HK1113907B - Potentiator for antibody against lymphoid tumor - Google Patents

Description

Enhancer of antibody to lymphocytic tumors

The divisional application is based on the divisional application of Chinese patent application with application number of 98810167.x, application date of 1998 of 10 month and 14 day, and invented name of 'enhancer of lymphocyte tumor antibody'.

Technical Field

The present invention relates to an enhancer comprising a Biological Response Modifier (BRM) as an active ingredient, as a drug for the treatment of lymphocytic tumors, and an antibody as an active ingredient, which is capable of specifically binding to a protein expressed in lymphocytic tumors.

Background

Lymphocytes are primarily responsible for immunity in organisms. Lymphocytes, all derived from the same stem cells in blood, undergo repeated differentiation by the action of various differentiation-inducing factors or growth factors in bone marrow or other organs, and are then released into peripheral blood. Lymphocytes are generally classified into B cells and T cells according to differentiation. It is generally accepted that B cells have the ability to produce antibodies, while T cells have the ability to present antigens, are cytotoxic, and have a variety of other capabilities. When cells undergo neoplastic transformation during the differentiation phase and begin to grow abnormally in bone marrow, lymphoid tissues and peripheral blood, they become so-called lymphocytic tumors.

Due to the recent introduction of new technologies, and in particular the technological advances in the differentiation of cell surface antigens using monoclonal antibodies, it is now possible to identify the origin and/or stage of lymphocytes. Currently, also for lymphocytic tumors, it is now possible to determine not only whether the origin of the tumor cell is a T cell or a B cell, but also the degree of maturation.

Lymphocytic tumors are generally classified as B cell tumors and T cell tumors, depending on the origin or maturity of the tumor cell. B cell tumors are classified by maturity as: acute B lymphocytic leukemia (B-ALL), chronic B lymphocytic leukemia (B-CLL), pre-B cell lymphoma, Burkitt's lymphoma, follicular cerebral cortical lymphoma, diffuse lymphoma, myeloma, and the like. T cell tumors are also classified according to maturity as: acute T-lymphocytic leukemia (T-ALL), chronic T-lymphocytic leukemia (T-CLL), adult T-cell leukemia (ATL), non-ATL peripheral T-lymphoma (RNTL), and the like (Zukai Rinsho [ Gan ] (clinical description [ cancer ]), Ser. No. 17, leukemia/lymphoma, Takashi Sugimura et al, medical observations (MEDICAL VIEW), 1987, B-cell tumors, Kiyoshi Kouzukukukukukaki, Nishimura Shoten, 1991).

Despite recent advances in medical technology, improvements in the treatment of lymphocytic tumors remain. For example, Acute Lymphoblastic Leukemia (ALL) is less than 20% cured. For lymphoma, the cure rate for B lymphoma is relatively high due to the progress of multiple drug combination therapy, but the cure rate for advanced stage is still about 50%. In addition, T lymphomas are more difficult to cure, with a cure rate of about 30% and less than 10% for adult T cell leukemia (ATL).

On the other hand, Goto, T. et al reported a monoclonal antibody (anti-HM 1.24 antibody) obtained by immunizing a mouse with human myeloma cells (Blood (1994)84, 1922-. When anti-HM 1.24 antibody is administered to a mouse transplanted with human myeloma cells, the antibody accumulates in tumor tissue in a specific manner (Masaaki Kosaka et al, Nippon Rinsho (Japanese clinical) (1995)53, 627-635), indicating that the anti-HM 1.24 antibody can be used for diagnosis of tumor localization by radioisotope labeling, missile therapy (e.g., radiotherapy), and the like.

Cancer patients, on the other hand, often have reduced immune function. Since this is thought to be associated with carcinogenesis, attempts to improve these functions are being made. Biological Response Modifiers (BRMs) are used to direct the biological ability to respond directly and/or indirectly to a tumour in a beneficial orientation and are used in therapy. They mainly enhance the functions of macrophages, T cells, etc., and can restore the immunological competence, etc. At the time of treatment and research using a nonspecific immune activating substance, the biological activities of various cytokines were studied, and cytokines considered to be possibly effective were produced in large quantities (Konnniti no Tiryoyaku (therapeutic drug today) (1995 edition), edited by yutamizashima and Akimasa Miyamoto, Nankodo k.k., 17 th edition, 3 rd print, published at 20 6 months 1995).

Disclosure of Invention

Currently used methods of treating lymphocytic tumors include various chemotherapies, radiation therapy, bone marrow transplantation, and the like. However, none of the above methods is ideal and there is a need for new therapeutic agents or methods that can alleviate lymphocytic tumors and prolong patient survival.

Therefore, it is an object of the present invention to provide a novel potentiator against lymphocyte tumor.

In an attempt to provide these therapeutic methods, the present inventors examined the combined use of an anti-HM 1.24 antibody (Goto, T. et al, blood (1994)84, 1922-.

Accordingly, the present invention provides an enhancer of antibodies for the treatment of lymphocytic tumors, the antibodies specifically binding to a peptide having the amino acid sequence as set forth in SEQ ID NO: 5 and having cytotoxicity, which comprises a biological response modifier as an active ingredient.

The present invention also provides a biological response modifier enhancer for the treatment of a lymphocytic tumor, the enhancer comprising as an active ingredient an antibody that specifically binds to a peptide having a sequence as set forth in seq id no: 5, and has cytotoxicity.

The present invention also provides the above-mentioned enhancer, wherein the lymphocytic tumor is a T cell tumor.

The present invention also provides the above-mentioned potentiator, wherein the lymphocytic tumor is a B cell tumor.

The present invention also provides the above enhancer wherein the B cell tumor is myeloma.

The present invention also provides the above-mentioned enhancer, wherein the antibody is a monoclonal antibody.

The present invention also provides the aforementioned enhancer, wherein the cytotoxicity is ADCC activity.

The present invention also provides the enhancer described above, wherein the antibody has a constant region cgamma of a human antibody.

The present invention also provides the enhancer as described above, wherein the constant region C.gamma.of the human antibody is C.gamma.1 or C.gamma.3.

The present invention also provides the above enhancer, wherein the antibody is anti-HM 1.24 antibody.

The present invention also provides the above-mentioned enhancer, wherein the antibody is a chimeric antibody or a humanized antibody.

The present invention also provides the aforementioned enhancer, wherein the antibody is a chimeric anti-HM 1.24 antibody.

The present invention also provides the aforementioned enhancer, wherein the antibody is a humanized anti-HM 1.24 antibody.

The present invention also provides the above enhancer, wherein the antibody specifically binds to an epitope recognized by the anti-HM 1.24 antibody.

The present invention also provides the above-mentioned potentiator, wherein the biological response modifier is interferon, OK432, lentinan (lentinan), sisofilan, pepstatin, coriolus versicolor polysaccharide, N-CWS, levamisole, G-CSF, IL-2, IL-10 or IL-15.

The present invention also provides the enhancer as described above, wherein the antibody has cytotoxicity with ADCC activity of less than 25% at an E/T ratio of 50.

The present invention also provides the above-mentioned enhancer, wherein the ADCC activity is measured by Cr (chromium) release assay.

The present invention also provides an enhancer according to any one of the above for use in vivo.

The present invention also provides a therapeutic agent for lymphocytic tumors, the agent comprising an antibody and a biological response modifier, wherein the antibody specifically binds to a peptide having the amino acid sequence as set forth in SEQ ID NO: 5, and has cytotoxicity.

Brief Description of Drawings

FIG. 1 is a graph showing that the humanized anti-HM 1.24 antibody shows strong cytotoxicity to RPMI8226 cells when cells derived from healthy human peripheral blood are used as effector cells.

FIG. 2 is a graph showing that the humanized anti-HM 1.24 antibody shows cytotoxicity to RPMI8226 cells when cells derived from peripheral blood of a myeloma patient are used as effector cells.

FIG. 3 is a graph showing that humanized anti-HM 1.24 antibody shows cytotoxicity to RPMI8226 cells when cells subjected to apheresis derived from myeloma patients treated with a large dose of cyclophosphamide are used as effector cells.

Embodiments for carrying out the invention

1. Antibody preparation

1-1 preparation of hybridomas

The hybridoma producing the antibody for use in the present invention can be constructed basically by a known method as described below. The HM1.24 antigen protein or cells expressing the HM1.24 antigen may be used as a sensitizing antigen and immunized according to conventional immunization methods. The immune cells thus obtained are fused with known parent cells by a conventional cell fusion method, and then screened by a conventional screening method to screen cells producing monoclonal antibodies.

For example, for HM1.24 antigen-expressing cells used as sensitizing antigens for obtaining antibodies, a human multiple myeloma cell line KPMM2 (Japanese unexamined patent application publication (Kokai) No. 7(1995) -236475) or KPC-32(Gotot et al, J.Clin.Hematol. (1991)32, 1400) can be used. In addition, for the sensitizing antigen, a peptide having seq id NO: 1 or a peptide or polypeptide comprising an epitope recognized by anti-HM 1.24 antibody.

As used herein, a polypeptide having the sequence of SEQ ID NO: 5 into the XbaI cleavage site of the vector pUC19, thereby preparing plasmid pRS38-pUC 19. Escherichia coli containing the plasmid has been deposited internationally under the provisions of the Budapest treaty by the national institute for bioscience and human technology, Industrial science and technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, on 1993 at 10.5.1993, as Escherichia coli DH5 α (pRS38-pUC19) under the accession number FERM BP-4434 (see Japanese unexamined patent application publication (Kokai) No. 7(1995) -196694). A peptide or polypeptide having an epitope recognized by anti-HM 1.24 antibody can be prepared by genetic engineering techniques using the cDNA fragment contained in the plasmid pRS38-pUC 19.

Preferably, the mammal immunized with the sensitizing antigen is selected for compatibility with the parent cell used in the cell fusion. They generally include, but are not limited to, rodents such as mice, rats, hamsters, and the like.

Immunization of animals with sensitizing antigen is carried out by a known method. For example, one common method involves intraperitoneal or subcutaneous administration of a sensitizing antigen to a mammal.

Specifically, a sensitizing antigen diluted or suspended with an appropriate amount of Phosphate Buffered Saline (PBS) or physiological saline or the like is mixed with an appropriate amount of Freund's complete adjuvant as desired, and after emulsification, it is preferably administered to a mammal several times every 4 to 21 days. Alternatively, a suitable carrier may be used in the immunization of the sensitizing antigen.

Following immunization and determination of an elevated level of the antibody of interest in serum, immune cells are taken from the mammal and subjected to cell fusion.

As other parent cells, mammalian myeloma cells that can be Cell-fused with the above-mentioned immunocytes preferably include various known Cell lines, such as P3X63Ag8.653 (J.Immunol.) (1979) 123: 1548-, R210(Galfre, G. et al, Nature (1979) 277: 131-.

Cell fusion of the above immunocytes with myeloma cells can be carried out basically according to known Methods such as those described in Milstein et al (Kohler, G., and Milstein, C., Methods (1981) 73: 3-46).

More specifically, the above cell fusion is carried out, for example, in a conventional nutrient broth in the presence of a cell fusion promoter. For example, polyethylene glycol (PFG), Sendai virus (HVJ) and the like can be used as a cell fusion promoter, and an adjuvant such as dimethyl sulfoxide can be added as desired to improve the fusion efficiency.

The preferred ratio of immune cells to myeloma cells used is, for example, 1-10 times more immune cells than myeloma cells. Examples of the medium which can be used for the above cell fusion include RPMI1640 medium and MEM medium suitable for the growth of the above myeloma cell line, and a conventional medium used for such cell culture, and in addition, a serum supplement such as Fetal Calf Serum (FCS) can be added.

When fusing cells, a predetermined amount of the above immune cells and myeloma cells are mixed well in the above culture solution, and a PEG solution previously heated to about 37 ℃ such as a PEG solution having an average molecular weight of about 1000-6000 is added thereto at a concentration of 30-60% (w/v), and the mixture is mixed to obtain desired fused cells (hybridomas). The cell fusion agent and the like which are not required for the growth of the hybridoma can then be removed by repeating the successive steps of addition of an appropriate culture solution and removal of the supernatant by centrifugation.

The hybridomas are selected by culturing in a conventional selective medium such as HAT medium (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT medium is usually continued for a period of time sufficient to achieve killing of cells other than the target hybridoma (non-fused cells), typically several days to several weeks. A conventional limiting dilution method is performed in which hybridomas producing the target antibodies are screened and monoclonally cloned.

In addition to the above hybridomas obtained by immunizing an animal other than a human with an antigen, it is also possible to sensitize human lymphocytes with HM1.24 antigen or cells expressing HM1.24 antigen in vitro, fuse the resulting sensitized lymphocytes with myeloma cells (e.g., U266), and obtain human antibodies of interest having an activity of binding to HM1.24 antigen or cells expressing HM1.24 antigen (see Japanese post-examination patent publication (Kokoku) No. 1(1989) -59878). Furthermore, a desired humanized antibody can be obtained by immunizing a transgenic animal containing all the components of the human antibody gene with the antigen, i.e., the HM1.24 antigen or the HM1.24 antigen-expressing cell, according to the above-described method (see International patent applications WO93-12227, WO92-03918, WO94-02602, WO94-25585, WO96-34096 and WO 96-33735).

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

In order to obtain a monoclonal antibody from the hybridoma, a method of culturing the hybridoma by a conventional method and obtaining the antibody in the form of supernatant or a method of obtaining the antibody in the form of ascites by administering the hybridoma to a mammal compatible with the hybridoma and growing the hybridoma therein can be mentioned. The foregoing method is suitable for obtaining antibodies of high purity, while the latter is suitable for large-scale production of antibodies.

In particular, hybridomas producing anti-HM 1.24 antibody can be constructed by the method of Goto, T. et al (blood (1994) 84: 1922-: wherein ascites is obtained by intraperitoneal injection into BALB/c mice (manufactured by CLEA, Japan) of a hybridoma producing an anti-HM 1.24 antibody, and the anti-HM 1.24 antibody is purified therefrom, which hybridoma is obtained by national institute of bioscience and human technology, Industrial science and technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki prof., Japan, International accession number FERM BP-5233, or by a method comprising: the hybridoma is cultured in a suitable medium such as RPMI1640 medium containing 10% fetal bovine serum and 5% BM-harmonized H1 (produced by Boehringer Mannheim), hybridoma SFM medium (produced by GIBCO-BRL), PFHM-II medium (produced by GIBCO-BRL), etc., and the anti-HM 1.24 antibody is purified from the supernatant.

1-2 recombinant antibodies

In the present invention, a recombinant antibody produced by recombinant gene technology can be used as a monoclonal antibody, wherein an antibody gene is cloned from a hybridoma, integrated into a suitable vector, and then introduced into a host (see, for example, Carl, A.K, Borrebaeck and James, W.Larrick, THERAPEUTIC monoclonal antibody (THERAPEUTIC MONOCLONALANTIBODIES), U.K. MACMILLAN PUBLISHERS LTD. publication, 1990).

Specifically, mRNA encoding the variable (V) region of the desired antibody is isolated from the antibody-producing hybridoma. For example, mRNA can be purified from total RNA by preparing the total RNA by a known method such as the guanidine ultracentrifugation method (Chirgwin, J.M. et al, Biochemistry (Biochemistry) (1979)18, 5294-. In addition, mRNA can be directly prepared using Quick Prep mRNA purification kit (produced by Pharmacia).

cDNA of the V region of the antibody can be synthesized from the mRNA thus obtained using reverse transcriptase. The cDNA can be synthesized using AMV reverse transcriptase first strand cDNA synthesis kit or the like. In addition, for the synthesis and amplification of cDNA, 5 '-Ampli FINDER kit (manufactured by Clontech) and 5' -RACE method (Frohman, M.A. et al, Proc. Natl.Acad.Sci.U.S.A.) (1988)85, 8998 9002; Belyavsky, A. et al, Nucleic acid research (Nucleic Acids Res.) (1989)17, 2919-. The target DNA fragment was purified from the obtained PCR product and ligated to the vector DNA. Further, a recombinant vector is thus constructed, and then introduced into E.coli or the like, from which colonies are selected to prepare a target recombinant vector. The nucleotide sequence of the target DNA can be confirmed by a known method such as the dideoxy method.

Once the DNA encoding the V region of the antibody of interest is obtained, it may be ligated to the DNA encoding the constant region (C region) of the antibody of interest and then incorporated into an expression vector.

To produce antibodies for use in the present invention, antibody genes are integrated into expression vectors as described below and expressed under the control of expression regulatory regions such as enhancers and/or promoters. Subsequently, the expression vector may be transformed into a host cell, where the antibody can then be expressed.

1-3. altered antibodies

In accordance with the present invention, artificially altered recombinant antibodies such as chimeric antibodies and humanized antibodies can be used in order to reduce heterologous antigenicity to humans.

Chimeric antibodies can be obtained by: the DNA encoding the V region of the antibody thus obtained is ligated with a DNA encoding the C region of a human antibody, which is then integrated into an expression vector and introduced into a host for the production of the antibody therein (see European patent application EP125023, and International patent application WO 96-02576). By this known method, a chimeric antibody that can be used in the present invention can be obtained.

For example, Escherichia coli harboring a plasmid containing the L chain V region or H chain of the chimeric anti-HM 1.24 antibody has been deposited by national institute of bioscience and human technology, the institute of Industrial science and technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, under the provisions of the Budapest treaty, as Escherichia coli DH5 α (pUC19-1.24L-g κ) and Escherichia coli DH5 α (pUC19-1.24L-g γ 1) with deposition numbers FERM BP-5646 and FERM BP-5644 (see International patent application WO98-14580) at 29.1996.

Humanized antibodies, also known as reshaped human antibodies, are prepared by grafting Complementarity Determining Regions (CDRs) of an antibody, other than human, of a mammal, such as a mouse antibody, into the CDRs of a human antibody. Common recombinant DNA techniques for the preparation of these antibodies are also well known (see European patent application EP125023 and International patent application WO 96-02576).

In particular, DNA sequences for linking the CDR of a mouse antibody to the Framework Region (FR) of a human antibody are synthesized from several separate oligonucleotides whose fragments overlap each other at their ends. The oligonucleotide is then synthesized as an integrated DNA. The DNA thus obtained is ligated with a DNA encoding a human antibody C region, which is then integrated into an expression vector and introduced into a host for antibody production (see European patent application EP239400 and International patent application WO 96-02576).

For the FRs of human antibodies linked by CDRs, complementarity determining regions are selected that form favorable antigen binding sites. If desired, amino acids in the framework regions of the antibody variable regions can be substituted so that the complementarity determining regions of the reshaped human antibody can form a suitable antigen binding site (Sato, K. et al, Cancer research (Cancer Res.) (1993)53, 851-.

For example, Escherichia coli harboring plasmids containing humanized anti-HM 1.24 antibody L chain a type and H chain r type has been deposited internationally by the national institute of bioscience and human technology, the institute of Industrial science and technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, under the provisions of the Budapest treaty, as Escherichia coli DH5 α (pUC19-RVLa-AHM-g κ) and Escherichia coli DH5 α (pUC19-RVHr-AHM-g γ 1) with deposition numbers FERM BP-5645 and FERM BP-5643 (International patent application WO98-14580), respectively, at 8/29.1996. In addition, Escherichia coli harboring a plasmid containing H chain s type has been deposited internationally under the provisions of the Budapest treaty, Escherichia coli DH5 α (pUC19-RVHs-AHM-g γ 1) with the accession number FERM BP-6127 by the national institute of Life sciences and human technology, Industrial science and technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, on 29.9.1997.

The amino acid sequence and nucleotide sequence of each of the V region of the L chain a form of the humanized anti-HM 1.24 antibody, the H chain r form of the humanized anti-HM 1.24 antibody and the H chain s form of the humanized anti-HM 1.24 antibody are shown in SEQ ID NOS: 2, 3 and 4. The amino acids at positions-15 to-1 are signal sequences.

For a chimeric antibody or a humanized antibody, the C region of a human antibody is used, and for the C region of a human antibody exhibiting cytotoxicity, human C γ such as C γ 1, C γ 2, C γ 3 and C γ 4 can be used. Among them, the antibody containing C γ 1 and C γ 3 particularly has strong cytotoxicity, i.e., ADCC activity and CDC activity, and is preferably used in the present invention.

The chimeric antibody is composed of a variable region derived from an antibody derived from a mammal other than human and a C region derived from a human antibody, and the humanized antibody is composed of a complementarity determining region derived from an antibody derived from a mammal other than human and antibody Framework Regions (FRs) and C regions derived from a human antibody. Therefore, antigenicity in humans is reduced therein, so that they can be used as an active ingredient of the therapeutic agent of the present invention.

A preferred embodiment of a humanized antibody for use in the present invention comprises a humanized anti-HM 1.24 antibody (see International patent application WO 98-14580).

1-4. expression and production

The antibody gene constructed as described above can be expressed and obtained by a known method. For mammalian cells, expression can be achieved using an expression vector containing a conventional effective promoter, an antibody gene to be expressed and a DNA in which a poly A signal is operably linked 3' downstream thereof or a vector containing the DNA. Examples of promoters/enhancers include the human cytomegalovirus immediate early promoter/enhancer.

In addition, as the promoter/enhancer which can be used for the expression of the antibody in the present invention, viral promoters/enhancers such as retrovirus, polyoma virus, adenovirus and simian virus 40(SV40), and promoters/enhancers derived from mammalian cells such as human elongation factor 1 α (HEF1 α) can be used.

For example, expression can be conveniently achieved as described by Mullingan et al (Nature (1979)277, 108) when using the SV40 promoter/enhancer, or Mizushima et al (nucleic acids Res. 1990)18, 5322) when using the HEF1 α promoter/enhancer.

For E.coli, expression can be performed as follows: the usual effective promoter, signal sequence for antibody secretion and antibody gene to be expressed are operably linked, followed by expression thereof. As the promoter, for example, lacz promoter and araB promoter can be mentioned. When the lacz promoter is used, the method of Ward et al (Nature (1098)341, 544-546; FASEB J. (1992)6, 2422-2427) can be used, and when the araB promoter is used, the method of Better et al (science (1988)240, 1041-1043) can be used.

As signal sequences for antibody secretion, the pelB signal sequence can be used when produced in the periplasm of E.coli (Lei, S.P. et al, J.Bacteriol. (1987)169, 4379). After isolation of the antibody produced in the periplasm, the structure of the antibody is suitably refolded prior to use (see, e.g., WO 96-30394).

As the replication origin, replication origins derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), and the like can be used. In addition, for amplification of gene copy number in a host cell system, the expression vector may comprise a selectable marker aminoglycoside transferase (APH) gene, Thymidine Kinase (TK) gene, escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase (dhfr) gene, and the like.

For the production of the antibody used in the present invention, any production system can be used. Production systems for antibody preparations include in vitro or in vivo production systems. As the in vitro production system, a production system using eukaryotic cells and a production system using prokaryotic cells can be mentioned.

When eukaryotic cells are used, there are production systems that use animal cells, plant cells and fungal cells. Known animal cells include (1) mammalian cells such as CHO cells, COS cells, myeloma cells, Baby Hamster Kidney (BHK) cells, HeLa cells, and Vero cells, (2) amphibian cells such as Xenopus cells, or (3) insect cells such as sf9, sf21, and Tn 5. Known plant cells include, for example, cells derived from the genus Nicotiana (Nicotiana), particularly tobacco (Nicotiana tabacum) which has been subjected to callus culture. Known fungal cells include yeasts such as yeasts (Saccharomyces) of the genus Saccharomyces, especially Saccharomyces cerevisiae, or filamentous fungi such as Aspergillus (Aspergillus) of the genus Aspergillus, especially Aspergillus niger.

When prokaryotic cells are used, there are production systems that use bacterial cells. Well-known bacterial cells include escherichia coli (e.coli) and Bacillus subtilis (Bacillus subtilis).

The antibody can be obtained by introducing the gene of the target antibody into these cells by transformation, and culturing the transformed cells in vitro. The culture is carried out according to a known method. For example, DMEM, MEM, RPMI1640, and IMDM can be used as the culture medium, and a serum supplement such as Fetal Calf Serum (FCS) can be used in combination. In addition, the antibody can also be produced in vivo by implanting cells into the abdominal cavity or the like of an animal into which the antibody gene has been introduced.

As other in vivo production systems, those using animals and those using plants can be mentioned. When animals are used, there are production systems that use mammals and insects.

For mammals, goats, pigs, sheep, mice and cattle can be used (Vicki Glaser, spectral Biotechnology Applications (spectra Biotechnology Applications), 1993). As the insect, silkworm can be used.

When plants are used, tobacco can be used, for example.

Antibody genes are introduced into these animals or plants, antibodies are produced in these animals or plants, and recovered. For example, a fusion gene is prepared by inserting an antibody gene into the middle of a gene encoding a protein naturally produced in milk, such as goat beta casein. A DNA fragment containing a fusion gene into which an antibody gene has been inserted is injected into a goat embryo, and the embryo is introduced into a female goat. Obtaining the target antibody from milk produced by the transgenic goat born by the goat receiving the embryo or the offspring thereof. In order to increase the amount of milk containing the target antibody produced by the transgenic goat, hormone may be suitably administered to the transgenic goat (Ebert, KM. et al, Bio/Technology (1994)12, 699-702).

When silkworms are used, the silkworms are infected with baculovirus into which a gene for a target antibody has been inserted, and the target antibody can be obtained from the body fluid of the silkworms (Susumu, M. et al, Nature (1985)315, 592-594). In addition, when tobacco is used, the target antibody gene is inserted into a plant expression vector such as pMON530, and then the vector is introduced into bacteria such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). The bacterium is then used to infect tobacco, such as tobacco, to obtain the antibody of interest from tobacco leaves (Julian, K-C.Ma et al, Eur. J Immunol (1994)24, 131-138).

When the antibody is produced in an in vitro or in vivo production system as described above, the DNA encoding the heavy chain (H chain) or light chain (L chain) of the antibody, respectively, may be integrated into an expression vector and the host transformed, or the DNA encoding the H chain or L chain may be integrated into a single expression vector and the host transformed therewith (see International patent application WO 94-11523).

Antibodies generated as described above can be used as modified antibodies in combination with a variety of molecules such as polyethylene glycol (PEG) "antibodies" as used herein include such modified antibodies. In order to obtain these modified antibodies, the obtained antibodies may be chemically modified. These methods have been established in the art.

2. Isolation and purification of antibodies

2-1 separation and purification of antibodies

The antibody produced and expressed as described above can be isolated from the inside or outside of the cell or host, and then can be purified to homogeneity. For the affinity chromatography column used herein, a protein A column and a protein G column can be mentioned. Examples of columns using protein a columns are Hyper D, POROS, Sepharose e.f.

The separation and purification of the antibody used in the present invention can be accomplished by appropriately combining chromatography, filtration, ultrafiltration, salting out, dialysis, and the like, in addition to the above-mentioned affinity chromatography. For example, chromatography includes ion exchange chromatography, hydrophobic chromatography, gel filtration, and the like.

2-2 determination of antibody concentration

The concentration of the antibody obtained in the above 2-1 can be measured by absorbance measurement or by ELISA or the like. Therefore, when the absorbance measurement is used, the antibody for the present invention or a sample containing the antibody is appropriately diluted with PBS (-), and then the absorbance is measured at 280nm, followed by calculation using an absorption coefficient of 1.35OD at 1 mg/ml. When the ELISA method was used, the measurement was performed as follows. Mu.l of goat anti-human IgG (produced by BIO SOURSE) diluted to 1. mu.g/ml with 0.1M bicarbonate buffer pH9.6 (produced by Nunc) was added to a 96-well plate, and incubated overnight at 4 ℃ to immobilize the antibody after blocking, 100. mu.l of each of the appropriately diluted antibodies of the present invention, or a sample containing the antibody, or 100. mu.l of human IgG of known concentration was added as a standard, incubated at room temperature for 1 hour, washed, 100. mu.l of alkaline phosphatase-labeled anti-human IgG antibody (produced by BIOSOURSE) diluted 5000-fold was added, and incubated at room temperature for 1 hour. After washing, a substrate solution was added for incubation, followed by measurement of absorbance at 405nm using a 3550 microplate reader (manufactured by Bio-Rad), and the concentration of the target antibody was calculated.

FCM analysis

The reactivity of the antibodies of the invention with lymphocytes can be detected by Flow Cytometry (FCM) analysis. As the cells, established cell lines or newly isolated cells can be used. As for the established cell lines, T cell lines such as RPMI8402(ATCC CRL-1995), acute lymphoblastic leukemia-derived CCRF-CEM (ATCC CCL119), acute lymphoblastic leukemia-derived HPB-ALL (FCCH1018), T lymphoma-derived HPB-MLT (FCCH1019), acute lymphoblastic leukemia-derived JM (FCCH1023), acute lymphoblastic leukemia-derived MOLT-4(ATCC CRL1582), acute lymphoblastic leukemia-derived Jurkat (FCCH1024), acute lymphoblastic leukemia-derived CCRF-HSB-2(ATCC CCL120.1), adult T cell leukemia-derived MT-1(FCCH1043) and Lennert lymphoma-derived KT-3(Shimizu, S.et al, blood (1988)71, 196-203) can be used, and B cell lines such as EB (EB) transformed with viruses such as CESS (ATCC CCL 190), TIST virus-derived SKW 6.SKB-derived cell (ATCC CRW 215) can be used, B lymphoma-derived MC116(ATCC CRL1649), acute lymphoblastic leukemia-derived CCRF-SB (ATCC CCL120), acute myelogenous leukemia patient-derived B cell RPMI6410(RCCH6047), Burkitt lymphoma-derived Daudi (ATCC CCL213), Burkitt lymphoma-derived EB-3(ATCC CCL85), Burkitt lymphoma-derived Jijoye (ATCC CCCL87) and Burkitt lymphoma-derived Raji (ATCC CCL86), and furthermore non-T non-B cell lines such as acute myelogenous leukemia-derived HL-60(ATCC CCL240), acute monocytic leukemia-derived THP-1(ATCC TIB202), histiocytic lymphoma-derived U-937(ATCC CRL1593), chronic myelogenous leukemia-derived K-562 (CCCL), myeloma-derived RPMI8226(ATCC CCL155), myeloma-derived U266B1 (TIB 38196), ATCC myeloma-derived MM2 (MM) of ATCC 38196), non-T non-B cell lines such as described above, can be used, Myeloma-derived KPC-32 and plasmacytoma-derived ARH-77(ATCC CRL1621).

After washing the above cells with PBS (-), 100. mu.l of an antibody or a control antibody diluted to 25. mu.g/ml with FACS buffer (PBS (-) -containing 2% fetal bovine serum and 0.05% sodium azide) was added thereto, followed by incubation on ice for 30 minutes. After washing with FACS buffer, 100. mu.l of 25. mu.g/ml FITC-labeled goat anti-mouse antibody (GAM, manufactured by Becton Dickinson) was added, followed by incubation on ice for 30 minutes. After washing with FACS buffer, the cells were suspended in 600. mu.l of 1ml FACS buffer, and the fluorescence intensity of each cell was measured by FACScan (produced by Becton Dickinson).

4. Cytotoxicity

4-1. determination of ADCC Activity

The antibody used in the present invention is an antibody having an activity such as ADCC as cytotoxicity.

According to the present invention, ADCC activity against lymphoid tumors can be measured by the following method. First, mononuclear cells are separated from human peripheral blood or bone marrow as effector cells by gravity centrifugation.

As for the target cells (target cells: T), RPMI8226(ATCC CCL-155), CCRF-CEM (ATCC CCL119), (ATCC CCL120.1), HPB-MLT (FCCH1019), EB-3(ATCC CCL-85), MC116(ATCC CRL1649), CCRF-SB (ATCC CCL120), K-562(ATCC CCL243), etc. are labeled with 51Cr, and target cells are prepared. Subsequently, an antibody to be tested for ADCC activity is added to the labeled target cells and incubated. Effector cells are then added in the appropriate ratio to the target cells and incubated.

After incubation, the supernatant is removed and radioactivity is measured using a gamma counter, the maximum free radioactivity being determined using 1% NP-40. Cytotoxicity (%) can be calculated as (A-C)/(B-C) X100, where A is the radioactivity released in the presence of antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released by antibody-free medium alone (cpm).

4-2 enhancement of cytotoxicity

In order to exhibit cytotoxicity such as ADCC activity, C γ (particularly, C γ 1 and C γ 3) is preferably used as a constant region (C region) of a human antibody. In addition, a stronger ADCC activity or CDC activity can be induced by adding, changing or modifying a part of amino acids in the C region of the antibody.

By way of example, the construction of IgM-like multimers of IgG by amino acid substitution (Smith, R.I.F. and Morrison, S.L., Bio/technology (1994)12, 683-, l. et al, European journal of immunology (1991)21, 2379-. The realization method can be realized by: oligomer site specific mutagenesis using primers, addition of base sequences using restriction enzyme cleavage sites, and the use of chemical modulators capable of generating covalent bonds.

5. Treatment object

The invention provides an antibody enhancer for treating lymphocyte tumor, the antibody can be specifically combined with a polypeptide with SEQ ID NO: 1, and is cytotoxic, which comprises a biological response modifier as an active ingredient. Although the subject is a human patient with a common lymphoid tumour, it will be apparent that the invention can also be used with human patients with reduced immune function, as described in the examples below. As used herein, "reduced immune function" means reduced cytotoxicity, such as ADCC.

In particular, it is particularly advantageous when the ADCC activity is only found to be less than 25% cytotoxic at an E/T ratio of 50. When the immune function is reduced by administration of an anticancer agent or the like, ADCC activity is found to be less than 25%. When the ADCC activity is measured, the incubation time (incubation time) is preferably 4 hours. As a method for measuring ADCC activity, Cr (chromium) release assay or trypan blue staining method is preferable.

6. Biological response modifier

The Biological Response Modifier (BRM) contained as an active ingredient in the present invention is a substance having an activity of activating immunity. Preferred examples of the biological response modifier include interferon, OK432, lentinan, sisofilan, pepstatin, coriolus versicolor polysaccharide, N-CWS, levamisole, G-CSF, IL-2, IL-10, IL-12 or IL-15(Konnnitino Tiryoyaku (therapeutic drugs today) (1995 edition), eds. Yutaka Mizushima and Akimasa Miyamoto, Nankodo K.K., 17 th edition, 3 rd printing, 20 th month in 1995; cytokine 94-Kisokara Saishin Jojomadad (cytokine 94-from basic to latest information), Shiei npKasakura, eds., Nihon Igakan KK, 1 st edition, 14 th month in 1994, 7 th month in 1994; Zusetsu Rinsho [ Gan ] (clinical statement [ cancer ]), Ser. No. 19, (clinical Advance of cancer, Gan to Menneki (cancer and immunization) (New edition) medical observations, Kk 8 th edition, 1993). Of these, IL-2 is particularly preferred.

These biological response modifiers are substances that act on effector cells in a cytotoxic response, thereby activating effector cell cytotoxicity. These biological response modifiers can be prepared by known methods and are commercially available.

7. Route of administration and pharmaceutical preparation

The enhancers of the invention may be administered systemically or locally by parenteral routes, such as intravenous (e.g., drip), intramuscular, intraperitoneal, and subcutaneous injection. The administration method can be appropriately selected depending on the age and condition of the patient, and the effective dose is selected to be 0.01mg to 100mg per kg body weight per administration. In addition, a dosage of 1-100mg, preferably 5-50mg per patient may also be selected. For biological response modifiers, the dosage is selected to be 1 unit to 1,000,000 units per administration.

According to the present invention, a biological response modifier that enhances the action of an antibody can be administered before or after the antibody is administered to a subject such as a tumor patient, as long as it enhances the action of the antibody wherein the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 1, and has cytotoxicity. Alternatively, it can be administered simultaneously with the antibody.

According to the present invention, the antibodies and biological response modifiers can be administered not only to tumor patients, but also for ex vivo treatment. Thus, after the effector cells are extracted from the patient's peripheral blood and activated for immune function with the bioresponse modifier, the effector cells are returned to the patient. The antibody may be administered before or after or simultaneously with returning the effector cells to the patient. The present invention can also be used for plasma separation and replacement of PBSCT (transplantation of peripheral blood stem cells). In PBSCT, the antibody and biological response modifier can be administered when returning extracted stem cells.

The present invention includes embodiments as described above, so long as the combined use of the antibody and the biological response modifier enhances its effect.

The enhancer of the invention may contain a pharmaceutically acceptable carrier or additive depending on the route of administration. Examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, Human Serum Albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, and the like. The additives used may be selected from, but are not limited to, the above materials or combinations thereof, depending on the dosage form.

The disease of interest for treatment according to the invention is a lymphocytic tumor, wherein the antibody used in the invention binds to an antigen on the target tumor cell. Specifically, multiple myeloma, acute B-lymphocytic leukemia (B-ALL), chronic B-lymphocytic leukemia (B-CLL), pre-B lymphoma, Burkitt's lymphoma, follicular cerebral cortical lymphoma, diffuse lymphoma, acute T-lymphocytic leukemia (T-ALL), chronic T-lymphocytic leukemia (T-CLL), adult T-cell leukemia (ATL), non-ATL peripheral T-lymphoma (PNTL), and the like. The potentiators of the present invention are useful as potentiators for the treatment of these lymphocytic tumors.

Examples

The invention will now be illustrated in more detail hereinafter with reference to the following examples. It should be noted, however, that the present invention is not limited in any way by these examples.

EXAMPLE measurement of ADCC Activity

ADCC (antibody dependent cellular cytotoxicity) Activity was determined according to the method described in "common protocols for immunology", Chapter 7, "immunological studies in humans", editor JohanE, Coligan et al, John Wiley & Sons, Inc., 1993.

1. Preparation of Effector cells

Then, to peripheral blood and bone marrow of healthy persons and multiple myeloma patients, an equal amount of PBS (-) was added, which was layered on Ficoll (Pharmacia) -Conrey (Daiichi pharmaceutical Co. Ltd.) (specific gravity 1.077), and centrifuged at 400g for 30 minutes. After collecting the monocyte layer, the cells were washed twice with RPMI1640 (produced by Sigma) supplemented with 10% fetal bovine serum (produced by Witaker), and 500U/ml of IL-2 (produced by Genzyme), 20ng/ml of IL-10 (produced by Genzyme), 20ng/ml of IL-12 (R) were added or not (medium alone) to the cells&D production), 20ng/ml IL15 (produced by Genzyme) or 5000U/ml M-CSF (produced by GreeCross K.K.), and cultured for 3 days. After washing each culture twice with the same medium, the same culture solution was used to prepare cells with a density of 5X 10⁶Cells per ml.

2. Preparation of target cells

A radiolabelled human myeloma cell line RPMI8226(ATCC CCL155) was prepared by mixing 0.1mCi with 10% fetal bovine serum (produced by Witaker) supplemented RPMI1640 (produced by Sigma)⁵¹Cr-sodium chromate incubated at 37 ℃ for 60 minutes after radiolabelling, cells were washed three times with Hanks Balanced Salt Solution (HBSS) and adjusted to 2X 10⁵Concentration per ml.

ADCC assay

To a 96-well U-bottom plate (produced by Corning), 50. mu.l of 2X 10 was added⁵Each target cell/ml, 1. mu.g/ml affinity-purified humanized anti-HM 1.24 antibody or control human IgG (manufactured by Serotec), was reacted at 4 ℃ for 15 minutes.

Then, 100. mu.l of 5X 10 was added⁵Effector cells/ml in CO₂Cultured in an incubator for 4 hours, in which the ratio (E: T) of effector cells (E) to target cells (T) was set to 50: 1.

mu.l of the supernatant was taken out and radioactivity released into the culture supernatant was measured by a gamma counter (ARC361, produced by Aloka). For the determination of maximum radioactivity, 1% NP-40 (produced by BRL) was used. Cytotoxicity (%) was calculated as (A-C)/(B-C). times.100, where A is the radioactivity released in the presence of antibody (cpm), B is the radioactivity released by NP-40 (cpm), and C is the radioactivity released by antibody-free medium alone (cpm).

As a result, when cells derived from healthy human peripheral blood were used as effector cells, the humanized anti-HM 1.24 antibody showed strong cytotoxicity to RPMI8226 cells while the control IgG showed almost no cytotoxicity (fig. 1). when cells derived from multiple myeloma patients with reduced immune function were used as effector cells, the humanized anti-HM 1.24 antibody showed cytotoxicity to RPMI8226 cells while the control IgG showed almost no cytotoxicity (fig. 2).

However, this activity is weaker than in the above healthy persons (less than 25%). The cytotoxicity of the humanized anti-HM 1.24 antibody was enhanced when IL-2, IL-10, IL-12 or IL-15 was added. Especially for IL-2, activity was enhanced to a level almost equal to that of healthy humans (FIG. 2).

Furthermore, when cells subjected to apheresis derived from myeloma patients treated with a large dose of cyclophosphamide were used as effector cells, the humanized anti-HM 1.24 antibody showed cytotoxicity to RPMI8226 cells, while the control IgG showed little cytotoxicity (fig. 3). However, this activity is weaker than in the above healthy persons (less than 25%). The cytotoxicity of the humanized anti-HM 1.24 antibody was enhanced when IL-2, IL-10 or IL-12 was added (FIG. 3).

This indicates that the humanized anti-HM 1.24 antibody has a weaker cytotoxicity on tumor cells, which are effector cells derived from patients with reduced immune function, than that of effector cells derived from normal humans or patients with non-reduced immune function. By adding BRM such as IL-2, cytotoxicity of effector cells can be enhanced, even to the level of healthy people.

Thus, the combined use of BRMs is expected to further enhance anti-tumor effects when the humanized anti-HM 1.24 antibody is administered in vivo or ex vivo to a patient with reduced immune function. Also, the combined use of BRM is expected to further enhance the anti-tumor effect when the humanized anti-HM 1.24 antibody is administered in vivo or ex vivo to a patient who has undergone a return of cells subjected to apheresis replacement at peripheral blood stem cell transplantation to the patient.

Reference example 1 preparation of hybridoma producing mouse anti-HM 1.24 monoclonal antibody

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

The plasmacytic cell line KPC-32 (1X 10) derived from bone marrow of human multiple myeloma patients was injected twice into the abdominal cavity of 6-week BALB/c mice (produced by Charles river)⁷Individual cells) (Goto, t. et al, japan journal of clinical hematology (1991)32, 1400).

Three days prior to animal killing, mice were injected with 1.5X 10 injections into their spleens⁶KPC-32 to further improve smallMurine antibody production capacity (Goto, t. et al, Tokushima journal of experimental medicine (1990)37, 89). After killing the animals, the spleens were removed and the removed organs were cell fused with myeloma cells SP2/0 as per Groth, dest. and Schreidegger (cancer research (1981)41, 3465).

Hybridoma culture supernatants were screened for antibodies by cell ELISA using KPC-32 (Posner, M.R. et al, J. Immunol. methods (1982)48, 23). Will be 5X 10⁴Each KPC-32 was suspended in 50ml of PBS, and then aliquoted into 96-well plates (U-bottom, manufactured by Corning, Iwaki) and air-dried at 37 ℃ overnight. After blocking with PBS containing 1% Bovine Serum Albumin (BSA), the culture supernatant of the hybridoma was added and incubated at 4 ℃ for 2 hours. Then, the reaction mixture was reacted with peroxidase-labeled anti-mouse IgG goat antibody (produced by Zymed) at 4 ℃ for 1 hour. After washing, the reaction mixture was reacted with an o-phenylenediamine substrate solution (manufactured by Sumitomo Bakelite) at room temperature for 30 minutes.

The reaction was terminated by adding 2N sulfuric acid, and the absorbance at 492nm was measured by using an ELISA plate reader (manufactured by Bio-Rad). In order to remove hybridomas producing anti-human immunoglobulin antibodies, the culture supernatants of positive hybridomas were adsorbed to human serum beforehand, and screened for reactivity with other cell lines by ELISA. Positive hybridomas were selected and their reactivity with various cells was studied by flow cytometry. The finally selected hybridoma colonies were cloned twice and injected into the abdominal cavity of pristane-treated BALB/c mice, from which ascites were obtained.

The monoclonal antibody was purified from ascites fluid of mice by ammonium sulfate precipitation and protein A affinity chromatography kit (Ampure PA, produced by Amersham.) the purified antibody was labeled with FITC using Quick Tag FITC binding kit (produced by Boehringer Mannheim).

As a result, monoclonal antibodies produced by 30 hybridoma colonies reacted with KPC-32 and RPMI 8226. After cloning, the culture supernatants of these hybridomas were investigated for reactivity with other cell lines or peripheral blood mononuclear cells.

Among the 3 clones, hybridoma clone which is most useful for flow cytometry and has CDC activity against RPMI8226 was selected and named HM 1.24. The subclass of monoclonal antibody produced by this hybridoma was determined by ELISA using a subclass-specific anti-mouse rabbit antibody (produced by Zymed). anti-HM 1.24 antibody has the subclass IgG2a K.hybridoma HM1.24 producing anti-HM 1.24 antibody was deposited internationally as defined in the Budapest treaty under the accession number FERM BP-5233 by the national institute of bioscience and human technology, the agency of Industrial science and technology, 1-3, Higashi1-chome, Tsukuba, Ibaraki pref, Japan, on 9/14.1995.

Reference example 2 preparation of humanized anti-HM 1.24 antibody

The humanized anti-HM 1.24 antibody is obtained according to the method described in International patent application WO 98-14580.

Total RNA was prepared from hybridoma HM1.24 prepared in reference example 1 by a conventional method. Thus, cDNA encoding the V region of the mouse antibody was synthesized and amplified by Polymerase Chain Reaction (PCR) and 5' -RACE. A DNA fragment containing the gene encoding the mouse V region was obtained, ligated with each plasmid pUC cloning vector, and then introduced into competent E.coli cells to obtain E.coli transformants. The above plasmid was obtained from the transformant. The base sequence of the cDNA coding region in the plasmid was determined by conventional methods and the Complementarity Determining Regions (CDRs) of each V region were determined.

In order to construct a vector expressing the chimeric anti-HM 1.24 antibody, cDNA encoding each of the L chain and H chain V regions of the mouse anti-HM 1.24 antibody is inserted into the HEF vector, and in addition, to construct a humanized anti-HM 1.24 antibody, the CDR of the V region of the mouse anti-HM 1.24 antibody is grafted to a human antibody by the CDR grafting method. The L chain of a human antibody is the L chain of a human antibody against the REI, the H chain of a human antibody against the Framework Region (FR)1-3 is the FR1-3 of a human antibody HG3, and the FR4 of a human antibody JH6 is used for FR 4. Substitution of amino acids in the FR of the H chain V region enables formation of a suitable antigen-binding site of the CDR-grafted antibody.

In order to express the thus constructed genes of the L chain and H chain of humanized anti-HM 1.24 antibody in mammalian cells, each gene was separately introduced into a HEF vector to construct a vector expressing the L chain or H chain of humanized anti-HM 1.24 antibody, respectively.

By introducing these two expression vectors into CHO cells simultaneously, a cell line producing humanized anti-HM 1.24 antibody was established. The antigen binding activity and binding inhibition activity of the humanized anti-HM 1.24 antibody obtained by culturing the human amniotic cell line WISH on the cell line were investigated by cell ELISA. The results showed that the humanized anti-HM 1.24 antibody had an antigen-binding activity equivalent to that of the chimeric antibody, and as to the binding inhibition activity studied using biotinylated mouse anti-HM 1.24 antibody, it had an activity equivalent to that of the chimeric antibody or mouse antibody.

Incidentally, Escherichia coli harboring a plasmid containing L chain or H chain of chimeric anti-HM 1.24 antibody has been deposited internationally by national institute of bioscience and human technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, under the provisions of the Budapest treaty, as Escherichia coli DH5 α (pUC19-1.24L-g κ) and Escherichia coli DH5 α (pUC19-1.24H-g γ 1) on 8/29.1996, with deposition numbers of FERM BP-5646 and FERM BP-5644, respectively.

In addition, Escherichia coli harboring plasmids containing humanized anti-HM 1.24 antibody L chain a type and H chain r type has been deposited internationally by the national institute of bioscience and human technology, agency of Industrial science and technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan, under the provisions of the Budapest treaty, as Escherichia coli DH5 α (pUC19-RVLa-AHM-gK) and Escherichia coli DH5 α (pUC19-RVHr-AHM-g γ 1) with accession numbers FERM BP-5645 and FERM BP-5643, respectively, at 8.29.1996. In addition, Escherichia coli harboring a plasmid containing humanized anti-HM 1.24 antibody H chain s type has been deposited internationally as Escherichia coli DH5 α (pUC19-RVHs-AHM-g γ 1) on 29.9.1997 by the national institute of bioscience and human technology, Industrial science and technology, 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki pref, Japan, under the Budapest treaty, under the number FERM BP-6127.

Reference example 3 cloning of cDNA encoding HM1.24 antigen protein

Construction of cDNA library

1) Preparation of Total RNA

cDNA encoding HM1.24 antigen protein that can be specifically recognized by mouse anti-HM 1.24 antibody was cloned as described below.

Total RNA was prepared from the human multiple myeloma cell line KPMM2 according to Chirgwin et al (biochemistry 18, 5294 (1979)). Then, the mixture was sufficiently homogenized in 20ml of 4M guanidine thiocyanate (manufactured by Nacalai Tesque Inc.)⁸And KPMM2.

The homogenate layer was applied to a 5.3M cesium chloride solution in a centrifuge tube and then centrifuged at 31,000 rpm for 24 hours at 20 ℃ in a Beckman SW40 rotor to pellet the RNA. The precipitate was washed with 70% ethanol and then dissolved in 300. mu.l of 10mM Tris-HCl (pH7.4) containing 1mM EDTA and 0.5% SDS. Pronase (manufactured by Boehringer) was added to a concentration of 0.5mg/ml, followed by incubation at 37 ℃ for 30 minutes, the mixture was extracted with phenol and chloroform, and RNA was precipitated with ethanol. The RNA pellet was then dissolved in 200. mu.l of 10mM Tris-HCl (pH7.4) containing 1mM EDTA.

2) preparation of poly (A) + RNA

Poly (A) + RNA was purified using Fast Track2.0mRNA isolation kit (manufactured by Invitrogen) according to the protocol attached to the kit, using 500. mu.g of total DNA prepared as described above as a starting material.

3) construction of cDNA library

Using a cDNA Synthesis kit TimeSaver cDNA Synthesis kit (produced by Pharmacia), double-stranded cDNA was synthesized using 10. mu.g of the above poly (A) + RNA as a starting material according to the protocol attached to the kit, and then ligated with the EcoRI linker provided in the kit using a directed cloning Toolbox (produced by Pharmacia) according to the protocol attached to the kit. Kination of EcoRI linker and restriction enzyme NotI treatment were performed according to the protocol attached to the kit. In addition, the adaptor-added double-stranded cDNA having a size of about 500bp or more was isolated and purified by 1.5% low melting point agarose gel (manufactured by Sigma), to obtain about 40. mu.l of adaptor-added double-stranded cDNA.

The thus-constructed double-stranded adaptor-added cDNA was ligated with the pCOS1 vector previously treated with restriction enzymes EcoRI and NotI and alkaline phosphatase (produced by Takara Shuzo) and T4DNA ligase (produced by GIBCO-BRL) to construct a cDNA library, the constructed cDNA library was transduced with E.coli DH 5. alpha. strain (produced by GIBCO-BRL), followed by estimation of about 2.5X 10⁶Total size independent clones. The plasmid pCOS1 was constructed by digesting the expression vector HEF-PMh-gGamma 1 (see International patent application WO92-19759) with EcoRI and SmaI, deleting the contained DNA, and ligating it with an EcoRI-NotI-BamHI linker (produced by Takara Shuzo).

2. Cloning by direct expression

1) Transfection of COS-7 cells

About 5X 10 of ampicillin in 2-YT medium (molecular cloning: A laboratory Manual, Sambrook et al, Cold spring harbor laboratory Press (1989)) containing 50. mu.g/ml of ampicillin⁵The above-mentioned transduced E.coli was cloned, cDNA was amplified, and plasmid DNA was recovered from E.coli by an alkaline lysis method (molecular cloning: A laboratory Manual, Sambrook et al, Cold spring harbor laboratory Press (1989)). The thus-obtained plasmid DNA was transfected into COS-7 cells by electroporation using a gene pulse generator (manufactured by Bio-Rad).

Then, 10 u g purified plasmid DNA was added to 0.8ml COS-7 cell solution, wherein the cells have been at 1X 10⁷Cells/ml were suspended in PBS and the mixture was pulsed at 1500V and 25. mu. FD capacitance. After 10 minutes of recovery at room temperature, the electroporated cells were cultured in DMEM medium (produced by GIBCO-BRL) containing 10% fetal bovine serum (produced by GIBCO-BRL) at 37 ℃ and 5% CO₂Cultured under the conditions for 3 days.

2) Preparation of elutriation vessel

The panning dishes coated with mouse anti-HM 1.24 antibody were prepared according to the method of B.seed et al (proceedings of the national academy of sciences USA, 84, 3365-. Then, mouse anti-HM 1.24 antibody was added to 50mM Tris-HCl (pH9.5) to a concentration of 10. mu.g/ml. 3 ml of the thus prepared antibody solution was added to a cell culture dish having a diameter of 60mm, and incubated at room temperature for 2 hours. After three washes with 0.15M NaCI solution, a Petri dish was added with a solution containing 5% fetal bovine serum, 1mM EDTA and 0.02% NaN₃The PBS (1). After blocking, the following cloning was performed.

3) cloning of cDNA library

COS-7 cells transfected as described above were lysed with PBS containing 5mM EDTA. After washing the cells once with PBS containing 5% fetal bovine serum, they were suspended in PBS containing 5% fetal bovine serum and 0.02% NaN₃To a concentration of about 1X 10 in PBS⁶Cells/ml. The suspension was added to the panning dishes prepared as described above and incubated for 2 hours at room temperature. The feed additive comprises 5% of fetal calf serum and 0.02% of NaN₃After three light washes with PBS, plasmid DNA was recovered from cells bound to the panning dish using a solution containing 0.6% SDS and 10mM EDTA.

The recovered plasmid DNA was transduced with E.coli DH5 α. After amplification as described above, plasmid DNA was recovered by alkaline lysis. The recovered plasmid DNA was transfected into COS-7 cells by electroporation, and the plasmid DNA was recovered from the cells bound as described above. The similar procedure was repeated once, and the recovered plasmid DNA was digested with restriction enzymes EcoRI and NotI to thereby determine the concentration of the insert of about 0.9kbp in size. In addition, a portion of the recovery of plasmid DNA transduction of Escherichia coli cells were inoculated to contain 50 u g/ml ampicillin 2-YT agar plate. After overnight incubation, plasmid DNA was recovered from individual colonies. Clone p3.19 was obtained by digestion with restriction enzymes EcoRI and NotI, the size of the insert being approximately 0.9 kbp.

The clones were subjected to a reaction using PRISM dye terminator cycle sequencing kit (manufactured by Perkin Elmer) according to the protocol attached to the kit, and their base sequences were determined using ABI373A DNA sequencer (manufactured by Perkin Elmer). The base sequence and the corresponding amino acid sequence are shown in SEQ ID NOs: 1 in (c).

Industrial applicability

By using the anti-HM 1.24 antibody in combination with the biological response modifier, the cellular activity of effector cells reduced in ADCC activity, in particular, is improved. This indicates that the combined use of the anti-HM 1.24 antibody and the biological response modifier results in an enhancement of the anti-tumor effect on lymphocyte tumors.

Reference is made to the microorganisms deposited under the rules of the patent Cooperation treaty 13-2, and to the name of the depository

Name: national institute of Life sciences and human technology, Industrial science and technology

Address: 1-3, Higashi1-chome, Tsukuba-shi, Ibaraki, Japan

Biology (1)

Name: escherichia coli DH5 alpha (pRS38-pUC19)

The preservation number is as follows: FERM BP-4434

The preservation date is as follows: 1993 at 10 months and 5 days

Biology (2)

Name: mouse-mouse hybridoma HM1.24

The preservation number is as follows: FERM BP-5233

The preservation date is as follows: 1995, 4 months and 27 days

Biology (3)

Name: escherichia coli DH5 alpha (pUC19-RVHr-AHM-g gamma 1)

The preservation number is as follows: FERM BP-5643

The preservation date is as follows: 1996, 8, 29 months

Biology (4)

Name: escherichia coli DH5 alpha (pUC19-1.24H-g gamma 1)

The preservation number is as follows: FERM BP-5644

The preservation date is as follows: 1996, 8, 29 months

Biology (5)

Name: escherichia coli DH5 alpha (pUC19-RVLa-AHN-g kappa)

The preservation number is as follows: FERM BP-5645

The preservation date is as follows: 1996, 8, 29 months

Biology (6)

Name: escherichia coli DH5 alpha (pUC19-1.24L-g kappa)

The preservation number is as follows: FERM BP-5646

The preservation date is as follows: 1996, 8, 29 months

Biology (7)

Name: escherichia coli DH5 alpha (pUC19-RVHs-AHM-g gamma 1)

The preservation number is as follows: FERM BP-6127

The preservation date is as follows: 9/29/1997

Sequence listing

SEQ ID NO：1

Sequence length: 1013

Sequence types: nucleic acids

Chain type: single strand

Topological structure: linearity

Molecular type: cDNA

Sequence of

SEQ ID NO：2

Sequence length: 379

Sequence types: nucleic acids

Topological structure: linearity

Molecular type: cDNA

Sequence of

SEQ ID NO：3

Sequence length: 418

Sequence types: nucleic acids

Topological structure: linearity

Molecular type: cDNA

Sequence of

SEQ ID NO：4

Sequence length: 418

Sequence types: nucleic acids

Topological structure: linearity

Molecular type: cDNA

Sequence of

Claims (8)

1. Use of a biological response modifier for the manufacture of a medicament for the treatment of a lymphocytic neoplasm in a human patient, wherein the medicament is for use in combination with a second medicament for the treatment of a lymphocytic tumor, the second medicament being administered to the patient before or after the response modifier, wherein the second medicament comprises, as an active ingredient, a humanized monoclonal antibody which specifically binds to an epitope recognized by the anti-HM 1.24 antibody produced by the hybridoma deposited as FERM BP-5233, and the biological response modifier is IL-10, wherein the human patient has reduced ADCC activity and the reduced ADCC activity in the human patient is less than 25% ADCC activity when at an E/T ratio of 50, wherein the biological response modifier enhances the cytotoxicity of the humanized monoclonal antibody.

2. Use according to claim 1, wherein the lymphocytic tumor is a T cell tumor.

3. Use according to claim 1, wherein the lymphocytic tumor is a B cell tumor.

4. Use according to claim 3, wherein the B cell tumour is a myeloma.

5. Use according to claim 1, wherein the cytotoxicity is ADCC activity.

6. Use according to claim 1, wherein the antibody has the constant region cy of a human antibody.

7. The use according to claim 6, wherein the constant region C γ of the human antibody is C γ 1 or C γ 3.

8. The use according to claim 1, wherein the antibody is a humanized anti-HM 1.24 antibody comprising an L chain encoded by FERM BP-5645 and an H chain encoded by FERM BP-5643 or FERM BP-6127.