JP2007093274A - Diagnostic method for cancer and therapeutic agent therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a diagnostic method for the presence of the metastasization of cancer, and a therapeutic agent for metastasized cancer. <P>SOLUTION: The diagnostic method of cancer a primary Ewing's sarcoma, Ewing's sarcoma metastatic tissue, metastatic melanoma tissue and liver cancer metastatic tissue is characterized by detecting GPC3 protein in a specimen to be examined. A caner therapeutic agent is also disclosed. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for diagnosing cancer selected from Ewing sarcoma primary lesion, Ewing sarcoma metastatic tissue, melanoma metastatic tissue and liver cancer metastatic tissue, and a therapeutic agent for the tissue and melanoma primary lesion.

  Cancer is the number one (30.3%) of death-causing diseases in Japan, greatly separating the causative diseases caused by other diseases, heart disease (15.3%) and brain disease (15.2%). Yes. On the other hand, cancer develops in many and various organs, and metastasis to various organs is observed as the pathology of the primary lesion progresses.

  Cancers in the primary organs have been developed, leading to early treatment due to the development of various diagnostic methods and early detection by diagnosis. However, in spite of the development of various diagnosis and treatment methods, there are many cases in which treatment has become difficult due to metastasis from the primary lesion, resulting in death. For example, in the case of Ewing sarcoma, the prognosis is extremely poor in cases with distant metastasis at the time of diagnosis even if the same treatment is performed as in cases without metastasis (Non-patent Document 1).

  Ewing sarcoma is a special type of bone tumor. It usually occurs during adolescent rapid bone growth, but is rare in children under 10 years of age. The 5-year survival rate of Ewing sarcoma patients in the US and Europe is about 60%, and the 5-year survival rate in Japan is 44.2%. Tumors often occur in the long bones of the limbs, especially the femur or pelvis, but are also found in the skull and the flat bones of the trunk. The main clinical symptoms are pain and swelling of the tumor site. In addition, fever is considered a bad prognostic sign. It is easy to metastasize to the lungs and other bones, and about one third of patients have metastasis at the time of diagnosis. There is no prophylaxis and diagnostic methods include X-rays, CT scans, and biopsies, but no specific diagnostic methods. Treatment includes chemotherapy, radiotherapy, and surgical resection (including primary and limb resections), but generally has a poor prognosis, with a 3-year disease-free survival rate of 71% in patients with tumor volumes less than 100 mL Is fatal with a 3-year disease-free survival rate of 31% in cases of 100 mL or more.

Cancer metastasis is observed in almost all types of cancer, but particularly in malignant lymphoma, lung cancer, breast cancer, etc., metastasis to the liver, lung, lymph node, adrenal gland, bone, bone marrow, etc. is observed.
Liver cancer has many metastases to the lung, lymph nodes, peritoneum, adrenal gland, bone, and bone marrow. In melanoma, metastasis to lymph nodes, lungs, liver, pancreas, heart, peritoneum, adrenal gland, kidney, brain, etc. is common.

  Diagnosis of liver cancer is performed by blood tests using AFP (alpha-type fetal protein) or PIVKA II as a marker, and diagnostic imaging using CT or ultrasonography. Tests with tumor markers are often false negatives and false positives due to other liver diseases, and are unreliable. In image diagnosis, the affected area may be difficult to see depending on the patient's condition and site. If these tests do not diagnose liver cancer, perform a needle biopsy. Needle biopsy has the risk of promoting bleeding and cancerous tissue enlargement. There are three main treatments for liver cancer: hepatectomy, hepatic artery embolization, and puncture therapy. Liver resection has many complications and there is a 1-2% mortality rate due to surgery. In some cases, surgery is not possible depending on the position of liver cancer and the state of liver function. Since hepatic artery embolization is less likely to be completely cured, it is necessary to repeat the same procedure. Puncture therapy includes percutaneous ethanol injection therapy and radiofrequency ablation therapy, but all of these treatments are small cancers and are most effective for cancers of 2 centimeters or less. Has been.

  To confirm the diagnosis of melanoma, a histopathological examination of the tumor is required, but in Japan, it is believed that when a skin biopsy is performed directly on a part of the melanoma, metastasis is induced. In principle, this diagnostic method has not been performed. If diagnosis is difficult due to clinical symptoms, the entire tumor is removed by surgery and histopathological examination is performed. In the case of serodiagnosis of melanoma, the test value of 5-S-cystinidupa is helpful. X-rays, ultrasound, scintigrams, and CT are used to diagnose metastases. The most important point in the treatment of melanoma is surgical surgery during early detection. At present, it is a general rule to remove a range several centimeters larger than the margin of the initial lesion. Radiation therapy for melanoma is often ineffective.

On the other hand, the following is known for GPC3 (glypican 3).
The presence of the glypican family as a new family of heparan sulfate proteoglycans present on the cell surface and, as members of that family, six types of glypicans (glypican 1, glypican 2, glypican 3, glypican 4, glypican 5 and glypican 6) Has been reported to exist. Members of this family have a uniform size (approximately 60 kDa) core protein, share a specific, well-retained cysteine sequence, and are bound to the cell membrane by glycosylphosphatidylinositol (GPI) anchors. Yes.

  GPC3 is deeply involved in the control of cell division and pattern in development and is known to be secreted into the blood, suggesting that it is cleaved between amino acids 358 and 359. When SDS-PAGE is carried out under reducing conditions, it is divided into the 1st to 358th fragments and the 359th to 580th fragments that have been macromolecularized by addition of heparan sulfate chains (Non-patent Document 2).

  On the other hand, it is known that the GPC3 gene is highly expressed in hepatoma cells and the GPC3 gene may be used as a hepatocellular carcinoma marker. Furthermore, Patent Literatures 1 to 3 and Non-Patent Literature 3 are already known as methods for diagnosing liver cancer by measuring GPC3.

  Furthermore, Non-Patent Document 4 reports the expression of GPC3 gene in Ewing sarcoma. Patent Document 4 describes that GPC3 gene and protein are expressed and produced in melanoma cells and are useful for early diagnosis of melanoma.

  However, all of the existing literature on liver cancer and melanoma are related to the primary lesion, and there are no reports on their metastasis. That is, it relates to the diagnosis and treatment of cancer in the primary organ. Any cancer is a cancer that easily metastasizes, and even if the primary cancer disappears by surgical treatment, the metastatic cancer in other organs does not disappear. In addition, metastatic cancer may be overlooked.

In Patent Document 4, it is described that blood GPC3 concentration falls below the measurement limit after surgery for a patient with high blood GPC3 concentration melanoma. However, in melanoma, there are many cases of metastasis, and it is often difficult to treat, but in this document, it is probably the case of surgery only for the primary lesion in patients who do not have metastasis.
WO 03/100429 WO 2004/018667 WO 2004/038420 WO 2005/039380 Makimoto, et al. Pediatric practice 67 (4): 607-614, 2004. J. Cell. Biol., 163 (3): 625-35. 2003 CANCER RESEARCH 64, 1-6, April 1, 2004 Int J Cancer. 2004 Jul 10; 110 (5): 687-94.

  There are various methods for diagnosing metastatic cancer that has grown large, but there are few methods for early diagnosis of the presence of small metastasis with low progression, and for melanoma and liver cancer, early diagnosis and treatment methods for metastatic cancer after primary surgery There was no. In particular, early diagnosis and development of a treatment method are desired for Ewing sarcoma, which has many metastases at the time of diagnosis of cancer and has a poor prognosis.

  From such a background, it is considered that knowing whether or not metastasis to other organs at an early stage after the operation of the primary lesion leads to the survival of cancer patients and the complete cure of cancer. Therefore, an early diagnosis method for metastatic tissue in cancer types with many metastases is desired.

  Therefore, the present inventor has conducted various studies to find an early diagnosis method for primary lesions and metastasis in liver cancer, melanoma and Ewing sarcoma. It was also found that there is a high correlation between the presence or absence of liver cancer metastatic tissue. Especially for Ewing sarcoma metastasis, melanoma metastasis, and liver cancer metastasis, there was no metastasis in cases where GPC3 concentration decreased after surgery for these primary cancers, and metastasis occurred in cases where GPC3 concentration was high even after surgery Therefore, it was found that the presence or absence of metastasis of these cancers can be diagnosed by detecting GPC3 protein in a biological sample. Furthermore, the present inventors have found that administration of anti-GPC3 antibody can treat Ewing sarcoma primary lesion, Ewing sarcoma metastatic tissue, melanoma primary lesion, melanoma metastatic tissue, and liver cancer metastatic tissue.

That is, the present invention provides a method for diagnosing cancer selected from Ewing sarcoma primary lesion, Ewing sarcoma metastasis tissue, melanoma metastasis tissue, and liver cancer metastasis tissue, characterized by detecting GPC3 protein in a test sample. .
The present invention also provides a diagnostic agent for cancer selected from primary Ewing sarcoma, Ewing sarcoma metastasis, melanoma metastasis, and liver cancer metastasis characterized by containing a GPC3 protein detection reagent.
Furthermore, the present invention provides a therapeutic agent for cancer selected from Ewing sarcoma primary lesion, Ewing sarcoma metastatic tissue, melanoma primary lesion, melanoma metastatic tissue and liver cancer metastatic tissue, characterized by containing an anti-GPC3 antibody. is there.

  According to the diagnostic method of the present invention, the presence or absence of Ewing sarcoma primary lesion, Ewing sarcoma metastasis tissue, melanoma metastasis tissue, or liver cancer metastasis tissue can be diagnosed at an early stage. In particular, the early diagnosis of the presence or absence of metastatic cancer after primary cancer surgery for these cancers is extremely important in terms of early treatment of metastatic cancer, prognosis management, and the like.

Hereinafter, the present invention will be described in detail.
Measurement includes quantitative or non-quantitative measurement. For example, non-quantitative measurement includes simply measuring whether or not GPC3 protein (hereinafter simply referred to as GPC3) is present. Measurement of presence or absence, measurement of comparing the amount of GPC3 with other samples (for example, control samples, etc.), etc. Quantitative measurement includes measurement of GPC3 concentration, amount of GPC3 Measurement etc. can be mentioned.

  The test sample is not particularly limited as long as it is a sample separated from a living body that may contain GPC3, but a sample collected from the body of a living organism such as a mammal is preferable, and a sample collected from a human is more preferable. It is. Specific examples of the test sample include blood, interstitial fluid, plasma, extravascular fluid, cerebrospinal fluid, synovial fluid, pleural fluid, serum, lymph fluid, saliva, urine, etc. Preference is given to blood, serum and plasma. A sample obtained from a test sample such as a culture solution of cells collected from the body of an organism is also included in the test sample of the present invention.

  As the test sample used in the present invention, blood, serum and plasma of Ewing sarcoma patients and patients after melanoma and liver cancer treatment are particularly preferable. Here, as a patient after treatment of melanoma, a patient after surgical treatment by primary surgery is particularly preferable. Patients after liver cancer treatment include patients after primary hepatectomy, radiofrequency ablation, ethanol injection, hepatic artery embolization, continuous arterial chemotherapy, etc., but after radical treatment That is, patients after hepatectomy or after radiofrequency ablation therapy or ethanol infusion therapy are particularly preferred.

  As a GPC3 detection means, an immunological detection means using an anti-GPC3 antibody is preferable. The anti-GPC3 antibody used in the present invention may be either a monoclonal antibody or a polyclonal antibody, but a monoclonal antibody is particularly preferred. In addition, the anti-GPC3 antibody is preferably an antibody against secretory GPC3, that is, an anti-soluble GPC3 antibody, from the viewpoint of using blood, serum, or plasma as a test sample. Furthermore, it is particularly preferable to use an anti-soluble GPC3 monoclonal antibody. Hereinafter, the case where the anti-GPC3 monoclonal antibody is used will be described in detail. On the other hand, a polyclonal antibody can be produced by a known method according to the method for producing a monoclonal antibody described below.

1. Preparation of anti-GPC3 monoclonal antibody The anti-GPC3 monoclonal antibody used in the present invention may be specifically bound to the GPC3 protein, regardless of its origin and shape. Specifically, known monoclonal antibodies such as mouse antibodies, rat antibodies, human antibodies, chimeric antibodies, and humanized antibodies can be used.
The anti-GPC3 monoclonal antibody immobilized on the support and the anti-GPC3 monoclonal antibody labeled with a labeling substance preferably recognize different epitopes of the GPC3 molecule, and the site is not particularly limited.

  The anti-GPC3 monoclonal antibody used in the present invention can be obtained using known means. The anti-GPC3 monoclonal antibody used in the present invention is particularly preferably a mammal-derived monoclonal antibody. Mammal-derived monoclonal antibodies include those produced by hybridomas and those produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques.

  A monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, GPC3, soluble GPC3 or a fragmented peptide thereof is used as a sensitizing antigen, immunized according to a normal immunization method, and the resulting immune cell is fused with a known parent cell by a normal cell fusion method. In this screening method, monoclonal antibody-producing cells can be screened.

Specifically, the monoclonal antibody can be prepared as follows.
First, GPC3 used as a sensitizing antigen for antibody acquisition is obtained by culturing HuH6, HepG2 cell lines, etc. and purifying natural GPC3 contained in the supernatant. It can also be obtained by expressing the GPC3 (MXR7) gene / amino acid sequence disclosed in Lage, H. et al., Gene 188 (1997), 151-156. That is, after inserting a gene sequence encoding GPC3 into a known expression vector system to transform an appropriate host cell, the target human GPC3 protein is purified from the host cell or culture supernatant by a known method. To do.
Next, this purified GPC3 is used as a sensitizing antigen, but a partial peptide of GPC3 can also be used as a sensitizing antigen. In this case, the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human GPC3, or part of the GPC gene can be incorporated into an expression vector, and further, natural GPC3 can be degraded by a proteolytic enzyme. Can also be obtained. The portion and size of GPC3 used as a partial peptide are not limited. In the present invention, full-length GPC3 is preferably used as the sensitizing antigen.

  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, guinea pigs, rabbits, monkeys, chickens and the like are used.

  In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline or the like, mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. The mammal is dosed several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen. In particular, when a partial peptide having a small molecular weight is used as a sensitizing antigen, it is desirable to immunize by binding to a carrier protein such as albumin or keyhole limpet hemocyanin.

  Thus, after immunizing a mammal and confirming that the desired antibody level rises in serum, immune cells are collected from the mammal and subjected to cell fusion. Cell.

  Mammalian myeloma cells are used as the other parent cell to be fused with the immune cells. This myeloma cell is known in various known cell lines such as P3 (P3 × 63Ag8.653) (J. Immnol. (1979) 123, 1548-1550), P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978)). 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. DH et al., Cell (1976) ) 8, 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, SF etal., J. Immunol. Methods (1980) 35, 1-21), S194 (Trowbridge, ISJ Exp. Med. (1978) 148, 313-323), R210 (Galfre, G. et al., Nature (1979) 277, 131-133) and the like are suitable. used.

  The cell fusion between the immune cells and myeloma cells is basically performed by a known method such as the method of Kohler and Milstein, C., Methods Enzymol. (1981) 73, 3- 46) etc.

  More specifically, the cell fusion is performed in a normal nutrient culture medium in the presence of a cell fusion promoter, for example. As the fusion promoter, for example, polyethylene glycol (PEG), Sendai virus (HVJ) or the like is used, and an auxiliary agent such as dimethyl sulfoxide can be used to enhance the fusion efficiency if desired.

  The use ratio of immune cells and myeloma cells can be arbitrarily set. 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, RPMI1640 culture medium suitable for growth of the myeloma cell line, MEM culture medium, and other normal culture liquids used for this type of cell culture can be used. Serum supplements such as fetal calf serum (FCS) can be used in combination.

  In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture solution, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60% (w / v) is added and mixed to form the desired fused cell (hybridoma). Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.

  The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fusion cells) to die. Subsequently, a normal limiting dilution method is performed, and screening and single cloning of a hybridoma that produces the target antibody are performed.

  Screening and single cloning of the target antibody may be performed by a screening method based on a known antigen-antibody reaction. For example, the antigen is bound to a carrier such as beads made of polystyrene or a commercially available 96-well microtiter plate, reacted with the culture supernatant of the hybridoma, washed with the carrier, and then reacted with an enzyme-labeled secondary antibody or the like. Thus, it can be determined whether or not the target antibody reacting with the sensitizing antigen is contained in the culture supernatant. A hybridoma producing the target antibody can be cloned by limiting dilution or the like. In this case, the antigen used for immunization may be used.

  In addition to immunizing animals other than humans to obtain the above hybridomas, human lymphocytes are sensitized to GPC3 in vitro, and the sensitized lymphocytes are fused with human-derived myeloma cells having a permanent division ability. A desired human antibody having a binding activity to GPC3 can also be obtained (see Japanese Patent Publication No. 1-59878). Furthermore, even if GPC3 as an antigen is administered to a transgenic animal having all repertoires of human antibody genes to obtain anti-GPC3 monoclonal antibody-producing cells, human antibodies against GPC3 can be obtained from the immortalized cells. Good (see WO 94/25585, WO 93/12227, WO 92/03918, WO 94/02602).

  The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.

  In order 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 therewith to proliferate, and as ascites is obtained. The method of obtaining is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.

  In the present invention, as a monoclonal antibody, an antibody gene is cloned from a hybridoma, incorporated into an appropriate vector, introduced into a host, and a recombinant type produced using gene recombination technology can be used. (See, for example, Vandamme, AM et al., Eur. J. Biochem. (1990) 192, 767-775, 1990). Specifically, mRNA encoding the variable (V) region of the anti-GPC3 monoclonal antibody is isolated from the hybridoma producing the anti-GPC3 monoclonal antibody. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC (Chomczynski, P. etal., Anal. Biochem. 1987) 162, 156-159), etc. to prepare total RNA, and the target mRNA is prepared using mRNA Purification Kit (Pharmacia). Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).

  The cDNA of the antibody V region is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis is performed 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 (Clontech) and 5'-RACE method using PCR (Frohman, MAet al., Proc. Natl. Acad. Sci. USA) (1988) 85, 8998-9002, Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932) and the like can be used.

  The target DNA fragment is purified from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method such as the dideoxynucleotide chain termination method.

  After obtaining the DNA encoding the V region of the desired anti-GPC3 monoclonal antibody, this is incorporated into an expression vector containing DNA encoding the desired antibody constant region (C region).

  In order to produce the anti-GPC3 monoclonal antibody used in the present invention, the antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody.

  The expression of the antibody gene may be carried out by co-transforming host cells by separately incorporating DNAs encoding antibody heavy chains (H chains) or light chains (L chains) into expression vectors, or H chains and L chains. Alternatively, a host cell may be transformed by incorporating a DNA encoding s into a single expression vector (see WO 94/11523).

  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 (such as goat β casein) inherently produced in milk to prepare a fusion gene. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and the 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 be used in transgenic goats as appropriate to increase the amount of milk containing the desired antibody produced from the transgenic goat (Ebert, KM et al., Bio / Technology (1994) 12, 699-702).

  In the present invention, in addition to the above-described antibodies, artificially modified gene recombinant antibodies such as chimeric antibodies and humanized antibodies can be used. These modified antibodies can be produced using known methods.

  A chimeric antibody is obtained by ligating the DNA encoding the antibody V region obtained as described above with DNA encoding the human antibody C region, incorporating it into an expression vector, introducing it into a host, and producing it. Using this known method, a chimeric antibody useful in the present invention can be obtained.

  A humanized antibody is also called a reshaped human antibody, which is a complementarity determining region (CDR) of a mammal other than a human, eg, a mouse antibody, to the complementarity determining region of a human antibody. It is transplanted and its general gene recombination technique is also known (see EP 125023, WO 96/02576).

  Specifically, the DNA sequence designed to link the CDR of a mouse antibody and the framework region (FR) of a human antibody has a portion that overlaps the terminal regions of both CDR and FR. The prepared oligonucleotides are used as primers to synthesize by PCR (see the method described in WO98 / 13388).

  As the framework region of the human antibody to be ligated via CDR, a region in which the complementarity determining region forms a favorable antigen binding site is selected. If necessary, framework region amino acids in the variable region of the antibody may be substituted so that the complementarity determining region of the reshaped human antibody forms an appropriate antigen binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

  For the C region of the chimeric antibody and humanized antibody, human antibodies are used. For example, Cγ1, Cγ2, Cγ3, Cγ4 can be used for the H chain, and Cκ, Cλ can be used for the L chain. In addition, the human antibody C region may be modified to improve the stability of the antibody or its production.

  A chimeric antibody consists of a variable region of a non-human mammal-derived antibody and a constant region derived from a human antibody. On the other hand, a humanized antibody consists of a complementarity determining region of a non-human mammal-derived antibody, and a framework region and C region derived from a human antibody.

  The antibody used in the present invention is not limited to the whole antibody molecule, and may be an antibody fragment or a modified product thereof as long as it binds to GPC3, and includes both a bivalent antibody and a monovalent antibody. For example, antibody fragments include Fab, F (ab ′) 2, Fv, Fab / c having one Fab and complete Fc, or a single chain in which Fv of H chain or L chain is linked by an appropriate linker. Examples include chain Fv (scFv). Specifically, the antibody is treated with an enzyme such as papain or pepsin to generate antibody fragments, or a gene encoding these antibody fragments is constructed and introduced into an expression vector, followed by appropriate host cells. (Eg, Co, MS et al., J. Immunol. (1994) 152, 2968-2976, Better, M. & Horwitz, AH Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc. Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 476-496, Academic Press, Inc., Lamoyi, E., Methods in Enzymology (1989) 121, 652-663, Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-669, Bird, RE et al., TIBTECH (1991) 9, 132-137).

  scFv is obtained by linking the H chain V region and 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. USA (1988) 85, 5879. -5883). The H chain V region and L chain V region in scFv may be derived from any of those described as antibodies herein. As the peptide linker that links the V regions, for example, any single chain peptide consisting of 12 to 19 amino acid residues is used.

  The DNA encoding scFv is the DNA encoding the H chain or H chain V region of the antibody, and the DNA encoding the L chain or L chain V region. Amplification by PCR using a primer pair that defines both ends of the DNA portion encoding as a template, and then further encoding the DNA that encodes the peptide linker portion, and both ends thereof linked to the H chain and L chain, respectively. Obtained by combining and amplifying primer pairs defined in 1.

  In addition, once a DNA encoding scFv is prepared, an expression vector containing them and a host transformed with the expression vector can be obtained according to a conventional method. ScFv can be obtained according to the method.

  These antibody fragments 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 includes fragments of these antibodies.

  As a modified antibody, an anti-GPC3 antibody bound to various molecules such as a labeling substance can also be used. The “antibody” in the present invention includes these modified antibodies. Such a modified antibody can be obtained by chemically modifying the obtained antibody. Antibody modification methods have already been established in this field.

  Furthermore, 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 GPC3 molecule, or one antigen binding site recognizes GPC3 and the other antigen binding site is labeled. Substances etc. may be recognized. Bispecific antibodies can be prepared by binding HL pairs of two types of antibodies, or by producing hybrid cells producing bispecific antibodies by fusing hybridomas producing different monoclonal antibodies. it can. Furthermore, bispecific antibodies can be produced by genetic engineering techniques.

  The antibody gene constructed as described above can be expressed and obtained by a known method. In the case of mammalian cells, it can be expressed by functionally binding a useful promoter commonly used, an antibody gene to be expressed, and a poly A signal downstream of the 3 ′ side thereof. For example, examples of the promoter / enhancer include human cytomegalovirus immediate early promoter / enhancer.

  In addition, other promoters / enhancers that can be used for the expression of the antibodies used in the present invention include viral promoters / enhancers such as retrovirus, polyomavirus, adenovirus, simian virus 40 (SV40), or human elongation factor 1a ( Examples include promoters / enhancers derived from mammalian cells such as HEF1a).

  When the SV40 promoter / enhancer is used, the method of Mulligan et al. (Nature (1979) 277, 108) is used. When the HEF1a promoter / enhancer is used, the method of Mizushima et al. (Nucleic Acids Res. (1990) 18, 5322). ) Allows easy gene expression.

  In the case of E. coli, the gene can be expressed by functionally combining a useful promoter commonly used, a signal sequence for antibody secretion, and an antibody gene to be expressed. Examples of the promoter include lacz promoter and araB promoter. When using the lacz promoter, the method of Ward et al. (Nature (1098) 341, 544-546; FASEBJ. (1992) 6, 2422-2427), or when using the araB promoter, the method of Better et al. (Science ( 1988) 240, 1041-1043).

  As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the periplasm of E. coli is produced. 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, polyomavirus, adenovirus, bovine papilloma virus (BPV), etc. can be used. Furthermore, the expression vector is a selection marker for gene copy number amplification in the host cell system. As an aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene and the like.

  Any expression system can be used for the production of the antibodies used in the present invention, for example, eukaryotic or prokaryotic cell systems. Examples of eukaryotic cells include established mammalian cell systems, insect cell systems, filamentous fungal cells, and animal cells such as yeast cells. Examples of 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 CHO, COS, myeloma, BHK, Vero, HeLa cells.
Next, the transformed host cell is cultured in vitro or in vivo to produce the desired antibody. Host cells are cultured according to a known method. For example, DMEM, MEM, RPMI1640, and IMDM can be used as the culture medium, and serum supplements such as fetal calf serum (FCS) can be used in combination.

  The antibody expressed and produced as described above can be isolated from cells and host animals and purified to homogeneity. Separation and purification of the antibody used in the present invention can be performed using an affinity column. For example, Hyper D, POROS, Sepharose F.F. (Pharmacia) etc. are mentioned as a column using a protein A column. In addition, it is only necessary to use a separation and purification method used in ordinary proteins, and the method is not limited at all. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns other than the affinity column, filters, ultrafiltration, salting out, dialysis and the like (Antibodies A Laboratory Manual. Ed Harlow, David Lane). Cold Spring Harbor Laboratory, 1988).

  Although the epitope of the antibody used in the present invention is not particularly limited, it may be sufficient to recognize full-length GPC3 or a peptide fragment thereof. More specifically, monoclonal antibodies and polyclonal antibodies produced by hybridomas described in WO03 / 100429, WO2004 / 018667, WO2004 / 038420 and the like can be used, but the polyclonal antibodies and monoclonal antibodies prepared in Examples 2 and 3 can be used. It is particularly preferred to use it.

2. Measurement of GPC3 GPC3 to be measured in the present invention may be a GPC3 protein to which heparan sulfate or the like has been added, or a GPC3 core protein.
GPC3 contained in the test sample is measured by an immunological method using an anti-GPC3 antibody. Examples of the immunological method include radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, luminescence immunoassay, immunoprecipitation method, immunoturbidimetric method, Western blot, immunostaining, immunodiffusion method, etc. An immunoassay, particularly preferred is an enzyme-linked immunosorbent assay (ELISA) (eg sandwich ELISA). The above-described immunological methods such as ELISA can be performed by methods known to those skilled in the art.

  As a general measurement method using an anti-GPC3 antibody, for example, an anti-GPC3 antibody is immobilized on a support, a test sample is added thereto, and incubation is performed to bind the anti-GPC3 antibody and the GPC3 protein, followed by washing. In addition, a method for measuring GPC3 protein in a test sample by measuring GPC3 protein bound to a support via an anti-GPC3 antibody can be mentioned.

  Examples of the support used in the present invention include insoluble polysaccharides such as agarose and cellulose, synthetic resins such as silicone resins, polystyrene resins, polyacrylamide resins, nylon resins, and polycarbonate resins, and insoluble supports such as glass. Can be mentioned. 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-GPC3 antibody and the support can be bound by a commonly used method such as chemical bonding or physical adsorption. All of these supports can be commercially available.

  The binding between the anti-GPC3 antibody and the GPC3 protein is usually performed in a buffer solution. As the buffer solution, for example, phosphate buffer solution, Tris buffer solution, citrate buffer solution, borate buffer solution, carbonate buffer solution and the like are used. Incubation is performed under conditions that are already often used, for example, incubation at 4 ° C. to room temperature for 1 to 24 hours. The washing after the incubation may be anything as long as it does not interfere with the binding between the GPC3 protein and the anti-GPC3 antibody. For example, a buffer containing a surfactant such as Tween 20 is used.

  In the GPC3 protein measurement method of the present invention, a control sample may be installed in addition to the test sample for which GPC3 protein is to be measured. Examples of the control sample include a negative control sample not containing GPC3 protein and a positive control sample containing GPC3 protein. In this case, it is possible to measure the GPC3 protein in the test sample by comparing the result obtained with the negative control sample not containing GPC3 protein and the result obtained with the positive control sample containing GPC3 protein. . In addition, a series of control samples with varying concentrations are prepared, the measurement results for each control sample are obtained as numerical values, a standard curve is created, and the test is performed based on the standard curve from the values of the test sample. It is also possible to quantitatively measure the GPC3 protein contained in the sample.

  A preferred embodiment of the measurement of the GPC3 protein bound to the support via the anti-GPC3 antibody includes a method using an anti-GPC3 antibody labeled with a labeling substance.

  For example, the test sample is brought into contact with the anti-GPC3 antibody immobilized on the support, and after washing, measurement is performed using a labeled antibody that specifically recognizes the GPC3 protein.

The labeling of the anti-GPC3 antibody can be performed by a generally known method. As the labeling substance, a labeling substance known to those skilled in the art, such as a fluorescent dye, an enzyme, a coenzyme, a chemiluminescent substance, and a radioactive substance, can be used. As specific examples, radioisotopes ( ³² P, ¹⁴ C, ¹²⁵ I, ³ H, ¹³¹ I, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase, horseradish peroxidase, glucoamylase, lysozyme, saccharide Examples include oxidase, microperoxidase, and biotin. 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. For binding of the labeling substance and the anti-GPC3 antibody, a known method such as glutaraldehyde method, maleimide method, pyridyl disulfide method or periodate method can be used.

  Specifically, a solution containing an anti-GPC3 antibody is added to a support such as a plate, and the anti-GPC3 antibody is fixed to the support. After washing the plate, it is blocked with, for example, BSA, gelatin, albumin, etc. to prevent nonspecific binding of proteins. Wash again and add the test sample to the plate. After incubation, wash and add labeled anti-GPC3 antibody. After moderate incubation, the plate is washed and the labeled anti-GPC3 antibody remaining on the plate is measured. The measurement 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 measured by liquid scintillation or RIA. In the case of labeling with an enzyme, a substrate is added, and an enzymatic change of the substrate, for example, color development, can be measured with 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 measured with a fluorometer.

  A particularly preferred embodiment of the GPC3 protein measurement method of the present invention includes a method using an anti-GPC3 antibody labeled with biotin and avidin.

  Specifically, a solution containing an anti-GPC3 antibody is added to a support such as a plate, and the anti-GPC3 antibody is immobilized. After washing the plate, it is blocked with, for example, BSA to prevent nonspecific binding of proteins. Wash again and add the test sample to the plate. After incubation, wash and add biotin-labeled anti-GPC3 antibody. After moderate incubation, the plate is washed and avidin conjugated with an enzyme such as alkaline phosphatase or peroxidase is added. After incubation, the plate is washed, a substrate corresponding to the enzyme bound to avidin is added, and GPC3 protein is measured using the enzymatic change of the substrate as an indicator.

  Other embodiments of the GPC3 protein measurement method of the present invention include a method using one or more primary antibodies that specifically recognize GPC3 protein and one or more secondary antibodies that specifically recognize the primary antibody. Can do.

  For example, a test sample is brought into contact with one or more types of anti-GPC3 antibodies immobilized on a support, incubated, washed, and bound GPC3 protein after washing is identified with the primary anti-GPC3 antibody and the primary antibody. Measure with one or more secondary antibodies that are recognized automatically. In this case, the secondary antibody is preferably labeled with a labeling substance.

  As another embodiment of the GPC3 protein measurement method of the present invention, a measurement method using an agglutination reaction can be mentioned. In this method, GPC3 can be measured using a carrier sensitized with an anti-GPC3 antibody. As the carrier for sensitizing the 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 erythrocytes and the like can be used, but it is preferable to use latex particles. As the latex particles, for example, polystyrene latex particles, styrene-butadiene copolymer latex particles, polyvinyl toluene latex particles and the like can be used, but it is preferable to use polystyrene latex particles. The sensitized particles are mixed with the sample and stirred for a certain time. The higher the concentration of anti-GPC3 antibody in the sample, the greater the degree of aggregation of the particles. Therefore, GPC3 can be measured by observing the aggregation with the naked eye. GPC3 can also be measured by measuring the turbidity due to aggregation with a spectrophotometer or the like.

  As another embodiment of the method for measuring GPC3 protein of the present invention, for example, a method using a biosensor utilizing the surface plasmon resonance phenomenon can be mentioned. A biosensor using the surface plasmon resonance phenomenon can observe a protein-protein interaction in real time as a surface plasmon resonance signal without labeling with a minute amount of protein. For example, it is possible to measure the binding between GPC3 protein and anti-GPC3 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 an anti-GPC3 antibody is immobilized, and the GPC3 protein that binds to the anti-GPC3 antibody can be measured as a change in resonance signal.

  The measurement method of the present invention can be automated using various automatic inspection apparatuses, and a large number of samples can be inspected at a time.

  The object of the present invention is to diagnose a cancer selected from Ewing sarcoma primary lesion, Ewing sarcoma metastasis tissue, melanoma metastasis tissue, and liver cancer metastasis tissue, and the diagnostic agent contains at least an anti-GPC3 antibody. When the diagnostic agent is based on the ELISA method, a carrier for immobilizing the antibody may be included, and the antibody may be bound to the carrier in advance. When the diagnostic agent is based on an agglutination method using a carrier such as latex, it may contain a carrier to which an antibody is adsorbed. The diagnostic agent may be a kit containing a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating a sample, and the like as appropriate.

  To diagnose primary Ewing sarcoma, blood GPC3 levels may be measured when abnormal swelling is observed in adolescent children. If the blood GPC3 level is high, it is diagnosed as Ewing sarcoma.

  In the diagnosis of Ewing sarcoma, liver cancer, and melanoma metastasis, the blood GPC3 concentration before and after treatment of these primary cancers may be measured. When the blood GPC3 concentration is reduced to the level of a healthy person by surgery, it is determined that there is no success of surgery and metastasis to other organs. However, even after surgery, if the blood GPC3 concentration is high, it is determined that there is metastasis to another organ.

  Furthermore, the anti-GPC3 antibody of the present invention can be used as a therapeutic agent for primary Ewing sarcoma and primary melanoma and as a therapeutic agent for metastatic cancers from these cancers and liver cancers, or in missile therapy specifically targeting metastatic cancers. .

  The therapeutic agent of the present invention can be formulated by mixing, dissolving, granulating, tableting, emulsifying, encapsulating, lyophilizing, etc. with a pharmaceutically acceptable carrier well known in the art.

  For oral administration, anti-GPC3 antibody, together with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., tablets, pills, dragees, soft capsules, hard capsules, solutions, It can be formulated into dosage forms such as suspensions, emulsions, gels, syrups, slurries and the like.

  For parenteral administration, anti-GPC3 antibody is combined with pharmaceutically acceptable solvents, excipients, binders, stabilizers, dispersants, etc., and solutions for injection, suspensions, emulsions, creams, ointments It can be formulated into dosage forms such as suppositories, inhalants, and suppositories. In injectable formulations, the anti-GPC3 antibody can be dissolved in an aqueous solution, preferably a physiologically compatible buffer such as Hanks's solution, Ringer's solution, or physiological saline buffer. Furthermore, the therapeutic agents of the present invention can take the form of suspensions, solutions, emulsions, etc. in oily or aqueous vehicles. Alternatively, the anti-GPC3 antibody may be produced in the form of a powder, and an aqueous solution or suspension may be prepared using sterilized water or the like before use. For administration by inhalation, the anti-GPC3 antibody can be powdered and made into a powder mixture with a suitable base such as lactose or starch. A suppository formulation can be produced by mixing an anti-GPC3 antibody with a conventional suppository base such as cocoa butter. Furthermore, the anti-GPC3 antibody can be encapsulated in a polymer matrix or the like and formulated as a sustained release preparation.

  The dosage and frequency of administration of the therapeutic agent of the present invention vary depending on the dosage form and administration route, and the patient's symptoms, age, and body weight, but generally about 0.001 mg to 1000 mg per kg body weight per day as an anti-GPC3 antibody. In the range of about 0.01 mg to 10 mg, preferably once to several times a day.

  The therapeutic agent of the present invention is usually administered by parenteral administration route, for example, injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.), transdermal, transmucosal, nasal, transpulmonary, and the like.

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

Example 1 Acquisition of Recombinant GPC3 Using a plasmid DNA containing a full-length human GPC3 cDNA, a flag-added soluble GPC3, that is, a recombinant GPC3 cDNA expression plasmid DNA was constructed. Downstream primer (5'-ATA GAA TTC CAC CAT GGC CGG GAC CGT GCG C -3 '(SEQ ID NO: 1)) designed to exclude the C-terminal hydrophobic region (564-580 amino acids) and EcoRI recognition sequence, Kozak PCR was performed using the upstream primer (5′-ATA GGA TCC CTT CAG CGG GGA ATG AAC GTT C -3 ′ (SEQ ID NO: 2)) to which the sequence was added. The obtained PCR fragment (1711 bp) was transferred to pCXND2-Flag. The cloned expression plasmid DNA was introduced into the CHO cell DXB11 strain, and selection with 500 μg / mL Geneticin gave a soluble GPC3 highly expressing CHO strain.

Using a 1700 cm ² roller bottle, the flag-added soluble GPC3 highly expressing CHO strain was cultured in large quantities, and the culture supernatant was collected and purified. The culture supernatant was charged into DEAE Sepharose Fast Flow (Amersham CAT # 17-0709-01), washed and then eluted with a buffer containing 500 mM NaCl. Next, affinity purification was performed using Anti-Flag M2 agarose affinity gel (SIGMA CAT # A-2220). Elution was performed with 200 μg / mL FLAG peptide. After concentration with Centriprep-10 (Millipore CAT # 4304), gel filtration with Superdex 200 HR 10/30 (Amersham CAT # 17-1088-01) was performed to remove the FLAG peptide. Finally, concentration was carried out using a DEAE Sepharose Fast Flow column, and at the same time, elution was performed with PBS containing Tween 20 (containing 500 mM NaCl) to obtain buffered recombinant GPC3.

Example 2 Production of anti-soluble GPC3 polyclonal antibody Using recombinant GPC3, a guinea pig polyclonal antibody was produced. A known method was used for the production. GPC3 emulsified with an adjuvant was administered subcutaneously for immunization. This was repeated several times. After confirming that the antibody titer had increased, blood was collected to obtain serum, and then polyclonal antibody (PPMX042) was obtained by ammonium sulfate precipitation.

Example 3 Production of anti-GPC3 monoclonal antibody
Five Balb / C mice (CRL) were immunized with GPC3. For the first immunization, the immunity protein was prepared to be 100 μg / animal and emulsified using FCA (Freund's complete adjuvant (H37 Ra), Difco (3113-60), Becton Dickinson (cat # 231131)) It was administered subcutaneously. What was prepared to become 50 μg / animal after 2 weeks was emulsified with FIA (Freund's incomplete adjuvant, Difco (0639-60), Becton Dickinson (cat # 263910)) subcutaneously. Thereafter, booster immunization was performed 5 times in total at 1-week intervals. The final immunization was diluted in PBS to 50 μg / mouse and administered into the tail vein. After confirming that the antibody titer in serum against GPC3 is saturated by ELISA using an immunoplate coated with GPC3 core protein, mouse myeloma cells P3U1 and mouse spleen cells are mixed, and PEG1500 (Roche Diagnostics, Cat # 783 641) was used for cell fusion. After seeding in a 96-well culture plate and selecting with HAT medium from the next day, the culture supernatant was screened by ELISA. Positive clones were monocloned by the limiting dilution method, expanded, and the culture supernatant was collected. Screening by ELISA was performed using the binding activity with the GPC3 core protein as an index, and an anti-soluble GPC3 monoclonal antibody having strong binding ability was obtained.

The antibody was purified using Hi Trap ProteinG HP (Amersham CAT # 17-0404-01). The hybridoma culture supernatant was directly charged onto the column, washed with a binding buffer (20 mM sodium phosphate (pH 7.0)), and then eluted with an elution buffer (0.1 M glycine-HCl (pH 2.7)). Elution was performed in a tube to which neutralization buffer (1M Tris-HCl (pH 9.0)) was added, and neutralized immediately. The antibody fractions were pooled, and dialyzed overnight with 0.05% Tween20 / PBS to replace the buffer. After adding NaN ₃ so that the purified antibody was 0.02%, it was stored at 4 ° C.
The resulting hybridomas were named PPMX008, PPMX009, PPMX0010 and PPMX031.
Among the obtained hybridomas, PPMX009, which is highly sensitive and specific in the ELISA shown below, was named M18D04 and deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (FERM P-20205). ).

Example 4 Analysis of Anti-soluble GPC3 Monoclonal Antibody The antibody concentrations used were goat anti-mouse IgG (gamma) (ZYMED CAT # 62-6600) and alkaline phosphatase-goat anti-mouse IgG (gamma) (ZYMED CAT # 62-6622). Mouse IgG sandwich ELISA was performed and quantified using a commercially available purified mouse IgG1 antibody (ZYMED CAT # 02-6100) as a standard.
For the isotyping of the anti-soluble GPC3 monoclonal antibody, ImmunoPure Monoclonal Antibody Isotyping Kit II (PIERCE CAT # 37502) was used, and the method followed the attached manual. The results of isotyping were all IgG1 type.

Example 5 Construction of Sandwich ELISA System In order to measure GPC3 in blood, many GPC3 sandwich ELISA systems were constructed, and a measurement system having excellent sensitivity and specificity was selected. The most excellent result was a system in which PPMX042 antibody, which is a polyclonal antibody, was coated on a 96-well plate, and M18D04 antibody was labeled with biotin. Scytek TMB was used for color development in order to achieve high measurement sensitivity.

  A 96-well immunoplate was coated with an anti-GPC3 monoclonal antibody or polyclonal antibody diluted with PBS (−) so as to be 10 μg / mL, and incubated at 4 ° C. overnight. The next day, after washing three times with 300 μL / well of PBS, 150 μL of ABI immunoassay stabilizer (ABI # 10-601-001) was added to perform blocking. After several hours at room temperature or overnight at 4 ° C, add purified protein, human serum, etc. diluted appropriately with dilution buffer (50 mM Tris-HCl pH 8.0, 0.15 M NaCl) containing animal serum, etc. Incubated for 2 hours at room temperature. Subsequently, biotin-labeled anti-soluble GPC3 monoclonal antibody or polyclonal antibody diluted to 20 μg / mL with PBS (−) containing animal serum or the like was added and incubated at room temperature for 2 hours. After discarding the reaction solution, use 3 ug of Vector Streptavidin-HRPO (Vector # SA-5004) using Cygnus Stab-ELISA-r (Cygnus # I-030) containing appropriate amount of animal serum. Diluted to / mL was added and incubated at room temperature for 0.5 hours. After washing 5 times with 300 μL / well washing buffer, color was developed using Scytek TMB (Cat # TM4999) according to the attached protocol, and the absorbance was measured with a microplate reader.

  For biotinylation of the antibody, Sulfo-NHS-LC-Biotin (CAT # 21335) manufactured by Pierce was used. The soluble GPC3 concentration in the sample was converted using a spreadsheet software GlaphPad PRISM (GlaphPad software Inc. ver. 3.0). As a result of creating a standard curve using purified soluble GPC3, it was possible to construct a measurement system that not only has a low absorbance when no standard product is added, but also has a high absorbance when GPC3 is added.

Example 6 Early Diagnosis of Liver Cancer Metastasis The relationship between blood GPC3 concentration and the presence or absence of liver cancer metastasis after primary hepatocellular carcinoma surgery and hepatic artery embolization was examined using a sandwich ELISA system described in Example 5.

Cases 1, 2 and 3 are examples of metastasis to the lung. In case 1, metastasis to the lung was observed about 2 months after the operation for liver cancer, but the cancer was removed from the lung, but it recurred in the liver about 4 months later.
In case 2, metastasis to the lung was observed about 3 months after liver cancer surgery, and cancer progressed in the lung of the metastasis destination. Case 3 had the same course as Case 1, but about 3 months after metastasis to the lung and about 1 year after recurrence in the liver.
On the other hand, cases 4, 5 and 6 are cases in which no metastasis was observed. There were no metastases after 59, 126, and 327 days, respectively.

  In 6 cases of liver cancer, blood was collected over time before and after surgery for liver cancer, and the GPC3 concentration in the blood was measured (0.4 ng / mL or more was determined to be positive for liver cancer). As a result, no cancer metastasis was observed in patients (n = 3) whose blood GPC3 concentration decreased after surgery and after hepatic artery embolization. On the other hand, cancer metastasis was confirmed later in patients (n = 3) whose blood GPC3 concentration increased or decreased little after surgery.

  From these results, it was suggested that the presence or absence of cancer metastasis can be diagnosed earlier than the conventional method by measuring the blood GPC3 concentration after surgery.

Claims (6)

  A method for diagnosing cancer selected from primary Ewing sarcoma, Ewing sarcoma metastasis, melanoma metastasis, and liver cancer metastasis characterized by detecting GPC3 protein in a test sample.   The diagnostic method according to claim 1, wherein the detection of GPC3 protein is immunological detection using an anti-GPC3 antibody.   The diagnostic method according to claim 1 or 2, wherein the test sample is blood, serum or plasma.   A diagnostic agent for cancer selected from primary Ewing sarcoma, Ewing sarcoma metastasis, melanoma metastasis, and liver cancer metastasis, comprising a GPC3 protein detection reagent.   The diagnostic agent according to claim 4, wherein the GPC3 protein detection reagent is a reagent containing an anti-GPC3 antibody. A therapeutic drug for cancer selected from primary Ewing sarcoma, Ewing sarcoma metastasis, melanoma metastasis, melanoma metastasis, and liver cancer metastasis, comprising an anti-GPC3 antibody.