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Konrad Sandhoff - One of the best experts on this subject based on the ideXlab platform.
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Ganglioside GM2 catabolism is inhibited by storage compounds of mucopolysaccharidoses and by cationic amphiphilic drugs.
Molecular genetics and metabolism, 2019Co-Authors: Susi Anheuser, Bernadette Breiden, Konrad SandhoffAbstract:The catabolism of Ganglioside GM2 is dependent on the lysosomal enzyme β-hexosaminidase A and a supporting lipid transfer protein, the GM2 activator protein. A genetically based disturbance of GM2 catabolism, leads to several subtypes of the GM2 gangliosidosis: Tay-Sachs disease, Sandhoff disease, the AB-variant and the B1-variant, all of them having GM2 as major lysosomal storage compound. Further on it is known that the Gangliosides GM2 and GM3 accumulate as secondary storage compounds in mucopolysaccharidoses, especially in Hunter disease, Hurler disease, Sanfilippo disease and Sly syndrome, with chondroitin sulfate as primary storage compound. The exact mechanism of Ganglioside accumulation in mucopolysaccaridoses is still a matter of debate. Here, we show that chondroitin sulfate strongly inhibits the catabolism of membrane-bound GM2 by β-hexosaminidase A in presence of GM2 activator protein in vitro already at low micromolar concentrations. In contrast, hyaluronan, the major storage compound in mucopolysaccharidosis IX, a milder disease without secondary Ganglioside accumulation, is a less effective inhibitor. On the other hand, hydrolysis of micellar-bound GM2 by β-hexosaminidase A without the assistance of GM2AP was not impeded by chondroitin sulfate implicating that the inhibition of GM2 hydrolysis by chondroitin sulfate is most likely based on an interaction with GM2AP, the GM2AP-GM2 complex or the GM2-carrying membranes. We also studied the influence of some cationic amphiphilic drugs (desipramine, chlorpromazine, imipramine and chloroquine), provoking drug induced phospholipidosis and found that all of them inhibited the hydrolysis of GM2 massively.
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membrane lipids regulate Ganglioside GM2 catabolism and GM2 activator protein activity
Journal of Lipid Research, 2015Co-Authors: Susi Anheuser, Bernadette Breiden, Günter Schwarzmann, Konrad SandhoffAbstract:Ganglioside GM2 is the major lysosomal stor- age compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with -hexosaminidase A and GM2 activator pro- tein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant cata- bolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of mem- brane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal trans- fer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Forster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. —Anheuser, S., B. Breiden, G. Schwarzmann, and K. Sandhoff. Membrane lipids regulate Ganglioside GM2 ca- tabolism and GM2 activator protein activity. J. Lipid Res. 2015. 56: 1747-1761.
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membrane lipids regulate Ganglioside GM2 catabolism and GM2 activator protein activity
Journal of Lipid Research, 2015Co-Authors: Susi Anheuser, Bernadette Breiden, Günter Schwarzmann, Konrad SandhoffAbstract:Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Forster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids.
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photoaffinity labelling of the human GM2 activator protein mechanistic insight into Ganglioside GM2 degradation
FEBS Journal, 2004Co-Authors: Michaela Wendeler, Günter Schwarzmann, Joerg Hoernschemeyer, Daniel Hoffmann, Thomas Kolter, Konrad SandhoffAbstract:The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of Ganglioside GM2 by lysosomal beta-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein result in a fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this glycoprotein cofactor, we synthesized the two photoaffinity labels [14C]C3-TPD-GM2 and [14C]C7-TPD-GM2. Incubation of GM2AP with these substrate analogues and subsequent irradiation led to covalent labelling of the protein. After separation of tryptic peptides by reverse-phase HPLC, the labelled peptide fractions were analysed by MALDI-TOF and sequenced by ESI-Q-TOF mass spectrometry. Both labels were found to be specifically photoincorporated into a part of the surface loop comprising residues V153-L163, a stretch of amino acids that was previously identified as the most flexible region in the crystal structure of the activator. Our results provide strong evidence that this loop constitutes the part of the activator protein that directly interacts with the Ganglioside substrate, suggesting that the hydrophobicity and the great structural mobility of this element are crucial for the extraction of the membrane-embedded glycolipid, its stabilization inside the spacious cavity and its guidance to the enzyme's active site. This study demonstrates that the approach of photoaffinity labelling in conjunction with accurate mass measurements can provide insight into substrate binding interactions that complements structural information. D-53121 Bonn, Germany.
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recombinant Ganglioside GM2 synthase expression in insect cells and enzyme assay
Methods in Enzymology, 2003Co-Authors: Michaela Wendeler, Günter Schwarzmann, Joerg Hoernschemeyer, Thomas Kolter, Helmut Reilaender, Konrad SandhoffAbstract:Publisher Summary This chapter investigates the recombinant Ganglioside G M2 synthase; and describes the expression of murine β -1,4- N -acetylgalactosaminyltransferase both as a complete membrane-bound enzyme and as a soluble form in the baculovirus insect -cell expression system. A novel assay based on radiochemically labeled G M3 prepared from the tissue Ganglioside, which permits the evaluation of potential UDP-GalNAc analogs is presented. Schematic protein structure of G M2 synthase showing the topology of type II transmembrane proteins is presented. For the expression of full-length G M2 synthase, the entire cDNA of G M2 synthase in the vector pCMV Blue is used as a template in the polymerase chain reaction (PCR) reaction. For expression of soluble G M2 synthase, the medium of infected cells is harvested 72 h postinfection by centrifugation (2000 rpm, 10 min) and concentrated 15-fold with a Vivaspin 4-ml concentrator with a PES membrane and 30,000 moleculor weight cut off (MWCO). This concentrated supernatant is prepared fresh for the assays and is always kept on ice. The chapter discusses the immunoblot analysis, standard glycosyltransferase assays, and kinetic analysis of recombinant G M2 synthase.
Seiji Yano - One of the best experts on this subject based on the ideXlab platform.
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genetically engineered humanized anti Ganglioside GM2 antibody against multiple organ metastasis produced by GM2 expressing small cell lung cancer cells
Cancer Science, 2011Co-Authors: Tadaaki Yamada, Hideaki Bando, Shinji Takeuchi, Kenji Kita, Wei Wang, Shiro Akinaga, Yasuhiko Nishioka, Saburo Sone, Seiji YanoAbstract:Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-Ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. (Cancer Sci 2011; 102: 2157–2163)
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genetically engineered humanized anti Ganglioside GM2 antibody against multiple organ metastasis produced by GM2 expressing small cell lung cancer cells
Cancer Science, 2011Co-Authors: Tadaaki Yamada, Hideaki Bando, Shinji Takeuchi, Kenji Kita, Wei Wang, Shiro Akinaga, Yasuhiko Nishioka, Saburo Sone, Seiji YanoAbstract:Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-Ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC.
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Humanized anti-Ganglioside GM2 antibody is effective to induce antibody-dependent cell-mediated cytotoxicity in mononuclear cells from lung cancer patients.
Cancer letters, 2001Co-Authors: Prahlad Parajuli, Seiji Yano, Hiroaki Yanagawa, Masaki Hanibuchi, Eiji Takeuchi, Toyokazu Miki, Saburo SoneAbstract:Abstract Ganglioside GM2 is one of the major Gangliosides expressed on the cell surface of human tumors including lung cancer. We have previously reported that a mouse–human chimeric monoclonal antibody (mAb), KM966, against GM2 promotes the lysis of lung cancer cells by human blood mononuclear cells (MNC) of healthy donors. In this study, we examined antibody-dependent cell-mediated cytotoxicity (ADCC) of MNC, using KM966 mAb and its humanized counterpart, KM8969, in 16 lung cancer patients and 18 control patients. The ADCC activity was assessed by 4-h 51Cr release from GM2 positive SBC-3 small cell lung cancer cells. MNC from lung cancer patients exhibited similar ADCC activity to those from control patients when KM966 and KM8969 were used as mAb. Moreover, effective ADCC activity was observed even in MNC from advanced lung cancer patients. These observations suggest the potential activity of humanized anti-GM2 mAb (KM8969), as well as chimeric KM966, in biological therapy for lung cancer patients.
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therapeutic efficacy of mouse human chimeric anti Ganglioside GM2 monoclonal antibody against multiple organ micrometastases of human lung cancer in nk cell depleted scid mice
International Journal of Cancer, 1998Co-Authors: Masaki Hanibuchi, Yasuhiko Nishioka, Seiji Yano, Hiroaki Yanagawa, Tetsuya Kawano, Saburo SoneAbstract:The development of distant metastases to multiple organs is a critical problem in the treatment of human lung cancer. In this study, we evaluated the therapeutic efficacy of a mouse-human chimeric anti-Ganglioside GM2 (GM2) monoclonal antibody (MAb), KM966 against metastasis formation of GM2-positive human lung cancer cells inoculated intravenously (i.v.) into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice. GM2-positive human small cell lung cancer (SCLC), SBC-3 cells (1 × 106), injected through a tail vein into NK cell-depleted SCID mice, formed large number of metastatic colonies in the liver, kidneys and lymph nodes by 42 days after inoculation (day 42). KM966, but not control MAb, given on days 2 and 7, almost completely inhibited metastasis formation of SBC-3 cells in the liver, kidneys and lymph nodes in a dose-dependent fashion. Moreover, treatment with KM966 at advanced stages of metastasis (even from day 28) significantly suppressed multiple organ metastases of SBC-3 cells. The anti-metastatic effect of KM966 in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction mediated by macrophages of the SCID mice. Our findings suggest that the mouse-human chimeric anti-GM2 MAb, KM966 may be useful for eradicating multiple organ micrometastases of lung cancer in humans.Int. J. Cancer 78:480–485, 1998. © 1998 Wiley-Liss, Inc.
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anti Ganglioside GM2 monoclonal antibody dependent killing of human lung cancer cells by lymphocytes and monocytes
Japanese Journal of Cancer Research, 1996Co-Authors: Masaki Hanibuchi, Yasuhiko Nishioka, Seiji Yano, Hiroaki Yanagawa, Saburo SoneAbstract:Ganglioside GM2 (GM2) frequently appears on the cell surface of human cancers of neuroendocrine origin. A mouse-human chimeric monoclonal antibody (mAb), KM966, against GM2 was previously found to promote the lysis of various cancer cells by human blood mononuclear cells (MNC). In this study, we analyzed the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against small cell lung cancer (SCLC) cells and examined the enhancing effect of various cytokines on the ADCC activity. The ADCC activity was assessed by 4-h 51Cr release assay. Highly purified lymphocytes (> 99%) and monocytes (> 90%) were separated by centrifugal elutriation from peripheral blood MNC of the same healthy donor. KM966 induced lysis of SCLC cells mediated by both lymphocytes and monocytes to similar extents, in a dose-dependent manner. Pretreatment of lymphocytes with various cytokines [interleukin (IL)-2, IL-12 and interferon-gamma] and that of monocytes with macrophage-colony-stimulating factor significantly augmented the killer activity against SCLC cells in the presence of KM966 mAb. KM966 was also effective for the lysis of non-small cell lung cancer cells in direct proportion to the GM2 expression levels. These findings suggest that combined treatment of KM966 mAb with cytokines may be therapeutically useful for in vivo killing of lung cancer cells expressing GM2 through the ADCC reaction.
Saburo Sone - One of the best experts on this subject based on the ideXlab platform.
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genetically engineered humanized anti Ganglioside GM2 antibody against multiple organ metastasis produced by GM2 expressing small cell lung cancer cells
Cancer Science, 2011Co-Authors: Tadaaki Yamada, Hideaki Bando, Shinji Takeuchi, Kenji Kita, Wei Wang, Shiro Akinaga, Yasuhiko Nishioka, Saburo Sone, Seiji YanoAbstract:Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-Ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. (Cancer Sci 2011; 102: 2157–2163)
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genetically engineered humanized anti Ganglioside GM2 antibody against multiple organ metastasis produced by GM2 expressing small cell lung cancer cells
Cancer Science, 2011Co-Authors: Tadaaki Yamada, Hideaki Bando, Shinji Takeuchi, Kenji Kita, Wei Wang, Shiro Akinaga, Yasuhiko Nishioka, Saburo Sone, Seiji YanoAbstract:Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-Ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC.
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Humanized anti-Ganglioside GM2 antibody is effective to induce antibody-dependent cell-mediated cytotoxicity in mononuclear cells from lung cancer patients.
Cancer letters, 2001Co-Authors: Prahlad Parajuli, Seiji Yano, Hiroaki Yanagawa, Masaki Hanibuchi, Eiji Takeuchi, Toyokazu Miki, Saburo SoneAbstract:Abstract Ganglioside GM2 is one of the major Gangliosides expressed on the cell surface of human tumors including lung cancer. We have previously reported that a mouse–human chimeric monoclonal antibody (mAb), KM966, against GM2 promotes the lysis of lung cancer cells by human blood mononuclear cells (MNC) of healthy donors. In this study, we examined antibody-dependent cell-mediated cytotoxicity (ADCC) of MNC, using KM966 mAb and its humanized counterpart, KM8969, in 16 lung cancer patients and 18 control patients. The ADCC activity was assessed by 4-h 51Cr release from GM2 positive SBC-3 small cell lung cancer cells. MNC from lung cancer patients exhibited similar ADCC activity to those from control patients when KM966 and KM8969 were used as mAb. Moreover, effective ADCC activity was observed even in MNC from advanced lung cancer patients. These observations suggest the potential activity of humanized anti-GM2 mAb (KM8969), as well as chimeric KM966, in biological therapy for lung cancer patients.
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therapeutic efficacy of mouse human chimeric anti Ganglioside GM2 monoclonal antibody against multiple organ micrometastases of human lung cancer in nk cell depleted scid mice
International Journal of Cancer, 1998Co-Authors: Masaki Hanibuchi, Yasuhiko Nishioka, Seiji Yano, Hiroaki Yanagawa, Tetsuya Kawano, Saburo SoneAbstract:The development of distant metastases to multiple organs is a critical problem in the treatment of human lung cancer. In this study, we evaluated the therapeutic efficacy of a mouse-human chimeric anti-Ganglioside GM2 (GM2) monoclonal antibody (MAb), KM966 against metastasis formation of GM2-positive human lung cancer cells inoculated intravenously (i.v.) into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice. GM2-positive human small cell lung cancer (SCLC), SBC-3 cells (1 × 106), injected through a tail vein into NK cell-depleted SCID mice, formed large number of metastatic colonies in the liver, kidneys and lymph nodes by 42 days after inoculation (day 42). KM966, but not control MAb, given on days 2 and 7, almost completely inhibited metastasis formation of SBC-3 cells in the liver, kidneys and lymph nodes in a dose-dependent fashion. Moreover, treatment with KM966 at advanced stages of metastasis (even from day 28) significantly suppressed multiple organ metastases of SBC-3 cells. The anti-metastatic effect of KM966 in vivo was mainly due to an antibody-dependent cell-mediated cytotoxicity (ADCC) reaction mediated by macrophages of the SCID mice. Our findings suggest that the mouse-human chimeric anti-GM2 MAb, KM966 may be useful for eradicating multiple organ micrometastases of lung cancer in humans.Int. J. Cancer 78:480–485, 1998. © 1998 Wiley-Liss, Inc.
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anti Ganglioside GM2 monoclonal antibody dependent killing of human lung cancer cells by lymphocytes and monocytes
Japanese Journal of Cancer Research, 1996Co-Authors: Masaki Hanibuchi, Yasuhiko Nishioka, Seiji Yano, Hiroaki Yanagawa, Saburo SoneAbstract:Ganglioside GM2 (GM2) frequently appears on the cell surface of human cancers of neuroendocrine origin. A mouse-human chimeric monoclonal antibody (mAb), KM966, against GM2 was previously found to promote the lysis of various cancer cells by human blood mononuclear cells (MNC). In this study, we analyzed the effector cells responsible for the chimeric mAb-dependent cell-mediated cytotoxicity (ADCC) against small cell lung cancer (SCLC) cells and examined the enhancing effect of various cytokines on the ADCC activity. The ADCC activity was assessed by 4-h 51Cr release assay. Highly purified lymphocytes (> 99%) and monocytes (> 90%) were separated by centrifugal elutriation from peripheral blood MNC of the same healthy donor. KM966 induced lysis of SCLC cells mediated by both lymphocytes and monocytes to similar extents, in a dose-dependent manner. Pretreatment of lymphocytes with various cytokines [interleukin (IL)-2, IL-12 and interferon-gamma] and that of monocytes with macrophage-colony-stimulating factor significantly augmented the killer activity against SCLC cells in the presence of KM966 mAb. KM966 was also effective for the lysis of non-small cell lung cancer cells in direct proportion to the GM2 expression levels. These findings suggest that combined treatment of KM966 mAb with cytokines may be therapeutically useful for in vivo killing of lung cancer cells expressing GM2 through the ADCC reaction.
Nobuo Hanai - One of the best experts on this subject based on the ideXlab platform.
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a humanized anti Ganglioside GM2 antibody biw 8962 exhibits adcc cdc activity against multiple myeloma cells and potent anti tumor activity in mouse xenograft models
Blood, 2008Co-Authors: Toshihiko Ishii, Asher A Chanankhan, Jazur Jafferjee, Noreen Ersing, Takeshi Takahashi, Masato Mizutani, Yukimasa Shiotsu, Nobuo HanaiAbstract:BIW-8962 is a humanized anti-Ganglioside GM2 (GM2) monoclonal antibody, produced by Poteligent technology to enhance ADCC activity. GM2 is expressed on many cancer cells including multiple myeloma (MM), small cell lung cancer and glioma cells. In this study, we evaluated the anti-myeloma activity of BIW-8962 in preclinical myeloma models both in vitro and in vivo. Expression of GM2 was analyzed in 15 human MM cell lines by FCM. Eleven out of 15 MM cell lines had positive surface expression of GM2. GM2 as a potential target was then verified in primary MM samples obtained from patients. Eleven out of 15 samples were positive for GM2. We then used two GM2 positive MM cell lines (U266B1 and KMS-11) and evaluated ADCC and CDC activity of BIW-8962 in vitro. BIW-8962 exhibited a potent ADCC and less potent CDC activity. In vivo anti-tumor activity of BIW-8962 was then examined using the standard subcutaneous xenograft model; KMS-11 was inoculated in the flank of SCID mice. BIW-8962 (intravenously administered biweekly for 3 weeks) exhibited a potent anti-tumor activity from as low a dose level as 0.1 mg/kg. Furthermore, in a more clinically relevant model, in which OPM-2/GFP (GM2 positive MM cell line) cells were intravenously inoculated into SCID mice with preferentially tumor growth within the bone marrow microenvironment, BIW-8962 (intravenously administered biweekly for 4 weeks, 10 mg/kg) suppressed OPM-2/GFP cell growth and serum M protein elevation, demonstrating in vivo anti-myeloma effect of BIW-8962. Our preclinical investigations rationalize clinical evaluation of BIW-8962 in patients with MM. Currently BIW-8962 is being investigated in a Phase 1 study in patients with multiple myeloma.
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dissection and optimization of immune effector functions of humanized anti Ganglioside GM2 monoclonal antibody
Molecular Immunology, 2000Co-Authors: Kazuyasu Nakamura, Kenya Shitara, Yuko Tanaka, Ikuko Fujino, Noriaki Hirayama, Nobuo HanaiAbstract:A mouse/human chimeric monoclonal antibody (MAb) KM966, specific for the cell-surface tumor antigen Ganglioside GM2, was humanized by the complementarity determining regions (CDRs) grafting method. Not only the amino acid residues in the CDRs but also several in the framework regions (FRs) were changed from the human to the murine residues. A humanized variant, huKM796H/Lm-28, containing eight and five amino acid alterations in variable light (VL) and variable heavy (VH) FRs, respectively, showed a 9-fold reduction in complement-dependent cytotoxicity (CDC) compared to the chimeric KM966, despite tight antigen binding and potent antibody-dependent cellular cytotoxicity (ADCC). Several additional variants were subsequently constructed to improve the CDC of the antibody. One of the variants, designated KM8969, which differs by three amino acids, exhibited a CDC within 3-fold of the chimeric KM966. In addition, humanized KM8969 bound GM2 antigen 1.25-fold more tightly than the chimeric KM966 and showed 5-fold higher ADCC than the chimeric KM966. These results clearly show that the humanized KM8969, having the optimized immune effector functions and theoretically minimal immunogenicity, is an ideal candidate to test the effectiveness of anti-GM2 MAb in human cancer therapy. Taken together, the results obtained here indicate that the ADCC and CDC of an antibody can be dissected independently via engineering of the antibody variable region.
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effect of a chimeric anti Ganglioside GM2 antibody on Ganglioside GM2 expressing human solid tumors in vivo
International Journal of Cancer, 1999Co-Authors: Hisao Fukumoto, Kazuto Nishio, So Ohta, Nobuo Hanai, Kazuya Fukuoka, Yuichiro Ohe, Kazumasa Sugihara, Tetsuro Kodama, Nagahiro SaijoAbstract:Ganglioside GM2 is expressed on the surface of neuroblastoma and glioblastoma cells, and may also be detected on lung cancer cells. We reported previously that anti-Ganglioside GM2 antibody exhibited strong in vitro anti-tumor activity against adriamycin-resistant cancer cells, which overexpressed Ganglioside GM2. In the present study, we examined the in vivo anti-tumor effect of the chimeric anti-Ganglioside GM2 antibody, KM966, against human lung and breast carcinoma cells, SBC-3 and MCF-7, and respective adriamycinresistant clones, SBC-3/ADM and AdrR MCF-7 in BALB/c nu/nu mice. Ratios of tumor volume (T/C) between KM966treated group and control group were 0.01 for SBC-3, 0.00 for SBC-3/ADM, 0.85 for MCF-7 and 0.34 for AdrR MCF-7 cells, respectively. Nude mice, which were pretreated with antiasialo GM1 antibody to remove natural killer cells, were transplanted with 4 3 10 7 of SBC-3 and SBC-3/ADM subcutaneously. Seven days later, when tumors had grown to a diameter of over 8 mm, mice began to receive intravenous treatment of 120 mg/mouse KM966 daily. Fourteen daily treatments induced regression to less than 4-mm diameter in 4/5 SBC-3 tumors and 5/5 of SBC-3/ADM tumors. All SBC-3/ ADM tumors disappeared completely, suggesting that KM966 exerts a strong in vivo anti-tumor effect on Ganglioside GM2-expressing cancer cells. In KM966-treated mice, the surface of the tumor cells stained positive with anti-human IgG. In addition, numerous leukocytes had infiltrated into the tumor mass. Antibody-dependent cell-mediated cytotoxicity (ADCC) of KM966 against tumor cells was examined in vitro by 51 Cr-release assay and revealed that KM966 induces ADCC activity against Ganglioside GM2-expressing tumors. Our results suggest that immunotherapy using KM966 may be useful for the treatment of Ganglioside GM2-expressing solid tumors. Int. J. Cancer 82:759‐764, 1999. r 1999 Wiley-Liss, Inc.
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reversal of adriamycin resistance with chimeric anti Ganglioside GM2 antibody
International Journal of Cancer, 1996Co-Authors: Hisao Fukumoto, Kazuto Nishio, So Ohta, Nobuo Hanai, Nagahiro SaijoAbstract:Ganglioside GM2 is one of the major cell-surface Gangliosides expressed in human tumors. We earlier established a mouse/human IgGI chimeric anti-GM2 antibody, KM966, which displayed anti-tumor activity in human tumor cells both in vitro and in vivo. In this study, we have screened for changes in Ganglioside expressions in several drug-resistant human cancer cell lines to examine the modulation of drug resistance by immunotherapy with anti-Ganglioside antibodies. Increased GM2 expression, detected by flow cytometry and thin-layer chromatography, was observed in the SBC-3/ADM and AdrR MCF7 adriamycin-resistant cell lines, in contrast with their parental lines. In other related Gangliosides, Ganglioside GD2 levels in AdrR MCF7 were higher than those in MCF7 cells. We confirmed increased N-acetylgalactosaminyltransferase mRNA in adriamycin-resistant cell lines, as compared with the parental cells, by Northern-blot analysis. Moreover, to investigate the possibility of exploiting the anti-tumor activity of KM966 in order to overcome resistance to adriamycin, we investigated the antibody-dependent cell-mediated cytotoxity of human peripheral mononuclear blood cells and the complement-dependent cytotoxity of human serum with KM966 against SBC-3, SBC-3/ADM, MCF7 and AdrR MCF7. Significantly higher killing via KM966 was observed in SBC-3/ADM and AdrR MCF7 cells as compared with the parental cells. This suggests that passive immunotherapy using KM966 against human adriamycin-resistant cancer may be useful for overcoming resistance to adriamycin. © 1996 Wiley-Liss, Inc.
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chimeric anti Ganglioside GM2 antibody with antitumor activity
Cancer Research, 1994Co-Authors: Kazuyasu Nakamura, Masamichi Koike, Kenya Shitara, Yoshihisa Kuwana, Kazumi Kiuragi, Shinobu Igarashi, Mamoru Hasegawa, Nobuo HanaiAbstract:Abstract Ganglioside GM2, which is one of the major Gangliosides expressed on the cell surface of human tumors of neuroectodermal origin, has been focused on as a target molecule for passive immunotherapy. GM2 is thought to be one of the T-cell-independent antigens and to elicit only IgM antibody responses in rodents and humans. We had previously established two murine anti-GM2 monoclonal antibodies with high specificity and strong binding activity, KM696 and KM697, both of which are of the IgM class. Variable heavy and light chain complementary DNAs of these two murine monoclonal antibodies were cloned and used in the construction of mouse/human IgG1 chimeric antibodies, KM966 and KM967, respectively, in this study. One of the chimeric antibodies, KM966, retained strong and specific reactivity with GM2 and showed the similarity of the binding activity with tumor cell lines to that of the original murine monoclonal antibody. Indirect immunofluorescence staining of tumor cell lines with the chimeric KM966 revealed that the antigen was expressed in substantial amounts on pulmonary tumor cells and leukemia cells as well as neuroectodermal origin tumor cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, respectively, chimeric KM966 was fully effective in killing GM2-expressing tumor cells. In addition, i.v. injection of chimeric KM966 markedly suppressed the establishment of human tumor xenografts in nude mice. Taken together, chimeric KM966 is the first antibody of the human IgG class to Ganglioside GM2 and has strong antitumor activity both in vitro and in vivo. It is likely that chimeric KM966 will be a useful agent for passive immunotherapy of human cancer.
Alan Spatz - One of the best experts on this subject based on the ideXlab platform.
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randomized phase iii trial comparing postoperative adjuvant Ganglioside GM2 klh qs 21 vaccination versus observation in stage ii t3 t4n0m0 melanoma final results of study eortc 18961
Journal of Clinical Oncology, 2010Co-Authors: Alexander M. M. Eggermont, Philippa Corrie, Poulam M. Patel, Alessandro Testori, Stefan Suciu, Piotr Rutkowski, Steinar Aamdal, J.r. Marsden, Paolo Antonio Ascierto, Alan SpatzAbstract:8505 Background: In the largest adjuvant phase III study in stage II melanoma to date, EORTC 18961 assessed the efficacy and toxicity of Ganglioside GM2-KLH/QS-21 vaccination versus observation (OBS). Methods: Patients (Pts) were observed or received vaccine sc once weekly weeks 1-4, every 3 months from week 12 for first 2 years and every 6 months during third year (total of 14 vaccinations). Stratification factors at randomization were: Breslow thickness, ulceration, being staged yes vs no by sentinel node or elective lymph node dissection, sex and institution. Relapse-free survival (RFS) was the prespecified primary endpoint; distant metastasis-free survival (DMFS) and overall survival (OS) were secondary endpoints. An intent-to-treat analysis was performed. Results: Between March 2002 and December 2005, 1,314 pts entered the trial. Patient demographics showed excellent balance for prognostic factors. The second interim analysis was performed when 267 RFS events were reported. For the primary endpoint, ...
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eortc 18961 post operative adjuvant Ganglioside GM2 klh21 vaccination treatment vs observation in stage ii t3 t4n0m0 melanoma 2nd interim analysis led to an early disclosure of the results
Journal of Clinical Oncology, 2008Co-Authors: Alexander M. M. Eggermont, Philippa Corrie, Poulam M. Patel, Alessandro Testori, Stefan Suciu, Steinar Aamdal, J.r. Marsden, Paolo Antonio Ascierto, W Ruka, Alan SpatzAbstract:9004 Background: EORTC 18961 assessed in the largest adjuvant phase III trial to date in stage II melanoma the efficacy and toxicity of Ganglioside GM2-KLH21 Vaccination (VAC) Treatment vs Observat...
