HK1079547B - Cell growth inhibitors containing anti-glypican 3 antibody - Google Patents

Description

Cell growth inhibitor containing anti-glypican 3 antibody

Technical Field

The present invention relates to a cell growth inhibitor containing an anti-glypican 3 antibody as an active ingredient.

Background

The family of glypicans has been reported as a new family of heparan sulfate proteoglycans present on the cell surface. To date, 5 glypicans (glypican 1, glypican 2, glypican 3, glypican 4, and glypican 5) have been reported as members of the glypican family. These family members have a core protein of uniform size (about 60kDa), share a specific and fully conserved cysteine sequence, and bind to the cell membrane via a Glycosylphosphatidylinositol (GPI) anchor.

Dally (delayed division) genes have been identified by screening genes for Drosophila melanogaster variants with abnormal cell division patterns in central nervous system development. It is known that the cDNA of Dally represents an Open Reading Frame (ORF) encoding a product whose sequence shows homology (24-26% homology) to the transmembrane proteoglycans (GRIP) of vertebrates containing all the characteristics of glypicans. And it has also been suggested that dally genes play a role in regulating dpp (decapentaplegia) receptor mechanisms. This suggests that mammalian glypicans can modulate signal transduction between TGF and BMP. In particular, glypicans have been suggested to act as co-receptors for some heparin-binding growth factors (e.g., EGF, PDGF, BMP2 and FGF).

Glypican 3 has been isolated as a transcript under developmental regulation in rat intestine (Filmus, J., Church, J.G. and Buick, R.n. (1988) mol.cell biol.8, 4243-. Also in humans, the Gene encoding glypican 3 has been isolated as MXR-7 from a human gastric cancer cell line (Hermann Lane et al, Gene 188(1997) 151-156). Glypican 3 has been reported to form a protein-protein complex with insulin-like growth factor-2 in order to regulate the action of growth factors (Pilia, G., et al, (1996) nat. Genet.12, 241-. This report suggests that glypican 3 does not always interact with growth factors containing heparan sulfate chains.

Glypican 3 has been reported to be useful as a marker for liver cancer (Hey-ChiHsu et al, CANCER RESERCH 57, 5179-5184 (1997)). However, no clear relationship was found to indicate the presence of glypican 3 and cancer cell proliferation.

In addition, it has been suggested that glypicans may function as receptors for endostatin, which is useful as an angiogenesis inhibitor (Molecular Cell (2001), 7, 811-one 822). However, the relationship between this function and cell proliferation is still not elucidated.

As described above, glypican 3 has been demonstrated to be involved in cell proliferation. However, the mechanism of cell proliferation and the like are unknown, and the use of glypican 3 for the regulation of cell proliferation has never been attempted.

Summary of The Invention

The object of the present invention is to provide a cell growth inhibitor containing an anti-glypican 3 antibody as an active ingredient.

As a result of intensive studies, it was found that the anti-glypican 3 antibody exerts a cell proliferation inhibitory activity by ADCC (antibody-dependent cell-mediated cytotoxicity) activity and CDC (complement-dependent cytotoxicity) activity, thereby completing the present invention. Furthermore, it is also predicted that the anti-glypican 3 antibody also exerts cell proliferation inhibitory activity by inhibiting the action of growth factors. Furthermore, the anti-glypican 3 antibody also exerts cell proliferation inhibitory activity by binding to a cytotoxic substance such as a radioisotope, a chemotherapeutic agent, or a bacterium-derived toxin.

The subject of the invention is as follows:

(1) a cell growth inhibitor comprising an anti-glypican 3 antibody as an active ingredient;

(2) the cytostatic agent of (1), wherein the anti-glypican 3 antibody has a cytotoxic activity;

(3) the cytostatic agent of (2), wherein the cytotoxic activity is antibody-dependent cell-mediated cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity;

(4) the cytostatic agent of any one of (1) to (3), wherein the cell is a cancer cell;

(5) the cell growth inhibitor according to (4), wherein the cells are selected from the group consisting of liver cancer cells, lung cancer cells, colon cancer cells, breast cancer cells, prostate cancer cells, leukemia cells, lymphoma cells, and pancreatic cancer cells;

(6) the cell growth inhibitor of (5), wherein the cell is a hepatoma cell;

(7) the cytostatic agent of any one of (1) to (6), wherein the antibody is a monoclonal antibody;

(8) the cytostatic agent of any one of (1) to (6), wherein the antibody is a humanized antibody or a chimeric antibody;

(9) an antibody to which glypican 3 binds;

(10) the antibody of (9), which has cytotoxic activity;

(11) the antibody of (10), which has a cytotoxic activity against hepatoma cells; and

(12) the antibody of (11), which has cytotoxic activity against a HuH-7 hepatoma cell line.

The present invention will be described in detail hereinafter.

The present invention is a cell growth inhibitor containing an anti-glypican antibody as an active ingredient. Further, the present invention is a cell growth inhibitor containing an anti-glypican antibody as an active ingredient, which is useful for the treatment of diseases based on abnormal cell proliferation, particularly for the treatment of cancer.

Examples of the anti-glypican 3 antibody of the invention include known antibodies such as a humanized antibody, a human antibody (WO96/33735), a chimeric antibody (Japanese patent publication (Kokai) No. 4-228089A (1992)) or a mouse antibody and an antibody disclosed in the invention. In addition, the antibody may be a polyclonal antibody, and preferably a monoclonal antibody.

The anti-glypican 3 antibody used in the invention may be derived from any origin, may be of any type (monoclonal or polyclonal antibody) and may be in any form as long as it can inhibit cell proliferation.

1. Anti-glypican 3 antibody

The anti-glypican 3 antibody used in the invention can be obtained in the form of a polyclonal antibody or a monoclonal antibody by a known route. Particularly preferred anti-glypican 3 antibodies for use in the invention are monoclonal antibodies derived from mammals. Examples of monoclonal antibodies derived from mammals include antibodies produced by hybridomas and antibodies produced by hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. The antibody binds to glypican 3 so as to inhibit cell proliferation.

An example of such an antibody is a monoclonal antibody produced by a hybridoma clone of the invention.

2. Antibody-producing hybridoma

The monoclonal antibody-producing hybridoma can be basically prepared by using the following known techniques. Namely, the hybridoma is prepared as follows: according to a standard immunization method, the glypican 3 is used as immunogen for immunization, the immune cells obtained in the way are fused with known parent cells by a standard cell fusion method, and then cells producing monoclonal antibodies are screened by a standard screening method.

Specifically, monoclonal antibodies can be prepared as follows.

Human glypican 3, which is to be used as an immunogen-inducing antibody, is first obtained by expressing the glypican 3(MXR7) Gene (amino acid sequence) as disclosed in Lage, H.et al (Gene 188(1997), 151-156). Specifically, the gene sequence encoding glypican 3 is inserted into a known expression vector system, appropriate host cells are transformed, and then the target human glypican 3 protein is purified from the host cells or culture supernatant by a known method.

Subsequently, the purified glypican 3 protein was used as an immunogen. Alternatively, a partial peptide of glypican 3 may 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 glypican 3.

The anti-glypican 3 antibody inhibits cell proliferation activity together with ADCC action, CDC action and activity of growth factors. In addition, the anti-glypican 3 antibody can also inhibit cell proliferation by binding to a cytotoxic substance such as a radioisotope, a chemotherapeutic agent, or a toxin derived from bacteria. Therefore, in the present invention, the epitope on the glypican 3 molecule is recognized by the anti-glypican 3 antibody, which is not limited to a specific epitope. The anti-glypican 3 antibody can recognize any epitope as long as the epitope is present on the glypican 3 molecule. Thus, any fragment may be used as an antigen to prepare the anti-glypican 3 antibody of the invention, as long as it contains the epitope on the glypican 3 molecule.

The mammal to be immunized with the immunogen is not particularly limited and is preferably selected in view of compatibility with the parent cell to be used 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, the immunogen is injected intraperitoneally or subcutaneously into a mammal and the immunization is carried out by this conventional method. Specifically, the immunogen is diluted or suspended with an appropriate volume of PBS (phosphate buffered saline), physiological saline, or the like; if desired, an appropriate volume of standard adjuvant such as Freund's complete adjuvant is mixed with the product; emulsification; the solution is then administered to the mammal several times every 4-21 days. In addition, suitable carriers may also be used with the immunogen at the time of immunization.

The mammal is immunized as described above and the increase in the titer of the desired antibody in the serum is then confirmed. Subsequently, immune cells are collected from the mammal and subjected to cell fusion. Preferred immune cells are especially spleen cells.

As partner cells to be fused with the above immune cells, mammalian myeloma cells are used. Examples of myeloma cells preferably used herein include various known Cell lines, such as P3(P3x63Ag8.653) (J.Immuol. (1979)123, 1548-1550), P3x63Ag8U.1(Current toppcs in Microbiology and Immunology (1978)81, 1-7), NS-1(Kohler.G. and Milstein, C.J.Immunol. (1976)6, 511-519), MPC-11(Margulies. D.H. et al, Cell (1976)8, 405-415), SP2/0(Shulman, M. et al, Nature (1978) 269-270), Eufo (St. Groth., S.F. et al, J.munol.35 (1980)35, 1-21), Trowr.276. 148, Nature (1978, Medfield. G.133, Nature) 23, 1979-67-11).

Cell fusion of the above immunocytes with myeloma cells can be carried out basically according to known Methods, for example, the Methods of Kohler and Milstein et al (Kohler. G. and Milstein, C., Methods Enzymol. (1981)73, 3-46).

More specifically, the above cell fusion is carried out in a standard nutrient medium containing, for example, a cell fusion accelerator. As the cell fusion accelerator, for example, polyethylene glycol (PEG), japanese Hemagglutination Virus (HVJ), or the like can be used. If desired, an adjuvant, such as dimethyl sulfoxide, may be added to further enhance the fusion efficiency.

Any ratio of immune cells to myeloma cells used herein can be set. For example, the number of immune cells is preferably 1 to 10 times higher than the number of myeloma cells. As a culture solution to be used for the above cell fusion, for example, RPMI1640 culture solution or MEM culture solution suitable for the growth of the above myeloma cell line, or other standard culture solutions used for such cell culture can be used. In addition, serum fluids such as Fetal Calf Serum (FCS) may be used in combination.

The method of cell fusion is as follows: a sufficient number of the above immune cells and myeloma cells are mixed in the above culture solution, a solution of PEG (for example, having an average molecular weight of about 1000-6000) previously heated at about 37 ℃ is added (concentration is generally 30-60% (w/v)), and then the above solutions are mixed to form target fused cells (hybridomas). Subsequently, an appropriate culture solution is successively added, and the step of removing the supernatant by centrifugation is repeated, thereby removing the agent for cell fusion and the like which are unfavorable for the growth of hybridoma.

The hybridomas obtained as described above are selected by culturing them in a standard selection medium such as HAT medium (medium containing hypoxanthine, aminopterin, thymidine). The incubation in HAT medium as described above is continued for a period of time sufficient to allow the cells (unfused cells) to die rather than the target hybridoma (typically days to weeks). Subsequently, hybridomas producing the target antibody are screened and monoclonal using standard limiting dilution methods.

In addition to the method of obtaining the above-mentioned hybridoma by immunizing a non-human animal with an antigen, a desired human antibody having a binding activity to glypican 3 can also be obtained by sensitizing human lymphocytes in vitro with glypican 3 and fusing the sensitized lymphocytes with human-derived myeloma cells having a potential to divide permanently (see Japanese patent publication (Kokoku) No. 1-59878B (1989)). In addition, glypican 3 is administered as an antigen to a transgenic animal having a gene pool of all human antibodies to obtain anti-glypican 3 antibody-producing cells, and then human antibodies to glypican 3 can be obtained from immortalized anti-glypican 3 antibody-producing cells (see international patent publication nos. WO 94/25585, WO 93/12227, WO 92/03918, and WO 94/02602).

The monoclonal antibody-producing hybridomas thus produced can be subcultured in a standard culture medium or can be stored in liquid nitrogen for a long period of time.

One example of a method for obtaining a monoclonal antibody from a hybridoma includes culturing the hybridoma according to a standard method and obtaining the monoclonal antibody in a culture supernatant. Another method comprises administering the hybridomas to a mammal compatible with the hybridomas to proliferate them, and obtaining monoclonal antibodies in ascites fluid. The former method is suitable for obtaining an antibody with high purity. The latter method is suitable for mass production of antibodies.

3. Recombinant antibodies

The monoclonal antibody which can be used in the invention is a recombinant monoclonal antibody, and the preparation method thereof is as follows: antibody genes are cloned from hybridomas using genetic engineering techniques, the genes are integrated into a suitable vector, the vector is introduced into a host, and the host is then allowed to produce recombinant monoclonal antibodies (see, e.g., Vandamm, A.M., et al, Eur.J.biochem. (1990)192, 767. sup. 775, 1990). Specifically, mRNA encoding the variable (V) region of the anti-glypican 3 antibody is isolated from the hybridoma producing the anti-glypican 3 antibody. methods for isolating mRNA are known, for example, the guanidine ultracentrifugation method (Chirgwin, J.M., et al, Biochemistry (1979)18, 5294-. Then, the target mRNA is prepared by using an mRNA purification kit (Pharmacia) or the like. Alternatively, mRNA can be prepared directly using the QuickPrepmRNA purification kit (Pharmacia).

cDNA for the antibody V region was synthesized from the mRNA prepared above using reverse transcriptase. The cDNA was synthesized using AMV reverse transcriptase first strand cDNA Synthesis kit (SEIKAGAKU CORPORATION). Furthermore, for the synthesis and amplification of cDNA, for example, PCR can be carried out using 5 '-Ampli FINDER RACE kit (Clontech) and 5' -RACE method (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).

The target DNA fragment is purified from the PCR product thus obtained, and then ligated to a vector DNA. Further, a recombinant vector is prepared from the product, and the vector is further introduced into Escherichia coli (Escherichia coli) or the like, and clones are selected, thereby preparing a desired recombinant vector. The nucleotide sequence of the target DNA is confirmed by a known method, for example, the dideoxynucleotide chain termination method.

After the DNA encoding the V region of the target anti-glypican 3 antibody is obtained, the DNA is integrated into an expression vector containing the DNA encoding the constant region (C region) of the desired antibody.

To produce the anti-glypican 3 antibody used in the invention, the antibody gene is integrated into an expression vector so that the gene is expressed under the regulation of a gene expression control region including, for example, an enhancer and a promoter. Next, the host cell is transformed with the expression vector, and the antibody is expressed by the host.

Expression of antibody Gene the DNA encoding the heavy chain (H chain) of the antibody or the DNA encoding the light chain (L chain) of the antibody can be incorporated into an expression vector, respectively, and then the two expression vectors are simultaneously transformed into a host cell; alternatively, the DNA encoding the H chain and the L chain may be incorporated into a single expression vector, which is then transformed into a host cell (see WO 94/11523).

In addition to the host cells described above, 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 uniquely produced in milk (e.g., goat β casein). 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 then introduced into a female goat. The desired antibodies are obtained from milk produced from offspring produced from the transgenic goat or goat that has received the embryo. In addition, in order to increase the amount of milk containing the desired antibody produced by the transgenic goat, a hormone may be administered to the transgenic goat (Ebert, K.M. et al, Bio/Technology (1994)12, 699-702).

4. Altered antibodies (altered antibodies)

In the present invention, in addition to the above-mentioned antibodies, artificially altered gene recombinant antibodies such as chimeric antibodies or humanized antibodies can be used, for example, to reduce xenogeneic antigenicity against humans. These altered antibodies can be produced by known methods.

The chimeric antibody was prepared as follows: the DNA encoding the V region of the above-mentioned antibody is ligated to the DNA encoding the C region of the human antibody, the product is integrated into an expression vector, and the vector is then introduced into a host to allow the host to produce the antibody. By using such a known method, a chimeric antibody useful in the present invention can be obtained.

Humanized antibodies are also constructed human antibodies, which are prepared by grafting the CDRs (complementarity determining regions) of an antibody of a mammal other than a human, such as a mouse, to the CDRs of a human antibody. The general gene recombination technique thereof is also known (see European patent application publication Nos. EP 125023 and WO 96/02576).

Specifically, the synthesis of a DNA sequence is carried out by a PCR method using, as primers, oligonucleotides which have been prepared in advance and have a portion in which the termination region of the CDR of a mouse antibody and the Framework Region (FR) of a human antibody overlap (see the method described in WO 98/13388).

Framework regions linked to CDRs with good antigen binding sites were selected. Amino acids in the framework regions of the antibody variable regions can be substituted, if desired, in order to reconstitute the CDRs of the human antibody to form a suitable antigen-binding site (Sato, K. et al, Cancer Res. (1993)53, 851-.

For the C region of the chimeric antibody and the humanized antibody, a human antibody region was used. For example, for an H chain, C γ 1, C γ 2, C γ 3, or C γ 4 may be used, while for an L chain, C κ or C λ may be used. In addition, the human antibody C region may be modified in order to improve the stability of the antibody or its production.

Chimeric antibodies consist of a variable region derived from an antibody of a mammal other than a human and a constant region derived from a human antibody. Meanwhile, a humanized antibody is composed of CDRs of an antibody derived from a mammal other than a human and a framework region and a C region derived from a human antibody. Since the antigenicity of the humanized antibody is intended to be low in human, it can be used as an active ingredient of the therapeutic agent of the present invention.

5. Modified antibodies

The antibody used in the present invention is not limited to the whole molecule as long as it can bind to glypican 3 and inhibit cell proliferation, and may be an antibody fragment or a modified product thereof. Both bivalent and monovalent antibodies are included. Examples of antibody fragments include Fab, F (ab') 2, Fv, Fab/c comprising one Fab and a complete Fc, and single chain Fv (scFv), wherein the Fv of H or L chain is linked to a suitable linker. Specifically, antibody fragments are synthesized by treating antibodies with enzymes such as papain or pepsin, or genes encoding these antibody fragments are constructed, introduced into expression vectors, and then expressed by appropriate host cells (see, for example, Co., M.S. et al, J.Immunol. (1994)152, 2968-.

The H chain V region of the antibody is linked to the L chain V region to obtain scFv. In scFv, the H chain V region and the L chain V region are joined by a linker or preferably by 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 derived from any of the antibodies as described in the present specification. As the peptide linker for connecting the V region, for example, any single-chain peptide having 12 to 19 amino acid residues is used.

DNA encoding scFv can be obtained as follows. Amplification is carried out by a PCR method using the whole or a part of the DNA encoding the desired amino acid sequence (the DNA encoding the H chain or H chain V region and the DNA encoding the L chain or L chain V region of the above-mentioned antibody) as a template and a primer pair defining both ends. Then, the amplification is further performed by using a DNA encoding a peptide linker moiety in combination with a primer pair defined to have both ends linked to the H chain and the L chain, respectively.

In addition, once the DNA encoding the scFv is prepared, an expression vector containing the DNA and a host transformed with the expression vector can be obtained according to standard methods. In addition, by using a host, scFv can be obtained according to standard methods.

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

As the modified antibody, an anti-glypican antibody conjugated with polyethylene glycol (PEG) or one of various molecules such as cytotoxic substances can be used. The "antibody" in the present invention also includes these modified antibodies. Such modified antibodies can be obtained by chemically modifying the resulting antibodies. Furthermore, antibody modification methods have also been established in the art.

Furthermore, the antibody used in the present invention may be a bispecific antibody. Bispecific antibodies can have antigen binding sites that recognize different epitopes on the glypican 3 molecule. Alternatively, one antigen binding site may recognize glypican 3, and the other antigen binding site may recognize a cytotoxic substance such as a chemotherapeutic agent, a cell-derived toxin, a radioactive substance, or the like. In this case, the cytotoxic substance is allowed to act directly on the cells expressing glypican 3 to specifically destroy the tumor cells, so that the proliferation of the tumor cells can be inhibited. Bispecific antibodies can be prepared by binding two antibodies of the H-L pair, it can also be through the fusion of different monoclonal antibody producing hybridoma to prepare bispecific antibody producing fusion cells. In addition, bispecific antibodies can also be prepared by genetic engineering techniques.

6. Expression and production of recombinant or modified antibodies

The antibody gene constructed as described above can be expressed and obtained by a known method. In the case of mammalian cells, the gene can be expressed by operably linking a commonly used useful promoter and an antibody gene to be expressed and by linking a polyA signal located downstream of the 3' end thereof. For example, the promoter/enhancer is the human cytomegalovirus immediate early promoter/enhancer.

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

Gene expression can be readily carried out by the method of Mullgan 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 E.coli, a commonly used useful promoter, a signal sequence for antibody secretion, and an antibody gene to be expressed are operably linked so that the gene can be expressed. Examples of the promoter include 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; FASEB J. (1992)6, 2422-2427), and 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, a pelB signal sequence can be used when the antibody is produced in the periplasm of E.coli (Lei, S.P. et al, J.Bacteriol. (1987)169, 4379). After the antibody produced in the periplasm is isolated, the structure of the antibody is suitably folded and used.

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

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

Preferably, the antibody for use in the present invention is expressed in mammalian cells such as CHO, COS, myeloma, BHK, Vero or Hela cells.

The transformed host cells are then cultured in vitro or in vivo to allow the host cells to produce the target antibody. The host cells are cultured according to known methods. For example, DMEM, MEM, RPMI1640, IMDM, or the like can be used as the culture medium. Serum fluids such as Fetal Calf Serum (FCS) may be used in combination.

7. Isolation and purification of antibodies

The antibodies expressed and produced as described above can be isolated from the cells or host animals and purified to a consistent level. The isolation and purification of antibodies to be used in the present invention may be carried out using an affinity column. Examples of using protein a columns are Hyper D, POROS, sepharose f.f. (Pharmacia). Other standard methods for protein isolation and purification can be used without limitation. For example, a chromatography column other than the above-mentioned affinity column, diafiltration, ultrafiltration, salting-out method, dialysis, etc. may be appropriately selected and used in combination for the purpose of separating and purifying Antibodies (Antibodies A Laboratory Manual. Ed harbor, David Lane, Cold spring harbor Laboratory, 1988).

8. Confirmation of antibody Activity

Known methods can be used to assay the Antibodies used in the present invention for antigen binding activity (Antibodies A Laboratory Manual. Ed Harlow, David Lane, Cold spring harbor Laboratory, 1988) and for activity in inhibiting ligand receptor binding (Harada, A. et al, International Immunology (1993)5, 681) 690).

As a method for measuring the antigen binding activity of the anti-glypican 3 antibody used in the present invention, ELISA (enzyme-linked immunosorbent assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluorescent antibody technique can be used. For example, when an enzyme immunoassay is used, a sample containing an anti-glypican 3 antibody, such as a culture supernatant of cells producing the anti-glypican 3 antibody, or a purified antibody is added to a plate coated with glypican 3. A second antibody labeled with an enzyme such as alkaline phosphatase is added. The plates are then incubated, washed, and an enzyme substrate, such as p-nitrophenyl phosphate, is added, followed by measurement of absorbance, in order to evaluate antigen binding activity.

To confirm the activity of the antibody used in the present invention, the neutralizing activity of the anti-glypican 3 antibody was measured.

9. Cytotoxic Activity

The antibody used in the present invention has ADCC activity or CDC activity as cytotoxic activity.

ADCC activity can be measured by mixing effector cells, target cells, and anti-glypican 3 antibody, and then detecting the level of ADCC. As the effector cells, for example, mouse spleen cells, human peripheral blood or single cells isolated from bone marrow may be used. As the target cell, for example, a human-established cell line such as HuH-7 human hepatoma cell line can be used. The target cells are used in advance⁵¹Cr labeling, anti-glypican 3 antibody was added to the cells, followed by incubation. Next, effector cells are added in an appropriate ratio of effector cells to target cells, followed by incubation. After incubation, the supernatant is collected and the radioactivity in the supernatant is counted, whereby ADCC activity can be determined.

The CDC activity was measured as follows: the labeled target cells and the anti-glypican 3 antibody are mixed, complement is added, incubated, cultured, and then radioactivity in the supernatant is counted.

Antibodies require an Fc portion to exert cytotoxic activity. When the cytostatic agent of the present invention utilizes the cytotoxic activity of an antibody, the anti-glypican 3 antibody used in the present invention needs to contain an Fc portion.

10. Inhibiting angiogenesis

The anti-glypican 3 antibody of the invention can be used for inhibiting angiogenesis.

11. Administration method and pharmaceutical preparation

The cytostatic agents of the invention are useful for treating or ameliorating conditions caused by diseases based on abnormal cell proliferation, particularly cancer.

Preferred examples of the target cancer cell of the cell growth inhibitor of the present invention include, but are not specifically limited to, liver cancer cells, lung cancer cells, colon cancer cells, breast cancer cells, prostate cancer cells, leukemia cells, lymphoma cells, and pancreatic cancer cells. Liver cancer cells are particularly preferred.

The effective dose is selected from the range of 0.001mg to 1000mg per kg body weight per administration. Alternatively, the dose range may be selected from 0.01 to 100000mg per patient. However, the therapeutic agent containing the anti-glypican 3 antibody of the invention is not limited to these dosages.

In addition, as the time of administration of the therapeutic agent of the present invention, the agent may be administered before or after the onset of clinical symptoms of the disease.

Therapeutic agents containing the anti-glypican 3 antibody of the invention as an active ingredient can be formulated according to standard methods (Remington's Pharmaceutical Science, latest edition, MarkPublishing Company, Easton, u.s.a.) and can contain pharmaceutically acceptable carriers and additives together.

Examples of such carriers and pharmaceutical additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, Human Serum Albumin (HSA), mannitol, sorbitol, lactose, and acceptable surfactants as pharmaceutical additives.

One or a suitable combination of the above additives is selected in practice according to the dosage form of the therapeutic agent of the present invention, but is not limited thereto. For example, a pharmaceutical agent useful as a pharmaceutical preparation for injection is prepared by dissolving a purified anti-glypican 3 antibody in a solvent such as physiological saline, a buffer solution or a glucose solution, and then adding an adsorption inhibitor such as Tween80, Tween20, gelatin or human serum albumin to the solution. Alternatively, the lyophilizates can be used to prepare dosage forms which are reconstituted for dissolution prior to use. As the excipient for lyophilization, for example, sugar alcohol such as mannitol or glucose or saccharide can be used.

Brief Description of Drawings

FIG. 1 shows ADCC activity of anti-glypican 3 antibody (K6534) on HuH-7 cells.

FIG. 2 shows CDC activity of anti-glypican 3 antibody (K6511) against HuH-7 cells.

FIG. 3 shows expression of glypican on HuH-7 cells.

Fig. 4A shows the results of FACS analysis expressing GPC3 from a human lung cancer cell line. They were analyzed with an anti-glypican 3 antibody (K6534).

FIG. 4B shows the results of FACS analysis of expression of GPC3 from human leukemia cell lines. They were analyzed with an anti-glypican 3 antibody (K6534).

FIG. 4C shows the results of FACS analysis of GPC3 expressed by human lymphoma cell line. They were analyzed with an anti-glypican 3 antibody (K6534).

FIG. 4D shows the results of FACS analysis of GPC3 expressed by human colon cancer cell lines. They were analyzed with an anti-glypican 3 antibody (K6534).

Fig. 4E shows the results of FACS analysis expressing GPC3 from human breast cancer cell line. They were analyzed with an anti-glypican 3 antibody (K6534).

Fig. 4F shows the results of FACS analysis expressing GPC3 from human prostate cancer cell line. They were analyzed with an anti-glypican 3 antibody (K6534).

Fig. 4G shows the results of FACS analysis expressing GPC3 from human pancreatic cancer cell line and human liver cancer cell line. They were analyzed with an anti-glypican 3 antibody (K6534).

Best Mode for Carrying Out The Invention

The invention will be further described with reference to the following examples. However, the technical scope of the present invention is not limited by these examples and the like.

EXAMPLE 1 preparation of monoclonal antibody against glypican-3 synthetic peptide

A peptide having the amino acid sequence of human glypican-3 protein (amino acids from 355 to 371) (RQYRSAYYPEDLFIDKK) was synthesized. The immunogen was prepared by binding a synthetic peptide to Keyhole Limpet Hemocyanin (KLH) using a Maleimide Benzoyloxy Succinimide (MBS) -type cross-linking agent. Mice (BALB/c, female, 6 weeks old) were immunized 3 times with immunogen at a dose of 100 μ g per mouse. Antibody titers in sera were analyzed. The antibody titer measurement method included reacting diluted serum with a peptide (0.5. mu.g) immobilized on a plate, reacting with an HRP-labeled anti-mouse antibody, adding a substrate, and then measuring the absorbance at 450nm of the developed color (peptide solid phase ELISA method). After confirmation of the antibody titer, spleen cells were collected and fused with myeloma cells (P3/X63-Ag8) (K ö hler, G, Milstein, C: Nature, 256: 495(1975)), thereby preparing hybridomas. The monoclonal antibodies produced by the 5 hybridomas were then purified. The binding activity to the peptide was measured by a peptide solid phase ELISA method, and then IgG1 antibody (hereinafter referred to as K6534) and IgG3 antibody (hereinafter referred to as K6511) having high binding activity were selected.

Example 2 inhibition of cell proliferation Using anti-glypican 3 antibody

ADCC (antibody-dependent cell-mediated cytotoxicity) activity and CDC (complement-dependent cytotoxicity) activity were determined according to the methods described in Current Protocols in Immunology, Chapter 7, Immunologic studiesin humans, John E, Cologan et al, eds., John Wiley & Sons, Inc., 1993.

1. Preparation of Effector cells

Spleens were excised from CBA/N mice (8 weeks old, male) and splenocytes were isolated in RPMI1640 medium (GIBCO). The cells were washed in the same medium containing 10% peptide bovine serum (FBS, HyClone) to give a cell concentration of 5X 10⁶mL, thereby preparing effector cells.

2. Preparation of complement solution

The complement solution was prepared by diluting young rabbit Complement (CEDARLANE) 10-fold in 10% FBS-containing DMEM medium (GIBCO).

3. Preparation of target cells

The HuH-7 Human liver cancer cell line (Japanese Collection of Research biosources (JCRB) No. JCRB0403, Human Science Research SupportBank (Human Science Kenkyu Shien Bank)) was used at 0.2mCi⁵¹Cr-sodium chromate (Amersham Pharmacia Biotech) was cultured in 10% FBS-containing DMEM medium at 37 ℃ for 1 hour, thereby performing radiolabelling. After radiolabelling, cells were washed 3 times in 10% FBS-containing RPMI1640 medium to give cell concentrations of2×10⁵mL, thereby preparing target cells.

4. Measurement of ADCC Activity

50. mu.l each of the target cells and the anti-glypican 3 antibody (K6534) were added to a 96-well U-shaped bottom plate (Beckton Dickinson) and reacted on ice for 15 minutes. Then, 100. mu.l of effector cells were added and cultured in a carbon dioxide gas incubator for 4 hours. The final concentration of the antibody was made to be O or 10. mu.g/mL. After incubation, 100. mu.L of the supernatant was collected and radioactivity was measured by a GAMMA counter (COBRA II AUTO-GAMMA, MODEL D5005, packard Instrument Company). The cytotoxic activity (%) was determined by the formula (A-C)/(B-C). times.100. Where "A" represents the radioactivity (cpm) in each sample, "B" represents the radioactivity (cpm) in the sample supplemented with 1% NP-40(nacalai tesque), and "C" represents the radioactivity (cpm) in the sample containing only target cells. The test was repeated twice and the average was calculated.

5. Determination of CDC Activity

50 μ l each of the target cells and the anti-glypican 3 antibody (K6511) were added to a 96-well flat-bottom plate (Beckton Dickinson) and reacted on ice for 15 minutes. Then, 100. mu.l of a complement solution was added, followed by incubation in a carbon dioxide gas incubator for 4 hours. The final concentration of antibody was 0 or 3. mu.g/mL. After incubation, 100 μ L of the supernatant was collected and radioactivity was measured using a γ counter. The cytotoxicity (%) was determined in the same manner as used in "4. measurement of ADCC activity". The test was repeated twice and the average was calculated.

Figure 1 shows ADCC activity, while figure 2 shows CDC activity. These results revealed that the anti-glypican 3 antibody exerts ADCC activity and CDC activity against the HuH-7 human liver cancer cell line, thereby inhibiting cell proliferation.

Example 3 determination of expression levels of glypican on HuH-7 cells

Will be about 5X 10⁵Individual HuH-7 cells were suspended in 100. mu.l FACS/PBS (by suspending 1g bovine serum albuminWhite (SIGMA) was prepared by dissolving in 1L CellWASH (Beckton Dickinson). Then, an anti-glypican 3 antibody (K6511) or mouse IgG2a (Biogenesis) was added as a control antibody at a concentration of 25. mu.g/mL, and the solution was allowed to stand on ice for 30 minutes. After washing with FACS/PBS, the product was suspended in 100. mu.l of FACS/PBS. Mu.l of goat anti-mouse Ig FITC (Becton Dickinson) was added and the solution was allowed to stand on ice for 30 minutes.

After washing twice with FACS/PBS, the product was suspended in 1ml of FACS/PBS. The fluorescence intensity of the cells was measured using a flow cytometer (EPICS XL, BECKMAN COULTER).

Fig. 3 shows the results of the flow cytometry assay. Glypican 3 was expressed on HuH-7 cells, suggesting that an anti-glypican 3 antibody binds to glypican 3 expressed on cells, thereby inhibiting cell proliferation (fig. 3).

Example 4 analysis of GPC3 expression on cancer cell groups Using FCM

Human cancer cell lines (lung, colon, rectum, breast, prostate, leukemia, lymphoma, myeloma, pancreas and liver) were analyzed for glypican 3(GPC3) expression using FCM.

Cells were cultured for 2 days and then analyzed. The attached cells were collected using 10-fold dilution of trypsin-EDTA (Cat. No. 25300-054, Cat. No. 14210, GIBCO) with cell dissociation buffer (Cat. No. 13150-016, Cat. No. 1098554, GIBCO). The collected cells were reacted on ice with an anti-glypican 3 antibody (K6534, 600. mu.g/ml) or mIgG2a antibody as a negative control (Biogenesis, M-IgG2a-i, batch No. EA990719A, 1mg/ml) (final antibody concentration 10. mu.g/ml). After washing, cells were reacted with FITC-labeled anti-mouse Ig antibody (cat 349031, BD PharMingen) on ice (2 μ l/test). After cell washing, the fluorescence intensity was measured using a flow cytometer (EPICS XL, BECKMAN COULTER). As a result, the expression of GPC3 was confirmed in the following cell lines: lung cancer cell lines (A549, NCI-H460, NCI-H23, NCI-H226, DMS114, EKVX, HOP-62 and NCI-H322M; leukemia cell lines P30/OHK, BALL-1, THP-1 and P39/TSU), lymphoma cell lines (MLMA, Ramos and U937) -, colon cancer cell lines (SW480, COLO205, Lo Vo and SW837), breast cancer cell lines (MDA-MB-231, SK-BR-3 and MDA-MB-468), prostate cancer cell lines (LNCaP and 22Rvl), pancreatic cancer cell lines (MIAPaCa-2) and liver cancer cell lines (HepG 2). From these results, it can be concluded that: the cell growth inhibitor of the present invention is useful in the treatment of lung cancer, colon cancer, breast cancer, prostate cancer, leukemia, lymphoma, pancreatic cancer, and the like.

Industrial applicability

According to the present invention, there is provided a cell growth inhibitor comprising an anti-glypican 3 antibody as an active ingredient. Further, according to the present invention, there is provided a cancer cell growth inhibitor containing an anti-glypican 3 antibody as an active ingredient. The anti-glypican 3 antibody binds to glypican 3 expressed on HuH-7 cells of a human liver cancer cell line, thereby inhibiting cell proliferation. Thus, agents containing anti-glypican 3 antibodies are useful as cytostatics, particularly as cancer cell inhibitors.

All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Therefore, those skilled in the art will readily appreciate that numerous modifications and variations are possible without departing from the technical concept and scope of the present invention as set forth in the appended claims. It is intended that the present invention include such modifications and variations.

Claims (6)

1. A cell growth inhibitor for cancer cells, which comprises an anti-glypican 3 antibody as an active ingredient.

2. The cytostatic agent of claim 1, wherein the anti-glypican 3 antibody has cytotoxic activity.

3. The cytostatic agent of claim 2, wherein the cytotoxic activity is an antibody-dependent cell-mediated cytotoxic activity or a complement-dependent cytotoxic activity.

4. The cytostatic agent of any one of claims 1-3, wherein the cell is a liver cancer cell.

5. The cytostatic agent of any one of claims 1-3, wherein the antibody is a monoclonal antibody.

6. The cytostatic agent of any one of claims 1-3, wherein the antibody is a humanized antibody or a chimeric antibody.