HK1105993B - Anti-a33 antibody - Google Patents

Description

anti-A33 antibodies

Technical Field

The present invention relates to anti-A33 antibodies that specifically bind to the A33 antigen. Further, the present invention relates to a prophylactic or therapeutic agent for a disease caused by a cell expressing a33, which comprises an anti-a 33 antibody as an active ingredient. The invention relates in particular to a therapeutic agent for malignant tumors.

Background

Cancer (tumor) is the leading cause of death in japan, and the number of cancer patients is increasing every year. Therefore, there is a strong need for drug development or therapeutic methods that can treat cancer with high efficiency and safety. Research studies conducted in 1999 have shown that, among various cancers, colorectal cancer accounts for 12.2% of the total number of cancers. The mortality rate for colorectal cancer ranks third in men and second in women. According to the significantly increasing cases of colorectal cancer in recent years, it is expected that the incidence or mortality of colorectal cancer may later surpass that of gastric cancer. In addition, investigations conducted in 1999 have shown that gastric cancer accounts for 17.4% of the total number of cancers, and that the mortality rate is second in men and first in women.

The use of antibodies as pharmaceuticals has been recognized as an important and valuable approach to the treatment of various pathological conditions (cancer types). The specificity of antibodies is very useful for the treatment of pathological conditions where tumor-specific antigens may exhibit heterologous cellular properties. Antibodies can effectively act on these cells by binding to tumor-specific antigens, proteins expressed on the cell surface. Currently, chimeric antibodies (Rituximab) against CD20 (receptor present on cell membrane), monoclonal antibodies such as humanized antibodies against Her2/neu, and the like have been used for the treatment of malignant diseases. The therapeutic effect thereof has been confirmed. Antibodies are characterized by a long blood half-life and high specificity for antigens, and therefore they are particularly suitable for use as antitumor agents. For example, for antibodies directed against tumor-specific antigens, it can be concluded that the administered antibody will accumulate in the tumor. In addition, it is also expected that the immune system of tumor cells can be attacked by complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC). Furthermore, by binding a drug (e.g., a radionuclide or a cytotoxic substance) to an antibody in advance, the bound drug can be efficiently delivered to a tumor site. At the same time, the amount of the medicine reaching other non-specific tissues is reduced, so that the side effect of the medicine can be reduced. Administration of antibodies with competitive activity when tumor-specific antigens have activity to induce cell death, or neutralizing activity when tumor-specific antigens are involved in cell growth or survival, can accumulate these tumor-specific antibodies and halt tumor growth or regress tumors by the activity of these antibodies. Therefore, they are considered to be suitable as antitumor agents based on the above characteristics of the antibodies.

Mice were used as target animals in early antibody production. However, the use of murine antibodies in vivo is limited by a number of factors. Murine antibodies recognized by the human host as foreign can induce "human anti-mouse antibodies", an induction known as the "HAMA" response (see Schiff et al, Canc. Res. (1985), 45, 879-885). In addition, the Fc portion of murine antibodies is not effective in stimulating human complement or cytotoxic effects.

Chimeric antibodies have been developed as a way to avoid these problems (see European patent application Nos.120694 and 125023). Chimeric antibodies comprise antibody portions derived from 2 or more species (e.g., the variable regions of a murine antibody and the constant regions of a human antibody). The chimeric antibody has the advantages of maintaining the characteristics of the murine antibody and stimulating human complement or cytotoxic effects due to the presence of human Fc. However, the chimeric antibody can still induce human anti-chimeric antibody; i.e., a "HACA" response (see Bruggemann, et al., j. exp. med., 170, 2153-.

In addition, recombinant antibodies have been developed in which only one of the alternative antibody portions is a complementarity determining region (i.e., "CDR") (british patent No. gb2188638a and U.S. patent No. 5585089). Antibodies comprising murine CDRs, human variable region frameworks, and constant regions (i.e., "humanized antibodies") have been produced by CDR grafting techniques (see Richmann, et al, Nature (1988), 332, 323-.

Murine anti-A33 antibodies, and tumor-specific antigens as well as humanized antibodies against members of the Ig superfamily, an antigen known as the type I cell membrane protein of "A33", have been reported (see the description of U.S. Pat. No. 5958412; King D.J.et., British J.cancer (1995)72, 1364. 1372; Welt S.et., J.clinical Oncology (1994), 12, 1561. 1571; Welt S.et., J.clinical Oncology (1996), 14, 1787. 1797; Welt S.et., Clinical Cancer Res. (2003), 9, 1338. 1346; and Welt S.et., Clinical Cancer Res. (2003), 9, 1347. 1353). Such antigens are known to be involved in colon and gastric cancers (see the specification of U.S. Pat. No. 5643550; the specification of U.S. Pat. No. 5160723; and Garin-Chesa P.G.et al, International J.Oncology (1996), 9, 465-471). In addition, in recent years, phase I Clinical trials of humanized A33 antibody have been conducted in colon Cancer patients (see Welt S.et al, Clinical Cancer Res. (2003), 9, 1338-. In previous reports on antibody administration alone, partial responses were observed in only 1 of 11 patients administered with antibody. In the subsequent reports on experiments using the antibody in combination with chemotherapy, partial responses were observed in 3 out of 12 patients administered with the antibody, of which 1 mixed response was observed. Even though Avastin (i.e., Bevacizumab; humanized anti-VEGF antibody) developed by Genentech in recent years, it has been reported that one of 12 patients showed a partial response in phase I clinical trials using Avastin in combination with standard chemotherapy (Margolin K.et al, J.Clin.Oncol. (2001)19, 851-. Therefore, based on the fact that administration of an antibody alone would cause one of 11 patients to exhibit a partial response, it is expected that the antibody will exhibit a broad anti-tumor effect against colorectal cancer.

As mentioned above, although the humanized a33 antibody showed a very significant tumor response in phase I clinical trials, there was still a high probability of up to 50% or even higher in both trials of production of human anti-humanized antibodies (i.e., "HAHAs"). Interestingly, no HAHA was observed in those patients who showed high tumor reactivity with the antibody.

Summary of The Invention

The present invention aims to provide an agent for preventing or treating various malignant tumors including solid tumors, which are currently difficult to treat, by developing an antibody that can bind to a33, specifically attacks a 33-expressing tumor cells by ADCC or CDC based on the immune system, and does not produce HAHA.

As described above, it has been considered that an antibody against the a33 antigen is suitable as an antitumor agent. In addition, by using such an antibody that does not produce HAHA, a higher antitumor effect can be obtained. Thus, through intensive studies on how the anti-a 33 antibody was produced, the present inventors have succeeded in obtaining a monoclonal antibody having an anti-tumor effect on a 33-expressing cancer cells and determining the sequence of the variable region of the monoclonal antibody, thereby completing the present invention.

The present invention will be described in detail below.

First, the present invention provides a monoclonal antibody that binds to a33, which is produced by a mouse-mouse hybridoma. In particular, a monoclonal antibody, preferably a human antibody, or a functional fragment thereof, produced by e.g. 263a17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA or 125R5AAAA is provided. The type of such monoclonal antibodies produced by 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5AAAA is human immunoglobulin G (IgG) type. Among the above-mentioned hybridomas, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were deposited at 24.8.2004 at the International Patent Organism Depositary (IPOD) of the national institute for Advanced Industrial Science and Technology (AIST) (Central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan) under accession numbers FERM BP-10107 (identified by M10), FERM BP-10106 (identified by M165), FERM-10108 (identified by M96), FERM BP-10109 (identified by N26), FERM BP-10104 (identified by Q47), FERM BP-10105 (identified by Q54), and FERM BP-3903 (identified by R5).

In one embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof containing the variable region of an antibody produced by one of the above hybridomas.

In another embodiment of the present invention, examples of antibodies of the present invention include those in modified subclasses. Specifically, the antibody of the present invention may be: an antibody or a functional fragment thereof produced by hybridoma 263a17 and belonging to subclass human IgG1, human IgG2, human IgG3 or human IgG 4; an antibody or a functional fragment thereof produced by hybridoma 125M10AA and belonging to the subclass human IgG1, human IgG2, human IgG3, or human IgG 4; an antibody or a functional fragment thereof produced by hybridoma 125M165DAAA and belonging to subclass human IgG1, human IgG2, human IgG3 or human IgG 4; an antibody or a functional fragment thereof produced by hybridoma 125M96ABA and belonging to subclass human IgG1, human IgG2, human IgG3 or human IgG 4; an antibody or a functional fragment thereof produced by hybridoma 125N26F6AA and belonging to subclass human IgG1, human IgG2, human IgG3 or human IgG 4; an antibody or a functional fragment thereof produced by hybridoma 125Q47BA and belonging to the subclass human IgG1, human IgG2, human IgG3, or human IgG 4; an antibody or a functional fragment thereof produced by hybridoma 125Q54AAAA and belonging to subclass human IgG1, human IgG2, human IgG3 or human IgG 4; an antibody or a functional fragment thereof produced by hybridoma 125R5AAAA and belonging to the subclass human IgG1, human IgG2, human IgG3 or human IgG 4.

In another aspect of the present invention, there is provided an antibody or a functional fragment thereof which binds to a33 and comprises the variable region of an antibody produced by hybridoma 263a17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5 AAAA.

In one embodiment of the invention, the antibody of the invention is an antibody having the variable region amino acid sequence of SEQ ID NOS: 23 and 25 or a functional fragment thereof. In another embodiment of the invention, an antibody of the invention is a variable region having the amino acid sequence of SEQ ID NOS: 27 and 29 or a functional fragment thereof. In another embodiment of the invention, an antibody of the invention is a variable region having the amino acid sequence of SEQ ID NOS: 31 and 33 or a functional fragment thereof. In another embodiment of the invention, the antibody of the invention is an antibody having a variable region amino acid sequence of SEQ id no: 35 and 37 or a functional fragment thereof. In another embodiment of the invention, an antibody of the invention is a variable region having the amino acid sequence of SEQ ID NOS: 39 and 41 or a functional fragment thereof. In another embodiment of the invention, an antibody of the invention is a variable region having the amino acid sequence of SEQ ID NOS: 43 and 45 or a functional fragment thereof. In another embodiment of the invention, an antibody of the invention is a variable region having the amino acid sequence of SEQ ID NOS: 47 and 49 or a functional fragment thereof. In another embodiment of the invention, an antibody of the invention is a variable region having the amino acid sequence of SEQ ID NOS: 51 and 53 or a functional fragment thereof. In another embodiment of the invention, the antibody of the invention is a full domain antibody having the amino acid sequence of SEQ ID NOS: 87 and 89, or a functional fragment thereof.

In another aspect, the present invention further provides the above-described antibody or a functional fragment thereof, which can inhibit the growth of a tumor (e.g., derived from the colorectal cancer cell line COLO205 implanted in a nude mouse). For tumor inhibition, the antibody or functional fragment thereof of the present invention is administered to a tumor-bearing experimental animal (e.g., a tumor-bearing experimental animal such as a mouse model having a tumor of colon cancer cells weighing 20 g) at a dose of 10. mu.g/body weight to 100. mu.g/body weight. For example, the dose is 100. mu.g/body weight or 5mg/kg, preferably 10. mu.g/body weight or 0.5 mg/kg.

In one embodiment of the invention, the antibody of the invention has one of the following characteristics.

(a) ADCC assay

The antibodies of the invention have antibody-dependent cellular cytotoxicity (ADCC) against a human cancer cell expressing a33 when normal human peripheral blood mononuclear cells are present.

(b) CDC experiment

The antibodies of the invention have complement-dependent cytotoxic effects against human cancer cells expressing a33 in the presence of human serum-derived complement.

(c) In vivo experiments

The antibody of the present invention has an antitumor effect against a non-human animal containing a human cancer cell expressing a 33.

(d) Competition experiment

The antibody of the present invention can (i) strongly compete (blocker), (ii) weakly compete (partial blocker) or (iii) not compete (non-blocker) with chimeric anti-A33 (consisting of the heavy chain variable region and the light chain variable region of the antibody produced by hybridoma ATCC HB-8779 and the heavy chain constant region and the light chain constant region of human IgG 1).

(e) Immunohistochemical assay

The antibodies of the invention stain human adult colon cancer tissue, human adult normal colon tissue, and human normal small intestine tissue in the presence of the antibodies.

In another aspect, the present invention further provides: a nucleic acid encoding an antibody possessed by a hybridoma selected from the group consisting of hybridomas 125M10AA (accession number FERM BP-10107), 125M165DAAA (accession number FERM BP-10106), 125M96ABA (accession number FERM BP-10108), 125N26F6AA (accession number FERM BP-10109), 125Q47BA (accession number FERM BP-10104), 125Q54AAAA (accession number FERM BP-10105) and 125R5AAAA (accession number FERM BP-10103), or a nucleic acid encoding a functional fragment of the antibody; a protein encoded by a nucleic acid; an expression vector containing the above nucleic acid; and a host selected from the group consisting of E.coli, yeast cells, insect cells, mammalian cells, plant cells, and mammals containing the expression vector.

In another aspect, the present invention further provides a method for producing an anti-a 33 monoclonal antibody or a functional fragment thereof, comprising: isolating a gene encoding an anti-a 33 monoclonal antibody (e.g., a gene encoding a variable region of a heavy chain amino acid sequence and a gene encoding a variable region of a light chain amino acid sequence) from a hybridoma selected from the group consisting of hybridomas 263a17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5 AAAA; constructing an expression vector containing the genes; introducing the expression vector into a host; culturing a host; expressing the monoclonal antibody; and recovering the anti-A33 monoclonal antibody or a functional fragment thereof from the obtained host or a culture such as a culture supernatant of the host, a secretion product of the host, or the like.

In another aspect, the present invention further provides a tumor preventive, therapeutic or diagnostic agent comprising the above antibody or a functional fragment thereof as an active ingredient.

Examples of the tumor that can be prevented or treated using the above-mentioned agent include at least one tumor selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, stomach cancer, pancreatic cancer, breast cancer, melanoma, renal cell carcinoma, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, prostate cancer, testicular cancer, and mesothelial cancer.

In another embodiment of the present invention, the antibody or functional fragment thereof of the present invention is characterized in that it has been confirmed that the above antibody or functional fragment thereof at a dose of 10. mu.g/body or 100. mu.g/body exhibits an inhibitory effect (after tumor transplantation) on tumors in tumor-bearing nude mice in which COLO205 cells are transplanted significantly higher than in a group of mice administered intravenously or in a group of mice administered with an anti-DNP-IgG 1 antibody.

The invention also relates to: an antibody that binds to a33, which recognizes the same epitope as an epitope recognized by an antibody produced by hybridoma M10 (deposited as FERM BP-10107); an antibody that binds to a33, which recognizes the same epitope as an epitope recognized by an antibody produced by hybridoma M96 (deposited as FERM BP-10108); an antibody that binds to a33, which recognizes the same epitope as that recognized by an antibody produced by hybridoma M165 (deposited under accession number FERM BP-10106); an antibody that binds to a33, which recognizes the same epitope as an epitope recognized by an antibody produced by hybridoma N26 (deposited as FERM BP-10109); an antibody that binds to a33, which recognizes the same epitope as an epitope recognized by an antibody produced by hybridoma Q47 (deposited as FERM BP-10104); an antibody that binds to a33, which recognizes the same epitope as an epitope recognized by an antibody produced by hybridoma Q54 (deposited as FERM BP-10105); an antibody that binds to a33, which recognizes the same epitope as an epitope recognized by an antibody produced by hybridoma R5 (deposited as FERM BP-10103);

this specification includes part or all of the contents disclosed in the specification and/or drawings of japanese patent application 2004-.

Brief description of the drawings

Figure 1A shows ADCC activity determined when COLO205 cells were acted on with each purified monoclonal antibody.

FIG. 1B shows CDC activity measured when COLO205 cells were allowed to interact with each purified monoclonal antibody.

FIG. 1C shows ADCC activity determined when NCI-H508 cells were treated with each purified monoclonal antibody.

FIG. 1D shows CDC activity measured when NCI-H508 cells were targeted using each purified monoclonal antibody.

FIG. 2A shows ADCC activity measured when COLO205 cells were treated with recombinant antibodies.

FIG. 2B shows CDC activity measured when COLO205 cells were allowed to act with recombinant antibodies.

FIG. 2C shows ADCC activity measured when NCI-H508 cells were treated with recombinant antibodies.

FIG. 2D shows CDC activity measured when NCI-H508 cells were treated with recombinant antibodies.

FIG. 3A is a photograph showing the results of Western blot analysis using purified and recombinant antibodies.

FIG. 3B is a photograph showing the results of Western blot analysis using purified and recombinant antibodies.

FIG. 4 is a photograph showing the results of immunohistostaining of human colon cancer tissue using purified and recombinant antibodies.

FIG. 5 is a photograph showing the results of immunohistostaining with human normal small intestine tissue using purified and recombinant antibodies.

FIG. 6 is a photograph showing the results of immunohistostaining with human normal colon tissue using purified and recombinant antibodies.

FIG. 7A shows the antitumor effects of recombinant antibodies cA33 and rec263 against a mouse tumor-bearing model in which COLO205 cells were transplanted.

FIG. 7B shows the antitumor effect of recombinant antibodies cA33 and rec263 against a mouse tumor-bearing model in which NCI-H508 cells were transplanted.

FIG. 7C shows the antitumor effects of purified hybridoma antibodies 125M10AA, 125M165DAAA, and 125M96ABA on a mouse tumor-bearing model implanted with COLO205 cells.

FIG. 7D shows the antitumor effects of the recombinant antibodies recN26 and recM165 on a mouse tumor-bearing model into which NCI-H508 cells and Matrigel were transplanted.

FIG. 7E shows the antitumor effects of recombinant antibodies recM10 and recQ54 on a mouse tumor-bearing model in which NCI-H508 cells and Matrigel were transplanted.

Preferred embodiments of the invention

The present invention will be described in detail below.

For a33, mouse anti-a 33 antibody and humanized anti-a 33 antibody have been obtained. Phase I Clinical experiments have also been reported in colon Cancer patients with mouse anti-A33 antibodies (see Welt S.et al, J.clinical Oncology (1994), 12, 1561- & gt 1571; Welt S.et al, J.clinical Oncology (1996), 14, 1787- & 1797) and humanized A33 antibodies (see Welt S.et al, Clinical Cancer Res. (2003), 9, 1338- & 1346; Welt S.et al, Clinical Cancer Res. (2003), 9, 1347- & 1353). However, the probability of producing HAMA or HAHA in patients receiving these antibodies is high, and no further clinical trials have been conducted thereafter. However, it is very interesting that, in clinical experiments with the humanized anti-a 33 antibody, no HAHA production was observed in patients who had been confirmed to exhibit a tumor response.

The novel human anti-A33 monoclonal antibody of the present invention is a fully human antibody. Therefore, the antibody of the present invention avoids in advance the problem of antigenicity against a portion containing a mouse sequence which always occurs when a mouse antibody or a humanized antibody is used. Specifically, in the above clinical experimental report, HAHA was generated by using a humanized antibody. However, since the novel human anti-A33 monoclonal antibody of the present invention is a fully human antibody, antigenicity of the antibody can be avoided, and HAHA is not produced. Therefore, it is expected that the antibody has a significant antitumor effect on colon cancer patients.

Examples of the types of antibodies used in the present invention include immunoglobulin G (IgG), A (IgA), E (IgE), and M (IgM). A preferred antibody class for use herein is IgG. In addition, IgG1, IgG2, IgG3, or IgG4 belonging to the IgG subclass can be used. Preferably, IgG1, IgG2 or IgG4 is used, more preferably IgG1 is used.

The present invention will be described in detail below by illustrating the meanings of terms and phrases used in the present invention.

A33 and anti-A33 antibodies

The antibody of the present invention, which is a member of the Ig superfamily, is a class I cell membrane protein and is an antibody against A33.

The "antibody binding to a 33" in the present invention refers to an antibody that can react with a portion of a33 or a33, or an antibody that recognizes a portion of a33 or a 33. "functional fragment" in the context of the present invention refers to a portion (partial fragment) of an antibody that retains 1 or more of the effects of the antibody on its corresponding antigen. Specific examples of such fragments include F (ab')₂Fab', Fab, Fv, disulfide stabilized Fv, single chain Fv (scFv) and multimers thereof (D.J.King., Applications and Engineering of Monoclonal antibodies, 1998T.J.International Ltd.). Alternatively, "functional fragment" refers to a fragment of an antibody that binds to an antigen.

The "human antibody" in the present invention means that the antibody is an expression product of an antibody gene derived from a human. As described below, human antibodies can be obtained by introducing human antibody loci and then administering the antigen to a transgenic animal that can produce antibodies derived from humans. One example of such a transgenic animal is a mouse. Methods for making such human antibody producing mice are described, for example, in WO 02/43478.

As described in the following examples, examples of the antibody of the present invention include various antibodies that can exert an anti-tumor effect on cancer cells expressing a33 even at low concentrations.

The antibody of the present invention also includes a monoclonal antibody having a heavy chain and/or a light chain of an amino acid sequence obtained by deleting, substituting or adding 1 or several amino acids from the amino acid sequence of the heavy chain or the light chain as a component of the antibody. The above-mentioned partial amino acid changes (deletion, substitution, insertion or addition) can be introduced into the amino acid sequence of the antibody of the present invention by partially changing the nucleotide sequence encoding the amino acid sequence. Such partial changes in nucleotide sequence can be introduced by using standard methods of site-directed mutagenesis known (Proc Natl Acad Sci U.S.A., 1984 Vol 81: 5662). In addition, the "antibody" in the present invention is an immunoglobulin in which all regions thereof, including a heavy chain variable region and a heavy chain constant region and a light chain variable region and a light chain constant region, are derived from a gene encoding the immunoglobulin.

Antibodies of the invention also include antibodies of any immunoglobulin type or isotype.

The anti-A33 antibody of the present invention can be produced by the production method described below. For example, a non-human mammal such as a human antibody-producing transgenic mouse is specifically immunized with a conjugate of a33, a portion of a33, or a portion of a33 with a suitable carrier substance (e.g., bovine serum albumin) that can enhance antigenicity, if necessary, together with an immunopotentiating agent (e.g., freund's complete or incomplete adjuvant). For A33, both native A33 and recombinant A33 can be used. Alternatively, an immune response can be elicited by introducing a gene encoding a33, followed by administration of animal cells that overexpress a33 on the cell surface. Monoclonal antibodies can be produced by: the antibody-producing cells obtained from the immunized animal are fused with myeloma cells incapable of producing any autoantibody, the obtained hybridomas are cultured, and then clones producing monoclonal antibodies having specific affinity for the antigen used for immunization are selected.

The antibody of the present invention also includes an antibody whose type is converted to a subclass different from the original subclass by known genetic engineering modification by those skilled in the art (see, for example, EP 314161). Specifically, an antibody subclass different from the original antibody subclass can be obtained by genetic engineering techniques using a DNA encoding the variable region of the antibody of the present invention.

ADCC refers to a type of cytotoxicity induced by activation of macrophages, NK cells, neutrophils, or other similar cells, which is recognized by binding of an antibody constant region to an Fc receptor expressed on the surface of the above-mentioned cells. In contrast, CDC refers to a type of cytotoxic action induced by activation of the complement system through binding of an antibody to an antigen. It is known that the intensity of these activating actions varies depending on the subclass of antibody. It is also known that this difference is due to structural differences in the constant regions of antibodies (Charles A. Janeway et al, immunology, 1997, Current Biology Ltd/Garland Publishing Inc.). For example, antibodies exhibiting low levels of binding to Fc receptors can be obtained by converting the antibody subclasses of the invention to IgG2 or IgG 4. In contrast, antibodies exhibiting high levels of binding to Fc receptors can be obtained by converting the antibody subclasses of the present invention to IgG1 or IgG 3. In addition, the degree of binding to Fc receptors can be altered by modifying the constant region amino acid sequence of the antibodies of the invention by genetic engineering techniques or by binding to constant region sequences having such amino acid sequences (see Janeway CA. Jr and polypeptides P. (1997), Immunobiology, third edition, Current Biology Ltd/Garland Publishing Inc.). Alternatively, the degree of binding to complement can be altered by the same method (see Mi-Hua Tao, et al.1993.J.Exp.Med.). For example, the extent of complement binding can be altered by mutating the sequence CCC of the heavy chain constant region part (encoding proline (P) at position 331) (according to the EU numbering system (see Sequences of proteins Immunological Interest, NIH Publication No.91-3242)) to TCC encoding serine (S), thus substituting proline for serine. For example, in the case of an anticancer agent, it is desirable that an antibody itself, which has an antitumor activity based on antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) mediated by Fc receptor, does not have an activity of inducing cell death. It may be more desirable for the antibody itself to have cell death-inducing activity, and for such antibodies to bind to a lesser extent to Fc receptors. In addition, for example, in the case of an immunosuppressive agent, it is desirable that an antibody only inhibits binding of a T cell to an antigen-presenting cell in three dimensions, and such an antibody lacks ADCC activity or CDC activity. In addition, when ADCC activity or CDC activity causes toxicity, it is necessary to mutate the Fc portion of the antibody or change its subclass in order to avoid the activity causing toxicity.

The present invention comprises the following steps for producing monoclonal antibodies: (1) purifying the biopolymer for use as an immunogen and/or preparing cells that overexpress antigenic proteins on the cell surface; (2) immunizing an animal by injecting an antigen, collecting blood, measuring an antibody titer, measuring a time for spleen extraction, etc., and then preparing antibody-producing cells; (3) preparing myeloma cells; (4) fusing antibody-producing cells with myeloma cells; (5) screening a hybridoma group producing the target antibody; (6) isolation into single cell clones (clones); (7) if necessary, culturing the hybridoma to produce the monoclonal antibody on a large scale, or breeding the hybridoma-transplanted animal; and (8) detecting the physiological activity and recognition specificity of the produced monoclonal antibody, or determining the product characteristics, for example, as a labeled reagent.

The polymorphism in A33 exists. The antibody of the present invention binds to a33 by recognizing all the polymorphisms of a33 known so far. The therapeutic or prophylactic agent comprising the antibody of the present invention can effectively act regardless of the difference in the A33 polymorphism in patients.

Hereinafter, a method for preparing the anti-a 33 monoclonal antibody will be described in detail according to the above procedure. However, the method for producing the antibody is not limited to the following method. For example, antibody-producing cells and myeloma cells other than spleen cells may be used.

(1) Antigen purification

The transformed cell line is prepared by integrating the DNA encoding A33 into an expression vector for animal cells, and then introducing the expression vector into animal cells. The transformed cell line thus obtained can be used directly as an antigen. In addition, since the primary structure of A33 is known (GenBank accession NP-005305, SEQ ID NO: 12), a person skilled in the art can chemically synthesize a polypeptide by a known method based on the amino acid sequence of A33, and the product can also be used as an antigen.

In addition, cells overexpressing A33 on the cell surface, which can be obtained by introducing full-length A33 into FM3A cells or L929 cells, are also effective as immunogens. p.DELTA.EGFP-N1-A33 was prepared by introducing the DNA encoding the A33 protein into an animal cell expression vector p.DELTA.EGFP-N1 (the region encoding the EGFP protein in modified p.DELTA.EGFP-N1 (produced by Becton Dickinson Bioscience Clontech) was deleted)). However, the DNA, vector, host and the like encoding A33 are not limited to these examples.

Specifically, a transformed cell line was obtained by transforming FM3A cells or L929 cells with p.DELTA.EGFP-N1-A33, followed by culture. The neomycin resistance profile obtained by cells inserted with the p.DELTA.EGFP-N1 vector was confirmed, and the expression of A33 was also confirmed with a mouse anti-human A33 antibody (ATCC No. HB-8779). Therefore, FM3A cells or L929 cells overexpressing A33 on the cell surface can be prepared using this confirmation result as an index.

(2) Step of preparing antibody-producing cells

Mixing the antibody obtained in (1) with Freund's complete or incomplete adjuvant or an adjuvant such as potassium aluminum sulfate. The experimental animals were immunized with the obtained mixture as an immunogen. For experimental animals, transgenic mice producing human antibodies are most suitable. Such mice are described in the literature of Tomizukat et al (Tomizuka. et al., Proc Natl Acad Sci U.S.A., 2000Vol 97: 722).

The route of administration of the immunogen upon immunization of a mouse may be subcutaneous injection, intraperitoneal injection, intravenous injection, intradermal injection, intramuscular injection, footpad injection, etc. Intraperitoneal injection, footpad injection or intravenous injection is preferred.

The immunization may be repeated 1 or several times at appropriate time intervals, preferably at intervals of 2 to 4 weeks. Then, the titer of antibodies against the antigen in the serum of the immunized animal is determined. The effect of the subsequent step can be enhanced by using an animal having a sufficiently high antibody titer as a source of antibody-producing cells. In general, it is preferred to use antibody-producing cells derived from animals 3 to 5 days after the last immunization for subsequent cell fusion.

Further, examples of the method for determining the antibody titer that can be employed include various known techniques such as a radioimmunoassay (hereinafter referred to as "RIA method"), an enzyme-linked immunosorbent assay (hereinafter referred to as "ELISA method"), a fluorescent antibody method, and a passive hemagglutination method. The RIA method and ELISA method are more suitable in view of detection sensitivity, rapidity, accuracy, possible automation of the process, and others.

For example, when the ELISA method is used, the antibody titer in the present invention can be determined by the procedure described below. First, an antigen against a human antibody is adsorbed on the solid phase surface of a 96-well plate for ELISA or the like. In addition, some regions on the solid phase surface to which no antigen is adsorbed are covered with a protein unrelated to the antigen (e.g., Bovine Serum Albumin (BSA)). After washing the surface, the surface is contacted with a serially diluted sample (e.g., mouse serum) that is used as a primary antibody. The anti-a 33 antibody in the sample binds to the antigen. An enzyme-labeled secondary antibody against the human antibody is added to bind to the human antibody. After washing, the enzyme substrate is added. The titer of the antibody was calculated by measuring the change in absorption and the like by measuring the color development due to the degradation of the substrate.

(3) Procedure for preparation of myeloma cells

For myeloma, cells derived from mammals such as mouse, rat, guinea pig, hamster, rabbit or human, which do not produce any autoantibody, can be used. In general, it is preferable to use mouse cells obtained from mice such as 8-azaguanine-resistant mouse (BALB/c-derived) myeloma cell line P3X63Ag8U.1(P3-U1) (Yelton, D.E.et al. Current copies in Microbiology and Immunology, 81, 1-7(1978)), P3/NSI/1-Ag4-1(NS-1) (Kohler, G.et al. European J.immunology, 6, 511-519(1976)), Sp2/O-Ag14(SP-2) (Shulma, M.et al., Nature, 276, 269-270(1978)), P3X63Ag8.653 (Kearney, J.F.et al., J.J.123, 123, 276, 269-270(1978)), and Harrisy cell lines (Harrisy. X3, 8, 63, 8, Harista, 63, Harison, Hakk, K-3, 1978)). These cell lines were subcultured in a suitable medium such as 8-azaguanine medium (prepared by adding 8-azaguanine to RPMI-1640 medium supplemented with glutamate, 2-mercaptoethanol, gentamicin, and fetal calf serum (hereinafter referred to as "FCS")), Iscove's Modified Dulbecco's medium (hereinafter referred to as "IMDM"), or Dulbecco's Modified Eagle medium (hereinafter referred to as "DMEM"). The cell lines are subcultured in a normal medium (e.g., DMEM medium containing 10% FCS) 3-4 days before cell fusion. During cell fusion, 2X 10 cells were maintained⁷Or more cells.

(4) Fusion of cells

Antibody-producing cells are plasma cells and lymphocytes as their precursor cells. Antibody-producing cells may be obtained from any part of a single body, typically from the spleen, lymph nodes, bone marrow, tonsils or peripheral blood, suitable combinations thereof, and the like. Splenocytes are the most commonly used.

After the last immunization, a site where antibody-producing cells are present, such as the spleen, is excised from a mouse for which the antibody titer has been determined in advance, thereby preparing spleen cells as antibody-producing cells. The spleen cells were then fused with myeloma cells. Currently, the most commonly used means for fusing splenocytes with myeloma cells obtained in step (3) is a polyethylene glycol method, which is characterized by relatively low cytotoxicity and simple cell fusion procedure. For example, the method includes the following procedures.

The spleen cells and myeloma cells are thoroughly washed with serum-free medium (e.g., DMEM) or phosphate buffered saline (hereinafter referred to as PBS), mixed so that the ratio of the number of spleen cells to myeloma cells is about 5: 1 to 10: 1, and then centrifuged. The supernatant was removed and the pelleted cell group was then loosened well. While stirring the solution, 1mL of serum-free medium containing 50% (w/v) polyethylene glycol (molecular weight 1000 to 4000) was added dropwise. Subsequently, 10mL of serum-free medium was gently added, followed by centrifugation. The supernatant was removed again. The precipitated cells are suspended in a normal medium (hereinafter, referred to as HAT medium) containing a suitable amount of hypoxanthine-aminopterin-thymidine (hereinafter, referred to as HAT) solution and human interleukin-6 (hereinafter, referred to as IL-6). The suspension was dispensed into the wells of a culture plate (hereinafter referred to as a plate), and then cultured in the presence of 5% carbon dioxide at 37 ℃ for about 2 weeks. During the culture period, HAT medium was appropriately supplemented.

(5) Screening of hybridoma groups

When the above myeloma cells belong to a cell line resistant to 8-azaguanine, more specifically, to a cell line deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT), then unfused myeloma cells and the fused cells of a plurality of myeloma cells cannot survive in a medium containing HAT. On the other hand, a fusion cell of a plurality of antibody-producing cells or a hybridoma of an antibody-producing cell and a myeloma cell can survive. However, the life of fused cells of such multiple antibody-producing cells is short. Therefore, only hybridomas that are fusion cells of antibody-producing cells and myeloma cells can survive by continuous culture on a medium containing HAT. Thus, such hybridomas can be screened.

For hybridomas that grow in a clonal form, HAT medium can be changed to medium from which aminopterin is removed (hereinafter referred to as HT medium). Subsequently, the supernatant fraction of the culture broth is collected, and the titer of the anti-A33 antibody is determined by, for example, an ELISA method. However, when the above fusion protein is used as an antigen for ELISA, a clone that produces an antibody specifically binding to the Fc region of human IgG should be removed to avoid screening of such a clone. The presence of such a clone can be confirmed by ELISA or the like using the Fc region of human IgG as an antigen.

As described above, the method using the 8-azaguanine-resistant cell line is described as an example. Other cell lines may be used depending on the method of screening for hybridomas, and the composition of the medium used in this case may also be varied.

(6) Cloning procedure

The antibody titer will be determined by a method similar to the method described in (2), and the hybridoma which has been confirmed to produce a specific antibody is transferred to another plate and then cloned. Examples of the cloning method used herein include a limiting dilution method in which only 1 hybridoma is contained in each well of a plate by dilution, and then the hybridomas are cultured; the soft agar method, that is, a method of culturing in soft agar, collecting clones, extracting single cells with a micromanipulator, and then culturing the single cells; and "sorting cloning" method, in which cells are separated one by one using a cell sorter. The limiting dilution method is the simplest and most commonly used.

For wells where antibody titers were observed, cloning was repeated 2 to 4 times by, for example, limiting dilution method. Cells in which antibody titers were stably observed were selected as hybridoma cell lines producing the anti-a 33 monoclonal antibody.

In addition, mouse-mouse hybridomas-125M 10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5 AAAA-which were deposited at the International Patent Organism Depositary (IPOD) of the national institute of Advanced Industrial Science and Technology (AIST) at 8.24.2004 (Central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan) and which were designated FERM BP-10107 (designated by M10 at the time of identification), FERM BP-10106 (designated by M165 at the time of identification), FERM BP-10108 (designated by M96 at the time of identification), FERM BP-10109 (designated by N26 at the time of identification), FERM-10104 (designated by Q47 at the time of identification), FERM BP-3505 (designated by Q54) and Q3R 5(at the time of identification) as human anti-A33 monoclonal antibody-producing cells according to the present invention.

(7) Preparation of monoclonal antibodies by culture of hybridomas

After cloning was completed, the HT medium was changed to the normal medium, and then the hybridomas were cultured. The large-scale culture is carried out by shaking culture in a large culture flask, spinner culture or culture in a hollow fiber system. The supernatant obtained from such large-scale culture is purified by a known method such as gel filtration by those skilled in the art, to thereby obtain a monoclonal antibody against a 33. Furthermore, hybridomas can be proliferated intraperitoneally in mice of the same line (e.g., BALB/c) or nu/nu mice, rats, guinea pigs, hamsters, rabbits, etc., and ascites containing the anti-A33 monoclonal antibody can be obtained on a large scale. As a simple purification method, a commercially available monoclonal antibody purification kit (e.g., MAbTrap GII kit; produced by Amersham Pharmacia Biotech) or the like can be used.

The monoclonal antibody thus obtained had high antigen specificity to A33.

(8) Determination of monoclonal antibodies

The isotype and subclass of the monoclonal antibody thus obtained can be determined by the following methods. Examples of the identification method include the Ouchterlony method, the ELISA method, and the RIA method. The Ouchterlony method is simpler, but requires a step of concentrating the concentration of monoclonal antibody at lower concentrations. Meanwhile, when the ELISA method or the RIA method is used, the culture supernatant is directly reacted with the antigen-adsorbed solid phase. The isotype and subclass of the monoclonal antibody can then be identified by using the isotype and subclass of the various immunoglobulins corresponding to the antibody as the secondary antibody.

In addition, protein can be measured by the Folin-Lowry method, which is calculated from absorbance at 280nm (1.4(OD280) ═ immunoglobulin (1 mg/mL)).

The antigenic determinants recognized by monoclonal antibodies can be identified by the following methods. First, various partial structures of molecules recognized by monoclonal antibodies are prepared. Examples of methods of making such partial structures include: methods for preparing various partial peptide chains of molecules using known oligopeptide synthesis techniques; and a method of integrating a DNA sequence encoding a partial peptide chain of interest into a suitable expression plasmid by a gene recombination technique and then producing the peptide chain intracellularly or extracellularly in a host such as Escherichia coli. Generally, these two methods can be used in combination in order to achieve the above object. For example, a series of polypeptides can be prepared by successively shortening the C-terminus or N-terminus of an antigenic protein by appropriate lengths using genetic recombination techniques known to those skilled in the art. The reactivity of the monoclonal antibody against each of these polypeptides is determined, and the approximate recognition site can be determined.

Various oligopeptides, oligopeptide variants, and the like associated with these sites are then further synthesized using oligopeptide synthesis techniques known to those skilled in the art. The antigenic determinant is determined by examining the binding ability of a monoclonal antibody (contained as an active ingredient in the prophylactic or therapeutic agent of the present invention) to these peptide chains, or by examining the competitive inhibition of the binding of the monoclonal antibody to an antigen by the peptide chains. Commercially available kits (for example, SPOTs kit (produced by GenoSys Biotechnology) and a series of various peptide chain synthesis kits using various synthesis methods (produced by Chiron corporation)) can be used as a convenient method for obtaining various types of oligopeptides.

In addition, a gene encoding the human monoclonal antibody is cloned from an antibody-producing cell such as a hybridoma, and then the gene is integrated into an appropriate vector. The vector is then introduced into a host (e.g., mammalian cell lines, E.coli, yeast cells, insect cells, and plant cells). Thus, recombinant Antibodies produced by genetic recombination techniques can also be prepared (P.J.Delves., Antibody Production engineering., 1997Wiley, P.Shepherd, and C.dean., Monoclonal Antibodies, 2000Oxford University Press, J.W.Goding., Monoclonal Antibodies: Principles and Practice, 1993Academic Press).

The present invention also includes nucleic acids comprising the gene sequences of the antibodies of the present invention, which are carried by the hybridomas that produce the antibodies. Specifically, nucleic acids of the heavy chain variable region and the light chain variable region of the antibody produced by the hybridoma of the present invention described below are also included. As used herein, "nucleic acid" includes DNA and RNA.

A method for preparing a gene encoding a monoclonal antibody derived from a hybridoma involves preparing DNAs encoding the L chain V region, L chain C region, H chain V region, and H chain C region of a monoclonal antibody by PCR or the like. As the primer, oligo DNAs designed based on the anti-A33 antibody gene or its amino acid sequence can be used. For the template, DNA prepared from a hybridoma can be used. These DNAs are integrated into a suitable vector, and the vector is then introduced into a host for expression. Alternatively, these DNAs are separately integrated into suitable vectors, and then co-expressed.

For the vector, a phage or plasmid that can autonomously replicate in the host microorganism is used. Examples of the plasmid DNA include plasmids derived from Escherichia coli, Bacillus subtilis or yeast, and the phage DNA may be lambda phage.

The hosts used for transformation are not particularly limited as long as they can express the desired gene. Examples of such hosts include bacteria (e.g., Escherichia coli and Bacillus subtilis), yeast, animal cells (e.g., COS cells and CHO cells), and insect cells.

Methods for introducing genes into hosts are known. Examples of such methods include any method such as a method using calcium ion, electroporation, protoplast, lithium acetate, calcium phosphate, and lipofection. In addition, examples of the method for introducing a gene into an animal described below include a microinjection method, a method for introducing a gene into ES cells by electroporation or lipofection, and a nuclear transfer method.

In the present invention, the anti-A33 antibody can be obtained by culturing the transformant and then recovering the antibody from the culture. "culture" means (a) a culture supernatant, (b) a cultured cell, a cultured microorganism or a cleavage product thereof, or (c) a secretion product of a transformant. For culturing the transformant, a medium suitable for the host is used, and a static culture method, a spinner flask culture method or the like is employed.

After the culture, when the antibody protein of interest is produced in the microorganism or the cell, the antibody is recovered by disrupting the microorganism or the cell. In addition, when the antibody of interest is produced outside the microorganism or cell, the culture solution is used as it is, or the microorganism or cell is removed by centrifugation or other methods. Then, the objective antibody is isolated and purified from the culture product by using a common biochemical method alone or by applying an appropriate combination of various types of biochemical methods for protein isolation and purification.

In addition, techniques for producing transgenic animals are used to produce animal hosts in which the antibody gene of interest is integrated into an endogenous gene, such as transgenic cattle, transgenic goats, transgenic sheep, or transgenic pigs. A large number of monoclonal antibodies derived from antibody genes can be obtained from the milk secreted by these transgenic animals (Wright, G., et al., (1991) Bio/Technology 9, 830-834). When a hybridoma is cultured in vitro, the hybridoma is proliferated, maintained and stored according to various conditions including the characteristics of the cell type to be cultured, the purpose of experiments and research, the culture method and other conditions. Such culture can be performed using a known rich medium for producing monoclonal antibodies in the culture supernatant, or various rich media summarized and prepared from a known basal medium.

(9) Antibody characterization

The antibody of the present invention has one of the following properties.

(a) ADCC assay

The antibodies of the invention exhibit antibody-dependent cellular cytotoxicity (ADCC) against a 33-expressing human cancer cells in the presence of normal human peripheral blood mononuclear cells.

(b) CDC experiment

The antibodies of the invention exhibit complement-dependent cytotoxic effects on human cancer cells expressing a33 in the presence of human serum-derived complement.

(c) In vivo experiments

The antibody of the present invention exhibits an antitumor effect on a non-human animal containing a human cancer cell expressing a 33.

(d) Competition experiment

The antibodies of the invention can (i) strongly compete (blocker), (ii) weakly compete (partial blocker) or (iii) do not compete (non-blocker) with chimeric anti-a 33 (consisting of the heavy and light chain variable regions of the antibody produced by hybridoma ATCCHB-8779 and the heavy and light chain constant regions of human IgG 1).

(e) Immunohistochemical assay

The antibodies of the invention stain human adult colon cancer tissue, human adult normal colon tissue, and human normal small intestine tissue in the presence of the antibodies.

Examples of such antibodies include antibodies produced by, for example, hybridomas 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5 AAAA. 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA are deposited at 24.8.2004 at International Patent Organism Depositary (IPOD) of the national institute of Advanced Industrial Science and Technology (AIST) (Central 6, 1-1, Rigashi 1, Tsukuba, Ibaraki, Japan) under accession numbers FERM BP-10107 (denoted by M10 for identification), FERM BP-10106 (denoted by M165 for identification), FERM BP-10108 (denoted by M96 for identification), FERM BP-10109 (denoted by N26 for identification), RMFERM BP-10104 (denoted by Q47 for identification), FERM BP-10105 (denoted by Q54 for identification) and FERM-10103 (denoted by R5 for identification).

2. Pharmaceutical composition

Pharmaceutical compositions containing the human anti-A33 antibody of the invention are also within the scope of the invention. Such formulations preferably contain, in addition to the antibody, a physiologically acceptable diluent or carrier, which may be a mixture of different antibodies or different drugs, such as antibiotic agents, incorporated therein. Examples of suitable carriers include, but are not limited to, saline, phosphate buffered saline glucose solution, and buffered saline. Alternatively, the antibody may be lyophilized and reconstituted, if desired, by addition of an aqueous buffer as described above. Such prophylactic or therapeutic agents can be administered by a variety of routes of administration. Examples of such routes of administration include oral administration by tablets, capsules, granules, powders, syrups or the like, and parenteral administration by injection, drops, suppositories or the like.

The dosage of such pharmaceutical compositions varies according to the symptoms, age, weight and other factors. Typically, for oral administration, an adult may administer a dose of between about 0.01mg and 1000mg once or several times a day. For parenteral administration, the dosage may be between about 0.01mg and 1000mg per administration by subcutaneous injection, intramuscular injection or intravenous injection.

The present invention also includes the above-mentioned methods for preventing or treating diseases using the antibody or the pharmaceutical composition of the present invention. In addition, the present invention also includes the use of the antibodies of the present invention for the manufacture of the above-mentioned prophylactic or therapeutic agents for the treatment of diseases.

Tumors that can be prevented or treated using the antibody or the functional fragment thereof of the present invention are colorectal cancer, colon cancer, rectal cancer, gastric cancer, pancreatic cancer, breast cancer, melanoma, renal cell carcinoma, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, prostate cancer, testicular cancer, mesothelial cancer, and the like. The number of tumor types on which the antibody of the present invention can act is not limited to a single type. The antibody can also be applied to medical records with various types of tumors in the same time.

3. Pharmaceutical preparation examples

The molecules of the invention may be used as a sterile solution for one injection or as a suspension prepared by dissolving the molecule in water or a pharmaceutically acceptable solution other than water. Alternatively, such a bolus may be filled with a sterile powdered pharmaceutical formulation (preferably, the molecule of the invention is lyophilized) and then diluted with a pharmaceutically acceptable solution at the time of use to form a bolus.

The present invention will be described in detail with reference to the following examples. However, the present invention is not limited to the embodiments.

EXAMPLE 1 preparation of mouse anti-A33 antibody

Mouse anti-A33 antibody was prepared and used as a positive control antibody in screening for hybridomas producing human monoclonal antibodies and in several experiments. AS33 hybridoma producing mouse anti-A33 antibody was purchased from ATCC (American type culture Collection (ATCC) No. HB-8779). Hybridomas were cultured according to the instructions attached to the ATCC product. The hybridomas are lyophilized. Subsequently, the A33 hybridoma was acclimatized in eRDF medium (produced by Kyokuto Pharmaceutical Industrial) containing 10% low IgG fetal bovine serum (produced by HyClone). The domesticated hybridomas are cryopreserved. Then, in order to purify the antibody, a part of the cryopreserved product was purified in a medium containing bovine insulin (5. mu.g/ml, produced by Gibco BRL), human transferrin (5. mu.g/ml, produced by Gibco BRL), ethanolamine (0.01mM, produced by Sigma), sodium selenite (2.5X 10)^-5mM, produced by Sigma) and 1% low IgG fetal bovine serum (produced by HyClone) were acclimated in eRDF medium (produced by Kyokuto Pharmaceutical Industrial). After culturing in the flask, the culture supernatant was collected. The concentration of the purified antibody derived from the hybridoma in the collected supernatant was calculated by measuring the absorbance at 280nm and calculating from 1.4OD the equivalent of 1mg/mL (antibody concentration).

EXAMPLE 2 preparation of chimeric anti-A33 antibody

Chimeric anti-A33 antibodies having human IgG1 heavy chain and human IgG1 light chain constant regions were prepared and used as positive control antibodies in screening hybridomas producing human monoclonal antibodies or in several experiments.

(1) cDNA clone of chimeric anti-A33 antibody gene and construction of expression vector

Hybridoma AS33 producing mouse anti-A33 antibody, purchased in example 1, was cultured in DMEM medium (produced by GibcoBRL) containing 10% fetal bovine serum (produced by HyClone), and then total RNA was purified using RNA extraction reagent ISOGEN (produced by NIPPONGEN) according to the relevant instructions. Next, polyA + RNA was purified from the total RNA using OligotexTM-dT30< Super > (produced by TAKARA BIO). Using the obtained polyA + RNA (2.5. mu.g) as a material, cloning experiments were carried out using SMART RACE cDNA amplification kit (Becton Dickinson Bioscience Clontech) according to the instructions attached thereto. Thereby obtaining cDNA of the antibody variable region.

1) Synthesis of first Strand cDNA

polyA+RNA (2.5μg)/3μl

5' -CDS primer 1. mu.l

SMART II A oligo 1μl

The reaction solution having the above composition was incubated at 70 ℃ for 2 minutes. The following reagents and enzymes were added, followed by incubation at 42 ℃ for 1.5 hours, followed by cDNA synthesis.

5 Xfirst Strand buffer 2. mu.l

DTT(20mM) 1μl

dNTP mix (10mM) 1. mu.l

PowerScript reverse transcriptase 1. mu.l

After completion of the reaction, 100. mu. l N- [ tris (hydroxymethyl) methyl ] glycine (tricine) buffer was added and incubated for 7 minutes at 72 ℃.

2) Amplification of heavy and light chain genes by PCR

PCR was performed using the obtained cDNA as a template. Primer pairs for PCR include: PCR primers (H chain: GPAHvR3Nhe (5'-GCC CTT GGT GCTAGC TGA AGA GAC GGT GAC CAG AGT CCC TTG-3') (SEQ ID NO: 1) or L chain: GPALvR3Bsi, (5'-GTG CAC GCC GCT GGT CAG GGC GCC TG-3') (SEQ ID NO: 2) specific for the 3 'end of mouse anti-A33 antibody heavy chain (hereinafter "heavy chain" may also be referred to as "H chain") or mouse anti-A33 antibody light chain (hereinafter "light chain" may also be referred to as "L chain") variable region DNA, and UPM primers (oligonucleotides complementary to the universal sequences, prepared at the 5' end of the synthesized cDNA) accompanying the SMARTRACE cDNA amplification kit, amplification of H chain leader sequence and variable region (hereinafter also referred to as "HV") by PCR cDNA amplification was performed, and then the following reaction solutions were prepared.

Sterilized Water 29.5. mu.l

cDNA 2.5μl

KOD-Plus buffer (10X) 5. mu.l

dNTP mix (2mM) 4. mu.l

MgSO₄(25mM) 2μl

KOD-Plus DNA polymerase (1 unit/. mu.l) 1. mu.l

Universal primer A mix (UPM) (10X) 5. mu.l

Gene Specific Primer (GSP) 1. mu.l

Total volume 50. mu.l

The thermocycling amplification reaction was performed under the following conditions.

5 cycles:

94 ℃ for 30 seconds

1 minute at 72 DEG C

5 cycles:

94 ℃ for 30 seconds

30 seconds at 70 DEG C

1 minute at 72 DEG C

25 cycles:

94 ℃ for 30 seconds

68 ℃ for 30 seconds

1 minute at 72 DEG C

The amplified PCR fragment was recovered by ethanol precipitation, recovered by agarose gel electrophoresis, and then purified by QIAquick gel extraction kit (produced by QIAGEN), which is a DNA purification kit using a membrane. The purified HV and LV amplified fragments were subcloned into PCR 4Blunt-TOPO vectors of Zero Blunt TOPO PCR cloning kit (produced by Invitrogen), respectively. The nucleotide sequence of the insert DNA on the plasmid DNA in the obtained clone was analyzed. For determination of the DNA nucleotide sequence, M13FW (5'-GTA AAA CGA CGG CCA GTG-3') (SEQ ID NO: 3) and M13RV (5'-CAG GAAACA GCT ATG AC-3') (SEQ ID NO: 4) were used as primers. The amino acid sequences of the antibodies encoded by the HV and LV gene regions determined matched exactly with the amino acid sequence of the mouse anti-A33 antibody (Br J cancer.1995Dec 72 (6): 1364-72) variable region reported by King D.J.et al.

(3) Construction of expression vector for chimeric anti-A33 antibody (N5KG 1_ mVhCA33)

HV of mouse anti-A33 antibody (94 ℃ C. for 3 minutes-94 ℃ C. for 10 seconds, 68 ℃ C. for 45 seconds (35 cycles) -72 ℃ C. for 7 minutes) was amplified by PCR using plasmid DNA containing the obtained HV chain of the antibody as a template, and primers (primer pairs for amplification: GPAHv2F5Sal (5'-AGA GAG AGG TCG ACC CAC CAT GAA CTT TGG GCT GAG CTT AGT T-3') (SEQ ID NO: 5) and GPAHvR3Nhe (SEQ ID NO: 1)) to which restriction enzyme sites were added at the ends for ligation by design. The amplified HV fragment was purified and then subcloned into the PCR 4Blunt-TOPO vector. The nucleotide sequence of the DNA of the insert part in the subclone was analyzed. Thereby screening plasmid DNA having a sequence that does not differ from the sequence of the template gene. Plasmid DNA was digested with the restriction enzymes SalI and Nhe I. DNA of about 440bp was obtained and purified by agarose gel electrophoresis. Meanwhile, N5KGl-Val Lark vector (modified vector of IDEC pharmaceuticals N5KG1 (U.S. Pat. No. 3, 6001358)) was treated similarly with restriction enzymes Sal I and Nhe I, and then dephosphorylated with alkaline phosphatase (E.coli C75) (produced by TAKARA BIO). Subsequently, about 8.9kb of DNA was recovered by agarose gel electrophoresis and DNA purification kit. These two fragments were subjected to ligation reaction using DNA ligation kit Ver2.1 (produced by TAKARA BIO), and then introduced into E.coli DH5 α to obtain a transformant. By selecting transformants, clone N5KG1_ GPA33Hv (clone # 2) into which the HV of interest was inserted was selected. To insert LV into the resulting N5KG1_ GPA33Hv, the plasmid DNA was subsequently cut with the restriction enzymes BglII and BsiWI and then dephosphorylated. Subsequently, about 9.2kb of vector DNA was purified. Meanwhile, the LV region was amplified by PCR using plasmid DNA containing LV, a mouse anti-A33 antibody, as a template.

GPALv2FBg1 (5'-AGA GAG AGA GAT CTC TCA CCA TGG GCA TCA AGA TGGAGT TTC AG-3') (SEQ ID NO: 6) and GPALvR3Bsi (SEQ ID NO: 2) were used as primer pairs for amplification. The purified and amplified LV fragments were subcloned into PCR 4 Blunt-TOPO. The nucleotide sequence of the DNA of the insert part in the subclone was analyzed. Thereby screening plasmid DNA having a sequence that does not differ from the sequence of the template gene. Plasmid DNA was digested with the restriction enzymes BglII and BsiWI. DNA of about 400bp was obtained and purified by agarose gel electrophoresis. The DNA was ligated to the above-described N5KG1_ A33Hv vector fragment which had been cleaved with the restriction enzymes BglII and BsiWI, and then introduced into E.coli DH 5. alpha. to obtain a transformant. By selecting transformants, the clone N5KG1_ GPA33HvLv (clone # 2) into which the target LV had been inserted was selected. The chimeric anti-A33 antibody expression plasmid DNA finally obtained was purified in large quantities. Thus, it was confirmed that no mutation occurred in the DNA fragment of the inserted L chain or H chain or in the DNA nucleotide sequence around the insertion site.

The DNAs encoding the chimeric anti-A33 heavy chain variable region and light chain variable region and the amino acid sequences of the heavy chain variable region and light chain variable region are shown below.

< chimeric anti-A33 heavy chain nucleic acid sequence (SEQ ID NO: 7)

10 20 30 40 50 60

ATGAACTTTG GGCTGAGCTT GATTTTCCTT GTCCTAATTT TAAAAGGTGT CCAGTGTGAA

70 80 90 100 110 120

GTGAAGCTGG TGGAGTCTGG GGGAGGCTTA GTGAAGCCTG GAGGGTCCCT GAAACTCTCC

130 140 150 160 170 180

TGTGCAGCCT CTGGATTCGC TTTCAGTACC TATGACATGT CTTGGGTTCG CCAGACTCCG

190 200 210 220 230 240

GAGAAGAGGC TGGAGTGGGT CGCAACCATT AGTAGTGGTG GTAGTTACAC CTACTATTTA

250 260 270 280 290 300

GACAGTGTGA AGGGCCGATT CACCATCTCC AGAGACAGTG CCAGGAACAC CCTATACCTG

310 320 330 340 350 360

CAAATGAGCA GTCTGAGGTC TGAGGACACG GCCTTGTATT ACTGTGCACC GACTACGGTA

370 380 390 400 410 420

GTCCCGTTTG CTTACTGGGG CCAAGGGACT CTGGTCACCG TCTCTTCAGC TAGC......

< chimeric anti-A33 heavy chain amino acid sequence (SEQ ID NO: 8)

10 20 30 40 50 60

MNFGLSLIFL VLILKGVQCE VKLVESGGGL VKPGGSLKLS CAASGFAFST YDMSWVRQTP

70 80 90 100 110 120

EKRLEWVATI SSGGSYTYYL DSVKGRFTIS RDSARNTLYL QMSSLRSEDT ALYYCAPTTV

130 140

VPFAYWGQGT LVTVSSAS..

< chimeric anti-A33 light chain nucleic acid sequence (SEQ ID NO: 9)

10 20 30 40 50 60

ATGGGCATCA AGATGGAGTT TCAGACCCAG GTCTTTGTAT TCGTGTTGCT CTGGTTGTCT

70 80 90 100 110 120

GGTGTTGATG GAGACATTGT GATGACCCAG TCTCAAAAAT TCATGTCCAC ATCAGTAGGA

130 140 150 160 170 180

GACAGGGTCA GCATCACCTG CAAGGCCAGT CAGAATGTTC GTACTGTTGT AGCCTGGTAT

190 200 210 220 230 240

CAACAGAAAC CAGGGCAGTC TCCTAAAACA CTGATTTACT TGGCCTCCAA CCGGCACACT

250 260 270 280 290 300

GGAGTCCCTG ATCGCTTCAC AGGCAGTGGA TCTGGGACAG ATTTCACTCT CACCATTAGC

310 320 330 340 350 360

AATGTGCAAT CTGAAGACCT GGCAGATTAT TTCTGTCTGC AACATTGGAG TTATCCTCTC

370 380 390 400

ACGTTCGGCT CGGGGACAAA GTTGGAAGTA AAACGT....

< chimeric anti-A33 light chain amino acid sequence (SEQ ID NO: 10)

10 20 30 40 50 60

MGIKMEFQTQ VFVFVLLWLS GVDGDIVMTQ SQKFMSTSVG DRVSITCKAS QNVRTVVAWY

70 80 90 100 110 120

QQKPGQSPKT LIYLASNRHT GVPDRFTGSG SGTDFTLTIS NVQSEDLADY FCLQHWSYPL

130 140

TFGSGTKLEV KR........

In the heavy chain nucleic acid sequence (SEQ ID NO: 7), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 408 and guanine (G) at position 409. In the heavy chain amino acid sequence (SEQ ID NO: 8), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 136 and alanine (A) at position 137. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 7), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and guanine (G) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 8), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 19 and glutamic acid (E) at position 20.

Thus, the variable region in the chimeric anti-A33 antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 7) ranging from guanine (G) at position 58 to adenine (A) at position 408. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 8) ranging from glutamic acid (E) at position 20 to serine (S) at position 136.

In the light chain nucleic acid sequence (SEQ ID NO: 9), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 393 and cytosine (C) at position 394. In the light chain amino acid sequence (SEQ ID NO: 10), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 131 and arginine (R) at position 132. In addition, in the light chain nucleic acid sequence (SEQ ID NO: 9), the boundary between the signal sequence and the antibody variable region is located between adenine (A) at position 72 and guanine (G) at position 73. In the light chain amino acid sequence (SEQ ID NO: 10), the boundary between the signal sequence and the antibody variable region is located between guanine (G) at position 24 and aspartic acid (D) at position 25.

Thus, the variable region in the chimeric anti-A33 antibody light chain has the nucleic acid sequence (SEQ ID NO: 9) ranging from guanine (G) at position 73 to adenine (A) at position 393. In addition, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 10) ranging from aspartic acid (D) at position 25 to lysine (K) at position 131.

The nucleic acid sequences of the synthesized DNAs are listed in Table 5 below.

The constructed expression vector of the chimeric anti-A33 recombinant antibody was transfected into a host cell to prepare a recombinant antibody-expressing cell. The host cells used for expression are CHO cells of the dhfr-deficient cell line (ATCC CRL-9096), such as CHO-Ras (Katakura Y., et al, Cytotechnology, 31: 103-109, 1999) or HEK293T (ATCC CRL-11268).

Host cells are transfected with the vector by electroporation, lipofection, or the like. About 2. mu.g of the antibody expression vector was linearized with restriction enzymes, and then subjected to electroporation using a Bio-Rad electrophoreter under conditions of 350V and 500. mu.F. Using gene pairs 4X 10⁶CHO cells were transfected and then seeded into 96-well culture plates. Lipofection was performed using LipofectAMINE Plus (produced by Gibco BRL) according to the instructions attached. After transfection with the vector, a drug corresponding to the selection marker used in the expression vector was added, and then culture was continued. After cloning was confirmed, antibody-expressing cell lines were screened by the method described in example 6. Antibody purification from the selected cells was performed as described in example 8.

EXAMPLE 3 preparation of antigen

In order to obtain cells overexpressing A33 (used as immunogen, for screening antibodies, etc.) on the cell membrane, a plasmid expression vector having the full-length A33 amino acid sequence was constructed. DNA encoding A33 was prepared by PCR.

a) Construction of full-Length A33 expression vector

A plasmid vector p.DELTA.EGFP-N1-GPA 33 containing cDNA encoding A33 was constructed to prepare an expression vector of full length A33. p.DELTA.EGFP-N1-GPA 33 was prepared as follows. Modification of full-Length A by Polymerase Chain Reaction (PCR)33 (GenBank DNANM-005814: SEQ ID NO: 11 or protein NP-005305: SEQ ID NO: 12) to add an EcoR I sequence at the 5 'end, a Not I sequence at the 3' end and a stop codon. Using Human Colon Marathon-Ready cDNA (purchased from Becton Dickinson Bioscience Clontech) as a template, synthesized A33-F25 '-GCAGACGAATTCAAGACCATGGTGGGGAAGAT-3' (SEQ ID NO: 13) and A33-R15 '-CTCGAGCGGCCGCTCTGCTGCTGGCCTGTCACTGGTCGAGGTG-3' (SEQ ID NO: 14) as primers, and KOD-plus DNA polymerase (produced by TOYOBO), a 30-cycle PCR reaction was performed (94 ℃ C. for 15 seconds, 60 ℃ C. for 30 seconds, 68 ℃ C. for 60 seconds). The synthesized sequence was then digested with EcoRI-Not I. The isolated product was an EcoR I-Not I fragment. The fragment was then ligated to p.DELTA.EGFP-N1 vector (modified pEGFP-N1 (produced by Becton Dickinson Bioscience Clontech) in which the region encoding the EGFP protein was deleted) which had been cleaved with the same enzyme. The resulting plasmid was named p.DELTA.EGFP-N1-GPA 33. A977 bp cDNA encoded by A33 was integrated into p.DELTA.EGFP-N1-GPA 33. Use ofPCR System 9700 (produced by PerkinElmer, Japan) was used to adjust the temperature of all PCR reactions in the examples below.

b) Preparation of A33 expressing cells

Transfecting the following two cell lines with p Δ EGFP-N1-GPA33 constructed in a): FM3A cell line (Japanese scientific biological resource Collection (JCRB) cell bank No.0701) and L929 cell line (ATCC No. CCL-1). Thus, two types of cells expressing a33 were prepared. The electroporation method was used for FM3A cells. Mu.g of p.DELTA.EGFP-N1-A33 vector was transfected with 5X 10 at 350V and 950. mu.F using an electrotransfer (produced by BTX)⁶FM3A cells. Cells were seeded in 6-well culture plates. After culturing at 37 ℃ for 48 hours in 5.0% carbonic acid gas, G418 (produced by Gibco BRL) was added to the cells to a concentration of 1mg/mL, and the cells were cultured for 1 week. Culture supernatants of AS33 hybridoma (ATCC NO. HB-8779) were used to confirm the A33 antigen expressed on the cell membrane surface. The supernatant of the AS33 hybridoma culture is used AS a primary antibody and labeledGoat anti-mouse Ig gamma F (ab')₂Antibody (produced by Dako) was subjected to flow cytometry (FCM: produced by Bectonv, Dickinson and company) as a secondary antibody. Thus, among transfected cells that acquired G418 resistance, cells expressing A33 on their cell membrane surface were selectively sorted.

L929 cells were transfected with Transs IT-LT1 (produced by TAKARA BIO INC). Transfection was performed according to the methods described in the relevant manual. After culturing at 37 ℃ for 24 hours under 5.0% carbonic acid gas, G418 (produced by Gibco BRL) was added to the cells to a concentration of 1mg/mL, and then the cells were cultured for 1 week. The a33 antigen expressed on the cell membrane surface was confirmed with AS33 hybridoma culture supernatant treated in the same manner AS FM3A cells. Goat anti-mouse Iggamma F (ab') labeled with R-phycoerythrin using AS33 hybridoma (ATCC No. HB-8779) AS primary antibody₂Antibody (produced by Dako) was analyzed by flow cytometry (FCM: produced by Bectonv, Dickinson and Company) as a secondary antibody. Thus, among transfected cells that acquired G418 resistance, cells expressing A33 on their cell membrane surface were selectively sorted.

A single clone expressing the full-length human a33 antigen at high levels can be obtained from both cell lines. FM3A cells and L929 cells expressing A33 antigen protein at high level were designated FM3A/A33 and L929/A33, respectively.

The shA33EX-hFc protein was prepared for use as an immunogen or for ELISA for antibody screening.

c) Construction of expression vector for soluble A33 human Fc fusion protein outside cell membrane

In order to construct an expression vector for the extracellular soluble A33 human Fc fusion protein (hereinafter referred to as shA33EX-hFc), a plasmid vector pTracer-CMV-humanFc-A33EXR containing cDNA encoding the extracellular A33 region was constructed. pTracer-CMV-humanFc-A33EXR was constructed as follows. The DNA (SEQ ID NO: 11) of the A33 cell membrane outer region containing the secretion signal sequence was modified by Polymerase Chain Reaction (PCR) to add an EcoR I sequence at its 5 'end and a Not I sequence and a stop codon at its 3' end. Using the cDNA of p Δ EGFP-N1-a33 prepared in a) as a template, a33-F2(SEQ ID NO: 13) and GPA-EXCRR25 '-CTCGAGCGGCCGCCAGTTCATGGAGGGAGATCTGACG-3' (SEQ ID NO: 15) as a primer, KOD-plus DNA polymerase (produced by TOYOBO) was added to carry out a 30-cycle PCR reaction (94 ℃ for 15 seconds, 60 ℃ for 30 seconds, 68 ℃ for 60 seconds). The synthesized sequence was then digested with EcoR I-Not I. The isolated product was an EcoR I-Not I fragment. The fragment was then ligated to pTracer-CMV-humanFc vector (a plasmid prepared by introducing FLAG and human IgG1Fc regions into the XbaI and Apa I sites of modified pTracer-CMV (produced by Invitrogen Life technologies)) which had been cleaved with the same enzyme. The resulting plasmid was designated pTracer-CMV-humanFc-A33 EXR.

Oligonucleotides such as PCR primers were all synthesized using an automatic DNA synthesizer (model 3948; manufactured by PerkinElmer, Applied Biosystems Division, Japan) according to the manual attached thereto (see Matteucci, M.D. and Caruthers, M.H. (1981) J.Am.chem.Soc.103, 3185-. After the synthesis is complete, the oligonucleotide is cleaved from the support and deprotected. The obtained solution was dried, solidified, and then dissolved in distilled water. The solution was stored at low temperature at-20 ℃ until use.

d) Expression and purification of shA33EX-hFc protein

Transfecting a host cell with the shA33EX-hFc protein expression vector constructed in c), thereby preparing a cell expressing the soluble A33 protein outside the cell membrane. The host cells used for expression are CHO cells of dhfr-deficient cell lines (ATCC CRL-9096), for example CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109, 1999) or HEK293T (ATCC CRL-11268).

Host cells are transfected with the vector by electroporation, lipofection, or other methods. About 2. mu.g of the shA33EX-hFc protein expression vector was linearized with restriction enzymes, and then subjected to electroporation using a Bio-Rad electrophoreter at 350V and 500. mu.F. Using gene pairs 4X 10⁶CHO cells were transfected and then seeded into 96-well culture plates. Using LipofectAMINEPLUS (produced by Gibco BRL) according to the attached protocolInstructions for lipofection. After transfection with the vector, a drug corresponding to the selection marker used in the expression vector was added, and then culture was continued.

The shA33EX-hFc protein was purified from the culture supernatant by the following method. The culture supernatant containing the shA33EX-hFc protein was subjected to affinity purification using Hitrap protein A FF (produced by Amersham pharmacia Biotech), PBS as an adsorption buffer, 20mN sodium acetate as an eluent, and 50mM sodium chloride (pH 2.7). The pH of the eluted fractions was adjusted to 5.5 by adding 50mM sodium phosphate solution (pH 7.0). The resulting extracellular soluble A33 protein solution was exchanged with PBS using Amicon Ultra-15 (manufactured by Amicon) and then sterilized by filtration using a membrane filter MILLEX-GV (manufactured by Millipore) having a pore size of 0.22. mu.m. Thus, a purified shA33EX-hFc protein was obtained. The concentration of the obtained shA33EX-hFc protein was calculated by measuring the absorbance at 280nm and calculating the 1.4OD corresponding to 1mg/mL (antibody concentration).

EXAMPLE 4 preparation of human antibody-producing mice

The mice used for immunization had a genetic background in which they were homozygotes with both the endogenous Ig heavy and kappa light chains disrupted. In addition, the mouse retains a chromosome 14 fragment (SC20) containing both the human Ig heavy chain gene locus and the human Ig kappa chain transgene (KCo 5). Such mice were produced by crossing mouse line a with the human Ig heavy chain gene locus with mouse line B with the human Ig kappa chain transgene. For example, as reported by Tomizuka et al (Tomizuka. et al., Procnnal Acad Sci U.S.A., 2000 Vol.97: 722), mouse line A is a homozygote in which both the endogenous Ig heavy and kappa light chains are disrupted and the endogenous chromosome 14 fragment (SC20) is retained. In addition, mouse line B is a homozygote with both the endogenous Ig heavy and kappa light chains disrupted. Furthermore, mouse line B, which retains the human Ig kappa chain transgene (KCo5), is a transgenic mouse as reported by Fishwild et al (Nat Biotechnol, (1996) 114: 856).

The mice used in the following immunization experiments were obtained by crossing male mouse line A with female mouse line B or crossing female mouse line A with male mouse line B, inHuman Ig heavy chain and kappa light chain (Ishida) can be detected simultaneously in mouse serum& Lonberg，IBS’s 11^thAntibody engineering, abstract 2000). In addition, the above-mentioned human antibody-producing mouse (hereinafter referred to as KM mouse) can be obtained from Kirin Brewery co.

Example 5 preparation of human monoclonal antibodies to A33

In this example, monoclonal antibodies were prepared according to the general method described in, for example, "Tan-Clone-Kotai-Jikken Manual" ("monoclonal antibody Experimental Manual") (Tamie ANDO et al, published by Kodansha scientific Press, Tokyo, Japan (1991)). A33 used as an immunogen was the A33-expressing FM3A cell or shA33EX-hFc protein prepared in example 1. The animal used for immunization was a mouse producing a human antibody (immunoglobulin) prepared in example 2.

To prepare a human monoclonal antibody against a33, a 33-expressing FM3A cells (1 × 10) prepared in example 3 were used⁷Cells/mouse) was mixed with RIBI adjuvant (manufactured by Corixa corporation), and then the human antibody-producing mouse was first immunized intraperitoneally with the mixture. After the first immunization, the animals were immunized 8 times per week with the same cells and RIBI adjuvant. 3 days before obtaining the spleen described below, the shA33EX-hFc protein was administered at 20. mu.g/mouse via the tail vein, and recombinant human IL-6 was administered at 5. mu.g/mouse subcutaneously.

In addition, the first immunization was carried out with a mixture of shA33EX-hFc protein (10. mu.g/mouse) and CpG adjuvant (produced by QIAGEN). After the first immunization, 2 immunizations were performed every 2 weeks with the same protein and CpG adjuvant. After 2 weeks, further immunization was performed using only this protein. 3 days before obtaining the spleen described below, the shA33EX-hFc protein was administered at 10. mu.g/mouse via the tail vein.

In addition, the shA33EX-hFc protein (10. mu.g/mouse) and FM3A cells (5X 10) expressing A33 were used⁶Cells/mice) and RIBI adjuvant were immunized intraperitoneally, then 1 to 4 times every 2 weeks. 4 days before acquisition of the spleen described below, by peritoneumThe shA33EX-hFc protein was administered at 5. mu.g/mouse.

Spleens were surgically obtained from each immunized mouse, and then 10mL of serum-free DMEM medium (produced by Gibco BRL, hereinafter referred to as serum-free DMEM medium) containing 350mg/mL of sodium bicarbonate, 50 units/mL of penicillin, and 50. mu.g/mL of streptomycin was added. The resultant was filtered through a filter with a mesh (cell filter: manufactured by Falcon) with a spatula. The cell suspension passed through the filter was centrifuged, thereby precipitating the cells. The cells were washed 2 times with serum-free DMEM medium, and then suspended in serum-free DMEM medium for cell counting. Meanwhile, myeloma cells SP2/0(ATCC No. CRL-1581) were cultured in the presence of 5% carbon dioxide at 37 ℃ in a DMEM medium (produced by Gibco BRL) containing 10% FCS (produced by Sigma; hereinafter referred to as serum-containing DMEM medium) so that the cell concentration thereof did not exceed 1X 10⁶cells/mL. Myeloma cells SP2/0(ATCC No. CRL-1581) were also washed with serum-free DMEM medium, and then suspended in serum-free DMEM medium for cell counting. The collected cell suspension was mixed with the mouse myeloma cell suspension at a ratio of 5: 1. After centrifugation, the supernatant was completely removed. To the precipitate, 1mL of 50% (w/v) polyethylene glycol 1500 (manufactured by Boehringer Mannheim) as a thawing agent was slowly added while stirring the solution with a pipette. 1mL of serum-free DMEM medium preheated at 37 ℃ was slowly added in two portions, and then 7mL of serum-free DMEM medium was added to the resulting solution. After centrifugation, the fused cells obtained by removing the supernatant were screened by the limiting dilution method described below. Selection of hybridomas was performed by culturing cells in DMEM medium containing 10% FCS, IL-6(10ng/mL) (or 10% hybridoma cloning factor (hereinafter, referred to as HCF; produced by BIOBASE)), hypoxanthine (H), aminopterin (A), and thymidine (T) (hereinafter, referred to as HAT; produced by Sigma). In addition, single clones were obtained by limiting dilution method using DMEM medium containing HT (produced by Sigma), 10% FCS and IL-6 (or 10% HCF). The culture was performed in a 96-well microtiter plate (produced by Becton, Dickinson and company). Enzyme-linked immunosorbent assay (EL) as described belowISA) and flow cytometry (FMC) to select (screen) hybridoma clones producing human monoclonal antibodies against a33 and to determine the properties of the human monoclonal antibodies produced by each hybridoma.

By the cell ELISA, protein ELISA and FMC analysis described in example 6, it was confirmed that a plurality of hybridomas producing human monoclonal antibodies having a human immunoglobulin gamma chain (higg) and a human immunoglobulin light chain kappa chain and specifically reactive with a33 were obtained. In addition, each hybridoma clone producing the human anti-a 33 monoclonal antibody of the present invention is designated by numbers and letters in all the following examples including the present example, and in all tables and figures showing experimental results in the examples. In addition, the numbers and letters of "antibody" attached to the following represent an antibody produced by the relevant hybridoma, or a recombinant antibody produced by a host cell containing an antibody gene (full-length or variable region) isolated from the relevant hybridoma. In addition, the name of a hybridoma clone may represent the name of an antibody, to the extent that the context is clear. The following hybridoma clones represent single clones: 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5 AAAA. 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA are deposited at 24.8.2004 at International Patent Organism Depositary (IPOD) of the national institute of Advanced Industrial Science and Technology (AIST) (Central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan) under accession numbers FERM BP-10107 (denoted by M10 for identification), FERM BP-10106 (denoted by M165 for identification), FERM BP-10108 (denoted by M96 for identification), FERM BP-10109 (denoted by N26 for identification), FERM BP-10104 (denoted by Q47 for identification), FERM BP-10105 (denoted by Q54 for identification) and FERM-10103 (denoted by R5 for identification).

Example 6 screening of clones producing human anti-A33 monoclonal antibody with human immunoglobulin gamma chain (hIg. gamma.) and human immunoglobulin light chain kappa (Ig. kappa.) (II)

Cell ELISA was performed as follows. FM3A/A33 prepared in example 3 at 1X 10 per well⁵Was added to a 96-well plate (produced by Falcon).Hybridoma supernatants were added and incubated at 4 ℃ for 30 minutes. The product was then washed 2 times with PBS containing 2% FCS, and goat anti-human IgG F (ab') labeled with horseradish peroxidase (50. mu.g/well: produced by IBL) was added₂Antibody, then at 4 degrees C temperature in 30 minutes. The product was washed 2 times with PBS containing 2% FCS, and then 100. mu.l of TMB chromogenic substrate (produced by DAKO) was added to each well, followed by incubation at room temperature for 20 minutes. To each well was added 0.5M sulfuric acid (100. mu.l/well) to stop the reaction. The absorbance at a wavelength of 450nm (reference wavelength: 570nm) was measured with a microplate reader (1420ARVO multi-label counter: produced by WALLAC). Antibody-producing clones showing a positive reaction were screened. Meanwhile, FM3A cells that do not express A33 antigen were used as negative controls. Specifically, culture supernatants that reacted with FM3A/A33 cells but not FM3A cells were screened as antibody-producing clones showing positive reactions.

In addition, protein ELISA was performed as follows. Mu.l of the shA33EX-hFc protein prepared in example 3 and adjusted to pH 9.4 with 1. mu.g/ml of carbonic acid buffer was added to each well of a 96-well microplate for ELISA (Maxisorp manufactured by Nunc). The incubation was performed at room temperature for 1 hour or overnight at 4 ℃ to adsorb the shA33EX-hFc protein to the microplate. Subsequently, the supernatant was removed, PBS containing 10% FCS was added to each well, and incubated at 37 ℃ for 1 hour. Thus, the shA33EX-hFc protein-free position can be blocked. In this manner, a microplate was prepared in which each well was coated with the shA33EX-hFc protein. Culture supernatants (50. mu.l) of each hybridoma were added to the wells and incubated at room temperature for 1 hour. Each well was washed 2 times with PBS containing 0.1% Tween20 (PBS-T). Subsequently, goat anti-human Ig kappa antibody labeled with horseradish peroxidase (50. mu.l/well, produced by The Bing Site) was diluted 2500-fold with PBS (PBS-T) containing 0.1% Tween 20. To each well 50. mu.l of the solution was added and incubated at 37 ℃ for 1 hour. The plate was washed 3 times with PBS-T. To each well, 100. mu.l of a TMB chromogenic substrate solution (produced by DAKO) was added, followed by incubation at room temperature for 20 minutes. To each well was added 0.5M sulfuric acid (100. mu.l/well) to stop the reaction. The absorbance at a wavelength of 450nm (reference wavelength: 570nm) was measured with a microplate reader (VersaMax manufactured by Molecular Devices Co.). Antibody-producing clones showing a positive reaction were screened.

In addition, FMC was performed as follows. The reactivity of the hybridoma culture supernatants to human colorectal cancer cell line COLO205 cells expressing the A33 antigen was examined. COLO205 cells at 2X 10⁶Suspension in a concentration of 0.1% NaN₃And 2% FCS in PBS Staining Buffer (SB). The cell suspension (50. mu.l/well) was dispensed into a 96-well round bottom plate (manufactured by Becton, Dickinson and Company). Hybridoma culture supernatant (50. mu.l) was added and incubated on ice for 30 minutes. Negative controls were prepared according to each subclass. Specifically, human IgG1 antibody (produced by Sigma) was adjusted to a concentration of 2. mu.g/ml with hybridoma culture medium, and then 50. mu.l of the solution was added, followed by incubation on ice for 30 minutes. The product was washed 2 times with SB, then 50. mu.l of RPE fluorescently labeled goat anti-human IgG F (ab')₂The antibody (produced by Southern Biotech) was then incubated on ice for 30 minutes. The product was washed 1 time with SB and then suspended in 300. mu.l FACS buffer. The mean fluorescence intensity of each cell line was determined by FACS (FACS caliber produced by Becton, Dickinson and company). The presence of antibodies binding to A33 expressed on the cells was confirmed because of their strong binding activity to cells of the COLO205 cell line.

Example 7 identification of each monoclonal antibody subclass in culture supernatants

ELISA (Maxisorp manufactured by Nunc) was performed by adding 50. mu.l of shA33EX-hFc protein prepared with 1. mu.g/ml of carbonic acid buffer solution (hereinafter referred to as PBS) to each well of a 96-well microplate. The incubation was carried out at room temperature for 1 hour or at 4 ℃ overnight to adsorb the shA33EX-hFc protein to the microplate. Then, the supernatant was removed, PBS containing 10% FCS was added to the wells, and incubated at room temperature for 1 hour or overnight at 4 ℃. Thus, the shA33EX-hFc protein-free position can be blocked. In this manner, a microplate was prepared in which each well was coated with the shA33EX-hFc protein. The plates were then washed 2 times with PBS containing 0.1% Tween20 (PBS-T). A goat anti-human IgG1 antibody labeled with horseradish peroxidase, a goat anti-human IgG2 antibody labeled with horseradish peroxidase, a goat anti-human IgG3 antibody labeled with horseradish peroxidase, or a goat anti-human IgG4 antibody labeled with horseradish peroxidase (diluted 1600, 6400, 25000, and 25000 times, respectively, produced by The Binding Site) was added at 50. mu.l/well. Incubate at room temperature for 1.5 hours. The plates were washed 3 times with PBS-T containing 0.1% Tween 20. Substrate buffer (TMB produced by DAKO) was added at 100. mu.l/well, followed by incubation at room temperature for 20 minutes. Then 0.5M sulfuric acid (100. mu.l/well) was added to stop the reaction. The absorbance at a wavelength of 450nm (reference wavelength: 570nm) was measured with a microplate reader (VersaMax manufactured by Molecular Devices Co.). The subclass of each clone was determined. Due to the high preference for ADCC and CDC, only human anti-a 33 antibodies of subclass IgG1 could be selected.

Table 1 shows only the reactivity of the final selected clones.

TABLE 1

Name of antibody	Subclass of	Reactivity to COLO205
			anti-DNP-IgG 1	IgG1	-
cA33	IgG1	+
			263A17	IgG1	+
125M10AA	IgG1	+
			125M165DAAA	IgG1	+
125M96ABA	IgG1	+
			125N26F6AA	IgG1	+
125Q47BA	IgG1	+
			125Q54AAAA	IgG1	+
125R5AAAA	IgG1	+

Example 8 preparation of Each type of antibody

The human anti-a 33 monoclonal obtained from the hybridoma culture supernatant described in example 6 was purified by the following method. Culture supernatants containing each type of human anti-A33 monoclonal antibody were cultured in SFM medium (produced by Invitrogen) containing 10% ultra-low IgG FBS (produced by Invitrogen). The culture supernatant was subjected to affinity purification using protein A Fast Flow gel (produced by Amersham Pharmacia Biotech) using PBS as an adsorption buffer and 0.02M glycine buffer (pH3.6) as an eluent. The eluted fractions were adjusted to about pH7.2 by adding 1M Tris (pH 8.0). The antibody solution thus prepared was exchanged with PBS using Sephadex G25 desalting column (NAP column; produced by Amersham Pharmacia Biotech), and then sterilized by filtration using a membrane filter MILLEX-GV (produced by Millipore) having a pore size of 0.22. mu.m. Thus, a purified human anti-A33 monoclonal antibody was obtained. The concentration of each purified antibody was calculated by measuring the 280nm absorbance and using 1.4OD corresponding to 1mg/mL (antibody concentration).

Example 9 testing the reactivity of each type of purified monoclonal antibody to A33-expressing cells

Each type of purified monoclonal antibody obtained in example 8 was tested for reactivity to human colorectal cancer cell line COLO205 cells, LoVo cells (ATCC No. CCL-229), LS174T cells (ATCC No. CL-188), and NCI-H508 cells (ATCC No. CCL-253) expressing the A33 antigen by FCM. Human colorectal cancer cell line HT-29 cells (ATCC No. HTB-38) that do not express the A33 antigen were also tested and used as negative control cells. 2X 10 per cell line⁶Suspension in a concentration of 0.1% NaN₃And 2% FCS in PBS Staining Buffer (SB). The cell suspension (50. mu.l/well) was dispensed into a 96-well round bottom plate (manufactured by Becton, Dickinson and Company). 50 μ l of each type of purified monoclonal antibody (adjusted to a concentration of 2000, 400, 80 or 16ng/ml using SB) was added, followed by incubation on ice for 30 minutes. Negative controls were prepared according to each subclass. Specifically, human IgG1 antibody (produced by Sigma) was adjusted to a concentration of 2000, 400, 80, or 16ng/ml with SB. Add 50. mu.l of solution and incubate for 30 min on ice. The product was washed 2 times with SB, then 50. mu.l of FITC fluorescently labeled goat anti-human IgG F (ab')₂The antibody (produced by Southern Biotech) was then incubated on ice for 30 minutes. The product was washed 1 time with SB and then suspended at 300 deg.Cμ l FACS buffer. The mean fluorescence intensity of each cell line was determined by FACS (FACScan, produced by Becton, Dickinson and company).

The results are shown in Table 2. For COLO205 cells, the half value of the mean fluorescence intensity was determined to be 90. For LoVo cells, the half value of the mean fluorescence intensity was determined to be 25. For LS174T cells, the half value of the mean fluorescence intensity was determined to be 125. For NCI-H508 cells, the half value of the mean fluorescence intensity was determined to be 125. When the concentration of antibody required to achieve this value is 10 < ═ X < 100ng/ml, reactivity is indicated by +++; when the concentration of antibody required to achieve this value is 100 < ═ X < 1000ng/ml, reactivity is expressed in + +; reactivity is expressed as + when the antibody concentration required to achieve this value is 1000 < ═ X < 10000 ng/ml. When no binding was observed, the reactivity was represented by-. Each type of purified monoclonal antibody showed binding to all cells expressing the a33 antigen.

EXAMPLE 10 Competition experiment of purified monoclonal antibody of each type with mouse anti-A33 antibody

Whether the antigenic determinant recognized by each type of purified monoclonal antibody obtained in example 8 was identical to that of the mouse anti-a 33 antibody was examined by competition experiments using FCM. COLO205 cell line at 2X 10⁶Suspension in a suspension containing 0.1% NaN₃And 2% FCS in PBS Staining Buffer (SB). The cell suspension (50. mu.l/well) was dispensed into a 96-well round bottom plate (manufactured by Becton, Dickinson and Company). To the wells containing each type of purified monoclonal antibody (1. mu.g/ml and 50. mu.l), the purified mouse anti-A33 antibody prepared in example 1 was added or not added at a concentration of 100. mu.g/ml, followed by incubation on ice for 30 minutes. A murine negative control (without added purified anti-a 33 antibody) was prepared as follows. Human IgG1 antibody (produced by Sigma) was adjusted to a concentration of 1. mu.g/ml with SB, and then added50 μ l of the solution, followed by incubation on ice for 30 minutes. The product was washed 2 times with SB, then 50. mu.l of FITC fluorescently labeled goat anti-human IgG F (ab')₂Antibody (produced by IBL) was then incubated on ice for 30 minutes. The product was washed 1 time with SB and then suspended in 300. mu.l FACS buffer. The mean fluorescence intensity of each cell line was measured by FACS (FACScan manufactured by Becton, Dickinson and Company). The inhibition (%) between each type of purified monoclonal antibody and the mouse a33 antibody was calculated according to the following formula:

inhibition (%) {100- (100 × average fluorescence intensity after preincubation with mouse a33 antibody)/(average fluorescence intensity after preincubation without mouse a33 antibody) }

Purified monoclonal antibodies having an inhibition rate (%) of 25% or less are classified as "non-blockers", those having an inhibition rate (%) of 25% or more but less than 90% are classified as "partial blockers", and those having an inhibition rate (%) of 90% or more are classified as "blockers". Thus, 263A17, 125M10AA and 125M96ABA were classified as "non-blockers", 125M165DAAA and 125N26F6AA were classified as "blockers", and 125Q47BA, 125Q54AAAA or 125R5AAAA were classified as "partial blockers". The results are shown in Table 3.

TABLE 3

Name of antibody	Inhibition ratio (%)	Classification
			cA33	94.2	Blocking object
263A17 125M10AA 125M165DAAA 125M96ABA 125N26F6AA 125Q47BA 125Q54AAAA 125R5AAAA	3.7 5.0 96.0 22.8 98.0 64.1 46.5 63.9	Non-blocker partial blocker

EXAMPLE 11 method for obtaining Normal human mononuclear leukocytes

First, normal human peripheral blood mononuclear cells were prepared according to a standard method using Ficoll (Ficoll-PaquePLUS: produced by Amersham pharmacia Biotech). Normal human blood was collected into a blood collection bag (produced by TERUMO) containing sodium citrate as an anticoagulant. Normal human blood is multi-layered in Ficoll, and then mononuclear cells are separated by specific gravity centrifugation (800G, room temperature, 15 minutes). The intermediate layer in the form of mononuclear leukocytes was extracted, and then the extracted mononuclear leukocytes were diluted with PBS. The dilution was centrifuged repeatedly 3 times at 200G for 10 minutes to remove the remaining platelets in the supernatant. Normal human peripheral blood mononuclear cells (hereinafter referred to as PBMCs) were obtained by the above method and then used as PBMCs in example 12. In addition, CD4+ T cells were isolated from PBMCs using CD4+ T cell isolation kit II (produced by Miltenyi Biotec) according to the attached instructions. The remaining cell population was also used as PBMCs in example 12.

EXAMPLE 12 cytotoxic Effect assay for each type of purified monoclonal antibody

Antibody-mediated cytotoxic effects were determined as described below. In the presence of cells having a killing activity such as NK cells or neutrophils and an antibody, cytotoxicity (antibody-dependent cytotoxicity, hereinafter referred to as ADCC) against a target cell is measured. In addition, in the presence of complement and antibodies, cytotoxic action against target cells (complement-dependent cytotoxic action, hereinafter referred to as CDC) was measured. The antibodies used here were: each type of purified monoclonal antibody prepared in example 8 and cA33 recombinant antibody as a control anti-a 33 antibody. In addition, an anti-DNP IgG1 antibody was used as a negative control.

The method is briefly described as follows. A compound of radioactive chromium (A), (B), (C), (⁵¹Cr) into the cytoplasm of the target cell and then determining the gamma dose to determine the release in the culture medium due to cell death⁵¹The amount of Cr.

Specifically, 10 is⁶Individual colorectal cancer cell lines COLO205(ATCC No. CCL-86) and 10⁶Each NCI-H508 cell (ATCC No. CCL-253) was suspended in 15. mu.l of Fetal Calf Serum (FCS) as a target cell. Add 50. mu.l (37MBq/mL)⁵¹Cr-labeled sodium chromate (manufactured by Perkinelmer; hereinafter referred to as⁵¹Cr), and then incubated at 37 ℃ for 1 hour. Next, 10mL of medium was added. The centrifugation was repeated 3 times to remove the medium, thereby removing those not integrated into the cells⁵¹Cr。

In the ADCC assay, 5,000 wells were incubated in V-bottom 96-well plates (produced by Coaster) containing antibodies at various concentrations and having a total volume of 200. mu.l⁵¹Cr-labeled target cells and 500,000 healthy human peripheral blood mononuclear leukocytes obtained by the method described in example 11 in the presence of 5% CO at 37 ℃₂Cultured for 4 hours under the conditions of (1).

In the CDC assay, 5,000 pieces of V-bottom 96-well plates (produced by Coaster) containing antibodies at various concentrations and having a total volume of 200. mu.l were cultured⁵¹Cr-labeled target cells and human serum-derived complement (produced by Sigma) at a final concentration of 5%, in the presence of 5% CO at 37 ℃₂Cultured for 4 hours under the conditions of (1).

In the ADCC and CDC assays, the plates are centrifuged after culturing to pellet the cells. Purified monoclonal antibodies of each type were prepared at a concentration of 0.4 to 500ng/ml, and 50. mu.l of the solution was added to a 96-well plate (Lumaplate. TM. -96; manufactured by Packard Instrument) containing a powder scintillator. The product was dried at 55 ℃ for 1.5 hours. After confirming that the plates were dry, the plates were covered with a special sealing film (TopSeal. TM. -A; 96-well microplate; manufactured by Packard Instrument). The gamma radiation dose was measured with a scintillation counter (TopCount; produced by Packard Instrument).

The results are shown in FIG. 1A to FIG. 1D and Table 4. For ADCC against COLO205 cells, the half value of the specific lysis (%) was determined to be 15%. The cytotoxic effect is indicated by +++ when the concentration of antibody required to achieve this value is 1 < ═ x < 10ng/ml, by + + when it is 10 < ═ x < 100ng/ml, by + when it is 100 < ═ x < 1000ng/ml, and by-when no specific lysis (%) is obtained. For ADCC against NCI-H508 cells, the half value of the specific lysis (%) was determined to be 15%. The cytotoxic effect is indicated by +++ when the concentration of antibody required to achieve this value is 1 < ═ x < 10ng/ml, by + + when it is 10 < ═ x < 100ng/ml, by + when it is 100 < ═ x < 1000ng/ml, and by-when no specific lysis (%) is obtained.

For CDC against COLO205 cells, the half value of specific lysis (%) was determined to be 10%. The cytotoxic effect is indicated by +++ when the antibody concentration required to achieve this value is 10 < ═ x < 100ng/ml, by + + when it is 100 < ═ x < 1000ng/ml, by + when it is x > - < 1000ng/ml, and by-when no specific lysis (%) is obtained. In addition, for CDC against NCI-H508 cells, the half value of the specific lysis (%) was determined to be 25%. The cytotoxic effect is indicated by +++ when the antibody concentration required to achieve this value is 10 < ═ x < 100ng/ml, by + + when it is 100 < ═ x < 1000ng/ml, by + when it is x > - < 1000ng/ml, and by-when no specific lysis (%) is obtained.

For ADCC, cA33 and 125Q54AAAA showed high cytotoxicity, while for CDC, 125M10AA showed high cytotoxicity.

Example 13 preparation of genes encoding Each type of monoclonal antibody

(1) cDNA Synthesis of Each type of monoclonal antibody

Hybridomas 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were cultured in DMEM medium (produced by Gibco BRL) containing 10ng/mL of IL-6 or 10% of HCF (produced by BIOBASE) and 10% of fetal bovine serum (produced by HyClone), respectively, after obtaining cells by centrifugation, ISOGEN (produced by NIPPON GENE) was added, and total RNA was extracted according to the relevant experiment. Antibody variable region cDNAs were cloned using SMART RACE cDNA amplification kit (produced by Becton Dickinson Bioscience Clontech) according to the attached instructions.

First strand cDNA was prepared using 5. mu.g of total RNA as a template.

Synthesis of first Strand cDNA

Total RNA 5. mu.g/3. mu.l

5’-CDS 1μl

SMART oligo 1μl

The reaction solution having the above composition was incubated at 70 ℃ for 2 minutes. The following components were then added followed by incubation at 42 ℃ for 1.5 hours.

5 Xbuffer 2. mu.l

DTT 1μl

DNTP mixture 1. mu.l

PowerScript reverse transcriptase 1. mu.l

In addition, 100. mu. l N- [ tris (hydroxymethyl) methyl ] glycine buffer was added, followed by incubation at 72 ℃ for 7 minutes to obtain first strand cDNA.

(2) PCR amplification of heavy and light chain genes and determination of nucleotide sequences

(2) -1: PCR amplification of the heavy and light chain genes of hybridoma 263A17

cDNA amplification was performed with KOD-Plus-DNA polymerase (manufactured by TOYOBO) by preparing the following reaction solution.

Sterilized Water 29.5. mu.l

cDNA 2.5μl

KOD-Plus buffer (10X) 5. mu.l

dNTP mix (2mM) 4. mu.l

MgSO₄(25mM) 2μl

KOD-Plus- (1 unit/. mu.l) 1. mu.l

Universal primer A mix (UPM) (10X) 5. mu.l

Gene Specific Primer (GSP) 1. mu.l

Total volume 50. mu.l

The reaction solution containing the above components was adjusted to a final volume of 50. mu.l with redistilled water, and then PCR was performed.

The 263A17 heavy chain gene was amplified by repeating the reaction cycle (98 ℃ for 1 second, 68 ℃ for 30 seconds) 30 times using the UPM primer and IgG1p primer (5'-TCTTGTCCACCTTGGTGTTGCTGGGCTTGTG-3') (SEQ ID NO: 16) attached to the SMART RACE cDNA amplification kit. Meanwhile, the 263A17 light chain gene was amplified by repeating the reaction cycle (98 ℃ for 1 second and 68 ℃ for 30 seconds) 30 times with the UPM primer and hk-2 (5'-GTT GAA GCT CTT TGT GAC GGG CGA GC-3') (SEQ ID NO: 17).

(2) -2: PCR amplification of heavy and light chain genes for hybridomas 125M10AA, 125M96ABA, 125Q47BA, 125Q54AAAA, and 125R5AAAA

The reaction conditions used here were the same as in (2) -1. The heavy chain genes of 125M10AA, 125M96ABA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were amplified by repeating the reaction cycle (98 ℃ for 1 second, 68 ℃ for 30 seconds) 30 times with UPM primers and IgG1p primers (SEQ ID NO: 16). In addition, 20 repeated reaction cycles (98 ℃ for 1 second and 68 ℃ for 30 seconds) were carried out using NUPM primers (SMARTRACE cDNA amplification kit; produced by Becton Dickinson Bioscience Clontech) and I gG2p/G134 primers (5'-TGC ACG CCG CTG GTC AGG GCG CCT GAG TTC C-3') (SEQ ID NO: 18) with 1. mu.l of the reaction solution as a template. Meanwhile, light chain genes of 125M10AA, 125M96ABA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were amplified by repeating the reaction cycle (98 ℃ for 1 second, 68 ℃ for 30 seconds) 30 times with UPM primers and hk-2(SEQ ID NO: 17). Furthermore, 20 repeated reaction cycles (98 ℃ C. for 1 second and 68 ℃ C. for 30 seconds) were carried out using 1. mu.l of the reaction solution as a template, NUPM primer and hk-5 primer (5'-AGG CAC ACA ACA GAG GCA GTT CCA GAT TTC-3') (SEQ ID NO: 19).

(2) -3: PCR amplification of the heavy and light chain genes of hybridomas 125M165DAAA and 125N26F6AA

The reaction conditions used here were the same as those of (2) -1. The 125M165DAAA and 125N26F6AA heavy chain genes were amplified by repeating the reaction cycle (98 ℃ for 1 second, 68 ℃ for 30 seconds) 30 times with the UPM primer and the hh-2 primer (5'-GCT GGA GGG CAC GGT CAC CAC GCT G-3') (SEQ ID NO: 20). Furthermore, 20 repeated reaction cycles (98 ℃ C. for 1 second and 68 ℃ C. for 30 seconds) were carried out using 1. mu.l of the reaction solution as a template, NUPM primer and hh-4 primer (5'-GGT GCC AGG GGG AAG ACC GAT GG-3') (SEQ ID NO: 21). Meanwhile, the 125M165DAAA and 125N26F6AA light chain genes were amplified by repeating the reaction cycle (98 ℃ for 1 second and 68 ℃ for 30 seconds) 30 times with UPM primers and hk-2(SEQ ID NO: 17). Furthermore, 20 repeated reaction cycles (98 ℃ C. for 1 second and 68 ℃ C. for 30 seconds) were carried out using 1. mu.l of the reaction solution as a template, and NUPM primer and hk-5 primer (SEQ ID NO: 19).

The PCR fragments of the heavy chain and the light chain amplified above were respectively recovered by ethanol precipitation, recovered by agarose gel electrophoresis, and then purified using a QIAquick gel extraction kit (manufactured by QIAGEN), which is a DNA purification kit using a membrane. The purified HV and LV amplified fragments were subcloned into PCR 4Blunt-TOPO vectors of Zero Blunt TOPO PCR cloning kit (produced by Invitrogen), respectively. The nucleotide sequence of the insert DNA on the plasmid DNA in the obtained clones was analyzed. The DNA nucleotide sequences were determined using M13FW (SEQ ID NO: 3) and M13RV (SEQ ID NO: 4) as primers.

DNAs encoding the 263A17 heavy chain variable region and the light chain variable region and the amino acid sequences of the heavy chain variable region and the light chain variable region are each shown below.

<263A17 heavy chain nucleic acid sequence > (SEQ ID NO: 22)

10 20 30 40 50 60

ATGGAGTTTG GGCTGAGCTG GCTTTTTCTT GTGGCTATTT TAAAAGGTGT CCAGTGTGAG

70 80 90 100 110 120

GTGCAGTTGT TGGAGTCTGG GGGAGGCTTG GTACAGCCTG GGGGGTCCCT GAGACTCTCC

130 140 150 160 170 180

TGTGCAGCCT CTGGATTCAC CTTTAGCAGC TATGCCATGA GCTGGATCCG CCAGGCTCCA

190 200 210 220 230 240

GGGAAGGGGC TGGAGTGGGT CTCAGCTATT AGTGCTAGTG GTGGTAGCAC ATACTACGCA

250 260 270 280 290 300

GACTCCGTGA AGGGCCGGTT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG

310 320 330 340 350 360

CAAATGAACA GCCTGAGAGC CGAGGACACG GCCGTATATT ACTGTGCGAA AGATCGGATA

370 380 390 400 410 420

GTGGGAGCTA CGAACTACTA CTACGGTATG GACGTCTGGG GCCAAGGGAC CACGGTCACC

430 440 450 460 470 480

GTCTCCTCAG CTAGC.....

<263A17 heavy chain amino acid sequence > (SEQ ID NO: 23)

10 20 30 40 50 60

MEFGLSWLFL VAILKGVQCE VQLLESGGGL VQPGGSLRLS CAASGFTFSS YAMSWIRQAP

70 80 90 100 110 120

GKGLEWVSAI SASGGSTYYA DSVKGRFTI S RDNSKNTLYL QMNSLRAEDT AVYYCAKDRI

130 140 150 160 170 180

VGATNYYYGM DVWGQGTTVT VS SAS.....

<263A17 light chain nucleic acid sequence > (SEQ ID NO: 24)

10 20 30 40 50 60

ATGGACATGA GGGTCCCCGC TCAGCTCCTG GGGCTCCTGC TGCTCTGGTT CCCAGGTTCC

70 80 90 100 110 120

AGATGCGACA TCCAGATGAC CCAGTCTCCA CCTTCCGTGT CTGCATCTGT AGGAGACAGA

130 140 150 160 170 180

GTCACCATCA CTTGTCGGGC GAGTCAGGGT ATTAGCAGCT GGTTAGCCTG GTATCAGCAT

190 200 210 220 230 240

AAACCAGGGA AAGCCCCAAA GCTCCTGATC TATGGTGCAT CCAGTTTGCA AAGTGGGGTC

250 260 270 280 290 300

CCATCAAGGT TCAGCGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG

310 320 330 340 350 360

CAGCCTGAAG ATTTTGCAAC TTACTATTGT CAACAGGCTA ATAGTTTCCC TATCACCTTC

370 380 390

GGCCAAGGGA CACGACTGGA GATTAAACGT

<263A17 light chain amino acid sequence > (SEQ ID NO: 25)

10 20 30 40 50 60

MDMRVPAQLL GLLLLWFPGS RCDIQMTQSP PSVSASVGDR VTITCRASQG ISSWLAWYQH

70 80 90 100 110 120

KPGKAPKLLI YGASSLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQANSFPITF

130

GQGTRLEIKR

In the heavy chain nucleic acid sequence (SEQ ID NO: 22), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 429 and guanine (G) at position 430. In the heavy chain amino acid sequence (SEQ ID NO: 23), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 143 and alanine (A) at position 144. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 22), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and guanine (G) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 23), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 19 and glutamic acid (E) at position 20.

Thus, the variable region in the 263A17 antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 22) ranging from guanine (G) at position 58 to adenine (A) at position 429. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 23) ranging from glutamic acid (E) at position 20 to serine (S) at position 143.

In the light chain nucleic acid sequence (SEQ ID NO: 24), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 387 and cytosine (C) at position 388. In the light chain amino acid sequence (SEQ ID NO: 25), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 129 and arginine (R) at position 130. Further, in the light chain nucleic acid sequence (SEQ ID NO: 24), the boundary between the signal sequence and the antibody variable region is located between cytosine (C) at position 66 and guanine (G) at position 67. In the light chain amino acid sequence (SEQ ID NO: 25), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 22 and aspartic acid (D) at position 23.

Thus, the variable region in the light chain of the 263A17 antibody has the nucleic acid sequence (SEQ ID NO: 24) ranging from guanine (G) at position 67 to adenine (A) at position 387. In addition, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 25) ranging from aspartic acid (D) at position 23 to lysine (K) at position 129.

DNAs encoding the 125M10AA heavy chain variable region and the light chain variable region and the amino acid sequences of the heavy chain variable region and the light chain variable region are each shown below.

<125M10AA heavy chain nucleic acid sequence > (SEQ ID NO: 26)

10 20 30 40 50 60

ATGGATCTCA TGTGCAAGAA AATGAAGCAC CTGTGGTTCT TCCTCCTGCT GGTGGCGGCT

70 80 90 100 110 120

CCCAGATGGG TCCTGTCCCA GCTGCAGGTG CAGGAGTCGG GCCCAGGACT GGTGAAGCCT

130 140 150 160 170 180

TCGGAGACCC TGTCCCTCAT CTGCACTGTC TCTGGTGGCT CCATCAGGAC CAGTGGTTAC

190 200 210 220 230 240

TACTGGGGCT GGTTCCGCCA GCCCCCAGGG AAGGGACTGG AGTGGATTGG GACTAGTCAT

250 260 270 280 290 300

AATAGTGGGA GCACCTACTA CAACCCGTCC CTCAAGAGTC GAGTCACCAT ATCCGTAGAC

310 320 330 340 350 360

ACGTCCAAGA ACCAGTTCTC CCTGAAGCTG AACTCTGTGA CCGCCGCAGA CACGGCTGTG

370 380 390 400 410 420

TATTACTGTG CGAGACAAGG TTACGATTTT AAAGTCAATA TAGACGTCTG GGGACAAGGG

430 440 450

ACCACGGTCA CCGTCTCCTC AGCTAGC...

<125M10AA heavy chain amino acid sequence > (SEQ ID NO: 27)

10 20 30 40 50 60

MDLMCKKMKH LWFFLLLVAA PRWVLSQLQV QESGPGLVKP SETLSLICTV SGGSIRTSGY

70 80 90 100 110 120

YWGWFRQPPG KGLEWIGTSH NSGSTYYNPS LKSRVTISVD TSKNQFSLKL NSVTAADTAV

130 140 150

YYCARQGYDF KVNIDVWGQG TTVTVSSAS.

<125M10AA light chain nucleic acid sequence > (SEQ ID NO: 28)

10 20 30 40 50 60

ATGGAAGCCC CAGCTCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA

70 80 90 100 110 120

GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC

130 140 150 160 170 180

CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTGGTACCA ACAGAAACCT

190 200 210 220 230 240

GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC

250 260 270 280 290 300

AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT

310 320 330 340 350 360

GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCCGCTCAC TTTCGGCGGA

370 380 390

GGGACCAAGG TGGAGATCAA ACGA......

<125M10AA light chain amino acid sequence > (SEQ ID NO: 29)

10 20 30 40 50 60

MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP

70 80 90 100 110 120

GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG

130

GTKVEIKR..

In the heavy chain nucleic acid sequence (SEQ ID NO: 26), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 441 and guanine (G) at position 442. In the heavy chain amino acid sequence (SEQ ID NO: 27), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 147 and alanine (A) at position 148. Further, in the heavy chain nucleic acid sequence (SEQ ID NO: 26), the boundary between the signal sequence and the antibody variable region is located between cytosine (C) at position 78 and cytosine (C) at position 79. In the heavy chain amino acid sequence (SEQ ID NO: 27), the boundary between the signal sequence and the antibody variable region is located between serine (S) at position 26 and glutamine (Q) at position 27.

Thus, the variable region in the 125M10AA antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 26) ranging from cytosine (C) at position 79 to adenine (A) at position 441. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 27) ranging from glutamine (Q) at position 27 to serine (S) at position 147.

In the light chain nucleic acid sequence (SEQ ID NO: 28), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 381 and cytosine (C) at position 382. In the light chain amino acid sequence (SEQ ID NO: 29), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 127 and arginine (R) at position 128. In addition, in the light chain nucleic acid sequence (SEQ ID NO: 28), the boundary between the signal sequence and the antibody variable region is located between adenine (A) at position 60 and guanine (G) at position 61. In the light chain amino acid sequence (SEQ ID NO: 29), the boundary between the signal sequence and the antibody variable region is located between glycine (G) at position 20 and glutamic acid (E) at position 21.

Thus, the variable region in the 125M10AA antibody light chain has the nucleic acid sequence (SEQ ID NO: 28) ranging from guanine (G) at position 61 to adenine (A) at position 381. Further, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 29) ranging from glutamic acid (E) at position 21 to lysine (K) at position 127.

DNAs encoding the 125M165DAAA heavy chain variable region and the light chain variable region and the amino acid sequences of the heavy chain variable region and the light chain variable region are each shown below.

<125M165DAAA heavy chain nucleic acid sequence > (SEQ ID NO: 30)

10 20 30 40 50 60

ATGGAGTTTG GGCTGAGCTG GGTTTTCCTC GTTGCTCTTT TAAGAGGTGT CCAGTGTCAG

70 80 90 100 110 120

GTGCAGCTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG GGAGGTCCCT GAGACTCTCC

130 140 150 160 170 180

TGTGCAGCGT CTGGATTCAC CTTCAGTTAT TATGGCATGC ACTGGGTCCG CCAGGCTCCA

190 200 210 220 230 240

GGCAAGGGGC TGGAGTGGGT GGCAGTTATA TGGTATGATG GAAGTAATAA ATACTATGCA

250 260 270 280 290 300

GACTCCGTGA AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAAAAC GCTGTATCTG

310 320 330 340 350 360

CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG AGATGGGCAT

370 380 390 400 410 420

AGCAGTGGCT GGGGGGACTT CCAGCACTGG GGC CAGGGCA CCCTGGTCAC CGTCTCCTCA

430

GCTAGC....

<125M165DAAA heavy chain amino acid sequence > (SEQ ID NO: 31)

10 20 30 40 50 60

MEFGLSWVFL VALLRGVQCQ VQLVESGGGV VQPGRSLRLS CAASGFTFSY YGMHWVRQAP

70 80 90 100 110 120

GKGLEWVAVI WYDGSNKYYA DSVKGRFTIS RDNSKKTLYL QMNSLRAEDT AVYYCARDGH

130 140 150

SSGWGDFQHW GQGTLVTVSS AS........

<125M165DAAA light chain nucleic acid sequence > (SEQ ID NO: 32)

10 20 30 40 50 60

ATGGAAGCCC CAGCTCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA

70 80 90 100 110 120

GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC

130 140 150 160 170 180

CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTCCTTAG CCTGGTACCA ACAGAAACCT

190 200 210 220 230 240

GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC

250 260 270 280 290 300

AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT

310 320 330 340 350 360

GAAGATTTTG CAATTTATTA CTGTCAGCAG CGTAGCAACT GGCCTCCGAC GTTCGGCCAA

370 380 390

GGGACCAAGG TGGAAATCAA ACGA......

<125M165DAAA light chain amino acid sequence > (SEQ ID NO: 33)

10 20 30 40 50 60

MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQSVS SSLAWYQQKP

70 80 90 100 110 120

GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTI SSLEP EDFAIYYCQQ RSNWPPTFGQ

130

GTKVEIKR..

In the heavy chain nucleic acid sequence (SEQ ID NO: 30), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 420 and guanine (G) at position 421. In the heavy chain amino acid sequence (SEQ ID NO: 31), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 140 and alanine (A) at position 141. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 30), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and cytosine (C) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 31), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 19 and glutamine (Q) at position 20.

Thus, the variable region in the 125M165DAAA antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 30) ranging from cytosine (C) at position 58 to adenine (A) at position 420. Further, the variable region in the heavy chain has the amino acid sequence (SEQ IU NO: 31) ranging from glutamine (Q) at position 20 to serine (S) at position 140.

In the light chain nucleic acid sequence (SEQ ID NO: 32), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 381 and cytosine (C) at position 382. In the light chain amino acid sequence (SEQ ID NO: 33), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 127 and arginine (R) at position 128. In addition, in the light chain nucleic acid sequence (SEQ ID NO: 32), the boundary between the signal sequence and the antibody variable region is located between adenine (A) at position 60 and guanine (G) at position 61. In the light chain amino acid sequence (SEQ ID NO: 33), the boundary between the signal sequence and the antibody variable region is located between glycine (G) at position 20 and glutamic acid (E) at position 21.

Thus, the variable region in the 125M165DAAA antibody light chain has the nucleic acid sequence (SEQ ID NO: 32) ranging from guanine (G) at position 61 to adenine (A) at position 381. Further, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 33) ranging from glutamic acid (E) at position 21 to lysine (K) at position 127.

DNAs encoding the 125M96ABA heavy chain variable region and the light chain variable region and the amino acid sequences of the heavy chain variable region and the light chain variable region are each shown below.

<125M96ABA heavy chain nucleic acid sequence > (SEQ ID NO: 34)

10 20 30 40 50 60

ATGAAGCACC TGTGGTTCTT CCTCCTGCTG GTGGCGGCTC CCAGATGGGT CCTGTCCCAA

70 80 90 100 110 120

CTGCAGCTGC AGGAGTCGGG CCCAGGACTG GTGAAGCCTT CGGAGACCCT GTCCCTCACC

130 140 150 160 170 180

TGCACTGTCT CTGGTGGCTC CATCAGCACT AGTAGTTACT ACTGGGGCTG GATCCGCCAG

190 200 210 220 230 240

CCCCCCGGGA AGGGCCTGGA ATGGATTGGG ACTATCTATT ATAATGGGAG CACCTACTAC

250 260 270 280 290 300

AGCCCGTCCC TCAAGAGTCG AGTCAGTATA TCCGTAGACA CGTCCAAGAA CCAGTTCTCC

310 320 330 340 350 360

CTGAAGCTGA GCTCTGTGAC CGCCGCAGAC ACGTCTGTGT ATTACTGTGC GAGACAAGGT

370 380 390 400 410 420

TACGATATTA AAATCAATAT AGACGTCTGG GGCCAAGGGA CCACGGTCAC CGTCTCCTCA

430

GCTAGC....

<125M96ABA heavy chain amino acid sequence > (SEQ ID NO: 35)

10 20 30 40 50 60

MKHLWFFLLL VAAPRWVLSQ LQLQESGPGL VKPSETLSLT CTVSGGSIST SSYYWGWIRQ

70 80 90 100 110 120

PPGKGLEWIG TIYYNGSTYY SPSLKSRVSI SVDTSKNQFS LKLSSVTAAD TSVYYCARQG

130 140 150

YDIKINIDVW GQGTTVTVSS AS........

<125M96ABA light chain nucleic acid sequence > (SEQ ID NO: 36)

10 20 30 40 50 60

ATGGAAGCCC CAGCTCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA

70 80 90 100 110 120

GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC

130 140 150 160 170 180

CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTGGTACCA ACAGAAACCT

190 200 210 220 230 240

GGCCAGGCTC CCAGGCTCCT CATCTATGTT GCATCCAACA GGGCCACTGG CATCCCAGCC

250 260 270 280 290 300

AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT

310 320 330 340 350 360

GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCCGCTCAC TTTCGGCGGA

370 380 390

GGGACCAAGG TGGAGATCAA ACGA......

<125M96ABA light chain amino acid sequence > (SEQ ID NO: 37)

10 20 30 40 50 60

MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP

70 80 90 100 110 120

GQAPRLLIYV ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG

130

GTKVEIKR..

In the heavy chain nucleic acid sequence (SEQ ID NO: 34), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 420 and guanine (G) at position 421. In the heavy chain amino acid sequence (SEQ ID NO: 35), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 140 and alanine (A) at position 141. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 34), the boundary between the signal sequence and the antibody variable region is located between cytosine (C) at position 57 and cytosine (C) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 35), the boundary between the signal sequence and the antibody variable region is located between serine (S) at position 19 and glutamine (Q) at position 20.

Thus, the variable region in the 125M96ABA antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 34) ranging from cytosine (C) at position 58 to adenine (A) at position 420. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 35) ranging from glutamine (Q) at position 20 to serine (S) at position 140.

In the light chain nucleic acid sequence (SEQ ID NO: 36), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 381 and cytosine (C) at position 382. In the light chain amino acid sequence (SEQ ID NO: 37), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 127 and arginine (R) at position 128. In addition, in the light chain nucleic acid sequence (SEQ ID NO: 36), the boundary between the signal sequence and the antibody variable region is located between adenine (A) at position 60 and guanine (G) at position 61. In the light chain amino acid sequence (SEQ ID NO: 37), the boundary between the signal sequence and the antibody variable region is located between glycine (G) at position 20 and glutamic acid (E) at position 21.

Thus, the variable region in the 125M96ABA antibody light chain has the nucleic acid sequence (SEQ ID NO: 36) ranging from guanine (G) at position 61 to adenine (A) at position 381. Further, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 37) ranging from glutamic acid (E) at position 21 to lysine (K) at position 127.

DNAs encoding the 125N26F6AA heavy chain variable region and light chain variable region and the amino acid sequences of the heavy chain variable region and light chain variable region are each shown below.

<125N26F6AA heavy chain nucleic acid sequence > (SEQ ID NO: 38)

10 20 30 40 50 60

ATGGAGTTTG GGCTGAGCTG GGTTTTCGTC GTTGCTCTTT TAAGAGGTGT CCAGTGTCAG

70 80 90 100 110 120

GTGCAGTTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG GGAGGTCCCT GAGACTCTCC

130 140 150 160 170 180

TGTGCAGCGT CTGGATTCAC CTTCAGTCAC TATGGCATGC ACTGGGTCCG CCAGGCTCCA

190 200 210 220 230 240

GGCAAGGGGC TGGAGTGGGT GGCACTTATA TGGTATGATG GAAGTAATAA ATACTATGCA

250 260 270 280 290 300

GACTCCGTGA AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG

310 320 330 340 350 360

CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG AGATCCCTTA

370 380 390 400 410 420

GCAGCTGGTA CGTCCTACTT TGACTACTGG GGCCAGGGAA CCCTGGTCAC CGTCTCCTCA

430

GCTAGC....

<125N26F6AA heavy chain amino acid sequence > (SEQ ID NO: 39)

10 20 30 40 50 60

MEFGLSWVFL VALLRGVQCQ VQLVESGGGV VQPGRSLRLS CAASGFTFSH YGMHWVRQAP

70 80 90 100 110 120

GKGLEWVALI WYDGSNKYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARDPL

130 140 150

AAGTSYFDYW GQGTLVTVSS AS........

<125N26F6AA light chain nucleic acid sequence > (SEQ ID NO: 40)

10 20 30 40 50 60

ATGTCGCCAT CACAACTCAT TGGGTTTCTG CTGCTCTGGG TTCCAGCCTC CAGGGGTGAA

70 80 90 100 110 120

ATTGTGCTGA CTCAGTCTCC AGACTTTCAG TCTGTGACTC CAAAGGAGAA AGTCACCATC

130 140 150 160 170 180

ACCTGCCGGG CCAGTCAGAG CATTGGTAGT AGCTTACACT GGTACCAGCA GAAACCAGAT

190 200 210 220 230 240

CAGTCTCCAA AGCTCCTCAT CAAGTATGCT TCCCAGTCCT TCTCAGGGGT CCCCTCGAGG

250 260 270 280 290 300

TTCAGTGGCA GTGGATCTGG GACAGATTTC ACCCTCACCA TCAATAGCCT GGAAGCTGAA

310 320 330 340 350 360

GATGCTGCAG CGTATTACTG TCATCAGAGT AGTAGTTTAC CATTCACTTT CGGCCCTGGG

370 380

ACCAAAGTGG ATATCAAACG A

<125N26F6AA light chain amino acid sequence > (SEQ ID NO: 41)

10 20 30 40 50 60

MSPSQLIGFL LLWVPASRGE IVLTQSPDFQ SVTPKEKVTI TCRASQSIGS SLHWYQQKPD

70 80 90 100 110 120

QSPKLLIKYA SQSFSGVPSR FSGSGSGTDF TLTINSLEAE DAAAYYCHQS SSLPFTFGPG

130

TKVDIKR...

In the heavy chain nucleic acid sequence (SEQ ID NO: 38), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 420 and guanine (G) at position 421. In the heavy chain amino acid sequence (SEQ ID NO: 39), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 140 and alanine (A) at position 141. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 38), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and cytosine (C) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 39), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 19 and glutamine (Q) at position 20.

Thus, the variable region in the 125M96ABA antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 38) ranging from cytosine (C) at position 58 to adenine (A) at position 420. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 39) ranging from glutamine (Q) at position 20 to serine (S) at position 140.

In the light chain nucleic acid sequence (SEQ ID NO: 40), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 388 and cytosine (C) at position 389. In the light chain amino acid sequence (SEQ ID NO: 41), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 126 and arginine (R) at position 127. In addition, in the light chain nucleic acid sequence (SEQ ID NO: 40), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and guanine (G) at position 58. In the light chain amino acid sequence (SEQ ID NO: 41), the boundary between the signal sequence and the antibody variable region is located between glycine (G) at position 19 and glutamic acid (E) at position 20.

Thus, the variable region in the 125M96ABA antibody light chain has the nucleic acid sequence (SEQ ID NO: 40) ranging from guanine (G) at position 58 to adenine (A) at position 388. In addition, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 41) ranging from glutamic acid (E) at position 20 to lysine (K) at position 126.

DNAs encoding the 125Q47BA heavy chain variable region and the light chain variable region and the amino acid sequences of the heavy chain variable region and the light chain variable region are each shown below.

<125Q47BA heavy chain nucleic acid sequence > (SEQ ID NO: 42)

10 20 30 40 50 60

ATGGAGTTTG GGCTGAGCTG GCTTTTTCTT GTGGCTATTT TAAAAGGTGT CCAGTGTGAG

70 80 90 100 110 120

GTGCAGCTGT TGGAGTCTGG GGGAGGCTTG GTACAGCCTG GGGGGTCCCT GAGACTCTCC

130 140 150 160 170 180

TGTGCAGCCT CTGGATTCAC CTTTAGCAGC TATGCCATGA GCTGGGTCCG CCAGGCTCCA

190 200 210 220 230 240

GGGAAGGGGC TGGAGTGGGT CTCAGATATT AGTGGTAGTG GTGGTTACAC ATACTACGCA

250 260 270 280 290 300

GACTCCGTGA AGGGCCGGTT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG

310 320 330 340 350 360

CAAATGAACA GCCTGAGAGC CGAGGACACG GCCGTATATT ACTGTGCGAA AACAGGCGAT

370 380 390 400 410 420

GGTTCGGGGA GTTATTCCCC TGACTCCTGG GGCCAGGGAA CCCTGGTCAC CGTCTCCTCA

430

GCTAGC....

<125Q47BA heavy chain amino acid sequence > (SEQ ID NO: 43)

10 20 30 40 50 60

MEFGLSWLFL VAILKGVQCE VQLLESGGGL VQPGGSLRLS CAASGFTFSS YAMTWVRQAP

70 80 90 100 110 120

GKGLEWVSDI SGSGGYTYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAKTGA

130 140 150

GSGSYSPDSW GQGTLVTVSS AS........

<125Q47BA light chain nucleic acid sequence > (SEQ ID NO: 44)

10 20 30 40 50 60

ATGGACATGA GGGTCCTCGC TCAGCTCCTG GGGCTCCTGC TGCTCTGTTT CCCAGGTGCC

70 80 90 100 110 120

AGATGTGACA TCCAGATGAC CCAGTCTCCA TCCTCACTGT CTGCATCTGT AGGAGACAGA

130 140 150 160 170 180

GTCACCATCA CTTGTCGGGC GAGTCAGGGT ATTAGCAGCT GGTTAGCCTG GTATCAGCAG

190 200 210 220 230 240

AAACCAGAGA AAGCCCCTAA GTCCCTGATC TATGCTGCAT CCAGTTTGCA AAGTGGGGTC

250 260 270 280 290 300

CCATCAAGGT TCAGCGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG

310 320 330 340 350 360

CAGCCTGAAG ATTTTGCAAC TTATTACTGC CAACAGTATA ATAGTTACCC GTACACTTTT

370 380 390

GGCCAGGGGA CCAAGCTGGA GATCAAACGA

<125Q47BA light chain amino acid sequence > (SEQ ID NO: 45)

10 20 30 40 50 60

MDMRVLAQLL GLLLLCFPGA RCDIQMTQSP SSLSASVGDR VTITCRASQG ISSWLAWYQQ

70 80 90 100 110 120

KPEKAPKSLI YAASSLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQYNSYPYTF

130

GQGTKLEIKR

In the heavy chain nucleic acid sequence (SEQ ID NO: 42), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 420 and guanine (G) at position 421. In the heavy chain amino acid sequence (SEQ ID NO: 43), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 140 and alanine (A) at position 141. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 42), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and guanine (G) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 43), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 19 and glutamic acid (E) at position 20.

Thus, the variable region in the 125Q47BA antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 42) ranging from guanine (G) at position 58 to adenine (A) at position 420. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 43) ranging from glutamic acid (E) at position 20 to serine (S) at position 140.

In the light chain nucleic acid sequence (SEQ ID NO: 44), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 387 and cytosine (C) at position 388. In the light chain amino acid sequence (SEQ ID NO: 45), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 129 and arginine (R) at position 130. Further, in the light chain nucleic acid sequence (SEQ ID NO: 44), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 66 and guanine (G) at position 67. In the light chain amino acid sequence (SEQ ID NO: 45), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 22 and aspartic acid (D) at position 23.

Thus, the variable region in the 125Q47BA antibody light chain has the nucleic acid sequence (SEQ ID NO: 44) ranging from guanine (G) at position 67 to adenine (A) at position 387. In addition, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 45) ranging from aspartic acid (D) at position 23 to lysine (K) at position 129.

DNAs encoding the 125Q54AAAA heavy chain variable region and the light chain variable region and the amino acid sequences of the heavy chain variable region and the light chain variable region are each shown below.

<125Q54AAAA heavy chain nucleic acid sequence > (SEQ ID NO: 46)

10 20 30 40 50 60

ATGGAGTTTG GGCTGAGCTG GCTTTTTCTT GTGGCTATTT TAAAAGGTGT CCAGTGTGAG

70 80 90 100 110 120

GTGCAGCTGT TGGAGTCTGG GGGAGGCTTG GTACAGCCTG GGGGGTCCCT GAGACTCTCC

130 140 150 160 170 180

TGTGCAGCCT CTGGATTCAC CTTTAGCAGC TATGCCATGA GCTGGGTCCG CCAGGCTCCA

190 200 210 220 230 240

GGGAAGGGGC TGGAGTGGGT CTCAGATATT AGTGGTAGTG GTGGTTACAC ATACTACGCA

250 260 270 280 290 300

GACTCCGTGA AGGGCCGGTT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG

310 320 330 340 350 360

CAAATGAACA GCCTGAGAGC CGAGGACACG GCCGTATATT ACTGTGCGAA AACAGGCGAT

370 380 390 400 410 420

GGTTCGGGGA GTTATTCCCC TGACTCCTGG GGCCAGGGAA CCCTGGTCAC CGTCTCCTCA

430

GCTAGC....

<125Q54AAAA heavy chain amino acid sequence > (SEQ ID NO: 47)

10 20 30 40 50 60

MEFGLSWLFL VAILKGVQCE VQLLESGGGL VQPGGSLRLS CAASGFTFSS YAMSWVRQAP

70 80 90 100 110 120

GKGLEWVSDI SGSGGYTYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAKTGD

130 140 150

GSGSYSPDSW GQGTLVTVSS AS........

<125Q54AAAA light chain nucleic acid sequence > (SEQ ID NO: 48)

10 20 30 40 50 60

ATGGACATGA GGGTCCTCGC TCAGCTCCTG GGGCTCCTGC TGCTCTGTTT CCCAGGTGCC

70 80 90 100 110 120

AGATGTGACA TCCAGATGAC CCAGTCTCCA TCCTCACTGT CTGCATCTGT AGGAGACAGA

130 140 150 160 170 180

GTCACCATCA CTTGTCGGGC GAGTCAGGGT ATTAGCAGGT GGTTAGCCTG GTATCAGCAG

190 200 210 220 230 240

AAACCAGAGA AAGCCCCTAA GTCCCTGATC TATGCTGCAT CCAGTTTGCA AAGTGGGGTC

250 260 270 280 290 300

CCATCAAGGT TCAGCGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG

310 320 330 340 350 360

CAGCCTGAAG ATTTTGCAAC TTATTACTGC CAACAGTATA ATAGTTACCC GTACACTTTT

370 380 390

GGCCAGGGGA CCAAGCTGGA GATCAAACGA

<125Q54AAAA light chain amino acid sequence > (SEQ ID NO: 49)

10 20 30 40 50 60

MDMRVLAQLL GLLLLCFPGA RCDIQMTQSP SSLSASVGDR VTITCRASQG ISRWLAWYQQ

70 80 90 100 110 120

KPEKAPKSLI YAASSLQSGV PSRFSGSGSG TDFTLTI SSL QPEDFATYYC QQYNSYPYTF

130

GQGTKLEIKR

In the heavy chain nucleic acid sequence (SEQ ID NO: 46), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 420 and guanine (G) at position 421. In the heavy chain amino acid sequence (SEQ ID NO: 47), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 140 and alanine (A) at position 141. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 46), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and guanine (G) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 47), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 19 and glutamic acid (E) at position 20.

Thus, the variable region in the 125Q54AAAA antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 46) ranging from guanine (G) at position 58 to adenine (A) at position 420. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 47) ranging from glutamic acid (E) at position 20 to serine (S) at position 140.

In the light chain nucleic acid sequence (SEQ ID NO: 48), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 387 and cytosine (C) at position 388. In the light chain amino acid sequence (SEQ ID NO: 49), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 129 and arginine (R) at position 130. Further, in the light chain nucleic acid sequence (SEQ ID NO: 48), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 66 and guanine (G) at position 67. In the light chain amino acid sequence (SEQ ID NO: 49), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 22 and aspartic acid (D) at position 23.

Accordingly, the variable region in the 125QS4AAAA antibody light chain has the nucleic acid sequence (SEQ ID NO: 48) ranging from guanine (G) at position 67 to adenine (A) at position 387. In addition, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 49) ranging from aspartic acid (D) at position 23 to lysine (K) at position 129.

DNAs encoding the 125R5AAAA heavy chain variable region and the light chain variable region and the amino acid sequences of the heavy chain variable region and the light chain variable region are each shown below.

<125R5AAAA heavy chain nucleic acid sequence > (SEQ ID NO: 50)

10 20 30 40 50 60

ATGGAGTTTG GGCTGAGCTG GCTTTTTCTT GTGGCTATTT TAAAAGGTGT CCAGTGTGAG

70 80 90 100 110 120

GTGCAGCTGT TGGAGTCTGG GGGAGGCTTG GTACAGCCTG GGGGGTCCCT GAGACTCTCC

130 140 150 160 170 180

TGTGCAGCCT CTGGATTCAC CTTTAGCAGC TATGCCATGA GCTGGGTCCG CCAGGCTCCA

190 200 210 220 230 240

GGGAAGGGGC TGGAGTGGGT CTCAGATATT AGTGGTAGTG GTGGTTACAC ATACTACGCA

250 260 270 280 290 300

GACTCCGTGA AGGGCCGGTT CACCATCTCC AGAGACAATT CCAAGAAAAC GCTGTATCTG

310 320 330 340 350 360

CAAATGAACA GCCTGAGAGC CGAGGACACG GCCGTATATT ACTGTGCGAA AACAGGCGAT

370 380 390 400 410 420

GGTTCGGGGA GTTATTCCCC TGACTACTGG GGCCAGGGAA CCCTGGTCAC CGTCTCCTCA

430

GCTAGC....

<125R5AAAA heavy chain amino acid sequence > (SEQ ID NO: 51)

10 20 30 40 50 60

MEFGLSWLFL VAILKGVQCE VQLLESGGGL VQPGGSLRLS CAASGFTFSS YAMSWVRQAP

70 80 90 100 110 120

GKGLEWVSDI SGSGGYTYYA DSVKGRFTIS RDNSKKTLYL QMNSLRAEDT AVYYCAKTGD

130 140 150

GSGSYSPDYW GQGTLVTVSS AS........

<125R5AAAA light chain nucleic acid sequence > (SEQ ID NO: 52)

10 20 30 40 50 60

ATGGACATGA GGGTCCTCGC TCAGCTCCTG GGGCTCCTGC TGCTCTGTTT CCCAGGTGCC

70 80 90 100 110 120

AGATGTGACA TCCAGATGAC CCAGTCTCCA TCCTCACTGT CTGCATCTGT AGGAGACAGA

130 140 150 160 170 180

GTCACCATCA CTTGTCGGGC GAGTCAGGGT ATTAGCAGCT GGTTAGCCTG GTATCAGCAG

190 200 210 220 230 240

AAACCAGAGA AAGCCCCTAA GTCCCTGATC TATGCTGCAT CCAGTTTGCA AAGTGGGGTC

250 260 270 280 290 300

CCATCAAGGT TCAGCGGCAG TGGATCTGGG ACAGATTTCA CTCTCACCAT CAGCAGCCTG

310 320 330 340 350 360

CAGCCTGAAG ATTTTGCAAC TTATTACTGC CAACAGTATA ATAGTTACCC GTACACTTTT

370 380 390

GGCCAGGGGA CCAAGCTGGA GATCAAACGA

<125R5AAAA light chain amino acid sequence > (SEQ ID NO: 53)

10 20 30 40 50 60

MDMRVLAQLL GLLLLCFPGA RCDIQMTQSP SSLSASVGDR VTITCRASQG ISSWLAWYQQ

70 80 90 100 110 120

KPEKAPKSLI YAASSLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQYNSYPYTF

130

GQGTKLEIKR

In the heavy chain nucleic acid sequence (SEQ ID NO: 50), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 420 and guanine (G) at position 421. In the heavy chain amino acid sequence (SEQ ID NO: 51), the boundary between the antibody variable region and the antibody constant region is located between serine (S) at position 140 and alanine (A) at position 141. In addition, in the heavy chain nucleic acid sequence (SEQ ID NO: 50), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 57 and guanine (G) at position 58. In the heavy chain amino acid sequence (SEQ ID NO: 51), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 19 and glutamic acid (E) at position 20.

Thus, the variable region in the 125R5AAAA antibody heavy chain has the nucleic acid sequence (SEQ ID NO: 50) ranging from guanine (G) at position 58 to adenine (A) at position 420. Further, the variable region in the heavy chain has the amino acid sequence (SEQ ID NO: 51) ranging from glutamic acid (E) at position 20 to serine (S) at position 140.

In the light chain nucleic acid sequence (SEQ ID NO: 52), the boundary between the antibody variable region and the antibody constant region is located between adenine (A) at position 387 and cytosine (C) at position 388. In the light chain amino acid sequence (SEQ ID NO: 53), the boundary between the antibody variable region and the antibody constant region is located between lysine (K) at position 129 and arginine (R) at position 130. Further, in the light chain nucleic acid sequence (SEQ ID NO: 52), the boundary between the signal sequence and the antibody variable region is located between thymine (T) at position 66 and guanine (G) at position 67. In the light chain amino acid sequence (SEQ ID NO: 53), the boundary between the signal sequence and the antibody variable region is located between cysteine (C) at position 22 and aspartic acid (D) at position 23.

Thus, the variable region in the 125R5AAAA antibody light chain has the nucleic acid sequence (SEQ ID NO: 52) ranging from guanine (G) at position 67 to adenine (A) at position 387. In addition, the variable region in the light chain has the amino acid sequence (SEQ ID NO: 53) ranging from aspartic acid (D) at position 23 to lysine (K) at position 129.

Example 14 determination of the full Length sequences of the constant regions of the human antibody heavy and light chain genes expressed by hybridomas 125N26F6AA and 125M10AA

The DNA nucleotide sequence and amino acid sequence of the variable region of each antibody were determined in example 13. For the hybridomas 125N26F6AA and 125M10AA derived from KM mice, the full-length sequences containing the constant regions were analyzed. According to example 13, cDNA synthesis was performed using SMART RACE cDNA amplification kit (produced by Becton Dickinson Bioscience Clontech) starting from total RNAs prepared from hybridomas 125N26F6AA and 125M10 AA.

Synthesis of first Strand cDNA

Total RNA 5. mu.g/3. mu.l

3' -CDS primer 1. mu.l

H₂O 1μl

The reaction solution having the above composition was incubated at 70 ℃ for 2 minutes, and then the following components were added, followed by incubation at 42 ℃ for 1.5 hours.

5 Xbuffer 2. mu.l

DTT 1μl

dNTP mix 1. mu.l

PowerScript reverse transcriptase 1. mu.l

In addition, 50. mu. l N- [ tris (hydroxymethyl) methyl ] glycine buffer was added, followed by incubation at 72 ℃ for 7 minutes to obtain first strand cDNA.

In order to obtain a DNA containing the entire coding region of the constant region, PCR amplification was carried out using the above-mentioned synthesized cDNA as a template, using the following primer pairs: synthetic DNA (5 'primer) whose sequence binds to the vicinity of ATG initiation codon at the 5' end of each antibody gene; and a synthetic DNA (3 'primer) specifically binding to the 3' untranslated region of a human antibody gene. By the amplification reaction, the full length sequence (cDNA) of the antibody gene from the ATG initiation codon to the 3' untranslated region including the stop codon can be obtained.

125N26F6AA heavy chain DNA was amplified 35 times by repeating the incubation cycle (94 ℃ for 15 seconds, 68 ℃ for 2 minutes) with primer pair-H chain 5 'primer N26H5Sal1(SEQ ID NO: 58) and H chain 3' primer H _3UTR1848 (5'-CGGGGTACGTGCCAAGCATCCTCGTG-3', SEQ ID NO: 74), or primer pair H chain 5 'primer N26H5Sal1(SEQ ID NO: 58) and H chain 3' primer H _3UTR1875 (5'-ATGCTGGGCGCCCGGGAAGTATGTAC-3', SEQ ID NO: 75). Meanwhile, the primer pair-L chain 5 'primer N26KA10Minor L Bgl (SEQ ID NO: 64) and L chain 3' primer: l _3UTR _823 (5'-GAAAGATGAGCTGGAGGACCGCAATA-3', SEQ ID NO: 76) to amplify 125N26F6AA light chain (. kappa.). Amplification was carried out using the same reaction solution as the components in example 13, (2) -1 except for the primers.

125M10AA heavy chain DNA was amplified with the primer pair H chain 5 'primer M10H5Sal (SEQ ID NO: 70) and H chain 3' primer H _3UTR1848(SEQ ID NO: 74), or the primer pair H chain 5 'primer M10H5Sal (SEQ ID NO: 70) and H chain 3' primer H _3UTR1875(SEQ ID NO: 75). The primers were prepared using the primer pair, L chain 5 'primer M10KBgl (SEQ ID NO: 66) and L chain 3' primer: l _3UTR _823(SEQ ID NO: 76) to amplify 125M10AA light chain (κ) DNA.

The amplified PCR fragments were each recovered by ethanol precipitation, recovered by agarose gel electrophoresis, and then purified using a QIAquick gel extraction kit (manufactured by QIAGEN).

The purified amplified fragments were subcloned into PCR 4Blunt-TOPO vectors of Zero Blunt TOPO PCR cloning kit (manufactured by Invitrogen), respectively. For the obtained clones, template DNAs for sequencing were prepared using a TempliPhi DNA amplification kit (produced by Amersham Biosciences) containing a reagent for preparing the template DNAs for sequencing according to the attached instructions. Thereby determining the nucleotide sequences of the inserted DNAs. Primers used for analysis of human antibody heavy chain DNA nucleotide sequences were M13FW (SEQ ID NO: 3), M13RV (SEQ ID NO: 4), hh4(SEQ ID NO: 21), hh1 (5'-CCAAGGGCCCATCGGTCTTCCCCCTGGCAC-3') (SEQ ID NO: 77), CMVH903F CMVH903F (5'-GACACCCTCATGATCTCCCGGACC-3') (SEQ ID NO: 78), CMVHR 1303 (5'-TGTTCTCCGGCTGCCCATTGCTCT-3') (SEQ ID NO: 79), hh-6 (5'-GGTCCGGGAGATCATGAGGGTGTCCTT-3') (SEQ ID NO: 80), hh-2(SEQ ID NO: 20), H _3UTR1848(SEQ ID NO: 74) and H _3UTR1875(SEQ ID NO: 75). Primers used for analysis of human antibody light chain (κ) DNA nucleotide sequences were M13FW (SEQ ID NO: 3), M13RV (SEQ ID NO: 4), hk-5(SEQ ID NO: 19) and hk-1 (5'-TGGCTGCACCATCTGTCTTCATCTTC-3') (SEQ ID NO: 81).

DNAs encoding the entire heavy chain region and the entire light chain region of the 125N26F6AA antibody and the amino acid sequences of the entire heavy chain region and the entire light chain region are shown below.

<125N26F6AA heavy chain nucleic acid sequence > (SEQ ID NO: 82)

10 20 30 40 50 60

ATGGAGTTTG GGCTGAGCTG GGTTTTCCTC GTTGCTCTTT TAAGAGGTGT CCAGTGTCAG

70 80 90 100 110 120

GTGCAGTTGG TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG GGAGGTCCCT GAGACTCTCC

130 140 150 160 170 180

TGTGCAGCGT CTGGATTCAC CTTCAGTCAC TATGGCATGC ACTGGGTCCG CCAGGCTCCA

190 200 210 220 230 240

GGCAAGGGGC TGGAGTGGGT GGCACTTATA TGGTATGATG GAAGTAATAA ATACTATGCA

250 260 270 280 290 300

GACTCCGTGA AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG

310 320 330 340 350 360

CAAATGAACA GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG AGATCCCTTA

370 380 390 400 410 420

GCAGCTGGTA CGTCCTACTT TGACTACTGG GGCCAGGGAA CCCTGGTCAC CGTCTCCTCA

430 440 450 460 470 480

GCCTCCACCA AGGGCCCATC GGTCTTCCCC CTGGCACCCT CCTCCAAGAG CACCTCTGGG

490 500 510 520 530 540

GGCACAGCGG CCCTGGGCTG CCTGGTCAAG GACTACTTCC CCGAACCGGT GACGGTGTCG

550 560 570 580 590 600

TGGAACTCAG GCGCCCTGAC CAGCGGCGTG CACACCTTCC CGGCTGTCCT ACAGTCCTCA

610 620 630 640 650 660

GGACTCTACT CCCTCAGCAG CGTGGTGACC GTGCCCTCCA GCAGCTTGGG CACCCAGACC

670 680 690 700 710 720

TACATCTGCA ACGTGAATCA CAAGCCCAGC AACACCAAGG TGGACAAGAA AGTTGAGCCC

730 740 750 760 770 780

AAATCTTGTG ACAAAACTCA CACATGCCCA CCGTGCCCAG CACCTGAACT CCTGGGGGGA

790 800 810 820 830 840

CCGTCAGTCT TCCTCTTCCC CCCAAAACCC AAGGACACCC TCATGATCTC CCGGACCCCT

850 860 870 880 890 900

GAGGTCACAT GCGTGGTGGT GGACGTGAGC CACGAAGACC CTGAGGTCAA GTTCAACTGG

910 920 930 940 950 960

TACGTGGACG GCGTGGAGGT GCATAATGCC AAGACAAAGC CGCGGGAGGA GCAGTACAAC

970 980 990 1000 1010 1020

AGCACGTACC GTGTGGTCAG CGTCCTCACC GTCCTGCACC AGGACTGGCT GAATGGCAAG

1030 1040 1050 1060 1070 1080

GAGTACAAGT GCAAGGTCTC CAACAAAGCC CTCCCAGCCC CCATCGAGAA AACCATCTCC

1090 1100 1110 1120 1130 1140

AAAGCCAAAG GGCAGCCCCG AGAACCACAG GTGTACACCC TGCCCCCATC CCGGGATGAG

1150 1160 1170 1180 1190 1200

CTGACCAAGA ACCAGGTCAG CCTGACCTGC CTGGTCAAAG GCTTCTATCC CAGCGACATC

1210 1220 1230 1240 1250 1260

GCCGTGGAGT GGGAGAGCAA TGGGCAGCCG GAGAACAACT ACAAGACCAC GCCTCCCGTG

1270 1280 1290 1300 1310 1320

CTGGACTCCG ACGGCTCCTT CTTCCTCTAC AGCAAGCTCA CCGTGGACAA GAGCAGGTGG

1330 1340 1350 1360 1370 1380

CAGCAGGGGA ACGTCTTCTC ATGCTCCGTG ATGCATGAGG CTCTGCACAA CCACTACACG

1390 1400 1410

CAGAAGAGCC TCTCCCTGTC TCCGGGTAAA TGA

<125N26F6AA heavy chain amino acid sequence > (SEQ ID NO: 83)

10 20 30 40 50 60

MEFGLSWVFL VALLRGVQCQ VQLVESGGGV VQPGRSLRLS CAASGFTFSH YGMHWVRQAP

70 80 90 100 110 120

GKGLEWVALI WYDGSNKYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARDPL

130 140 150 160 170 180

AAGTSYFDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS

190 200 210 220 230 240

WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP

250 260 270 280 290 300

KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW

310 320 330 340 350 360

YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS

370 380 390 400 410 420

KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

430 440 450 460 470 480

LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

<125N26F6AA light chain nucleic acid sequence > (SEQ ID NO: 84)

10 20 30 40 50 60

ATGTCGCCAT CACAACTCAT TGGGTTTCTG CTGCTCTGGG TTCCAGCCTC CAGGGGTGAA

70 80 90 100 110 120

ATTGTGCTGA CTCAGTCTCC AGACTTTCAG TCTGTGACTC CAAAGGAGAA AGTCACCATC

130 140 150 160 170 180

ACCTGCCGGG CCAGTCAGAG CATTGGTAGT AGCTTACACT GGTACCAGCA GAAACCAGAT

190 200 210 220 230 240

CAGTCTCCAA AGCTCCTCAT CAAGTATGCT TCCCAGTCCT TCTCAGGGGT CCCCTCGAGG

250 260 270 280 290 300

TTCAGTGGCA GTGGATCTGG GACAGATTTC ACCCTCACCA TCAATAGCCT GGAAGCTGAA

310 320 330 340 350 360

GATGCTGCAG CGTATTACTG TCATCAGAGT AGTAGTTTAC CATTCACTTT CGGCCCTGGG

370 380 390 400 410 420

ACCAAAGTGG ATATCAAACG AACTGTGGCT GCACCATCTG TCTTCATCTT CCCGCCATCT

430 440 450 460 470 480

GATGAGCAGT TGAAATCTGG AACTGCCTCT GTTGTGTGCC TGCTGAATAA CTTCTATCCC

490 500 510 520 530 540

AGAGAGGCCA AAGTACAGTG GAAGGTGGAT AACGCCCTCC AATCGGGTAA CTCCCAGGAG

550 560 570 580 590 600

AGTGTCACAG AGCAGGACAG CAAGGACAGC ACCTACAGCC TCAGCAGCAC CCTGACGCTG

610 620 630 640 650 660

AGCAAAGCAG ACTACGAGAA ACACAAAGTC TACGCCTGCG AAGTCACCCA TCAGGGCCTG

670 680 690 700

AGCTCGCCCG TCACAAAGAG CTTCAACAGG GGAGAGTGTT AG

<125N26F6AA light chain amino acid sequence > (SEQ ID NO: 85)

10 20 30 40 50 60

MSPSQLIGFL LLWVPASRGE IVLTQSPDFQ SVTPKEKVTI TCRASQSIGS SLHWYQQKPD

70 80 90 100 110 120

QSPKLLIKYA SQSFSGVPSR FSGSGSGTDF TLTINSLEAE DAAAYYCHQS SSLPFTFGPG

130 140 150 160 170 180

TKVDIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKYD NALQSGNSQE

190 200 210 220 230

SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC

DNAs encoding the entire heavy chain region and the entire light chain region of the 125M10AA antibody and the amino acid sequences of the entire heavy chain region and the entire light chain region are shown below.

<125M10AA heavy chain nucleic acid sequence > (SEQ ID NO: 86)

10 20 30 40 50 60

ATGGATCTCA TGTGCAAGAA AATGAAGCAC CTGTGGTTCT TCCTCCTGCT GGTGGCGGCT

70 80 90 100 110 120

CCCAGATGGG TCCTGTCCCA GCTGCAGGTG CAGGAGTCGG GCCCAGGACT GGTGAAGCCT

130 140 150 160 170 180

TCGGAGACCC TGTCCCTCAT CTGCACTGTC TCTGGTGGCT CCATCAGGAC CAGTGGTTAC

190 200 210 220 230 240

TACTGGGGCT GGTTCCGCCA GCCCCCAGGG AAGGGACTGG AGTGGATTGG GACTAGTCAT

250 260 270 280 290 300

AATAGTGGGA GCACCTACTA CAACCCGTCC CTCAAGAGTC GAGTCACCAT ATCCGTAGAC

310 320 330 340 350 360

ACGTCCAAGA ACCAGTTCTC CCTGAAGCTG AACTCTGTGA CCGCCGCAGA CACGGCTGTG

370 380 390 400 410 420

TATTACTGTG CGAGACAAGG TTACGATTTT AAAGTCAATA TAGACGTCTG GGGACAAGGG

430 440 450 460 470 480

ACCACGGTCA CCGTCTCCTC AGCCTCCACC AAGGGCCCAT CGGTCTTCCC CCTGGCACCC

490 500 510 520 530 540

TCCTCCAAGA GCACCTCTGG GGGCACAGCG GCCCTGGGCT GCCTGGTCAA GGACTACTTC

550 560 570 580 590 600

CCCGAACCGG TGACGGTGTC GTGGAACTCA GGCGCCCTGA CCAGCGGCGT GCACACCTTC

610 620 630 640 650 660

CCGGCTGTCC TACAGTCCTC AGGACTCTAC TCCCTCAGCA GCGTGGTGAC CGTGCCCTCC

670 680 690 700 710 720

AGCAGCTTGG GCACCCAGAC CTACATCTGC AACGTGAATC ACAAGCCCAG CAACACCAAG

730 740 750 760 770 780

GTGGACAAGA AAGTTGAGCC CAAATCTTGT GACAAAACTC ACACATGCCC ACCGTGCCCA

790 800 810 820 830 840

GCACCTGAAC TCCTGGGGGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC

850 860 870 880 890 900

CTCATGATCT CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC

910 920 930 940 950 960

CCTGAGGTCA AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG

970 980 990 1000 1010 1020

CCGCGGGAGG AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC

1030 1040 1050 1060 1070 1080

CAGGACTGGC TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC

1090 1100 1110 1120 1130 1140

CCCATCGAGA AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC

1150 1160 1170 1180 1190 1200

CTGCCCCCAT CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA

1210 1220 1230 1240 1250 1260

GGCTTCTATC CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC

1270 1280 1290 1300 1310 1320

TACAAGACCA CGCCTCCCGT GCTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC

1330 1340 1350 1360 1370 1380

ACCGTGGACA AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG

1390 1400 1410 1420 1430

GCTCTGCACA ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA ATGA

<125M10AA heavy chain amino acid sequence > (SEQ ID NO: 87)

10 20 30 40 50 60

MDLMCKKMKH LWFFLLLVAA PRWVLSQLQV QESGPGLVKP SETLSLICTV SGGSIRTSGY

70 80 90 100 110 120

YWGWFRQPPG KGLEWIGTSH NSGSTYYNPS LKSRVTI SVD TSKNQFSLKL NSVTAADTAV

130 140 150 160 170 180

YYCARQGYDF KVNIDVWGQG TTVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF

190 200 210 220 230 240

PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK

250 260 270 280 290 300

VDKKVEPKSC DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMI SRTPEVT CVVVDVSHED

310 320 330 340 350 360

PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA

370 380 390 400 410 420

PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN

430 440 450 460 470 480

YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK

<125M10AA light chain nucleic acid sequence > (SEQ ID NO: 88)

10 20 30 40 50 60

ATGGAAGCCC CAGCTCAGCT TCTCTTCCTC CTGCTACTCT GGCTCCCAGA TACCACCGGA

70 80 90 100 110 120

GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC

130 140 150 160 170 180

CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTGGTACCA ACAGAAACCT

190 200 210 220 230 240

GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC

250 260 270 280 290 300

AGGTTCAGTG GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG CCTAGAGCCT

310 320 330 340 350 360

GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCCGCTCAC TTTCGGCGGA

370 380 390 400 410 420

GGGACCAAGG TGGAGATCAA ACGAACTGTG GCTGCACCAT CTGTCTTCAT CTTCCCGCCA

430 440 450 460 470 480

TCTGATGAGC AGTTGAAATC TGGAACTGCC TCTGTTGTGT GCCTGCTGAA TAACTTCTAT

490 500 510 520 530 540

CCCAGAGAGG CCAAAGTACA GTGGAAGGTG GATAACGCCC TCCAATCGGG TAACTCCCAG

550 560 570 580 590 600

GAGAGTGTCA CAGAGCAGGA CAGCAAGGAC AGCACCTACA GCCTCAGCAG CACCCTGACG

610 620 630 640 650 660

CTGAGCAAAG CAGACTACGA GAAACACAAA GTCTACGCCT GCGAAGTCAC CCATCAGGGC

670 680 690 700

CTGAGCTCGC CCGTCACAAA GAGCTTCAAC AGGGGAGAGT GTTAG

<125M10AA light chain amino acid sequence > (SEQ ID NO: 89)

10 20 30 40 50 60

MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP

70 80 90 100 110 120

GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPLTFGG

130 140 150 160 170 180

GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ

190 200 210 220 230

ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC

EXAMPLE 15 construction of vectors for expressing recombinant antibodies

Vectors for expressing 263A17, 125M10AA, 125M165DAAA, 125N26F6AA and 125Q54AAAA recombinant antibodies were constructed.

For the 263A17, 125M165DAAA, or 125N26F6AA antibodies, plasmid DNA containing HV chains of each type of antibody obtained was used as a template. The primers used here were designed to add restriction enzyme sites (SalI at the 5 'end and NheI at the 3' end) at the ends for ligation. Specifically, the primers used herein are as follows.

263A17；

HV strand 5' primer: A332-6A2H VH3-23 Sal I (SEQ ID NO: 54)

5’-GCG ACT AAGTCG ACC ATG GAG TTT GGG CTG AGC TG-3’

A332-6A2H VH3-23Nhe I(SEQ ID NO：55)

5’-TGG GCC CTT GGT GCT AGC TGA GGA GAC GGT GAC CG-3’

125M165DAAA；

HV strand 5' primer: M165H5Sal (SEQ ID NO: 56)

5’-AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGG TTT-3’

HV strand 3' primer: M165H3Nhe (SEQ ID NO: 57)

5’-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TGC-3’

125N26F6AA；

HV strand 5' primer: N26H5Sall (SEQ ID NO: 58)

5’-AGA GAG AGAGGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGG TTT-3’

HV strand 3' primer: N26H3Nhel (SEQ ID NO: 59)

5’-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC CC-3’

Each type of A33 antibody HV was amplified by PCR (94 ℃ 3 min-94 ℃ 10 sec, 68 ℃ 45 sec (35 cycles) -72 ℃ 7 min). The amplified DNA fragment was digested with SalI and NheI. The digested fragment was then inserted into an N5KG1-Val Lark vector (a modified vector of IDEC Pharmaceuticals, N5KG1 (U.S. Pat. No. 3, 6001358)) which had been cleaved with the same enzyme. The sequence of the insert was confirmed by sequencing using the vector as a template to be identical to the nucleotide sequence of the subcloned HV as determined by DNA nucleotide analysis.

Then, LV was inserted into the obtained HV-inserted plasmid vector. Plasmid DNA containing the LV chain of each type of antibody obtained was used as a template. The primers used here were designed to add restriction enzyme sites (BglII at the 5 'end and BsiW I at the 3' end) at the ends for ligation. Specifically, the primers used herein are as follows.

263A17；

LV strand 5' primer: A332-6A2K L19 BglII (SEQ ID NO: 60)

5’-ATC ACA GAT CTC TCA CCA TGG ACA TGA GGG TCC CC-3’

LV strand 3' primer: A332-6A2K L19 Bs iWI (SEQ ID NO: 61)

5’-ACA GAT GGT GCA GCC ACC GTA CGT TTA ATC TCC AG-3’

125M165DAAA；

LV strand 5' primer: M165K5L6Bgl 2(SEQ ID NO: 62)

5’-AGA GAG AGA GAG ATC TCA CCA TGG AAG CCC CAG CTC AGC TTC TCT-3’

LV strand 3' primer: M165K3L6Bs iW1(SEQ ID NO: 63)

5’-AGA GAG AGA GCG TAC GTT TGA TTT CCA CCT TGG TCC CTT GGC-3’

125N26F6AA；

LV strand 5' primer: n26KA10Minor L Bgl (SEQ ID NO: 64)

5’-AGA GAG AGA GAT CTC TCA CCA TGT CGC CAT CAC AAC TCA TTG GG-3’

LV strand 3' primer: n26KA10Minor L Bsi (SEQ ID NO: 65)

5’-AGA GAG AGA GCG TAC GTT TGA TAT CCA CTT TGG TCC CAG GG-3’、

Each type of A33 antibody LV was amplified by PCR (94 ℃ 3 min-94 ℃ 10 sec, 68 ℃ 45 sec (35 cycles) -72 ℃ 7 min). The amplified DNA fragment was digested with Bgl II and BsiWI. The digested fragments were then inserted into an N5KG1-HV vector that had been cut with the same enzyme. The inserted sequence was confirmed by sequencing using the vector as a template to be identical to the nucleotide sequence of subcloned LV as determined by DNA nucleotide analysis.

For the 125M10AA or 125Q54AAAA antibody, plasmid DNA containing the LV chain of each type of antibody obtained was used as a template. The primers used here were designed to add restriction enzyme sites (Bgl II at the 5 'end and BsiWI at the 3' end) at the ends for ligation. Specifically, the primers used herein are as follows.

125M10AA；

LV strand 5' primer: m10KBgl (SEQ ID NO: 66)

5’-AGAGAGAGAGAGATCTCACCATGGAAGCCCCAGCTCAGCTTCTCT-3’

LV strand 3' primer: m10KBsi (SEQ ID NO: 67)

5’-AGAGAGAGAGCGTACGTTTGATCTCCACCTTGGTCCCTCCG-3’

125Q54AAAA；

LV strand 5' primer: Q54K5Bgl (SEQ ID NO: 68)

5’-AGAGAGAGAGAGATCTCACCATGGACATGAGGGTCCTCGCTCAGC-3’

LV strand 3' primer: Q54K3Bs i (SEQ ID NO: 69)

5’-AGAGAGAGAGCGTACGTTTGATCTCCAGCTTGGTCCCCTGG-3’

Each type of A33 antibody LV was amplified by PCR (94 ℃ 3 min-94 ℃ 10 sec, 68 ℃ 45 sec (35 cycles) -72 ℃ 7 min). The amplified DNA fragment was digested with BglH and BsiWI. The digested fragment was then inserted into the N5KG1-Val Lark vector which had been cleaved with the same enzyme. The inserted sequence was confirmed by sequencing using the vector as a template to be identical to the nucleotide sequence of subcloned LV as determined by DNA nucleotide analysis.

Then, HV was inserted into the obtained plasmid vector into which LV had been inserted. Plasmid DNA containing HV chains of each type of antibody obtained was used as a template. The primers used here were designed to add restriction enzyme sites (SalI at the 5 'end and NheI at the 3' end) at the ends for ligation. Specifically, the primers used herein are as follows.

125 M10AA；

HV strand 5' primer: M10H5Sal (SEQ ID NO: 70)

5’-AGA GAG AGA GGT CGA CCA CCA TGG ATC TCA TGT GCA AGA AAA TGA AGC -3

HV strand 3' primer: M10H3Nhe (SEQ ID NO: 71)

5’AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCG TGG TCC CT-3’

125 Q54AAAA；

HV strand 5' primer: Q54H5Sal (SEQ ID NO: 72)

5’-AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGC TTT-3’

HV strand 3' primer: Q54H3Nhe (SEQ ID NO: 73)

5’-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC CC-3’

Each type of A33 antibody HV was amplified by PCR (94 ℃ 3 min-94 ℃ 10 sec, 68 ℃ 45 sec (35 cycles) -72 ℃ 7 min). The amplified DNA fragment was digested with Sal I and NheI. The digested fragment was then inserted into the N5KG1-LV vector which had been cleaved with the same enzyme. The sequence of the insert was confirmed by sequencing using the vector as a template to be identical to the nucleotide sequence of the subcloned HV as determined by DNA nucleotide analysis.

Table 5 lists the nucleotide sequences of the synthesized DNAs. Table 6 lists the names of the recombinant vectors and the names of the antibodies produced.

TABLE 6

Antibodies	Name of vector	Subclass of	Recombinant antibody name
				263A17	N5KG1-Val Lark	IgG1	rec263
125M10AA	N5KG1-Val Lark	IgG1	recM10
				125M165DAAA	N5KG1-Val Lark	IgG1	recM165
125N26F6AA	N5KG1-Val Lark	IgG1	recN26
				125Q54AAAA	N5KG1-Val Lark	IgG1	recQ54

EXAMPLE 16 preparation of recombinant antibodies

The recombinant antibody-expressing cells were prepared by transfecting host cells with the recombinant antibody-expressing vector constructed in example 15. The host cells used for expression are, for example, CHO cells of the dhfr-deficient cell line (ATCC CRL-9096), CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109, 1999) or HEK293T (ATCC CRL-11268).

Host cells are transfected with each type of vector by electroporation, lipofection, or the like. About 2. mu.g of the antibody expression vector was linearized with restriction enzymes, and then subjected to electroporation using a Bio-Rad electrophoreter under conditions of 350V and 500. mu.F. With 4X 10 pairs of genes of each type⁶CHO cells were transfected and then seeded into 96-well culture plates. Lipofection was performed using LipofectAMINE Plus (produced by Gibco BRL) according to the instructions attached. After transfection with the vector, drugs corresponding to the selection markers used in the vector are added,then, the culture was continued. After the clones were confirmed, cell lines expressing the antibodies were screened by the method described in example 6. Using a 3.2X 10cm column of Mab-screening protein A (produced by Amersham pharmacia Biotech), antibodies were purified from the cells selected by washing 2 times with PBS after adsorption and then eluting with 20mM (glycine) sodium citrate and 50mM NaCl (pH2.7) buffer. The eluate was neutralized with 50mM sodium phosphate buffer (pH 7.0). Then, purification was performed using a Hitrap Q HP dextran column (produced by Amersham Pharmacia Biotech), which is an anion exchange column, and then the same operation was performed using a Hitrap SP HP dextran column (produced by Amersham Pharmacia Biotech), which is a cation column. The antibody solution thus prepared was exchanged with PBS using a dialysis membrane (cut-off 10000, manufactured by Spectrum laboratories), and then sterilized by filtration using a membrane filter MILLEX-GV (manufactured by Millipore) having a pore diameter of 0.22. mu.m. The purified antibody thus obtained has a purity of at least 95% or more and an endotoxin level of 0.1EU/mg or less. The concentration of each type of the recombinant purified anti-A33 antibody was obtained by measuring the absorbance at 280nm and converting it into 1.4OD equivalent to 1mg/mL (antibody concentration).

Example 17 reactivity test of recombinant antibody

Each type of the recombinant antibodies obtained in example 16 was assayed for reactivity to COLO205 cells, LS174T cells (ATCC No. CL-188) or NCI-H508 cells (ATCC No. CCL-253) of a human colorectal cancer cell line expressing the A33 antigen by FCM. The reactivity of human colorectal cancer cell line HT-29 cells (ATCC No. HTB-38) that do not express the A33 antigen as negative control cells was also determined. The experimental procedure used here was the same as in example 9.

The results are shown in Table 7. For COLO205 cells, the half value of the mean fluorescence intensity was determined to be 45. For LA174T cells, the half value of the mean fluorescence intensity was determined to be 100. For NCI-H508 cells, the half value of the mean fluorescence intensity was determined to be 175. Reactivity is expressed as +++ when the concentration of antibody required to achieve this value is 10 < ═ x < 100ng/ml, + + when it is 100 < ═ x < 1000ng/ml, + when it is 1000 < ═ x < 10000ng/ml, and-when no binding is observed. Each type of recombinant antibody appeared to bind to all cells expressing the a33 antigen.

EXAMPLE 18 Competition assay for Each type of recombinant antibody with mouse anti-A33 antibody

Whether each type of recombinant antibody obtained in example 16 recognized the same epitope as the mouse anti-a 33 antibody was examined by competition experiments with FCM. The experimental procedure used here was the same as in example 10.

As with the results for each type of purified monoclonal antibody in example 10, rec263 and recM10 were classified as "non-blockers", recM165 and recN26 were classified as "blockers", and recQ54 was classified as "partial blockers". Table 8 shows the results.

TABLE 8

Name of antibody	Inhibition ratio (%)	Classification
			cA33	94.6	Blocking object
rec263	15.3	Non-blocking substance
			recM10	0.2	Non-blocking substance
recM165	93.4	Blocking object
			recN26	95.3	Blocking object
recQ54	44.6	Partial block

EXAMPLE 19 cytotoxic Effect of recombinant antibodies

ADCC and CDC of the recombinant antibody obtained in example 16 were measured. In the ADCC assay, 5,000 wells were incubated in V-bottom 96-well plates (produced by Coaster) containing antibodies at various concentrations and having a total volume of 200. mu.l⁵¹Cr-labeled target cells and 500,000 healthy human peripheral blood mononuclear leukocytes obtained by the method described in example 11 in the presence of 5% CO at 37 ℃₂Cultured for 4 hours under the conditions of (1).

In the CDC assay, 5,000 pieces of V-bottom 96-well plates (produced by Coaster) containing antibodies at various concentrations and having a total volume of 200. mu.l were cultured⁵¹Cr-labeled target cells andhuman serum-derived complement (produced by Sigma) at a concentration of 5%, in the presence of 5% CO at 37 ℃₂Cultured for 4 hours under the conditions of (1).

The experimental procedure used here was the same as in example 12.

The results are shown in fig. 2A to 2D and table 9. For ADCC against COLO205 cells as target cells, the half value of the specific lysis (%) was determined to be 12.5%. For ADCC against NCI-H508 cells as target cells, the half value of the specific lysis (%) was determined to be 30%. The cytotoxic effect is indicated by +++ when the concentration of antibody required to achieve this value is 1 < ═ x < 10ng/ml, by + + when it is 10 < ═ x < 100ng/ml, by + when it is 100 < ═ x < 1000ng/ml, and by-when no specific lysis (%) is obtained. For CDC against COLO205 cells as target cells, the half value of specific lysis (%) was determined to be 7%. For CDC against NCI-H508 cells as target cells, the half value of the specific lysis (%) was determined to be 20%. The cytotoxic effect is indicated by +++ when the antibody concentration required to achieve this value is 10 < ═ x < 100ng/ml, by + + when it is 100 < ═ x < 1000ng/ml, by + when it is x > - < 1000ng/ml, and by-when no specific lysis (%) is obtained. For ADCC, cA33, recM10 and recQ54 exhibit high cytotoxic effects, while for CDC, recM10 exhibits high cytotoxic effects.

Example 20 Western blot analysis of purified and recombinant antibodies

Mouse anti-a 33 and humanized a33 antibodies have been reported to recognize conformational epitopes. Specifically, for these antibodies, it has been reported that no reactivity was observed by Western blot analysis under reducing conditions (5%. beta. -mercaptoethanol). Therefore, Western blot analysis can be used to test the reactivity of purified human anti-a 33 and recombinant antibodies.

The shA33EX-hFc protein prepared in example 3 was separated by SDS-PAGE using a 10% to 20% polyacrylamide gradient gel (produced by Daiichi pure Chemicals) under reducing (5%. beta. -mercaptoethanol) and non-reducing conditions. At this time, the shA33EX-hFc protein was diluted to 2.5ng per lane. Meanwhile, biotinylated SDS-PAGE Standard BroadRange (produced by Bio-Rad Laboratories) was loaded on one lane as a marker. The shA33EX-hFc protein was transferred to the PVDF membrane by transferring it for 1 hour at 150 mA/membrane using a PantherSemidy Electroblotter (Daiichi Pure Chemicals). The protein-transferred membrane was washed with TBS buffer and TBS (TTBS) containing 0.05% Tween. Blocking was performed with BlockAce (by Dainippon Pharmaceutical). Wash 2 times with TTBS. Purified antibodies from 125M10AA, 125Q54AAAA, 125M96ABA, 125Q47BA, and 125R5AAAA hybridomas of each type (1. mu.g/ml) were reacted for 60 minutes at room temperature. Meanwhile, each type (1. mu.g/ml) of recombinant antibodies, including chimeric anti-A33 antibody, 125M165DAAA (i.e., recM165), and 125N26F6AA (i.e., recN26), was reacted at room temperature for 60 minutes. After washing with TTBS, goat anti-human Kappa chain F (ab') labeled with horseradish peroxidase (diluted 1000-fold) was added₂An antibody (produced by Biosource) was used as the antibody for detection. At the same time, streptavidin labeled with horseradish peroxidase (diluted 3000 times) was also added for reaction to detect the marker. After washing 2 times with TTBS and once with PBS, band detection was performed using ECL-plus (produced by Amersham Biosciences) which is a Western blotting detection system. Chemiluminescence was integrated with an image analyzer LAS-100 (manufactured by Fuji Photo Film), followed by image processing.

The results are shown in FIGS. 3A and 3B. This result indicates that only 125Q54AAAA reacts with a protein band of approximately 67kD even under reducing conditions. In the case of other antibodies, the reaction occurred only under non-reducing conditions, as with the chimeric anti-A33 antibody.

Example 21 immunohistochemical analysis of purified and recombinant antibodies

To assess whether each type of human anti-a 33 antibody is comparable in specificity and selectivity to the chimeric anti-a 33 antibody, tumor tissue sections and normal tissue sections were analyzed by immunohistochemical analysis.

(1) Fluorescent labeling of purified and recombinant antibodies

With Alexa Fluor^TM488 (produced by Molecular Probe) directly labels each type of purified monoclonal antibody prepared in example 8. Each type of recombinant antibody (rec263, 125M10AA, recM165, recN26, and 125Q54AAAA) prepared in example 16 was also directly labeled as such. Similarly, chimeric anti-A33 antibody was directly labeled as a positive control, and anti-DNP-IgG 1 antibody was directly labeled as a negative control. Fluorescent labeling of purified and recombinant antibodies is described below. Alexa Fluor was prepared according to the attached instructions^TM488 binds to each type of anti-a 33 antibody prepared in example 8 or 16. To 2mg/ml of each type of purified antibody and 0.5ml of each type of recombinant antibody was added 50. mu.l of 1M carbonate buffer. The solution was mixed with Alexa Fluor^TM488, and then reacted at room temperature for 1 hour while stirring the solution. Hydroxylamine was added to terminate the reaction. The mixed solution was applied to a gel filtration column (NAP5, by Amersham Pharmacia Biotech) to remove AlexaFluor not bound to the antibody^TM488. Under these conditions, 4-6 fluorescent substances were bound to 1 molecule of antibody. The fluorescent-labeled antibody was able to bind to COLO205 cells and exhibited binding activity comparable to that of the unlabeled antibody.

(2) Immunohistochemistry

The tissue sections used herein were frozen human adult colon cancer tissue sections (produced by BioChain), frozen human adult normal colon tissue sections (produced by BioChain), frozen human adult small intestine tissue sections (produced by BioChain), and frozen human adult stomach tissue sections (produced by BioChain). Using 10% sheep serum (composed ofProduced by Gibco BRL) was blocked at room temperature for 1 to 2 hours. Wash 2 times with PBS. Alexa Fluor used in example 21(1)^TMEach type of purified monoclonal or recombinant antibody labeled 488 was reacted at 1. mu.g/ml for 30 to 60 minutes at room temperature. Then, a section was made and observed under a fluorescence microscope (BX51, produced by Olympus). The image was analyzed with DP70 (produced by Olympus).

The results are shown in fig. 4 to 6. As with the results of immunohistological staining of colorectal cancer tissues reported by Garin-Chesa P et al (int.J. Oncology19969: 465-471), extensive, uniform and strong staining of glandular epithelial cells or heteroglandular structures of colon cancer tissues was observed for chimeric anti-A33 antibodies, rec263, 125M10AA, recM165, recN26 and 125Q54AAAA (FIG. 4). In addition, it was observed that normal small intestine tissue (FIG. 5) and normal colon tissue (FIG. 6) were also stained in the same manner as the chimeric anti-A33 antibody. In contrast, even in the presence of antibodies, normal stomach tissue was not stained, which is the same as the results reported in the previous literature. In addition, the anti-DNP-IgG 1 antibody as a negative control was not stained in any tissue.

EXAMPLE 22 Effect of antibodies purified from hybridomas and recombinant antibodies on mouse tumor-bearing models

The effect of the human anti-A33 recombinant antibody obtained in example 16 was examined using a mouse tumor-bearing model according to the following method. The colorectal cancer cell lines used herein were COLO205 cells and NCI-H508 cells.

The method for preparing a tumor-bearing murine model using the COLO205 cell line is described below. Cells of colorectal cancer cell line COLO205 at 5X 10⁶Mice were subcutaneously transplanted to the back of 6-week-old Balb/c nude mice (purchased from CLEA, Japan). On days 1, 2, 7 and 10 after the engrafting of the cells, the chimeric anti-A33 or rec263 antibody (dissolved in 200. mu.l of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10. mu.g/mouse or 100. mu.g/mouse to tumor-bearing mice (10 mice per group). Tumor sizes were determined on days 7, 9, 11, 14, 17, and 21 after the engrafting of the cells. Human anti-DNP-Ig in the same amount as the above antibodyThe G1 antibody was a negative control antibody. "vehicle" means 1% nude mouse serum-containing PBS (200. mu.l) as a medium for dispersing the amount of antibody administered.

The method for preparing a tumor-bearing murine model using the NCI-H508 cell line is described below. Cells of the colorectal cancer cell line NCI-H508 and Matrigel (produced by Becton Dickinson Bioscience) containing mouse malignant sarcoma (promoting survival and proliferation of tumor cells) were treated at 1X 10⁷Mice were subcutaneously transplanted to the back of 6-week-old Balb/c nude mice (purchased from CLEA, Japan) at a ratio of NCI-H508 to Matrigel of 1: 1. On days 1, 4 and 7 after the engrafting of the cells, the chimeric anti-A33 or rec263 antibody (dissolved in 200. mu.l of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10. mu.g/mouse or 100. mu.g/mouse to tumor-bearing mice (10 mice per group). Tumor sizes were determined on days 7, 11, 14, 18, 21, 27, 33, 40, 48, 55, and 62 after the engrafting of the cells. The negative control antibody was human anti-DNP-IgG 1 antibody in the same amount as the above antibody. "vehicle" means 1% nude mouse serum-containing PBS (200. mu.l) as a medium for dispersing the amount of antibody administered.

FIG. 7 shows the results of the above experiment.

For the mouse line transplanted with the COLO205 cell line, significant tumor suppression (p < 0.05) was observed on days 7, 9 and 11 after the engrafting of the cells in the case of the group to which the rec263 antibody had been administered at 10. mu.g/mouse, compared with the group to which the vehicle had been administered. Significant differences in tumor volume (p < 0.05) were observed after 7, 9, 11 and 14 days of engrafting of the cells, compared with the group administered with anti-DNP-IgG 1 antibody. In addition, significant tumor suppression (p < 0.05) was observed on days 14 and 17 after the engrafting of the cells in the case of the group to which the rec263 antibody was administered at 100. mu.g/mouse, compared with the group to which the anti-DNP-IgG 1 antibody was administered. In contrast, significant tumor suppression (p < 0.05) was observed on days 7 and 11 after the engrafting of the cells in the case of the group to which the chimeric anti-A33 recombinant antibody had been administered at 10. mu.g/mouse, compared with the group to which the vehicle had been administered. Compared with the group to which the anti-DNP-IgG 1 antibody was administered, significant tumor suppression (p < 0.05) was observed on days 7, 9, 11, 14, 17, and 21 after the engrafting of the cells. In addition, significant tumor suppression (p < 0.05) was observed on days 7, 9, and 14 after the engrafting of the cells in the case of the group to which the chimeric anti-A33 recombinant antibody was administered at 100. mu.g/mouse, compared with the group to which the vehicle was administered. Compared with the group to which the anti-DNP-IgG 1 antibody was administered, significant tumor suppression (p < 0.05) was observed on days 7, 9, 11, 14, 17, and 21 after the engrafting of the cells (fig. 7A).

On the other hand, for the mouse line into which the NCI-H508 cell line was transplanted, the group to which the rec263 antibody was administered at 10. mu.g/mouse had no antitumor effect. In contrast, significant tumor suppression (p < 0.05) was observed at day 18 after the engrafting of the cells in the case of the group to which the rec263 antibody had been administered at 100. mu.g/mouse, compared with the group to which the vehicle had been administered. In addition, significant tumor suppression (p < 0.05) was observed on almost all detection days, compared with the group to which the anti-DNP-IgG 1 antibody had been administered. In contrast, significant tumor suppression (p < 0.05) was observed on days 21, 55, and 62 after the engrafting of the cells in the case of the group to which the chimeric anti-A33 recombinant antibody had been administered at 10. mu.g/mouse, compared with the group to which the vehicle had been administered. In addition, significant tumor suppression (p < 0.05) was observed on days 7, 21 and 33 after the engrafting of the cells, compared with the group to which the anti-DNP-IgG 1 antibody had been administered. Furthermore, significant tumor suppression (p < 0.05) was observed on almost all the days of detection in the group administered with the chimeric anti-A33 recombinant antibody at 100. mu.g/mouse, compared with the group administered with the vehicle. In addition, significant tumor suppression (p < 0.05) was observed at day 33 after the engrafting of the cells, compared with the group to which the anti-DNP-IgG 1 antibody had been administered.

As described above, the results showed that the antibody of the present invention has a high antitumor effect on all of the tumor-bearing mouse models prepared using 2 types of colorectal cancer cell lines (FIG. 7B).

The effect of the antibody purified from the hybridoma producing human anti-A33 and the human anti-A33 recombinant antibody was examined using a mouse tumor-bearing model. The colorectal cancer cell lines used herein were COLO205 cells and NCI-H508 cells.

(anti-tumor Effect of antibodies purified from 125M10AA, 125M165DAAA, and 125M96ABA hybridomas on COLO cell lines)

Cells of colorectal cancer cell line COLO205 at 5X 10⁶Mice were subcutaneously transplanted to the back of 6-week-old Balb/c nude mice (purchased from CLEA, Japan). On days 1, 3, 7, 10, 14 and 17 after the engrafting of the cells, 125M10AA, 125M165DAAA or 125M96ABA antibody (dissolved in 200. mu.l of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 20. mu.g/mouse to tumor-bearing mice (15 mice per group to the group to which the vehicle was administered, and 10 mice per group in other cases). Tumor sizes were determined on days 7, 10, 12, 14 and 17 after the engrafting of the cells. "vehicle" means 1% nude mouse serum-containing PBS (200. mu.l) as a medium for dispersing the amount of antibody administered.

Fig. 7C shows the results of the above experiment. In fig. 7C, M10 represents the 125M10AA antibody, M96 represents the 125M96ABA antibody, and M165 represents the 125M165DAAA antibody. Significant tumor suppression (p < 0.05) was observed on all days tested in the group administered with the 125M10AA antibody compared to the group administered with vehicle. In addition, significant tumor suppression (p < 0.05) was observed on days 12, 14, and 17 after the engrafting of the cells in the group to which the 125M165DAAA antibody was administered, compared to the group to which the vehicle was administered. In contrast, significant tumor suppression (p < 0.05) was observed on days 12 and 14 after the engrafting of the cells in the group to which the 125M96ABA antibody had been administered, compared with the group to which the vehicle had been administered.

(anti-tumor Effect of recombinant antibodies N26 and M165 on COLO205 and NCI-H508 cell lines)

Cells of colorectal cancer cell line COLO205 at 5X 10⁶Mice were subcutaneously transplanted to the back of 6-week-old Balb/c nude mice (purchased from CLEA, Japan). On days 1, 3, and 6 after the engrafting of the cells, recN26 or recM165 antibody (dissolved in 200. mu.l of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10. mu.g/mouse or 100. mu.g/mouse to tumor-bearing mice (10 mice per group). Tumor sizes were determined on days 8, 10, 13, 15, 17, 20 and 23 after the engrafting of the cells.

Fig. 7D shows the results of the above experiment. In FIG. 7D, M165-10 indicates the recM165 antibody (administered at 10. mu.g/mouse), M165-100 indicates the recM165 antibody (administered at 100. mu.g/mouse), N26-10 indicates the recN26 antibody (administered at 10. mu.g/mouse), and N26-100 indicates the recN26 antibody (administered at 100. mu.g/mouse). Significant tumor suppression (p < 0.05) was observed on days 10 and 13 after the engrafting of the cells in the case of the group to which the recN26 antibody had been administered at 10. mu.g/head, compared with the group to which the vehicle had been administered. In addition, significant tumor suppression (p < 0.05) was observed at days 8, 10, 13, 15, 17, and 20 after the engrafting of the cells in the case of the group to which the recN26 antibody had been administered at 100. mu.g/head, compared with the group to which the vehicle had been administered. In addition, significant tumor suppression (p < 0.05) was observed on days 8, 10, 13, 15, 17, and 20 after the engrafting of the cells in the case of the group to which the recM165 antibody had been administered at 100. mu.g/head, compared with the group to which the vehicle had been administered.

Cells of the colorectal cancer cell line NCI-H508 and Matrigel (produced by Becton Dickinson Bioscience) containing murine malignant sarcoma (promoting survival and proliferation of transplanted tumor cells) were treated at 1X 10⁷Mice were subcutaneously transplanted to the back of 6-week-old Balb/c nude mice (purchased from CLEA, Japan) at a ratio of NCI-H508 to Matrigel of 1: 1. On days 1, 4 and 7 after the engrafting of the cells, the recN26 or recM165 antibody (dissolved in 200. mu.l of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10. mu.g/mouse or 100. mu.g/mouse to tumor-bearing mice (12 mice per group to the group to which the vehicle was administered, and 10 mice per group in other cases). Tumor sizes were determined on days 11, 18, 28, 36, 43, 50, 57 and 64 after the engrafting of the cells.

FIG. 7E shows the results of the above experiment. In FIG. 7E, N26-10 indicates the recN26 antibody (administered at 10. mu.g/mouse), N26-100 indicates the recN26 antibody (administered at 100. mu.g/mouse), M165-10 indicates the recM165 antibody (administered at 10. mu.g/mouse), and M165-100 indicates the recM165 antibody (administered at 100. mu.g/mouse). For the mouse line transplanted with the NCI-H508 cell line, significant tumor suppression (p < 0.05) was observed on days 11, 18, 36 and 43 after the engrafting of the cells in the case of the group to which the recN26 antibody had been administered at 10. mu.g/mouse, compared with the group to which the vehicle had been administered. In addition, significant tumor suppression (p < 0.05) was observed on days 11, 18, 28, 36, and 50 after the engrafting of the cells in the case of the group to which the recN26 antibody had been administered at 100. mu.g/mouse, compared with the group to which the vehicle had been administered. In contrast, significant tumor suppression (p < 0.05) was observed on days 11 and 18 after the engrafting of the cells in the case of the group to which the recM165 antibody had been administered at 10. mu.g/mouse, compared with the group to which the vehicle had been administered. Significant tumor suppression (p < 0.05) was observed on all days of examination in the group administered with recM165 antibody at 100. mu.g/mouse, compared with the group administered with the vehicle.

(antitumor Effect of M10 and Q54 recombinant antibodies on the NCI-H508 cell line)

Cells of the colorectal cancer cell line NCI-H508 and Matrigel (produced by Becton Dickinson Bioscience) containing murine malignant sarcoma (promoting survival and proliferation of transplanted tumor cells) were treated at 1X 10⁷Mice were subcutaneously transplanted to the back of 6-week-old Balb/c nude mice (purchased from CLEA, Japan) at a ratio of NCI-H508 to Matrigel of 1: 1. On days 1, 4 and 7 after the engrafting of the cells, recM10 or recQ54 antibody (dissolved in 200. mu.l of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10. mu.g/mouse or 100. mu.g/mouse to tumor-bearing mice (10 mice per group). Tumor sizes were determined after 14, 21, 28, 35, 42, 49, 56 and 63 days of engrafting the cells.

Fig. 7F shows the results of the above experiment. In FIG. 7F, M10-10 indicates recM10 antibody (administered at 10. mu.g/mouse), M10-100 indicates recM10 antibody (administered at 100. mu.g/mouse), Q54-10 indicates recQ54 antibody (administered at 10. mu.g/mouse), and Q54-100 indicates recQ54 antibody (administered at 100. mu.g/mouse). For the mouse line transplanted with the NCI-H508 cell line, significant tumor suppression (p < 0.05) was observed on days 14, 21, 28, 42, 49 and 56 after the engrafting of the cells in the case of the group to which the recM10 antibody had been administered at 10. mu.g/mouse, compared with the group to which the vehicle had been administered. In addition, significant tumor suppression (p < 0.05) was observed on days 14, 21, 28, 35, 42, 49 and 56 after the engrafting of the cells in the case of the group to which the recM10 antibody had been administered at 100. mu.g/mouse, compared with the group to which the vehicle had been administered. In contrast, significant tumor suppression (p < 0.05) was observed on days 28 and 42 after the engrafting of the cells in the case of the group to which the recQ54 antibody had been administered at 10. mu.g/mouse, compared with the group to which the vehicle had been administered. In addition, significant tumor suppression (p < 0.05) was observed on days 14, 21, 28, 35, 42, and 56 after the engrafting of the cells in the case of the group to which the recQ54 antibody had been administered at 100. mu.g/mouse, compared with the group to which the vehicle had been administered.

All references cited herein are incorporated by reference. It is clearly understood by those skilled in the art that various modifications and changes may be made to the present invention within the technical spirit and scope of the present invention as disclosed in the appended claims. Such modifications and variations are also included in the scope of the present invention.

Industrial applicability

According to the present invention, there is provided a prophylactic or therapeutic agent against a disease caused by a cell expressing a33, and in particular, a molecule for treating a malignant tumor in a patient suffering from a33 polymorphism.

It is known that A33mRNA has 9 polymorphisms, and 7 of these polymorphisms are present in the untranslated region. In addition, one of the remaining 2 polymorphisms was present in codon 3. Thus, the polymorphism is a silent mutation and no amino acid substitution occurs. While the other of the remaining 2 polymorphisms had an amino acid substitution but was present in the signal sequence. Therefore, the antibody of the present invention is an effective therapeutic or prophylactic agent regardless of the a33 polymorphism.

All documents, patents and patent applications cited herein are incorporated by reference.

Claims (13)

1.A human antibody that binds to a33, produced by hybridoma N26 with accession number FERM BP-10109.

2. The human antibody of claim 1, having an amino acid sequence consisting of SEQ ID NO: 39 and the heavy chain variable region amino acid sequence represented by SEQ ID NO: 41, or a light chain variable region amino acid sequence represented by seq id no.

3. A pharmaceutical composition comprising the human antibody according to claim 1 or 2 as an active ingredient.

4. A prophylactic or therapeutic agent for tumor comprising the human antibody according to claim 1 or 2 as an active ingredient.

5. The prophylactic or therapeutic agent for tumor according to claim 4, wherein the tumor is a tumor comprising cancer cells expressing A33.

6. The prophylactic or therapeutic agent for tumor according to claim 4 or 5, wherein the tumor is selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, gastric cancer, pancreatic cancer, breast cancer, melanoma, renal cell carcinoma, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, prostate cancer, testicular cancer, and mesothelial cancer.

7. A hybridoma which is N26 deposited under accession number FERM BP-10109.

8. A method for producing an antibody, comprising culturing the hybridoma of claim 7 and obtaining the antibody from the culture product.

9. A method for producing an antibody, comprising isolating a gene encoding an antibody that binds to A33 from the hybridoma of claim 7, constructing an expression vector containing the gene, introducing the expression vector into a host, culturing the host, and obtaining the antibody from the culture product.

10. A method for producing an antibody, comprising isolating genes encoding the heavy chain variable region and the light chain variable region of an antibody which binds to A33 from the hybridoma of claim 7, constructing expression vectors containing the genes, introducing the expression vectors into a host, culturing the host, and obtaining the antibody from the culture product.

11. A method of producing an antibody comprising constructing a polypeptide comprising a sequence consisting of SEQ ID NO: 38 and the heavy chain variable region nucleic acid sequence represented by SEQ ID NO: 40, introducing the expression vector into a host, culturing the host, and then obtaining the antibody from the culture product.

12. The production method as claimed in any one of claims 9 to 11, wherein the host is selected from the group consisting of Escherichia coli, yeast cells, insect cells, mammalian cells and plant cells.

13. The production method as claimed in any one of claims 9 to 11, wherein the host is selected from the group consisting of plants and non-human mammals.