# Patent

## Title
Anti-A33 antibody

## Abstract

The present invention provides: an antibody or a antibody fragment thereof, which can bind to A33, which specifically attacks A33-expressing tumor cells with the use of ADCC and CDC based on the immune system, and for which no HAHA is produced; and a preventive or therapeutic agent for various malignant tumors including solid tumors that are currently treated with difficulty, which contains the antibody or an antibody fragment thereof. Specifically, the antibody or a functional fragment thereof is capable of binding to A33 and is produced by a hybridoma M10 (accession No. FERM BP-10107), M96 (accession No. FERM BP-10108), M165 (accession No. FERM BP-10106), N26 (accession No. FERM BP-10109), Q47 (accession No. FERM BP-10104), Q54 (accession No. FERM BP-10105), or R5 (accession No. FERM BP-10107). The preventive or therapeutic agent for tumors contains the antibody or a functional fragment thereof.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application is a Continuation-In-Part of U.S. application Ser. No. 11/629,779 filed Dec. 15, 2006, which is a National Phase Entry of PCT/JP2005/016576 filed Sep. 2, 2005, which claims foreign priority of Japanese Application No. JP-2004-259090 filed Sep. 6, 2004, all incorporated herein by reference in its entirety.


TECHNICAL FIELD
The present invention relates to an anti-A33 antibody that specifically binds to an A33 antigen. Furthermore, the present invention relates to a preventive or therapeutic agent for diseases due to A33-expressing cells, which comprises the anti-A33 antibody as an active ingredient. The present invention particularly relates to a therapeutic agent for malignant tumors.
BACKGROUND ART
Cancer (tumor) is the leading cause of death in Japan and the number of cancer patients is increasing each year. Hence, the development of drugs or therapeutic methods against cancer that have high effectiveness and safety has been strongly desired. Among various types of cancer, colorectal cancer accounted for 12.2% of total cancer cases, as shown in the survey conducted in 1999. The colorectal cancer mortality rates ranked third in the case of men and second in the case of women. Based on the significantly increased number of colorectal cancer cases in recent years, colorectal cancer morbidity or mortality is predicted to surpass the same for gastric cancer in the future. Furthermore, gastric cancer accounted for 17.4% of total cancer cases, as shown in the survey conducted in 1999, and the mortality rates thereof ranked second in the case of men and first in the case of women.
The use of an antibody as a remedy is being recognized as an important and valuable approach for the treatment of various pathological conditions (cancer types). Antibody specificity is useful for treating pathological conditions wherein a tumor-specific antigen exhibits the properties of heterologous cells. Antibodies effectively target such cells through binding to tumor-specific antigens, which are proteins to be expressed on cell surfaces. Currently, a chimeric antibody (Rituximab) that targets at CD20 (the receptor existing on the cell membranes), a monoclonal antibody such as a humanized antibody that targets at Her2/neu, and the like have been used against malignant tumors as subject diseases. The therapeutic effects thereof have been recognized. Antibodies are characterized by long blood half-life and high specificity to antigens, and they are particularly useful as antitumor agents. For example, in the case of an antibody that targets a tumor-specific antigen, it is inferred that the administered antibody is accumulated in tumors. Furthermore, the immune system that attacks against cancer cells through the use of complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) can be expected. Moreover, previous binding of a drug (such as a radioactive nuclide or a cytotoxic substance) to the antibody enables efficient delivery of the bound drug to tumor sites. Simultaneously, since the amounts of such drugs that reach other non-specific tissues are decreased, alleviation of side effects can also be expected. Through administration of an antibody having agonistic activity when a tumor-specific antigen has activity of inducing cell death or through administration of an antibody having neutralization activity when a tumor-specific antigen is involved in the growth and survival of cells, the accumulation of such tumor-specific antibody and the suspension of tumor growth or the degeneration of tumors through the activity of such antibody can be expected. It is thought that antibodies are appropriate for use as antitumor agents because of their features as described above.
Mice have been used as target animals in early antibody production. However, the use of mouse antibodies in vivo is limited for many reasons. Mouse antibodies that are recognized by human hosts as extraneous matter induce “human anti-mouse antibodies,” with such induction referred to as the “HAMA” response (see Schiff et al., Canc. Res. (1985), 45, 879-885). Furthermore, the Fc portions of mouse antibodies are not effective for stimulation of human complements or cytotoxicity.
A chimeric antibody has been developed as an approach to avoid such problem (see European Patent Application Nos. 120694 and 125023). A chimeric antibody contains parts of antibodies derived from 2 or more species (e.g., a mouse antibody variable region and a human antibody constant region). Such chimeric antibody has the advantage of retaining the features of a mouse antibody and can stimulate a human complement or cytotoxicity because of its human Fc. However, such chimeric antibody still induces a “human anti-chimeric antibody;” that is, “HACA” response (see Bruggemann, et al., J. Exp. Med., 170, 2153-2157, 1989).
Furthermore, a recombinant antibody has been developed, wherein only one substituted antibody portion is a complementarity determining region (that is, “CDR”) (British Patent No. GB2188638A and U.S. Pat. No. 5,585,089). An antibody comprising mouse CDR, a human variable framework, and a constant region (that is, a “humanized antibody”) has been produced using CDR-grafting technology (see Riechmann, et al., Nature (1988), 332, 323-327).
A mouse anti-A33 antibody against an antigen that is a class I cell membrane protein referred to as “A33,” a member of the Ig superfamily, and a tumor-specific antigen and a humanized antibody have been reported (see U.S. Pat. No. 5,958,412, Description; King D. J. et al., British J. Cancer (1995) 72, 1364-1372; Welt S. et al., J. Clinical Oncology (1994), 12, 1561-1571; Welt S. et al., J. Clinical Oncology (1996), 14, 1787-1797; Welt S. et al., Clinical Cancer Res. (2003), 9, 1338-1346; and Welt S. et al., Clinical Cancer Res. (2003), 9, 1347-1353). Involvement of such antigen in colon cancer and gastric cancer is known (see U.S. Pat. No. 5,643,550, Description; U.S. Pat. No. 5,160,723, Description; and Garin-Chesa P. G. et al., International J. Oncology (1996), 9, 465-471). Furthermore, in recent years, phase I clinical tests using the humanized A33 antibody have been conducted for colon cancer patients (see Welt S. et al., Clinical Cancer Res. (2003), 9, 1338-1346 and Welt S. et al., Clinical Cancer Res. (2003), 9, 1347-1353). In the former report about the administration of the antibody alone, partial reactions were observed in 1 out of 11 patients to which the antibody could be administered. Moreover, in the latter report about the test using the antibody and chemotherapy in combination, partial reactions were observed in 3 out of 12 patients to which the antibody could be administered, and a mixed reaction was observed in 1 of the same. Even in the case of Avastin (Bevacizumab; humanized anti-VEGF antibody) under development by Genentech in recent years, there is a report that 1 out of 12 patients showed partial reactions in the phase I clinical test using Avastin and standard chemotherapy in combination (Margolin K. et al., J. Clin. Oncol. (2001) 19, 851-856). Accordingly, based on the result that 1 out of 11 patients showed partial reactions as a result of the administration of the antibody alone, the antibody is expected to exert extensive antitumor effects against colorectal cancer.
Although the humanized A33 antibody showed very significant tumor reactions in the phase I clinical tests as described above, human anti-humanized antibodies (that is, “HAHAs”) were produced in both tests with probabilities as high as 50% or more. Interestingly, no HAHA was observed in patients in which the antibody showed high tumor reactivity.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide a preventive or therapeutic agent for various malignant tumors, including solid tumors, that are currently treated with difficulty through the development of an antibody that can bind to A33, specifically attacks A33-expressing tumor cells with the use of ADCC or CDC based on the immune system, and does not cause the production of HAHA.
As described above, it is thought that an antibody that targets the A33 antigen is appropriate for use as an antitumor agent. In addition, with the use of such an antibody by which no HAHA is produced, even higher antitumor effects may be obtained. Hence, as a result of intensive studies concerning the production of an antibody against A33, the present inventors have succeeded in obtaining a monoclonal antibody showing antitumor effects against A33-expressing cancer cells and have specified the sequences of the variable regions of the monoclonal antibody. Thus, the present inventors have completed the present invention.
Specifically, the present invention is as described below.
In the first aspect, the present invention provides a monoclonal antibody binding to A33, which is produced by a mouse-mouse hybridoma. Specifically, a monoclonal antibody, which is preferably a human antibody, or a functional fragment thereof produced by 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5AAAA, for example, is provided. The type of such monoclonal antibody produced by 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5AAAA is human immunoglobulin G (IgG) type. Among the above hybridomas, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were deposited on Aug. 24, 2004, with the International Patent Organism Depositary (IPOD) (Central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan), the National Institute of Advanced Industrial Science and Technology (AIST) under accession Nos. FERM BP-10107 (denotation for identification: M10), FERM BP-10106 (denotation for identification: M165), FERM BP-10108 (denotation for identification: M96), FERM BP-10109 (denotation for identification: N26), FERM BP-10104 (denotation for identification: Q47), FERM BP-10105 (denotation for identification: Q54), and FERM BP-10103 (denotation for identification: R5), respectively.
In an embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof, which has the variable regions of an antibody produced by one of the above hybridomas.
In another embodiment of the present invention, examples of the antibody of the present invention include antibodies of modified subclasses. Specifically, the antibody of the present invention may be: an antibody or a functional fragment thereof which is produced by hybridoma 263A17 and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4; an antibody or a functional fragment thereof which is produced by hybridoma 125M10AA and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4; an antibody or a functional fragment thereof which is produced by hybridoma 125M165DAAA and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4; an antibody or a functional fragment thereof which is produced by hybridoma 125M96ABA and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4; an antibody or a functional fragment thereof which is produced by hybridoma 125N26F6AA and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4; an antibody or a functional fragment thereof which is produced by hybridoma 125Q47BA and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4; an antibody or a functional fragment thereof which is produced by hybridoma 125Q54AAAA and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4; or an antibody or a functional fragment thereof which is produced by hybridoma 125R5AAAA and is of the subclass human IgG1, human IgG2, human IgG3, or human IgG4.
Furthermore, in another aspect of the present invention, the present invention provides an antibody or a functional fragment thereof, which binds to A33 and contains the variable regions of an antibody produced by hybridoma 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, or 125R5AAAA.
In an embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 23 and 25. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 27 and 29. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 31 and 33. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 35 and 37. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 39 and 41. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 43 and 45. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 47 and 49. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the variable regions of the amino acid sequences represented by SEQ ID NOS: 51 and 53. In another embodiment of the present invention, the antibody of the present invention is an antibody or a functional fragment thereof having the entire region of both of the amino acid sequences represented by SEQ ID NOS: 87 and 89.
In another aspect, the present invention further provides the above antibody or a functional fragment thereof that suppresses tumor growth (e.g., derived from colorectal cancer cell line COLO205 cells implanted in nude mice). Upon the suppression of tumors, the antibody or a functional fragment thereof of the present invention is administered to a tumor-bearing animal to be tested (e.g., a tumor-bearing experimental animal such as a colon cancer cell tumor-bearing mouse model having a body weight of 20 g) in an amount between 10 μg/body and 100 μg/body. The dose is 100 μg/body or 5 mg/kg, and preferably 10 μg/body or 0.5 mg/kg, for example.
In an embodiment of the present invention, the antibody of the present invention has one of the following properties.
(a) ADCC Test
In the presence of normal human peripheral blood mononuclear cells, the antibody of the present invention exerts antibody-dependent cellular cytotoxicity (ADCC) against A33-expressing human cancer cells.
(b) CDC Test
In the presence of human serum-derived complements, the antibody of the present invention exerts complement-dependent cytotoxicity (CDC) against A33-expressing human cancer cells.
(c) In Vivo Test
The antibody of the present invention exerts antitumor effects against non-human animals bearing A33-expressing human cancer cells.
(d) Competition Test
The antibody of the present invention is (i) strongly competitive (blocker), (ii) weakly competitive (partial blocker), or (iii) not competitive (non-blocker) with chimeric anti-A33 (comprising the heavy chain variable region and the light chain variable region of an antibody produced by hybridoma ATCC HB-8779 and the heavy chain constant region and the light chain constant region of human IgG1).
(e) Immunohistochemical Test
The presence of the antibody of the present invention results in the staining of human adult colon cancer tissues, human adult normal colon tissues, and human normal small intestine tissues.
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 No. FERM BP-10107), 125M165DAAA (accession No. FERM BP-10106), 125M96ABA (accession No. FERM BP-10108), 125N26F6AA (accession No. FERM BP-10109), 125Q47BA (accession No. FERM BP-10104), 125Q54AAAA (accession No. FERM BP-10105), and 125R5AAAA (accession No. FERM BP-10103) or a nucleic acid encoding a functional fragment of the antibody; a protein encoded by the nucleic acid; an expression vector having the above nucleic acid; and a host selected from the group consisting of Escherichia coli, a yeast cell, an insect cell, a mammalian cell, a plant cell, and a mammal having the expression vector.
In another aspect, the present invention further provides a method for producing an anti-A33 monoclonal antibody or a functional fragment thereof, which comprises: isolating a gene encoding an anti-A33 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, and 125R5AAAA; constructing an expression vector having such gene; introducing the expression vector into a host; culturing the host; causing the expression of the monoclonal antibody; and harvesting the anti-A33 monoclonal antibody or a functional fragment thereof from the thus obtained host or culture products such as the culture supernatant of the host, the secretion product of the host, or the like.
In another aspect, the present invention further provides a preventive, therapeutic, or diagnostic agent for tumors, which contains the above antibody or a functional fragment thereof as an active ingredient.
Examples of tumors that can be prevented or treated with the use of the above agent include at least one tumor selected from the group consisting of colorectal cancer, colon cancer, rectal cancer, gastric cancer, pancreatic cancer, breast cancer, melanoma, renal cell cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, prostatic cancer, testicular cancer, and mesothelial cancer.
In another embodiment of the present invention, the antibody or a functional fragment thereof of the present invention is characterized in that a dose of 10 μg/body or 100 μg/body of the above antibody or a functional fragment thereof is confirmed to exhibit suppression (after tumor grafting) of the tumors of tumor-bearing nude mice wherein COLO205 cells are implanted to a more significant degree than that in the case of a group of the mice to which a vehicle is administered or a group of the mice to which an anti-DNP-IgG1 antibody is administered.
The present invention also relates to: an antibody binding to A33, which recognizes the same epitope as that recognized by an antibody that is produced by hybridoma M10 (accession No. FERM BP-10107); an antibody binding to A33, which recognizes the same epitope as that recognized by an antibody that is produced by hybridoma M96 (accession No. FERM BP-10108); an antibody binding to A33, which recognizes the same epitope as that recognized by an antibody that is produced by hybridoma M165 (accession No. FERM BP-10106); an antibody binding to A33, which recognizes the same epitope as that recognized by an antibody that is produced by hybridoma N26 (accession No. FERM BP-10109); an antibody binding to A33, which recognizes the same epitope as that recognized by an antibody that is produced by hybridoma Q47 (accession No. FERM BP-10104); an antibody binding to A33, which recognizes the same epitope as that recognized by an antibody that is produced by hybridoma Q54 (accession No. FERM BP-10105); and an antibody binding to A33, which recognizes the same epitope as that recognized by an antibody that is produced by hybridoma R5 (accession No. FERM BP-10103).
This description includes part or all of the contents as disclosed in the description and/or drawings of Japanese Patent Application No. 2004-259090, which is a priority document of the present application.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows ADCC activity determined when COLO205 cells were targeted using each purified monoclonal antibody.
FIG. 1B shows CDC activity determined when COLO205 cells were targeted using each purified monoclonal antibody.
FIG. 1C shows ADCC activity determined when NCI-H508 cells were targeted using each purified monoclonal antibody.
FIG. 1D shows CDC activity determined when NCI-H508 cells were targeted using each purified monoclonal antibody.
FIG. 2A shows ADCC activity determined when COLO205 cells were targeted using recombinant antibodies.
FIG. 2B shows CDC activity determined when COLO205 cells were targeted using recombinant antibodies.
FIG. 2C shows ADCC activity determined when NCI-H508 cells were targeted using recombinant antibodies.
FIG. 2D shows CDC activity determined when NCI-H508 cells were targeted using recombinant antibodies.
FIG. 3A is a photograph showing the results of Western blot analysis using a purified antibody and recombinant antibodies.
FIG. 3B is a photograph showing the results of Western blot analysis using a purified antibody and recombinant antibodies.
FIG. 4 shows photographs showing the results of immunohistostaining of human colon cancer tissues using a purified antibody and recombinant antibodies.
FIG. 5 shows photographs showing the results of immunohistostaining of human normal small intestine tissues using a purified antibody and recombinant antibodies.
FIG. 6 shows photographs showing the results of immunohistostaining of human normal colon tissues using a purified antibody and recombinant antibodies.
FIG. 7A shows the antitumor effects of recombinant antibodies cA33 and rec263 against a mouse tumor-bearing model wherein COLO205 cells were implanted.
FIG. 7B shows the antitumor effects of recombinant antibodies cA33 and rec263 against a mouse tumor-bearing model wherein NCI-H508 cells were implanted.
FIG. 7C shows the antitumor effects of purified hybridoma antibodies 125M10AA, 125M165DAAA, and 125M96ABA against a mouse tumor-bearing model wherein COLO205 cells were implanted.
FIG. 7D shows the antitumor effects of recombinant antibodies recN26 and recM165 against a mouse tumor-bearing model wherein NCI-H508 cells were implanted.
FIG. 7E shows the antitumor effects of recombinant antibodies recN26 and recM165 against a mouse tumor-bearing model wherein NCI-H508 cells were implanted with Matrigel.
FIG. 7F shows the antitumor effects of recombinant antibodies recM10 and recQ54 against a mouse tumor-bearing model wherein NCI-H508 cells were implanted with Matrigel.
FIG. 8A shows anti-GPA33 antibody (cA33) binding to cynomolgus rectum.
FIG. 8B shows anti-GPA33 antibody (M10) binding to cynomolgus rectum.
FIG. 8C shows anti-GPA33 antibody (M165) binding to cynomolgus rectum.
FIG. 8D shows anti-GPA33 antibody (N26) binding to cynomolgus rectum.
FIG. 8E shows anti-GPA33 antibody (Q54) binding to cynomolgus rectum.
FIG. 8F shows anti-DNP antibody (negative control) binding to cynomolgus rectum.
FIG. 9A shows anti-GPA33 antibody (cA33) binding to cynomolgus colon.
FIG. 9B shows anti-GPA33 antibody (M10) binding to cynomolgus colon.
FIG. 9C shows anti-GPA33 antibody (M165) binding to cynomolgus colon.
FIG. 9D shows anti-GPA33 antibody (N26) binding to cynomolgus colon.
FIG. 9E shows anti-GPA33 antibody (Q54) binding to cynomolgus colon.
FIG. 9F shows anti-DNP antibody (negative control) binding to cynomolgus colon.
FIG. 10 shows the reactivity of anti-GPA33 antibody to cynomolgus A33 outer cell membrane region recombinant protein.
FIG. 11 shows the binding activity of anti-GPA33 antibody in cynomolgus A33 forced expression cell line.



PREFERRED EMBODIMENTS OF THE INVENTION
Hereafter, the present invention is described in detail.
For A33, a mouse anti-A33 antibody and a humanized anti-A33 antibody have already been obtained. Phase I clinical tests conducted on colon cancer patients using the mouse anti-A33 antibody (see Welt S. et al., J. Clinical Oncology (1994), 12, 1561-1571; Welt S. et al., J. Clinical Oncology (1996), 14, 1787-1797) and the humanized A33 antibody (see Welt S. et al., Clinical Cancer Res. (2003), 9, 1338-1346; Welt S. et al., Clinical Cancer Res. (2003), 9, 1347-1353) have been reported. However, HAMA or HAHA was produced at very high probabilities in patients receiving the antibody, and no further clinical tests were conducted following these. However, very interestingly, no HAHA production was observed in the cases of patients confirmed to have shown tumor reactivity in the clinical test using the humanized anti-A33 antibody.
The novel human anti-A33 monoclonal antibody of the present invention is a complete human antibody. Hence, the antibody of the present invention avoids in advance the problem that always arises upon the use of a mouse antibody or a humanized antibody regarding antigenicity against the portion comprising the mouse sequence. Specifically, in the above clinical test report, HAHA was produced because of the use of the humanized antibody. However, since the novel human anti-A33 monoclonal antibody of the present invention is a complete human antibody, the antigenicity of an antibody can be avoided, and no HAHA is produced. Thus, significant antitumor effects of the antibody can be expected for colon cancer patients.
Examples of antibody classes used herein include immunoglobulin G(IgG), A(IgA), E(IgE), and M(IgM). A preferable antibody class to be used herein is IgG. Furthermore, as an IgG subclass, IgG1, IgG2, IgG3, or IgG4 is used. Preferably, IgG1, IgG2, or IgG4 is used and further preferably, IgG1 is used.
Hereafter, the present invention will be described in detail through clarification of the meanings of terms and phrases used in the present invention.
1. A33 and Anti-A33 Antibody
The antibody of the present invention is a class I cell membrane protein and is an antibody against A33, which is a member of the Ig superfamily.
“Antibody binding to A33” in the present invention refers to an antibody that has reactivity to A33 or a part of A33 or an antibody that recognizes A33 or a part of A33. “Functional fragment” in the present invention refers to a portion (partial fragment) of an antibody, which retains 1 or more actions of an antibody on its corresponding antigen. Specific examples of such fragment include F(ab′)2, Fab′, Fab, Fv, disulfide-stabilized Fv, single-chain Fv(scFv), and multimers thereof (D. J. King., Applications and Engineering of Monoclonal Antibodies., 1998 T. J. International Ltd). Alternatively, “functional fragment” refers to a fragment of an antibody, which is capable of binding to an antigen.
“Human antibody” in the present invention refers to an antibody that is an expression product of a human-derived antibody gene. As described later, a human antibody can be obtained by introducing a human antibody gene locus and then administering an antigen to a transgenic animal capable of producing a human-derived antibody. An example of such transgenic animal is a mouse. A method for producing such mouse capable of producing a human antibody is described in International Patent Publication WO02/43478, for example.
Examples of the antibody of the present invention include, as described in the following examples, various antibodies that exert antitumor effects against A33-expressing cancer cells, even in low concentrations.
The antibody of the present invention also encompasses a monoclonal antibody comprising a heavy chain and/or light chain having an amino acid sequence derived from the amino acid sequence of a heavy chain or light chain constituting an antibody by deletion, substitution, or addition of 1 or several amino acids. The above-mentioned partial amino acid modification (deletion, substitution, insertion, or addition) can be imparted to the amino acid sequence of the antibody of the present invention by partially modifying the nucleotide sequence encoding the amino acid sequence. Such partial modification of a nucleotide sequence can be imparted by a standard method using known forms of site-specific mutagenesis (Proc Natl Acad Sci U.S.A., 1984 Vol 81: 5662). In addition, an “antibody” in the present invention is an immunoglobulin wherein all regions including the heavy chain variable region and the heavy chain constant region as well as the light chain variable region and the light chain constant region, are derived from a gene encoding an immunoglobulin.
The antibody of the present invention also encompasses an antibody of any immunoglobulin class or isotype.
The anti-A33 antibody of the present invention can be produced by a production method as described below. Specifically, for example, nonhuman mammals such as human-antibody-producing transgenic mice are immunized with A33, a part of A33 or a conjugate of the part of A33 and an appropriate carrier substance (e.g., bovine serum albumin) for enhancing antigenicity together with, if necessary, an immuno-augmenting agent (e.g., Freund's complete or incomplete adjuvant). As A33, both natural A33 and recombinant A33 can be used. Alternatively, immunization can be performed by introducing a gene encoding A33 and then administering animal cells that overexpress A33 on their cell surfaces. A monoclonal antibody can be obtained by fusing antibody-producing cells obtained from immunized animals to myeloma cells incapable of producing any autoantibody, culturing the thus obtained hybridomas, and then selecting clones that produce the monoclonal antibody showing specific affinity for an antigen used for immunization.
The antibody of the present invention also encompasses an antibody, the class of which is converted to a subclass differing from the original subclass by genetic engineering modification (e.g., see EP314161) known by persons skilled in the art. Specifically, an antibody subclass differing from the original antibody subclass can be obtained by a genetic engineering technique using a DNA encoding a variable region of the antibody of the present invention.
ADCC refers to a type of cytotoxicity induced by activation of macrophages, NK cells, neutrophil cells, or the like that are recognized through the binding of antibody constant regions to Fc receptors expressed on the surfaces of the above cells. In contrast, CDC refers to a type of cytotoxicity induced by activation of a complement system that occurs through binding of an antibody to an antigen. It is known that the intensities of these activities vary depending on antibody subclasses. It is also known that such differences are due to structural differences among antibody constant regions (Charles A. Janeway et al., Immunobiology, 1997, Current Biology Ltd/Garland Publishing Inc.). For example, an antibody exhibiting a low degree of binding to Fc receptor can be obtained by converting the antibody subclass of the present invention to IgG2 or IgG4. In contrast, an antibody exhibiting a high degree of binding to Fc receptor can be obtained by converting the antibody subclass of the present invention to IgG1or IgG3. Furthermore, the degree of binding to Fc receptor can be varied by modifying the amino acid sequence of a constant region of the antibody of the present invention by a genetic engineering technique or the binding of a constant region sequence having such amino acid sequence (see Janeway Calif. Jr. and Travers P. (1997), Immunobiology, Third Edition, Current Biology Ltd/Garland Publishing Inc). Alternatively, a degree of binding to a complement can also be varied by the same method (see Mi-Hua Tao, et al. 1993. J. Exp. Med). For example, the degree of binding to a complement can be varied by mutating a sequence CCC (that encodes proline (P) at position 331 (based on the EU numbering system (see Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242))) of a heavy chain constant portion to TCC encoding serine (S), so as to substitute proline with serine. In the case of an anticancer agent, for example, it is desired that when an antibody alone is unable to exert activity inducing cell death, such antibody have anti-tumor activity based on antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) mediated by Fc receptor. It may be more desired when an antibody alone is able to exert activity inducing cell death, such antibody have a low degree of binding to Fc receptor. Furthermore, in the case of an immunosuppressive agent, for example, it is desired that when an antibody three-dimensionally inhibits only the binding of T cells to antigen-presenting cells, such antibody lack ADCC activity or CDC activity. Moreover, when ADCC activity or CDC activity can cause toxicity, an antibody wherein the Fc portion is mutated or the subclass thereof is altered so as to avoid activity that causes toxicity may also be desired.
The present invention encompasses the following steps upon production of monoclonal antibodies: (1) purification of a biopolymer to be used as an immunogen and/or preparation of cells overexpressing an antigen protein on the cell surfaces; (2) immunization of animals by injection of an antigen, collection of blood, determination of the antibody titer, determination of timing for extracting the spleen and the like, and then preparation of antibody-producing cells; (3) preparation of myeloma cells; (4) cell fusion of antibody-producing cells and myeloma cells; (5) selection of a hybridoma group producing a target antibody; (6) division into a single cell clone (cloning); (7) if necessary, culture of the hybridoma for producing the monoclonal antibody in a large amount, or breeding of animals wherein the hybridoma is implanted; and (8) examination of the physiological activity and the recognition specificity of the thus produced monoclonal antibody or determination of the properties of the product as a labeling reagent, for example.
Polymorphisms are present in the case of A33. The antibody of the present invention binds to A33 by recognizing all A33 polymorphisms that are currently known. Regardless of differences of A33 polymorphisms among patients, a therapeutic or preventive agent containing the antibody of the present invention can act effectively.
Hereinafter, a method for preparing an anti-A33 monoclonal antibody will be described in detail based on the above steps. However, such method for preparing the antibody is not limited to the following methods. For example, antibody-producing cells and myeloma cells other than splenocytes can also be used.
(1) Purification of Antigen
Transformed cell lines are prepared by incorporating a DNA encoding A33 into an expression vector for animal cells and then introducing the expression vector into animal cells. The thus obtained transformed cell line can be directly used as an antigen. Furthermore, since the primary structure of A33 protein is known (GenBank accession No. NP—005305, SEQ ID NO: 12), a peptide is chemically synthesized from the amino acid sequence of A33 by a method known by persons skilled in the art, following which the product can also be used as an antigen.
Furthermore, cells overexpressing A33 on their cell surfaces can also be effective as immunogens, which are prepared by introducing full-length A33 into FM3A cells or L929 cells. pΔEGFP-N1-A33 can be prepared by incorporating a DNA encoding the A33 protein into an expression vector pΔEGFP-N1 for animal cells (where a region encoding an EGFP protein of modified pEGFP-N1 (produced by Becton Dickinson Bioscience Clontech) is deleted). However, such DNA encoding A33, vector, host, and the like are not limited to these examples.
Specifically, a transformed cell line is obtained by transforming FM3A cells or L929 cells with pΔEGFP-N-1-A33, followed by culturing. The trait of neomycin resistance acquired by cells into which pΔEGFP-N1 vectors are inserted is confirmed, and the expression of A33 is also confirmed using a mouse anti-human A33 antibody (ATCC No. HB-8779). Hence, FM3A cells or L929 cells overexpressing A33 on the cell surfaces can be prepared using such confirmation results as indices.
(2) Step for Preparing Antibody-Producing Cells
The antigen obtained in (1) is mixed with Freund's complete or incomplete adjuvant or an adjuvant such as potassium aluminum sulfate. Experimental animals are immunized with the thus obtained mixture as an immunogen. As experimental animals, transgenic mice capable of producing a human-derived antibody are most suitably used. Such mice are described in document of Tomizuka et al (Tomizuka. et al., Proc Natl Acad Sci U.S.A., 2000 Vol 97: 722).
The route of administration of an immunogen upon immunization of mice may be subcutaneous injection, intraperitoneal injection, intravenous injection, intracutaneous injection, intramuscular injection, footpad injection, or the like. Intraperitoneal injection, footpad injection, or intravenous injection is preferred.
Immunization can be performed once or several times repeatedly at appropriate intervals (preferably at 2-week to 4-week intervals). Subsequently, antibody titers against the antigen in the sera of immunized animals are determined. The effects of the subsequent procedures can be increased with the use of an animal with a sufficiently elevated antibody titer as a supply source of antibody-producing cells. In general, it is preferable to use antibody-producing cells derived from an animal 3 to 5 days after final immunization for subsequent cell fusion.
Examples of methods for determining antibody titers that may be employed herein include various known techniques such as a radioimmunoassay method (hereinafter referred to as the “RIA method”), an enzyme-linked immunosorbent assay method (hereinafter referred to as the “ELISA method”), a fluorescence antibody method, and a passive haemagglutination method. In view of detection sensitivity, rapidity, accuracy, possible automation of procedures, and the like, the RIA method or the ELISA method is more suitable.
Antibody titer can be determined in the present invention by procedures described below, when the ELISA method is employed, for example. First, an antigen against a human antibody is adsorbed on the solid phase surface of a 96-well plate for ELISA or the like. Furthermore, some areas on the solid phase surface at which the antigen is not adsorbed are covered with a protein independent of the antigen (e.g., bovine serum albumin (BSA)). After the surface is washed, the surface is caused to come into contact with a sample (e.g., mouse serum) serially diluted as a primary antibody. An anti-A33 antibody in a sample is caused to bind to the above antigen. An enzyme-labeled secondary antibody against a human antibody is added to bind to the human antibody. After washing, the substrate of the enzyme is added. Antibody titer is calculated through determination of changes or the like in absorbance due to color development based on substrate degradation.
(3) Step for Preparing Myeloma Cells
As myeloma cells, cells incapable of producing any autoantibody derived from mammals such as a mouse, rat, guinea pig, hamster, rabbit, or human can be used. In general, it is preferable to use established cell lines obtained from mice, such as 8-azaguanine-resistant mouse (derived from BALB/c) myeloma cell lines P3X63Ag8U.1 (P3-U1) (Yelton, D. E. et al. Current Topics 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) (Shulman, M. et al., Nature, 276, 269-270 (1978)), P3X63Ag8.653 (653) (Kearney, J. F. et al., J. Immunology, 123, 1548-1550 (1979)), and P3X63Ag8 (X63) (Horibata, K. and Harris, A. W. Nature, 256, 495-497 (1975)). These cell lines are sub-cultured in appropriate medium such as 8-azaguanine medium (prepared by adding 8-azaguanine to RPMI-1640 medium supplemented with glutamine, 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”). 3 to 4 days before cell fusion, the cell lines are sub-cultured in normal medium (e.g., DMEM medium containing 10% FCS). On the day of cell fusion, 2×107 or more cells are maintained.
(4) Cell Fusion
Antibody-producing cells are plasma cells and the lymphocytes that are the precursor cells thereof. Antibody-producing cells may be obtained from any sites of an individual body and can be obtained generally from the spleen, lymph nodes, bone marrow, tonsil, or peripheral blood, an appropriate combination thereof, or the like. Splenocytes are most generally used.
After final immunization, a site at which antibody-producing cells are present, such as the spleen, is excised from a mouse for which a predetermined antibody titer has been obtained, so that splenocytes, which are antibody-producing cells, are prepared. Next, the splenocytes are fused to myeloma cells. Currently, the most generally conducted means for fusing such splenocytes to myeloma cells obtained in step (3) is a method using polyethylene glycol, which is characterized by relatively low cytotoxicity and simple procedures for cell fusion. This method comprises the following procedures, for example.
Splenocytes and myeloma cells are washed well with serum-free culture medium (e.g., DMEM) or phosphate buffered saline (hereinafter, referred to as PBS), mixed until the ratio of the number of cells of splenocytes to myeloma cells reaches approximately 5:1 to 10:1, and then subjected to centrifugation. The supernatant is removed and then precipitated cell groups are loosened well. 1 mL of serum-free culture medium containing 50% (w/v) polyethylene glycol (with a molecular weight between 1000 and 4000) is added dropwise while agitating the solution. Subsequently, 10 mL of serum-free culture medium is gently added, followed by centrifugation. The supernatant is discarded again. The precipitated cells are suspended in normal medium (hereinafter, referred to as HAT medium) containing an appropriate amount of a hypoxanthine-aminopterin-thymidine (hereinafter, referred to as HAT) solution and human interleukin-6 (hereinafter, referred to as IL-6). The suspension is dispensed to each well of a culture plate (hereinafter, referred to as plate), followed by approximately 2 weeks of culture in the presence of 5% carbonic acid gas at 37° C. During the culture period, HAT medium is appropriately supplemented.
(5) Selection of Hybridoma Groups
When the above myeloma cells are of a 8-azaguanine-resistant cell line, and more specifically, of a hypoxanthine guanine phosphoribosyltransferase (HGPRT)-deficient cell line, non-fused myeloma cells and the fusion cells of multiple myeloma cells are unable to survive in medium containing HAT. On the other hand, fusion cells of multiple antibody-producing cells or hybridomas of antibody-producing cells and myeloma cells can survive. However, such fusion cells of multiple antibody-producing cells have limited lifetime. Accordingly, through continuation of culture in medium containing HAT, only hybridomas that are the fusion cells of antibody-producing cells and myeloma cells survive. As a result, such hybridomas can be selected.
For hybridomas that grow in the form of colonies, HAT medium is exchanged with medium from which aminopterin is excluded (hereinafter, referred to as HT medium). Subsequently, a portion of the culture supernatant is collected and then anti-A33 antibody titer is determined by the ELISA method, for example. However, when the above fusion protein is used as an antigen for ELISA, clones that produce an antibody specifically binding to the Fc region of human IgG should be eliminated so as to avoid selection of such clones. The presence or the absence of such clones 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 can also be used, depending on the method used for selecting a hybridoma, and the composition of medium to be used in such a case will vary.
(6) Cloning Step
Hybridomas that are revealed to produce a specific antibody as a result of determination of antibody titers by a method similar to the method described in (2) are transferred to another plate, followed by cloning. Examples of a cloning method to be used herein include a limiting dilution method that involves performing dilution so that each well of a plate then contains one hybridoma, and culturing the hybridomas; a soft agar method that involves culturing in soft agar medium and then harvesting colonies, a method that involves extracting each cell using a micromanipulator and then culturing each cell; and a “sorter clone” method that involves separating cells one-by-one with the use of a cell sorter. The limiting dilution method is convenient and is often used.
For wells for which antibody titers are observed, cloning is repeated 2 to 4 times by a limiting dilution method, for example. Cells for which antibody titers are stably observed are selected as cells of the hybridoma line producing the anti-A33 monoclonal antibody.
In addition, mouse-mouse hybridomas that are the human anti-A33 monoclonal antibody-producing cells of the present invention-125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA-were deposited on Aug. 24, 2004, with the International Patent Organism Depositary (IPOD) (Central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan), National Institute of Advanced Industrial Science and Technology (AIST), under accession Nos. FERM BP-10107 (denotation for identification: M10), FERM BP-10106 (denotation for identification: M165), FERM BP-10108 (denotation for identification: M96), FERM BP-10109 (denotation for identification: N26), FERM BP-10104 (denotation for identification: Q47), FERM BP-10105 (denotation for identification: Q54), and FERM BP-10103 (denotation for identification: R5), respectively.
(7) Preparation of Monoclonal Antibodies by Culture of Hybridomas
After completion of cloning, HT medium is exchanged with normal medium and then hybridomas are cultured. Large-scale culture is performed by rotation culture using large culture bottle, spinner culture, or culture using a hollow fiber system or the like. The supernatant obtained by such large-scale culture is purified by a method known by persons skilled in the art, such as gel filtration, so that the anti-A33 monoclonal antibody can be obtained. Furthermore, the hybridomas are proliferated intraperitoneally in mice (e.g., BALB/c) of the same line or nu/nu mice, rats, guinea pigs, hamsters, rabbits, or the like, so that ascites containing the anti-A33 monoclonal antibody in large amounts can be obtained. As a convenient method for purification, a commercial monoclonal antibody purification kit (e.g., a MAbTrap GII kit; produced by Amersham Pharmacia Biotech) or the like can also be used.
The thus obtained monoclonal antibody has high antigen specificity against A33.
(8) Determination of Monoclonal Antibody
The isotype and subclass of the thus obtained monoclonal antibody can be determined as follows. Examples of an identification method include an Ouchterlony method, an ELISA method, and an RIA method. The Ouchterlony method is convenient, but requires a procedure for condensing the concentration of a monoclonal antibody when the concentration is low. In the meantime, when the ELISA method or the RIA method is used, the culture supernatant is directly caused to react with antigen-adsorbed solid phase. The isotype and the subclass of a monoclonal antibody can then be identified using antibodies corresponding to various immunoglobulin isotypes and subclasses as secondary antibodies.
Furthermore, protein determination can be performed by a Folin-Lowry method, which is a method that involves calculation based on absorbance at 280 nm (1.4 (OD 280)=immunoglobulin (1 mg/mL)).
Identification of an epitope that is recognized by a monoclonal antibody can be performed as described below. First, various partial structures of molecules that a monoclonal antibody recognizes are prepared. Examples of a method for preparing such partial structures include: a method by which various partial peptides of a molecule are prepared using a known oligopeptide synthesis technique; and a method by which a DNA sequence encoding a target partial peptide is incorporated into a suitable expression plasmid using a gene recombination technique and then the peptide is produced within or outside a host such as Escherichia coli. In general, both methods are used in combination for the above purpose. For example, a series of polypeptides are prepared through sequential shortening of the C-terminus or the N-terminus of an antigen protein by appropriate lengths using a gene recombination technique known by persons skilled in the art. The reactivity of a monoclonal antibody against each of these polypeptides is examined, so that a rough recognition site is determined.
Various oligopeptides corresponding to such sites, variants of such peptides, and the like are further subsequently synthesized using an oligopeptide synthesis technique known by persons skilled in the art. An epitope is determined through examination of the binding of a monoclonal antibody (contained as an active ingredient in a preventive or therapeutic agent of the present invention) to such peptides or through examination of the competitive inhibition activity of a peptide against the binding of such monoclonal antibody to an antigen. As a convenient method for obtaining various types of oligopeptides, commercial kits (e.g., SPOTs kit (produced by GenoSys Biotechnology) and a series of multipin peptide synthesis kits (produced by Chiron Corporation) using a multipin synthesis method) can be used.
Furthermore, a gene encoding a human monoclonal antibody is cloned from antibody-producing cells such as hybridomas, and then the gene is incorporated into an appropriate vector. The vector is then introduced into a host (e.g., mammalian cell lines, Escherichia coli, yeast cells, insect cells, and plant cells). Hence, a recombinant antibody produced using a gene recombination technique can also be prepared (P. J. Delves., Antibody Production Essential Techniques., 1997 Wiley, P. Shepherd, and C. Dean., Monoclonal Antibodies, 2000 Oxford University Press, J. W. Goding., Monoclonal Antibodies: Principles and Practice, 1993 Academic Press).
The present invention also encompasses nucleic acids containing the gene sequences of the antibodies of the present invention, which are possessed by hybridomas producing the antibodies. In particular, it also encompasses the nucleic acids of the heavy chain variable regions and the light chain variable regions of the antibodies produced by the hybridomas of the present invention described later. “Nucleic acid(s)” used herein include DNA and RNA.
A method employed for preparation of a gene encoding a monoclonal antibody from a hybridoma involves preparing DNAs encoding monoclonal antibody L chain V region, L chain C region, H chain V region, and H chain C region by the PCR method, or the like. As primers, oligo DNAs designed from the anti-A33 antibody gene or the amino acid sequence thereof can be used. As a template, a DNA prepared from a hybridoma can be used. These DNAs are incorporated into one appropriate vector, and then the vector is introduced into a host for expression. Alternatively, these DNAs are separately incorporated into appropriate vectors, followed by co-expression of the DNAs.
As a vector, a phage or a plasmid that is autonomously replicable in host microorganisms is used. Examples of a plasmid DNA include plasmids derived from Escherichia coli, Bacillus subtilis, or yeast, while the phage DNA may be λ phage.
Hosts that are used for transformation are not particularly limited as long as they can express a target 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 arbitrary methods such as a method using calcium ions, an electroporation method, a spheroplast method, a lithium acetate method, a calcium phosphate method, and a lipofection method. In addition, examples of a method for introducing genes into animals described later include a microinjection method, a method by which genes are introduced into ES cells using the electroporation or lipofection method, and a nuclear transplantation method.
In the present invention, the anti-A33 antibody can be obtained by culturing a transformant and then harvesting the antibody from the culture product. “Culture product(s)” refers to (a) culture supernatant, (b) cultured cells, cultured microbial bodies or disrupted products thereof, or (c) secretion products of the transformant. To culture a transformant, medium appropriate for a host is used and a static culture method, a roller bottle culture method, or the like is employed.
After culture, when a target antibody protein is produced within microbial bodies or cells, the antibody is harvested by disrupting the microbial bodies or cells. In addition, when a target antibody is produced out of microbial bodies or cells, the culture solution is used intact, or microbial bodies or cells are removed by centrifugation or the like. Thereafter, a target antibody can be isolated and purified from the culture product by a single general biochemical method or an appropriate combination of such biochemical methods using various types of chromatography, which are used for protein isolation and purification.
Furthermore, a technique for producing transgenic animals is used to produce an animal host wherein the gene of a target antibody is incorporated in an endogenous gene, such as transgenic cattle, transgenic goats, transgenic sheep, or transgenic pigs. A monoclonal antibody derived from the antibody gene can also be obtained in large amounts from milk secreted from such 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 in accordance with various conditions, including the properties of cell types to be cultured, the purposes of experiment and research, culture methods, and the like. Such culture can be performed using known nutrition medium that is used for production of a monoclonal antibody in culture supernatant or all types of nutrition medium induced and prepared from known basic medium.
(9) Antibody Properties
The antibody of the present invention has one of the following properties.
(a) ADCC Test
In the presence of normal human peripheral blood mononuclear cells, the antibody of the present invention exerts antibody-dependent cellular cytotoxicity (ADCC) against A33-expressing human cancer cells.
(b) CDC Test
In the presence of human serum-derived complements, the antibody of the present invention exerts complement-dependent cytotoxicity (CDC) against A33-expressing human cancer cells.
(c) In Vivo Test
The antibody of the present invention exerts antitumor effects against non-human animals bearing A33-expressing human cancer cells.
(d) Competition Test
The antibody of the present invention is (i) strongly competitive (blocker), (ii) weakly competitive (partial blocker), or (iii) not competitive (non-blocker) with chimeric anti-A33 (comprising the heavy chain variable region and the light chain variable region of an antibody produced by hybridoma ATCC HB-8779 and the heavy chain constant region and the light chain constant region of human IgG1).
(e) Immunohistochemical Test
The antibody of the present invention results in the staining of human adult colon cancer tissues, human adult normal colon tissues, and human normal small intestine tissues.
Examples of such antibodies include antibodies produced by hybridomas 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA, for example. 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were deposited on Aug. 24, 2004 with the International Patent Organism Depositary (IPOD) (Central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan), the National Institute of Advanced Industrial Science and Technology (AIST) under accession Nos. FERM BP-10107 (denotation for identification: M10), FERM BP-10106 (denotation for identification: M165), FERM BP-10108 (denotation for identification: M96), FERM BP-10109 (denotation for identification: N26), FERM BP-10104 (denotation for identification: Q47), FERM BP-10105 (denotation for identification: Q54), and FERM BP-10103 (denotation for identification: R5), respectively.
2. Pharmaceutical Compositions
A pharmaceutical composition containing the human anti-A33 antibody of the present invention is also within the scope of the present invention. Such formulation preferably contains a physiologically acceptable diluent or carrier in addition to the antibody, and may be a mixture thereof with a different antibody or a different drug such as an antibiotic agent. Examples of an appropriate carrier include, but are not limited to, physiological saline, phosphate buffered physiological saline, a phosphate buffered physiological saline glucose solution, and buffered physiological saline. Alternatively, the antibody may be freeze-dried and, when needed, reconstituted by the addition of an aqueous buffer as described above. Such preventive or therapeutic agent can be administered through various routes for administration. Examples of such routes for administration include oral administration with the use of tablets, capsules, granules, powders, syrups, or the like and parenteral administration with the use of injections, drops, suppositories, or the like.
The dose of such pharmaceutical composition differs depending on symptom, age, body weight, and the like. Generally, in the case of oral administration, a dose ranging from approximately 0.01 mg to 1000 mg per day for an adult and can be administered once or several separate times. In the case of parenteral administration, a dose ranging from approximately 0.01 mg to 1000 mg per administration can be administered via subcutaneous injection, intramuscular injection, or intravenous injection.
The present invention also encompasses the above method for preventing or treating diseases using the antibody or the pharmaceutical composition of the present invention. Furthermore, the present invention also encompasses the use of the antibody of the present invention for production of the above preventive or therapeutic agent for diseases.
Tumors that can be prevented or treated with the use of the antibody or a functional fragment thereof of the present invention are colorectal cancer, colon cancer, rectal cancer, gastric cancer, pancreatic cancer, breast cancer, melanoma, renal cell cancer, cervical cancer, endometrial cancer, ovarian cancer, esophageal cancer, prostatic cancer, testicular cancer, mesothelial cancer, and the like. The number of tumor type to which the antibody of the present invention is applied is not limited to a single type. The antibody can also be applied to a case where tumors of a plurality of types are developed simultaneously.
3. Pharmaceutical Preparation Examples
The molecule of the present invention can be used as an ampule of a sterile solution or a suspension prepared by dissolving the molecule in water or a pharmacologically acceptable solution other than water. Moreover, such ampule is filled with a sterile powdery pharmaceutical preparation (preferably, the molecule of the present invention is freeze-dried) and then the ampule may be diluted with a pharmacologically acceptable solution when it is used.
4. Cross-Reactivity to Primates
Currently there are over five hundred mAb drug products that have been developed worldwide. It is thought that a complete human antibody has a potential to avoid immunogenicity problem. However, there are many cases that a complete human antibody does not react with rodent. In these cases, we have to conduct toxicity study of drug product with primates, such as chimpanzee, rhesus macaque, and cynomolgus. On the other hand, for a complete human antibody that reacts with rodent, in order to apply new drug to FDA (U.S. Food and Drug Administration), we should perform toxicity study by different genus/species, such as rodent and primates in many cases. Furthermore, it is easy to predict drug toxicity in clinical study by conducting prior toxicity study with primates than with rodent.
As just described, there are many cases to conduct toxicity study with primates, but in the case that the drug product only cross-reacts with chimpanzee, we place restrictions on toxicity study. The reason for the restriction are:

    
    
        (a) limitation of practicable research plant to conduct chimpanzee study;
        (b) HIV infection rate of chimpanzee is high, and the industrial health problem of the pursuer; and
        (c) impossibility to conduct anatomical study and reproductive toxicity study with chimpanzee.

Consequently, it is useful to develop drug product that is characterized by cross-reactivity with rhesus macaque or cynomolgus.

    
    


Example of methods for determining cross-reactivity of antibody are immunohistochemistry, enzyme-linked immunosorbent assay method (hereinafter referred to as the “ELISA method”), and flow cytometry (hereinafter referred to as the “FMC”). But the methods are not limited to the these known methods.
The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to embodiments described in the examples.
Example 1
Preparation of Mouse Anti-A33 Antibody
A mouse anti-A33 antibody was prepared for use as a positive control antibody in screening for hybridomas producing human monoclonal antibodies and various experiments. An AS33 hybridoma (American Type Culture Collection (ATCC) No. HB-8779) producing the mouse anti-A33 antibody was purchased from ATCC. The hybridoma was cultured according to instructions included in the ATCC product. The hybridoma was freeze-dried. Subsequently, the AS33 hybridoma was acclimatized in eRDF medium (produced by Kyokuto Pharmaceutical Industrial) containing 10% Low IgG Fetal Bovine Serum (produced by HyClone). The acclimatized hybridoma was cryopreserved. Next, for the purpose of antibody purification, a part of the cryopreserved product was acclimatized in eRDF medium (produced by Kyokuto Pharmaceutical Industrial) containing cattle insulin (5 μg/ml, produced by Gibco BRL), human transferrin (5 μg/ml, produced by Gibco BRL), ethanolamine (0.01 mM, produced by Sigma), sodium selenite (2.5×105 mM, produced by Sigma), and 1% Low IgG Fetal Bovine Serum (produced by HyClone). After culture in a flask, the culture supernatant was collected. The concentration of the purified antibody derived from the hybridoma in the collected supernatant was obtained by measurement of absorbance at 280 nm and calculation with 1.4 OD being equivalent to 1 mg/mL (antibody concentration).
Example 2
Preparation of Chimeric Anti-A33 Antibody
A chimeric anti-A33 antibody having human IgG1 heavy chain and human IgG1 light chain constant regions was prepared as a positive control antibody for use in screening for hybridomas producing human monoclonal antibodies or various experiments.
(1) cDNA Cloning of Chimeric Anti-A33 Antibody Gene and Construction of Expression Vector
The hybridoma AS33 producing the mouse anti-A33 antibody purchased in Example 1 was cultured in DMEM medium (produced by Gibco BRL) containing 10% Fetal Bovine Serum (produced by HyClone), and then total RNA was purified using an RNA extraction reagent ISOGEN (produced by NIPPON GENE) according to the relevant protocols. Next, polyA+ RNA was purified from the total RNA using OligotexTM-dT30<Super> (produced by TAKARA BIO). Cloning experiments were conducted using the thus obtained polyA+RNA (2.5 μg) as a material and a SMART RACE cDNA Amplification Kit (Becton Dickinson Bioscience Clontech) according to the instructions included therein. The cDNA of a variable region of the antibody gene was thus obtained.
1) Synthesis of 1st Strand cDNA
















polyA + RNA
(2.5 μg)/3 μl



5′-CDS primer
1 μl



SMART II A oligo
1 μl










A reaction solution having the above composition was subjected to 2 minutes of incubation at 70° C. The following reagents and enzymes were added, and then incubation was performed for 1.5 hours at 42° C., followed by cDNA synthesis.
















5X First-Strand buffer
2 μl



DTT (20 mM)
1 μl



dNTP Mix (10 mM)
1 μl



PowerScript Reverse Transcriptase
1 μl










After completion of the reaction, 100 μl of Tricine Buffer was added and then incubation was performed at 72° C. for 7 minutes.
2) Amplification of Heavy Chain and Light Chain Genes by PCR
The thus obtained cDNA was used as a template for PCR. A primer set used for PCR was composed of: the PCR primer (for H chain: GPAHvR3Nhe (5′-GCC CTT GGT GCT AGC TGA AGA GAC GGT GAC CAG AGT CCC TTG-3′) (SEQ ID NO: 1) or for L chain: GPALvR3Bsi (5′-GTG CAC GCC GCT GGT CAG GGC GCC TG-3′) (SEQ ID NO: 2)) specific to the 3′ terminus of the mouse anti-A33 antibody heavy chain (hereinafter, a “heavy chain” may also be referred to as an “H chain”) variable region DNA or the 3′ terminus of the mouse anti-A33 antibody light chain (hereinafter, a “light chain” may also be referred to as an “L chain”) variable region DNA; and a UPM primer (oligonucleotide complementary to the common sequence, which is prepared at the 5′ terminus of the synthesized cDNA) included in a SMART RACE cDNA Amplification Kit. The H chain leader sequence and variable region (hereinafter, also referred to as “HV”) were amplified by PCR. Further, the L chain leader sequence and variable region (hereinafter, also referred to as “LV”) were amplified by PCR. cDNA amplification was performed using KOD-Plus-DNA polymerase (produced by TOYOBO), and the following reaction solution was prepared.

















Sterile H2O
29.5 μl




cDNA
2.5 μl



KOD-Plus-buffer (10X)
5 μl



dNTP Mix (2 mM)
4 μl



MgSO4 (25 mM)
2 μl



KOD-Plus-DNA polymerase (1 unit/μl)
1 μl



Universal primer A mix (UPM) (10X)
5 μl



Gene specific primers (GSP)
1 μl



Total volume
50 μl










A thermal cycling amplification reaction was performed under the following conditions.
5 cycles:
94° C. for 30 sec
72° C. for 1 min
5 cycles:
94° C. for 30 sec
70° C. for 30 sec
72° C. for 1 min
25 cycles:
94° C. for 30 sec
68° C. for 30 sec
72° C. for 1 min
The thus amplified PCR fragments were harvested by ethanol precipitation, harvested by agarose gel electrophoresis, and then purified using a QIAquick Gel Extraction Kit (produced by QIAGEN), which is a DNA purification kit using a membrane. The thus purified HV and LV amplified fragments were separately subcloned into a PCR 4 Blunt-TOPO vector of a Zero Blunt TOPO PCR Cloning Kit (produced by Invitrogen). The nucleotide sequences of the insert DNAs were analyzed for the plasmid DNAs of the thus obtained clones. For determination of the DNA nucleotide sequences, M13FW (5′-GTA AAA CGA CGG CCA GTG-3′) (SEQ ID NO: 3) and M13RV (5′-CAG GAA ACA GCT ATG AC-3′) (SEQ ID NO: 4) were used as primers. The antibody amino acid sequences encoded by the thus determined HV and LV gene regions completely matched the amino acid sequences of the mouse anti-A33 antibody (Br J. Cancer. 1995 Dec 72 (6): 1364-72) variable regions reported by King D. J. et al.
(3) Construction of Expression Vector (N5KG1_mVhCA33) for Chimeric Anti-A33 Antibody
The mouse anti-A33 antibody HV was amplified by PCR (94° C. for 3 minutes→94° C. for 10 seconds and 68° C. for 45 seconds (35 cycles)→72° C. for 7 minutes) using a plasmid DNA containing the thus obtained antibody HV chain as a template and primers (of a primer set 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)) designed to add restriction enzyme sites to the termini for ligation. The amplified HV fragments were purified, and then subcloning was performed into PCR 4 Blunt-TOPO vectors. The DNA nucleotide sequence of the inserted portion was analyzed for the subclones. Thus, plasmid DNAs each having a sequence designed not to differ from the gene sequence used as a template were selected. The plasmid DNAs were digested with restriction enzymes Sal I and Nhe I. An approximately 440-bp DNA was harvested and purified by agarose gel electrophoresis. In the meantime, an N5KG1-Val Lark vector (a modified vector of IDEC Pharmaceuticals, N5KG1 (U.S. Pat. No. 6,001,358)) was similarly treated with restriction enzymes Sal I and Nhe I and then subjected to dephosphorylation using alkaline phosphatase (E. coli C75) (produced by TAKARA BIO). Subsequently, an approximately 8.9-kb DNA was harvested by agarose gel electrophoresis and a DNA purification kit. These two fragments were subjected to a ligation reaction using a DNA ligation kit Ver 2.1 (produced by TAKARA BIO) and then introduced into Escherichia coli DH5α, thereby obtaining transformants. Through screening of the transformants, a clone N5KG1_GPA33Hv (clone #2), into which the target HV had been inserted, was selected. For insertion of LV into the thus obtained N5KG1_GPA33Hv, the plasmid DNA was cleaved sequentially with restriction enzymes Bgl II and BsiW I and then subjected to dephosphorylation. A vector DNA of approximately 9.2 kb was then purified. Meanwhile, the LV region was amplified by PCR using a plasmid DNA containing the mouse anti-A33 antibody LV as a template. GPALv2FBgl (5′-AGA GAG AGA GAT CTC TCA CCA TGG GCA TCA AGA TGG AGT TTC AG-3′) (SEQ ID NO: 6) and GPALvR3Bsi (SEQ ID NO: 2) were used as a primer set for amplification. The thus purified and amplified LV fragments were subcloned into PCR 4 Blunt-TOPO. The DNA nucleotide sequence of the inserted portion was analyzed for the subclones. Thus, plasmid DNAs each having a sequence designed not to differ from the gene sequence used as a template were selected. The DNAs were digested with restriction enzymes Bgl II and BsiW I. An approximately 400-bp DNA was then harvested and purified by agarose gel electrophoresis. The DNA was ligated to the above N5KG1_A33Hv vector fragment that had been cleaved with restriction enzymes Bgl II and BsiW I, and was then introduced into Escherichia coli, thereby obtaining transformants. Through screening of the transformants, a clone N5KG1_GPA33HvLv (clone #2), into which the target LV had been inserted, was selected. The finally obtained chimeric anti-A33 antibody expression plasmid DNA was purified in a large quantity. Thus, it was confirmed that no mutations had occurred in the inserted L chain or H chain DNA fragments, or in the DNA nucleotide sequences of the peripheries of the insertion sites.
DNAs encoding the chimeric anti-A33 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.








<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
AAACTCTCC


 


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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, 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. Further, 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 following Table 5 lists the nucleotide sequences of synthetic DNAs.
Host cells were transfected with the thus constructed expression vector for the chimeric anti-A33 recombinant antibody, thereby preparing recombinant-antibody-expressing cells. Host cells to be used for expression were CHO cells of a dhfr-deficient cell line (ATCC CRL-9096), CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109, 1999), or HEK293T (ATCC CRL-11268), for example.
Host cells were transfected with the vector by electroporation, lipofection, or the like. Approximately 2 μg of the antibody expression vector was linearized using restriction enzymes and then subjected to electroporation using a Bio-Rad electrophoreter under conditions of 350 V and 500 μF. 4×106 CHO cells were thus transfected with the gene and then the cells were inoculated on a 96-well culture plate. Lipofection was performed using LipofectAMINE Plus (produced by Gibco BRL) according to the manual included therein. 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 confirmation of colonies, an antibody-expressing cell line was selected by the method described in Example 6. Antibody purification was performed from the selected cells as described in Example 8.
Example 3
Preparation of Antigen
To obtain cells overexpressing A33 (to be used as an immunogen, for screening for an antibody, or the like) on the cell membranes, a plasmid expression vector for the full-length A33 amino acid sequence was constructed. A DNA encoding A33 was prepared by the PCR method.
a) Construction of Full-Length A33 Expression Vector
A plasmid vector pΔEGFP-N1-GPA33 retaining A33-encoding cDNA was constructed to prepare a full-length A33 expression vector. pΔEGFP-N1-GPA33 was constructed by the following method. The complete full-length A33 DNA (GenBank DNA NM—005814: SEQ ID NO: 11 or protein NP—005305: SEQ ID NO: 12) was modified by performing a polymerase chain reaction (PCR) so as to add an EcoR I sequence to the 5′ terminus and to add a Not I sequence and a termination codon to the 3′ terminus. 30 cycles of a PCR reaction (94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 60 seconds) were performed using Human Colon Marathon-Ready cDNA (purchased from Becton Dickinson Bioscience Clontech) as a template, A33-F2
5′-GCAGACGAATTCAAGACCATGGTGGGGAAGAT-3′ (SEQ ID NO: 13) and A33-R1
5′-CTCGAGCGGCCGCTCTGCTGCTGGCCTGTCACTGGTCGAGGTG-3′ (SEQ ID NO: 14) synthesized as primers, and KOD-plus DNA polymerase (produced by TOYOBO). The thus synthesized sequence was digested with EcoR I-Not I. The resultant was isolated as an EcoR I-Not I fragment. The fragment was then ligated to pΔEGFP-N1-vector (from which the region encoding EGFP protein of modified pEGFP-N1 (produced by Becton Dickinson Bioscience Clontech) had been deleted) that had been cleaved with the same enzymes. The thus obtained plasmid was named pΔEGFP-N1-GPA33. A 977-bp cDNA was encoded in A33 incorporated in pΔEGFP-N1-GPA33. Hereinafter, GeneAmp® PCR System 9700 (produced by PerkinElmer Japan) was used for regulating the temperatures for all PCR reactions in the examples.

b) Preparation of A33-Expressing Cells

The two following cell lines were transfected with pΔEGFP-N1-GPA33 constructed in a): FM3A cell line (Japanese Collection of Research Bioresources (JCRB) Cell Bank No. 0701) and the L929 cell line (ATCC No. CCL-1). Thus, 2 types of A33-expressing cells were prepared. The electroporation method was employed for FM3A cells. 5×106 FM3A cells were transfected with 20 μg of the pΔEGFP-N1-A33 vector using an electrophoreter (produced by BTX) under conditions of 350 V and 950 μF. The cells were inoculated on a 6-well culture plate. After 48 hours of culture at 37° C. under 5.0% carbonic acid gas, G418 (produced by Gibco BRL) was added to the cells at a concentration of 1 mg/mL and then the cells were cultured for 1 week. The A33 antigen expressed on the cell membrane surfaces was confirmed using the culture supernatant of the AS33 hybridoma (ATCC No. HB-8779). Flowcytometer (FCM: produced by Becton, Dickinson and Company) analysis was performed using the culture supernatant of the AS33 hybridoma as a primary antibody and a goat anti-mouse Ig gamma F(ab′)2 antibody (produced by Dako) labeled with R-phycoerythrin as a secondary antibody. Thus, among transfected cells that had acquired the trait of G418 resistance, cells expressing A33 on their cell membrane surfaces were selectively sorted.
L929 cells were transfected using Trans IT-LT1 (produced by TAKARA BIO INC). Transfection was performed by a method described in the related manual. After 24 hours of culture at 37° C. under 5.0% carbonic acid gas, G418 (produced by Gibco BRL) was added to the cells at a concentration of 1 mg/mL and then culture was performed for 1 week. In a manner similar to that in the case of FM3A cells, the A33 antigen expressed on the cell membrane surfaces was confirmed using the culture supernatant of the AS33 hybridoma. Flowcytometer (FCM: produced by Becton, Dickinson and Company) analysis was performed using the AS33 hybridoma (ATCC No. HB-8779) as a primary antibody, a goat anti-mouse Ig gamma F(ab′)2 antibody (produced by Dako) labeled with R-phycoerythrin as a secondary antibody. Thus, among transfected cells that had acquired the trait of G418 resistance, cells expressing A33 on their cell membrane surfaces were selectively sorted.
Single clones expressing the full-length human A33 antigen at high levels could be obtained from both cell lines. FM3A cells and L929 cells, which had expressed the A33 antigen protein at high levels, were named FM3A/A33 and L929/A33, respectively.
shA33EX-hFc protein was prepared for use as an immunogen or in ELISA upon screening for an antibody.
c) Construction of an Expression Vector for a Soluble A33 Human Fc Fusion Protein Outside the cell membrane
To construct an expression vector (hereinafter referred to as shA33EX-hFc) for a soluble A33 human Fc fusion protein outside the cell membrane, a plasmid vector pTracer-CMV-humanFc-A33EXR retaining cDNA encoding the A33 region outside the cell membrane was constructed. pTracer-CMV-humanFc-A33EXR was constructed by the following method. The DNA of the A33 region outside the cell membrane (SEQ ID NO: 11) containing a secretory signal sequence was modified via a polymerase chain reaction (PCR) so as to add an EcoR I sequence to the 5′ terminus and to add a Not I sequence and a termination codon to the 3′ terminus. 30 cycles of a PCR reaction (94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 60 seconds) were performed using pΔEGFP-N1-A33 cDNA prepared in a) as a template, A33-F2 (SEQ ID NO: 13) and GPA-EXCRR25′-CTCGAGCGGCCGCCAGTTCATGGAGGGAGATCTGACG-3′ (SEQ ID NO: 15) synthesized as primers, and KOD-plus DNA polymerase (produced by TOYOBO). The thus synthesized sequence was digested with EcoR I-Not I. The resultant was isolated as an EcoR I-Not I fragment. The fragment was then ligated to a pTracer-CMV-humanFc vector (plasmid prepared by introducing FLAG and a human IgG1Fc region at the Xba I and Apa I sites of modified pTracer-CMV (produced by Invitrogen Life Technologies)) that had been cleaved with the same enzymes. The thus obtained plasmid was named pTracer-CMV-humanFc-A33EXR.
Oligonucleotides such as PCR primers were all synthesized using a DNA autosynthesizer (model 3948; produced by PerkinElmer Japan, Applied Biosystems Division) according to the manual included therewith (see Matteucci, M. D. and Caruthers, M. H. (1981) J. Am. Chem. Soc. 103, 3185-3191). After completion of synthesis, each oligonucleotide was cleaved from support medium and subjected to deprotection. The thus obtained solution was dried, solidified, and then dissolved in distilled water. This solution was cryopreserved at −20° C. until use.
d) Expression and Purification of shA33EX-hFc Protein
Host cells were transfected with the shA33EX-hFc protein expression vector constructed in c), thereby preparing cells expressing the soluble A33 protein outside the cell membrane. Host cells to be used for expression were CHO cells of a dhfr-deficient cell line (ATCC CRL-9096), CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109, 1999), or HEK293T (ATCC CRL-11268), for example.
Host cells were transfected with the vector by electroporation, lipofection, or the like. Approximately 2 μg of the shA33EX-hFc protein expression vector was linearized using restriction enzymes and then subjected to electroporation using a Bio-Rad electrophoreter under conditions of 350 V and 500 μF. 4×106 CHO cells were transfected with the gene, and then the cells were inoculated on a 96-well culture plate. Lipofection was performed using LipofectAMINE Plus (produced by Gibco BRL) according to the manual included therewith. After transfection with the vector, a drug corresponding to the selection marker for 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, 20 mM sodium citrate as an elution buffer, and 50 mM sodium chloride (pH 2.7). The eluted fraction was adjusted at pH 5.5 by the addition of a 50 mM sodium phosphate solution (pH 7.0). The thus prepared solution of the soluble A33 protein outside the cell membrane was substituted with PBS using Amicon Ultra-15 (produced by Amicon) and then sterilization by filtration was performed using a membrane filter MILLEX-GV (produced by Millipore) with a pore size of 0.22 μm. A purified shA33EX-hFc protein was thus obtained. The concentration of the shA33EX-hFc protein was found by measurement of absorbance at 280 nm and calculation with 1.4 OD being equivalent to 1 mg/mL (antibody concentration).
Example 4
Production of Human-Antibody-Producing Mice
Mice used for immunization had genetic background such that they were homozygous for both disrupted endogenous Ig heavy chain and κ light chain. Furthermore, the mice simultaneously retained a chromosome 14 fragment (SC20) containing the human Ig heavy chain gene locus and a human Igκ chain transgene (KCo5). The mice had been produced by crossing mice of line A having the human Ig heavy chain gene locus with mice of line B having the human Igκ chain transgene. The mice of line A were homozygous for both disrupted endogenous Ig heavy chain and κ light chain and retained the inheritable chromosome 14 fragment (SC20), as described in the report of Tomizuka et al (Tomizuka. et al., Proc Natl Acad Sci U.S.A., 2000 Vol. 97: 722), for example. Furthermore, the mice of line B were homozygous for both disrupted endogenous Ig heavy chain and κ light chain. Furthermore, the mice of line B retaining the human Igκ chain transgene (KCo5) were transgenic mice, as described in the report of Fishwild et al (Nat Biotechnol, (1996) 114: 845).
Mice used in the following immunization experiments were obtained by crossing male mice of line A with female mice of line B or by crossing female mice of line A with male mice of line B, in the mouse sera of which the human Ig heavy chain and κ light chain were simultaneously detected (Ishida & Lonberg, IBC's 11th Antibody Engineering, Abstract 2000). In addition, the above human-antibody-producing mice (referred to as KM mice) are available from Kirin Brewery Co., LTD. by contract.
Example 5
Preparation of Human Monoclonal Antibody Against A33
Monoclonal antibodies were prepared in this example according to a general method described in “Tan-Clone-Kotai-Jikken-Manual (“Experimental Manual for Monoclonal Antibody”) (written by Tamie ANDO et al. and published by Kodansha Scientific, Ltd., Tokyo, Japan (1991)), for example. A33 used as an immunogen was the A33-expressing FM3A cell prepared in Example 1 or the shA33EX-hFc protein. Animals used for immunization were the human-antibody (immunoglobulin)-producing mice produced in Example 2.
For the purpose of preparing the anti-A33 human monoclonal antibody, A33-expressing FM3A cells (1×107 cells/mouse) prepared in Example 3 were mixed with an RIBI adjuvant (produced by Corixa Corporation), and then human-antibody-producing mice were initially immunized intraperitoneally with the mixture. Following the initial immunization, the animals were immunized with the same cells and the RIBI adjuvant every week for 8 times in total. 3 days before obtainment of the spleen described later, the shA33EX-hFc protein was administered at 20 μg/mouse via the caudal vein and recombinant human IL-6 was subcutaneously administered at 5 μg/mouse.
Furthermore, initial immunization was performed using the mixture of the shA33EX-hFc protein (10 μg/mouse) and a CpG adjuvant (produced by QIAGEN). Following the initial immunization, the same protein and the CpG adjuvant were used for immunization twice every 2 weeks. 2 weeks later, immunization was further performed using only such protein. 3 days before obtainment of the spleen described later, the shA33EX-hFc protein was administered at 10 μg/mouse via the caudal vein.
Furthermore, immunization was performed intraperitoneally with the shA33EX-hFc protein (10 μg/mouse), A33-expressing FM3A cells (5×106 cells/mouse), and the RIBI adjuvant, followed by 1 to 4 times of immunization every 2 weeks. 4 days before obtainment of the spleen described later, the shA33EX-hFc protein was administered intraperitoneally at 5 μg/mouse.
The spleen was surgically obtained from each of the thus immunized mice and then added to 10 mL of serum-free DMEM medium (produced by Gibco BRL; (hereinafter, referred to as serum-free DMEM medium) containing 350 mg/mL sodium hydrogencarbonate, 50 units/mL penicillin, and 50 μg/mL streptomycin. The resultant was strained through a strainer with a mesh (cell strainer: produced by Falcon) using a spatula. The cell suspension that had passed through the strainer was centrifuged, so as to precipitate the cells. The cells were washed twice with serum-free DMEM medium and then suspended in serum-free DMEM medium, followed by determination of cell count. In the meantime, myeloma cells SP2/0 (ATCC No. CRL-1581) were cultured in DMEM medium (produced by Gibco BRL) containing 10% FCS (produced by Sigma; hereinafter, referred to as serum-containing DMEM medium) at 37° C. in the presence of 5% carbonic acid gas, so as not to exceed the cell concentration of 1×106 cells/mL. The myeloma cells SP2/0 (ATCC No. CRL-1581) were similarly washed with serum-free DMEM medium and then suspended in serum-free DMEM medium, followed by determination of a cell count. The thus collected cell suspension was mixed with the mouse myeloma cell suspension at a 5:1 ratio in terms of cell count. After centrifugation, the supernatant was completely removed. 1 mL of 50% (w/v) polyethylene glycol 1500 (produced by Boehringer Mannheim) was slowly added as a fusion agent to the pellets while agitating the solution using the tip of a pipette. 1 mL of serum-free DMEM medium pre-heated at 37° C. was slowly added in twice and then 7 mL of serum-free DMEM medium was further added to the resultant. After centrifugation, fusion cells obtained by removal of the supernatant were subjected to screening using a limiting dilution method as described below. Hybridoma selection was performed by culturing the cells in DMEM medium containing 10% FCS, IL-6 (10 ng/mL) (or 10% hybridoma cloning factor (hereinafter, referred to as HCF; produced by B10BASE)), hypoxanthine (H), aminopterin (A), and thymidine (T) (hereinafter, referred to as HAT; produced by Sigma). Furthermore, single clones were obtained by a limiting dilution method using DMEM medium containing HT (produced by Sigma), 10% FCS, and IL-6 (or 10% HCF). Culture was performed in a 96-well microtiter plate (produced by Becton, Dickinson and Company). Selection (screening) of hybridoma clones producing the anti-A33 human monoclonal antibody and characterization of the human monoclonal antibody produced by each hybridoma were performed through measurement by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FMC), as described later.
Many hybridomas producing the human monoclonal antibody having the human immunoglobulin γ chain (hlgγ) and the human immunoglobulin light chain κ and having reactivity specific to A33 were obtained as confirmed by Cell ELISA, protein ELISA, and FMC analyses described in Example 6. In addition, each hybridoma clone producing the human anti-A33 monoclonal antibody of the present invention was named using numbers and letters in all the following examples, including this example, and in all tables or figures showing the test results in the examples. Furthermore, numbers and letters with “antibody” attached to one end thereof represent antibodies that are produced by the relevant hybridomas or recombinant antibodies produced by host cells retaining antibody genes (full-length or a variable region) isolated from the relevant hybridoma. Furthermore, within a contextually clear range, the name of a hybridoma clone may represent the name of an antibody. The following hybridoma clones represent single clones: 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA. 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were deposited on Aug. 24, 2004, with the International Patent Organism Depositary (IPOD) (Central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan) at the National Institute of Advanced Industrial Science and Technology (AIST) under accession Nos. FERM BP-10107 (denotation for identification: M10), FERM BP-10106 (denotation for identification: M165), FERM BP-10108 (denotation for identification: M96), FERM BP-10109 (denotation for identification: N26), FERM BP-10104 (denotation for identification: Q47), FERM BP-10105 (denotation for identification: Q54), and FERM ABP-10103 (denotation for identification: R5), respectively.
Example 6
Selection of Clones Producing Human Anti-A33 Monoclonal Antibody Having the Human Immunoglobulin γ Chain (hIgγ) and the Human Immunoglobulin Light Chain κ (Igκ)
Cell ELISA was performed as follows. FM3A/A33 prepared in Example 3 was added at 1×105 per well to a 96-well plate (produced by Falcon). Hybridoma supernatant was added and then incubation was performed at 4° C. for 30 minutes. Subsequently, the resultants were washed twice with PBS containing 2% FCS and then a goat anti-human IgG F(ab′)2 antibody labeled with horseradish peroxidase (50 μg/well: produced by IBL) was added, followed by 30 minutes of incubation at 4° C. The resultants were washed twice with PBS containing 2% FCS and then 100 μL of a TMB color development substrate (produced by DAKO) was added to each well, followed by 20 minutes of incubation at room temperature. 0.5 M sulfuric acid (100 μL/well) was added to each well to stop the reaction. Absorbance at a wavelength of 450 nm (reference wavelength: 570 nm) was measured using a microplate reader (1420 ARVO multilabel counter: produced by WALLAC). Antibody-producing clones showing positive reactions were selected. At this time, FM3A cells expressing no A33 antigen were used as negative controls. Specifically, culture supernatants that had reacted with FM3A/A33 cells but not with FM3A cells were selected as antibody-producing clones that had shown positive reactions.
Furthermore, protein ELISA was performed as follows. 50 μl of the shA33EX-hFc protein prepared in Example 3 and adjusted at pH 9.4 using a 1 μg/ml carbonate buffer was added to each well of a 96-well microplate for ELISA (Maxisorp produced by Nunc). Incubation was performed at room temperature for 1 hour or 4° C. overnight, so that the shA33EX-hFc protein was adsorbed to the microplate. Subsequently, the supernatant was discarded, and PBS containing 10% FCS was added to each well, followed by 1 hour of incubation at 37° C. Thus, sites where no shA33EX-hFc protein had bound were blocked. In this manner, the microplate was prepared, wherein each well had been coated with the shA33EX-hFc protein. The culture supernatant (50 μl) of each hybridoma was added to each well, followed by 1 hour of reaction at room temperature. Each well was washed twice with PBS (PBS-T) containing 0.1% Tween20. Subsequently, a sheep anti-human Igκ antibody (50 μl/well, produced by The Binding Site) labeled with horseradish peroxidase was diluted 2500 fold using PBS (PBS-T) containing 0.1% Tween20. 50 μl of the solution was added to each well, followed by 1 hour of incubation at 37° C. The microplate was washed three times with PBS-T. 100 μl of a TMB color development substrate solution (produced by DAKO) was added to each well, followed by 20 minutes of incubation at room temperature. 0.5 M sulfuric acid (100 μl/well) was added to each well so as to stop the reaction. Absorbance at a wavelength of 450 nm (reference wavelength: 570 nm) was measured using a microplate reader (VersaMax produced by Molecular Devices Corporation). Antibody-producing clones showing positive reactions were selected.
Furthermore, FMC was performed as follows. The reactivity of hybridoma culture supernatants against human colorectal cancer cell line COLO205 cells expressing the A33 antigen was examined. The COLO205 cell line was suspended at a concentration of 2×106/ml in a PBS staining buffer (SB) containing 0.1% NaN3 and 2% FCS. The cell suspension (501l/well) was dispensed in a 96-well round bottom plate (produced by Becton, Dickinson and Company). The culture supernatant (50 μl) of each hybridoma was added, followed by 30 minutes of incubation on ice. A negative control was prepared depending on each subclass. Specifically, a human IgG1 antibody (produced by Sigma) was adjusted at a concentration of 2 μg/ml with hybridoma culture medium and then 50 μl of the solution was added, followed by 30 minutes of incubation on ice. The resultant was washed twice with SB and then 50 μl of RPE fluorescence-labeled goat anti-human IgG F(ab′)2 antibody (produced by Southern Biotech) was added, followed by 30 minutes of incubation on ice. The resultant was washed once with SB and it was then suspended in 300 μl of a FACS buffer. The mean fluorescence intensity of each cell line was measured by FACS (FACS caliber produced by Becton, Dickinson and Company). As a result, the presence of antibodies binding to A33 expressed on cells was demonstrated because of their strong activity of binding to the cells of the COLO205 cell line.
Example 7
Identification of Each Monoclonal Antibody Subclass in the Culture Supernatant
50 μl of the shA33EX-hFc protein prepared using a 1 μg/ml carbonate buffer (hereinafter, referred to as PBS) was added to each well of a 96-well microplate for ELISA (Maxisorp produced by Nunc). Incubation was performed at room temperature for 1 hour or 4° C. overnight, so that the shA33EX-hFc protein was adsorbed to the microplate. Subsequently, the supernatant was discarded, and PBS containing 10% FCS was added to each well, followed by 1 hour of incubation at room temperature or at 4° C. overnight. Thus, sites where no shA33EX-hFc protein had bound were blocked. In this manner, the microplate was prepared, with each well having been coated with the shA33EX-hFc protein. Subsequently, the microplate was washed twice with PBS (PBS-T) containing 0.1% Tween20. A sheep anti-human IgG1 antibody labeled with horseradish peroxidase, a sheep anti-human IgG2 antibody labeled with horseradish peroxidase, a sheep anti-human IgG3 antibody labeled with horseradish peroxidase, or a sheep anti-human IgG4 antibody labeled with horseradish peroxidase (diluted 1600-, 6400-, 25000-, and 25000-fold, respectively and produced by The Binding Site) was added at 50 μL/well. Incubation was performed for 1.5 hours at room temperature. The microplate was washed three times with PBS-T containing 0.1% Tween20. A substrate buffer (TMB produced by DAKO) was added at 100 μL/well, followed by 20 minutes of incubation at room temperature. Subsequently, 0.5 M sulfuric acid (100 μl/well) was added to stop the reaction. Absorbance at a wavelength of 450 nm (reference wavelength: 570 nm) was measured using a microplate reader (VersaMax produced by Molecular Devices Corporation). The subclass of each clone was determined. Only human anti-A33 antibodies of subclass IgG1 were selected because a high priority was placed on ADCC and CDC.
Table 1 shows the reactivity of only the finally selected clones.











TABLE 1







Antibody name
Subclass
Reactivity to COLO205









Anti-DNP-IgG1
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-A33 monoclonal antibodies obtained from the culture supernatants of hybridomas as described in Example 6 were purified by the following methods. A culture supernatant containing each type of human anti-A33 monoclonal antibody was 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), PBS as an adsorption buffer, and a 0.02 M glycine buffer (pH 3.6) as an elution buffer. The eluted fraction was adjusted at around pH 7.2 by the addition of 1M Tris (pH 8.0). The thus prepared antibody solution was substituted 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) with a pore size of 0.22 μm. Thus, purified human anti-A33 monoclonal antibodies were obtained. The concentration of each purified antibody was obtained by measurement of absorbance at 280 nm and calculation with 1.4 OD being equivalent to 1 mg/mL (antibody concentration).
Example 9
Test of the Reactivity of each Type of Purified Monoclonal Antibody to A33-Expressing Cells
The reactivity of each type of purified monoclonal antibody obtained in Example 8 to A33-antigen-expressing 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) was examined by FCM. Human colorectal cancer cell line HT-29 cells (ATCC No. HTB-38) expressing no A33 antigen were also examined as negative control cells. Each cell line was suspended at a concentration of 2×106/ml in a PBS Staining Buffer (SB) containing 0.1% NaN3 and 2% FCS. The cell suspension (50 μl/well) was dispensed in a 96-well round bottom plate (produced by Becton, Dickinson and Company). 50 μl of each type of purified monoclonal antibody (adjusted using SB at a concentration of 2000, 400, 80, or 16 ng/ml) was added, and then incubation was performed on ice for 30 minutes. A negative control was prepared depending on each subclass. Specifically, a human IgG1 antibody (produced by Sigma) was adjusted using SB at a concentration of 2000, 400, 80, or 16 ng/ml. 50 μl of the solution was added, followed by 30 minutes of incubation on ice. The resultant was washed twice with SB and then 50 μl of an FITC fluorescence-labeled goat anti-human IgG F(ab′)2 antibody (produced by Southern Biotech), followed by 30 minutes of incubation on ice. The resultant was washed once with SB and then the resultant was suspended in 300 μl of a FACS buffer. The mean fluorescence intensity of each cell line was measured by FACS (FACScan produced by Becton, Dickinson and Company).
Table 2 shows the results. 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 an antibody concentration required to reach such value was 10<=x<100 ng/ml, the reactivity was represented by +++, when the same was 100<=x<1000 ng/ml, the reactivity was represented by ++, and when the same was 1000<=x<10000 ng/ml, the reactivity was represented by +. 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.












TABLE 2







Antibody name
COLO205
LoVo
LS174T
NCI-H508
HT-29





Anti-DNP-IgG1
−
−
−
−
−


cA33
+++
+++
++
++
−


263A17
++
++
++
++
−


125M10AA
+++
+++
+++
+++
−


125M165DAAA
+++
+++
++
++
−


125M96ABA
+++
+++
++
++
−


125N26F6AA
+++
+++
++
++
−


125Q47BA
+++
+++
++
++
−


125Q54AAAA
+++
+++
++
++
−


125R5AAAA
+++
+++
++
++
−



Half value of mean
Half value of mean
Half value of mean
Half value of mean



fluorescence intensity 90
fluorescence intensity 25
fluorescence intensity 125
fluorescence intensity 125



10<=x<100 ng/ml; +++
10<=x<100 ng/ml; +++
10<=x<100 ng/ml; +++
10<=x<100 ng/ml; +++



100<=x<1000 ng/ml; ++
100<=x<1000 ng/ml; ++
100<=x<1000 ng/ml; ++
100<=x<1000 ng/ml; ++



1000<=x<10000 ng/ml; +
1000<=x<10000 ng/ml; +
1000<=x<10000 ng/ml; +
1000<=x<10000 ng/ml; +



No binding; −
No binding; −
No binding; −
No binding; −









Example 10
Competition Test Regarding Each Type of Purified Monoclonal Antibody and the Mouse Anti-A33 Antibody
Whether or not each type of purified monoclonal antibody obtained in Example 8 recognizes an epitope similar to that of the mouse anti-A33 antibody was examined by a competition test using FCM. The COLO205 cell line was suspended at a concentration of 2×106/ml in a PBS Staining Buffer (SB) containing 0.1% NaN3 and 2% FCS. The cell suspension (50 μl/well) was dispensed in a 96-well round bottom plate (produced by Becton, Dickinson and Company). The purified mouse anti-A33 antibody prepared in Example 1 was not added or added at a concentration of 100 μg/ml to the wells containing each type of purified monoclonal antibody (1 μg/ml and 50 μl), followed by 30 minutes of incubation on ice. A mouse negative control (containing no purified anti-A33 antibody added thereto) was prepared as follows. A human IgG1 antibody (produced by Sigma) was adjusted at a concentration of 1 μg/ml with SB and then 50 μl of the solution was added, followed by 30 minutes of incubation on ice. The resultant was washed twice with SB and then 50 μl of FITC fluorescence-labeled goat anti-human IgG F(ab′)2 antibody (produced by IBL) was added, followed by 30 minutes of incubation on ice. The resultant was washed once with SB and then the resultant was suspended in 300 μl of a FACS buffer. The mean fluorescence intensity of each cell line was measured by FACS (FACScan produced by Becton, Dickinson and Company). The inhibition (%) between each type of purified monoclonal antibody and the mouse A33 antibody was calculated by the following formula:

Inhibition (%)={100−(100×mean fluorescence intensity after preincubation with mouse A33 antibody)/(mean fluorescence intensity after preincubation without mouse A33 antibody)}.

A purified monoclonal antibody showing inhibition (%) of 25% or less was classified as a “non-blocker,” one showing inhibition (%) of 25% or more and less than 90% was classified as a “partial blocker,” and one showing inhibition (%) of 90% or more was classified as a “blocker.” As a result, 263A17, 125M10AA, and 125M96ABA were classified as “non-blockers,” 125M165DAAA and 125N26F6AA were classified as “blockers,” and 125Q47BA, 125Q54AAAA, and 125R5AAAA were classified as “partial blockers.” Table 3 shows the results.











TABLE 3







Antibody name
Inhibition (%)
Classification




















cA33
94.2
Blocker



263A17
3.7
Non-blocker



125M10AA
5.0
Non-blocker



125M165DAAA
96.0
Blocker



125M96ABA
22.8
Non-blocker



125N26F6AA
98.0
Blocker



125Q47BA
64.1
Partial blocker



125Q54AAAA
46.5
Partial blocker



125R5AAAA
63.9
Partial blocker










Example 11
Method for Obtaining Normal Human Mononuclear Leukocytes
First, normal human peripheral blood mononuclear cells were prepared using Ficoll (Ficoll-PaquePLUS: produced by Amersham Pharmacia Biotech) according to a standard method. Normal human blood was collected in a blood collection bag (produced by TERUMO) containing a sodium citrate solution as an anticoagulant. The normal human blood was multi-layered in Ficoll and then specific gravity centrifugation (800G, room temperature, and 15 minutes) was performed to separate monocytes. The intermediate layer was extracted in the form of mononuclear leukocytes, and the extracted mononuclear leukocytes were then diluted with PBS. The diluted solution was repeatedly centrifuged at 200 G for 10 minutes three times, thereby removing blood platelets remaining in the supernatant. Normal human peripheral blood mononuclear cells (hereinafter referred to as PBMC) were obtained by the above method and then used as PBMC for Example 12. Furthermore, CD4+ T cells were isolated from PBMC using a CD4+ T cell Isolation kit II (produced by Miltenyi Biotec) according to the instructions included therein. The remaining cell group was also used as PBMC for Example 12.
Example 12
Test of the Cytotoxicity of Each Type of Purified Monoclonal Antibody
Antibody-mediated cytotoxicity was determined as follows. Cytotoxicity (antibody-dependent cellular cytotoxicity, hereinafter referred to as ADCC) against target cells was determined in the presence of cells having killer activity, such as NK cells or neutrophils, and antibodies. Furthermore, cytotoxicity (complement-dependent cytotoxicity, hereinafter referred to as CDC) against target cells was determined in the presence of complements and antibodies. The antibodies used herein were: each type of purified monoclonal antibody prepared in Example 8 and a cA33 recombinant antibody as a control anti-A33 antibody. Furthermore, an anti-DNP IgG1 antibody was used as a negative control.
The method is simply described as follows. Target cells were caused to incorporate radioactive chromium (51Cr) into the cytoplasms and γ-dose was measured to find the amounts of 51Cr that are released in culture solutions because of cell death.
Specifically, 106 cells of colorectal cancer cell line COLO205 (ATCC No. CCL-86) and 106NCI-H508 cells (ATCC No. CCL-253) were separately suspended as target cells in 15 μL of fetal calf serum (FCS). 50 μL (37 MBq/mL) of 51Cr-labeled sodium chromate (produced by PerkinElmer; hereinafter referred to as 51Cr) was added, followed by 1 hour of culture at 37° C. Next, 10 mL of medium was added. Discarding of medium by centrifugation was repeated 3 times, thereby eliminating 51Cr that had not been incorporated within the cells.
In the ADCC assay, 5,000 51Cr-labeled target cells and 500,000 healthy human peripheral blood mononuclear leukocytes obtained by the method described in Example 11 were cultured in a V-bottom 96-well plate (produced by Coaster) at a total volume of 200 μL with antibody having each concentration at 37° C. in the presence of 5% CO2 for 4 hours.
In the CDC assay, 5000 51Cr-labeled target cells and human serum-derived complements (produced by Sigma) at a final concentration of 5% were cultured in a V-bottom 96-well plate at a total volume of 200 μL with antibody having each concentration at 37° C. in the presence of 5% CO2 for 4 hours.
In both ADCC and CDC assays, the plate was subjected to centrifugation after culture to cause the cells to precipitate. Each type of purified monoclonal antibody was prepared at a concentration of 0.4-500 ng/ml and then 50 μL of the solution was transferred to a 96-well plate (LumaplateTM-96; produced by Packard Instrument) including a powder scintillator. The resultant was dried at 55° C. for 1.5 hours. After confirming that the plate had dried, the plate was covered with a special cover (TopSealTM-A; 96-well microplates; produced by Packard Instrument). The γ-ray dose was measured with a scintillation counter (TopCount; produced by Packard Instrument).
FIG. 1A to FIG. 1D and Table 4 show the results. In the case of ADCC against COLO205 cells, the half value of the specific lysis (%) was determined to be 15%. When an antibody concentration required to reach such value was 1<=x<10 ng/ml, the cytotoxicity was represented by +++, when the same was 10<=x<100 ng/ml, the cytotoxicity was represented by ++, when the same was 100<=x<1000 ng/ml, the cytotoxicity was represented by +, and when no specific lysis (%) was obtained, the cytotoxicity was represented by −. In the case of ADCC against NCI-H508 cells, the half value of the specific lysis (%) was determined to be 15%. When an antibody concentration required to reach such value was 1<=x<10 ng/ml, the cytotoxicity was represented by +++, when the same was 10<=x<100 ng/ml, the cytotoxicity was represented by ++, when the same was 100<=x<1000 ng/ml, the cytotoxicity was represented by +, and when no specific lysis (%) was observed, the cytotoxicity was represented by −.
In the case of CDC against COLO205 cells, the half value of the specific lysis (%) was determined to be 10%. When an antibody concentration required to reach such value was 10<=x<100 ng/ml, the cytotoxicity was represented by +++, when the same was 100<=x<1000 ng/ml, the activity was represented by ++, when the same required to reach such value was x>=1000 ng/ml, the activity was represented by +, and when no specific lysis (%) was obtained, the activity was represented by −. Furthermore, in the case of CDC against NCI-H508 cells, the half value of the specific lysis (%) was determined to be 25%. When an antibody concentration required to reach such value was 10<=x<100 ng/ml, the cytotoxicity was represented by +++, when the same was 100<=x<1000 ng/ml, the cytotoxicity was represented by ++, when the same was x>=1000 ng/ml, the cytotoxicity was represented by +, and when no specific lysis (%) was obtained, the cytotoxicity was represented by −.
In the case of ADCC, cA33 and 125Q54AAAA showed high cytotoxicity, while in the case of CDC, 125M10AA showed high cytotoxicity.









TABLE 4







Cell name
COLO205
NCI-H508











Antibody name
ADCC
CDC
ADCC
CDC





Anti-DNP-IgG1
−
−
−
−


cA33
+++
−
+++
−


263A17
++
++
+++
++


125M10AA
++
+++
+++
+++


125M165DAAA
++
+
+++
++


125M96ABA
+
++
++
+++


125N26F6AA
++
−
++
+


125Q47BA
++
++
++
++


125Q54AAAA
+++
++
+++
++


125R5AAAA
++
++
+++
++



Half value of specific
Half value of specific
Half value of specific
Half value of specific



lysis (%) 15%
lysis (%) 10%
lysis (%) 15%
lysis (%) 25%



1<=x<10 ng/ml; +++
10<=x<100 ng/ml; +++
1<=x<10 ng/ml; +++
10<=x<100 ng/ml; +++



10<=x<100 ng/ml; ++
100<=x<1000 ng/ml; ++
10<x<100 ng/ml; ++
100<x<1000 ng/ml; ++



100<=x<1000 ng/ml; +
x>=1000 ng/ml; +
100<=x<1000 ng/ml; +
x>=1000 ng/ml; +



No specific lysis; −
No specific lysis; −
No specific lysis; −
No specific lysis; −









Example 13
Preparation of the Gene Encoding Each Type of Monoclonal Antibody
(1) cDNA Synthesis for Each Type of Monoclonal Antibody
Hybridomas 263A17, 125M10AA, 125M165DAAA, 125M96ABA, 125N26F6AA, 125Q47BA, 125Q54AAAA, and 125R5AAAA were separately cultured in DMEM medium (produced by Gibco BRL) containing 10 ng/mL IL-6 or 10% HCF (produced by B10BASE) and 10% Fetal Bovine Serum (produced by HyClone). After the cells were harvested by centrifugation, ISOGEN (produced by NIPPON GENE) was added and then total RNAs were extracted according to the relevant instruction manual. Variable regions of antibody cDNAs were cloned using a SMART RACE cDNA amplification kit (produced by Becton Dickinson Bioscience Clontech) according to instructions included therein.
1st strand cDNA was prepared using 5 μg of total RNA as a template.
1st Strand cDNA Synthesis

















Total RNA
5 μg/3 μl




5′CDS
1 μl



SMART oligo
1 μl










A reaction solution with the above composition was incubated at 70° C. for 2 minutes, and then the following components were added, followed by 1.5 hours of incubation at 42° C.
















5 × Buffer
2 μl



DTT
1 μl



DNTP mix
1 μl



PowerScript Reverse Transcriptase
1 μl










Furthermore, 100 μl of a tricine buffer was added and then incubation was performed at 72° C. for 7 minutes, thereby obtaining 1st strand cDNA.
(2) PCR Amplification of Heavy Chain and Light Chain Genes and Confirmation of the Nucleotide Sequences
(2)-1: PCR Amplification of the Heavy Chain and Light Chain Genes of Hybridoma 263A17
cDNA amplification was performed by preparing the following reaction solution using KOD-Plus-DNA polymerase (produced by TOYOBO).

















Sterile H2O
29.5 μl




cDNA
2.5 μl



KOD-Plus- buffer (10X)
5 μl



dNTP Mix (2 mM)
4 μl



MgSO4 (25 mM)
2 μl



KOD-Plus- (1 unit/μl)
1 μl



Universal primer A mix (UPM) (10X)
5 μl



Gene specific primers (GSP)
1 μl



Total volume
50 μl










The reaction solution with the above composition was adjusted to a final volume of 50 μl with re-distilled water and then subjected to PCR.
The 263A17 heavy chain gene was amplified by repeating a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) 30 times using the UPM primer included in a SMART RACE cDNA Amplification Kit and a IgG1p primer (5′-TCTTGTCCACCTTGGTGTTGCTGGGCTTGTG-3′) (SEQ ID NO: 16). Meanwhile, the 263A17 light chain gene was amplified by repeating a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) 30 times using the UPM primer and an hk-2 (5′-GTT GAA GCT CTT TGT GAC GGG CGA GC-3′) (SEQ ID NO: 17) primer.
(2)-2: PCR Amplification of Heavy Chain and Light Chain Genes of Hybridomas 125M10AA, 125M96ABA, 125Q47BA, 125Q54AAAA, and 125R5AAAA
Reaction conditions employed herein were similar to those in (2)-1. The 125M10AA, 125M96ABA, 125Q47BA, 125Q54AAAA, and 125R5AAAA heavy chain genes were amplified by repeating a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) 30 times using the UPM primer and the IgG1p primer (SEQ ID NO: 16). Furthermore, a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) was repeated 20 times using 1 μl of the reaction solution as a template, a NUPM primer (SMART RACE cDNA amplification Kit; produced by Becton Dickinson Bioscience Clontech), and a IgG2p/G134 primer (5′-TGC ACG CCG CTG GTC AGG GCG CCT GAG TTC C-3′) (SEQ ID NO: 18). Meanwhile, the 125M10AA, 125M96ABA, 125Q47BA, 125Q54AAAA, and 125R5AAAA light chain genes were amplified by repeating a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) 30 times using the UPM primer and the hk-2 primer (SEQ ID NO: 17). Furthermore, a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) was repeated 20 times using 1 μl of the reaction solution as a template, the NUPM primer, and an 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 Chain and Light Chain Genes of Hybridomas 125M165DAAA and 125N26F6AA
Reaction conditions employed herein were similar to those in (2)-1. 125M165DAAA and 125N26F6AA heavy chain genes were amplified by repeating a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) 30 times using a UPM primer and an hh-2 primer (5′-GCT GGA GGG CAC GGT CAC CAC GCT G-3′) (SEQ ID NO: 20). Furthermore, a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) was repeated 20 times using 1 μl of the reaction solution as a template, a NUPM primer, and an 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 a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) 30 times using the UPM primer and the hk-2 primer (SEQ ID NO: 17). Furthermore, a reaction cycle (98° C. for 1 second and 68° C. for 30 seconds) was repeated 20 times using 1 μl of the reaction solution as a template, an NUPM primer, and the hk-5 primer (SEQ ID NO: 19).
PCR fragments of the heavy and light chains amplified as described above were separately harvested by ethanol precipitation, harvested by agarose gel electrophoresis, and then purified using a QIAquick Gel Extraction Kit (produced by QIAGEN), which is a DNA purification kit that uses membranes. The thus purified HV and LV amplified fragments were separately subcloned into a PCR 4 Blunt-TOPO vector of a Zero Blunt TOPO PCR Cloning Kit (produced by Invitrogen). For the plasmid DNAs of the thus obtained clones, the nucleotide sequences of the insert DNAs were analyzed. M13FW (SEQ ID NO: 3) and M13RV (SEQ ID NO: 4) were used as primers for determination of the DNA nucleotide sequences.
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
DSVKGRFTIS
RDNSKNTLYL
QMNSLRAEDT
AVYYCAKDRI


 


130
140
150
160
170
180


VGATNYYYGM
DVWGQGTTVT
VSSAS. . .


 







<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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, the variable region in the 263A17 antibody light chain has the nucleic acid sequence (SEQ ID NO: 24) ranging from guanine (G) at position 67 to adenine (A) at position 387. Further, 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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, 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
GGCCAGGGCA
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
FILTISSLEP
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. Furthermore, 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.
Accordingly, 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 ID 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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, the variable region in the 125M165DAAA 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 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.








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













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


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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, 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. Further, 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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, 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. Further, 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
TDFTLTISSL
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. Furthermore, 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.
Accordingly, 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. Furthermore, 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 125Q54AAAA 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. Further, 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. Furthermore, 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.
Accordingly, 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. Furthermore, 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.
Accordingly, 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. Further, 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 Full-length Sequences Containing Constant Regions of Human Antibody Heavy Chain and Light Chain Genes Expressed by Hybridomas 125N26F6AA and 125M10AA
The DNA nucleotide sequence and the amino acid sequence of the antibody variable region of each antibody were determined in Example 13. For the hybridomas 125N26F6AA and 125M10AA derived from KM mice, full-length sequences containing constant regions were analyzed. cDNA synthesis was performed according to Example 13 using as materials total RNAs prepared from the hybridomas 125N26F6AA and 125M10AA and a SMART RACE cDNA amplification Kit (produced by Becton Dickinson Bioscience Clontech).
1st Strand cDNA Synthesis

















Total RNA
5 μg/3 μl




3′CDS primer
1 μl



H2O
1 μl










A reaction solution with the above composition was incubated at 70° C. for 2 minutes and then the following components were added, followed by 1.5 hours of incubation at 42° C.
















5 × Buffer
2 μl



DTT
1 μl



dNTP mix
1 μl



PowerScript Reverse Transcriptase
1 μl










Furthermore, 50 μl of a tricine buffer was added and then incubation was performed at 72° C. for 7 minutes, thereby obtaining 1st strand cDNA.
To obtain a DNA containing the entire coding region of a constant region, a PCR amplification reaction was performed using the above synthesized cDNA as a template and a primer set of: a synthetic DNA (5′ primer) having a sequence binding around the ATG initiation codon at the 5′ end of each antibody gene; and a synthetic DNA (3′ primer) specifically binding to the 3′ non-translation region of a human antibody gene. As a result of the amplification reaction, the full-length sequence of an antibody gene (cDNA) ranging from the ATG initiation codon to the 3′ non-translation region, including the stop codon, could be obtained.
The 125N26F6AA heavy chain DNA was amplified by repeating an incubation cycle (94° C. for 15 seconds and 68° C. for 2 minutes) 35 times using a primer set of an H chain 5′ primer: N26H5Sal1 (SEQ ID NO: 58) and an H chain 3′ primer: H—3UTR1848 (5′-CGGGGTACGTGCCAAGCATCCTCGTG-3′, SEQ ID NO: 74) or a primer set of the H chain 5′ primer: N26H5Sal1 (SEQ ID NO: 58) and an H chain 3′ primer: H—3UTR1875 (5′-ATGCTGGGCGCCCGGGAAGTATGTAC-3′, SEQ ID NO: 75). Meanwhile, the 125N26F6AA light chain (κ) was amplified using a primer set of an L chain 5′ primer: N26KA10 Minor L Bgl (SEQ ID NO: 64) and an L chain 3′ primer: L—3UTR—823 (5′-GAAAGATGAGCTGGAGGACCGCAATA-3′, SEQ ID NO: 76). Except for primers, amplification was performed with a reaction solution having the same composition as that employed in Example 13 (2)-1.
For amplification of the 125M10AA heavy chain DNA, a primer set of an H chain 5′ primer: M10H5Sal (SEQ ID NO: 70) and the H chain 3′ primer: H—3UTR1848 (SEQ ID NO: 74) or a primer set of the H chain 5′ primer: M10H5Sal (SEQ ID NO: 70) and the H chain 3′ primer: H—3UTR1875 (SEQ ID NO: 75) was used. For amplification of the 125M10AA light chain (K) DNA, a primer set of an L chain 5′ primer: M10KBgl (SEQ ID NO: 66) and the L chain 3′ primer: L—3UTR—823 (SEQ ID NO: 76) were used.
The amplified PCR fragments were separately harvested by ethanol precipitation, harvested by agarose gel electrophoresis, and then purified using a QIAquick Gel Extraction Kit (produced by QIAGEN). The thus purified amplified fragments were separately subcloned into a PCR 4 Blunt-TOPO vector of a Zero Blunt TOPO PCR Cloning Kit (produced by Invitrogen). For the thus obtained clones, sequencing template DNAs were prepared using a TempliPhi DNA Amplification Kit (produced by Amersham Biosciences) including reagents for preparation of sequencing template DNA according to the protocols included therein. Thus, the nucleotide sequences of the insert DNAs were determined. Primers used for analyses of the DNA nucleotide sequences of the human antibody heavy chains were M13FW (SEQ ID NO: 3), M13RV (SEQ ID NO: 4), hh4 (SEQ ID NO: 21), hhl (5′-CCAAGGGCCCATCGGTCTTCCCCCTGGCAC-3′) (SEQ ID NO: 77), CMVH903F (5′-GACACCCTCATGATCTCCCGGACC-3′) (SEQ ID NO: 78), CMVHR1303 (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 analyses of the DNA nucleotide sequences of the human antibody light chains (κ) 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 125N26F6AA antibody heavy chain region and the entire light chain region and the amino acid sequences of the entire heavy chain region and the entire light chain region are each 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
REAKVQWKVD
NALQSGNSQE


 


190
200
210
220
230


SVTEQDSKDS
TYSLSSTLTL
SKADYEKHKV
YACEVTHQGL
SSPVTKSFNR
GEC






DNAs encoding the entire 125M10AA antibody heavy chain region and the entire light chain region and the amino acid sequences of the entire heavy chain region and the entire light chain region are each 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
LKSRVTISVD
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
LMISRTPEVT
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 Expression of Recombinant Antibodies
Vectors for expression of 263A17, 125M10AA, 125M165DAAA, 125N26F6AA, and 125Q54AAAA recombinant antibodies were constructed.
For the 263A17, 125M165DAAA, or 125N26F6AA antibody, a plasmid DNA containing the HV chain of each type of obtained antibody was used as a template. Primers used herein were designed to add restriction enzyme sites (Sal I on the 5′ terminus and Nhe I on the 3′ terminus) to the termini for ligation. Specifically, the primers used herein are as follows.
263A17:
5′ primer for HV chain: A33 2-6A2H VH3-23 Sal I (SEQ ID NO: 54)
5′-GCG ACT AAG TCG ACC ATG GAG TTT GGG CTG AGC TG-3′
3′ primer for HV chain: A33 2-6A2H VH3-23 Nhe I (SEQ ID NO: 55)
5′-TGG GCC CTT GGT GCT AGC TGA GGA GAC GGT GAC CG-3′
125M165DAAA:
5′ primer for HV chain: M165H5Sal (SEQ ID NO: 56)
5′-AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGG TTT-3′
3′primer for HV chain: M165H3Nhe (SEQ ID NO: 57)
5′-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TGC-3′
125N26F6AA:
5′ primer for HV chain: N26H5Sal1 (SEQ ID NO: 58)
5′-AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGG TTT-3′
3′ primer for HV chain: N26H3Nhe1 (SEQ ID NO: 59)
5′-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC CC-3′
The HV of each type of A33 antibody was amplified by PCR (94° C. for 3 minutes 94° C. for 10 seconds and 68° C. for 45 seconds (35 cycles) 72° C. for 7 minutes). The amplified DNA fragment was digested with Sal I and Nhe I. The digested fragment was then introduced into an N5KG1-Val Lark vector (IDEC Pharmaceuticals, a modified vector of N5KG1 (U.S. Pat. No. 6,001,358)) that had been cleaved with the same enzymes. It was confirmed by sequencing with the use of the vector as a template that the inserted sequence was identical to the nucleotide sequence of the subcloned HV as determined by DNA nucleotide sequence analysis.
Subsequently, LV was inserted into the thus obtained plasmid vector into which the HV had been inserted. A plasmid DNA containing the LV chain of each type of obtained antibody was used as a template. Primers used herein were designed to add restriction enzyme sites (Bgl II on the 5′ terminus and BsiW I on the 3′ terminus) to the termini for ligation. Specifically, primers used herein are as follows.
263A17:
5′ primer for LV chain: A33 2-6A2 K L19 Bgl II (SEQ ID NO: 60)
5′-ATC ACA GAT CTC TCA CCA TGG ACA TGA GGG TCC CC-3′
3′ primer for LV chain: A33 2-6A2 K L19 BsiW I (SEQ ID NO: 61)
5′-ACA GAT GGT GCA GCC ACC GTA CGT TTAATC TCC AG-3′
125M165DAAA:
5′ primer for LV chain: M165K5L6Bgl2 (SEQ ID NO: 62)
5′-AGA GAG AGA GAG ATC TCA CCA TGG AAG CCC CAG CTC AGC TTC TCT-3′
3′ primer for LV chain: M165K3L6BsiW1 (SEQ ID NO: 63)
5′-AGA GAG AGA GCG TAC GTT TGA TTT CCA CCT TGG TCC CTT GGC-3′
125N26F6AA:
5′ primer for LV chain: N26KA10 Minor L Bgl (SEQ ID NO: 64)
5′-AGA GAG AGA GAT CTC TCA CCA TGT CGC CAT CAC AAC TCA TTG GG-3′
3′ primer for LV chain: N26KA10 Minor L Bsi (SEQ ID NO: 65)
5′-AGA GAG AGA GCG TAC GTT TGA TAT CCA CTT TGG TCC CAG GG-3′,
The LV of each type of A33 antibody was amplified by PCR (94° C. for 3 minutes→94° C. for 10 seconds and 68° C. for 45 seconds (35 cycles)→72° C. for 7 minutes). The amplified DNA fragment was digested with Bgl II and BsiW I. The digested fragment was then introduced into an N5KG1-HV vector that had been cleaved with the same enzymes. It was confirmed by sequencing with the use of the vector as a template that the inserted sequence was identical to the nucleotide sequence of the subcloned LV as determined by DNA nucleotide sequence analysis.
For the 125M10AA or 125Q54AAAA antibody, a plasmid DNA containing the LV chain of each type of obtained antibody was used as a template. Primers used herein were designed to add restriction enzyme sites (Bgl II on the 5′-terminus and BsiW I on the 3′ terminus) to the termini for ligation. Specifically, primers used herein are as follows.
125M10AA:
5′ primer for LV chain: M10KBgl (SEQ ID NO: 66)
5′-AGAGAGAGAGAGATCTCACCATGGAAGCCCCAGCTCAGCTTCTCT-3′
3′ primer for LV chain: M10KBsi (SEQ ID NO: 67)
5′-AGAGAGAGAGCGTACGTTTGATCTCCACCTTGGTCCCTCCG-3′
125Q54AAAA:
5′ primer for LV chain: Q54K5Bgl (SEQ ID NO: 68)
5′-AGAGAGAGAGAGATCTCACCATGGACATGAGGGTCCTCGCTCAGC-3′
3′ primer for LV chain: Q54K3Bsi (SEQ ID NO: 69)
5′-AGAGAGAGAGCGTACGTTTGATCTCCAGCTTGGTCCCCTGG-3′
The LV of each type of A33 antibody was amplified by PCR (94° C. for 3 minutes→94° C. for 10 seconds and 68° C. for 45 seconds (35 cycles)→72° C. for 7 minutes). The amplified DNA fragment was digested with Bgl II and BsiW I. The digested fragment was then introduced into a N5KG1-Val Lark vector that had been cleaved with the same enzymes. It was confirmed by sequencing with the use of the vector as a template that the inserted sequence was identical to the nucleotide sequence of the subcloned LV as determined by DNA nucleotide sequence analysis.
Subsequently, HV was inserted into the thus obtained plasmid vector into which the LV had been inserted. A plasmid DNA containing the HV chain of each type of obtained antibody was used as a template. Primers used herein were designed to add restriction enzyme sites (Sal I on the 5′ terminus and Nhe I on the 3′ terminus) to the termini for ligation. Specifically, primers used herein are as follows.
125 M10AA:
5′ primer for HV chain: M10H5Sal (SEQ ID NO: 70)
5′-AGA GAG AGA GGT CGA CCA CCA TGG ATC TCA TGT GCA AGA AAA TGA AGC-3′
3′ primer for HV chain: M10H3Nhe (SEQ ID NO: 71)
5′-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCG TGG TCC CT-3′
125 Q54AAAA:
5′ primer for HV chain: Q54H5Sal (SEQ ID NO: 72)
5′-AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGC TTT-3′
3′ primer for HV chain: Q54H3Nhe (SEQ ID NO: 73)
5′-AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC CC-3′
The HV of each type of A33 antibody was amplified by PCR (94° C. for 3 minutes→94° C. for 10 seconds and 68° C. for 45 seconds (35 cycles)→72° C. for 7 minutes). The amplified DNA fragment was digested with Sal I and Nhe I. The digested fragment was then introduced into an N5KG1-LV vector that had been cleaved with the same enzymes. It was confirmed by sequencing with the use of the vector as a template that the inserted sequence was identical to the nucleotide sequence of the subcloned HV as determined by DNA nucleotide sequence analysis.
Table 5 lists the nucleotide sequences of the synthetic DNAs. Table 6 lists the names of the recombinant vectors and the names of the antibodies produced.











TABLE 5





No
Primer name
Sequence (5′ to 3′)
Length
SEQ ID NO:



















1
GPAHvR3Nhe
GCC CTT GGT GCT AGC TGA AGA GAC GGT GAC CAG AGT CCC TTG
42
1


2
GPALvR3Bsi
GTG CAC GCC GCT GGT CAG GGC GCC TG
26
2


3
M13FW
GTA AAA CGA CGG CCA GTG
18
3


4
M13RV
CAG GAA ACA GCT ATG AC
17
4


5
GPAHv2F5Sal
AGA GAG AGG TCG ACC CAC CAT GAA CTT TGG GCT GAG CTT AGT T
43
5


6
GPALv2FBgl
AGA GAG AGA GAT CTC TCA CCA TGG GCA TCA AGA TGG AGT TTC AG
44
6


7
A33-F2
GCA GAC GAA TTC AAG ACC ATG GTG GGG AAG AT
32
13


8
A33-R1
CTC GAG CGG CCG CTC TGC TGC TGG CCT GTC ACT GGT CGA GGT G
43
14


9
GPA-EXCRR2
CTC GAG CGG CCG CCA GTT CAT GGA GGG AGA TCT GAC G
37
15


10
lgG1p
TCT TGT CCA CCT TGG TGT TGC TGG GCT TGT G
31
16


11
hk-2
GTT GAA GCT CTT TGT GAC GGG CGA GC
26
17


12
lgG2p/G134
TGC ACG CCG CTG GTC AGG GCG CCT GAG TTC C
31
18


13
hk-5
AGG CAC ACA ACA GAG GCA GTT CCA GAT TTC
30
19


14
hh-2
GCT GGA GGG CAC GGT CAC CAC GCT G
25
20


15
hh-4
GGT GCC AGG GGG AAG ACC GAT GG
23
21


16
A33 2-6A2 H
GCG ACT AAG TCG ACC ATG GAG TTT GGG CTG AGC TG
35
54



VH3-23 Sall


17
A33 2-6A2 H
TGG GCC CTT GGT GCT AGC TGA GGA GAC GGT GAC CG
35
55



VH3-23 Nhel


18
M165H5Sal
AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGG TTT
45
56


19
M165H3Nhe
AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TGC
39
57


20
N26H5Sal1
AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGG TTT
45
58


21
N26H3Nhe1
AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC CC
41
59


22
A33 2-6A2 K
ATC ACA GAT CTC TCA CCA TGG ACA TGA GGG TCC CC
35
60



L19 Bglll


23
A33 2-6A2 K
ACA GAT GGT GCA GCC ACC GTA CGT TTA ATC TCC AG
35
61



L19 BsiWl


24
M165K5L6Bgl2
AGA GAG AGA GAG ATC TCA CCA TGG AAG CCC CAG CTC AGC TTC TCT
45
62


25
M165K3L6BsiW1
AGA GAG AGA GCG TAC GTT TGA TTT CCA CCT TGG TCC CTT GGC
42
63


26
N26KA10Minor L Bgl
AGA GAG AGA GAT CTC TCA CCA TGT CGC CAT CAC AAC TCA TTG GG
44
64


27
N26KA10Minor L Bsi
AGA GAG AGA GCG TAC GTT TGA TAT CCA CTT TGG TCC CAG GG
41
65


28
M10KBgl
AGA GAG AGA GAG ATC TCA CCA TGG AAG CCC CAG CTC AGC TTC TCT
45
66


29
M10KBsi
AGA GAG AGA GCG TAC GTT TGA TCT CCA CCT TGG TCC CTC CG
41
67


30
Q54K5Bgl
AGA GAG AGA GAG ATC TCA CCA TGG ACA TGA GGG TCC TCG CTC AGC
45
68


31
Q54K3Bsi
AGA GAG AGA GCG TAC GTT TGA TCT CCA GCT TGG TCC CCT GG
41
69


32
M10H5Sal
AGA GAG AGA GGT CGA CCA CCA TGG ATC TCA TGT GCA AGA AAA TGA AGC
48
70


33
M10H3Nhe
AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCG TGG TCC CT
41
71


34
Q54H5Sal
AGA GAG AGA GGT CGA CCA CCA TGG AGT TTG GGC TGA GCT GGC TTT
45
72


35
Q54H3Nhe
AGA GAG AGA GGC TAG CTG AGG AGA CGG TGA CCA GGG TTC CC
41
73


36
H_3UTR1848
CGG GGT ACG TGC CAA GCA TCC TCG TG
26
74


37
H_3UTR1875
ATG CTG GGC GCC CGG GAA GTA TGT AC
26
75


38
L_3UTR_823
GAA AGA TGA GCT GGA GGA CCG CAA TA
26
76


39
hh-1
CCA AGG GCC CAT CGG TCT TCC CCC TGG CAC
30
77


40
CMVH903F
GAC ACC CTC ATG ATC TCC CGG ACC
24
78


41
CMVHR1303
TGT TCT CCG GCT GCC CAT TGC TCT
24
79


42
hh-6
GGT CCG GGA GAT CAT GAG GGT GTC CTT
27
80



















TABLE 6





Antibody name
Vector name
Subclass
Recombinant antibody







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
Host cells were transfected with the vectors for expression of recombinant antibodies constructed in Example 15, thereby preparing recombinant-antibody-expressing cells. Host cells to be used for expression were CHO cells of a dhfr-deficient cell line (ATCC CRL-9096), CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109, 1999), or HEK293T (ATCC CRL-11268), for example.
Host cells were transfected with each type of vector by electroporation, lipofection, or the like. Approximately 2 μg of the antibody expression vector was linearized using restriction enzymes and then subjected to electroporation using a Bio-Rad electrophoreter under conditions of 350 V and 500 μF. 4×106 CHO cells were thus transfected with each type of gene, and then the cells were inoculated on a 96-well culture plate. Lipofection was performed using LipofectAMINE Plus (produced by Gibco BRL) according to the manual included therein. After transfection with such vector, a drug corresponding to the selection marker used in the expression vector was added, and then culture was continued. After confirmation of colonies, antibody-expressing cell lines were selected by the method described in Example 6. Antibody purification was performed from the selected cells by washing twice with PBS after adsorption and then eluting using 20 mM (glycine) sodium citrate and a 50 mM NaCl (pH 2.7) buffeu sing a Mab Select Protein A 3.2×10 cm column (produced by Amersham Pharmacia Biotech). The eluted solution was neutralized with a 50 mM sodium phosphate buffer (pH 7.0). Next, purification was performed using a Hitrap Q HP Sepharose column (produced by Amersham Pharmacia Biotech), which is an anion exchange column, and then similarly performed using a Hitrap SP HP Sepharose column (produced by Amersham Pharmacia Biotech) which is a cation column. The thus prepared antibody solution was substituted with PBS using a dialysis membrane (10000 cut-off produced by Spectrum Laboratories) and then sterilized by filtration using a membrane filter MILLEX-GV (produced by Millipore) with a pore size of 0.22 μm. Thus purified antibodies with purity of at least 95% or higher and with endotoxin levels of 0.1 EU/mg or less endotoxin were obtained. The concentration of each type of recombinant purified anti-A33 antibody was obtained by measurement of absorbance at 280 nm and calculation with 1.4 OD being equivalent to 1 mg/mL (antibody concentration).
Example 17
Test of the Reactivity of Recombinant Antibodies
The reactivity of each type of recombinant antibody obtained in Example 16 to A33-antigen-expressing human colorectal cancer cell line COLO205 cells, LS174T cells (ATCC No. CL-188), or NCI-H508 cells (ATCC No. CCL-253) was examined by FCM. The reactivity of human colorectal cancer cell line HT-29 cells (ATCC No. HTB-38) expressing no A33 antigen was also examined as negative control cells. The test method conducted herein was similar to that in Example 9.
Table 7 shows the results. For COLO205 cells, the half value of the mean fluorescence intensity was determined to be 45. For LS174T 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. When an antibody concentration required to reach such value was 10<=x<100 ng/ml, the reactivity was represented by +++, when the same was 100<=x<1000 ng/ml, the reactivity was represented by ++, and when the same was 1000<=x<10000 ng/ml, the reactivity was represented by +. When no binding was observed, the reactivity was represented by −. Each type of recombinant antibody showed binding to all cells expressing the A33 antigen.











TABLE 7





Antibody name
COLO205
LS174T
NCI-H508
HT-29







Anti-DNP-IgG1
−
−
−
−


cA33
+++
+++
+++
−


rec263
++
++
++
−


recM10
+++
+++
+++
−


recM165
++
++
++
−


recN26
++
+++
+++
−


recQ54
+++
+++
+++
−






Half value of the mean
Half value of the mean
Half value of the mean



fluorescence intensity 45
fluorescence intensity 100
fluorescence intensity 175



10<=x<100 ng/ml; +++
10<=x<100 ng/ml; +++
10<x<100 ng/ml; +++



100<=x<1000 ng/ml; ++
100<=x<1000 ng/ml; ++
100<=x<1000 ng/ml; ++



1000<=x<10000 ng/ml; +
1000<=x<10000 ng/ml; +
1000<=x<10000 ng/ml; +



No binding; −
No binding; −
No binding; −






Example 18
Competition Test Regarding each type of Recombinant Antibody and Mouse Anti-A33 Antibody
Whether or not each type of type of recombinant antibody obtained in Example 16 recognizes an epitope similar to that of the mouse anti-A33 antibody was examined by a competition test using FCM. The test method conducted herein was similar to that in Example 10.
Similar to the results for each type of purified monoclonal antibody in Example 10, rec263 and recM 10 were classified as “non-blockers,” recM165 and recN26 were classified as “blockers,” and recQ54 was classified as “a partial blocker.” Table 8 shows the results.











TABLE 8







Antibody name
Inhibition (%)
Classification




















cA33
94.6
Blocker



rec263
15.3
Non-blocker



recM10
0.2
Non-blocker



recM165
93.4
Blocker



recN26
95.3
Blocker



recQ54
44.6
Partial blocker










Example 19
Test of the Cytotoxicity of Recombinant Antibodies
ADCC and CDC of the recombinant antibodies obtained in Example 16 were determined. In the ADCC assay, 5,000 51Cr-labeled target cells (COLO205 or NCI-H508) and 500,000 healthy human peripheral blood mononuclear leukocytes obtained by the method described in Example 11 were cultured in a V-bottom 96-well plate (produced by Coaster) at a total volume of 200 μL with antibody having each concentration at 37° C. in the presence of 5% CO2 for 4 hours.
In the CDC assay, 5000 51Cr-labeled target cells (COLO205 or NCI-H508) and human serum-derived complements (produced by Sigma) at a final concentration of 5% were cultured in a V-bottom 96-well plate at a total volume of 200 μL with antibody having each concentration at 37° C. in the presence of 5% CO2 for 4 hours.
The test method conducted herein was similar to that in Example 12.
FIG. 2A to FIG. 2D and Table 9 show the results. In the case of ADCC against COLO205 cells as target cells, the half value of the specific lysis (%) was determined to be 12.5%. In the case of ADCC against NCI-H508 cells as target cells, the half value of the specific lysis (%) was determined to be 30%. When an antibody concentration required to reach such value was 1<=x<10 ng/ml, the cytotoxicity was represented by +++, when the same was 10<=x<100 ng/ml, the cytotoxicity was represented by ++, when the same was 100<=x<1000 ng/ml, the cytotoxicity was represented by +, and when no specific lysis (%) was obtained, the cytotoxicity was represented by −. In the case of CDC against COLO205 cells as target cells, the half value of the specific lysis (%) was determined to be 7%. In the case of CDC against NCI-H508 cells as target cells, the half value of the specific lysis (%) was determined to be 20%. When an antibody concentration required to reach such value was 10<=x<100 ng/ml, the cytotoxicity was represented by +++, when the same was 100<=x<1000 ng/ml, the cytotoxicity was represented by ++, when the same was x>=1000 ng/ml, the cytotoxicity was represented by +, and when no specific lysis (%) was observed, the cytotoxicity was represented by −. In the case of ADCC, cA33, recM10, and recQ54 showed high cytotoxicity, while in the case of CDC, recM10 showed high cytotoxicity.









TABLE 9







Cell name
COLO205
NCI-H508











Antibody name
ADCC
CDC
ADCC
CDC





Anti-DNP-IgG1
−
−
−
−


cA33
+++
±
++
++


rec263
++
++
++
++


recM10
+++
+++
++
+++


recM165
++
+
++
++


recN26
++
+
++
++


recQ54
+++
++
++
++






Half value of specific
Half value of specific
Half value of specific
Half value of specific



lysis (%) 12.5%
lysis (%) 7%
lysis (%) 30%
lysis (%) 20%



1<=x<10 ng/ml; +++
10<=x<100 ng/ml; +++
1<=x<10 ng/ml; +++
10<=x<100 ng/ml; +++



10<=x<100 ng/ml; ++
100<=x<1000 ng/ml; ++
10<=x<100 ng/ml; ++
100<=x<1000 ng/ml; ++



100<=x<1000 ng/ml; +
1000<=x<10000 ng/ml; +
100<=x<1000 ng/ml; +
1000<=x<10000 ng/ml; +



No specific lysis; −
x>=10000 ng/ml; ±
No specific lysis; −
x>=10000 ng/ml; ±




No specific lysis; −

No specific lysis; −






Example 20
Western Blot Analysis of the Purified Antibodies and Recombinant Antibodies
Mouse anti-A33 and humanized A33 antibodies have been reported to recognize conformational epitopes. Specifically, in the cases of these antibodies, it has been reported that no reactivity was observed via Western blot analysis under reducing conditions (5% β-mercaptoethanol). Hence, Western blot analysis was performed in order to examine the reactivity of the purified human anti-A33 and recombinant antibodies.
The shA33EX-hFc protein prepared in Example 3 was separated by SDS-PAGE using 10% to 20% polyacrylamide gradient gel (produced by Daiichi Pure Chemicals) under reducing (5% β-mercaptoethanol) and non-reducing conditions. At this time, the shA33EX-hFc protein was diluted to 2.5 ng per lane. In the meantime, biotinylated SDS-PAGE Standard BroadRange (produced by Bio-Rad Laboratories) was also applied to one lane as a marker. The shA33EX-hFc protein was blotted onto a PVDF membrane using Panther Semidry Electroblotter (Daiichi Pure Chemicals) at 150 mA/membrane for 1 hour. The membrane onto which the protein had been blotted was washed using a TBS buffer and TBS (TTBS) containing 0.05% Tween. Blocking was performed using BlockAce (produced by Dainippon Pharmaceutical). Washing was performed twice with TTBS. Each type (1 μg/ml) of antibody purified from 125M10AA, 125Q54AAAA, 125M96ABA, 125Q47BA, and 125R5AAAA hybridomas was caused to react at room temperature for 60 minutes. In the meantime, each type (1 μg/ml) of recombinant antibody including the chimeric anti-A33 antibody, 125M165DAAA (that is, recM165), and 125N26F6AA (that is, recN26), was caused to react at room temperature for 60 minutes. After washing with TTBS, a goat anti-human Kappa chain F(ab′)2 antibody (produced by Biosource) labeled with horseradish peroxidase (diluted 1000-fold) was used as an antibody for detection. At this time, streptavidin labeled with horseradish peroxidase (diluted 3000 fold) was also added and caused to react so as to detect markers. After washing twice with TTBS and then once with PBS, band detection was performed using Western blotting detection system ECL-plus (produced by Amersham Biosciences). Chemoluminescence was incorporated using an image analyzer LAS-100 (produced by Fuji Photo Film) and then image processing was performed.
FIG. 3A and FIG. 3B show the results. It was found as a result that only 125Q54AAAA reacted with a protein band of approximately 67 kD even under reducing conditions. In the cases of the other antibodies, reaction was observed only under non-reducing conditions, similar to the case of the chimeric anti-A33 antibody.
Example 21
Immunohistochemical Analysis of the Purified Antibodies and Recombinant Antibodies
To evaluate whether or not each type of human anti-A33 antibody is equivalent to the chimeric anti-A33 antibody in terms of specificity and selectivity, the reactivity of tumor tissue sections and that of normal tissue sections were analyzed by immunohistochemical analysis.
(1) Fluorescent Labeling of the Purified Antibodies and Recombinant Antibodies
Each type of purified monoclonal antibody prepared in Example 8 was directly labeled with Alexa FluorTM 488 (produced by Molecular Probe). Each type of recombinant antibody (rec263, 125M10AA, recM165, recN26, and 125Q54AAA) prepared in Example 16 was also directly labeled with the same. Similarly, the chimeric anti-A33 antibody was directly labeled as a positive control and an anti-DNP-IgG1 antibody was directly labeled as a negative control. Fluorescent labeling was performed for the purified and recombinant antibodies as described below. Alexa FluorTM 488 was bound to each type of anti-A33 antibody prepared in Example 8 or 16 according to the instructions included therein. 50 μl of a 1 M carbonate buffer was added to 2 mg/ml of each type of purified antibody and to 0.5 ml of each type of recombinant antibody. The solutions were mixed with Alexa FluorTM488, followed by 1 hour of reaction at room temperature while stirring of the solutions took place. Hydroxylamine was added to stop the reactions. Each of the mixed solutions was applied to a gel filtration column (NAP5, produced by Amersham Pharmacia Biotech), thereby removing Alexa FluorTM488 that had not bound to the antibodies. 4 to 6 fluorescent substances bound to 1 molecule of antibody under these conditions. The fluorescent-labeled antibodies bound to COLO205 cells and showed binding activity equivalent to those of unlabeled antibodies.
(2) Immunohistochemistry
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). Blocking was performed for 1 to 2 hours at room temperature using PBS containing 10% goat serum (produced by Gibco BRL). Washing was performed twice with PBS. Each type of purified monoclonal or recombinant antibody labeled with Alexa FluorTM 488 in Example 21 (1) was caused to react at 1 μg/ml at room temperature for 30 to 60 minutes. Subsequently, the sections were mounted and then observed under a fluorescence microscope (BX51 produced by Olympus). The images were analyzed using DP70 (produced by Olympus).
FIGS. 4 to 6 show the results. Similar to the results of immunohistostaining of colorectal cancer tissues as reported by Garin-Chesa P et al. (Int. J. Oncology 1996 9: 465-471), extensive, uniform, and strong staining of glandular epithelium cells or dysplastic gland structures of colon cancer tissues were observed in the cases of the chimeric anti-A33 antibody, rec263, 125M10AA, recM165, recN26, and 125Q54AAA (FIG. 4). Moreover, it was observed that normal small intestine tissues (FIG. 5) and normal colon tissues (FIG. 6) had been stained in a manner similar to that in the case of the chimeric anti-A33 antibody. In contrast, no staining of normal gastric tissues was observed even in the cases of the antibodies, similar to the results of the aforementioned paper. Furthermore, in the case of the anti-DNP-IgG1 antibody as a negative control, staining was not observed in any tissues.
Example 22
Effects of Antibodies Purified from Hybridomas and Recombinant Antibodies on Mouse Tumor-Bearing Models
The effects of the human anti-A33 recombinant antibodies obtained in Example 16 were examined using mouse tumor-bearing models according to the method described below. The cells of colorectal cancer cell lines used herein were COLO205 cells and NCI-H508 cells.
A method for preparing a mouse tumor-bearing model using the COLO205 cell line is described below. The cells of the colorectal cancer cell line COLO205 were subcutaneously engrafted at 5×106/mouse in the dorsal regions 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 μl of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10 μg/mouse or 100 μg/mouse to tumor-bearing mice (10 mice per group). Tumor sizes were measured on days 7, 9, 11, 14, 17, and 21 after the engrafting of the cells. A human anti-DNP-IgG1 antibody was used as a negative control antibody in amounts identical to those of the above antibodies. “Vehicle” denotes 1% nude mouse serum-containing PBS (200 μl) that was used as medium for dissolution upon antibody administration.
A method for preparing a mouse tumor-bearing model using the NCI-H508 cell line is described below. The cells of the colorectal cancer cell line NCI-H508 and Matrigel (produced by Becton Dickinson Bioscience) comprising mouse malignant sarcoma (which improves the survival and proliferation of tumor cells) were subcutaneously engrafted at 1×107/mouse in the dorsal regions of 6-week-old Balb/c nude mice (purchased from CLEA Japan), so that the ratio of NCI-H508 to Matrigel would be 1:1. The chimeric anti-A33 or rec263 antibody (dissolved in 200 μl of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10 μg/mouse or 100 μg/mouse to tumor-bearing mice (10 mice per group) on days 1, 4, and 7 after the engrafting of the cells. Tumor sizes were measured on days 7, 11, 14, 18, 21, 27, 33, 40, 48, 55, and 62 after the engrafting of the cells. A human anti-DNP-IgG1 antibody was used as a negative control antibody in amounts identical to those of the above antibodies. “Vehicle” denotes 0.1% nude mouse serum-containing PBS (200 μl) that was used as a medium for dissolution upon antibody administration.
FIG. 7 shows the results of the above experiment.
Regarding the mouse line in which the COLO205 cell line had been engrafted, 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 μg/mouse, compared with the group to which the vehicle had been administered. Compared with the group to which the anti-DNP-IgG1 antibody had been administered, significant differences in tumor size (p<0.05) were observed on days 7, 9, 11, and 14 after the engrafting of the cells. Furthermore, compared with the group to which the anti-DNP-IgG1 antibody had been administered, 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 had been administered at 100 μg/mouse. 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 μg/mouse, compared with the group to which the vehicle had been administered. Compared with the group to which the anti-DNP-IgG1 antibody had been administered, significant tumor suppression (p<0.05) was observed on days 7, 9, 11, 14, 17, and 21 after the engrafting of the cells. Moreover, 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 had been administered at 100 μg/mouse, compared with the group to which the vehicle had been administered. Compared with the group to which the anti-DNP-IgG1 antibody had been 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, regarding the mouse line in which the NCI-H508 cell line had been engrafted, no antitumor effects were exerted in the case of the group to which the rec263 antibody had been administered at 10 μg/mouse. In contrast, significant tumor suppression (p<0.05) was observed more than 18 days after the engrafting of the cells in the case of the group to which the rec263 antibody had been administered at 100 μg/mouse, compared with the group to which the vehicle had been administered. Moreover, compared with the group to which the anti-DNP-IgG1 antibody had been administered, significant tumor suppression (p<0.05) was observed on nearly all measuring days. 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 μg/mouse, compared with the group to which the vehicle had been administered. Furthermore, compared with the group to which the anti-DNP-IgG1 antibody had been administered, significant tumor suppression (p<0.05) was observed on days 7, 21, and 33 after the engrafting of the cells. Moreover, significant tumor suppression (p<0.05) was observed on nearly all measuring days in the case of the group to which the chimeric anti-A33 recombinant antibody had been administered at 100 μg/mouse, compared with the group to which the vehicle had been administered. Furthermore, compared with the group to which the anti-DNP-IgG1 antibody had been administered, significant tumor suppression (p<0.05) was observed by day 33 after the engrafting of the cells.
As described above, it was demonstrated that the antibodies of the present invention had high antitumor effects on the mouse tumor-bearing models using 2 types of the colorectal cancer cell lines (FIG. 7B).
The effects of the antibodies purified from human anti-A33-producing hybridomas and the human anti-A33 recombinant antibodies were examined using the mouse tumor-bearing models. The cells of colorectal cancer cell lines used herein were COLO205 cells and NCI-H508 cells. (Antitumor effects of the antibodies purified from 125M10AA, 125M165DAAA and 125M96ABA hybridomas on the COLO205 cell line)
The cells of the colorectal cancer cell line COLO205 were subcutaneously engrafted at 5×106/mouse in the dorsal regions 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, the 125M10AA, 125M165DAAA, or 125M96ABA antibody (dissolved in 200 μl of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 20 μg/mouse to tumor-bearing mice (15 mice per group in the case of the group to which the vehicle was administered and 10 mice per group in the other cases). Tumor sizes were measured on days 7, 10, 12, 14, and 17 after the engrafting of the cells. “Vehicle” denotes 1% nude mouse serum-containing PBS (200 μl) that was used as medium for dissolution upon antibody administration.
FIG. 7C shows the results of the above experiment. In FIG. 7C, M10 denotes the 125M10AA antibody, M96 denotes the 125M96ABA antibody, and M165 denotes 125M165DAAA antibody. Significant tumor suppression (p<0.05) was observed on all measuring days after the engrafting of the cells in the case of the group to which the 125M10AA antibody had been administered, compared with the group to which the vehicle had been administered. Furthermore, significant tumor suppression (p<0.05) was observed on days 12, 14, and 17 after the engrafting of the cells in the case of the group to which the 125M165DAAA antibody had been administered, compared with the group to which the vehicle had been administered. In contrast, significant tumor suppression (p<0.05) was observed on days 12 and 14 after the engrafting of the cells in the case of the group to which the 125M96ABA antibody had been administered, compared with the group to which the vehicle had been administered.
(Antitumor Effects of N26 and M165 Recombinant Antibodies on COLO205 and NCI-H508 Cell Lines)
The cells of the colorectal cancer cell line COLO205 were subcutaneously engrafted at 5×106/mouse in the dorsal regions of 6-week-old Balb/c nude mice (purchased from CLEA Japan). On days 1, 3, and 6 after the engrafting of the cells, the recN26 or recM165 antibody (dissolved in 200 μl of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10 μg/mouse or 100 ag/mouse to tumor-bearing mice (10 mice per group). Tumor sizes were measured 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 denotes the recM165 antibody (administered at 10 μg/mouse), M165-100 denotes the recM165 antibody (administered at 100 μg/mouse), N26-10 denotes the recN26 antibody (administered at 10 μg/mouse), and N26-100 denotes the recN26 antibody (100 μg/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 μg/head, compared with the group to which the vehicle had been administered. Furthermore, 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 recN26 antibody had been administered at 100 μg/head, compared with the group to which the vehicle had been administered. Furthermore, 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 μg/head, compared with the group to which the vehicle had been administered.
The cells of the colorectal cancer cell line NCI-H508 and Matrigel (produced by Becton Dickinson Bioscience) comprising mouse malignant sarcoma (that improves the survival and proliferation of engrafted tumor cells) were subcutaneously engrafted at 1×107/mouse to the dorsal regions of 6-week-old Balb/c nude mice (purchased from CLEA Japan), so that the ratio of NCI-H508 to Matrigel would be 1:1. The recN26 or recM165 antibody (dissolved in 200 μl of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10 μg/mouse or 100 μg/mouse to tumor-bearing mice (12 mice per group in the case of the group to which the vehicle was administered and 10 mice per group in the other cases) on days 1, 4, and 7 after the engrafting of the cells. Tumor sizes were measured 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 denotes the recN26 antibody (administered at 10 μg/mouse), N26-100 denotes the recN26 antibody (administered at 100 μg/mouse), M165-10 denotes the recM165 antibody (administered at 10 μg/mouse), and M165-100 denotes the recM165 antibody (administered at 100 μg/mouse). Regarding the mouse line to which the NCI-H508 cell line had been engrafted, 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 μg/mouse, compared with the group to which the vehicle had been administered. Furthermore, compared with the group to which the vehicle had been administered, 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 μg/mouse. 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 μg/mouse, compared with the group to which the vehicle had been administered. Compared with the group to which the vehicle had been administered, significant tumor suppression (p<0.05) was observed on all measuring days after the engrafting of the cells in the case of the group to which the recM165 antibody had been administered at 100 μg/mouse.
(Antitumor Effects of the M10 and Q54 Recombinant Antibodies on the NCI-H508 Cell Line)
The cells of the colorectal cancer cell line NCI-H508 and Matrigel (produced by Becton Dickinson Bioscience) comprising mouse malignant sarcoma (that improves the survival and proliferation of engrafted tumor cells) were subcutaneously engrafted at 1×107/mouse to the dorsal regions of 6-week-old Balb/c nude mice (purchased from CLEA Japan), so that the ratio of NCI-H508 to Matrigel would be 1:1. The recM10 or recQ54 antibody (dissolved in 200 μl of 1% nude mouse serum-containing PBS) was administered intraperitoneally at 10 μg/mouse or 100 μg/mouse to tumor-bearing mice (10 mice per group) on days 1, 4, and 7 after the engrafting of the cells. Tumor sizes were measured on days 14, 21, 28, 35, 42, 49, 56, and 63 after the engrafting of the cells.
FIG. 7F shows the results of the above experiment. In FIG. 7F, M10-10 denotes the recM10 antibody (administered at 10 μg/mouse), M10-100 denotes the recM10 antibody (administered at 100 μg/mouse), Q54-10 denotes the recQ54 antibody (administered at 10 μg/mouse), and Q54-100 denotes the recQ54 antibody (administered at 100 μg/mouse). Regarding the mouse line to which the NCI-H508 cell line had been engrafted, 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 μg/mouse, compared with the group to which the vehicle had been administered. Furthermore, compared with the group to which the vehicle had been administered, 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 μg/mouse. 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 μg/mouse, compared with the group to which the vehicle had been administered. Furthermore, compared with the group to which the vehicle had been administered, 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 μg/mouse.
Example 23
Preparation of Cynomolgus A33 Outer Cell Membrane Region Recombinant Protein
a) Construction of Full-Length Cynomolgus A33 Expression Vector
A plasmid vector pΔEGFP-N1-GPA33 retaining A33-encoding cDNA was constructed to prepare a full-length A33 expression vector. pΔEGFP-N1-GPA33 was constructed by the following method. The complete full-length cynomolgus A33 cDNA (GenBank DNA AB168694: SEQ ID NO: 90 or protein BAE00805: SEQ ID NO: 91) was modified by performing a polymerase chain reaction (PCR) so as to add an EcoR I sequence to the 5′ terminus and to add a Not I sequence and a termination codon to the 3′ terminus. 30 cycles of a PCR reaction (94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 60 seconds) were performed using cynomolgus Colon cDNA (purchased from BioChain) as a template, A33-F2 5′-GCAGACGAATTCAAGACCATGGTGGGGAAGAT-3′ (SEQ ID NO: 13) and A33-R2 5′-CTCGAGCGGCCGCTCTGCTGCTGGCCTGTCACTGGTCGAGGTGGTC-3′ (SEQ ID NO: 92) synthesized as primers, and KOD-plus DNA polymerase (produced by TOYOBO). The thus synthesized sequence was digested with EcoR I-Not I. The resultant was isolated as an EcoR I-Not I fragment. The fragment was then ligated to pΔEGFP-N1-vector (from which the region encoding EGFP protein of modified pΔEGFP-N1 (produced by Becton Dickinson Bioscience Clontech) had been deleted) that had been cleaved with the same enzymes. The thus obtained plasmid was named pΔEGFP-N1-cynoGPA33. A 980-bp cDNA was encoded in cynomolgus A33 incorporated in pΔEGFP-N1-cynoGPA33. Hereinafter, GeneAmp® PCR System 9700 (produced by PerkinElmer Japan) was used for regulating the temperatures for all PCR reactions in the examples.
b) Preparation of Cynomolgus A33-Expressing Cells
HcT116 human colorectal carcinoma cells were transfected with pΔEGFP-N1-GPA33 constructed in a): HcT116 cell line (ATCC No. CCL-247) were transfected using Lipofectamine 2000 (produced by Invitrogen). Transfection was performed by a method described in the related manual. After 24 hours of culture at 37° C. under 5.0% carbonic acid gas, G418 (produced by Gibco BRL) was added to the cells at a concentration of 0.5 mg/mL and then culture was performed for 1 week. The A33 antigen expressed on the cell membrane surfaces was confirmed using the Goat anti human A33 polyclonal antibodies (produced by R&D). Flowcytometer (FCM: produced by Becton, Dickinson and Company) analysis was performed using the Goat anti human A33 polyclonal antibodies as a primary antibody, a donkey anti-goat Ig gamma F(ab′)2 antibody (produced by Invitrogen) labeled with Alexa FluorTM 488 as a secondary antibody. Thus, among transfected cells that had acquired the trait of G418 resistance, cells expressing A33 on their cell membrane surfaces were selectively sorted.
Single clones expressing the full-length cynomolgus A33 antigen at high levels could be obtained from this cell lines. HcT116 cells, which had expressed the cynomolgusA33 antigen protein at high levels, were named HcT116/cyno A33.
scynoA33EX-hFc protein was prepared for use as an immunogen or in ELISA upon screening for an antibody.
c) Construction of an Expression Vector for a Soluble Cynomolgus A33 Fc Fusion Protein Outside the Cell Membrane
To construct an expression vector (hereinafter referred to as scynoA33EX-hFc) for a soluble A33 cynomolgus Fc fusion protein outside the cell membrane, a plasmid vector pTracer-CMV-humanFc-A33EXR retaining cDNA encoding the A33 region outside the cell membrane was constructed. pTracer-CMV-humanFc-cynoA33EXR was constructed by the following method. The DNA of the A33 region outside the cell membrane (SEQ ID NO: 11) containing a secretory signal sequence was modified via a polymerase chain reaction (PCR) so as to add an EcoR I sequence to the 5′ terminus and to add a Not I sequence and a termination codon to the 3′ terminus. 30 cycles of a PCR reaction (94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 60 seconds) were performed using pΔEGFP-N1-A33 cDNA prepared in a) as a template, A33-F2 (SEQ ID NO: 13) and GPA-EXCRR25′-CTCGAGCGGCCGCCAGTTCATGGAGGGAGATCTGACG-3′ (SEQ ID NO: 15) synthesized as primers, and KOD-plus DNA polymerase (produced by TOYOBO). The thus synthesized sequence was digested with EcoR I-Not I. The resultant was isolated as an EcoR I-Not I fragment. The fragment was then ligated to a pTracer-CMV-humanFc vector (plasmid prepared by introducing FLAG and a human IgG1Fc region at the Xba I and Apa I sites of modified pTracer-CMV (produced by Invitrogen Life Technologies)) that had been cleaved with the same enzymes. The thus obtained plasmid was named pTracer-CMV-humanFc-cynoA33EXR.
Oligonucleotides such as PCR primers were all synthesized using a DNA autosynthesizer (model 3948; produced by PerkinElmer Japan, Applied Biosystems Division) according to the manual included therewith (see Matteucci, M. D. and Caruthers, M. H. (1981) J. Am. Chem. Soc. 103, 3185-3191). After completion of synthesis, each oligonucleotide was cleaved from support medium and subjected to deprotection. The thus obtained solution was dried, solidified, and then dissolved in distilled water. This solution was cryopreserved at −20° C. until use.
d) Expression and Purification of shA33EX-hFc Protein
Host cells were transfected with the scynoA33EX-hFc protein expression vector constructed in c), thereby preparing cells expressing the soluble A33 protein outside the cell membrane. Host cells to be used for expression were CHO cells of a dhfr-deficient cell line (ATCC CRL-9096), CHO-Ras (Katakura Y., et al., Cytotechnology, 31: 103-109, 1999), or HEK293T (ATCC CRL-11268), for example.
Host cells were transfected with the vector by electroporation, lipofection, or the like. Approximately 2 μg of the scynoA33EX-hFc protein expression vector was linearized using restriction enzymes and then subjected to electroporation using a Bio-Rad electrophoreter under conditions of 350 V and 500 pF. 4×106 CHO cells were transfected with the gene, and then the cells were inoculated on a 96-well culture plate. Lipofection was performed using LipofectAMINE Plus (produced by Gibco BRL) according to the manual included therewith. After transfection with the vector, a drug corresponding to the selection marker for the expression vector was added and then culture was continued.
The scynoA33EX-hFc protein was purified from the culture supernatant by the following method. The culture supernatant containing the scynoA33EX-hFc protein was subjected to affinity purification using Hitrap Protein A FF (produced by Amersham Pharmacia Biotech), PBS as an adsorption buffer, 20 mM sodium citrate as an elution buffer, and 50 mM sodium chloride (pH 2.7). The eluted freaction was adjusted at pH 5.5 by the addition of a 50 mM sodium phosphate solution (pH 7.0). The thus prepared solution of the soluble A33 protein outside the cell membrane was substituted with PBS using Amicon Ultra-15 (produced by Amicon) and then sterilization by filtration was performed using a membrane filter MILLEX-GV (produced by Millipore) with a pore size of 0.22 μm. A purified scynoA33EX-hFc protein was thus obtained. The concentration of the scynoA33EX-hFc protein was found by measurement of absorbance at 280 nm and calculation with 1.4 OD being equivalent to 1 mg/mL (antibody concentration).
Example 24
Cross-Reactivity Study of the Recombinant Antibodies
To evaluate whether or not each type of human anti-A33 antibody is cross-react to cynomolgus, cross-reactivity study were performed by following method.
(1) Immunohistochemistry
Tissue sections used herein were cynomolgus A33 colon tissue sections (produced by Shin Nippon Biomedical Laboratories, Ltd.), and cynomolgus A33 rectum tissue sections (produced by Shin Nippon Biomedical Laboratories, Ltd).
Blocking was performed for 1 to 2 hours at room temperature using PBS containing 2% rabbit serum (produced by Gibco) and 100 μg/ml human IgG (produced by Sigma). Washing was performed twice with PBS. Each type of recombinant antibody labeled with Alexa FluorTM488 (produced by Invitrogen) in Example 21 (1) was caused to react at 1 μg/ml at room temperature for 30 to 60 minutes. Subsequently, the sections were mounted and then observed under a fluorescence microscope (BX51 produced by Olympus). The images were imported and analyzed using DP70 (produced by Olympus).
FIGS. 8A to 8F and 9A to 9F show the results. FIGS. 8A to 8F show the results of anti-GPA33 antibody binding to cynomolgus rectum and FIGS. 9A to 9F show the results of anti-GPA33 antibody binding to cynomolgus colon.
Strong staining of cynomolgus A33 colon tissue and cynomolgus A33 rectum tissue were observed in the cases of the chimeric anti-A33 antibody, recN26, recM10. In the case of the recM165, Q54, anti-DNP-IgG1 antibody, as a negative control, staining was not observed in any tissues.
(2) ELISA
Protein ELISA was performed as follows. 501 of the cynomolgus A33 outer cell membrane region recombinant protein prepared in Example 23 and adjusted at pH 9.4 using a 50 μg/ml carbonate buffer was added to each well of a 96-well microplate for ELISA (Maxisorp produced by Nunc). Incubation was performed at 4° C. for overnight, so that the cynomolgus A33 outer cell membrane region recombinant protein was adsorbed to the microplate.
Subsequently, the supernatant was discarded, and SuperBlock (produced by PIERCE) was added to each well, followed by 30 minutes of incubation at room temperature. Thus, sites where no cynomolgus A33 outer cell membrane region recombinant protein had bound were blocked. In this manner, the microplate was prepared, wherein each well had been coated with the cynomolgus A33 outer cell membrane region recombinant protein.
Each antibody diluted in 10% BlockAce containing 0.1% Tween 20 TBS (produced by dainihonseiyaku) (TTBS) was added to each well, followed by 1 hour of reaction at room temperature. Each well was washed twice with TTBS.
Subsequently, a sheep anti-human Igκ antibody (50 μl/well, produced by Dako) labeled with horseradish peroxidase was diluted 500 folds using 10% BlockAce containing TTBS. 50 μl of the solution was added to each well, followed by 1 hour of incubation at room temperature. The microplate was washed three times with TTBS. 50 μl of a TMB color development substrate solution (produced by DAKO) was added to each well, followed by 2 minutes of incubation at room temperature. 0.5 M sulfuric acid (50 μl/well) was added to each well so as to stop the reaction. Absorbance at a wavelength of 450 nm (reference wavelength: 570 nm) was measured using a microplate reader (produced by Colona). FIG. 10 shows the results.
The reactivity of each type of purified monoclonal antibody cA33, recM10, recM165, recN26, Q54, DNP (negative control), M96, Q47, R5, rec263 was examined by ELISA.
Strong reactivity was observed in the cases of antibody recM10 and M96. Moderately-strong reactivity was observed in the cases of antibody cA33, and recN26. Weakly reactivity was observed in the case of antibody recM165.
(3) FMC
FMC was performed as follows. 105 of the cynomolgus A33 forced expression cell line was suspended at a concentration of 2×106/ml in a PBS staining buffer (SB) containing 0.1% NaN3 and 2% FCS. The cell suspension (50 μl/well) was dispensed in a 96-well round bottom plate (produced by Becton, Dickinson and Company). Each diluted antibody (50 μl) was added, followed by 15 minutes of incubation on ice on ice. A negative control was prepared depending on each subclass. Specifically, a human IgG1 antibody (produced by Sigma) was adjusted at a concentration of 400 or 2000 ng/ml with SB and then 50 μl of the solution was added, followed by 15 minutes of incubation on ice on ice. The resultant was washed twice with SB and then 50 μl of FITC fluorescence-labeled goat anti-human IgG F(ab′)2 antibody (produced by IBL) was added, followed by 15 minutes of incubation on ice on ice. The resultant was washed twice with SB and it was then suspended in 300 μl of SB. The mean fluorescence intensity of each cell line was measured by FCM (FACS caliber produced by Becton, Dickinson and Company). FIG. 11 shows the result.
The reactivity of each type of purified monoclonal antibody cA33, recM10, recM165, recN26, Q54, DNP (negative control), M96, Q47, R5, rec263A1 was examined by FMC.
Strong reactivity was observed in the cases of antibody cA33 and recN26. Moderately-strong reactivity was observed in the cases of antibody recM10, and M96. Weakly reactivity was observed in the case of antibody recM165.
The results of example 24 indicate that recN26 takes good effects.
All publications cited herein are incorporated herein in their entirety. Persons skilled in the art would easily understand that various modifications and changes of the present invention are feasible within the technical idea and the scope of the invention as disclosed in the attached claims. The present invention is intended to include such modifications and changes.
INDUSTRIAL APPLICABILITY
According to the present invention, a preventive or therapeutic agent for diseases due to A33-expressing cells, and in particular, a molecule that is also useful as a malignant tumor remedy for patients with A33 polymorphism, are provided.
9 polymorphisms are currently known for A33 mRNA and 7 such polymorphisms are present in the non-translation region. Furthermore, one of the remaining 2 polymorphisms is present in the 3rd codon. Hence, this polymorphism is a silent mutation that does not undergo amino acid substitution. Furthermore, the other one of the remaining two polymorphisms undergoes amino acid substitution, but is present within the signal sequence. Accordingly, the antibody of the present invention is effective for therapeutic or preventive agents, regardless of A33 polymorphism.
All publications, patents, and patent applications cited in this description are herein incorporated by reference in their entirety.

## Claims

1. An isolated recombinant human antibody, which has the heavy chain variable region sequence of SEQ ID NO: 39 and the light chain variable region sequence of SEQ ID NO: 41.

2. The recombinant human antibody according to claim 1, wherein the class of the antibody heavy chain constant region is IgG.

3. A pharmaceutical composition, which contains the recombinant human antibody according to claim 1 as an active ingredient.