CZ285499A3 - Pharmaceutical preparation for lymphatic tumors, antibody, chimeric antibody and modified antibody - Google Patents

Abstract

Therapeutic agent for lymphatic vessels (except rryelorrhea), which they contain, as their active ingredient, an antibody that is specifically it binds to a protein whose amino acid sequence is shown in SEQ IDNO: 1 has cytotoxic activity

Description

The present invention relates to therapeutic agents for lymphatic tumors (excluding myelomas) which contain as an active ingredient antibodies that specifically bind to proteins expressed by these lymphatic tumors. The present invention also relates to therapeutic agents for T cell or B cell tumors (excluding myelomas).

Furthermore, the present invention relates to antibodies that specifically bind to proteins expressed in lymphatic tumors and which have cytotoxic activity.

BACKGROUND OF THE INVENTION

Lymph cells are mainly responsible for the immunity of living organisms. All lymphoid cells are derived from the same hematopoietic stem cells that are released into peripheral blood after repeated differentiation that occurs under the influence of various differentiation inducing factors or growth factors in bone marrow or other organs. Due to differences in this differentiation, lymphocytes are divided into B cells and T cells. B cells are those having the ability to produce antibodies, while T cells are those having the ability to present antigens, act cytotoxically, and the like. When these cells undergo tumor changes for some reason or during certain stages of their differentiation and start to proliferate in an uncontrolled manner in bone marrow, lymphatic tissues, blood, and the like, this condition is called lymphatic tumor.

As a result of the introduction of new technologies, particularly advanced technologies that use monoclonal antibodies against cell surface differentiation antigens, it has become possible to determine the origin and / or differentiation status of lymphatic cells. At the same time, it has also become possible to determine not only whether such tumor cells are derived from T cells or B cells, but also to determine the degree of maturity of the tumor cells.

The basic classification of lymphatic tumors is B cell tumors and T cell tumors, which is based on the origin and degree of maturity of the tumor cells. Based on the degree of maturity of tumor cells, B cell tumors are classified as acute B lymphatic leukemia (B-ALL), chronic B lymphatic leukemia (B-CLL), pre-B lymphoma, Burkitt lymphoma, follicular lymphoma, follicular envelope lymphoma, diffuse lymphoma and alike. On

on the other hand, based on the degree of maturity of the tumor cells, T cell tumors are classified as acute T lymphocytic leukemia (T-ALL), chronic T lymphocytic leukemia (T-CLL), mature T cell leukemia (ATL), non-ATL peripheral T lymphoma (PNTL) ) and the like (Zukai Rinsho (GAN) (Illuminated Clinical: Cancer), Series 17 Leukemia and lymphoma, Takashi Sugimura et al., Medical View Co., Ltd., 1987, B cell tumors, Kiyioshi Takatsuki, Nishimura Shoten, 1991).

It is true that, despite recent advances in medical techniques, the treatment of lymphatic tumors is not satisfactory. E.g. the cure rate of acute lymphatic leukemia (ALL) is still 20% or less, and the lymphomas are advanced in the advanced state of about 50%, although the cure rate for B lymphomas is said to be relatively high due to advances in multidrug therapy. In addition, T-cell lymphoma is more resistant and its cure rate is about 30%, the percentage of successful cure in adult T cell leukemia (ATL) is currently less than 10%.

On the other hand, Goto, T. et al. described a monoclonal antibody (antiHM1.24 antibody), which was obtained by immunizing mice with human myeloma cells (Blood (1994) 84, 1922-1930). When mice were transplanted with human myeloma cells, the antibody accumulated specifically in tumor tissue (Masaaki Kosaka et al., Nippon Rinsho (Japan Clinical) (1995) 53, 627-635), suggesting that the anti-HM1 antibody. 24 can be used in the diagnosis of tumor location by radioisotope labeling, in targeted therapies such as radioimmunotherapy, etc. However, it is not known whether an anti-HM1.24 antibody can be used to treat other lymphatic tumors.

SUMMARY OF THE INVENTION

Treatment methods for lymphatic tumors currently used include various types of chemotherapy, bone marrow transplantation, and the like. However, as noted above, none of them are sufficiently satisfactory and hence revolutionary therapeutic agents or methods that are able to alleviate lymphatic tumors and prolong patient survival are still expected.

It is an object of the present invention to provide a new therapeutic agent for lymphatic tumors, in addition to myelomas.

In order to provide such a therapeutic agent, the inventors have conducted extensive in vitro studies, including flow cytometry (FCM) analysis, determination of

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444 444 444 44 44 44 cytotoxic activities such as ADCC activity, CDC activity etc. and in vivo studies of anti-tumor effects using anti-HM1.24 antibodies (Goto, T. et al., Blood (1994) 84, 1922-1939) and a study of isolation of the antigenic protein to which the antiHM1.24 antibody specifically binds. As a result, it has been found that the antigenic protein recognized by the anti-HM1.24 antibody is expressed on lymphatic tumors and that the antiHM1.24 antibody has an antitumor effect on lymphatic tumors and this constitutes the material of the present invention.

Thus, the present invention provides a therapeutic agent for lymphatic tumors (excluding myelomas) comprising, as an active ingredient, an antibody that specifically binds to a protein having the amino acid sequence as set forth in SEQ ID No: 1 and having cytotoxic activity.

The present invention also provides a T cell tumor therapeutic agent or a B cell tumor therapeutic agent (excluding myeloma) comprising as an active ingredient an antibody that specifically binds to a protein having the amino acid sequence as set forth in SEQ ID No: 1 and which has cytotoxic activity.

The present invention also provides a T cell tumor therapeutic agent or a B cell tumor therapeutic agent (excluding myeloma) comprising as its active ingredient a monoclonal antibody that specifically binds to a protein having the amino acid sequence as set forth in SEQ ID No : 1 and which has cytotoxic activity.

The present invention also provides a T cell tumor therapeutic agent or a B cell tumor therapeutic agent (excluding myeloma) comprising as its active ingredient a monoclonal antibody that specifically binds to a protein having the amino acid sequence as set forth in SEQ ID No And which has ADCC activity or CDC activity as its cytotoxic activity.

The present invention also provides a T cell tumor therapeutic agent or a B cell tumor therapeutic agent (excluding myeloma) comprising as its active ingredient a monoclonal antibody that specifically binds to a protein having the amino acid sequence as set forth in SEQ ID No 1, which has cytotoxic activity and which has a Cy from the human antibody as a constant region.

The present invention also provides a T cell tumor therapeutic agent or a B cell tumor therapeutic agent (excluding myeloma) comprising, as an active ingredient, a chimeric antibody or an altered antibody that specifically binds.

9999 99 9 9 99 to a protein having an amino acid sequence as shown in SEQ ID No: 1 and having cytotoxic activity.

The present invention also provides a T cell tumor therapeutic agent or a B cell tumor therapeutic agent (excluding myeloma) comprising, as an active ingredient, an antibody that specifically binds to a cepitope recognized by an anti-HM1.24 antibody.

The present invention also provides a therapeutic agent for T cell tumors or a therapeutic agent for B cell tumors (excluding myelomas), which comprises as an active ingredient an anti-HM1.24 antibody.

Furthermore, the present invention relates to an antibody that specifically binds to a protein expressed on lymphatic tumors and which has cytotoxic activity. Methods used in carrying out the invention.

1. Preparation of antibodies

1-1. Preparation of hybridomas

The antibody-producing hybridomas used in the present invention can be essentially generated using the known procedure described below. Thus, the HM1.24 antigen protein or cells expressing it can be used as a sensitizing antigen and are used for immunization by a conventional immunization procedure. The thus obtained immune cells are then fused to known parent cells, using a conventional cell fusion method, after which a bulk screening is performed using a conventional screening procedure to select cells producing monoclonal antibodies.

More specifically, monoclonal antibodies can be obtained as follows. E.g. KPMM2 cell line from multiple myelomas (Japanese Unexamined Patent Publication (Kokai) No. 7-236475) or KPC-32 cell line (Goto, T.) can be used as cells expressing HM1.24 antigen that is sensitizing antigen to obtain an antibody. et al., Jpn J. Clin Hematol (1991) 3, 1400). Optionally, a protein having the amino acid sequence shown in SEQ ID NO: 1 or a peptide or polypeptide comprising an epitope recognized by an anti-HM1.24 antibody may be used as the sensitizing antigen.

Here, cDNA encoding a protein having the amino acid sequence shown in SEQ ID No: 1 was inserted into the XbaI cleavage site of pUC19 vector to construct plasmid pRS38-pUC19. E. coli containing this plasmid was deposited for international use under the provisions of the Budapest Treaty as Escherichia coli DH5a (pRS38-pUC19) on October 5, 1993 at the National Institute of Bioscience and Human-Technology, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, as FERM BP-4434 (see Japanese Unexamined Patent Publication (Kokai) No. 7-196694). The cDNA fragment contained in this plasmid pRS38-pUC19 can be used using genetic engineering technology to prepare a peptide or polypeptide comprising an epitope recognized by an anti-HM1.24 antibody.

Predominantly mammals are used for immunization with a sensitizing antigen, and their compatibility with parent cells in cell fusion is considered. Generally, they are, inter alia, rodents such as mice, rats and the like.

Immunization of mammals with a sensitizing antigen is accomplished by conventional techniques. A general procedure, eg, involves intraperitoneal or subcutaneous administration of a sensitizing antigen to a mammal. More specifically, the sensitizing antigen, which has been diluted and suspended in a suitable amount of phosphate buffered saline (PBS) or saline, etc., is admixed as desired with an appropriate amount of complete Freud's adjuvant. After emulsification, it is preferably administered to mammals several times every 4 to 21 days. Alternatively, a suitable carrier may be used at the time of immunization with the sensitizing antigen.

After immunization and confirmation that the desired antibody levels in the serum have increased, the immune cells are collected from the animal and are subjected to cell fusion, preferably using spleen cells as the immune cells.

Preferably, various known cell lines, such as P3X63AG8.653) are used as mammalian myeloma cells as second parent cells with which cell fusion with the above immune cells is performed (J. Immunol. (1979) 123: 15481550), P3X63AG8U 1 (Current Topics in Microbiology and Immunology (1978) 81: 1-7) NS-1 (Kohler, G. and Milstein, C., Eur. J. Immunol. (1976) 6: 511-519), MPC- 11 (Margulies, DH et al., Cell (1976) 8: 405-415), SP2 / 0 (Shulman, M. et al., Nature (1978) 276: 269-270), FO (de St. Groth, SF et al., J. Immunol. Methods (1980) 35: 121), S194 (Trowbridge, IS, J. Exp. Med. (1978) 148: 313-323), R210 (Galfre, G. et al. Nature (1979) 277: 131-133) and the like.

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Cellular fusion between the above immune cells and myeloma cells can be essentially performed according to a known procedure as described by Milstein et al. (Kohler, G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46) or something like that.

More specifically, the aforementioned cell fusion is performed in a conventional nutrient broth, in the presence, eg, of a cell fusion accelerator. For example, polyethylene glycol (PEG), Sendai virus (HVJ) may be used as a cell fusion accelerator, and an adjuvant such as dimethylsulfoxide etc. may be added to increase fusion efficiency.

The preferred ratio of immune to myeloma cells to be used is, for example, one to ten times more immune cells than myeloma cells. Examples of culture media used for the above cell fusion are RPM11640 and MEM media suitable for the growth of the above myeloma cell lines, and the conventional culture medium used for this type of cell culture may additionally be added as a serum supplement such as eg fetal calf serum (FCS).

In cell fusion, predetermined amounts of immune cells and myeloma cells are mixed thoroughly in said culture medium to which a PEG solution preheated to 37 ° C has been added, e.g. a PEG solution having an average molecular weight of 1000 to 6000 is added at a concentration 30 to 60% (w / v) and mixed until the desired cell fusion (hybridomas) is achieved. Thereafter, by repeating the sequential addition of a suitable culture medium and centrifugation after which the supernatant is removed, the cell fusion promoting agent, which is undesirable for hybridoma growth, may be removed.

Said hybridomas are selected by cultivation in a conventional selective medium, eg HAT culture medium (culture solution containing hypoxanthine, aminopterin and thymidine). Cultivation in said HAT culture medium generally takes place for a time sufficient to kill cells other than the desired hybridomas (unfused cells), which is generally several days to several weeks. A conventional end-dilution method is then carried out to select hybridomas that produce the desired antibodies and are further monoclonally cloned.

In addition to obtaining the above hybridomas by immunizing non-human animals with an antigen, it is also possible to sensitize human lymphocytes in vitro with an antigen or with cells expressing the HM1.24 antigen, and the resulting sensitized lymphocytes are fused to human myeloma cells, e.g. to obtain the desired human antibody having the ability to bind to HM1.24 antigens or to cells expressing the HM1.24 antigen (see Japanese Postexamined Patent Publication (Kohoku) No. 1-59878). Alternatively, a transgenic animal having a set of all human anti-gene genes is immunized with an antigen, i.e., HM1.24 or with cells expressing the HM1.24 antigen, to obtain the desired human antibody as previously described (see International Patent Publication). Applications WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).

The monoclonal antibody-producing hybridomas thus constructed can be cultured in a conventional culture medium or can be stored for a long time in liquid nitrogen.

To obtain monoclonal antibodies from said hybridomas, a method may be mentioned in which the hybridoma is cultured in a conventional manner and the antibodies are obtained in a supernatant, or a method in which the hybridoma is administered and cultured in an animal body compatible with said hybridoma and antibodies are derived from ascites . The former is suitable for obtaining highly pure antibodies, while the latter is suitable for large-scale production of antibodies.

More specifically, hybridomas producing anti-HM1.24 antibodies can be constructed by the method of Goto T. et al., (Blood (1994) 84: 1922-1930). This can be done in that the hybridoma producing anti-HM1.24 antibodies, which was deposited for international use under the terms of the Budapest Treaty as FERM BP-5233 on September 14, 1995 by the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology of 1-3, Higashi 1chome, Tsukuba city, Ibaraki pref., Japan, is administered intraperitoneally to BALB / c mice (grown in CLEA Japan) to obtain ascites from which the anti-HM1.24 antibody is purified: or a method wherein the above hybridoma is cultured in a suitable culture medium such as RPMI 1640 medium containing 10% fetal calf serum and 5% BM-Condimed H1 (manufactured by Boehringer-Mannheim), SFM hybridoma medium (manufactured by GIBCI-BRL), PFHM-II medium (manufactured by GIBCI-BRL) or the like, and antibodies can be purified from the supernatant.

1-2. Recombinant antibodies

A recombinant antibody that has been produced by recombinant gene technology, in which the antibody gene has been cloned from hybridomas and incorporated into a suitable vector, which has then been introduced into a host, can be used in the present invention. 9 · 9 ·) 99999999999

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used as a monoclonal antibody (see, e.g., Carl, A.K., Borrebaeck, and James, W. Larrick, THERAPEUTICAL MONOCLONAL ANTIBODIES, published in MACMILLAN PUBLISHERS LTD. 1990, UK).

More specifically, the mRNA encoding the variable region (V region, V region) of the antibody of interest is isolated from the hybridoma that produces the antibody. Isolation of mRNA is accomplished by preparing total RNA using, for example, a known method such as the guanidine ultracentrifugation method (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), the AGPC method (Chomczynski, P. et al. (Analytical Biochemistry (1987) 162,156-159) and then mRNA from total RNA is purified using an mRNA Purification kit (manufactured by Pharmacia) or the like. Alternatively, mRNA can be prepared using a Quick Prep mRNA kit (manufactured by Pharmacia).

The cDNA for the antibody V region can be synthesized from the mRNA thus obtained by reverse transcriptase. The cDNA can be synthesized using the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit or the like. Another option is to use the 5'-Ampli FINDER RACE Kit (manufactured by Clontech) for cDNA synthesis and amplification, and the 5'-RACE method (Frohman, MA et al., Proc. Natl. Acad. Sci. USA (1988) 85) can be used. Belyavski, A. et al., Nucleic Acids Res. (1989) 17, 29192932), which utilizes a polymerase chain reaction (PCR). The desired DNA fragment is purified from the obtained PCR product and can be ligated into vector DNA. A recombinant vector is then constructed therefrom and is then introduced into E. coli, etc., from whose selected colonies the desired recombinant vector is prepared. The nucleotide sequence of the DNA of interest can be confirmed by a known method, such as the dideoxy method.

Once the DNA encoding the V region of the desired antibody is obtained, it can be ligated into DNA encoding the constant region (C region) of the desired antibody, which is then incorporated into an expression vector. Another possibility is to incorporate the DNA encoding the antibody V region into an expression vector that already contains the DNA encoding the antibody C region.

In order to produce the antibody used in the present invention, the antibody gene is incorporated as described below into an expression vector such that it is expressed under the control of an expression regulatory region, eg, a transcriptional enhancer or promoter. Subsequently, the expression vector may be transformed into a host cell, and the antibody expressed therein.

1-3. Altered antibodies

According to the present invention, an artificially altered recombinant antibody, such as a chimeric antibody or an altered antibody, can be used to reduce heterologous antigenicity against humans. These altered antibodies can be generated by known methods.

The chimeric antibody can be obtained by ligating the obtained DNA encoding the antibody V region into DNA encoding the human antibody C region which is then inserted into an expression vector and introduced into a host suitable for antibody production (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576). Using these known methods, chimeric antibodies useful in the present invention can be obtained.

E.g. E. coli carrying a plasmid containing DNA encoding the L chain V region or the H chain V region of the anti-HM 1.24 chimeric antibody was deposited for international use under the terms of the Budapest Treaty as Escherichia coli DH5α (pUC19-1.24L-gK) and Escherichia coli DH5a (pUC19-1.24H-gYl), on 29 August 1996 by the National Institute of Bioscience and Human Technology, 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. Japan, such as FERM BP-5646 and FERM BP-5644 (see Japanese Patent Application No. 9-271536).

The altered antibody, also referred to as altered human antibody, was generated by attaching a region that determines complementarity (CDR) in a non-human mammal antibody, eg, a mouse, to the CDR of a human antibody. General DNA recombinant technology for preparing such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96/02576).

In more detail, a DNA sequence designed to link the CDR of a murine antibody to a framework region (FR) of a human antibody is synthesized by PCR from several separate oligonucleotide regions that overlap each other at their ends. The DNA thus obtained is linked to DNA encoding the human antibody C region and then inserted into an expression vector which is then introduced into a host (see European Patent Application EP 239400 and International Patent Application WO 96/02576).

The framework region (FR) of the human antibody, joined by complementarity determining regions (CDRs), is selected so that the

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Complementarity created a site that is capable of binding the desired antigen. If desired, amino acids in the support regions of the variable portions of the antibodies can be exchanged such that the complementarity determining regions in the altered antibodies form a site capable of binding the desired antigen (Sato, K. et al., Cancer Res. (1993) 53, 851). -856).

For example, E. coli carrying a plasmid that contains a DNA encoding the V region of the α (SEQ ID NO: 2) L chain and E. coli carrying a plasmid that contains a DNA encoding the r (SEQ ID NO: 3) V region of the V region the altered anti-HM 1.24 antibodies were deposited for international use under the terms of the Budapest Treaty as Escherichia coli DH5α (pUC19-RVLa-AHM-gK) and Escherichia coli DH5α (pUC19-RVHr-AHM-gYl) on 29 August 1996 by the National Institute of Bioscience and Human-Technology, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, such as FERM BP-5645 and FERM BP-5643 (see Japanese Patent Application No. 9). E. coli carrying a plasmid containing a DNA encoding a version of the (SEQ ID NO: 4) H chain The V region of the altered anti-HM 1.24 antibody was deposited for international use under the terms of the Budapest Treaty as Escherichia coli DH5α (pUC19). -RVHs-AHM-gYl) on August 29, 1997 orga of the National Institute of Bioscience and Human-Technology

1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, as FERM BP-6127 (see Japanese Patent Application No. 9-271536).

For the chimeric antibody or the altered antibody, the C region of a human antibody is used, most preferably it is used as a constant region of a human Cy antibody such as Cy1, Cy2, Cy3 and Cy4. Of the antibodies thus prepared, antibodies that contain γγ1 and Cy3 have potent cytotoxic activity, i.e., ADCC activity and CDC activity, and are therefore preferably used in the present invention.

Chimeric antibodies comprise an antibody variable region derived from a mammalian species (but not human) and a C region derived from a human antibody, while the altered antibody has complementarity determining regions derived from a mammalian species (but not human) and a support region (FRs) together with C regions of an antibody derived from a human antibody. Accordingly, their antigenicity in the human body is reduced to be useful as active ingredients of the therapeutic agents prepared according to the present invention.

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A preferred embodiment of the altered antibody suitable for use in the present invention includes altered anti-HM 1.24 antibodies (see Japanese Patent Application No. 9-271536). A preferred embodiment of the L chain of the V region of the altered anti-HM 1.24 antibody comprises those having the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 2. A preferred embodiment of the H chain of the V region of the altered anti-HM 1.24 antibody comprises those having an amino acid sequence encoded by a base sequence that is set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

-4. Expression and production

Antibody genes constructed as described above can be expressed to obtain antibodies, all by known methods. In the case of mammalian cells, expression can be achieved using an expression vector containing a commonly used useful promoter, an antibody gene to be expressed and DNA in which the poly-A signal is operably linked 3 'of that gene (in the transcription direction) ), or a vector comprising said DNA. An example of a cytomegalovirus promoter / enhancer is the immediate early promoter / enhancer of cytomegalovirus.

Furthermore, viral promoters / enhancers such as retrovirus, polyomavirus, adenovirus and simian virus 40 (SV40), as well as mammalian cell-derived promoters / enhancers such as human elongation, may be used as promoter / enhancer for use in the present invention. Factor 1a (HEF1a).

E.g. expression can be readily achieved by the Mulligan et al. method (Nature (1979) 277, 108) using the SV40 promoter / enhancer or by the Mizushima et al. method (Nucleic Acids Res. (1990) 18, 5322) where a HEF1a promoter / amplifier is used.

In the case of E. coli, expression can be achieved by operably linking a commonly used promoter, an antibody secretion signal sequence, and the antibody gene to be expressed, followed by self-expression of the gene. As an example of a promoter, mention may be made of the lacz promoter and the araB promoter. The method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427) is applicable using the lacz promoter, while the method of Better et al. (Science (1988) (240, 1041-1043) can be used using the araB promoter.

The pelB signal sequence can be used as a signal sequence for antibody secretion when produced into the periplasmic space of E. coli (Lei, S. P. and 0000 • 9).

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00 et al., J. Bacteriol. (1987) 169, 4379). After separation of the antibody produced into the periplasmic space, the structure of the antibody is suitably modified before use (see, eg, WO 96/30394).

As the origin of replication, the origin of replication derived from SV40, polyomavirus, adenovirus, bovine papillomavirus (BPV) and the like can be used. In addition, to multiply the gene copy number in the host cell system, the expression vector may comprise as a selectable marker an aminoglycoside transferase (APH) gene, a thymidine kinase gene, an E. coli xanthine guanine phosphoryl transferase (Ecogpt) gene, and the like.

Any production system can be used to produce the antibodies used in the present invention. The production system for preparing antibodies comprises an in vitro and in vivo production system. As the in vitro production system, mention may be made of a production system that utilizes eukaryotic cells and a production system that utilizes prokaryotic cells.

When eukaryotic cells are used, these are production systems that utilize animal cells, plant cells or fungal cells. Known animal cells include (1) mammalian cells such as CHO cells, COS cells, myeloma cells, baby hamster cells (BHK), HeLa cells and Vero cells, (2) amphibian cells such as Xenopus oocytes or (3) insect cells such as sf9, sf21 and Tn5. Known plant cells include, for example, those derived from the genus Nicotiana, more particularly cells derived from Nicotiana tabacum. Known fungal cells include yeast, such as the genus Saccharomyces, more specifically Saccharomyces cereviceae, or filamentous fungi, such as the genus Aspergillus, more particularly Aspergillus niger.

When prokaryotic cells are used, these are production systems that utilize bacterial cells. Known bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis.

By transforming the gene of the desired antibody into these cells and transforming the cells so transformed in vitro, antibody production can be achieved. The cultivation is carried out by known methods. E.g. DMEM, MEM, RPM11640 and IMDM may be used as culture media, and fetal calf serum (FCS) may be used as serum supplement. Further, the antibodies can be produced in vivo such that cells into which the antibody gene has been introduced are implanted into the abdominal cavity of the animal.

• · • ·

Other in vivo production systems include those using animals and those using plants. When animals are used, these are production systems that use mammals and insects.

Among mammals, goats, pigs, sheep, mice and cattle can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). As far as insects are concerned, silkworms may be used.

For plants, tobacco can be used, for example.

Antibody genes are introduced into these animals or plants, antibodies are produced in and derived from these animal or plants. E.g. the antibody gene is inserted into the middle of a gene encoding a protein that is naturally produced in milk, such as goat β casein, in which way a fusion gene is generated. DNA fragments that contain a fusion gene into which the antibody gene has been inserted are injected into goat embryos and these embryos are inserted into a goat. The antibody of interest is obtained from milk produced by a transgenic goat that has been born to a goat receiving this embryo or from the milk of its offspring. In order to increase the amount of milk containing the desired antibody and produced by the transgenic goat, hormones can be administered, if appropriate. (Ebert, K. M. et al., Bio / Technology (1994) 12, 699702).

When a silkworm is used, the baculovirus in which the gene for the desired antibody has been inserted is infected with the silkworm and the desired antibody can be obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985) 315, 592-). 594). Further, when tobacco is used, the gene for the desired antibody is inserted into a plant expression vector, eg, pMON 530, and then the vector is introduced into a bacterium such as Agrobacterium tumefaciens. Tobacco such as Nicotiana tabacum is further infected with this bacterium to obtain the desired antibody from tobacco leaves (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994) 24, 131-138).

When the antibody is produced in vitro or in vivo by production systems as described above, the DNA encoding the heavy chain (H chain) or light chain (L chain) of the antibody can be inserted into expression vectors separately but the host cells are transformed simultaneously, or DNA the H chain and the L chain encoding can be linked in a single expression vector and transformed by the host cells (see International Patent Application WO 94/11523).

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The antibody produced as described above can be bound to various molecules such as polyethylene glycol (PEG) and used as a modified antibody. The term "antibody" as used herein includes these modified antibodies. In order to obtain such a modified antibody, the obtained antibody may be chemically modified. Such methods have already been established in the art.

2. Separation and purification of antibody.

2-1. Separation and purification of antibody

Antibodies that are produced and expressed as described above can be separated from within the cell, from the cell or from the host and then purified to a homogeneous state. Affinity chromatography can be performed to separate and purify the antibody for use in the present invention. Columns suitable for such affinity chromatography include a Protein A column and a Protein G column. Examples of carriers used for the Protein A column are Hyper A, POROS, Sepharosa.

F.F. and the like.

Alternatively, other methods of separation and purification commonly used for proteins can be used without limitation. Separation and purification of the antibody for use in the present invention may be accomplished by various combinations of other suitable chromatographic techniques other than affinity chromatography, filtration, ultrafiltration, salting-out, dialysis, and the like. Chromatography includes, for example, ion-exchange chromatography, hydrophobic chromatography, gel filtration, and the like. These chromatographic procedures can be performed by HPLC. Another possibility is reverse phase chromatography.

2-2. Determination of antibody concentration

The concentration of the antibodies obtained in the above-mentioned 2-1 can be determined by absorbance measurement, or by an enzyme-linked immunosorbent assay (ELISA) and the like. Thus, when absorbance measurement is used, an antibody, for use in the invention, or a sample, containing the antibody is appropriately diluted with PBS (-), then absorbance is measured at 280 nm followed by a calculation using an absorption coefficient of 1.35 OD at a concentration of 1 mg / ml. When ELISA is used to measure antibody concentration, the procedure proceeds as follows. One hundred μΙ of goat anti-human IgG (manufactured by BIO SOURCE) diluted to 1 pg / ml in 0.1 M sodium bicarbonate buffer, pH 9.6, is added to a 96-well plate (manufactured by NUNC) and incubated overnight at temperature 4 ° C to bind the antibodies.

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After blocking, 100 μΙ of a suitably diluted antibody prepared according to the invention or a sample containing the antibody, or 100 μΙ of human IgG of known concentration (as a standard) is added and incubated for 1 hour at room temperature. After washing, 100 μ fosf of 5000-fold diluted alkaline phosphatase-labeled anti-human IgG antibody (manufactured by BIO SOURCE) is added and incubated for 1 hour at room temperature. After washing, substrate solution is added, incubated, followed by absorbance measurement at 405 nm on a MICROPLATE READER Model 3550 (manufactured by BioRad), based on which the concentration of the desired antibody was calculated.

3. FCM analysis

The reactivity of an antibody suitable for use in the present invention for reaction with lymphatic tumor cells can be determined by the flow cytometry analysis (FCM) method. Either cells from established cell lines or cells freshly isolated can be used. Among established cell lines, for example, RPMI 8402 (ATCC CRL-1994), CCRFCEM (ATCC CCL-119) derived from acute lymphoblastic leukemia, HPB-ALL (FCCH1018) derived from acute lymphatic leukemia, HPB-MLT may be used as a T cell line (FCCH1019) derived from T lymphoma, JM (FCCH1023) derived from acute lymphatic leukemia, MOLT-4 (ATCC CRL-1582) derived from acute lymphoblastic leukemia, Jurkat (FCCH1024) derived from acute lymphatic leukemia, CCRF-HSB-2 (ATCC CCL -120.1) derived from acute lymphoblastic leukemia, MT-1 (FCCH1043) derived from mature T cell leukemia, KT3 derived from Lennert's lymphomas (Shimizu, S. et al., Blood (1988) 71, 196-203) and the like, as B cell line, e.g., EB virus (ATCC TIB-190) transformed CESS cells, SKW 6.4 EB-positive B cell (ATCC TIB-215), B lymphoma-derived MC116 (ATCC CRL-1649), CCRF-SB (ATCC CCL- 120) derived from acute lymphoblastic leukemia, RPMI 6410 B cells (FCCH6047) derived from a patient with acute myelocytic leukemia, Daudi (ATCC CCL-213) derived from Burkitt's lymphoma, Jijoye (ATCC CCL-87) derived from Burkitt's lymphoma, Paradise (ATCC CCL-86) derived from Burkitt's lymphoma, and as non-T, non-B HL-60 cell line (ATCC CCL-240) derived from acute myelocytic leukemia, THP-1 (ATCC TIB-202) derived from acute monocytic leukemia, U-937 (ATCC CRL-1593) derived from connective lymphomas, K-562 ( ATCC (CCL-243) derived from chronic myelocytic leukemia, and the like.

«···«

· · · · · · ««

9 • 9

9 9 • 9 9 99

999

99

999

After washing the above cells in PBS (-), 100 μΙ of antibodies or control antibodies diluted to 25 gg / ml in FACS buffer (PBS (-) containing 2% fetal bovine serum and 0.1% sodium azide) is added and incubated for ice 30 minutes. After washing with FACS buffer, 100 μΙ of 25 pg / ml FITC-labeled goat anti-mouse antibody (GAM, manufactured by Becton Dickinson) is added and incubated on ice for 30 minutes. After washing with FACS buffer, cells are suspended in 600 μΙ or 1 ml of FACS buffer and fluorescence intensity can be measured for each cell using a scanning instrument (FACScan, manufactured by Becton Dickinson).

From the measured fluorescence intensity value for each cell, the reactivity of the antibody used in the present invention with each individual cell can be determined. Thus, from the measured fluorescence intensity value of each cell, it can be ascertained whether the HM1.24 antigen is expressed on individual cells (the cell is positive or negative) and also the degree of expression. The presence and intensity of HM1.24 antigen expression in lymphatic tumor cells is described in Example 2.2. FCM analysis below.

Tumor cells of lymphatic tumors, which may be a therapeutic target of the present invention, express the HM1.24 antigen. More specifically, tumor cells of lymphatic tumors are preferably those in which the percentage of positivity for the HM1.24 antigen is not less than 5%. More specifically, tumor cells of lymphatic tumors are preferably those in which the percentage of positivity to the HM1.24 antigen is 20% or greater. More specifically, tumor cells of lymphatic tumors are preferably those in which the percentage of positivity to the HM1.24 antigen is 50% or greater. More specifically, tumor cells of lymphatic tumors are preferably those in which the percentage of positivity to the HM1.24 antigen is 80% or greater.

4. Cytotoxic activity

4-1. Measurement of CDC activity

For example, an antibody suitable for use in the present invention is one having CDC activity as a cytotoxic activity.

The CDC activity of the lymphatic tumor treatment agent prepared according to the present invention can be measured as follows. First, target cells are prepared in a suitable medium, e.g. RPM11640 medium containing 10% fetal bovine serum (manufactured by GIBCO-BRL) at a concentration of 4 × 10 ⁵ cells / ml. CCRF-CEM (ATCC CCL-119), CCRF-HSB-2 (ATCC CCL120.1), HPB-MLT (FCCH1019), EB-3 (ATCC CCL-85), M116 (ATCC CRL-1649) may be used as target cells. ) CCRF-

SB (ATCC CCL-120), K-562 (ATCC CCL-243), and the like. Fifty μΙ of these cells is added to a 96-well flat-bottom plate (manufactured by FALCON) and the plate is incubated overnight in a CO ₂ incubator at 37 ° C.

Then, the antibody whose CDC activity is measured is added, incubation is continued for 60 minutes, and then appropriately diluted complement, eg, Baby Rabitt Complement (manufactured by CEDARLANE) is added and incubated for 2 hours. 10 μΙ Alamar Bule (manufactured by BIO SOURCE) is added to each well, incubated for 4 hours and then fluorescence intensity (excitation wavelength 530 nm, emission wavelength 590 nm) is measured using a CytoFluor 2350 measurement system (manufactured by MILLIPORE). Cytotoxic activity (%) can be calculated as (A-C) / (B-C) x 100, where A is the fluorescence intensity when incubating in the presence of antibody, B is the fluorescence intensity when incubating the antibody-free medium alone and C is fluorescence intensity in the cell-free well.

4-2. Measurement of ADCC activity

For example, an antibody suitable for use in the present invention is one having ADCC activity as a cytotoxic activity.

The ADCC activity of a lymphatic tumor treatment agent prepared according to the present invention can be measured as follows. First, mononuclear cells are isolated from human peripheral blood or bone marrow by centrifugation and will be used as effector cells in the assay. As target cells are CCRF-CEM (ATCC CCL-119), CCRF-HSB-2 (ATCC CCL-120.1), HPB-MLT (FCCH1019), EB-3 (ATCC CCL-85), M116 (ATCC CRL-1649). CCRF-SB (ATCC CCL-120), K-562 (ATCC CCL-243) or the like labeled ⁵¹ Cr. Then, the antibody to which the ADCC activity is measured and incubated is added to the labeled target cells. Effector cells are then added to the target cells in an appropriate ratio and incubation is continued.

After incubation, the supernatant is collected and its activity measured using a gamma counter, with 1% NP-40 being used to measure the maximum free radioactivity. Cytotoxic activity (%) can be calculated as (AC) / (BC) x 100, where A is the radioactivity (cpm) released in the presence of the antibody, B is the radioactivity (cpm) released by NP-40 and C is the radioactivity (cpm) released by the medium without antibodies.

4-3. Enhancement of cytotoxic activity

In order to exhibit cytotoxic activity, such as ADCC activity and CDC activity, it is advantageous to use Cy as a constant region (C region) in humans, especially Cy1 and e.

¹⁸

Cy3. Further, it is possible to induce stronger ADCC activity or CDC activity by adding, replacing or modifying a portion of the amino acids in the antibody C region.

As an example, the construction of an IgM-like IgM polymer (IgM-like) by amino acid substitution (Smith, RIF & Morrison, SL BIO / TECHNOLOGY (1994) 12, 683-688), the construction of an IgM-mimetic IgG (IgM) -like) by adding amino acids (Smith, RIF et al., J. Immunology (1995) 154, 2226-2236), expressing a tandem-linked gene encoding the L chain (Shuford, W. et al., Science (1991) 252, 724-727), dimerization of IgG by amino acid substitution (Caron PC et al., J. Exp. Med. (1992) 176, 1191-1195, Shopes, B., J. Immunology (1992) 148, 2918-2922), dimerization of IgG by chemical modification (Wolff, EA et al., Cancer Res. (1993) 53, 2560-2565) and introduction of effector function by altering the amino acid (s) in the hinge region of the antibody (Norderhaug,

L. et al., Eur. J. Immunol. (1991) 21, 2379-2384) and the like. These approaches can be accomplished either by using site-specific mutagenesis by a primer, by adding a base sequence by a restriction cleavage site, by using a chemical modifying agent that creates a covalent bond.

5. Confirmation of therapeutic effects

The effects of the therapeutic agent used in the present invention for lymphatic tumors can be confirmed by administering to the animals transplanted with lymphatic tumor cells the antibody used in the present invention and evaluating its anti-tumor effect on these animals.

As lymphatic tumor cells administered to animals, cells from established cell lines as well as freshly isolated cells can be used. From established cell lines, CCRF-CEM (ATCC CCL-119), HPB-MLT (FCCH1019), MOLT-4 (ATCC CRL-1582), CCRF-HSB-2 (ATCC CCL-120.1), and the like can be used as T cell lines, and CESS (ATCC TIB-190), SKW 6.4 (ATCC TIB-215), CCRF-SB (ATCC CCL-120), RPMI 6410 (FCCH6047), EB-3 (ATCC CCL-85) and the like, as a B cell line.

In animals transplanted, immunological functions are predominantly reduced or absent. For example, nude mice, SCID mice, beige mice, nude rats and the like can be used. The antitumor effects to be evaluated can be confirmed by measuring the volume and weight of the tumor or the survival time of the animal or the like.

As shown in the examples below, administration of anti-HM1.24 antibody results in suppression of tumor volume increase and, in addition, prolongation of survival time in tumor transplanted mice. These facts indicate that the anti-HM1.24 antibody has an antitumor effect on lymphatic tumors.

6. Method of administration and pharmaceutical preparation

The therapeutic agents for lymphatic tumors prepared according to the present invention can be administered either systemically or locally, by the parenteral route, eg by intravenous injection such as drop infusion, intramuscular injection, intraperitoneal injection and subcutaneous injection. The route of administration may be selected depending on the age and condition of the patient. The effective dosage is selected from the range of 0.01 mg to 100 mg per kg body weight per dose. Optionally, a dosage in the range of 1 to 1000 mg, more preferably 5 to 50 mg per patient may be selected.

Therapeutic agents for lymphatic tumors prepared according to the present invention may contain pharmaceutically acceptable carriers or additives depending on the mode of administration. Examples of such carriers or additives are water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylic acid, sodium alginate, water-soluble dextran, carboxymethyl starch, pectin, carboxymethylcellulose, ethylcellulose, xcellulose, xcellulose. arabic gum, casein, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose and pharmaceutically acceptable surfactants and the like. The additives to be used are selected, inter alia, from the aforementioned substances or combinations thereof, depending on the dosage form.

The diseases treated according to the present invention are lymphatic tumors (except myelomas) that have antigens on the tumor cells to which the antibodies used in the present invention bind. Specifically, acute B lymphatic leukemia (B-ALL), chronic B lymphatic leukemia (B-CLL), pre-B lymphoma, Burkitt lymphoma, follicular lymphoma, follicular envelope lymphoma, diffuse lymphoma, acute T lymphatic leukemia (T-ALL) , chronic T lymphatic leukemia (T-CLL), mature T cell leukemia (ATL), non-ATL peripheral T lymphoma (PNTL), and the like. The therapeutic agents prepared according to the present invention are useful as therapeutic agents for these lymphatic tumors.

9 9 9 9

9 · 9 9 9 9

Description of the picture

Giant. 1 shows a histogram of said B cell line, which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 2 shows a histogram of said B cell line, which was analyzed by FCM by indirect method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 3 shows a histogram of said B cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 4 shows a histogram of said B cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 5 shows a histogram of said B cell line which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 6 shows a histogram of said B cell line, which was analyzed by indirect FCM using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 7 shows a histogram of said B cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 8 shows a histogram of said B cell line, which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 9 shows a histogram of said B cell line, which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 10 shows a histogram of said T cell line which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 11 shows a histogram of said T cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 12 shows a histogram of said T cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 13 shows a histogram of said T cell line which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 14 shows a histogram of said T cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 15 shows a histogram of said T cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 16 shows a histogram of said T cell line which was analyzed by the indirect FCM method using anti-HMI24 antibody and control mouse IgG2a.

• · · · i

Giant. 17 shows a histogram of said T cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 18 shows a histogram of said T cell line which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 19 shows a histogram of said T cell line which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 20 shows a histogram of said non-T, non-B cell line that was analyzed by indirect FCM using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 21 shows a histogram of said non-T, non-B cell line, which was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 22 shows a histogram of said non-T, non-B cell line that was analyzed by the indirect FCM method using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 23 shows a histogram of said non-T, non-B cell line, which was analyzed by indirect FCM using anti-HM1.24 antibody and control mouse IgG2a.

Giant. 24 is a graph showing that the anti-HM1.24 antibody acts cytotoxically on CCRF-CEM, CCRF-HSB-2, and HPB-MLT T cell tumor lines, with a dose-dependent effect.

Giant. 25 is a graph showing that anti-HM1.24 antibody acts cytotoxically on EB cell lines EB-3, MC116 and CCRF-SB, with dose-dependent effect.

Giant. 26 is a graph showing that tumor volume increase in human lymphatic tumor transplanted mice is suppressed in the antibody-treated group as compared to the IgG2a control mouse group.

Giant. 27 is a graph showing that the survival time in mice transplanted with human lymphatic tumor was prolonged in the antiHM1.24 antibody group compared to the IgG2a control mouse group.

AND

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in more detail in the following examples. It is emphasized that the present invention is by no means limited to these examples.

Example 1. Construction of an anti-HM1.24 antibody.

1. Preparation of murine ascites containing anti-HM1.24 antibody

Hybridomas producing anti-HM 1.24 antibody were obtained according to the method of Goto, T. et al., (Blood (1994) 84, 1922-1930).

BALB / c mice (grown in CLEA Japan), which received 500 μΙ 2,6,10,14-tetramethylpentadecane (manufactured by Waco Pure Chemical Industries, Ltd.) intraperitoneally 11 and 3 days in advance, were injected intraperitoneally with 5 x 10 ⁶ hybridoma cells. . From day 10 post hybridoma cell injection, ascites that accumulated in the abdominal cavity of mice were collected using a Happycas # 19 needle (manufactured by Medikit), introduced over a longer period of time. The collected ascites were centrifuged twice at 1000 and 3000 rpm in a low-speed RLX-131 centrifuge (manufactured by Tomy Seiko) to remove hybridomas and contaminants such as blood cells and the like.

2. Purification of anti-HM 1.24 antibodies from mouse ascites

Purification of the anti-HM 1.24 antibody from the above murine ascites was performed as follows. After adding the same amount of PBS (-) to mouse ascites, the mixture was filtered on a Mediaprep hollow fiber filter (manufactured by MILLIPORE) and then affinity purified using a ConSep LC1000 high speed antibody purification apparatus (manufactured by MILLIPORE) and a Hyper D Protein A column (column volume). 20 ml, manufactured by Nihon Gaisi) using PBS (-) as absorption buffer and 0.1 M sodium citrate buffer (pH 4) as elution buffer according to the enclosed instructions. The eluted fractions were immediately adjusted to pH 7.4 by addition of 1 M Tris-HCl buffer (pH 8.0), then concentrated while simultaneously buffering the buffer with PBS (-) using a Centriprep 10 centrifugal ultrafiltration concentrator, further sterilized by filtration on a MILLEXGV membrane filter. (manufactured by MILLIPORE) with a pore size of 0.22 gm to obtain purified anti-HM 1.24 antibody.

• ·

* • ·· · • · • • • ·· • • • •

3. Determination of antibody concentration

The concentration of purified antibody was determined by absorbance measurement. Thus, the purified antibody was diluted in PBS (-), the absorbance at 280 nm was measured, and the concentration was calculated assuming that 1.35 OD corresponds to 1 mg / ml.

Example 2. Study of reactivity of anti-HM 1.24 antibody with lymphatic tumor cells

1. Purification of control mouse IgG2a

Control mouse IgG2a was purified as follows. Commercially available IgG2a (KAPPA) (UPC 10) ascites (manufactured by CAPPEL) were dissolved in purified water and PBS (-). The solution was filtered on an Acrodisc membrane filter (manufactured by Gelman Sciences) with a pore size of 0.2 µm a) and then affinity purified using a ConSep LC1000 high speed antibody purification apparatus (manufactured by MILLIPORE) and a Hyper D Protein A column (20 ml column volume, manufactured). Nihon Gaisi) using PBS (-) as absorption buffer and 0.1 M sodium citrate buffer (pH 4) as elution buffer according to the enclosed instructions. The eluted fractions were immediately adjusted to pH 7.4 by addition of 1 M Tris-HCl buffer (pH 8.0), then concentrated and simultaneously exchanged buffer with PBS (-) using a Centriprep 10 centrifugal ultrafiltration concentrator, further sterilized by filtration on a MILLEX membrane filter. -GV (manufactured by MILLIPORE) having a pore size of 0.22 µm, thereby obtaining purified control mouse IgG2a.

Determination of the concentration of control mouse IgG2a was performed as described in section 3 above. Determination of antibody concentration.

2. FCM analysis

The reactivity of the anti-HM 1.24 antibody to lymphatic tumor cells was examined by flow cytometric analysis (FCM). After washing of T cell lines RPMI 8402 (ATCC CRL-1195), CCRF-CEM (ATCC CCL-119) derived from acute lymphoblastic leukemia, HPB-ALL (FCCH1018) HPB-MLT (FCCH1019) derived from T lymphoma, JM (FCCH1023) derived from acute lymphoblastic leukemia, MOLT-4 (ATCC CRL-1582) derived from acute lymphoblastic leukemia, Jurkat (FCCH1024) derived from acute lymphoblastic leukemia, CCRF-HSB-2 (ATCC CCL-120.1) derived from acute lymphoblastic leukemia, MT- 1 (FCCHG1043) derived from mature T cell leukemia and Lennert's lymphoma-derived KT-3 (Shimizu, S. et al., Blood (1988) 71,196-203) as EB cell transformed CESS line (ATCC) can be used as B cell line TIB-190), EB virus-positive B cell SKW 6.4 (ATCC TIB-215), MC116 (ATCC CRL-1649) derived from B lymphoma, CCRF-SB (ATCC • fl · flfl ·· ·

CCL-120) derived from acute lymphoblastic leukemia, RPMI 6410 B cell (FCCH6047) derived from acute myelocytic leukemia patient, Daudi (ATCC CCL213) derived from Burkitt's lymphoma, Jijoye (ATCC CCL-87) from Burkitt's lymphoma, Paradise (ATCC CCL- 86) derived from Burkitt's lymphoma, and as a non-T, non-B cell line HL-60 (ATCC CCL-240) derived from acute myelocytic leukemia, THP1 (ATCC TIB-202) derived from acute monocytic leukemia, U-937 ( ATCC CRL-1593) derived from connective lymphoma and K-562 (ATCC CCL-243) derived from chronic myelocytic leukemia in PBS (-), is added to 100 μΙ of antibodies or purified, diluted to 25 gg / ml in FACS buffer (PBS ( -) containing 2% fetal bovine serum and 0.1% sodium azide) and incubated on ice for 30 minutes.

After washing with FACS buffer, 100 μΙ 25 μg / ml FITC-labeled goat anti-mouse antibody (GAM) is added and incubated on ice for 30 minutes. After washing with FACS buffer, cells were suspended in 600 μΙ or 1 ml of FACS buffer and fluorescence intensity was measured for each cell suspension using a FACScan (manufactured by Becton Dickinson). The results shown in Figures 1 to 23 confirm that all T cell lines and all B cell lines (except Daudi and Paradise that do not respond) reacted with anti-HM

1.24 antibody and strongly expressed HM 1.24 antigen. On the other hand, no non-T, non-B cell line reacted with anti-HM 1.24 antibody and HM antigen expression

1.24 was not detected in any of them.

In the histograms shown in Figures 1 to 23, the histogram conditions were adjusted such that the negative cells formed 98% and the positive cells 2% when stained with control mouse IgG2a. According to these histogram conditions, the percentage of HM 1.24 antigen positive cells was calculated when an anti-HM 1.24 antibody was used and the result is shown in Table 1. According to the percentage of HM 1.24 antigen positive cells, the expression level was differentiated into five states: , +/-, +, ++, and +++. It was confirmed that all T cell lines and B cell lines (except Daudi and Paradise) strongly expressed HM 1.24 antigen, as shown in the results in Figures 1 to 23. Also in all cases of non-T, non-B cell lines was the percentage of HM 1.24 antigen positive cells is zero or less than 5%, indicating that the expression of the antigen is low or very low.

·· · 0 * 0 0 · 0 ··· 000

Table 1

Cell name Expression level

B cell lines CESS +++ 94.5 ENW 6.4 +++ 92.8 MC116 ++ 65.0 CCRF-SB +++ 98.4 RPMI 6410 +++ 94.5 EB-3 +++ 88.3 Jijoye +++ 92.3 Daudi - 2.8 Paradise - 2,0 T cell lines RPMI 8402 +++ 94.0 CCRF-CEM +++ 97.8 HPB-ALL ++ 63.8 HPB-MLT +++ 94.6 JM +++ 99.6 MOLT-4 +++ 84.1 Jurkat ++ 70.9 CCRF-HSB-2 +++ 100.0 MT-1 +++ 95.9 KT-3 +++ 96.0 Non-T, Non-B HL-60 - 2.9 cell line THP-1 - 1.5 U-297 - 1.1 K-562 3. 9 -. <5%; +/-, 5-20%; +, 20-50%; ++, 50-80%; +++,> 80%

Example 3. Determination of CDC activity

The CDC activity of the anti-HM 1.24 antibody against lymphatic tumor cells was determined as follows.

♦ ΦΦΦ φ>>>>>> ► ►

1. Preparation of target cells

CCRF-CEM (ATCC CCL-119) derived from acute lymphoblastic leukemia, CCRF-HSB-2 (ATCC CCL-120.1) derived from acute lymphoblastic leukemia, HPB-MLT (FCCH1019) derived from T lymphoma, EB-3 were prepared as target cells (ATCC CCL-85) derived from Burkitt's lymphoma, MC116 (ATCC CRL-1649) derived from B lymphoma, CCRF-SB (ATCC CCL-120) derived from acute lymphoblastic leukemia and K562 (ATCC CCL-243) derived from acute myelocytic leukemia at concentration 4 × 10 ⁵ cells / ml in RPMI1640 medium containing 10% fetal bovine serum (manufactured by GIBCO-BRL). Fifty μΙ of each cell suspension was added to a 96-well flat-bottomed plate (manufactured by FALCON) and incubated in 5% CO ₂ in a high humidity incubator (manufactured by TABAI) overnight at 37 ° C.

2. Preparation of anti-HM 1.24 antibody

The purified anti-HM 1.24 antibody obtained in Example 1 above was prepared at 0, 0.2, 2 and 20 gg / ml in RPMI1640 medium containing 10% fetal bovine serum (manufactured by GIBCO-BRL) and 50 µΙ of these antibodies were was added to the 96-well (flat bottom) plate prepared in the above paragraph 1. After incubation of the plate in 5% CO ₂ in a high humidity air incubator (manufactured by TABAI) at 37 ° C for 60 minutes, it was centrifuged in a low-turn. centrifuge 05PR-22 (manufactured by Hitachi) at 1000 rpm for 5 minutes and then 50 μΙ of the supernatant was removed.

3. Preparation of complement

Baby Rabbit Complement (manufactured by CEDARLANE) was dissolved in purified water of 1 ml per vial, which was further diluted in 5 ml of RPMI1640 medium (manufactured by GIBCO-BRL) that did not contain fetal calf serum (FCS). Fifty μΙ of this solution was expanded into a 96-well (flat-bottomed) plate prepared in paragraph 2 above and incubated in 5% CO ₂ in a high humidity incubator (manufactured by TABAI) at 37 ° C for 2 hours.

4. Determination of CDC activity

At the end of the incubation, 10 μΙ Alamar Bule (manufactured by BIO SOURCE), prepared in paragraph 3 above, was added to each well of a 96-well (flat-bottomed) plate and incubated in 5% CO ₂ in a high humidity incubator (manufactured by TABAI). ) at 37 ° C for 4 hours. Fluorescence intensity (excitation wavelength 530 nm, emission wavelength 590 nm) was then measured for each well using a CytoFluor 2350 measurement system (manufactured by MILLIPORE). Cytotoxic activity (%) can be calculated as (AC) / (BC) x 100, where A is the fluorescence intensity when incubating in the presence of antibody, B is the fluorescence intensity when incubating the antibody-free medium alone, and C is the fluorescence intensity in the cell-free well.

The results showed, as shown in Figures 24 and 25, that K562 cells that did not react with anti-HM 1.24 antibody in FCM analysis showed no cytotoxic activity even after the addition of anti-HM 1.24 antibody, while CCRF-CEM, CCRF-HSB- 2, HPB-MLT, EB-3, MC116 and CCRF-SB, which react with anti-HM 1.24 antibody, show cytotoxic activity, the extent of which is dependent on the concentration of anti-HM 1.24 antibody. This clearly demonstrates that the anti-HM 1.24 antibody exhibits CDC activity against lymphatic tumors having on their cell surface an antigenic protein to which anti-HM

1.24 the antibody specifically binds.

Example 4. Anti-tumor effects of anti-HM 1.24 antibody in mice transplanted with human lymphatic tumor

1. Preparation of an antibody for administration

1-1. Preparation of anti-HM 1.24 antibody

The purified anti-HM 1.24 antibody obtained in Example 1 above was

1- 2. Preparation of control mouse IgG2a

The purified antibody obtained in Example 2 above was prepared at a concentration of 1 mg / ml in PBS (-) sterilized by filtration and used in the following experiments.

2. Anti-tumor effects of anti-HM 1.24 antibody in mice transplanted with human lymphatic tumor

2- 1. Preparation of mice transplanted with human lymphatic tumor

Human lymphatic tumor transplanted mice were prepared as follows. CCRF-HSB-2 cells (ATCC CCL-120.1) derived from acute lymphoblastic leukemia that were grown in vivo in SCID mice (CLEA Japan) were diluted to a concentration of 1 x 10 ⁸ cells / ml in RPMI1640 medium containing 10% fetal bovine serum (manufactured by GIBCO-BRL). The cell suspensions thus prepared were administered subcutaneously in the abdomen of SCID mice (male, 6 weeks old), each of whom received 100 μΙ anti-asialo GM1 (Wako Pure Chemical Industries, Ltd.) intraperitoneally the previous day.

φ · φ φ φ · · · · · · · · φ • • · φ · φ φ φ φ φ · φ φ φ φ φ φ φ φ φ φ φ φ φφφ φφφ φ φ • φ φφ

2-2. Administration of antibodies

On day 7 after tumor transplantation, the tumor diameter at the site where CCRF-HSB-2 cells were transplanted was measured by caliper. After counting the tumor volume, the animals were grouped so that the average tumor volume in each group was almost the same (8 animals per group, 3 groups). From the same day, each group was administered intraperitoneally with 100 μΙ anti-HM 1.24 antibody at concentrations of 1 mg / ml or 200 pg / ml, or 1 mg / ml of control mouse IgG2a prepared as described in paragraph 1 above. were given twice a week, a total of 19 times, always in a similar manner. During this time, the tumor diameter was measured twice a week by caliper and its volume calculated.

2-3. Evaluation of anti-tumor effect of anti-HM 1.24 antibody in mice transplanted with human lymphatic tumor

The anti-tumor effect of the anti-HM 1.24 antibody was evaluated by changes in tumor volume and survival time in mice. As a result, as shown in Figure 26, the increase in tumor volume was suppressed in the antiHM 1.24 antibody-treated group as compared to the control mouse IgG2a-treated group. Also, as shown in Figure 27, an increase in survival time was observed in the anti-HM 1.24 antibody treated group compared to the control mouse IgG2a group. These facts indicate that the anti-HM 1.24 antibody has an anti-tumor effect in mice transplanted with a human lymphatic tumor.

Reference Example 1. Preparation of hybridomas that produce a murine anti-HM 1.24 monoclonal antibody

Hybridomas producing a murine anti-HM 1.24 antibody were prepared according to the method of Goto, T. et al., Blood (1994) 84, 1992-1930).

The plasma cell line of KPC-32 (1 x 10 ⁷ ), derived from the bone marrow of a patient with multiple human myeloma (Goto, T. et al., Jpn. J. Clin. Hematol. (1991) 32, 1400), was each. injected six weeks into the abdominal cavity of BALB / c mice (manufactured by Charles River).

Three days before sacrifice, 1.5 x 10 ⁶ KPC-32 cells were injected into the spleen to further enhance the ability to produce antibodies (Goto, T. et al., Tokushima J. Exp. Med. (1990) 37, 89). After the animals were sacrificed, the spleen was removed and the cell was fused to the SP2 / 0 myeloma cells according to the Groth, de St. &Amp; Schreidegger (Cancer Research (1981) 41, 3465).

· · Ta ta ta ta ta ta ta ta ta ta ta ta ta ta * * a a a a a a a a a a

By using an ELISA assay using KPC-32 cells (Posner, MR et al., J. Immunol. Methods (1982) 48, 23), the hybridoma culture supernatant was tested for the presence of antibodies. 5 x 10 ⁴ KPC-32 cells were suspended in 50 ml PBS and then dispensed into a 96-well plate (U-bottom, Corning, manufactured by Iwaki), which was then air-dried at 37 ° C overnight. After blocking with PBS containing 1% bovine serum albumin (BSA), hybridoma culture supernatant was added and incubated at 4 ° C for 2 hours. The reaction was then performed for 1 hour at 4 ° C with a peroxidase-labeled goat anti-mouse IgG antibody (manufactured by ZYMED). After washing, a solution of o-phenylenediamine (manufactured by Sumimoto Bakelite) was added and the reaction was allowed to proceed at room temperature for 30 minutes.

The reaction was stopped by the addition of 2 N sulfuric acid and the absorbance was measured at 492 nm on an ELISA reader plate reader (manufactured by Bio-Rad). To remove the hybridoma that generates antibodies against human immunoglobulin, the positive hybridoma supernatant was pre-absorbed with human serum and reactivity with other cell lines was determined by ELISA. Positive hybridomas were selected and their reactivity with different cell lines was determined by flow cytometry. The last selected hybridoma clone was cloned twice and injected into the abdominal cavity of the BALB / c mice treated with the landing, and ascites were obtained therefrom.

Monoclonal antibodies from mouse ascites were purified by ammonium sulfate precipitation and Protein A affinitt chromatography kit (Ampure PA, manufactured by Amersham). Purified antibodies were labeled with FITC using a Quick Tag FITC biding kit (manufactured by Boehringer Mannheim).

As a result, monoclonal antibodies produced by 30 hybridoma clones reacted with KPC-32 and RPMI 8226 cells.

After cloning, the reactivity of the supernatant of these hybridomas with other cell lines or peripheral blood mononuclear cells was examined.

Three of these clones produced monoclonal antibodies that specifically reacted with plasma cells. Of these three clones, the clone that was most useful for flow cytometric analysis and had CDC activity against RPMI 8226 was selected and designated as HM1.24. The monoclonal antibody subclass produced by this hybridoma was determined by ELISA using rabbit anti-mouse antibodies specific for each subclass (manufactured by Zymed). The anti-HM 1.24 antibody was a subclass of IgG2a to. Hybridoma HN1.24, which produces anti-HM 1.24 antibodies, was deposited for international use under the provisions of the Budapest Treaty as FERM BP-5233 on September 14, 1995 at the National Institute of Bioscience and Human- Technology, Industrial Science and Technology Agency, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan.

Reference Example 2. Preparation of an altered anti-HM 1.24 antibody

The altered anti-HM 1.24 antibody was obtained as follows.

Total RNA was prepared from the HM1.24 hybridoma prepared in Reference Example 1 in a conventional manner. Accordingly, cDNA encoding the cDNA was synthesized

V region of mouse antibody, amplified by polymerase chain reaction (PCR) and 5'-RACE method. A DNA fragment containing the gene encoding the mouse was obtained

In the region, it was ligated into the pUC cloning vector and then introduced into competent E. coli cells to obtain E. coli transformants. The above plasmid was obtained from transformants. The nucleotide sequence of the cDNA coding regions in the plasmid was determined by a conventional procedure and the complementarity determining region (CDR) was determined for each V region.

In order to construct a vector expressing the chimeric anti-HM 1.24 antibody, a cDNA encoding the V region of each L chain and H chain of the murine anti-HM 1.24 antibody was inserted into the HEF vector. Further, in order to construct the altered antiHM 1.24 antibody, the V region of the CDR of the murine anti-HM 1.24 antibody was linked to a human antibody. The human antibody L chain L1 was used as the human antibody L chain, FR 1-3 human HG3 antibodies were used as framework regions (FRs) 1-3 human antibody H chain, and FR4 human JH6 antibody was used for FR4. Some amino acids in the FR of the H chain V region have been changed so that the CDRs of the altered antibody can create a suitable antigen binding site.

In order to express the L chain and H chain genes and thereby construct the altered anti-HM 1.24 antibody in a mammalian cell, each gene was separately introduced into the HEF vector to generate vectors expressing either the L chain or the H chain of the altered anti-HM 1.24 antibody .

By co-introducing these two expression vectors into CHO cells, a cell line was produced that produced the altered anti-HM 1.24 antibody. The antigen-binding activity and the binding inhibitory activity of the reshaped anti-HM 1.24 antibody obtained by culturing this cell line for the human amnion cell line WISH was determined by ELISA using cells. The results showed that the altered anti-HM 1.24 antibody has the same antigen binding activity as the chimeric antibody, and using the biotinylated mouse anti-HM 1.24 antibody also showed the same binding inhibitory activity as the chimeric antibody or mice antibodies.

Incidentally, E. coli carrying a plasmid containing DNA encoding the L chain V region of the chimeric antibody and E. coli carrying a plasmid containing DNA encoding the H chain V region of the chimeric antibody were deposited for international use under the provisions of the Budapest Treaty as Escherichia coli DH5a (pUC19-1.24L-gK) and Escherichia coli DH5a (pUC19-1.24H-gy1) on August 29, 1996 at the National Institute of Bioscience and Human-Technology, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, such as FERM BP-5646 and FERM BP5644. Furthermore, an E. coli carrying a plasmid containing DNA encoding the α version (SEQ ID NO:

2) In the L chain region and E. coli carrying a plasmid containing the DNA encoding version r (SEQ ID NO: 3) In the H chain region, the altered anti-HM 1.24 antibodies were deposited for international use under the provisions of the Budapest Treaty as Escherichia coli DH5a (pUC19-RVLa-AHM-gK) and Escherichia coli DH5a (pUC19-RVHr-AHM-gYl) on August 29, 1996 at the National Institute of Bioscience and Human-Technology, Higashi 1-chome, Tsukuba city , Ibaraki pref., Japan, such as FERM BP-5645 and FERM BP-5643. Furthermore, an E. coli carrying a plasmid containing a DNA encoding a version with (SEQ ID NO: 4) of the L chain V region altered by anti-HM

1.24 the antibody was deposited for international use under the provisions of the Budapest Treaty as Escherichia coli DH5a (pUC19-RVHs-AHM-gy1) on September 29, 1997 at the National Institute of Bioscience and Human-Technology, Higashi 1-chome, Tsukuba city, Ibaraki pref., Japan, as FERM BP-6127.

Reference Example 3. Cloning of cDNA encoding HM1.24 antigen protein cDNA encoding HM1.24 antigen protein that is specifically recognized by anti-HM

1.24 antibody was cloned.

1. Construction of cDNA library

1) Preparation of total RNA

Total RNA was derived from the human multiple myeloma cell line KPMM2 by total RNA using the method of Chirgwin et al., (Biochemistry, 18, 5294 (1979)). Thus, 2 x 10 ⁸ KPMM2 cells were completely homogenized in 20 ml of 4 M guanidine isothiocyanate (manufactured by Nacalai Tesque Inc.). The homogenate was layered on 5.3 M cesium chloride in a centrifuge tube, which was then centrifuged in the rotor.

Backman SW40 at 31,000 rpm at 20 ° C for 24 hours to precipitate RNA. The RNA precipitate was washed with 70% ethanol and dissolved in 300 μ110 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 0.5% SDS. Pronase (manufactured by Boehringer) was added to a concentration of 0.5 mg / ml and incubated for 30 minutes at 37 ° C. The mixture was extracted with phenol-chloroform and RNA was precipitated with ethanol. The RNA precipitate was then dissolved in 200 µl of 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA.

2) Preparation of poly (A) + RNA

Poly (A) + RNA was purified using 500 µg of total RNA material prepared as described above using the Fast Track 2.0 mRNA Isolation Kit (manufactured by Invitrogen) according to the instructions included with the kit.

3) Construction of a cDNA library

The double stranded cDNA was synthesized and 10 µg of the above poly (A) + RNA was used as material using the TimeSaver cDNA Synthesis Kit (manufactured by Pharmacia) according to the kit instructions, then ligated to the EcoRI adapter included in the kit Directional Cloning Toolbox (manufactured by Pharmacia) according to the instructions included with the kit. Kinase and digestion of the EcoRI adapter with a NotI restriction enzyme were performed according to the instructions included with the kit. Further, a double-stranded adapter-attached cDNA of 500 base pairs or more was separated and purified using a 1.5% low melting agarose gel (manufactured by Sigma) to obtain a 40 μΙ double-stranded adapter-attached cDNA.

The double stranded cDNA constructed with the attached adapter was ligated with T4 ligase (manufactured by GIBCO-BRL) to the pCOS1 vector (Japanese Patent Application 8-255196), which had been previously treated with EcoRI and Notl and alkaline phosphatase (manufactured by Takara Shuzo) a cDNA library constructed. The constructed cDNA library was introduced into E. coli strain DH5α (manufactured by GIBCO-BRL) and subsequently established as an independent clone having a total size of 2.5 χ 10 ⁶ .

2. Cloning by direct expression method

1) Transfection of COS-7 cells

Approximately 5 10 10 ⁵ clones of the above transduced E. coli were grown in 2-YT medium (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring). »» ····

44 44 44 444444 • · · · · · · ·

444 444 444 44 44 4 · ³³

Harbor Laboratory Press (1989)), containing 50 µg / ml ampicillin to cause cDNA amplification, was then processed by the alkaline method (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press (1989)) to plasmid DNA from E. coli. The plasmid DNA thus obtained was electroporated into COS-7 cells using a Gene Pulser (manufactured by Bio-Rad).

Ten µg of purified plasmid DNA was added to 0.8 ml of a COS-7 cell solution in which the cells were suspended in PBS at a concentration of 1 x 10 ⁷ cells / ml and the mixture was subjected to pulses of 1500 V and a capacity of 25 pFD. After a 10-minute recovery period at room temperature, electroporated cells were grown in DMEM culture medium (manufactured by GIBCO-BRL) containing 10% fetal bovine serum (manufactured by GIBCO-BRL) at 37 ° C and 5% CO ₂ for 3 days. .

2) Preparation of adsorption plate

An adsorption plate coated with a mouse anti-HM 1.24 antibody was prepared by the method of B. Seed et al., (Proc. Natl. Acad. Sci. USA, 84, 3365-3369 (1987)). Mouse anti-HM

1.24 antibody was added to 50 mM Tris-HCl, pH 9.5 at a concentration of 10 µg / ml. Three ml of the thus prepared antibody solution was added to a 60 mm diameter tissue culture plate and incubated at room temperature for 2 hours. Then the plate was washed three times with 0.15 M NaCl solution and PBS containing 5% fetal bovine serum, 1 mM EDTA, 0.02% NaN ₃ was added. After blocking, the plate thus prepared was used for subsequent cloning.

3) Cloning the cDNA library

COS-7 cells transfected as described above were dispersed in PBS containing 5 mM EDTA. After one wash with PBS containing 5% fetal bovine serum, cells were suspended in PBS containing 5% fetal bovine serum and 0.02% NaN ₃ to a concentration of 1 x 10 ⁶ cells / ml. The suspension was then added to the adsorption plate prepared as described above and incubated at room temperature for 2 hours. After three careful washes with PBS containing 5% fetal bovine serum and 0.02% NaN ₃ , plasmid DNA was isolated from cells bound to an adsorption plate, using a solution containing 0.6% SDS and 10 mM EDTA.

The obtained plasmid DNA was transduced into Escherichia coli DH5α cells.

After amplification of the plasmid DNA as described above, the DNA was isolated by an alkaline method. The obtained plasmid DNA was transfected by electroporation into COS-7 cells and plasmid DNA isolated from adsorbed cells as described above. A similar procedure was repeated once more and the plasmid DNA obtained was digested with the restriction enzymes EcoRI and NotI. Finally, a 0.9 kbp insert concentration was confirmed. Further, E. coli cells transduced with a portion of the obtained plasmid DNA were plated on 2-YT agar plates containing 50 gg / ml ampicillin. After overnight culture, DNA was isolated from one colony. This was digested with the restriction enzymes EcoRI and NotI, and a clone p3.19 bearing the 0.9 kbp insert was obtained.

The base sequence of this clone was determined using the PRISM, Dye Terminator Cycle Sequencing Kit (manufactured by Perkin Eimer) according to the instructions provided with the kit and the DNA sequencer ABI 373A (manufactured by Perkin Eimer). Its nucleotide sequence and corresponding amino acid sequence are shown in SEQ ID NO: 1.

Industrial applicability

The results of FCM analysis showed that the anti-HM 1.24 antibody reacts strongly with most cells derived from human lymphatic tumors, suggesting that in many lymphatic tumors the polypeptide containing the epitope recognized by the anti-HM 1.24 antibody is strongly expressed. Furthermore, in mice transplanted with a human tumor that reacts with an anti-HM 1.24 antibody, administration of the anti-HM 1.24 antibody results in suppression of the increase in tumor volume and, in addition, an increase in the animal's survival time. These facts indicate that an anti-HM 1.24 antibody or an antibody recognizing a polypeptide that contains an epitope recognized by an anti-HM 1.24 antibody has cytotoxic activity for many lymphatic tumors. This suggests that the antibody may be useful for treating patients with lymphatic tumors.

• * • φ • φ · · · · 49 • 49

List of organisms deposited under the Patent Cooperation Treaty, Rule 13-2 and the name of the depositing institute

Title: National Institute of Bioscience and Human Technology

Industrial Science and Technology, MITI

Address: Higashi 1-chome, Tsukuba City, Ibalaki Pref., Japan

Microorganism (1)

Name: Escherichia coli DH5 (pRS38-pUC19)

Accession No: FERM BP-4434 Date of storage: 5 October 1993

Microorganism (2)

Name: hybridoma HM1.24 Accession No: FERM BP-5233 Date of storage: 14 September 1995

Microorganism (3)

Name: Escherichia coli DH5 (pUC19-RVHr-AHM-gy1)

Accession No: FERM BP-5643 Date of storage: 29 August 1996

Microorganism (4)

Name: Escherichia coli DH5 (pUC19-1.24H-gy1)

Accession No: FERM BP-5644 Date of storage: 29 August 1996

Microorganism (5)

Name: Escherichia coli DH5 (pUC19-RVLa-AHM-gK)

Accession No: FERM BP-5645 Date of storage: 29 August 1996

Microorganism (6)

Name: Escherichia coli DH5 (pUC19-1.24L-gK)

Accession No .: FERM BP-5646 • · · · · ··

Date of deposit: August 29, 1996

Microorganism (7)

Name: Escherichia coli DH5a (pUC19-RVHs-AHM-gy1) Accession No .: FERM BP-6127 Date Added: September 29, 1997 • · · · · ·· · · · · · · · · · · ♦ ·· · ·· · 9 · »

SEQUENCE LIST <110> CHUGAI SEIYAKU KABUSHIKI KAISHA <120> Lymphatic Tumor Treatment Agent <130> E908 <140> PCT / JP98 / 00568 <141> 1998-02-12 <150> JP 9-41410 <151> 1997-02 -02 <160> 8 <210> 1 <211> 1013 <212> DNA <213> Homosapiens <223> Nucleotide sequence encoding HM 1.24 antigen <400> 1 gaattcggca cgagggatct gg atg gca tet act tcg tat gac tat tgc 49

Met Ala Ser Thr Ser 5 Tyr Asp Tyr Cys aga gtg CCC atg gaa gac ggg gat aag ege tgt aag ctt ctg ctg ggg 97 Arg Wall For Met Glu Asp Gly Asp Lys Arg Cys Lys Leu Leu Leu Gly 10 15 Dec 20 May 25 ata gga att ctg gtg ctc ctg atc atc gtg att ctg ggg gtg CCC ttg 145 Ile Gly Ile Leu Wall Leu Leu Ile Ile Wall Ile Leu Gly Wall For Leu 30 35 40 att atc ttc acc atc aag gcc aac age gag gcc tgc cgg gac ggc ctt 193 Ile Ile Phe Thr Ile Lys Ala Asn Ser Glu Ala cys Arg Asp Gly Leu 45 50 55 cgg gca gtg atg gag tgt ege aat gtc acc cat ctc ctg caa caa gag 241 Arg Ala Wall Met Glu Cys Arg Asn Wall Thr His Leu Leu Gin Gin Glu 60 65 70 ctg acc gag gcc cag aag ggc ttt cag gat gtg gag gcc cag gcc gcc 289 Leu Thr Glu Ala Gin Lys Gly Phe Gin Asp Wall Glu Ala Gin Ala Ala 75 80 85

• a a · · a a a · ·

acc tgc aac cac act gtg atg gcc cta atg gct tcc ctg gat gca gag 337 Thr Cys Asn His Thr Val Met Ala Leu Met Ala Ser Leu 90 95 100 105 aag gcc caa gga caa aag aaa gtg gag gag gtt gag gag gag atc act 385 Lys Ala Gin Gly Gin Lys Lys Val Glu Glu Glu Glu Glu Glu Ile Thr 110 115 120 aca tta aac cat aag ctt cag gac gcg tet gca gag gg ega ctg 433 Thr Leu Asn His Lys Leu Gin Asp Ala Ser Glu Val Glu Arg Leu 125 130 135 aga aga gaa aac cag gtc tta agc gtg aga atc gcg aac aag tac 481 Arg Arg Glu Asn Gin Val Leu Ser Wall Arg Ile Ala Lys Lys Tyr 140 145 150 tray ccc agc tcc cag gac tcc agc tcc gct gcg gcg ccc cag ctg ctg 529 Tyr Pro Ser Ser Gin Asp Ser Ser Ser Ala Ala Ala For Gin Leu Leu 155 160 165 att gtg ctg ctg ggc ctc agc gct ctg ctg cag tga gatcccagga 575 Ile Val Leu Gly Leu Ser Ala Leu Leu Gin 170 175 180

agctggcaca tcttggaagg tccgtcctgc tcggcttttc gcttgaacat tcccttgatc 635 tcatcagttc tgagcgggtc atggggcaac acggttagcg gggagagcac ggggtagccg 695 gagaagggcc tctggagcag gtctggaggg gccatggggc agtcctgggt ctggggacac 755 agtcgggttg acccagggct gtctccctcc agagcctccc tccggacaat gagtcccccc 815 tcttgtctcc caccctgaga ttgggcatgg ggtgcggtgt ggggggcatg tgctgcctgt 875 tgttatgggt tttttttgcg gggggggttg cttttttctg gggtctttga gctccaaaaa 935 aataaacact tcctttgagg gagagcacac cttaaaaaaa aaaaaaaaaa aaaaaaaaaa 995 aaaattcggg cggccgcc 1013 <210> 2 <211> 379 <212> DNA <213> Artificial sequence <220>

<221>

<222>

<223> Nucleotide DNA sequence encoding the V chain of the V region and the altered anti-HM 1.24 antibody <400> 2

atg gga tgg agc tgt atc atc ctc tcc ttg gta gca aca gct aca ggt 48 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Wall Ala Thr Ala Thr Gly -15 -10 -5 gtc cac tcc gac atc cag atg acc cag agc approx agc agc ctg agc gcc 96 Wall His Ser Asp Ile Gin Met Thr Gin Ser For Ser Ser Leu Ser Ala -1 1 5 10 agc gtg ggt gac aga gtg acc atc acc tgt aag gct agt cag gat gtg 144 Ser Wall Gly Asp Arg Wall Thr Ile Thr Cys Lys Ala Ser Gin Asp Wall 15 Dec 20 May 25 aat act gct gta gcc tgg tray cag cag aag approx gga aag gct approx aag 192 Asn Thr Ala Wall Ala Trp Tyr Gin Gin Lys For Gly Lys Ala For Lys 30 35 40 45

* · · · · · · · · · · · · · · · · · · · ·

ctg ctg atc tray tcg gca tcc aac cgg tray act ggt gtg approx agc aga 240 Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Wall For Ser Arg 50 55 60 ttc agc ggt agc ggt agc ggt acc gac ttc acc ttc acc atc agc agc 288 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 ctc cag approx gag gac atc gct acc tray tray tgc cag caa cat melt agt 336 Leu Gin For Glu Asp Ile Ala Thr Tyr Tyr Cys Gin Gin His Tyr Ser 80 85 90 act approx ttc acg ttc ggc caa ggg acc aag gtg gaa atc aaa C 379 Thr For Phe Thr Phe Gly Gin Gly Thr Lys Wall Glu Ile Lys

100 105 <210> 3 <211> 418 <212> DNA <213> Artificial sequence <220>

<221>

<222>

<223> Nucleotide DNA sequence encoding the H chain V region of version r of the modified anti-HM 1.24 antibody <400> 3

atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct approx ggt 48 Met Asp Trp Thr Arg Wall Phe Phe Leu Val Ala For Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tet ggg gct gag gtg aag aag 96 Ala His Ser Gin Val Gin Leu Wall Gin Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gca tet gga tac acc ttc 144 Pro Gly Ala Ser Lys Wall Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 Dec 20 May 25 act ccc tac tgg atg cag tgg gtg ega cag gcc cct gga caa ggg ctt 192 Thr Pro Tyr Trp Met Gin Trp Wall Arg Gin Ala For Gly Gin Giy Leu 30 35 40 45 gag tgg atg gga tet att ttt cct gga gat ggt gat act agg tray agt 240 Glu Trp Met. Gly Ser Ile Phe For Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gtc acc atg acc gca gac aag tcc acg agc 288 Gin Lys Phe Lys Arg Wall Thr Met Thr Ala Asp Lys Ser Thr Ser 65 70 75 aca gcc tac atg gag ctg agc agc ctg aga tet gag gac acg gcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Wall 80 85 90 tat tac tgt gcg aga gga tta ega ega ggg ggg tac tac ttt gac tray 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr

100 tgg ggg caa gg acc acg gtc acc gtc Trp Gly Gin Thr Thr Val

105 tcc tca g Ser

120

418

• MO 0 · 0 00 00 0 0 0 «0 0 0 0 0 0 0 0 • 00 • 0 0 • » 0 1 • 0 0 0 0 0 • 0 0 0 0 0 1 0 0 0 0 0 0 • 0 0 0 0 0 0 0 » 0 0 0 «

<210> 4 <211> 418 <212> DNA <213> Artificial sequence <220>

<221>

<222>

<223> Nucleotide DNA sequence encoding the H chain V region of the anti-HM 1.24 antibody <400> 4 version

atg gac tgg acc tgg agg gtc ttc ttc ttg ctg gct gta gct approx ggt 48 Met Asp Trp Thr Trp Arg Wall Phe Phe Leu Leu Gly -15 -10 -5 gct cac tcc cag gtg cag ctg gtg cag tet ggg gag gag gag aag aag 96 Ala His Ser Gin Val Gin Leu Wall Glu Ser Gly Ala Glu Val Lys Lys -1 1 5 10 cct ggg gcc tca gtg aag gtt tcc tgc aag gca tet gga tac acc ttc 144 For Gly Ala Ser Val Lys Wall Ser Cys Lys Ala Gly Tyr Thr Phe 15 Dec 20 May 25 act CCC tac tgg atg cag tgg gtg ega cag gc cct gga caa ggg ctt 192 Thr For Tyr Trp Met Gin Trp Wall Arg Gin Ala For Gly Gin Gly Leu 30 35 40 45 gag tgg atg gga tet att ttt cct gga gat ggt gat act agg tac agt 240 Glu Trp Met Gly Ser Ile Phe For Gly Asp Thr Arg Tyr Ser 50 55 60 cag aag ttc aag ggc aga gtc acc atac acc gca gac aag tcc acg age 288 Gin Lys Phe Lys Gly Arg Wall Thr Ile Thr Ala Lys Ser Thr Ser 65 70 75 and ca gcc tac atg gag ctg age age ctg aga tet gag gac acg gcc gtg 336 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg. Ser Glu Asp Thr Ala Wall 80 85 90 melt tray tgt gcg aga gga tta ega egg ggg ggg tac tac ttt gac tray 384 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 ALIGN! 105 tgg ggg caa ggg acc acg gtc acc gtc tcc tca g 418 Trp Gly Gin Gly Thr Thr Wall Thr Val Ser. Ser 110 115 120

<210> 5 <211> 180 <212> PRT <213> Homosapiens <223> HM 1.24 antigen amino acid sequence <400> 5

Met Ala Ser Thr Ser Tyr Asp Tyr Cys 9 99 9 9 9 9 9 9

999 999

9

9 99

Arg Wall For Met Glu Asp Gly Asp Lys Arg Cys Lys Leu Leu Leu Gly 10 15 Dec 20 May 25 Ile Gly Ile Leu Wall Leu Leu Ile Ile Wall Ile Leu Gly Wall For Leu 30 35 40 Ile Ile Phe Thr Ile Lys Ala Asn Ser Glu Ala Cys Arg Asp Gly Leu 45 50 55 Arg Ala Wall Met Glu Cys Arg Asn Wall Thr His Leu Leu Gin Gin Glu 60 65 70 Leu Thr Glu Ala Gin Lys Gly Phe Gin Asp Wall Glu Ala Gin Ala Ala 75 80 85 Thr Cys Asn His Thr Wall Met Ala Leu Met Ala Ser Leu Asp Ala Glu 90 95 105 Lys Ala Gin Gly Gin Lys LysVal Glu Glu Leu Glu Glu Glu Ile Thr 110 115 120 Thr Leu Asn His Lys Leu Gin Asp Ala Ser Ala Glu Wall Glu Arg Leu 125 130 135 Arg Arg Glu Asn Gin Wall Leu Ser Wall Arg Ile Ala Asp Lys Lys Tyr 140 145 150 Tyr For Ser Ser Gin Asp Ser Ser Ser Ala Ala Ala For Gin Leu Leu 155 160 165 Ile Wall Leu Leu Gly Leu Ser Ala Leu Leu Gin

<210>

<211>

<212>

<213>

<220>

<221>

<222>

<223>

126

PRT

Artificially prepared sequence

The amino acid sequence of the L chain V region version and the altered anti-HM 1.24 antibody

<400> 6 Met Gly Trp Ser Cys Ile Ile Leu Ser Leu Wall Ala Thr Ala Thr Gly -15 -10 -5 Val His Ser Asp Ile Gin Met Thr Gin Ser For Ser Ser Leu Ser Ala -1 1 5 10 Ser Val Gly Asp Arg Wall Thr Ile Thr Cys Lys Ala Ser Gin Asp Wall 15 Dec 20 May 25 Asn Thr Ala Wall Ala Trp Tyr Gin Gin Lys For Gly Lys Ala For Lys 30 35 40 45 Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Wall For Ser Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 Leu Gin For Glu Asp Ile Ala Thr Tyr Tyr Cys Gin Gin His Tyr Ser 80 85 90 Thr Pro Phe Thr Phe Gly Gin Gly Thr Lys Wall Glu Ile Lys 95 100 ALIGN! 105

999 99 99 999 99 999 999 999 9 9 9 9 9 <210> 7 <211> 139 <212> PRT <213> Artificially prepared sequence < 220>

<221>

<222>

<223> The amino acid sequence of the H chain V region of the revised anti-HM 1.24 antibody <400> 7

Met Asp Trp Thr Trp Arg Val Phe Phe Leu Leu Gly -15 -10 -5 Ala His Ser Gin Wall Gin Leu Wall Gin Ser Gly Ala Glu Wall Lys Lys -1 1 5 10 For Gly Ala Ser Wall Lys Wall Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 Dec 20 May 25 Thr For Tyr Trp Met Gin Trp Wall Arg Gin Ala For Gly Gin Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe For Gly Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gin Lys Phe Lys Gly Arg Wall Thr Met Thr Ala Asp Lys Ser Thr Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Wall 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 ALIGN! 105 Trp Gly Gin Gly Thr Thr Wall Thr Wall Ser Ser

110 115 120 <210> 8 <211> 139 <212> PRT <213> Artificial sequence <220>

<221>

<222>

<223> The amino acid sequence of the H chain V region of the modified anti-HM 1.24 antibody <400> 8

Met Asp Trp Thr Trp Arg Wall Phe Phe Leu Leu Ala Wall Ala For Gly -15 -10 -5 Ala His Ser Gin Wall Gin Leu Wall Gin Ser Gly Ala Glu Wall Lys Lys -1 1 5 10 For Gly Ala Ser Wall Lys Wall Ser Cys Lys Ala Ser Gly Tyr Thr Phe 15 Dec 20 May 25

• · · · · ···

Thr Pro Tyr Trp Met Gin Gin Ala For Gly Gin Gly Leu 30 35 40 45 Glu Trp Met Gly Ser Ile Phe Pro Asp Gly Asp Thr Arg Tyr Ser 50 55 60 Gin Lys Phe Lys Gly Arg Thr Ala Asp Lys Ser Thr Ser 65 70 75 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg. Ser Glu Asp Thr Ala Wall 80 85 90 Tyr Tyr Cys Ala Arg Gly Leu Arg Arg Gly Gly Tyr Tyr Phe Asp Tyr 95 100 ALIGN! 105 Gly Gin Gly Thr Thr Thr Val Thr Thr Ser Ser 100 ALIGN! 115 120

9 ** BB ··· BB ··················· · 9 · BB

999 BBB ·· BB »·

Claims (16)

PATENT CLAIMS A therapeutic agent for lymphatic tumors (excluding myelomas) comprising, as an active ingredient, an antibody that specifically binds to a protein having the amino acid sequence as shown in SEQ ID NO: 1 and having cytotoxic activity. The therapeutic agent of claim 1, wherein the lymphatic tumor is a T cell tumor. The therapeutic agent of claim 1, wherein the lymphatic tumor is a B cell tumor (excluding myelomas). The therapeutic agent of claim 1, wherein the antibody is a monoclonal antibody. The therapeutic agent according to claim 1, wherein the cytotoxic activity is ADCC activity. The therapeutic agent of claim 1, wherein the cytotoxic activity is CDC activity. The therapeutic agent according to claim 4, wherein the antibody has a constant region Cy of a human antibody. The therapeutic agent of claim 7, wherein the human Cy antibody constant region is Cy1 or Cy3. The therapeutic agent of claim 4, wherein the antibody is an anti-HM 1.24 antibody. The therapeutic agent of claim 4, wherein the antibody is a chimeric antibody or an altered antibody. 9 99 · · 9 9 99 99 9,990 «999 999 999 9999 99 9 9 9 · 999 999 9 9 9 9 9 999 999 99 99 99 The therapeutic agent of claim 9, wherein the antibody is a chimeric anti-HM 1.24 antibody. The therapeutic agent of claim 9, wherein the antibody is an altered anti-HM 1.24 antibody. The therapeutic agent of claim 9, wherein the antibody specifically binds to an epitope recognized by the anti-HM 1.24 antibody. 14. An antibody which specifically binds to a protein having the amino acid sequence shown in SEQ ID NO: 1 and having cytotoxic activity. 15. The antibody of claim 13, wherein the cytotoxic activity is ADCC activity. The antibody of claim 13, wherein the cytotoxic activity is CDC activity. Giant. 1 Measured radioactivity B-1 • ·· # 49 * 94 9 43 4 4 4 4 9 4 4,441 4 4 4 9 4 4 1 4,444 1 4 4 4 494 949 4 4 9 4 449 494 944 41 94 99 Giant. 2 B-2 Measured radioactivity