibody.

(7) The method according to (6), which comprises using an anti-GPC 3 antibody immobilized on a carrier and an anti-GPC 3 antibody labeled with a labeling substance.

(8) The method according to (7), wherein the labeling substance is biotin.

(9) A cancer diagnostic agent comprising an anti-GPC 3 antibody.

(10) The diagnostic reagent according to (9), comprising an anti-GPC 3 antibody using a carrier and an anti-GPC 3 antibody labeled with a labeling substance.

(11) The diagnostic reagent according to (9) or (10), wherein the cancer is liver cancer.

(12) The diagnostic agent according to any one of (9) to (11), wherein the anti-GPC 3 antibody recognizes an N-terminal peptide of GPC 3.

(13) A diagnostic kit comprising an anti-GPC 3 antibody, and

(14) the diagnostic kit according to (13), comprising an anti-GPC 3 antibody immobilized on a carrier, and an antibody labeled with a labeling substance.

The present invention is described in detail below.

The present invention relates to a method for detecting cancer by detecting soluble glypicans in a test sample.

The meaning of "detecting" includes both quantitative and qualitative detection. Examples of qualitative assays include determining the presence or absence of GPC3 protein alone, determining whether the GPC3 protein content is greater than a baseline level, and determining the level of GPC3 protein in a comparative test sample to another sample (e.g., a control sample). Examples of quantitative determinations include determination of the concentration of GPC3 protein and determination of the level of GPC3 protein.

Test samples include any sample that may contain GPC3 protein. The test sample is preferably taken from an organism such as a mammal, and further preferably from a human. Specific examples of test samples include blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph, saliva, and urine. Preferred test samples are blood, serum and plasma. The test samples of the present invention also include test samples obtained from a test sample, for example from a culture medium of cells of an organism.

The cancer to be diagnosed in the present invention includes, but is not limited to, liver cancer, pancreatic cancer, lung cancer, colon cancer, breast cancer, prostate cancer, leukemia and lymphoma, preferably liver cancer.

1. Preparation of anti-GPC 3 antibody

The anti-GPC 3 antibody used in the present invention can be obtained from any source and can be of any type (monoclonal or polyclonal) and in any form, so long as it specifically binds to the GPC3 protein. Specifically, a known antibody, such as a mouse antibody, a rat antibody, a human antibody, a chimeric antibody or a humanized antibody, can be used.

The antibody may be a polyclonal antibody, but is preferably a monoclonal antibody.

Further, the anti-GPC 3 antibody immobilized on a carrier and the anti-GPC 3 antibody labeled with a labeling substance may recognize the same epitope on the GPC3 molecule, but preferably recognize different epitopes.

Preferably, the epitope recognized by the antibody is present on the N-terminal fragment of the GPC3 protein (from amino acid Met at position 1 to amino acid Arg at position 358, or from amino acid Met at position 1 to amino acid Lys at position 374).

The anti-GPC 3 antibody used in the present invention may be a polyclonal or monoclonal antibody obtained using known techniques. In particular, as the anti-GPC 3 antibody, a monoclonal antibody obtained from a mammal is preferably used in the present invention. Examples of monoclonal antibodies obtained from mammals include antibodies produced by hybridomas and antibodies produced by hosts transformed by genetic engineering techniques using expression vectors containing antibody genes.

Hybridomas producing monoclonal antibodies can be prepared using known techniques as follows. Hybridomas are prepared by immunizing animals with GPC3 as the immunogen according to standard immunization protocols to obtain immune cells, which are then fused to known parental cells by standard cell fusion procedures. The fused cells were used for monoclonal antibody-producing cell screening by standard screening methods.

Specifically, the monoclonal antibody can be prepared by the following procedure.

GPC3 was first obtained as an immunogen for increasing antibodies by expressing the GPC3(MXR7) Gene disclosed by Lane, H.et al (Gene 188 (1997)), 151-156. Specifically, a gene sequence encoding GPC3 is inserted into a known expression vector system, a suitable host cell is transformed, and then the objective human GPC3 protein is purified from the host cell or culture supernatant by a known method.

In addition, naturally occurring GPC3 may also be purified and used.

The purified GPC3 protein is then used as an immunogen. Alternatively, a partial peptide of GPC3 may be used as the immunogen. In this case, by chemical synthesis based on the amino acid sequence of human GPC 3; incorporating a portion of the GPC3 gene into an expression vector; or degrading natural GPC3 with proteolytic enzyme to obtain partial peptide. The region of GPC3 used as a partial peptide is not limited to any specific region. To obtain an antibody recognizing an epitope present on the N-terminal fragment, a peptide ranging from Met at amino acid 1 to Arg at amino acid 358 of GPC3 or a peptide ranging from Met at amino acid 1 to Lys at amino acid 374 can be used. Peptides containing epitopes from these regions but smaller than the above-mentioned peptides may also be used.

The mammal immunized with the immunogen is not particularly limited, and is preferably in view of compatibility with the partner cell for cell fusion. For example, rodents such as mice, rats, hamsters or rabbits, or monkeys are commonly used.

Animals are immunized with the immunogen according to known methods. For example, immunization can be carried out by intraperitoneal or subcutaneous injection of the immunogen into the mammal. Specifically, the immunogen is diluted or suspended with an appropriate volume of PBS (phosphate buffered saline), physiological saline, or the like; if necessary, mixed with an appropriate volume of standard adjuvant such as Freund's complete adjuvant; emulsification; and then administered to the mammal several times every 4 to 21 days. In addition, after immunization with the immunogen, a suitable carrier may also be used. Especially when peptide fragments having a small molecular weight are used as the immunogen, the peptides are preferably bound to a carrier protein such as albumin or Keyhole limpet hemocyanin (Keyhole limpet hemocyanin), and then used for immunization.

The mammal immunized as described above is assayed for an increased titer of the antibody of interest in its serum. Subsequently, immune cells are collected from the mammal, followed by cell fusion. Particularly preferred immune cells are spleen cells.

Mammalian myeloma cells are used as the partner cells for fusion with the above immune cells. Examples of myeloma Cell lines preferably used herein include various known Cell lines such as P3(P3x63Ag8.653) (J.Imnoll. (1979)123, 1548-containing 1550), P3x63Ag8U.1(Current Topics in Microbiology and Immunology (1978)81, 1-7), NS-1(Kohler.G. and Milstein, C.Eur.J.Immunol. (1976)6, 511-containing 519), MPC-11(Margulies.D.H. et al, Cell (1976)8, 405-containing 415), SP2/0(Shulman, M. et al, Nature (1978) 269, 270), St.Groth, Ims.F. et al, J.munol.35 (1980)35, 1-21), Nature (1978) 269, 11-containing FO (St.Groth., S.F. et al, J.11. J.M. 79-containing J.11, Nature (1978, Medof. S.194, S.J.11-containing J.S.S.S.133, 1978, 1979, J.23-containing No. 9, 1979, 1978, 1979, 1978, and Medof. 1-11).

The cell fusion of the above-mentioned immune cells with myeloma cells can be carried out basically according to known methods, for example,and Milstein et al (G. And Milstein, c., Methods Enzymol. (1981)73, 3-46).

More specifically, the above-described cell fusion is carried out in a standard nutrient medium in the presence of, for example, a cell-fusion promoter. The cell-fusion promoter includes, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and the like. If desired, adjuvants such as dimethyl sulfoxide can also be added to further enhance the fusion efficiency.

Any ratio of immune cells to myeloma cells may be used herein. For example, it is preferable that the number of immune cells is 1 to 10 times greater than the number of myeloma cells. As a medium for the above cell fusion, for example, RPMI1640 medium or MEM medium suitable for the growth of the above myeloma cell line, or other standard media for this type of cell culture can be used. In addition, serum such as Fetal Calf Serum (FCS) may be used in combination.

Cell fusion was performed as follows: the above immune cells and myeloma cells are mixed well in a certain amount in the above culture medium, a solution of PEG (e.g., having an average molecular weight of about 1000 to 6000) preheated at about 37 ℃ is added (at a concentration of 30 to 60% (w/v)), and then the solution is mixed to form fused cells (hybridomas). Subsequently, a suitable medium is sequentially added, the supernatant is removed by centrifugation, and these steps are repeated to remove cell fusion agents, etc., which are unfavorable for the growth of hybridomas.

The thus obtained hybridomas are selected by culturing them in a standard selective medium such as HAT medium (a medium containing hypoxanthine, aminopterin and thymidine). Culturing in the HAT medium described above is continued for a period of time (typically, several days to several weeks) sufficient to kill non-confluent cells (other than the hybridoma of interest). Subsequently, those hybridomas producing the desired antibody were screened and monoclonal by standard limiting dilution.

Screening and monoclonality of a hybridoma producing an antibody of interest can be performed by a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to a carrier such as polystyrene magnetic beads or the like or a commercially available 96-well microtiter plate, and the culture supernatant of the hybridoma is added to a microplate to react with the antigen. After washing the carrier, an enzyme-labeled secondary antibody or the like is added to determine whether the antibody reactive with the immunogen is contained in the culture supernatant. The hybridoma producing the target antibody can be cloned by limiting dilution or the like. Antigens used for immunization can be used in this screen. To obtain antibodies against the N-terminal fragment of GPC3, the N-terminal fragment can be used as an antigen for screening.

In addition to the above-described method for obtaining a hybridoma by immunizing a non-human animal with an antigen, a human-derived antibody of interest having a GPC3 binding activity can also be obtained by sensitizing human lymphocytes in vitro with GPC3 and causing the sensitized lymphocytes to fuse with myeloma cells obtained from a human, which have the ability to divide permanently (see Japanese patent publication (Kokoku) No.1-59878B (1989)). Alternatively, the GPC3 antigen can be administered to a transgenic animal with an all human antibody gene bank to obtain anti-GPC 3 antibody-producing cells, and then human anti-GPC 3 antibodies can be obtained from the inactivated anti-GPC 3 antibody-producing cells (see international patent publications WO94/25585, WO93/12227, WO92/03918, and WO 94/02602).

The monoclonal antibody-producing hybridomas thus prepared may be subcultured in standard medium, or may be stored in liquid nitrogen for a long period of time.

One example of a method for obtaining monoclonal antibodies from hybridomas includes culturing hybridomas according to standard methods and obtaining monoclonal antibodies in the culture supernatant. Another method involves administering the hybridoma to a suitable mammal and obtaining monoclonal antibodies from ascites fluid thereof. The former method is suitable for obtaining highly purified antibodies, while the latter method is suitable for mass production of antibodies.

In the present invention, recombinant monoclonal antibodies produced by genetic engineering techniques can also be used as monoclonal antibodies. Recombinant monoclonal antibodies are prepared by cloning antibody genes from hybridomas, introducing the genes into a suitable vector, introducing the vector into a host, and then causing the host to produce a recombinant monoclonal antibody (see, e.g., Vandamm, A.M., et al, Eur.J.biochem. (1990)192, 767. SP. 775, 1990). Specifically, mRNA encoding the variable (V) region of the anti-GPC 3 antibody was isolated from a hybridoma producing anti-GPC 3 antibody. Total RNA was isolated by known methods such as the guanidine ultracentrifugation method (Chirgwin, J.M., et al, Biochemistry (1979)18, 5294-. Then, mRNA is prepared from the total RNA using an mRNA purification kit (Pharmacia) or the like. Alternatively, mRNA can be directly prepared using QuickPrep mRNA purification kit (Pharmacia).

cDNA for the variable (V) region of the antibody is synthesized from the mRNA thus obtained using reverse transcriptase. For example, cDNA is synthesized using AMV reverse transcriptase first strand cDNA Synthesis kit (SeIKAAKU, Inc.). For the synthesis and amplification of cDNA, for example, the 5 '-Ampli FINDER RACE kit (Clontech) and the 5' -RACE method using PCR (Frohman, M.A. et al, Proc. Natl. Acad. Sci. USA (1988)85, 8998-9002, Belyavsky, A. et al, Nucleic Acids Res. (1989)17, 2919-2932) can be used.

The DNA fragment of interest is purified from the obtained PCR product, and then ligated with a vector DNA to prepare a recombinant vector. Then, the vector is introduced into E.coli or the like, and colonies are selected to prepare a desired recombinant vector. The nucleotide sequence of the DNA fragment is determined by a known method, such as the dideoxynucleotide chain termination method.

Once the DNA encoding the V region of the anti-GPC 3 antibody is obtained, the DNA is introduced into a DNA expression vector containing the constant region (C region) encoding the antibody of interest.

To produce the anti-GPC 3 antibody for use in the present invention, an antibody gene is inserted into an expression vector so that the gene is expressed under the regulation of gene expression regulatory regions, such as enhancers and promoters. Subsequently, the host cell is transformed with the expression vector, and the antibody is expressed by the host cell.

The antibody gene can be expressed by inserting DNA encoding the heavy chain (H-chain) or encoding the light chain (L-chain) of the antibody into an expression vector, respectively, and then simultaneously transforming a host cell with the vector; or by inserting DNAs encoding H-chain and L-chain into a separate expression vector and then transforming the host cell with the vector to express the antibody gene (see WO 94/11523).

In addition to the above host cells, transgenic animals may also be used to produce recombinant antibodies. For example, a fusion gene is prepared by inserting an antibody gene into a gene encoding a protein produced only in milk (e.g., goat β casein). DNA fragments containing fusion genes including antibody genes were injected into goat embryos, which were then introduced into female goats. The antibody of interest is obtained from milk produced by a transgenic goat that has received an embryo or its offspring. In order to increase the amount of milk containing the antibody of interest produced by the transgenic goat, hormones may be administered to the transgenic goat as needed (Ebert, K.M., et al, Bio/Technology (1994)12, 699-702).

In the present invention, in addition to the above-mentioned antibodies, artificially modified recombinant antibodies such as chimeric antibodies or humanized antibodies can also be used. These engineered antibodies can be prepared using known methods.

Chimeric antibodies can be prepared as follows: the DNA encoding the V-region of the above antibody is ligated with the DNA encoding the C-region of the human antibody, and the product is introduced into an expression vector, which is then introduced into a host to allow the host to produce the antibody. Using this known method, a chimeric antibody useful in the present invention is obtained.

Humanized antibodies, also known as engineered human antibodies, are prepared by grafting CDRs (complementarity determining regions) of an antibody from a non-human mammal, such as a mouse, to the CDRs of a human antibody. Conventional genetic recombination techniques are well known in the art (see European patent application publication Nos. EP125023 and WO 96/02576).

Specifically, a DNA sequence designed to link mouse antibody CDRs with a Framework Region (FR) of a human antibody is synthesized by PCR using, as primers, several oligonucleotides having partially overlapping terminal regions of the mouse antibody CDRs and the Framework Region (FR) (see the method described in WO 98/13388).

The framework regions of the human antibody are selected for linkage by the CDRs, which will therefore form good antigen binding sites. Amino acids in the framework regions of antibody variable regions may be substituted as desired so that the CDRs of the engineered human antibody form suitable antigen binding sites (Sato, K., et al, cancer Res. (1993)53, 851-.

The C region obtained from a human antibody is used for the C region of a chimeric antibody and a humanized antibody. For example, C.gamma.1, C.gamma.2, C.gamma.3 or C.gamma.4C can be used for the H chain, and C.kappa.or C.lambda.can be used for the L chain. Furthermore, the human antibody C-region may be modified in order to improve the stability of the antibody or its production method.

Chimeric antibodies include the variable regions of an antibody obtained from a non-human mammal and the constant regions obtained from a human antibody, while humanized antibodies include the CDRs of an antibody obtained from a non-human mammal, and the framework regions and C regions obtained from a human antibody. Since the antigenicity of the humanized antibody is expected to decrease in the human body, it can be used as an active ingredient of the therapeutic agent of the present invention.

The antibody used in the present invention is not limited to the whole antigen molecule, but includes an antibody fragment or a modified product thereof as long as it binds to GPC 3. Both bivalent and monovalent antibodies are included. Examples of antibody fragments include Fab, F (ab') 2, Fv, Fab/c with one Fab and one complete Fc, and single chain Fv (scFv) in which the Fv of H-and L-chains are linked by a suitable linker. Specifically, antibody fragments can be prepared by treating antibodies with enzymes such as papain or pepsin, or by a method in which genes encoding these antibody fragments are constructed, introduced into an expression vector, and then expressed in suitable host cells (see, for example, Co., M.S. et al, J.Immunol. (1994)152, 2968-2976, Better, M. & Horwitz, A.H.methods in Enzymology (1989)178, 496, Academic Press, Inc., Pluechhun, A. & Skerra, A.methods in Enzymology (1989)178, 476-496, Academic-476, Lamoyi, E., Methods in Enzymology (1989)121, 652, Rousseau., J.J.in Enzymology (1989)121, 663, Methods in 1989, 1989) 132, and BTE.132).

scFv was obtained by linking the H-chain V-region and L-chain V-region of an antibody. In scFv, the H-chain V-region and the L-chain V-region are linked by a linker, preferably a peptide linker (Huston, J.S. et al, Proc. Natl.Acad.Sci.U.S.A. (1988)85, 5879-. The H-chain V-region and the L-chain V-region in the scFv may be obtained from any of the antibodies described in the present specification. As the peptide linker linking the V-region, for example, a single-chain peptide comprising any of 12 to 19 amino acid residues can be used.

DNA encoding scFv can be obtained by the following procedure. Amplification is carried out by PCR using the whole or part of the DNA encoding the amino acid sequence of interest (DNA encoding the H-chain or H-chain V-region of the above-mentioned antibody, and DNA encoding the L-chain or L-chain V-region) as a template and using a primer pair specific for both ends. Further amplification is then performed using a primer pair that encodes a peptide linker moiety and is specific for each end attached to the H-and L-strands.

Furthermore, once the DNA encoding the scFv has been prepared, an expression vector containing the DNA, and a host transformed with the expression vector, can be obtained according to standard methods. Further, by using a host, scFv can be obtained according to a standard method.

These antibody fragments can be produced by obtaining the genes thereof and then expressing the genes in a host in a manner similar to the above-described method. The "antibody" in the present invention includes such an antibody fragment.

Anti-glypican antibodies bound to various molecules such as a labeling substance can be used as the modified antibody. The "antibody" in the present invention also includes these modified antibodies. Such a modified antibody can be obtained by chemically modifying an antibody obtained as described above. Methods of modifying antibodies are known in the art.

Furthermore, the antibody used in the present invention may be a bispecific antibody. Bispecific antibodies may have antigen binding sites that recognize different epitopes on the GPC3 molecule. Alternatively, one antigen binding site may recognize GPC3 and another antigen binding site may recognize a labeling substance, and so forth. Bispecific antibodies can be prepared by binding H-L pairs of two types of antibodies, or by fusing hybridomas that produce different monoclonal antibodies. In addition, it can also be prepared by genetic engineering techniques.

The antibody can be expressed by an antibody gene constructed by a known method as described above. In the case of mammalian cells, the antibody may be expressed by operably linking a useful conventional promoter, the antibody gene to be expressed, and a polyA signal 3' downstream thereof. Promoters/enhancers include, for example, the human cytomegalovirus immediate early promoter/enhancer.

In addition, another example of a promoter/enhancer that can be used for the expression of the antibody of the present invention includes a viral promoter/enhancer such as retrovirus, polyoma virus, adenovirus or simian virus 40(SV40), or a promoter/enhancer obtained from a mammalian cell such as human elongation factor 1a (HEF1 a).

Antibodies can be expressed more readily by the method of Mulligan et al (Nature (1979)277, 108) when the SV40 promoter/enhancer is used, and by the method of Mizushima et al (Nucleic Acids Res (1990)18, 5322) when the HEF1a promoter/enhancer is used.

In the case of using E.coli, a useful conventional promoter, a signal sequence for antibody secretion and an antibody gene are operably linked so that the gene is expressed. The promoter includes lacz promoter and araB promoter. When the lacz promoter is used, the antibody gene can be expressed by the method of Ward et al (Nature (1098)341, 544-546; FASEBJ. (1992)6, 2422-2427), or when the araB promoter is used, the antibody gene can be expressed by the method of Better et al (Science (1988)240, 1041-1043).

As a signal sequence for antibody secretion, the pelB signal sequence can be used when the antibody is produced in the periplasm of Escherichia coli (Lei, S.P. et al, J.Bacteriol. (1987)169, 4379). After the antibodies produced in the periplasm of the egg are isolated, the structure of the antibodies is properly refolded and used.

The origin of replication may be derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV) and the like. In addition, to amplify the copy number of genes in the host cell system, the expression vector may contain an aminoglycoside transferase (APH) gene, a Thymidine Kinase (TK) gene, an E.coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, and the like as a selection marker.

For the production of antibodies for use in the present invention, any expression system may be used, such as eukaryotic cell systems or prokaryotic cell systems. Examples of eukaryotic cells include animal cells such as cells of established mammalian cell lines or insect cell lines, as well as filamentous fungal cells and yeast cells. Prokaryotic cells include bacterial cells such as E.coli cells.

Preferably, the antibodies used in the present invention are expressed in mammalian cells such as CHOC, OS, myeloma, BHK, Vero or HeLa cells.

The transformed host cell is then cultured in vitro or in vivo such that the host cell produces the antibody of interest. The host cell can be cultured according to known methods. For example, DMEM, MEM, RPMI1640, IMDM, or the like can be used as the medium. Serum such as Fetal Calf Serum (FCS) may be used in combination.

The antibody expressed and produced as described above can be isolated from the cell or host animal and purified to homogeneity. The separation and purification of the antibody used in the present invention can be performed using an affinity column. Examples of protein a columns are HyperD, POROS, sepharose f.f. (Pharmacia). Any other standard method of isolating and purifying proteins may be used. For example, a chromatography column other than the above-mentioned affinity column, filtration, ultrafiltration, salting out, dialysis and the like may be appropriately selected and used in combination to isolate and purify Antibodies (Antibodies A Laboratory Manual. EdHarlow, David Lane, Cold Spring Harbor Laboratory, 1988).

Detection of GPC3

The GPC3 detected in the present invention is not particularly limited, and may be full-length GPC3 or a fragment thereof. When detecting fragments of GPC3, the fragments may be N-terminal or C-terminal fragments, and are preferably N-terminal fragments. Alternatively, the GPC3 to be detected may be attached GPC3 protein such as heparan sulfate, or GPC3 core protein.

The method of detecting the GPC3 protein in the test sample is not particularly limited. Preferably, the GPC3 protein is detected immunologically using anti-GPC 3 antibodies. Examples of immunological methods include radioimmunoassays, enzyme immunoassays, fluorescence immunoassays, luminescence immunoassays, immunoassay precipitation methods, immuno-turbidimetry, Western blots, immunostaining, and immunodiffusion techniques. A preferred detection method is an enzyme immunoassay, and enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich ELISA) is particularly preferred. The above immunological method such as ELISA can be performed by a method known to those skilled in the art.

An example of a conventional detection method using an anti-GPC 3 antibody includes immobilizing an anti-GPC 3 antibody on a carrier, adding a test sample to the carrier, incubating the carrier to bind GPC3 protein to the anti-GPC 3 antibody, washing, and then detecting GPC3 protein in the test sample by detecting GPC3 protein bound to the carrier through the anti-GPC 3 antibody.

Examples of the carrier of the present invention include insoluble carriers such as insoluble polysaccharides (e.g., agarose or cellulose), synthetic resins (e.g., silicone resins, polystyrene resins, polyacrylamide resins, nylon resins or polycarbonate resins), and glass. These carriers can be used in the form of magnetic beads or microplates. In the case of magnetic beads, the column or the like may be filled with magnetic beads. In the case of a microplate, a multi-well microplate (e.g., a 96-well multi-well microplate), a biosensor chip, or the like may be used. The anti-GPC 3 antibody can be bound to the support by any conventional method such as chemical binding or physical adsorption. Most of the carriers used herein are commercially available.

The binding of anti-GPC 3 antibody to GPC3 protein is typically performed in buffer. The buffer includes, for example, phosphate buffer, Tris buffer, citrate buffer, borate buffer, carbonate buffer, and the like. The incubation is carried out under conditions which have been conventionally used, for example, at a temperature of from 4 ℃ to room temperature for from 1 to 24 hours. After incubation, washing was performed with any solution that did not interfere with the binding of GPC3 protein to anti-GPC 3 antibody. For example, a buffer containing a surfactant such as Tween20 is used.

In the method for detecting GPC3 protein according to the present invention, a control sample may be provided in addition to a test sample containing the GPC3 protein to be detected. Examples of control samples include a negative control sample that does not contain GPC3 protein and a positive control sample that contains GPC3 protein. In this case, the results obtained from the test sample were compared with the results of the negative control containing no GPC3 protein and the results of the positive control sample containing GPC3 protein, thereby detecting the GPC3 protein in the test sample. In addition, a series of control samples having successively different concentrations were prepared, and the detection result of each control sample was obtained as a numerical value to prepare a standard curve. GPC3 protein contained in a test sample can be quantitatively determined from the values obtained from the test sample according to a standard curve.

One preferred embodiment for detecting the GPC3 protein bound to the carrier by the anti-GPC 3 antibody is a detection method using an anti-GPC 3 antibody labeled with a labeling substance.

For example, the test sample is allowed to contact anti-GPC 3 antibody immobilized on a support. After washing, the GPC3 protein was detected using a labeled antibody that specifically recognizes the GPC3 protein.

The anti-GPC 3 antibody can be labeled by a known conventional method. Labeling substances are well known to those skilled in the art, such as fluorescent dyes, enzymes, co-enzymes, chemiluminescent substances or radioactive substances may be used. Specific examples of the labeling substance include radioactive isotopes (e.g.,³²p，¹⁴C，¹²⁵I，³h and¹³¹I) luciferin, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, beta-galactosidase, beta-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, carbohydrate oxidase, microperoxidase and biotin. When biotin is used as the labeling substance, it is preferable to further add avidin bound to an enzyme such as alkaline phosphatase after adding the biotin-labeled antibody. For binding of the labeling substance to the anti-GPC 3 antibody, any known method such as the glutaraldehyde method, the maleimide method, the pyridyl disulfide method or the periodic acid method can be used.

Specifically, a solution containing the anti-GPC 3 antibody is added to a support such as a microplate to immobilize the anti-GPC 3 antibody on the support. After washing the microplate, blocking is performed with, for example, BSA, gelatin, albumin, etc. to avoid non-specific binding of proteins. After rinsing the microplate again, the test sample is added to the microplate. After incubation, the plates were washed and then labeled anti-GPC 3 antibody was added. After appropriate incubation, the plates were washed and the labelled anti-GPC 3 antibody remaining on the plate was detected. Detection is carried out by methods well known to those skilled in the art. For example, in the case of labeling with a radioactive substance, the labeled antibody is detected by a liquid scintillation method or a RIA method. In the case of labeling with an enzyme, a substrate is added, and the result of an enzymatic reaction of the substrate such as color development is detected by a spectrophotometer. Specific examples of the substrate include 2, 2-azinebis (3-ethylbenzothiazoline-6-sulfonate) diamine salt (ABTS), 1, 2-phenylenediamine (o-phenylenediamine), and 3, 3 ', 5, 5' -Tetramethylbenzidine (TME). In the case of labeling with a fluorescent substance, the labeled antibody can be detected by a fluorometer.

A particularly preferred embodiment of the method of the present invention for detecting PC3 protein utilizes a biotinylated anti-GPC 3 antibody and avidin.

Specifically, a solution containing the anti-GPC 3 antibody is added to a microplate such as a microplate to allow the anti-GPC 3 antibody to be immobilized on the microplate. The microplate is washed and blocked with BSA or the like to avoid non-specific binding of proteins. After rinsing the microplate again, the test sample is then added to the microplate. After incubation, the plates were washed and then biotinylated anti-GPC 3 antibody was added. After appropriate incubation, the microplate is washed and avidin conjugated to an enzyme such as alkaline phosphatase or peroxidase is added. After incubation, the microplate is washed, a substrate corresponding to the enzyme bound to avidin is added, and then GPC3 protein is detected using an indicator showing enzymatic alteration of the substrate.

Another embodiment of the present invention of the GPC3 protein detection method involves the use of a primary antibody that specifically recognizes the GPC3 protein, and a secondary antibody that specifically recognizes the primary antibody.

For example, a test sample is contacted with an anti-GPC 3 antibody immobilized on a support. After incubation and washing, binding of GPC3 protein after washing was detected using a primary anti-GPC 3 antibody and a secondary antibody that specifically recognizes the primary antibody. In this case, the secondary antibody is preferably labeled with a labeling substance.

Specifically, a solution containing the anti-GPC 3 antibody is added to a carrier such as a microplate to immobilize the anti-GPC 3 antibody to the microplate. The microplate is washed and blocked with BSA or the like to avoid non-specific binding of proteins. After rinsing the microplate again, the test sample is then added to the microplate. After incubation and washing, primary anti-GPC 3 antibody was added. After appropriate incubation, the microplate was washed. Subsequently, a secondary antibody that specifically recognizes the primary antibody is added. After appropriate incubation, the microplate is washed and then the secondary antibodies remaining on the microplate are detected. The secondary antibody can be detected by the methods described above.

Another embodiment of the present invention for detecting GPC3 protein involves the use of agglutination reactions. In this method, GPC3 was detected using a carrier sensitized with an anti-GPC 3 antibody. Any carrier may be used for sensitization of the antibody as long as it is insoluble, does not cause nonspecific reactions and is stable. For example, latex particles, bentonite, collodion, kaolin or immobilized sheep red blood cells may be used. Latex particles are preferably used. The latex particles used in the present invention include, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles or polyvinyl toluene latex particles. Polystyrene latex particles are preferably used. The sensitising particles are mixed with the sample and the mixture is then stirred for a period of time to observe agglutination. The higher the concentration of GPC3 antibody contained in the sample, the greater the degree of aggregation of the particles observed. Thus, GPC3 can be detected by visual observation of agglutination. GPC3 can also be measured by measuring the turbidity caused by agglutination using a spectrophotometer or the like.

Another embodiment of the GPC3 protein detection method of the present invention comprises a biosensor using the surface plasmon resonance phenomenon. By using the biosensor of the surface plasmon resonance phenomenon, it is possible to observe protein-protein interactions in real time in the form of a surface plasmon resonance signal using only trace amounts of proteins without labels. For example, binding of GPC3 protein to anti-GPC 3 antibody can be detected by using a biosensor of BIAcore (Pharmacia) or the like. Specifically, a test sample is brought into contact with a sensor chip to which an anti-GPC 3 antibody has been fixed, and then a GPC3 protein bound to an anti-GPC 3 antibody can be detected from a change in resonance signal.

The detection method of the present invention can also be automatically performed using various automatic test systems, so that a large number of samples can be tested simultaneously.

It is another object of the present invention to provide a diagnostic reagent or kit for detecting GPC3 protein in a test sample to diagnose cancer. The diagnostic reagent or kit of the present invention contains at least an anti-GPC 3 antibody. When the diagnostic reagent or kit is based on an ELISA method, the reagent or kit may contain a carrier for immobilizing the antibody, or the antibody may have been previously bound to the carrier. When the diagnostic reagent or kit is based on an agglutination method using a carrier such as latex, the reagent or kit may contain a carrier having an antibody adsorbed thereon. In addition, the kit may also suitably contain a blocking solution, a reaction terminating solution, a reagent for treating a sample, and the like.

Brief description of the drawings

FIG. 1 shows the results of GPC3mRNA expression analysis using a gene chip. FIG. 1A shows the expression of GPC3, and FIG. 1B shows the expression of alpha-fetoprotein (AFP). NL, CH, LC, WD, MD and PD on the abscissa represent normal liver, chronic hepatitis site, cirrhosis site, highly differentiated malignant tumor, moderately-differentiated malignant tumor and lowly-differentiated malignant tumor, respectively.

Figure 2 shows purified heparan sulfate-attached GPC3 and GPC3 core protein CBB stained images.

FIG. 3 shows the expression of GPC3 gene in human liver cancer.

FIG. 4 shows the results of Western blotting using anti-GPC 3 antibody soluble core protein.

FIG. 5 shows the principle of a sandwich ELISA using anti-GPC 3 antibody.

FIG. 6 shows a standard curve for GPC3 sandwich ELISA using M6B1 and M18D 4.

FIG. 7 is a schematic diagram showing the structure of GPC 3.

FIG. 8 shows a combination of anti-GPC 3 antibodies in an ELISA.

Figure 9 shows a standard curve for a GPC3 sandwich ELISA system using different anti-GPC 3 antibody combinations.

Best mode for carrying out the invention

The present invention is described in detail below. However, the present invention is not limited to these examples.

In the examples described in the specification of the present application, the following materials were used.

As expression vectors for soluble GPC3 and soluble GPC3 core protein, pCXND2 and pCXND3, which have been prepared by introducing DHFR gene and neomycin-resistance gene into pCAGGS, were used.

DXB11 cells used herein were purchased from ATCC. For the culture, 5% FBS (GIBCO BRL CAT #10099-141, LOT # A0275242)/minimal essential Medium alpha Medium (alpha MEM (+) (GIBCO BRL CAT # 12571-071)/1% penicillin-streptomycin (GIBCO BRL CAT #15140-122) was used. For screening DXB11 cells, 500. mu.g/mL Geneticin (GIBCO BRL CAT # 10131-027)/5% FBS/(α MEM without ribonucleosides and deoxyribonucleosides (GIBCO BRL CAT #12561-056) (. alpha.MEM (-)/PS) was used alone or with MTX supplemented to a final concentration of 25 nM.

HepG2 cells used herein were purchased from ATCC and cultured in 10% FBS/Dulbecco's Modified Eagle Medium (DMEM) (GIBCO BRLCAT # 11995-.

The hybridomas were cultured in 10% FBS/RPMI1640/1 XHAT feed medium, (SIGMACAT # H-0262)/0.5 XBM-conditioned H1 hybridoma clone feed medium (RocheCAT # 1088947).

Example 1 cloning and expression analysis of human GPC (GPC3) cDNA

Cloning of full-Length cDNA encoding human glypican 3 (hereinafter abbreviated as GPC3)

A full-length cDNA encoding human GPC3 was amplified by PCR reaction using first strand cDNA prepared from a colon cancer cell line Caco2 as a template by standard methods and an Advantage2 kit (CLONTECH, Cat. No8430-1). Specifically, 50. mu.l of a reaction solution containing 2. mu.l of cDNA obtained from Caco2, 1. mu.l of sense primer (SEQ ID NO: 1), 1. mu.l of antisense primer (SEQ ID NO: 2), 5. mu.l of Advantage 210 XPCR buffer, 8. mu.l of NTP mix (1.25mM), and 1.0. mu.l of Advantage polymerase mix was used for 35 cycles of a reaction cycle consisting of 94 ℃ for 1 minute, 63 ℃ for 30 seconds, and 68 ℃ for 3 minutes. The PCR amplification product (inserted in the TA vector pGEM-T Easy using pGEM-T Easy vector System I (Promega, Cat. No. A1360)) was sequenced using the ABI3100DNA sequencer to confirm that the cDNA encoding full-length human GPC3 was isolated. Sequence SEQ ID NO: 3 represents the nucleotide sequence of the human GPC3 gene, and the sequence SEQ ID NO: 4 represents the amino acid sequence of human GPC3 protein.

SEQ ID NO：1：GATATC-ATGGCCGGGACCGTGCGCACCGCGT

SEQ ID NO：2：GCTAGC-TCAGTGCACCAGGAAGAAGAAGCAC

Analysis of human GPC3mRNA expression Using Gene chips

Using GeneChip^TMUG95A target (Affymetrix) analyzed mRNA expression in samples of 24 liver cancer cases (highly differentiated malignancy: WD; moderately differentiated malignancy: MD; lowly-differentiated malignancy: PD), 16 non-liver cancer cases (chronic hepatitis fraction: CH; cirrhosis fraction: LC) and 8 normal liver cases (NL). These samples were obtained from the university of tokyo, institute of medical research and medical school, and the Saitama tumor center, with informed consent. Specifically, total RNA was prepared from the above-mentioned various tissues using ISOGEN (Nippon Gene Co., Ltd.), and then gene expression analysis was performed on 15. mu.g of each total RNA according to the expression analysis technical manual (Affymetrix).

As shown in FIG. 1, it was observed that the expression of mRNA of human GPC3 gene (probe set ID: 39350_ at) was higher in tumor tissue than in normal liver tissue in many cases regardless of the differentiation stage of liver cancer. In addition, the mRNA expression level of human GPC3 gene was compared with that of alpha-fetoprotein (Probe series ID: 40114_ at), which is currently the most frequently used diagnostic marker for liver cancer. As a result, GPC3 was observed to sufficiently enhance mRNA expression in high-degree differentiated cancer cases, with little expression of alpha fetoprotein mRNA observed, and it showed that the incidence of enhanced mRNA expression of GPC3 was higher than AFP. Based on the above results, we considered that GPC3 assay is useful as an early diagnosis method for liver cancer.

Example 2 preparation of anti-GPC 3 antibody

Preparation of soluble human GPC3

Soluble GPC3 protein lacking a hydrophobic region on the C-terminal side was prepared as a material for preparing an anti-GPC 3 antibody.

Soluble GPC3cDNA expression plasmid DNA was constructed using plasmid DNA containing full-length human GPC3cDNA, provided by the top scientific and technical research center of tokyo university. PCR was performed using a downstream primer (5'-ATA GAA TTC CAC CAT GGCCGG GAC CGT GCG C-3' (SEQ ID NO: 5)) designed to eliminate the C-terminal side hydrophobic region (amino acid 564 to amino acid 580) and an upstream primer (5'-ATA GGA TCC CTT CAG CGG GGA ATGAAC GTT C-3' (SEQ ID NO: 6)) containing an EcoR I recognition sequence and a Kozak sequence. The PCR fragment thus obtained (1711bp) was cloned into pCXND 2-Flag. The expression plasmid DNA thus prepared was introduced into the CHO cell line DXB 11. CHO cell lines highly expressing soluble GPC3 were obtained by selection using 500. mu.g/mL geneticin.

Using 1700cm²Shake flasks were cultivated on a large scale on a CHO cell line highly expressing soluble GPC 3. The culture supernatant was collected and purified. The culture supernatant was applied to a DEAE Sepharose fast flow column (Amersham CAT # 17-0709-01). After washing, the mixture was washed with a solution containing 500mM NaCl to elute the product. Then, the product was affinity-purified using anti-Flag M2 agarose affinity gel (SIGMACAT # A-2220) and eluted through FLAG peptide at 200. mu.g/mL. After concentration using Centriprep-10(Millipore CAT #4304), the FLAG peptide was removed by gel filtration using Superdex 200HR 10/30(Amersham CAT # 17-1088-01). Finally, the protein was concentrated using a DEAE Sepharose fast flow column and eluted by buffer exchange using PBS (containing 500mM NaCl) without Tween 20.

Preparation of soluble human GPC3 core protein

cDNA in which 495 th serine and 509 th serine were substituted with alanine was prepared by assembly PCR using the wild-type human GPC3cDNA as a template. At this time, the primer was designed to have a His tag added to its C-terminal. The cDNA thus obtained was cloned into pCXND3 vector. The expression plasmid DNA thus prepared was introduced into DXB11 cell line. CHO cell lines highly expressing the soluble GPC3 core protein were obtained by screening using 500. mu.g/mL geneticin.

Using 1700cm²Shake flasks for large scale culture. The culture supernatant was collected and purified. The culture supernatant was applied to a Q-Sepharose fast flow column (Amersham CAT # 17-0510-01). After washing, the product was eluted with a phosphate buffer containing 500mM NaCl. The product was then affinity purified using a chelating agarose fast flow column (Amersham CAT # 170575-01) and eluted with a gradient of 10 to 150mM imidazole. Finally, the product was concentrated using a Q-Sepharose fast flow column and then eluted using a phosphate buffer containing 500mM NaCl.

SDS polyacrylamide gel electrophoresis showed a faint band of 50 to 300kDa and a band of approximately 40 kDa. Fig. 2 shows the result of electrophoresis. GPC3 is a proteoglycan with a heparan sulfate addition sequence at the 69kDa C-terminus. The faint bands were considered to be GPC3 modified with heparan sulfate. Amino acid sequencing showed that the approximately 40kDa band contained a fragment from the terminal side of GPC3N, indicating that GPC3 had undergone some cleavage.

To remove the anti-heparan sulfate antibodies in the following hybridoma screen, soluble GPC3 core protein was prepared. That is, the two amino acid residues serine 495 and serine 509 used as heparin sulfate addition signal sequences were substituted with alanine. A CHO cell line highly expressing the protein was prepared as described above, and then the culture supernatant was affinity purified using His-tag. SDS polyacrylamide gel electrophoresis showed three bands of 70kDa, 40kDa and 30 kDa. Amino acid sequencing revealed that the 30kDa band was a fragment on the C-terminal side of GPC3, indicating that GPC3 had been enzymatically cleaved between 358 arginine and 359 serine. No 30kDa band was observed in heparan sulfate-attached GPC3, probably because the attachment of heparan sulfate to GPC3 caused band blurring. The fact that GPC3 is enzymatically cleaved at a particular amino acid sequence is a novel finding and its biological significance has not been elucidated.

From these results, we assumed that GPC3 on the membrane was also lysed in liver cancer patients, and soluble type GPC3 was secreted in blood. GPC3 in early stage liver cancer patients had higher levels of gene expression compared to AFP, which is a liver cancer tumor marker (fig. 1). Thus, to investigate the ability of GPC3 as a possible new tumor marker with higher clinical utility than AFP, anti-GPC 3 antibody was prepared and a sandwich ELISA system was constructed according to the methods described in example 2 and the examples that follow.

Preparation of anti-GPC 3 antibody

Since human GPC3 and mouse GPC3 have a high homology of 94% at the amino acid level, it is considered that it is difficult to obtain an anti-GPC 3 antibody when a normal mouse is immunized with human GPC 3. Thus, mice with the autoimmune disease MRL/lpr were used for immunization. 5 MRL/lpr mice (CRL) were immunized with soluble GPC 3. 100 μ g/mouse of the immunizing protein was prepared for the first immunization and then emulsified with FCA (Freund's complete adjuvant (H37Ra), Difco (3113-60), Becton Dickinson (cat # 231131)). The emulsified product is administered subcutaneously. Two weeks later, 50. mu.g/mouse protein was prepared and then emulsified with FIA (Freund's incomplete adjuvant, Difco (0639-60), Becton Dickinson (cat # 263910)). The emulsified product is administered subcutaneously. Subsequently, the immunization was boosted a total of 5 times at 1-week intervals. For final immunization, the protein was diluted in PBS at 50 μ g/mouse and then administered via the gluteal vein. After confirming the saturated serum antibody titer against GPC3 by ELISA using an immunoplate coated with GPC3 core protein, cells were fused by mixing mouse splenocytes with P3U1 mouse myeloma cells in the presence of PEG1500(Roche Diagnostics, cat # 783641). The fused cells were seeded on 96-well culture dishes and selected from the next day using HAT medium, and then culture supernatants were screened by ELISA. Positive clones were monocloned by limiting dilution followed by expansion culture and culture supernatant was collected. By performing ELISA screening using the binding activity to GPC3 core protein as an indicator, 6 anti-GPC 3 antibodies having strong binding ability were obtained.

The antibody was purified using Hi Trap protein G HP (Amersham CAT # 17-0404-01). Hybridoma culture supernatants were applied directly to the column. After washing with binding buffer (20mM sodium phosphate, pH 7.0), the antibody was eluted with elution buffer (0.1M glycine-HCl, pH 2.7). The eluate was collected in a tube containing a neutralization buffer (1M Tris-HCl (pH9.0)) so that the product was immediately neutralized. Antibody fractions were collected and buffer replaced by dialysis overnight against 0.05% Tween20 PBS. Adding 0.02% NaN₃The purified antibody was added, and the mixture was stored at 4 ℃.

Analysis of anti-GPC 3 antibody

Mouse IgG sandwich ELISA was performed with goat anti-mouse IgG (γ) (ZYMED CAT #62-6600) and alkaline phosphatase-goat anti-mouse IgG (γ) (ZYMED CAT # 62-6622). Antibody concentrations were determined using a commercially available purified mouse IgG1 antibody (ZYMED CAT #02-6100) as a standard.

Isotyping of anti-GPC 3 antibodies was performed using ImmunoPure monoclonal antibody isotype kit II (PIERCECAT #37502) according to the attached instructions. The results of isotyping indicate that all antibodies are of the IgG1 type.

Epitope classification of anti-GPC 3 antibodies was performed by Western blotting using GPC3 core protein. Soluble GPC3 core protein was used at 100 ng/lane in 10% SDS-PAGEMINII (TEFCO CAT # 01-075). After electrophoresis (60V 30 min, 120V 90 min), cells (BIO-RAD) were transferred to immobilon-P (Milliporea CAT # IPVHR8510) using transfer-blot semidry electrophoresis (15V 60 min). The membrane was briefly rinsed with TBS-T (0.05% Tween20, TBS) and then shaken for 1 hour (at room temperature) or overnight (4 ℃) with TBS-T containing 5% skim milk. After shaking with TBS-T for about 10 minutes, various anti-GPC 3 antibodies diluted to 0.1 to 10. mu.g/mL with TBS-T containing 1% skim milk were added, followed by shaking for 1 hour. After washing with TBS-T (10 min. times.3), HRP-anti-mouse IgG antibody (Amersham CAT # NA931) diluted to 1/1000 with 1% skim milk containing TBS-T was added. After 1 hour of shaking, the membrane was rinsed with TBS-T (10 min. times.3), developed with ECL-Plus (Amersham RPN2132), and imaged on HyperfileECL (Amersham CAT # RPN 2103K). FIG. 4 shows the results of Western blot analysis. Antibodies are classified according to the fact that the antibody reacts with a 40kDa band recognizing an epitope on the N-terminus, and the antibody reacts with a 30kDa band recognizing an epitope on the C-terminus. Antibodies recognizing M6B1, M18D4, and M19B11 on the N-terminal side, and antibodies recognizing M3C11, M13B3, and M3B8 on the C-terminal side were obtained. The KD values for each antibody were between 0.2 and 17.6nM using BIACORE analysis.

Example 3 detection of soluble GPC3

Mouse xenograft model

3,000,000 HepG2 human hepatoma cells were transplanted subcutaneously into the abdomen of 6-week-old female SCID mice (Fox CHARE C.B-17/Icr-scidJcl, CLEA Japan, Inc.) and nude mice (BALB/cAJcl-nu, CLEA Japan, Inc.). After 53 days (when the tumor mass had formed sufficiently), whole blood was collected via the posterior vena cava of HepG 2-transplanted SCID mice #1, 3 and 4. Plasma was prepared using a Niprono tube (vacuum blood-collection tube, NIPRO, NT-EA0205) in the presence of EDTA-2Na and aprotinin, and then stored at-20 ℃ until assayed. In addition, whole blood was collected via the posterior vena cava from HepG 2-transplanted SCID mouse #2 at day 62 after HepG2 transplantation, and HepG 2-transplanted nude mice #1 and 2 at day 66 after transplantation. As a control, plasma was also prepared from normal SCID mice of the same age by a similar procedure.

Sandwich ELISA

To detect soluble GPC3 in blood, a sandwich ELISA system for GPC3 was constructed. 96-well microplates were coated with M6B1 and GPC3 bound to M6B1 was detected by conjugation with biotinylated M18D4 antibody. For color development, ampak (dako) was used to achieve high sensitivity detection.

anti-GPC 3 antibody coated 96-well immunoplates were treated with 10. mu.g/mL coating buffer (0.1M NaHCO)₃(pH9.6)，0.02％(w/v)NaN₃) Dilutions were made and incubated overnight at 4 ℃. On the following day, the wells were washed 3 times with 300. mu.L/well washing buffer (0.05% (v/v) Tween20, PBS), and 200. mu.l of dilution buffer (50mM Tris-HCl (pH8.1), 1mM MgCl) was added₂，150mM NaCl，0.05％(v/v)Tween20，0.02％(w/v)NaN₃1% (w/v) BSA). The plate was maintained at room temperature for several hours or overnight at 4 ℃, and mouse plasma or culture supernatant diluted appropriately with dilution buffer was added and incubated at room temperature for 1 hour. After washing 3 times with 300. mu.L/well of RB, 10. mu.g/ml of biotin-labeled anti-GPC 3 antibody diluted with dilution buffer was added and incubated at room temperature for 1 hour. After washing 3 times with 300. mu.L/well of RB, AP-streptavidin (ZYMED) diluted to 1/1000 with dilution buffer was added and incubated at room temperature for 1 hour. After washing 5 times with 300. mu.L/well of washing buffer, color development was carried out using AMPAK (DAKO CAT # K6200) according to the attached protocol. The absorbance was measured using a microplate reader.

The biotin labeling kit (CAT # 1418165, Roche) was used for biotinylation of antibodies. The concentration of soluble GPC3 in the sample was calculated using the GlaphPad PRISM data sheet program (GlaphPad software inc.ver.3.0). FIG. 5 shows the principle of the sandwich ELISA in these examples.

A standard curve was prepared using purified soluble GPC3 to construct a system with a detection limit of several ng/mL. FIG. 6 shows a standard curve for GPC3 sandwich ELISA using M6B1 and M18D 4. Using these systems, detection of GPC3 in the culture supernatant of HepG2 described above and mouse serum into which HepG2 human liver cancer cells had been transplanted was attempted. Soluble GPC3 was detected in the culture supernatant of HepG2 and in the serum of mice that had been transplanted with HepG2 human liver cancer cells, while the level of soluble GPC3 in the serum of control media and control mice was below the limit of detection. Concentrations in the HepG2 culture supernatant were 1.2. mu.g/mL when expressed as the concentration of purified soluble GPC3, and 23 to 90ng/mL in mouse serum (Table 1).

Structure of secretory GPC3

GPC3 was investigated whether it was cleaved between 358 arginine and 359 serine and whether secretion was carried out as previously assumed. If secretory GPC3 is an N-terminal fragment, it is not possible to detect such GPC3 with a sandwich ELISA using a combination of an antibody recognizing the N-terminus and an antibody recognizing the C-terminus. Sandwich ELISA systems with different antibody combinations were constructed using three types of antibodies recognizing the N-terminal fragment and recognizing the C-terminal fragment. Fig. 7 shows the structure of secreted soluble GPC3, and fig. 8 shows the combination of antibodies. FIG. 9 shows a standard curve for a sandwich ELISA. Table 1 shows the results of the measurement. As shown in table 1, high levels of secretory GPC3 were detected in the culture supernatant of HepG2 and in the serum of mice that had been transplanted with HepG2 human liver cancer cells using a combination of antibodies, both of which recognize the N-terminal fragment. In contrast, the detection results obtained with the system comprising an antibody recognizing a C-terminal fragment were below the detection limit in many mice. Thus, it is expected that the N-terminal fragment is predominant in secretory GPC3 based on the findings of the present invention.

Industrial applicability

As shown in these examples, it can be shown that GPC3 is highly expressed in liver cancer cells, and a part of GPC3 exists in the blood as a secreted protein. Since GPC3 was observed to express genes at an earlier stage in cancer tissues than that of liver cancer marker AFP, detection of GPC3 was considered useful for cancer diagnosis. GPC3 has also been found to be expressed in cell lines of cancers other than liver cancer, such as lung cancer, colon cancer, malignancies, prostate cancer, pancreatic cancer or lymphomas. Therefore, GPC3 can also be used for the diagnosis of cancers other than liver cancer.

Furthermore, the possibility here shown is that the N-terminal fragment cleaved between arginine 358 and serine 359 is present predominantly in secretory GPC 3. Accordingly, we believe that antibodies that recognize N-terminal fragments can be used as diagnostic antibodies. Furthermore, if an antibody recognizing a C-terminal fragment is used as an antibody having ADCC activity and CDC activity for the treatment of liver cancer, it is possible to efficiently reach liver cancer cells without being captured by secretory GPC3 in blood.

All publications, patents and patent applications cited herein are incorporated by reference in their entirety. Accordingly, those skilled in the art will readily appreciate that numerous modifications and variations may be made thereto without departing from the spirit and scope of the present invention as set forth in the appended claims. It is intended that the present invention includes such modifications and variations.

Sequence listing

<110> Yinxian protein science of Kabushiki Kaisha (PERSEUS PROTEOMICS INC.)

<120> method for diagnosing cancer by detecting GPC3 (A method for diagnosing cancer by detecting

GPC3)

<130>SCT050747-47

<140>PCT/JP03/11320

<141>2003-09-04

<150>PCT/JP02/08997

<151>2002-09-04

<160>6

<170>PatentIn Ver.2.1

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gatatcatgg ccgggaccgt gcgcaccgcg t 31

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<213>Artificial Sequence

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<223>Description of Artificial Sequence：Synthetic DNA

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gctagctcag tgcaccagga agaagaagca c 31

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<213>Homo sapiens

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cagcacgtct cttgctcctc agggccactg ccaggcttgc cgagtcctgg gactgctctc 60

gctccggctg ccactctccc gcgctctcct agctccctgc gaagcagg atg gcc ggg 117

Met Ala Gly

1

acc gtg cgc acc gcg tgc ttg gtg gtg gcg atg ctg ctc agc ttg gac 165

Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu Ser Leu Asp

5 10 15

ttc ccg gga cag gcg cag ccc ccg ccg ccg ccg ccg gac gcc acc tgt 213

Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys

20 25 30 35

cac caa gtc cgc tcc ttc ttc cag aga ctg cag ccc gga ctc aag tgg 261

His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp

40 45 50

gtg cca gaa act ccc gtg cca gga tca gat ttg caa gta tgt ctc cct 309

Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro

55 60 65

aag ggc cca aca tgc tgc tca aga aag atg gaa gaa aaa tac caa cta 357

Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu

70 75 80

aca gca cga ttg aac atg gaa cag ctg ctt cag tct gca agt atg gag 405

Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu

85 90 95

ctc aag ttc tta att att cag aat gct gcg gtt ttc caa gag gcc ttt 453

Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe

100 105 110 115

gaa att gtt gtt cgc cat gcc aag aac tac acc aat gcc atg ttc aag 501

Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys

120 125 130

aac aac tac cca agc ctg act cca caa gct ttt gag ttt gtg ggt gaa 549

Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu

135 140 145

ttt ttc aca gat gtg tct ctc tac atc ttg ggt tct gac atc aat gta 597

Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val

150 155 160

gat gac atg gtc aat gaa ttg ttt gac agc ctg ttt cca gtc atc tat 645

Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr

165 170 175

acc cag cta atg aac cca ggc ctg cct gat tca gcc ttg gac atc aat 693

Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn

180 185 190 195

gag tgc ctc cga gga gca aga cgt gac ctg aaa gta ttt ggg aat ttc 741

Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe

200 205 210

ccc aag ctt att atg acc cag gtt tcc aag tca ctg caa gtc act agg 789

Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg

215 220 225

atc ttc ctt cag gct ctg aat ctt gga att gaa gtg atc aac aca act 837

Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr

230 235 240

gat cac ctg aag ttc agt aag gac tgt ggc cga atg ctc acc aga atg 885

Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met

245 250 255

tgg tac tgc tct tac tgc cag gga ctg atg atg gtt aaa ccc tgt ggc 933

Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly

260 265 270 275

ggt tac tgc aat gtg gtc atg caa ggc tgt atg gca ggt gtg gtg gag 981

Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly Val Val Glu

280 285 290

att gac aag tac tgg aga gaa tac att ctg tcc ctt gaa gaa ctt gtg 1029

Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val

295 300 305

aat ggc atg tac aga atc tat gac atg gag aac gta ctg ctt ggt ctc 1077

Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu

310 315 320

ttt tca aca atc cat gat tct atc cag tat gtc cag aag aat gca gga 1125

Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly

325 330 335

aag ctg acc acc act att ggc aag tta tgt gcc cat tct caa caa cgc 1173

Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg

340 345 350 355

caa tat aga tct gct tat tat cct gaa gat ctc ttt att gac aag aaa 1221

Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys

360 365 370

gta tta aaa gtt gct cat gta gaa cat gaa gaa acc tta tcc agc cga 1269

Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu Ser Ser Arg

375 380 385

aga agg gaa cta att cag aag ttg aag tct ttc atc agc ttc tat agt 1317

Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser

390 395 400

gct ttg cct ggc tac atc tgc agc cat agc cct gtg gcg gaa aac gac 1365

Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp

405 410 415

acc ctt tgc tgg aat gga caa gaa ctc gtg gag aga tac agc caa aag 1413

Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys

420 425 430 435

gca gca agg aat gga atg aaa aac cag ttc aat ctc cat gag ctg aaa 1461

Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys

440 445 450

atg aag ggc cct gag cca gtg gtc agt caa att att gac aaa ctg aag 1509

Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys

455 460 465

cac att aac cag ctc ctg aga acc atg tct atg ccc aaa ggt aga gtt 1557

His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val

470 475 480

ctg gat aaa aac ctg gat gag gaa ggg ttt gaa agt gga gac tgc ggt 1605

Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly

485 490 495

gat gat gaa gat gag tgc att gga ggc tct ggt gat gga atg ata aaa 1653

Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys

500 505 510 515

gtg aag aat cag ctc cgc ttc ctt gca gaa ctg gcc tat gat ctg gat 1701

Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp

520 525 530

gtg gat gat gcg cct gga aac agt cag cag gca act ccg aag gac aac 1749

Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn

535 540 545

gag ata agc acc ttt cac aac ctc ggg aac gtt cat tcc ccg ctg aag 1797

Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser Pro Leu Lys

550 555 560

ctt ctc acc agc atg gcc atc tcg gtg gtg tgc ttc ttc ttc ctg gtg 1845

Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe Phe Leu Val

565 570 575

cac tga ctgcctggtg cccagcacat gtgctgccct acagcaccct gtggtcttcc 1901

His

580

tcgataaagg gaaccacttt cttatttttt tctatttttt tttttttgtt atcctgtata 1961

cctcctccag ccatgaagta gaggactaac catgtgttat gttttcgaaa atcaaatggt 2021

atcttttgga ggaagataca ttttagtggt agcatataga ttgtcctttt gcaaagaaag 2081

aaaaaaaacc atcaagttgt gccaaattat tctcctatgt ttggctgcta gaacatggtt 2141

accatgtctt tctctctcac tccctccctt tctatcgttc tctctttgca tggatttctt 2201

tgaaaaaaaa taaattgctc aaataaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2261

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2300

<210>4

<211>580

<212>PRT

<213>Homo sapiens

<400>4

Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu

1 5 10 15

Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp

20 25 30

Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly

35 40 45

Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val

50 55 60

Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys

65 70 75 80

Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala

85 90 95

Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln

100 105 110

Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala

115 120 125

Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe

130 135 140

Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp

145 150 155 160

Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro

165 170 175

Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu

180 185 190

Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe

195 200 205

Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln

210 215 220

Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile

225 230 235 240

Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu

245 250 255

Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys

260 265 270

Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly

275 280 285

Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu

290 295 300

Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu

305 310 315 320

Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys

325 330 335

Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser

340 345 350

Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile

355 360 365

Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu

370 375 380

Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser

385 390 395 400

Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala

405 410 415

Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr

420 425 430

Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His

435 440 445

Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp

450 455 460

Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys

465 470 475 480

Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly

485 490 495

Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly

500 505 510

Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr

515 520 525

Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro

530 535 540

Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser

545 550 555 560

Pro Leu Lys Leu Leu Thr Ser Met Ala Ile Ser Val Val Cys Phe Phe

565 570 575

Phe Leu Val His

580

<210>5

<211>31

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：Synthetic DNA

<400>5

atagaattcc accatggccg ggaccgtgcg c 31

<210>6

<211>31

<212>DNA

<213>Artificial Sequence

<220>

<223>Description of Artificial Sequence：Synthetic DNA

<400>6

ataggatccc ttcagcgggg aatgaacgtt c 31

Claims (6)

1. A diagnostic reagent for cancer, comprising an anti-GPC 3 antibody capable of detecting an N-terminal peptide of GPC3 protein in a test sample, wherein the N-terminal peptide of GPC3 is a peptide having an amino acid sequence consisting of amino acid 1 to amino acid 358 of GPC 3.

2. The diagnostic reagent according to claim 1, wherein the anti-GPC 3 antibody is immobilized on a carrier, and wherein the reagent further comprises an antibody labeled with a labeling substance.

3. The diagnostic reagent according to claim 2, wherein the labeling substance is biotin.

4. A diagnostic reagent according to any one of claims 1 to 3 wherein the cancer is liver cancer.

5. A diagnostic kit for cancer comprising an anti-GPC 3 antibody capable of detecting an N-terminal peptide of GPC3 protein, wherein the N-terminal peptide of GPC3 is a peptide having an amino acid sequence consisting of amino acid 1 to amino acid 358 of GPC 3.

6. The diagnostic kit according to claim 5, wherein the anti-GPC 3 antibody is immobilized on a carrier, and wherein the kit further comprises an antibody labeled with a labeling substance.