The tool used in the treatment of tumors of lymphatic tissue

 

(57) Abstract:

The invention relates to medicine. The essence of it is the drug used in the treatment of tumors of lymphatic tissue (excluding myeloma) comprising as an active ingredient an antibody that specifically binds to a protein having the amino acid sequence specified in SEQ ID NO: 1, in the description, and which has cytotoxic activity. The specified antibody contains constatou area C or is a chimeric antibody against NM. The technical result - expanding Arsenal of tools against tumors of lymphatic tissue. 2 C. and 17 C. p. F.-ly, 27 ill. , 1 table.

The technical field to which the present invention

The invention relates to the means used in the treatment of tumors of the lymphatic tissue (excluding myeloma) comprising as an active ingredient antibodies that specifically bind to proteins expressed in these tumors of lymphatic tissue. The present invention relates also to the means used in the treatment of tumors of T cells or tumor cells (excluding myeloma). In addition, the present invention is x tissues, and that possess cytotoxic activity.

Background of the invention

For the immunity of living beings are responsible primarily lymphatic cells. The lymph cells include all cells that originate from the same hematopoietic stem cells, which are released into the peripheral blood after repeated stages of differentiation under the influence of various factors that induce differentiation, including factors called growth factors present in the bone marrow or other organs. On the basis of differences in the differentiation of lymphocytes are classified into two major groups of b-cells and T-cells. It is believed that B-cells have the ability to produce antibodies, while T-cells are related to the manifestation of antigenicity, cytotoxicity and other properties. In this state, when such cells for one reason or another are changes associated with the formation of tumors at various stages of differentiation and begin to proliferate uncontrollably in the bone marrow, lymphatic tissue, blood or etc., referred to tumor lymphatic tissue.

Through the use of new technologies, in particular those advantages is possible to identify the start and/or stage of differentiation of lymphatic cells. In this case, it became possible not only to determine which T-cells or b-cells, occur such tumor cells, but also to identify the degree of maturity of tumor cells.

Tumors of lymphatic tissue on the basis of origin and maturity of tumor cells are divided into two major classes: tumor cells and tumor T cells. Depending on the degree of maturity of tumor cells tumor cells then classify acute-lymphocytic leukemia (B-ALL), chronic b-lymphocytic leukemia (b-CLL), pre-B-lymphoma, lymphoma Burkitt, follicular lymphoma, follicular lymphoma of the cerebral cortex, diffuse lymphoma, etc. on the other hand, tumor T cells, depending on the degree of maturity of tumor cells to classify acute-lymphocytic leukemia (B-ALL), chronic b-lymphocytic leukemia (b-CLL), a disease related to the human virus of T cells (LAT), lymphoma peripheral T cells do not ATL-type (PNTL) and others (Zukai Rinso [Gan] (Illustrated Clinical: Cancer), series 17 the Leukemia and lymphoma, Takashi Sugimura et al. , Medical View Co. Co., Ltd. , 1987, In cell tumors, Kiyoshi Takatsuki, Nishimura Shoten, 1991).

However, despite recent advances in medical technology, treatment of tumors of lymphatic tissue has not achieved satisfactory results. Stepova disease about 50%, this relatively high degree of cure In-lymphoma associated with the progress of multiple therapy. In addition, T-lymphoma are more likely to penetrate other tissues and is characterized by the degree of cure of about 30%, whereas the degree of cure of a disease associated with the human virus of T cells (LAT), currently stands at 10%.

In addition, goto et al. (Goto, A. et al. ) reported receiving monoclonal antibodies (antibodies to NM.24) after immunization of mice myeloma cells (Blood, 1994, 84, 1922-1930). With the introduction of mice transplanted with cells of the myeloma human antibodies to NM.24, the antibody specifically accumulates in tumor tissues (Masaakai Kosaka et al. , Nippon Rinsho (Japan Clinical) 1995, 53, 627-635), which suggests the possibility of using antibodies to NM.24 for the diagnosis of tumor localization with radioisotope tagging in therapy of particles, such as radioimmunotherapy, etc. But no known evidence that antibody to NM.24 is used to treat tumors of the lymphatic tissue.

The invention

The currently used method of treatment of tumors of the lymphatic tissue includes various types of chemotherapy, radiotherapy, transmetallation in the treatment of these diseases and, in this regard, there is still the need for a breakthrough in the creation of medicines and the development of methods that could facilitate the course of cancer of the lymphatic tissue and lengthen the life expectancy of patients.

In connection with the foregoing, the present invention is the development of medicines for use in case of tumors of the lymphatic tissue, except for myeloma.

To create such a drug, the inventors have conducted extensive studies in vitro, which included analysis by flow cytometry, the determination of the cytotoxic activity, such as antiteloobrazovanie cleocinonline cytotoxicity (ADCC/OCC activity), complementability cytotoxicity (CDC/CSC cytotoxicity) and others , in vivo studies to determine the antitumor effect of using antibodies to NM.24 (Goto et al. Blood, 1994, 84, 1922-1930), as well as work on the selection of the protein antigen with which the antibody specifically binds to NM.24. As a result of the studies the authors showed that lymphatic tumors expressed protein antigen specifically recognized by antibody to NM.24, with the indicated antibody to Nastojashego invention.

In this regard, the present invention relates to a medicinal product used in the tumors of lymphatic tissue (excluding myeloma), which includes as an active ingredient, an antibody that specifically binds to a protein having the amino acid sequence shown in the sequence SEQ ID NO: 1, and which has cytotoxic activity (sequence listing is provided at the end of the description).

The present invention also relates to a medicinal product used in the tumors, T-cell, or a drug that is used for tumors In the cells (excluding myeloma) comprising as an active ingredient an antibody that specifically binds to a protein having the amino acid sequence shown in the sequence SEQ ID NO: 1, and which has cytotoxic activity.

The present invention also relates to a medicinal product used in the tumors, T-cell, or a drug that is used for tumors In the cells (excluding myeloma) comprising as an active ingredient a monoclonal antibody that specifically svyazyvaete who gives cytotoxic activity.

The present invention also relates to a medicinal product used in the tumors, T-cell, or a drug that is used for tumors In the cells (excluding myeloma) comprising as an active ingredient an antibody that specifically binds to a protein having the amino acid sequence shown in the sequence SEQ ID NO: 1, and which has AOCC or CSC activity as cytotoxic activity.

The present invention also relates to a medicinal product used in the tumors, T-cell, or a drug that is used for tumors In the cells (excluding myeloma) comprising as an active ingredient an antibody that specifically binds to a protein having the amino acid sequence shown in the sequence SEQ ID NO: 1, and in which the constant region contains human antibodies.

The present invention also relates to a medicinal product used in the tumors, T-cell, or a drug that is used for tumors In the cells (excluding myeloma) comprising as an active ingredient chimeric anti the sequence, specified in the sequence of SEQ ID NO: 1, and which has cytotoxic activity.

The present invention also relates to a medicinal product used in the tumors, T-cell, or a drug that is used for tumors In the cells (excluding myeloma) comprising as an active ingredient an antibody that specifically binds to the epitope recognized by the antibody to NM.24.

The present invention also relates to a medicinal product used in the tumors, T-cell, or a drug that is used for tumors In the cells (excluding myeloma) comprising as an active ingredient the antibody to NM.24.

In addition, the present invention relates to an antibody that specifically binds to a protein expressed in tumors of lymphatic tissue, and which has cytotoxic activity.

Brief description of drawings

In Fig. 1 shows a histogram obtained by analysis by flow cytometry indicated In cell lines by indirect method using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 2 shows a histogram of the criminal code is Itanium antibodies to NM.24 and the control mouse IgG2a.

In Fig. 3 shows a histogram of the specified b-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 4 shows a histogram of the specified b-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 5 shows a histogram of the specified b-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 6 shows a histogram of the specified b-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 7 shows a histogram of the specified b-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 8 shows a histogram of the specified b-cell line, which was investigated by an indirect method by way of flow cytometry using an the AI, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 10 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 11 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 12 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 13 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 14 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

On pyrotechny cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 16 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 17 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 18 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 19 shows a histogram of the indicated T-cell line, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 20 shows a histogram of the specified cell line, is not either the T-or b-type, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 21 shows a histogram of the specified cell line, is not either the T-or b-type, which was not investigated what P CLASS="ptx2">

In Fig. 22 shows a histogram of the specified cell line, is not either the T-or b-type, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 23 shows a histogram of the specified cell line, is not either the T-or b-type, which was investigated by an indirect method by way of flow cytometry using antibodies to NM.24 and the control mouse IgG2a.

In Fig. 24 is a graph showing that antibody to NM.24 has a cytotoxic effect on the cell line tumor T cells CCRF-CEM, CCRF-HSB-2 and HPB-MLT dependent on dose.

In Fig. 25 is a graph showing that antibody to NM.24 has a cytotoxic effect on the cell lines of tumor cells EB-3, MS and CCRF-SB-dependent dose.

In Fig. 26 is a graph showing that the introduction of antibodies to NM.24 experimental group of mice with transplanted lymphatic tumor of man, is the suppression of increase in tumor volume compared to control animals, which were injected IgG2a.

In Fig. 27 is a graph showing that the introduction of antimalaria survival compared with the control group of animals, which was introduced IgG2a.

The embodiments of the invention

1. Antibodies

1-1. Getting hybridoma

Hybridoma that produce antibodies used in the present invention, can be constructed mainly using well-known procedures below. Thus, the protein antigen NM.24 or cells that Express the antigen NM.24, can be used as a sensitizing antigen and then used for immunization in the framework of the conventional method of immunization. The obtained immune cells merge with known parent cells in the normal course of merge cells, and then conduct screening using traditional screening method for selection of cells that produce monoclonal antibodies.

Specifically, monoclonal antibodies can be obtained in the following way. So, for example, as cells expressing the antigen NM.24, which represents a sensitizing antigen for obtaining antibodies, can be used multiple myeloma cell line human CRM (pending patent application of Japan (Kokai) 7-236475) or CRS-32 (Goto, T. et. al. , Jpn. J. Clin. Hematol. , 1991, 32, 1400). Alternatively, as CE is th below in SEQ ID NO: 1, or painted, or polypeptide containing the epitope recognized by the antibody against NM.24.

In the framework of the present description cDNA, which encodes a protein having the amino acid sequence specified in SEQ ID NO: 1 was inserted into the cleavage site XbaI vector pUC19 to obtain plasmid pRS38-pUC19. E. coli carrying this plasmid deposited with the International Collection in accordance with the regulations of the Budapest Treaty as Escherichia coli DH5 (pRS38-pUC19) of 5 October 1993 by the National Institute of Bioscience and human technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) as FERM BP-4434 (see unexamined patent application of Japan (Kokai) 7-196694). A fragment of the cDNA contained in the plasmid pRS38-pUC19, can be used to obtain a peptide or polypeptide containing the epitope recognized by the antibody against NM.24, using genetic engineering techniques.

Preferably, the mammals, which are subjected to immunization sensitizing antigen, selected based on their compatibility with the parent cell in the procedure of cell fusion. Mostly they include, but are not limited to, rodents such as mice, rats, hamsters, etc.

Immunizati, what, for example, intraperitoneal or subcutaneous administration sensitizing antigen to the mammal. Specifically, the sensitizing antigen, which was diluted and suspended in an appropriate amount of a phosphate buffer solution (FBI) or saline solution, etc. are mixed, if desired, with a suitable adjuvant's adjuvant. After emulsification it is preferably administered to the mammal multiple times every day 4-21. An alternative may be used a suitable carrier for administration at the time of immunization sensitizing antigen.

After immunization, and proof of increasing the level of the desired antibodies in the serum of immune cells taken from the body of the mammal and carry out cell fusion, in which the preferred immune cells include, in particular, cells of the spleen.

The myeloma cells of mammals, as an example of the other parent cells, which are subjected to the procedure of cell merging with these immune cells, preferably include various other cell lines, such as Rad.653 (J. Immunol. 1979, 123: 1548-1550), P3X63Ag8U. l (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 (Margu, . Exp. Med. , 1978, 148: 313-323), R210 (Galfre, G. et. al. , Nature, 1979, 277: 131-133), etc.

The procedure of cell fusion between immune cells and myeloma cells can be performed essentially in accordance with the known method, such as described by Milshtein et al. (Kohler, G. and Milstein, C. , Methods Enzymol. , 1981, 73: 3-46) and other

More specifically, the above procedure merge cells is carried out in nutrient broth in the presence of, for example, accelerator merge cells. As accelerator merge cells using, for example, polyethylene glycol (PEG), Sendai virus (HVJ) and, in addition, may be optionally added adjuvant such as dimethyl sulfoxide, and others , to improve the activity of the merger.

Preferably, the ratio used of immune cells and myeloma cells corresponds to, for example, 1-10 times the number of immune cells in comparison with myeloma. An example of cultural media that can be used to implement the above process of merging cells is a medium RPMI1640 culture medium MEM, suitable for the growth of these myeloma cell lines, and conventional culture medium used for culturing the specified type is T, FSC).

In the process of merging cells specified number of these immune cells and myeloma cells are thoroughly mixed in the culture medium, to which is added a solution of PEG, pre-heated to approximately 37oWith, for example, a solution of PEG with an average molecular weight from about 1000 to 6000, at a concentration of from 30% to 60% (weight/volume) and stirred for obtaining the desired cell fusion (hybrid). Then, by repeated successive addition of a suitable culture medium and centrifugation, removing the supernatant, the agents needed to merge cells, etc. that are undesirable for the growth of hybridomas can be deleted.

The selection of these hybrid conducted under cultivation in traditional breeding environment, such as culture medium HAT (liquid culture containing gipoksantin, aminopterin and thymidine). Cultivation in the specified culture medium continue, mainly in the period of time sufficient for cell death that is different from the desired hybrid (which are not cells merge), and, typically, it ranges from several days to several weeks. Usually use the traditional method of serial dilutions within kotorogo CLASS="ptx2">

In addition to obtaining this hybridoma through immunization, the antigen of the animal other than man, it is also possible to sensitize human lymphocytes in vitro antigen NM.24 or cells expressing antican NM.24, and then the sensitized lymphocytes merge with myeloma cells such as U266, to obtain the desired human antibody having the activity of binding to the antigen NM.24 or cells expressing the antigen NM.24 (see publication for patent application in Japan (Kokoku) 1-59878). In addition, the transgenic animal carrying a range of all of antibody genes of the person subjected to immunization with antigen, for example an antigen NM.24, or cells, espressioni antigen NM.24, to obtain the desired gumanitarnogo antibodies according to the above method (see application for international patent WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).

Designed to hybridoma producing monoclonal antibody can be subjected to subculturing in traditional culture medium or may be stored for a long period of time in liquid nitrogen.

To obtain monoclonal antibodies from a specified hybridoma COI is in the supernatant, or method, according to which hybridoma injected for subsequent growth in the body of a mammal compatible with the specified hybridomas, and then receive antibodies from ascites. The first method is suitable for obtaining antibodies of high purity, while the latter is suitable for large-scale process of obtaining antibodies.

In particular, hybridoma producing antibody against NM.24, can be obtained using the method of goto (Goto, T. , et. al. , Blood, 1994, 84: 1922-1930). It can also be obtained by the method in which hybridoma producing antibody against NM.24, which was deposited in the International Collection in accordance with the provisions of the Budapest Treaty as FERM BP-5233 14 September 1995 by the National Institute of Bioscience and human technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) is injected intraperitoneally to mice of BALB/C (production CLEA, Japan) to produce ascites, which is isolated and purified antibodies against NM.24, or use the method, according to which the specified hybridoma cultivated in a suitable culture medium such as RPMI1640 containing 10% amniotic si is I (production GIBCO-BRL) and other and in this case, the antibody against NM.24 can be isolated and purified from the supernatant.

1-2. Recombinant antibody

Recombinant antibody obtained in accordance with the technology of manipulation of recombinant genes in which the gene for the antibody clone from hybridoma and integrate into an appropriate vector, which is then injected into the host organism, can be used according to the present invention as monoclonal antibody (see , for example, Carl, A. K. , orrebaeck, James, W. Larrick, Therapeutic Monoclonal Antibodies, published in the UK, Macmillan Publishers LTD. , 1990).

Specifically, mRNA encoding the variable region (V region) of the desired antibodies, separated from hybridoma producing the antibody. For isolation of mRNA receive total RNA using, for example, a known method such as a method of ultracentrifugation in guanidine (Chirgwin, J. M et al. , Biochemistry, 1979, 18: 5294-5299), the AGPC method (Chomczynski, P. et al. , Analytical Biochemistry, 1987, 162: 156-159), and then allocate the mRNA from the total RNA using a kit for purification of mRNA (production Pharmacia) and other Alternative mRNA can be directly retrieved using a set of Quick Prep for the purification of mRNA production Pharmacia.

cDNA to the V-region of the antibodies can be synthesized using including reverse transcriptase for cDNA synthesis AMV Reverse Transcriptase First-strand cDNA Synthesis Kit and other Alternatively, for the synthesis and amplification of cDNA can be used to set the 5'-Ampli Finder Race Kit (production Clontech) and the 5'-Race (Frohman, M. A. et al. , Proc. Natl. Acad. Sci. USA, 1988, 85: 8998-9002; Belyavsky, A. et al. , Nucleic Acids Res. , 1989, 17: 2919-2932), which utilizes polymerase chain reaction (PCR). The desired DNA fragment is purified from the obtained PCR product and then it can be Legerova with vector DNA. In addition, on the basis of the design of recombinant vector and injected it into E. coli and selection of colonies to obtain the desired recombinant vector. The nucleotide sequence of a given DNA can be confirmed by using a known method such as dimethoxylated.

When obtaining DNA encoding the V region of the desired antibodies, it can be ligitamate with DNA encoding the constant region (C-region) of the desired antibodies, and then the resulting product is integrated into an expression vector. Alternatively, DNA encoding the V region of the antibody, can be integrated into an expression vector that contains DNA encoding the C-region of the antibody.

For producing antibodies used in the present invention, the antibody gene is integrated, as will be described below, the expression vector so that h is. the consequence of the expression vector may be brought by the transformation into the host cell, where the antibody can be expressed.

1-3. The modified antibody

In accordance with the present invention artificially modified recombinant antibody such as a chimeric antibody and humanitariannet antibody can be used for the purpose of lowering heterologous antigenicity against humans. Such modified antibodies can be obtained using known methods.

A chimeric antibody can be obtained by legirovanii obtained as described above, DNA encoding the V region of the antibody with DNA coding-region of human antibodies, and then the resulting product is inserted into an expression vector and introduced into a host organism for production of antibodies in it (see the application for the European patent EP 125023 and application for international patent WO 96/02576). Chimeric antibody used in the present invention, can be obtained using a known method.

For example, E. coli carrying a plasmid that contains the DNA encoding the L-chain V-region or H-chain V-region of the chimeric antibody against NM.24, was deposited in the International Collection in accordance with clause the 996 National Institute of Bioscience and human technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) as FERM BP-5646 and FERM BP-5644, respectively (see patent application Japan 9-271536).

Humanitariannet antibody, which is also called the restored human antibody is obtained by transplantation hypervariable segment (HLG) antibody of a mammal other than human, such as mouse antibodies, HLG human antibodies. In General, the technology based on the preparation of recombinant DNA to produce such antibodies are also known (see application for the European patent EP 125023 and application for international patent WO 96/02576).

Specifically, PCR synthesized DNA sequence, which should be suitable for ligating HLG mouse antibodies with frame area (KO) human antibodies using several separate oligonucleotides bearing on the ends of the sections overlapping one another. Thus obtained DNA are ligated with DNA encoding the C-region of a human antibody, and then embed the resulting product into the expression vector which is then introduced into the host organism for the production of antibodies (see application for the European patent EP 239400 and application for international patent WO 96/02576).

For example, E. coli carrying a plasmid which contains a DNA encoding a variant (SEQ ID NO: 2) L-chain V-region and a variant of r (SEQ ID NO: 2) L-chain V-region gumanitarnogo antibodies against NM.24, was deposited in the International Collection in accordance with the provisions of the Budapest Treaty as Escherichia coli DH5 (pUC19-RVLa-AHM-gk) and Escherichia coli DH5 (U19-Vr-ASM-gyl), respectively, on 29 August 1996, the National Institute of Bioscience and human technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) as FERM BP-5645 and FERM BP-5643, respectively (see patent application Japan 9-271536). In addition, E. coli carrying a plasmid that contains the DNA encoding the variant s (SEQ ID NO: 4) H-chain V-region gumanitarnogo antibodies against NM.24, was deposited in the International Collection in accordance with the provisions of the Budapest Treaty as Escherichia coli DH5 (pUC19-RVHs-AHience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) as FERM BP-6127 (patent application Japan 9-271536).

In the case of chimeric or gumanitarnogo antibodies used-region of a human antibody, and most preferably in the constant region of human antibodies may be used, such as C1, C2, C3 and C4. Among these antibodies, containing C1 and C3, have potent cytotoxic activity, i.e. AOCC and CSC activity, and in this regard, they are preferably used in the present invention.

Chimeric antibody comprises the variable region of antibody derived from a mammal other than human, and C-region derived from human antibody, while humanitariannet antibody comprises hypervariable sites of antibody derived from a mammal other than human, the frame region and C-region of antibodies derived from human antibodies. In accordance with their antigenicity in the human body is reduced, so that they can be used as an active ingredient of medicines according to the present invention.

The preferred option gumanitarnogo antibodies for IP is ponii 9-271536). A preferred variant of the L-chain V-region gumanitarnogo antibodies against NM.24 includes the one which has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2. A preferred variant of the H-chain V-region gumanitarnogo antibodies against NM.24 includes the one that has the amino acid sequence encoded by the sequence of bases shown in SEQ ID NO: 3 or 4.

1-4. Expression and production

Genes for antibodies, constructed as described above, can be expressed, resulting in using a known method can be obtained antibody. In the case of mammalian cells expression can be carried out using the expression vector containing commonly used promoter, the antibody gene that will be expressed, and DNA, in which the poly And the signal was quickly sewn on the 3' end in the direction of replication, or a vector containing this DNA. Examples of promoter/enhancer include early promoter/enhancer of the human cytomegalovirus.

In addition, as a promoter-enhancer, which can be used for expression of the antibodies in the present image is the USA polimi, adenovirus and simian vacuolating virus 40 (SV40), and promoters/enhancers derived from mammalian cells such as human elongation factor 1 (HEF1).

For example, expression can be easily carried out by the method of Milligan et al. (Milligan et al. , Nature, 1979, 277, 108) within which the promoter/enhancer of SV40, or by the method of Mizushima et al. (Mizushima et al. , Nucleic Acids Res. , 1990, 18, 5322), which uses the promoter/enhancer HEF1.

In the case of E. coli expression can be carried out in the operational binding is commonly used promoter, a signal sequence for secretion of antibodies and antibody gene to be expressed, followed by its expression. As a promoter, for example, to mention the lacz promoter and promoter Agabus. In the case of promoter lacz you can use the method of ward et al. (Ward et al. , Nature, 1998, 341, 544-546; Faseb J. 1992, 6, 2422-2427), and in the case of promoter agaves can find application method Better et al. (Better et al. , Science, 1988, 240, 1041-1043).

As a signal sequence for secretion of antibodies when producing it in periplasm E. Li can be used to signal the pelB sequence (Lei, S. P. et al. , J. Bacteri the training before use (see for example, WO 96/30394).

As customers start replication can be used such sites, which are derived from SV40, a virus polyoma, adenovirus, virus, bovine papillomavirus (BPV), etc. in Addition, amplification of gene copies in the host cell system, the expression vector may include as a breeding marker gene aminoglycosides (ARN), gene timedancing (TC) and gene contingenciesliabilities E. Li (Ecogpt) gene digidrofolatreduktazy (dhfr), etc.

For producing antibodies used in the present invention, it is possible to apply any system of production. System for production of antibodies may be based on a system of production in vitro and in vivo. As a system of production in vitro may be mentioned the production system, which uses eukaryotic cells, and the system of production, which are prokaryotic cells.

System of production based on eukaryotic cells include animal cells, plant cells and fungal cells. Known animal cells include (1) mammalian cells such as cells of Cho, COS cells, myeloma cells, kidney cells, baby hamsters (KSS), He is zvezdnye plant cells include, for example, those that come from the genus Nicotiana and more specifically, cells derived from Nicotiana tabacum, which form a callus culture. Known fungal culture include yeasts such as the genus Saccharomyces, more specifically Saccharomyces cereviceae or filamentous fungi such as the genus Aspergillus, and more specifically Aspergillus niger.

System of production on the basis of prokaryotic cells include bacterial cells. Known bacterial cells include Escherichia coli (E. coli) and Bacillus subtilis.

The antibody can be obtained by introduction through the process of transformation of the gene of the desired antibodies in these cells and further culturing the transformed cells in vitro. The cultivation is carried out with known methods. For example, can be used culture medium such as DMEM, MEM, RPMI1640, which may be of serum supplements such as amniotic calf serum (BCS). In addition, antibodies can be produced in vivo upon implantation of cells into which has been introduced a gene antibodies in the abdominal cavity of the animal, etc.

As other systems of production can be mentioned those that use animals, and those in which and insects.

As mammals can be used goats, pigs, sheep, mice and cattle (Vicki Glaser, Spectrum Biotechnology applications, 1993). As insects can be used silkworms.

In the case of plants you can find application in the tobacco plant.

Gene antibodies injected into the body of these animals and plants, and in these animals and plants are produced antibodies. For example, the antibody gene is introduced into the middle of the gene encoding a protein that is produced in milk, such as goat-casein, to obtain the fused genes. DNA fragments containing the fused gene, which was inserted gene antibody is injected into a goat embryo, and then the embryo is introduced into the body of the goat. The desired antibody is obtained from milk produced by the transgenic goat, which was the goat, which introduced the specified embryo or her offspring. In order to increase the amount of milk containing the desired antibody produced by the transgenic goat, such transgenic goat, in case of acceptance, may be injected hormones (Ebert, K. M. et al. , Bio/Technology, 1994, 12, 699-702).

In the case of silkworm to infect his use of the baculovirus, which is incorporated gene preferably is al. , Nature 1985, 315, 592-594). And in the case of tobacco plants, the gene of the desired antibody are inserted into suitable for plant expression vector, for example a PMOs 530, and then the vector is introduced into a bacterium such as Agrobacterum tumefaciens. Specified bacteria then used for infection of tobacco plants, such as Nicotiana tabacum, to obtain the desired antibodies from tobacco leaves (Julian, K.-C. MA et al. , Eur. J. Immunol. , 1994, 24, 131-138).

When the production of antibodies in production systems in vitro and in vivo, as described above, DNA encoding a heavy chain (H chain) or light chain (L chain) of antibody may be separately introduced into the expression vector, which then simultaneously transform the host cell, in another case, a DNA encoding the H chain and L chain may be integrated into a single expression vector and used for transformation of the host organism (see application for international patent WO 94/11523).

The antibody produced by the above method, may be associated with various molecules such as polyethylene glycol (PEG), to obtain for the purpose of subsequent use of the modified antibodies. "Antibody" in the context of the present description includes such modified antibodies. In order palua level of technology.

2. Separation and purification of antibodies

2-1. Separation and purification of antibodies

Antibodies are produced and expressed, as described above, can be separated from vorena or external environment of the cell or isolated from the host organism and further they can be purified to homogeneity. Isolation and purification of antibodies for later use in the present invention can be carried out using affinity chromatography. As the column used in such an affinity chromatography column can be used with protein a and column with protein G. Examples of carriers used in the column And are Hyper d , Poros, Sepharose F. F. etc.

Alternatively, without any restrictions can be used traditional methods of isolation and purification of proteins. Isolation and purification of antibodies for use further in the present invention can be carried out when linking properly in the process of chromatography was carried out, different from the above-mentioned affinity chromatography, filter, ultrafiltration, salting out, dialysis and other Chromatography includes, for example, ion exchange chromatography, hydrophobic chromatography, gel-filtration and other Such types of chromatography can br> 2-2. The concentration of antibodies

The concentration of the antibodies obtained in the above section 2-1, can be determined by measuring the absorption or by using enzyme linked immunosorbent assay (IFTF), etc. So that when it is used to measure the level of absorption, the antibody of the present invention or a sample containing the antibody, diluted appropriately FBI(-) and then measure the absorption at 280 nm, followed by a calculation using the absorbance (optical density) equal to 1.35, at a concentration of 1 mg/ml by using the method of IFTTA measurements carried out as follows. 100 μl of goat against human IgG (manufactured by Bio Source) diluted to a concentration of 1 μg/ml in 0.1 M bicarbonate buffer, pH 9,6, bring on 96-alopecia microtiter plate (manufactured Nunc) and incubated overnight at a temperature of 4oFor immobilization of antibodies.

After blocking, 100 μl of each diluted appropriately antibodies of the present invention or a sample containing the antibody, or 100 μl of human IgG of known concentration, taken as standard, add in the cell and carry out incubation at room the ESA, labeled with alkaline phosphatase (manufactured by Bio Source), and carry out incubation at room temperature for 1 hour. After wash add substrate solution and incubated, and then, after measuring the absorption at 405 nm using the Microplate reader model 3550 (manufactured by Bio-Rad), shall count the concentration of the desired antibody.

3. Analysis by flow cytometry

Reactivity of the antibodies used in the present invention against tumor cells in lymphatic tissue may be analyzed by flow cytometry. Used cells may be an established cell lines or freshly isolated cells. As established cell lines can be used, for example, a line of T-cells and RPMI 8402 (ATSS CRL 1994), CCRF-CEM (ATSS CCL-119), obtained from cells in acute lymphoblastic leukemia, HPB-ALL (FCCH1018) obtained from cells in acute leukemia, HPB-MLT (FCCH1019) obtained from cells in T-lymphoma, JM (FCCH1023) obtained from cells in acute leukemia, MOLT-4 (ATSS CRL-1582), obtained from cells in acute lymphoblastic leukemia, Jurkat (FCCH1024) obtained from cells in acute leukemia, CCRF-HSB-2 (ATSS CCL-120.1), obtained from cells in acute lampolla, obtained from cell lymphoma, Lennart (Shimizu, S. et al. , Blood, 1988, 71, 196-203), and others ; and as the line b-cell line (ATSS TIB-190), transformed by EB virus, positive for the virus EV line b-cells SKW 6.4 (ATSS TIB-215), MS (ATCC CRL-1649), obtained from cells In lymphoma, CCRF-SB (ATCC CCL-120) derived from cells in acute lymphoblastic leukemia, line b-cells RPMI 6410 (FCCH6047) obtained from a patient with acute miliitary leukemia, Daudi (ATCC CCL-213), obtained from cell lymphoma Burkitt, EB-3 (ATCC CCL-85) was obtained from cell lymphoma Burkitt, Jijoye (ATCC CCL-87) was obtained from cell lymphoma Burkitt, Raji (ATCC CCL-86), obtained from cell lymphoma Burkitt, and as cell lines, is not either the T-or b-type, line HL-60 (ATCC CCL-240) was obtained from cells in acute miliitary leukemia, TNR-1 (ATCC TIB-202) was obtained from cells in acute monocytic leukemia U-937 (ATCC CRL-1593), obtained from cells at histiocytomas lymphoma, K-562 (ATCC CCL-243), obtained from cells in chronic miliitary leukemia and other

After washing the above-mentioned cells in the FBI(-) to add 100 ál of antibody or control antibody, diluted to a concentration of 25 μg/ml in FACS buffer FBI ( -) containing 2% amniotic calf serum and 0.1% Aleut 100 ál protivorechivogo goat antibodies labeled FITZ (GAM, production Becton Dickinson), at a concentration of 25 μg/ml and then incubated the mixture on ice for 30 minutes. After washing in FACS buffer, the cells are suspended in 600 ál or 1 ml FACS buffer and measure the intensity of fluorescence of all cells using FACScan instrument (manufacture Becton Dickinson).

On the basis of obtained for each cell type the values of the fluorescence intensity is calculated reactivity of antibodies from the point of view of use of the present invention, each type of cells. So, based on the value of fluorescence intensity for each cell type can be defined, whether expressed antigen NM.24 on the cells of each type (positive or negative) or may be determined by the degree of expression. Data on the presence and intensity of expression of antigen NM.24 in the tumor cells of lymphatic tissue below in example 2.2, is devoted to the analysis method of flow cytometry.

Tumor cells in the tumors of lymphatic tissue, which can be targeted for treatment by the present invention that Express the antigen NM.24. More specifically, tumor cells in the tumor lymphatic tissue preferably relate to those cells, which is e 5%. More specifically, tumor cells in tumors of lymphatic tissue preferably represent those in which positive percentage manifestations of antigen NM.24 is 20% or higher.

More specifically, tumor cells in tumors of lymphatic tissue are preferably those in which a positive percentage manifestations of antigen NM.24 is 50% or higher. More specifically, tumor cells in tumors of lymphatic tissue preferably represent those in which positive percentage manifestations of antigen NM.24 is 80% or higher.

4. Cytotoxic activity

4-1. Measurement CSC activity

The antibody used in the present invention, represents such an antibody, which has, for example, CSC activity as cytotoxic activity.

CSC activity medicines used for tumors of the lymphatic tissue of the present invention, can be measured as follows. First prepare the target cells at a concentration of 4105cells/ml in an appropriate environment, for example in RPMI1640 medium containing 10% serum amniotic calf (production Gibco-BRL). As the glue is CL-120), K-562 (ATCC CCL-243) and the other Fifty μl of these cells contribute to 96-alopecia microtiter plate with flat bottom (Falcon production) and the plate incubated in the incubator feeding CO2when the temperature of the 37oWith during the night.

Then add the antibody CSC activity of which is to be measured, all incubated for 60 minutes and then add the appropriate diluted complement, for example, the complement of a baby rabbit (Baby Rabbit Complement, production Cedarlane) and are incubated for 2 hours. Then, for each cell, add 10 ál of Alamar Bule (manufactured by Bio Source) and are incubated for 4 hours and then measure the intensity of fluorescence (wavelength excitation 530 nm, the wavelength of emission 590 nm) using a system to measure fluorescence CytoFluor 2350 (production of Millipor). Cytotoxic activity (%) can be calculated by the formula (a-C)/(b-C)100, where a represents the intensity of fluorescence during incubation in the presence of antibody, indicates the intensity of the fluorescence during incubation in the medium containing no antibody, and represents the intensity of fluorescence of cells not containing cells.

4-2. Measurement OCC activity

The antibody ispovesti as cytotoxic activity.

OCC activity medicines used in the treatment of tumors of the lymphatic tissue of the present invention, can be measured as follows. First, menagerie cells secrete as effector cells from peripheral blood or from bone marrow gravity centrifugation. As target cells take CCRF-CEM (ATSS CCL-119), CCRF-HSB-2 (ATCC CCL-120.1), HPB-MLT (FCCH1019), EB-3 (ATSC CCL-85), MS ATSC CRL-1649), CCRF-SB (ATSS CCL-120), K-562 (ATSS CCL-243) or others who mark51SG obtaining drugs In the target cells. Then to labeled target cells add antibody, the activity of which is to be measured, and spend incubation. Effector cells in a suitable ratio then add to the target cells and carry out incubation.

After incubation, collect the supernatant and measure the radioactivity using a gamma counter for measuring maximum of released radioactivity can be used 1% NP-40. Cytotoxic activity (%) is calculated by the formula (a-C)/(b-C)100, where a represents the radioactivity (counts/min), released in the presence of antibodies, represents the radioactivity (counts/min), released in the presence of NP-40, and C indicates radioactive the particular

To achieve cytotoxic activity, such as OCC activity and CSC activity, it is preferable to use, in particular C1 and C3, as the constant region (C-region) of human antibodies. In addition, enhanced OCC activity or CSC activity can be induced when you add, change or modification of the amino acids in the C-region of the antibody.

As an example, you can specify on the construction of IgM-like polymer IgG at amino acid substitution (Smith, R. I. F. and Morrison, S. L. , Bio/Technology, 1994, 12, 683-688), the construction of IgM-like polymer IgG adding amino acids (Smith, R. I. F. et al. , J. Immunology, 1995, 154, 2226-2236), the expression of tandemly-very gene encoding the L-chain (Shuford, W. , et al. , Science, 1991, 252, 724-727), dimerization of IgG at amino acid substitution (Sagap, R. C. et al. , J. Exp. Med. , 1992, 176, 1191-1195; Shopes, B. , J. Immunology, 1992, 148, 2918-2922), dimerization of IgG by chemical modification (Wolff, E. A. et al. , Cancer Res. , 1993, 53, 2560-2565) and the introduction of effector functions when you change the amino acid(s) in the hinge region of antibodies (Norderhaug, L. , et al. , Eur. J. Immunol. , 1991, 21, 2379-2384), etc. All these procedures can be performed using site-specific mutagenesis using a primer, adding nucleotide sequence at site R is valentno link.

5. Confirm therapeutic effect

therapeutic effect of the medicinal product used in the present invention, when tumors of lymphatic tissue can be confirmed with the introduction of antibodies used in the present invention, an animal that transplanted tumor cells in the lymphatic tissues and the subsequent evaluation of the antitumor effect of money on animals.

As a lymphatic tumor cells injected animal, can be used established cell lines or freshly isolated cells. As an established cell line can be used CCRF-CEM (ATCC CCL-119), HPB-MLT (FCCH1019), MOLT-4 (ATCC CRL-1582), CCRF-HSB-2 (ATCC CCL-120.1), etc. as a 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) etc. as b-cell line.

Animals that spent transplantation, preferably those whose immune function is reduced or absent. For example, use a mouse, devoid of hair, mouse, SCID-beige mice, rats, deprived of hair, and other Specific anti-tumor effect can be confirmed by measuring the volume and weight of tumors ylidene antibodies against NM.24 leads to the suppression of the growth of the tumor and, in addition, to increase the period of survival of mice with transplanted tumors. These facts indicate that the antibody against NM.24 has an antitumor effect on tumor lymphatic tissue.

6. The route of administration and pharmaceutical preparations

Medicines for the treatment of tumors of the lymphatic tissue of the present invention can be administered either systemically or topically, parenterally way, for example by intravenous injection such as drip infusion, intramuscular injection, intraperitoneal injection, and subcutaneous injection. Method of introduction may be selected as indicated depending on the age and condition of the patient. Effective dosage is chosen in the range from 0.01 mg to 100 mg per kilogram of body weight per administration. Alternatively, it may be selected dosage in the range from 1 to 1000 mg, preferably from 5 to 50 mg per patient.

Medicines for the treatment of tumors of the lymphatic tissue of the present invention may contain pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, pharmaceutically acceptable organic raseel-cellulose, sodium salt of polyacrylic acid, sodium alginate, water-soluble dextran, sodium carboxymethylate starch, pectin, methylcellulose, ethylcellulose, xanthan gum, Arabic gum, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin (CSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactant and other additives are selected from these funds or their combinations, are not limited to, depending on the shape of the applied dosage.

Diseases to be treated according to the method of the present invention, are tumors of lymphatic tissue (excluding myeloma) that carry the antigen on the tumor cells and associated with the antibody used in the present invention. As specific diseases such can be noted In acute-lymphocytic leukemia (B-ALL), chronic b-lymphocytic leukemia (b-CLL), pre-B-lymphoma, lymphoma Burkitt, follicular lymphoma, follicular lymphoma of the cerebral cortex, diffuse lymphoma, acute T-lymphocytic leukemia (T-ALL), chronic T-lymphocytic leukemia (T-CLL), a disease associated with people) and other Drugs of the present invention are used as medicines in the treatment of tumors of the lymphatic tissue.

Examples

The following examples serve for a more detailed description of the present invention. It should be noted that the present invention is not limited to these examples in any way.

Example 1. Designing antibodies against NM.24

1. Getting mouse ascites containing antibody against NM.24

Get hybridoma producing antibodies against NM.24 in accordance with the method of goto et al. (Goto, T. et al. , Blood, 1994, 84, 1922-1930).

Mice of BALB/C (received from the company CLEA, Japan), which had previously administered intraperitoneally with 500 µl 2,6,10,14-tetramethylpentane (manufactured Wako Pure Chemical Industries, Ltd. ), 11 and 3 days before the experiment is injected intraperitoneally 5106hybridoma cells. Starting 10 days after the injection of hybridoma cells, harvested ascites, which has accumulated in the abdominal cavity of a mouse, using a constant needle 19 size Narusis (production Medikit). The collected ascites centrifuged twice at a speed of 1000 and 3000 rpm using a low-speed centrifuge RLX-131 (manufactured by Tomy Seiko) to remove stitov

Purification of antibodies against NM.24 from the above mouse ascites carried out as follows. After adding an equal number, FBI(-) to mouse ascites the mixture is filtered using a fiber filter Mediaprep (production Millipore) and then subjected to affinity purification using high-speed device for the purification of antibodies ConSep LC100 (production Millipore) and column Hyper D Protein A (column volume of 20 ml production Nihon Gaisi), FBI(-) as the adsorption buffer and 0.1 M sodium citrate buffer (pH 4) as an eluting buffer, in accordance with the attached instructions. Eluruume fractions are immediately brought to a pH of 7.4 by adding 1 M Tris-Hcl (pH 8.0), and then subjected to concentration and replaces the buffer with the FBI(-) using ultrafiltration concentrator Centriprep 10, followed by sterile filtration through a membrane filer Millex-GV (production Millipore) with a pore size of 0.22 μm to obtain purified antibodies against NM.24.

3. The concentration of antibodies

The concentration of purified antibody is determined at the absorption measurement. Thus, the purified antibody diluted in the FBI(-), measured by absorption at 280 nm and calculate the concentration using coefficie the th ability of antibodies against NM.24 with lymphatic tumor cells

1. Cleaning the control mouse IgG2a.

Control mouse IgG2a purified as follows. Commercially available IgG2a (KAPPA) (UpC 10) from ascites (production Cappel) was dissolved in purified water and the FBI(-). The solution is filtered using a membrane Acrodisc filter (production Gelman Sciences) with a pore size of 0.2 μm, and then purified affinity method using a high-speed device for the purification of antibodies ConSep LC 100 (production Millipore), column Hyper D Protein A (column volume of 20 ml, the production of Nihon Gaisi), FBI(-) as the adsorption buffer and 0.1 M sodium citrate buffer (pH 4) as an eluting buffer, in accordance with the attached instructions.

Eluruume fractions are immediately brought to a pH of 7.4 by adding 1 M Tris-Hcl (pH 8.0) and then subjected to concentration and replaces the buffer FBI(-) using centrifugal ultrafiltration concentrator Centriprep 10, followed by sterile filtration through a membrane filter Millex GV (production Millipore) with a pore size of 0.22 μm to obtain a purified control mouse IgG2a.

Determining the concentration of a control mouse IgG2a carried out in accordance with the procedure described earlier in section 3. Determination of concentration is in NM.24 with lymphatic tumor cells examined by flow cytometry. After washing the FBI(-) T-cell line RPMI 8402 (ATSS CRL 1995), CCRF-CEM (ATCC CRL-119), obtained from cells in acute lymphoblastic leukemia HPB-ALL (FCCH1018) obtained from cells in acute leukemia, HPB-MLT (FCCH1019) obtained from cells in T-lymphoma, JM (FCCH1023) obtained from cells in acute leukemia, MOLT-4 (ATSS CRL-1582), obtained from cells in acute lymphoblastic leukemia, Jurkat (FCCH1024)obtained from cells in acute leukemia, CCRF-HSB-2 (ATSS CCL-120.1), obtained from cells in acute lymphoblastic leukemia, MT-1 (FCCH1043) obtained from the cells during disease associated with human virus to T cells, and CT-3, obtained from cell lymphoma, Lennart (Shimizu, S. et al. , Blood, 1988, 71, 196-203), as well as In cell lines CESS cells (ATCC TIB-190), transformed by EB virus positive by EB virus In cells SKW 6.4 (ATSS TIB-215), MC116 (ATCC CRL-1649), obtained by In-lymphoma, CCRF-SB (ATCC CCL-120) derived from cells in acute lymphoblastic leukemia, b-cell RPMI 6410 (FCCH6047) obtained from a patient with acute miliitary leukemia, Daudi (ATCC CCL-213), obtained from cell lymphoma Burkitt, EB-3 (ATSC CCL-85) was obtained from cell lymphoma Burkitt, Jijoye (ATCC CCL-87) was obtained from cell lymphoma Burkitt, Raji (ATCC CCL-86), obtained from cell lymphoma Burkitt and the quality is ary leukemia, TNR-1 (ATCC TIB-202) was obtained from cells in acute monocytic leukemia U-937 (ATCC CRL-1593), obtained from cells at histiocytomas lymphoma, and K-562 (ATCC CCL-243), obtained from cells in chronic miliitary leukemia, add 100 ál of antibody against NM.24 cleaned or control murine IgG2 antibody, diluted to a concentration of 25 μg/ml in FACS buffer (FBI ( -) containing 2% amniotic calf serum and 0.1% sodium azide), and then all incubated on ice for 30 minutes.

After washing in FACS buffer add 100 ál of goat protivorechivogo antibodies (GAM), labeled FITZ, at a concentration of 25 μg/ml and then incubated the mixture on ice for 30 minutes. After washing in FACS buffer, the cells are suspended in 600 ál or 1 ml FACS buffer and each cell suspension measure the intensity of fluorescence using a FACScan instrument (manufacture Becton Dickinson). The results presented in Fig. 1-23, confirm that all T-cell lines and In all cell lines (except for Daudi and Raji, which do not react) react with the antibody against NM.24 and highly Express the antigen NM.24. On the other hand, none of the cell lines that are not related to T - or b-type, does not react with the antibody against NM.24 and not expressively mouse IgG2a negative cells are 98% positive cells make up 2%. Based on the pattern obtained with the specified histogram markers was calculated the percentage of positive antigen NM.24 cells using antibodies against NM.24, and the result is presented in the table. The percentage of positive antigen NM.24 cells the level of expression of the antigen NM.24 was divided into 5 levels: -, +/-, +, ++ and +++. In the result, it was confirmed that all T-cell lines and cell lines (except for Daudi and Raji) highly Express the antigen NM.24, which is similar to the results shown in Fig. 1-23. In all cases, the use of cell lines that are not related to T - and b-types, the percentage of positive antigen NM.24 cells was negative or less than 5%, which indicates that the expression of the antigen is absent or is at a very low level.

Example 3. Definition CSC activity

CSC activity of antibodies against NM.24 in relation to lymphatic tumor cells is determined as follows:

1. Obtaining target cells

As target cells prepared suspension CCRF-CEM (ATSS CCL-119), obtained from cells in acute leukemia, CCRF-HSB-2 (ATSS CCL-120.1), obtained from cells in acute lymphoblastic leukemia, HPB-MLT (FCCH1019) obtained from cell lymphoma Burkitt, CCRF-SB (ATSS CCL-120) derived from cells in acute leukemia, and C ATS CCL-243), obtained from cells in chronic miliitary leukemia, at a concentration of 4105cells/ml in RPMI1640 medium (manufactured Gibco BRL) containing 10% amniotic calf serum (Gibco BRL). Fifty μl of each of these cell suspensions contribute 96-alopecia microtiter plate with flat bottom (Falcon production) and carry out the incubation in the incubator with high humidity in the atmosphere of 5% CO2(production Tabai) overnight at 37oC.

2. Obtaining antibodies against NM.24

Purified antibody against NM.24 obtained in the above Example 1 is prepared in concentrations of 0 to 0.2, 2 and 20 μg/ml in RPMI1640 medium containing 10% serum amniotic calf (production Gibco BRL), and 50 ál of the mixture to a 96-alopecia microtiter plate prepared as described above in Section 1. After incubation the plates in the incubator with high humidity in the atmosphere of 5% CO2(production Tabai) at a temperature of 37oC for 60 minutes, perform centrifugation at low speed centrifuge 05PR-22 (manufactured by Hitachi) at 1000 rpm for 5 minutes and take 50 ál of the supernatant.

3. Cooking com is the 1 ml vial, and further diluted in 5 ml of RPMI1640 medium (manufactured Gibco-BRL), not containing amniotic calf serum. Fifty μl of this mixture was dispensed into 96-alopecia microtiter plate with a flat bottom, prepared as described above in Section 2, and carry out the incubation in the incubator with high humidity in the atmosphere of 5% CO2(production Tabai) for 2 hours at 37oC.

4. Definition CSC activity

After the incubation, add 10 ál of Alamar Bule (manufactured by Bio Source) to each cell of the 96-alopecia microtiter tablet with a flat bottom that is described in Section 3, and carry out the incubation in the incubator with high humidity in the atmosphere of 5% CO2(production Tabai) for 4 hours at 37oC. Then each cell promerium on the intensity of fluorescence (wavelength excitation 530 nm, the wavelength of emission 590 nm) using a system to measure fluorescence CytoFluor 2350 (production Millipore). Cytotoxic activity (%) is calculated by the formula (A-C)/(b-C)100, where a represents the intensity of fluorescence during incubation in the presence of antibody, indicates the intensity of the fluorescence during incubation only in the medium without the addition of antibodies, With the seat is, 24 and 25, that K, which does not react with the antibody against NM.24 the results of analysis by flow cytometry, showed no cytotoxicity even adding antibodies against NM.24, while CCRF-CEM, CCRF-HSB-2, HPB-MLT, EB-3, MC-116 and CCRF-SB, which interact with the antibody against NM.24 demonstrate zitotoksicnogo way, dependent on the concentration of antibodies against NM.24. This result indicates that the antibody against NM.24 shows CSC activity against lymphatic tumor bearing on cell surface protein antigen with which the antibody against NM.24 specifically binds.

Example 4. Antitumor activity of antibodies against NM.24 in respect of mice with transplanted lymphatic tumor man

1. Obtaining antibodies for insertion

1-1. Obtaining antibodies against NM.24

Purified antibody against NM.24 obtained in the above example 1 is prepared in a concentration of 1 mg/ml and 200 μg/ml in sterile filtered FBI(-) and used in the following experiments.

1-2. Receiving control mouse IqG2a

The purified antibody obtained in the above example 2 is prepared in a concentration of 1 mg/ml in sterile filtered FBI(-) and transplantirovannam lymphatic tumor man

2-1. Getting mice with transplanted lymphatic tumor man

Mice with transplanted lymphatic tumor person receive the following way. Cells CCRF-HSB-2 (ATSS CCL-120.1), resulting in acute lymphoblastic leukemia and who had subculturally in vivo using SCID mice (Clea, Japan), prepared as a suspension in a concentration of 1108cells/ml in RPMI1640 medium containing 10% serum amniotic calf (production Gibco-BRL). Cell suspension obtained as described above, is injected subcutaneously into the abdominal cavity of SCID mice (6-week-old males), the day after introducing them intraperitoneally with 100 μl antikvarnogo drug GM1 (Waco Pure Chemical Industries).

2-2. Introduction antibodies

On the 7th day after transplantation of the tumor is measured with a compass tumor diameter, formed in the place where these mice transplanted CCRF-HSB-2 with lymphatic tumor person. After determining the tumor volume of animals are grouped so that each group the mean value of the tumor volume was approximately the same (8 animals per group, all 3 groups). Starting with one and the same day, the animals in each group injected intraperitoneally with 100 μl of antibodies against NM.24 in concentration ie held twice weekly, a total of 19 times in the same way. During this period measured by the compass tumor diameter twice a week and calculate tumor volume.

2-3. Evaluation of the antitumor effect of antibodies against NM.24 mice with transplanted lymphatic tumor man

Antitumor effect of antibodies against NM.24 appreciate the change of tumor volume and period survival rate of mice. As a result, as shown in Fig. 26, it was found that the increase in the volume of the tumor is suppressed with the introduction of animal antibodies against NM.24 in comparison with the control group of animals who were injected murine IgG2a. That also can be seen from Fig. 27, with the introduction of the group of animals antibodies against NM.24 there is an increase of the period of survival of animals compared with the control group of animals who enter murine IgG2a. These facts indicate that the antibody against NM.24 hide antitumor activity in mice with transplanted lymphatic tumor of man.

Referential example 1. Development of hybridomas that produce mouse monoclonal antibody against NM.24

According to the method of goto et al. (Goto, T. et al. , Blood, 1994, 84, 1922-1930) get hybridoma that produce mouse MES is the main brain of the patient with multiple myeloma person (Goto, So et al. , Jpn. J. Clin. Hematol. , 1991, 32, 1400) is injected twice into the abdominal cavity of mice BALB/c (production of Charles River) every six weeks.

3 days before the killing of the animal in the spleen of a mouse injected 1,5106cells CRS-32 to enhance antibody productive capacity of the mouse (Goto, T. et al. , Tokushima J. Exp. Med. , 1990, 37, 89). After killing the animal extracted spleen, and extracted the authority conducting the procedure of cell fusion with a myeloma cell SP2/0 (method Groth, de St. and Schreidegger, Cancer Research, 1981, 41, 3465).

According to the method of IFTTA cells (Posner, M. R. et al. , J. Immunol. Methods, 1982, 48, 23) using cattle-32 conduct screening the culture supernatant hybrid for the presence of antibodies. 5104cells CRS-32 is suspended in 50 ml of the FBI, an aliquot is transferred to a 96-alopecia microtiter plate (U-shaped granular bottom, production Iwaki) and then dried in air at a temperature of 37oWith during the night. After blocking the reaction by adding the FBI, containing 1% bovine serum albumin (BSA), make the culture supernatant hybrid and carry out incubation at 4oC for 2 hours. This is followed by a reaction at 4oC for 1 hour with goat antibodies against mouse IgG labeled with peroxy the elite) at room temperature for 30 minutes.

The reaction is stopped by adding 2 N. sulfuric acid and measure the absorption at 492 nm using a counter IFCFG (production Bio-Rad). For selection of hybridomas which produce antibodies against human immunoglobulin, culture supernatant positive hybridomas initially adsorb on human serum, followed by screening for reactivity against other cell lines using the method of IFTTA. Selected positive hybridoma and their reactivity to different cells is determined using the method of flow cytometry. The last selected hybridoma clone clone twice and injected into the abdominal cavity of mice BALB/c mice treated with dock and then collect from them ascites.

From ascites of a mouse get monoclonal antibodies and purified their precipitation with ammonium sulfate and set for affinity chromatography protein A (Ampure PA, production Amersham). Purified antibodies mark FITZ with a set of Quick Tag FITC biding kit (production Boehringer Mannheim).

Research shows that monoclonal antibodies produced 30 hybridoma clones that react with CRS-3 and RPMI 8226. After cloning explore reacciona leccami peripheral blood.

Of these 3 clones producing monoclonal antibodies that specifically react with a plasma cell. Of these 3 clones were selected and designated as NM.24 hybridoma clone, which is most suitable for analysis by flow cytometry and which has CSC activity to RPMI 8226.

The subclass of the monoclonal antibodies produced by these hybridomas, identify method of IFTTA using protivorechivogo antibodies rabbit, specific to this subclass (production Zymed). Antibody against NM.24 refers to the IgG2a subclass. Hybridoma NM.24, which produces antibody against NM.24, was deposited in the International Collection in accordance with the provisions of the Budapest Treaty as FERM BP-5233 14 September 1995 by the National Institute of Bioscience and human technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan).

Referential example 2. Getting gumanitarnogo antibodies against NM.24

Humanitariannet antibody against NM.24 receive in accordance with the following method.

From hybridoma NM.24 obtained in reference example 1, receive total RNA using traditionally what ispolzovaniem polymerase chain reaction (PCR) and by the method of 5'-RACE. Get a DNA fragment containing the gene encoding murine V-region, which are ligated to a cloning vector based on the plasmid pUC and then introduced into competent cells of E. Li obtaining transformant E. Li. The indicated plasmid derived from transformant. Define the traditional method the nucleotide sequence encoding section cDNA in the plasmid and identify hypervariable segment (HLG) in each V-region.

To construct a vector expressing a chimeric antibody against NM.24, HEF vector insert cDNA encoding V region of each of the L-chain and H chain of the mouse antibody against NM.24. Next to design gumanitarnogo antibodies against NM.24 HLG V-region of a mouse antibody against NM.24 transplanted into a human antibody according to the method of transplantation HLG. L-chain of the human antibody REI used as an L-chain of human antibodies for frame areas (KO) 1-3 N-chain of the human antibody is used TO 1-3 human antibody HG3 and CO of human antibodies JH6 used for CO. Some amino acids in KO from V-N-chain is substituted so that HLG-transplantirovannam antibody could form a suitable site for binding antigen.

M1.24 in mammalian cells each gene separately injected into the vector HEF for design vector, to Express the L-chain or H-chain gumanitarnogo antibodies against NM.24, respectively.

With the simultaneous introduction of these 2 vectors for expression in Cho cells is established cell line, which produces humanitariannet antibody against NM.24. Antigennegative activity and inhibition of binding activity against the cell line WISH cells amnion person gumanitarnogo antibodies against NM.24, obtained by cultivation of the specified cell line,

were investigated cell method IFTTA. The result shows that humanitariannet antibody against NM.24 has antigennegative activity equal to that of the chimeric antibody, and in regard to activity by inhibition of binding using biotinidase mouse antibodies against NM.24, it also shows activity equal to the activity of the chimeric antibody or mouse antibodies.

E. coli carrying a plasmid that contains the DNA encoding the V region of the L-chain and DNA encoding V region of H chain chimeric antibody against NM.24, was deposited in the International Collection in accordance with the provisions of the Budapest Treaty as Escherichia col technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) as FERM BP-5646 and FERM BP-5644 respectively. In addition, E. coli carrying a plasmid which contains a DNA encoding a variant (SEQ ID NO: 2) V region of the L-chain or r (SEQ ID NO: 3) V-N-chain gumanitarnogo antibodies against NM.24, was deposited in the International Collection in accordance with the provisions of the Budapest Treaty as Escherichia coli DH5 (pUC19-RVLa-AHM-gk) and Escherichia coli DH5 (pUC19-RVHr-gl), respectively, on 29 August 1996, the National Institute of Bioscience and human technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) as FERM BP-5645 and FERM BP-5643, respectively. Next, E. coli carrying a plasmid that contains the DNA encoding the variant s (SEQ ID NO: 4) V-N-chain gumanitarnogo antibodies against NM.24, was deposited in the International Collection in accordance with the provisions of the Budapest Treaty as Escherichia coil DH5 (pUC19-RVHs-AHM-gl) September 29, 1997, the National Institute of Bioscience and human technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, of 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. , Japan) as FERM BP-6127.

Reference example 3. Cloning of a cDNA that encodes a protein antigen NM.24

>/P>1. Construction of cDNA library

1) Obtaining total RNA

From cell lines of multiple myeloma person CRMM get total RNA according to the method Chirgwin et al. (Chirgwin et al. , Biochemistry, 18, 5294, 1979). In accordance with the specified method, 2,2108cells CRM completely homogenized in 20 ml of 4 M guanidinoacetate (production Nacalai Tesque Inc. ). The homogenate was layered 5.3 M solution of caesium chloride in the centrifuge tube, which was then centrifuged using a rotor Beckman SW40 speed 31000 rpm at 20oC for 24 hours to precipitate RNA. The precipitated RNA was washed with 70% ethanol and then dissolved in 300 μl of 10 mm Tris-Hcl (pH 7.4) containing 1 M EDTA and 0.5% - ordinator. Add pronase (production Boehringer) to a concentration of 0.5 mg/ml and then carry out incubation at a temperature of 37oC for 30 minutes. The mixture is extracted with phenol and chloroform and the RNA precipitated with ethanol. The precipitated RNA was dissolved in 200 μl 10 mm Tris-Hcl (pH 7.4) containing 1 mm EDTA.

2) Obtaining a poly(A)+RNA

Poly(A)+RNA purified using as source material 500 µg total RNA, obtained by the above method using the dial to highlight the Fast Track mRNA of 2.0 mRNA Isolation Kit (manufactured Invitrogen) in accordance with the attached is use as a starting material above 10 ág of poly(A)+RNA using a kit for synthesis of cDNA TimeSaver cDNA Synthesis Kit (manufactured Pharmacia), in accordance with the attached to the kit instructions, and then spend ligation with EcoRI adapter supplied in the kit, using Directional Cloning Toolbox (production Pharmacia), in accordance with the attached to the kit instructions. Caching and processing restriction enzyme NotI EcoRI adapter carried out in accordance with the instruction attached to the kit. Next, double-stranded cDNA attached to the adapter, having a size of 500 NP or more, separated and purified using a 1.5% low-melting agarose gel (Sigma production) with a 40 µl added to the adapter chain cDNA.

Thus obtained is added to the adapter, double-stranded cDNA are ligated using vector pCOSl (patent application Japan 8-255196) and DNA ligase T4 (production Gibco-BRL), which was pre-treated restrictase EcoRI and NotI and alkaline phosphatase (manufactured by Takara Shuzo) to create a cDNA library. Constructed a cDNA library using transduction is introduced into the strain E. coli DH5 (production Gibco-BRL), was subsequently established that this is an independent clone, which total approximately 2,5106.

2. The cloning method of direct expression

1) Transvestia COS-7 cells

When the ory Manual, Sambrook et al. , Cold Spring Harbor Laboratory Press, 1989) containing 50 μg/ml ampicillin, for amplification of cDNA, which is subjected to processing by the alkaline method (Molecular Cloning: A Laboratory Manual, Sambrook et al. , Cold Spring Harbor Laboratory Press, 1989), with the recovery of plasmid DNA from E. Li. Thus obtained plasmid DNA transfer method transfection in COS-7 cells by electroporation method using the apparatus Gene Pulser (manufactured by Bio-Rad).

In accordance with the specified method, 10 µg of purified plasmid DNA is added to 0.8 ml of a solution of COS-7 cells, in which cells were suspended in the FBI at a concentration of 1107cells/ml, and the resulting mixture is exposed to pulses of 1500 V at a capacitance of 25 μf. At the end of the 10-minute recovery period at room temperature, the cells after electroporation were cultured in culture medium DMEM (production Gibco-BRL) containing 10% serum amniotic calf (production Gibco-BRL) at a temperature of 37oC in an atmosphere of 5% CO2within 3 days.

2) Preparation of the cell

The cuvette on which is applied a murine antibody against NM.24, prepared by the method of SIDA et al. (B. Seed et al. , Proc. Natl. Acad. Sci. USA, 84, 3365-3369, 1987). In accordance with the specified method murine antibody against NM the OPA antibody is added to the Cup with cell culture with a diameter of 60 mm and incubated at room temperature for 2 hours. After washing 3 times with 0.15 M NaCl to a bowl add the FBI, containing 5% amniotic calf serum, 1 mm EDTA and 0.02% NN3. The mixture after blocking used for subsequent cloning.

3) Cloning of the cDNA library

COS-7 cells subjected to transfection by the above method, process, FBI, containing 5 mm EDTA. After a single washing of the cells of the FBI, containing 5% amniotic calf serum, suspended in the FBI, containing 5% amniotic calf serum and 0.02% NaN3to a concentration of approximately 1106cells/ml Suspension contribute to the cell prepared as described above, and carry out incubation at room temperature for about 2 hours. After careful triple rinsing the FBI, containing 5% amniotic calf serum and 0.02% NN3allocate plasmid DNA from cells associated with the cuvette, using a solution containing 0.6% of LTOs and 10 mm EDTA.

The recovered plasmid DNA is transferred by the method of transduction in E. coli DH5. After amplification by the above method plasmid DNA restore the alkaline method. The recovered plasmid DNA is transferred by transfection in COS-7 cells using electroy procedure and recovered plasmid DNA cleaved by restrictase. EcoRI and NotI, which confirms the presence of the insert having a size of about 0.9 thousand nm. Cells of E. coli, in which a portion of the recovered plasmid DNA was transferred by transduction, inoculant in a Cup with 2-YT agar containing 50 μg/ml ampicillin. After culturing overnight distinguish from one colony plasmid DNA. It split restrictase EcoRI and NotI to obtain a clone P3.19, in which the size of the insert is approximately About 9 thousand NP.

Conducting the reaction of the specified clone using the kit for sequencing PRISM Dye Terminator Cycle Sequencing kit (manufactured Perkin Elmer) in accordance with the supplied set of instructions and determine the nucleotide sequence using a DNA sequencing machine (ABI A DNA Sequencer (manufactured Perkin Elmer). The specified nucleotide sequence and corresponding amino acid sequence presented in SEQ ID NO: 1.

Industrial applicability

The results of the analysis by flow cytometry showed that the antibody against NM.24 interacts with the majority of cells derived from tumors of the lymphatic tissue, indicating that in the case of many tumors of lymphatic tissue polypeptide having an epitope recognized entitycache tumor human which reacts with the antibody NM.24, antibodies against NM.24 leads to growth inhibition of tumor volume and increased survival time. These facts show that the antibody against NM.24 or antibodies recognized, a polypeptide having an epitope recognized by the antibody against NM.24, possess cytotoxic activity against many tumors of lymphatic tissue and this suggests that this antibody can be used for the treatment of patients with tumors of lymphatic tissue.

Link to microorganisms deposited in accordance with the Treaty Patent cooperation (Patent Cooperation Treaty), the Position 13-2 and the name of the Depositing Institution.

Depositing Institute

Name: national Institute of Bioscience and human technology. Agency of industrial science and technology (National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology)

Address: 1-3, Higashi 1-chome, Tsukuba city, Ibaraki pref. Japan

The microorganism (1)

Name: Escherichia coli DH5 (pRS38-pUC19)

The number of storage: FERM BP-4434

Date of Deposit: October 5, 1993

The microorganism (2)

Name: Hybridoma NM.24

The number of storage: FERM BP-5233

Date Dept is R storage: FERM BP-5643

Date of Deposit: August 29, 1996

The microorganism (4)

Name: Escherichia coil DH5 (pUC19-l. 24H-gl)

The number of storage: FERM BP-5644

Date of Deposit: August 29, 1996

The microorganism (5)

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

The number of storage: FERM BP-5645

Date of Deposit: August 29, 1996

The microorganism (6)

Name: Escherichia coli DH5 (pUC19-l. 24L-gk)

The number of storage: FERM BP-5646

Date of Deposit: August 29, 1996

The microorganism (7)

Name: Escherichia coli DH5 (pUC19-RVHs-AHM-gl)

The number of storage: FERM BP-6127

Date of deposition: September 29, 1997

1. The drug used in the treatment of tumors of lymphatic tissue (excluding myeloma) comprising as an active ingredient an antibody that specifically binds to a protein having the amino acid sequence specified in SEQ ID NO : 1, and which has cytotoxic activity.

2. Drug under item 1, characterized in that said tumor lymphatic tissue is a tumor T cells.

3. Drug under item 1, characterized in that said tumor lymphatic tissue is particularly the indicated antibody is a monoclonal antibody.

5. Drug under item 1, characterized in that the cytotoxic activity is antiteloobrazovania cleocinonline cytotoxicity (ADCC/OCC activity).

6. Drug under item 1, characterized in that the cytotoxic activity is complementability cytotoxicity (CDC/CSC activity)

7. Drug under item 4, characterized in that the antibody contains a constant region of human antibodies.

8. Drug under item 7, characterized in that the constant region of human antibodies represents a C1 or C3.

9. Drug under item 7, characterized in that the antibody is an antibody against NM.24, which is produced by hybridoma FERM BP-5233.

10. Drug under item 4, characterized in that said antibody is a chimeric antibody or humanitariannet antibody.

11. Drug under item 9, characterized in that said antibody is a chimeric antibody against NM.24.

12. Drug under item 9, characterized in that the and the. 1, characterized in that said antibody specifically binds to the epitope recognized by the antibody against NM.24.

14. The antibody that specifically binds to a protein having the amino acid sequence specified in SEQ ID NO : 1, and which has cytotoxic activity.

15. The antibody under item 14, characterized in that the cytotoxic activity is antiteloobrazovania cleocinonline cytotoxicity (ADCC/OCC activity).

16. The antibody under item 14, characterized in that the cytotoxic activity is complementability cytotoxicity (CDC/CSC activity).

17. Drug under item 4, characterized in that said antibody is a human antibody.

18. Drug under item 2, characterized in that said tumor T cells is selected from the group consisting of acute T-lymphocytic leukemia (T-ALL), chronic T-lymphocytic leukemia (T-CLL), a disease associated with the human virus of T cells (LAT), lymphoma peripheral T cells do not ATL-type (PNTL).

19. Drug under item 3, wherein the tumor cells to vybirayutsa Burkitt, follicular lymphoma, follicular lymphoma of the cerebral cortex, diffuse lymphoma.

 

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