JP5798307B2 - Monoclonal antibody specifically recognizing globotriaosylceramide and its production method - Google Patents

Description

  The present invention relates to a method for producing an antibody (anti-Gb3 / CD77 antibody) specifically recognizing globotriosylceramide (Gb3 / CD77) which is a globo-based glycolipid and its use. Specifically, the present invention provides an efficient method for producing an anti-Gb3 / CD77 monoclonal antibody, an anti-Gb3 / CD77 monoclonal antibody, and uses thereof.

  Since Kohler and Milstein established technology for establishing hybridomas for the production of monoclonal antibodies (Non-Patent Document 1), monoclonal technology has spread dramatically and has been widely used in various biological research fields around the world. Not only B cell hybridomas but also T cell hybridomas have been developed and widely used for immunological analysis (Non-patent Document 2). Monoclonal antibodies have become one of the most useful techniques for promoting modern biology and medical research (Non-patent Document 3). Animal immunization and hybridoma screening are most important in the production of monoclonal antibodies. Antibody diagnostics such as specificity of antibody binding, CDC activity (complement dependent cytotoxic activity) or ADCC activity (antibody dependent cytotoxic activity) for disease diagnosis and cancer immunotherapy Very important. Various attempts have been made regarding the preparation of immunogens to improve the effect of immunity. For example, binding of a hapten, addition of an adjuvant to an antigen, embedding in a liposome, utilization of a peptide deduced from the configuration of each protein, and the like. However, there are no reports on specific mice useful for the production of efficient monoclonal antibodies.

  In order to produce monoclonal antibodies that react with glycolipids, the method of injecting tumor cells into normal mice was an immunization method widely distributed to many laboratories. Immortalization of B cells by EB virus was particularly effective in the production of human monoclonal antibodies that did not have good partner cells for fusion (Non-patent Documents 4 and 5). Later, purified or recombinant proteins were widely used for the production of monoclonal antibodies. Peptides of various functional molecules have also been used as immunogens for various biomolecules. Purified gangliosides were used as immunogens to produce a set of useful monoclonal antibodies against gangliosides (Non-Patent Documents 6-8), based on past reports (Non-Patent Document 9). Specific mouse strains were used. For the production of anti-ganglioside monoclonal antibodies, knockout mice lacking complex gangliosides were used, and good antibody production was shown (Non-patent Document 10). The research group of the present inventors also made GD1a (Non-patent Document 11) or GD3 and GD1b (Non-patent Document 12) by using GM2 / GD2 synthetase-deficient (knockout) mice or GD3 synthetase (knockout) mice. We reported that we were able to produce useful antibodies that react. These anti-ganglioside antibodies showed higher specificity and affinity for the missing structure in the mutant mice used for immunization. However, there are no reports on the use of knockout mice that lack neutral glycolipids such as globo-based glycolipids.

G. Kohler, C. Milstein, Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256 (1975) 495-497. P.N.Nelson, G.M.Reynolds, E.E.Waldron, E. Ward, K. Giannopoulos, P.G.Murray, Monoclonal antibodies, Mol.Pathol. 53 (2000) 111-117. R.H.Kennett, Hybridomas: a new dimension in biological analyzes, In Vitro 17 (1981) 1036-1050. Y. Nagatsuka, S. Hanazawa, T. Yasuda, Y. Ono, Anti-glycolipid antibodies produced by Epstein-Barr virus (EBV) -transformed oligoclonal B cell lines obtained from normal persons and autoimmune disease patients, Hum. Antibodies Hybridomas 5 ( 1994) 183-186. H. Yamaguchi, K. Furukawa, SR Fortunato, PO Livingston, KO Lloyd, HF Oettgen, LJ Old, Cell-surface antigens of melanoma recognized by human monoclonal antibodies, Proc. Natl. Acad. Sci. USA 84 (1987) 2416- 2420. I. Kawashima, N. Tada, S. Ikegami, S. Nakamura, R. Ueda, T. Tai, Mouse monoclonal antibodies detecting disialogangliosides on mouse and human T lymphomas, Int. J. Cancer 41 (1988) 267-274. M. Kotani, H. Ozawa, I. Kawashima, S. Ando, T. Tai, Generation of one set of monoclonal antibodies specific for a-pathway ganglio-series gangliosides, Biochim. Biophys. Acta 1117 (1992) 97-103. H. Ozawa, M. Kotani, I. Kawashima, T. Tai, Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides, Biochim. Biophys. Acta. 1123 (1992) 184-190. I. Kawashima, O. Nakamura, T. Tai, Antibody responses to ganglio-series gangliosides in different strains of inbred mice, Mol. Immunol. 29 (1992) 625-632. MP Lunn, LA Johnson, SE Fromholt, S. Itonori, J. Huang, AA Vyas, JE Hildreth, JW Griffin, RL Schnaar, KA Sheikh, High-affinity anti-ganglioside IgG antibodies raised in complex ganglioside knockout mice: reexamination of GD1a immunolocalization. J. Neurochem. 75 (2000) 404-412. BJ Willison, D. Nicholl, ER Wagner, K. Townson, C. Goodyear, K. Furukawa, K. Furukawa, J. Conner, HJ Willison, Innate murine B cells produce anti-disialosyl antibodies reactive with Campylobacter jejuni LPS and gangliosides that are polyreactive and encoded by a restricted set of unmutated V genes, J. Neuroimmunol. 152 (2004) 98-111. J. Boffey, M. Odaka, D. Nicoll, ER Wagner, K. Townson, T. Bowes, J. Conner, K. Furukawa, HJ Willison, Characterization of the immunoglobulin variable region gene usage encoding the murine anti-ganglioside antibody repertoire , J. Neuroimmunol. 165 (2005) 92-103.

  Gb3 / CD77 is expressed in a part of germinal center B cells, kidney, small intestine and vascular endothelial cells of human (reference 6) and mouse (reference 7), Burkitt lymphoma (reference 6) and colon cancer It is known to be overexpressed in tumor cells such as (References 9 and 10). Many studies have reported that Gb3 / CD77 functions as a receptor for bacterial toxins (Ref. 11), but little is known about its physiological role. Therefore, a useful means for elucidating the function of Gb3 / CD77 is desired. In addition, if we summarize the previous reports, it is clear that Gb3 / CD77 will be an important target for medical purposes, and the creation of diagnostic and therapeutic technologies targeting Gb3 / CD77 is expected. Under such circumstances, an object of the present invention is to provide a means for efficiently producing a competent antibody targeting Gb3 / CD77. Another object of the present invention is to use the antibody obtained by this technique (provide use).

  In order to solve the above-mentioned problems, the present inventors expect that if a mutant mouse lacking a globo-series glycolipid is used, a monoclonal antibody against a known or unknown sugar chain structure lost in the mouse can be efficiently produced. We proceeded with examination. Specifically, using knockout mice lacking the α-1.4 galactose transferase gene essential for the synthesis of globo-series glycolipids, the production of monoclonal antibodies that react with the glycosphingolipid globotriosylceramide (Gb3) Tried. As a result, it was proved that a monoclonal antibody that was clearly better than the conventional method by immunization of wild-type mice could be produced. Specifically, a larger number of clones were obtained than in the conventional method, and half of the obtained clones were IgG class (all 3 clones obtained by the conventional method were all IgM class). Moreover, some of the antibodies obtained exhibited ADCC activity in addition to CDC activity. In addition, an antibody named c.52 showed aggregation activity against Burkitt lymphoma cell lines. Interestingly, during aggregation, c.52 antibody binding induced tyrosine phosphorylation of several proteins similar to those seen upon binding of anti-B cell receptor antibodies. In particular, binding of the c.52 antibody caused a strong phosphorylation of a 120 kDa protein named c-Cbl known as E3 ubiquitin ligase. It was found that when B cells were pretreated with anti-Gb3 monoclonal antibody before stimulation with anti-B cell receptor antibody, Gb3-mediated signals suppressed antigen pseudosignals from B cell receptors.

As described above, results exceeding expectations were brought about, and it was shown that α-1.4 galactose transferase gene-deficient mice are extremely effective in producing monoclonal antibodies against globo-based glycolipids. In addition, interesting findings were obtained that were useful in using anti-Gb3 / CD77 monoclonal antibodies. This finding supports the usefulness of the anti-Gb / CD77 monoclonal antibody and shows that the present monoclonal production method can efficiently produce an anti-Gb / CD77 monoclonal antibody that exhibits its physiological functions well. The present invention listed below is mainly based on the above-mentioned results.
[1] Method for producing anti-Gb3 / CD77 monoclonal antibody, comprising the following steps (1) to (5):
(1) immunizing a non-human mammal lacking the α1,4-galactosyltransferase gene with Gb3 / CD77;
(2) recovering antibody-producing cells from the non-human mammal after immunization;
(3) immortalizing the recovered antibody-producing cells;
(4) a step of monoclonalizing a cell that produces an anti-Gb3 / CD77 antibody among immortalized antibody-producing cells; and (5) a step of recovering an antibody produced by a clone obtained by the monoclonalization.
[2] The production method according to [1], wherein the non-human mammal is a mouse.
[3] The production method according to [2], wherein the mouse is an A / J strain.
[4] The production method according to any one of [1] to [3], wherein the immunization in step (1) is performed using a liposome containing Gb3 / CD77, lipid A, cholesterol and diparamitoylphosphatidylcholine as an immunogen. .
[5] The production method according to any one of [1] to [4], wherein the antibody-producing cells are spleen cells.
[6] The production method according to any one of [1] to [5], wherein step (3) is performed by fusion of antibody-producing cells and myeloma cells.
[7] The production method according to any one of [1] to [6], wherein in step (4), cells producing an IgG class anti-Gb3 / CD77 antibody are monoclonalized.
[8] An isolated monoclonal antibody that specifically recognizes Gb3 / CD77 and exhibits complement-dependent cytotoxic activity and / or antibody-dependent cytotoxic activity.
[9] The isolated monoclonal antibody according to [8], which exhibits an aggregating activity against Burkitt lymphoma cell line.
[10] The isolated monoclonal antibody according to [8] or [9] obtained by the production method according to any one of [1] to [7].
[11] The isolated monoclonal antibody according to [9] or [10], which is a human antibody or a humanized antibody.
[12] The isolated monoclonal antibody according to any one of [9] to [11], which is an IgG, Fab, Fab ′, F (ab ′) 2, scFv, or dsFv antibody.
[13] A research reagent targeting a cell expressing Gb3 / CD77, comprising the isolated monoclonal antibody according to any one of [9] to [12].
[14] A composition for diagnosis or treatment targeting a malignant tumor overexpressing Gb3 / CD77, comprising the isolated monoclonal antibody according to any one of [9] to [12].
[15] The composition according to [14], wherein the malignant tumor is Burkitt lymphoma, colon cancer, teratoma or megakaryoblastic leukemia.

The figure which shows the specificity and reactivity of the monoclonal antibody obtained by immunizing A / J mouse | mouth with Gb3 / CD77. (A) An example of the result of ELISA performed on a purified glycolipid (20 ng / well) using a monoclonal antibody (hybridoma supernatant) diluted in two steps. After washing the supernatant, an HRP-labeled secondary antibody was added and allowed to stand for 1 hour. After washing, a substrate solution was added, and binding of the monoclonal antibody was detected. After 10-20 minutes, the degree of color development was recorded by a scanner. (B) Specificity of monoclonal antibodies obtained from A / J mice for various glycolipids by TLC immunostaining. TLC immunostaining was performed as described in <Materials and Methods> with the resulting monoclonal antibody (1: 2 dilution of hybridoma supernatant). TLC was performed in a developing solvent of C: M: DW (60: 35: 8). Glycolipid (0.5 μg / well) was used. Antibody binding was detected with the ABC complex ^™ kit and Konica Immunostain HRP 1000 ^™ . The figure which shows the specificity and reactivity of the monoclonal antibody obtained from Gb3 synthetase knockout mouse. TLC immunostaining was performed as described in <Materials and Methods> using the obtained monoclonal antibody (1: 2 dilution of hybridoma supernatant). TLC was performed in a developing solvent system C: M: DW (60: 35: 8). Glycolipid (0.5 μg / well) was used. Antibody binding was detected with ABC complex ^™ and Konica Immunostain HRP 1000 ^™ . Since monoclonal antibody c.76 could not be used in this assay system, the results of studies on 7 antibodies are shown. The figure which shows the specificity and reactivity with respect to various glycolipids of the monoclonal antibody obtained from the Gb3 synthetase knockout mouse. The obtained monoclonal antibody (hybridoma supernatant) was serially diluted 2-fold and subjected to ELISA. After washing, an HRP-labeled secondary antibody was added. After washing, binding of the monoclonal antibody was detected by adding a substrate solution. The figure which shows the anti-tumor activity of the monoclonal antibody with respect to Ramos cell. Complement dependent cytotoxic activity. After standing with the set of monoclonal antibodies obtained from Ramos cells (ascites diluted x20 to x10240), rabbit serum was added as a complement source and allowed to stand. Complement-dependent cytotoxic activity was performed by WST-1 assay as described in <Materials and Methods>. Solid line: no serum, wide dotted line: serum (1:10 dilution), narrow dotted line: serum (1: 2 dilution). The figure which shows antibody-dependent cytotoxic activity. Ramos cells or SK-MEL-28 cells were allowed to stand with monoclonal antibodies c.52, c.95, c.37 or R24, respectively, and then peripheral blood mononuclear cells were added and allowed to stand for 1 hour. Control IgG was used as a control. ADCC activity was detected by measuring the released LDH using CytoTox 96 Non-Radioactive Cytotoxicity Assay ^™ as described in <Materials and Methods>. ○: control IgG, ●: monoclonal antibody c.52 or monoclonal R24. The figure which shows the effect of the cell pretreatment of an anti- Gb3 / CD77 monoclonal antibody with respect to the apoptosis induction by verotoxin. (A) Results of Western immunoblotting of caspase-3. Ramos cells were allowed to stand with VT-2 for various times and the cell lysates were used for immunoblotting as described in <Materials and Methods>. The original caspase 3 (32 kDa) degradation product appeared 3 hours after addition of verotoxin. (B) Effect of anti-Gb3 / CD77 antibody pretreatment on apoptosis. Ramos cells were reacted with anti-Gb3 / CD77 antibody at 4 ° C. for 2 hours and then allowed to stand in the presence of VT2 (200 ng / ml) for 3 hours. As a result of immunoblotting, it was found that when treated with monoclonal antibodies c.168, c.52 and c.95, the original caspase 3 band remained slightly. The relative strength of the caspase 3 band is shown. That is, the ratio of non-degraded caspase 3 to the sum of non-degraded and degraded caspase 3 was calculated from the band intensity of B and plotted. Similar results were obtained in repeated experiments. The figure which shows the influence of the anti- Gb3 / CD77 monoclonal antibody with respect to the form of Ramos cell. (A, B) Ramos cells were allowed to stand with monoclonal antibody c.52, c.95 or c.220. PBS was used as a negative control. Only cells that were allowed to stand for 1 minute with monoclonal antibody c.52 caused aggregation. (C) Ramos cells (1 × 10 ⁷ cells / ml) were reacted with monoclonal antibodies c.52, c.220 or c.95 (20 μg / ml). A WST-1 assay was performed at each time indicated in the figure. The same experiment was repeated several times with similar results. The figure which shows the tyrosine phosphorylation of the protein induced | guided | derived by the monoclonal antibody c.52. Tyrosine phosphorylation of the protein induced by monoclonal antibody c.52 was examined. Ramos cells (10 ⁷ cells / ml) were stimulated with monoclonal antibodies c.52 (20 μg / ml) or c.220 (20 μg / ml) at 37 ° C. for the times indicated in the figure. A cell lysate was prepared and used for immunoblotting analysis with PY20. Proteins that are tyrosine phosphorylated by monoclonal antibody c.52 are similar to proteins that are phosphorylated by anti-μ antibodies. (A) Western immunoblotting result of tyrosine phosphorylated protein induced by treatment with monoclonal antibody c.52. Ramos cells were reacted with monoclonal antibody c.52 (0-40 μg / ml) for 5 minutes, and the cell lysate was used for immunoblotting with PY20. (B) Western immunoblotting of tyrosine phosphorylated protein with anti-μAb. After reacting Ramos cells with anti-μ antibody (0-40 μg / ml), the lysate was used for Western immunoblotting with PY20. A similar experiment was performed with a lower concentration of anti-μ antibody (B-2). The figure which shows that p120 is identification of c-Cbl, and the kinetics of phosphorylation. (A) Immunoprecipitation was performed using PY20 or anti-c-Cbl antibody before and after treatment with monoclonal antibody c.52 for 5 minutes. The immunoprecipitate was used for immunoblotting with PY20 or anti-c-Cb antibody. (B) Time course of phosphorylation of c-Cbl by stimulation with monoclonal antibody c.52. Immunoprecipitation with anti-c-Cbl antibody was performed using a lysate of Ramos cells (derived from 1 × 10 ⁷ cells) stimulated with monoclonal antibody c.52. Thereafter, the immunoprecipitate was immunoblotted with HRP-PY20 or anti-c-Cbl antibody and HRP-labeled anti-rabbit IgG. The figure which shows the influence with respect to the protein phosphorylation pattern by anti-microbody antibody of the pretreatment by the monoclonal antibody c.52. (A) Western immunoblotting of cell lysate. Ramos cells were reacted with monoclonal antibody c.52 or PBS for the time indicated in the figure, and then reacted with anti-μ antibody for 5 minutes. A cell lysate was prepared and used for immunoblotting with PY20. (B) The relative intensity of the p120 band of A was measured by NIH image 1.61, corrected for β-actin values, and plotted on a graph. Similar results were obtained in repeated experiments. The figure which shows the intracellular localization of Gb3 / CD77 and a BCR complex. (A) Molecular floating patterns were examined by immunoblotting using fractions fractionated by sucrose density gradient ultracentrifugation of extracts using Triton X-100. GM1 and lipid raft markers were all present in the third to fourth fractions corresponding to lipid rafts. Gb3 was also localized to lipid rafts as reported previously. (B) Localization change of BCR on Ramos cells by treatment with monoclonal antibody c.52 or anti-μ antibody. As shown in A, immunoblotting of each fraction was performed using a lysate of Ramos cells treated without treatment (top), treated with monoclonal antibody c.52 (middle), and treated with anti-μ antibody (bottom). Was performed with anti-BCR antibody.

(the term)
For convenience of explanation, definitions and meanings of some terms used in the present specification are summarized below.
A “monoclonal antibody” is an antibody obtained from a clone derived from a single antibody-producing cell, and exhibits a much higher specificity than a polyclonal antibody that is a mixture of a plurality of types.
The term “isolated” means that the substance has been removed from its original environment (for example, the natural environment in the case of natural substances), that is, exists in a state different from the original state by human manipulation. Means. Therefore, an “isolated antibody” includes a natural antibody that has not been subjected to any external manipulation (artificial manipulation), that is, an antibody that is produced in a body of an individual and remains there. Is not included.
Globotriosylceramide is a globo-series glycolipid (globo-type glycolipid) which is a kind of glycosphingolipid and is abbreviated as Gb3. Since Gb3 is also called alias CD77 antigen, globotriaosylceramide is sometimes referred to as “Gb3 / CD77” in the present specification.

1. Method for Producing Anti-Gb3 / CD77 Monoclonal Antibody The first aspect of the present invention provides a method for producing a monoclonal antibody that specifically recognizes Gb3 / CD77, ie, an “anti-Gb3 / CD77 monoclonal antibody”. In the present invention, an immunological technique is used. Specifically, the following steps (1) to (5) are performed.
(1) Step of immunizing non-human mammal deficient in α1,4-galactosyltransferase gene with Gb3 / CD77 (2) Step of recovering antibody-producing cells from non-human mammal after immunization (3) Recovered antibody Step of immortalizing production cells (4) Step of monoclonalizing cells that produce anti-Gb3 / CD77 antibody among immortal antibody-producing cells (5) Collecting antibodies produced by clones obtained by monoclonalization Step

  In step (1), first, a non-human mammal deficient in the α1,4-galactosyltransferase gene is prepared. The non-human mammal can be produced according to a previously reported method (Reference Document 11). The outline of the production method (an example when using a mouse) is explained. Neo resistance of a genomic fragment containing the gene fragment of α1,4-galactosyltransferase by insertion or substitution into a region encoding a site important for enzyme activity. Create a targeting vector incorporating the gene. This vector is introduced into cultured ES cells by electroporation to obtain a neo-resistant clone, and then a homologous gene recombinant is obtained and injected into a blastocyst. From the resulting chimeric mice, heterozygous and homozygous mice are prepared. Non-human mammals that can be used as immunized animals are used. For example, mouse, rat, rabbit, goat and the like are employed as the non-human mammal here. Preferably, a mouse is used. In particular, A / J strain mice can be used because of their good reactivity with glycolipids (especially gangliosides). A / J mice can be purchased from Japan Charles River or Japan SLC.

  On the other hand, an antigen (immunogen) for immunization is prepared. Gb3 / CD77 is used as the antigen. Gb3 / CD77 may be a purified product derived from a living body or a synthetic product. For example, the biological product Gb3 / CD77 is available from Larodan AB. As an antigen, a liposome containing Gb3 / CD77, lipid A, cholesterol and diparamitoylphosphatidylcholine (DPPC) is preferably used as an immunogen. The reason is to bring the antigen's existence mode closer to the natural state. The liposome may be prepared, for example, according to the method already reported (reference document 13). The outline of the preparation method (one example) will be described. 0.5 μmol of DPPC, cholesterol, 10 μg of lipid A and 100 μg of ganglioside are dissolved in C (chloroform): M (methanol) (2: 1) and evaporated. Subsequently, dissolve in 0.5 ml PBS and stir well after treatment at 50 ° C for 1 minute.

  Immunization is repeated as necessary, and when a sufficient increase in antibody titer is observed, antibody-producing cells are removed from the immunized animal and collected. Typically, the spleen or a part thereof is removed and antibody-producing cells are collected. However, as long as subsequent operations are possible, the source of antibody-producing cells is not limited to the spleen.

  Next, the obtained antibody-producing cells are immortalized. Typically, antibody-producing cells are fused with myeloma cells to obtain hybridomas. If the antibody-producing cell species is a mouse, NS-1 myeloma cells, P3X63-Ag8653, Sp2 / 0-Ag14, PAI, and the like can be used.

  Subsequently, among the immortalized antibody-producing cells, the cells producing the anti-Gb3 / CD77 antibody are monoclonalized. For the monoclonalization, selective culture using a HAT medium (medium containing hypoxanthine, aminopterin, and thymidine) and a limiting dilution method are generally used. In this technique, hybridomas in which antibody-producing cells and tumor cells are fused are selected by culturing in HAT medium, and hybridomas that produce the desired antibody (anti-Gb3 / CD77 antibody) are cloned using the limiting dilution method. . In one embodiment of the present invention, cells that produce IgG class antibodies that are more useful are monoclonalized. In other words, screening is performed using whether or not an IgG class anti-Gb3 / CD77 antibody is produced as an index to obtain an immortalized antibody-producing cell clone. According to the production method of the present invention, clones producing not only IgM class but also IgG class antibodies can be obtained at a high rate (see Examples below), and thus such screening is possible and effective. is there.

  The antibody of interest is obtained by recovering the antibody produced by the clone obtained by monoclonalization. For example, the antibody is recovered by purifying the culture medium of the selected clone. On the other hand, after the selected clone is grown to a desired number or more, it is transplanted into the abdominal cavity of an animal (for example, a mouse) and grown in ascites to obtain the desired antibody. it can. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

Various modifications can be made to the obtained antibody on condition that the specific binding property to Gb3 / CD77 is maintained. For example, a fusion antibody or a labeled antibody can be constructed by fusing or binding a low molecular weight compound, protein, labeling substance or the like. Examples of the labeling substance include fluorescent dyes such as fluorescein, rhodamine, Texas red, oregon green, enzymes such as horseradish peroxidase, microperoxidase, alkaline phosphatase, β-D-galactosidase, chemical or bioluminescent compounds such as luminol, acridine dye, etc. , ³² P, ¹³¹ I, ¹²⁵ I and the like, and biotin. It is also possible to perform chemical modification (for example, acetylation, PEGylation, phosphorylation, amidation of some amino acids).

2. Anti-Gb3 / CD77 monoclonal antibody and use thereof The second aspect of the present invention is the fact that a plurality of anti-Gb3 / CD77 monoclonal antibodies exhibiting CDC activity have been successfully obtained, and anti-Gb3 / CD77 monoclonal antibody also exhibits ADCC activity in addition to CDC activity Based on the fact that the Gb3 / CD77 monoclonal antibody has been successfully obtained, an isolated antibody that specifically recognizes Gb3 / CD77 and exhibits CDC activity and / or ADCC activity is provided. The antibody of the present invention is useful, for example, as a component of a research reagent for cells expressing Gb3 / CD77 or as an active component of a diagnostic or therapeutic reagent targeting a malignant tumor overexpressing Gb3 / CD77.

The antibody of the present invention is typically prepared by the antibody production method of the present invention. The antibodies of the present invention include antibodies from non-human animals such as mice and rats, chimeric antibodies in which a part of the region has been substituted with those from other animals (including humans), humanized antibodies, and human antibodies. The origin, type, class, form and the like of the antibody of the present invention are not particularly limited, but are preferably IgG class antibodies. For example, the antibodies belong to mouse antibody subclass IgG1, IgG2a, IgG2b or IgG3, or human antibody subclass IgG1, IgG2, IgG3 or IgG4.

  One form of the antibody of the present invention is a humanized antibody. “Humanized antibody” refers to an antibody having a structure similar to that of a human antibody. A human chimeric antibody (for example, an antibody in which a part of the antibody is humanized, an antibody in which the CH2 region is humanized, Fc Humanized antibody, constant region humanized antibody), and human-type CDR grafting (CDR-grafted) in which parts other than the CDR (complementarity determining region) present in the constant region and variable region are humanized ) Antibodies (PTJohons et al., Nature 321,522 (1986)), fully humanized antibodies and the like. In order to enhance the antigen binding activity of human CDR-grafted antibody, a method of selecting human antibody FR having high homology with mouse antibody, a method of producing humanized antibody having high homology, and further after transplanting mouse CDR to human antibody Techniques for improving the method of substituting amino acids in FR have already been developed (US Patent No. 5585089, US Patent No. 5693761, US Patent No. 5693762, US Patent No. 6180370, European Patent No. 451216, European Patent No. 682040) , Refer to Japanese Patent No. 2828340, etc.) and can also be used to prepare the human CDR-grafted antibody of the present invention.

  The antibodies of the invention can also be prepared as antibody fragments such as Fab, Fab ′, F (ab ′) 2, scFv, or dsFv antibodies. Fab is a fragment with a molecular weight of about 50,000, which is obtained by digesting an IgG antibody with papain in the presence of cysteine and consisting of an L chain, an H chain variable region, and an H chain fragment consisting of a CH1 domain and a part of the hinge part. It is. If there is a desired IgG antibody, Fab can be obtained by digesting it with papain. Alternatively, a Fab can be prepared from a transformant obtained by incorporating a DNA encoding a part of the H chain and the L chain into an appropriate vector and transforming using the vector.

Fab ′ is a fragment having a molecular weight of about 50,000 obtained by cleaving a disulfide bond between the H chains of F (ab ′) ₂ described later. If there is a desired IgG antibody, Fab ′ can be obtained by digesting it with pepsin and cleaving the disulfide bond with a reducing agent. Similarly to Fab, it can also be prepared by genetic engineering using DNA encoding Fab ′.

F (ab ′) ₂ is a fragment (Fab ′) that is obtained by pepsin digestion of an IgG antibody and consists of an L chain, an H chain variable region, and an H chain fragment consisting of the CH1 domain and part of the hinge region. ) Is a fragment having a molecular weight of about 100,000 bonded by a disulfide bond. If there is a desired IgG antibody, F (ab ′) ₂ can be obtained by digesting it with pepsin. Similarly to Fab, it can be prepared by genetic engineering using DNA encoding F (ab ′) ₂ .

scFv is an antibody fragment in which an Fv comprising an H chain variable region and an L chain variable region is made into a single chain by linking the C terminus of one chain and the other N terminus with an appropriate peptide linker. As the peptide linker, for example, (GGGGS) ₃ having high flexibility can be used. For example, construct a scFv antibody-encoding DNA using DNA encoding the H-chain variable region and L-chain variable region and DNA encoding a peptide linker, incorporate it into an appropriate vector, and transform using the vector An scFv can be prepared from the transformed product.

  dsFv is an Fv fragment in which a Cys residue is introduced at an appropriate position in the H chain variable region and the L chain variable region, and the H chain variable region and the L chain variable region are stabilized by disulfide bonds. The position of Cys residue introduction in each chain can be determined based on the three-dimensional structure predicted by molecular modeling. For example, a three-dimensional structure is predicted from the amino acid sequences of the H chain variable region and the L chain variable region, and DNAs encoding the H chain variable region and L chain variable region into which mutations are introduced are constructed based on such prediction, DsFv can be prepared from a transformant that is incorporated into a vector and transformed using the vector.

(Research use)
The present invention further provides, as a use of the antibody of the present invention, a research reagent containing the antibody of the present invention, targeting cells expressing Gb3 / CD77. The research reagent of the present invention is widely applicable to research targeting or using cells expressing Gb3 / CD77 (for example, a part of B cells, kidney cells, small intestinal cells, vascular endothelial cells, etc.). In particular, it is useful in studies of malignant tumors in which Gb3 / CD77 overexpression is observed, such as Burkitt lymphoma and colorectal cancer, and studies aimed at elucidating the physiological role of Gb3 / CD77. The labeled antibody of the present invention is one of the forms suitable for the research use. In addition to the antibody of the present invention, the research reagent of the present invention can contain a solvent, a buffer solution, a stabilizer, a preservative, and the like.

(Diagnosis / treatment)
By using the antibody of the present invention, it is possible to specifically detect and identify cells expressing Gb3 / CD77. Therefore, application of the antibody of the present invention to the diagnosis and treatment of malignant tumors such as Burkitt lymphoma and colorectal cancer in which overexpression of Gb3 / CD77 is observed is also expected. Therefore, the present invention further provides a composition comprising the antibody of the present invention that can be used for diagnosis or treatment targeting a malignant tumor that overexpresses Gb3 / CD77. Examples of targeted malignancies are Burkitt lymphoma, colon cancer, teratomas, megakaryoblastic leukemia. In one embodiment of the therapeutic composition of the present invention, an anti-Gb3 / CD77 monoclonal antibody exhibiting CDC activity or ADCC activity is contained as an active ingredient. Preferably, an anti-Gb3 / CD77 monoclonal antibody exhibiting ADCC activity is used. In the therapeutic composition of this embodiment, a therapeutic effect can be obtained by cell damage utilizing ADCC activity.

  In another embodiment of the therapeutic composition of the present invention, an anti-Gb3 / CD77 monoclonal antibody is utilized as a carrier for DDS. That is, this embodiment provides an immune complex obtained by binding an anti-Gb3 / CD77 monoclonal antibody with a drug (such as a cytotoxin) or a radioisotope (collectively referred to as “active ingredient”). An immune complex containing a drug (cytotoxin) having a cell-killing or cytotoxic activity is generally called an immunotoxin. Examples of cytotoxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitromycin, actino Mention may be made of mycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof. As an active ingredient contained in the immune complex, a protein or peptide having a desired biological activity may be used. Proteins that can be used for such purposes include abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin, tumor necrosis factor, interferon-γ, interleukin-1 (IL-1), interleukin-2 ( IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF) lymphokine. Techniques for conjugating active moieties to antibodies are known, e.g. Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.), Pp. 243-56 (Alan R. Liss, Inc. 1985), Controlled Drug Delivery (2nd edition.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987), Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 ( Academic Press 1985), Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982).

  The therapeutic composition can be formulated according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used. The dosage form for formulation is not particularly limited, and can be prepared as, for example, tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.

  In the treatment using the therapeutic composition of the present invention, the therapeutic composition of the present invention is administered to a subject (patient) having a target malignant tumor. The therapeutic composition of the present invention is applied to a subject (patient) by oral administration or parenteral administration (intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal injection, direct introduction into target cells, etc.) depending on the form. obtain. The dosage varies depending on symptoms, patient age, sex, body weight, and the like, but those skilled in the art can appropriately set an appropriate dosage. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In setting the administration schedule, it is possible to consider the patient's medical condition, the duration of effect of the active ingredient and the like.

<Materials and methods>
(1) Animals
A / J mice, BALB / c ^{nu / nu} mice and KSN ^{nu / nu} mice were purchased from Nippon Charles River. An α-1,4 galactose transferase gene (A4galt-) deficient mouse was established in our laboratory (Reference 11). All protocols for animal experiments are in line with the `` National Institutes of Health Guide for the Care and Use of Laboratory Animals (1966) ''. Approved by the experimental committee, all animals were maintained under specific pathogen-free conditions.

(2) Cell culture The myeloma cell line NS-1 and Burkitt lymphoma cell Ramos used in this study were cultured in RPMI 1640 containing 10% fetal calf serum (FCS). The L cells transfected with cDNA were maintained in a culture medium in which 7.5% FCS and G418 (300 μg / ml) were added to Dulbecco's modified eagle's medium (DMEM). Burkitt lymphoma cell lines Ramos, HS-Sultan, Daudi and Raji were obtained from the Japan Health Science Foundation. A4galt-introduced L cells (LVTR-1) and vector control cell lines (Lneo-2) were established as previously reported (Reference 12).

(3) Production of monoclonal antibodies against Gb3 / CD77 Three Gb3 / CD77 glycolipids (100 μg), lipid A (10 μg), cholesterol (0.5 μmol), diparamitoyl against three 8-week-old A / J mice Immunization was performed subcutaneously every two weeks using liposomes (reference document 13) containing phosphatidylcholine (dipalmitoyl-phosphatidylcholine; DPPC) (0.5 μmol). Antibody titer in mouse serum was determined by immunofluorescence (IF). Three days after the fifth immunization, spleen cells were removed and fused with NS-1 myeloma cells using polyethylene glycol (PEG). After selection with HAT selection medium (Sigma, St Louis, MO), antibodies in the culture supernatant were detected by immunofluorescence (IF) and enzyme-linked-immuno-solvent assay (ELISA) . Positive clones were subcloned by limiting dilution, and their specificity was further confirmed by TLC-immunostaining. As for A4galt-deficient mutant mice, 8-week-old mice were used, and the liposomes having the above constitution were subcutaneously immunized on the 0th, 4th, 7th, and 11th days. Immunization was performed. The antibody titer of each mouse was measured by IF using a FITC-labeled anti-mouse IgG antibody (Zymed Lab. Inc., San Francisco, Calif.). ELISA and / or IF method was performed using the culture supernatant of the hybridoma as a sample, subcloning was performed on positive clones, and specificity was further confirmed by TLC-immunostaining. The antibody in the culture supernatant of the positive clone was purified by an affinity column using protein A-agarose 4 fast flow according to the attached protocol (Amersham Biosciences, Bucks, UK).

(4) ELISA
Commercially available glycolipids (20 ng / 10 μl) dissolved in methanol were plated on 60-well Terasaki plates (Greiner BioOne, Frickenhausen, Germany). After air drying for 20 minutes, 5% BSA-containing phosphate buffer (PBS) was added to the plate, and blocking was performed at room temperature for 2 hours. After washing twice, serially diluted antibody was added to the plate and allowed to stand at room temperature for 2 hours. After washing 5 times, HRP-labeled anti-mouse IgGs (Amersham Biosciences) was added as a secondary antibody. After 5 washes, o-phenylenediamine (ortho-phenylenediamine) (2 mg) (Wako Junyaku, Osaka, Japan) and H ₂ O ₂ (8 μl) (Wako) in 5 ml citrate-phosphate (citrate-phosphate substrate solution (10 μl) dissolved in buffer) was added to the plate. After standing in the dark for an appropriate time, the degree of color development was recorded using a scanner.

(5) IF assay
LVTR-1 or Lneo-2 cells were added to the Terasaki plate (500 cells / well) and allowed to stand overnight. After removing the culture supernatant, serially diluted culture supernatant or ascites was added to the plate and allowed to stand at room temperature for 1 hour. After washing twice, a FITC-labeled anti-mouse IgG (H + L) antibody (Cappel, Durham, NC) was added and allowed to stand for 1 hour in the dark at room temperature. After washing twice, antibody binding was observed under a fluorescence microscope (BX51, OLYMPUS, Tokyo, Japan).

(6) TLC immunostaining
TLC was carried out using high performance TLC plates (Merck, Darmstadt, Germany) using chloroform / methanol / 0.22% CaCl ₂ (60: 35: 8) as a developing solvent. Specificity and reactivity to Gb3 / CD77 were examined using silica plates (Merck) of aluminum plates as previously reported (Reference 14). After TLC, the plates were blotted against PVDF (polyvinylidene difluoride) membranes as previously reported. After blocking with PBS containing 5% BSA, the plate was attached to the culture supernatant, and antibody binding was performed using ABC kit ^TM (Vector Laboratories, Burlingame, CA) and Konica immunostaining HRP-1000 ^TM (Konica, Tokyo). Detected.

(7) Flow cytometry Gb3 / CD77 on the cell surface was examined with FACSCaliver ^™ (Becton Dickinson, Mountain View, CA). In summary, cells were allowed to stand for 60 minutes on ice with diluted ascites or purified antibody, and then reacted with FITC-labeled goat anti-mouse IgG (H + L) (Cappel, Durham, NC) antibody for 45 minutes on ice. Cells prepared using only the secondary antibody were used as control cells for flow cytometry. The CELLQuest ^™ program was used for quantification of positive cells.

(8) Proliferation test and growth inhibition test In order to examine the growth inhibition effect of the prepared antibody, a WST-1 test was conducted. As previously reported (Reference 12), the absorbance at 450 nm was detected with an ELISA reader (Immuno Mini NJ-2300 (System Instruments, Tokyo)) to measure the reduction of WST-1 to formazan. In summary, cells (1 × 10 ⁴ ) were seeded in 48-well plates and cultured overnight in RPMI 1640 containing 10% FCS. Thereafter, diluted ascites or purified antibody was added to the plate and cultured. WST-1 tests were performed every 24 hours in triplicate using Ramos cells. The growth inhibitory effect of the anti-Gb3 antibody was examined in comparison with that of the control antibody.

(9) Inhibitory effect of antibody on toxicity of verotoxin Cells (1 × 10 ⁶ ) were seeded in a 48-well plate and cultured overnight in RPMI 1640 containing 10% FCS. Thereafter, the diluted antibody was added to the plate and allowed to stand on ice for 1 hour. Subsequently, the minimum amount of verotoxin that could sufficiently kill the cells was added without washing and allowed to stand. Thereafter, a cell lysate was prepared and used for immunoblotting to examine the inhibitory effect on toxin toxicity.

(10) CDC test
After washing the cells with 5% FCS containing PBS in order to remove the RPMI, were plated in 48-well plates ^{(1x10 4 / 50μl (5%} FCS -containing PBS)). 50 μl of PBS containing 5% FCS and various amounts of ascites was added to each well and allowed to stand at room temperature for 1 hour. Thereafter, 200 μl of PBS containing 5% FCS and rabbit serum (30 μl: 1/10 or 150 μl: 1/2) was added to each well, finally at a volume of 300 μl / well at 37 ° C. for 1 hour. Left to stand. After the CDC reaction, cell proliferation measurement reagent WST-1 (Roche, Manheim, Germany) was added to each well (30 μl / well). After standing for 2 hours, the plate was sufficiently shaken for 1 minute, and the absorbance at 450 nm of the sample was measured using a microplate reader (ImmunoMini NJ-2300, ImmunoMini NJ-2300, InterMed, Tokyo, Japan). .

(11) ADCC test The target cells (1x10 ⁶ ) were allowed to stand on ice for 1 hour in a diluted antibody and a Falcon tube, washed twice with 10% FCS-containing RPMI, and then plated on each well. Human peripheral mononuclear cells were separated from normal human heparinized blood (25 ml) by centrifugation according to the manufacturer's instructions in a Ficoll-Hypaque gradient (Amersham Biosciences Inc.). These cells were added to a microtiter plate at a predetermined ratio (target / effector) and allowed to stand at 37 ° C. for 4 hours. To measure the amount of LDH released by ADCC, CytoTox 96 Non-Radioactive Cytotoxicity Assay ^™ (Promega, Madison, Wis.) Was used and examined according to the manufacturer's instructions.

(12) Immunoblotting and immunoprecipitation Cells were lysed with a lysis buffer (20 mM Tris-HCl, pH 7.4, containing the following reagents: 0.15 M NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (Sigma) and 5 μg. / ml aprotinin). Cell lysate (derived from 4.0 × 10 ⁶ cells) was subjected to SDS-PAGE and then transferred to a PVDF membrane (Millipore, Bedford, Mass.). After blocking with PBS containing 5% BSA for 1 hour at room temperature, the PVDF membrane was reacted with the primary antibody for 1 hour at room temperature. Thereafter, the plate was washed with PBS containing 0.05% Tween 20 three times, horseradish peroxidase-labeled anti-rabbit IgG (H + L) (BD transduction Laboratories, San Jose, Calif.) Was added, and the mixture was allowed to stand for 1 hour and caspase-3 (caspase- 3) p38 and phosphorylated p38 were detected. FAK, Lyn and c-Cbl were detected using horseradish peroxidase-labeled anti-mouse IgG (H + L) antibody (Zymed Laboratories Inc. San Francisco, Calif.). Detection was performed according to the manufacturer's instructions using ECL system ^™ (Amersham Biosciences Inc.). For immunoprecipitation, cell lysates (derived from 4 × 10 ⁶ cells) were immunoprecipitated with polyclonal antibodies against PY20, FAK, Lyn or c-Cbl and Protein G Sepharose 4 first flow ^™ (Amersham Biosciences). After washing the immune complex, SDS sample buffer was added and boiled for 3 minutes.

(13) B cell stimulation by anti-B cell receptor (BCR) antibody or mAb c.52
Ramos cells were suspended in RPMI containing 10% FCS at a concentration of 10 ⁷ / mL. Cells were left for 1 hour at 37 ° C. before stimulation. Thereafter, stimulation was carried out for a predetermined time using goat anti-human IgM (10 μg / ml) (Fc5μ fragment specific antibody, Jackson Immuno-Laboratory, West Grove, PA) or monoclonal antibody c.52 (10 μg / ml).

<Result>
First, A / J mice were immunized with liposomes embedded with Gb3. Among the immunized mice, the spleen cells of the mice that showed the highest antibody titer against Gb3 were used for fusion with the myeloma cell line NS1. Screening by ELISA and fluorescent antibody method was performed. As a result of screening 400 clones, three hybridomas c.37, c.61, and c.80 were established. The specificity of the antibodies produced by these hybridoma clones for Gb3 / CD77 was analyzed by ELISA and TLC immunostaining (FIGS. 1A and 1B). All 13 clones responded exclusively to Gb3 / CD77 against 13 different glycolipids. None of the three clones reacted with lactosylceramide, GlcCer, GM3, and Gb4, suggesting that α4-galactose at the non-reducing end is essential for the resulting antibody to react. Regarding other glycolipids, no reactivity to GalCer, GD1b, GD3, GA1, GT1b, GA2, GM1 and GD1a was observed (data not shown). Similar reaction specificity was also observed by TLC immunostaining (FIG. 1B). mAbs 61 and 80 also reacted only with Gb3 (data not shown). The isotype of the 3 clone antibodies was determined by ELISA and found to be of IgM class (data not shown).

  A4galt-deficient mice lacking all globo-based glycolipids were treated with Gb3-embedded liposomes as an immunogen in the wild, aiming to establish antibodies with reactivity to Gb3 / CD77 and higher performance in terms of antitumor effects. Immunization was performed as in the case of type mice. Using the mouse that showed the highest antibody titer against Gb3 / CD77 among the immunized mice, the spleen cells were fused with mouse NS-1 myeloma cells. As a result of screening by ELISA and immunofluorescence, hybridomas of 323 to 8 clones were established based on the reactivity to Gb3 / CD7. The specificity of the resulting monoclonal antibody for Gb3 / CD77 was determined by ELISA and TLC immunostaining (FIGS. 2 and 3). Of 13 glycolipids, all clones reacted only with Gb3 / CD77. The results of ELISA for various glycolipids are shown in FIG. 3 (results for GalCer, GD1b, GD3, GA1, GA2, GT1b, GM1 and GD1a are omitted). The antibody isotypes of 8 clones were determined by ELISA and found to be IgM (4 clones), IgG3 (3 clones) and IgG2b (1 clone) (data not shown).

  Next, in order to analyze the antitumor activity of the obtained clone, CDC activity was examined using Burkitt lymphoma Ramos cells as a target. Three clones of IgM class monoclonal antibodies obtained using A / J mice and IgM class monoclonal antibodies obtained using A4galt-deficient mice showed strong CDC activity, but the antibodies alone induce cytotoxicity. Did not (Figure 4). Four clones of IgG class antibodies obtained using A4galt-deficient mice similarly induced obvious CDC activity, but their strength was weaker than that of IgM type monoclonal antibodies. Next, the ADCC activity of these antibodies was examined. Monoclonal antibody c.52 induced clear ADCC activity against Ramos cells using human peripheral blood mononuclear cells as an effector (FIG. 5). IgM or IgG2b class monoclonal antibodies showed no or very little ADCC activity (FIG. 5).

  From the fact that multiple IgG class antibodies were obtained from A4galt-deficient mice, it was considered necessary to search for other functions of antibodies such as high-performance competitive role for verotoxin receptors. As shown in FIG. 6, when Ramos cells were allowed to stand for 3 hours with verotoxin 2, degradation of caspase-3 was induced, indicating cytotoxicity caused by verotoxin. When Ramos cells were treated for 2 hours on ice in the presence of anti-Gb3 / CD77 antibody before addition of verotoxin 2 (2 ng / ml) (minimum amount required to induce cell death), caspase 3 Degradation was partially inhibited by monoclonal antibodies c.168 (IgM), c.52 (IgG3) and c.95 (IgG2b). However, it was not suppressed by monoclonal antibody c.220 (IgG3) or R24 (control) (FIG. 6B). Although the degradation of caspase 3 was surely suppressed in part or not by the monoclonal antibody, the direction of cell death was clearly not suppressed.

  Gb3 / CD77 is expressed in a subpopulation of germinal center B lymphocytes and may be required for the process of affinity maturation of antibodies. Therefore, we thought that there was an unknown ligand for Gb3 / CD77, which might trigger a signal in germinal center B cells. To examine this hypothesis, Ramos cells were treated with monoclonal antibodies and analyzed for morphological changes and intracellular protein phosphorylation. Among the clones studied, monoclonal antibody c.52 caused calcium-dependent cell aggregation, but monoclonal antibody c.220 did not (Figs. 7A and 7B). Monoclonal antibody c.52 also showed mild but reliable cytostatic activity against Ramos cells (FIG. 7C). As a result of immunoblotting of phosphorylated proteins using cells after addition of these antibodies, it was shown that dramatic changes occurred in the phosphorylation pattern of intracellular proteins (FIG. 8). These changes were also observed in cells treated with monoclonal antibody c.220. However, the degree of phosphorylation was milder than that with the monoclonal antibody c.52 (right side of FIG. 8). Given that monoclonal antibody c.220 did not induce cell aggregation, phosphorylation by monoclonal antibody c.52 would not be due to cell aggregation. Protein phosphorylation elicited by monoclonal antibody c.52 was similar to the anti-BCR antibody phosphorylation by cross-linking when compared to the signal mediated by the anti-μ antibody via the B cell receptor (Fig. 9A and 9B). Among several phosphorylated proteins, the 120 kDa protein appeared to be a molecule that was phosphorylated in common by both stimuli (FIG. 9). As described above, monoclonal antibody c.52 shows mild but obvious growth-inhibitory activity against Ramos cells, and Gb3 / CD77-mediated signals are negative for existing signals such as BCR-mediated signals and survival signals. This suggests the possibility of bringing

  To identify molecules with a 120 kDa band and to determine whether c-Cbl (ref. 15) was phosphorylated based on Gb3 / CD77 cross-linking by monoclonal antibody c.52 Immunoprecipitation (IP) -immunoblotting (IB) analysis was performed. As a result, it was shown that at least a part of the 120 kDa band was c-Cbl, which was phosphorylated by Gb3 / CD77 cross-linking with monoclonal antibody c.52 (FIG. 10A). The time course of c-Cbl phosphorylation was shown (FIG. 10B). Effect of Gb3 / CD77 on protein phosphorylation pattern when Ramos cells were pretreated with monoclonal antibody c.52 before anti-μ antibody was added to examine whether Gb3 / CD77 acts as a negative regulator of BCR signal It was investigated. As expected, pretreatment with monoclonal antibody c.52 significantly suppressed protein phosphorylation by anti-μ antibody compared to control IgG (FIGS. 11A and 11B).

<Discussion>
In this study, it was shown that anti-Gb3 antibodies can be produced efficiently when Gb3 / CD77 synthase-deficient (knockout) mice are used for immunization. The production efficiency of the monoclonal antibody and the quality of the established antibody function were clearly better than those obtained using normal mice. In past attempts, purified Igb3 was used as an immunogen and exclusively IgM monoclonal antibodies were produced (Ref. 23). Conventional methods generally provide IgM class monoclonal antibodies, but in this study, IgG class monoclonal antibodies that react with Gb3 were obtained at a relatively high rate.

  Gb3 is the starting molecule for the synthesis of all globo-based glycolipids, as a tumor marker for Burkitt lymphoma (Ref. 8), as a receptor for Verotoxin (Refs. 11, 24), or cell death of the B cell lineage As a marker (reference document 25), it has attracted attention from researchers. The role of globo glycolipids under physiological and pathological conditions is currently being analyzed in our laboratory using mutant mice lacking globo glycolipids (Reference 11). In any case, anti-Gb3 antibodies having various functions are extremely useful for elucidating the functions of glycolipids.

  In fact, caspase 3 degradation was partially inhibited by these antibodies. However, the direction of cell death was not clearly suppressed although the degradation of caspase 3 was significantly suppressed. These results suggest that the binding of these antibodies is not strong enough to suppress the death signal, even though it caused a delay in the apoptotic signal.

  In terms of the biological function of monoclonal antibodies, the anti-Gb3 antibody obtained in this study is very interesting. This is because some of them bound to the cell surface and induced activation of the B cell signal transduction pathway in the same manner as in the case of cross-linking with anti-μ antibody (Ref. 26). . There are several components, including c-Cbl, that undergo tyrosine phosphorylation when treated with monoclonal antibody c.52. c-Cbl is known as E3 ubiquitin ligase (Ref. 27), and induces molecules such as Syk to degrade in the proteasome system, thereby reducing the signal via BCR (Ref. 28). In analyzing the role of signals from Gb3 / anti-Gb3 antibodies in BCR-mediated signals, pretreatment of Ramos cells with monoclonal antibody c.52 reduced the level of tyrosine phosphorylation by anti-μ antibody slightly . From this result, it was found that c-Cbl activation actually acts on the regulation of BCR-mediated signal. Therefore, the most interesting problem here is whether there is an endogenous factor that interacts with Gb3, and if so, what.

  Since Gb3 / CD77 is expressed in a subpopulation of B lymphocytes at the germinal center and binding of monoclonal antibodies to Burkitt lymphoma cells induces cell aggregation and protein tyrosine phosphorylation, we It was speculated that the endogenous ligand of CD77 was present and might be involved in signal control of germinal center B cells. When Ramos cells were pretreated with monoclonal antibodies, cell growth was suppressed. This is because our signal that Gb3 / CD77 has a negative effect on existing signals such as BCR signals by BCR cross-linking. It supported the assumption that it might be introduced. It is well known that c-Cbl and E3 ubiquitin ligases play a negative regulator of BCR signals based on degradation by the ubiquitin proteasome system of signal molecules located upstream of BCR-mediated signal transduction pathways such as Syk, SFK, and Tec. (References 28 and 29). When activated by BCR cross-linking, they are phosphorylated. As shown in this study, c-Cbl was phosphorylated by monoclonal antibody c.52, and pretreatment of Ramos cells with c.52 suppressed cell proliferation as well as attenuated BCR signal. These results are also related to the general function of c-Cbl in the developmental process (Ref. 30) and its significance in pathology (Ref. 31).

  It was well known that BCR complexes migrate from non-lipid rafts to lipid rafts after cross-linking BCRs with similar antigens or anti-μ antibodies (references 32, 33) (FIG. 12). Since Gb3 / CD77 is also localized in lipid rafts, we hypothesized that Gb3 / CD77 acts in negative regulation of BCR signals in lipid rafts. Indeed, monoclonal antibody c.52 significantly suppressed phosphorylation induced by anti-μ antibody. Furthermore, binding of the monoclonal antibody c.52 caused BCR to migrate to lipid rafts in Ramos cells (FIG. 12). This fact not only suggests that the transition of BCR from non-lipid raft to lipid raft leads to effective signal transduction of BCR, but also through the ubiquitin proteasome based on the regulation of Gb3 / CD77-mediated signaling It also suggests the possibility of initiating negative signaling such as degradation and dephosphorylation of key molecules.

  According to the monoclonal antibody production method of the present invention, a monoclonal antibody that specifically recognizes Gb3 / CD77, which is a globo-based glycolipid, can be efficiently produced. That is, the monoclonal antibody production method of the present invention is useful as a means for efficiently producing an anti-Gb3 / CD77 monoclonal antibody. The monoclonal antibody production method of the present invention is particularly useful as a means for obtaining an IgG class monoclonal antibody. The anti-Gb3 / CD77 monoclonal antibody produced by the present invention is expected to be used and utilized not only for research purposes but also in various fields such as diagnostic uses and therapeutic uses.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

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Claims (6)

Gb3 / CD77 specifically recognizes, complement dependent cytotoxicity and antibody-dependent cellular cytotoxicity shows, and shows the aggregation activity against Burkitt lymphoma cell line, isolated monoclonal antibodies. It is a human or humanized antibody, the isolated monoclonal antibody of claim 1. The isolated monoclonal antibody according to claim 1 or 2 , which is an IgG, Fab, Fab ', F (ab') 2, scFv, or dsFv antibody. A research reagent targeting cells expressing Gb3 / CD77, comprising the isolated monoclonal antibody according to any one of claims 1 to 3 . A composition for diagnosis or treatment targeting a malignant tumor overexpressing Gb3 / CD77, comprising the isolated monoclonal antibody according to any one of claims 1 to 3 . The composition according to claim 5 , wherein the malignant tumor is Burkitt lymphoma, colon cancer, teratoma or megakaryoblastic leukemia.

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