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Jin-ichi Inokuchi - One of the best experts on this subject based on the ideXlab platform.
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altered expression of Ganglioside GM3 molecular species and a potential regulatory role during myoblast differentiation
Journal of Biological Chemistry, 2017Co-Authors: Lucas Veillon, Alessandro Prinetti, Sandro Sonnino, Maria Grazia Ciampa, Laura Mauri, Chihiro Sato, Ken Kitajima, Jin-ichi InokuchiAbstract:Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of Gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given Ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into Ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of Ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of Gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process.
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identification of Ganglioside GM3 molecular species in human serum associated with risk factors of metabolic syndrome
PLOS ONE, 2015Co-Authors: Lucas Veillon, Akemi Suzuki, Wakana Matsuyama, Mika Nagasaki, Yutaka Yatomi, Jin-ichi InokuchiAbstract:Serum GM3 molecular species were quantified in 125 Japanese residents using tandem mass spectrometry multiple reaction monitoring. Individuals were categorized by the presence or absence of metabolic disease risk factors including visceral fat accumulation, hyperglycemia and dyslipidemia. A total of 23 GM3 molecular species were measured, of these, eight were found to be significantly elevated in individuals with visceral fat accumulation and metabolic disease, defined as the presence of hyperglycemia and dyslipidemia. All of the GM3 molecular species were composed of the sphingoid base sphingosine (d18:1 (Δ4)) and, interestingly, six of the eight elevated GM3 molecular species contained a hydroxylated ceramide moiety. The hydroxylated GM3 species were, in order of decreasing abundance, d18:1-h24:0 ≈ d18:1-h24:1 > d18:1-h22:0 » d18:1-h20:0 > d18:1-h21:0 > d18:1-h18:1. Univariate and multiple linear regression analyses were conducted using a number of clinical health variables associated with obesity, type 2 diabetes, metabolic disease, atherosclerosis and hypertension. GM3(d18:1-h24:1) was identified as the best candidate for metabolic screening, proving to be significantly correlated with intima-media thickness, used for the detection of atherosclerotic disease in humans, and a number of metabolic disease risk factors including autotaxin, LDL-c and homeostatic model assessment insulin resistance (HOMA-IR).
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Running title. Sialylation and Sulfation of LacCer in Malignancy
2014Co-Authors: Satoshi Uemura, Yasuyuki Igarashi, Kazuya Kabayama, Mariko Noguchi, Jin-ichi InokuchiAbstract:To investigate the significance of sialylation and sulfation of lactosylceramide in transformed cells, we established Ganglioside GM3- and lactosylsulfatide (SM3)-reconstituted cells by transfecting cDNAs of GM3 synthase and cerebroside sulfotransferase into the J5 subclone of 3LL Lewis lung carcinoma cells. The J5 clone was selected for the transfection of these genes, since it lacks GM3 and SM3 but accumulates lactosylceramide. The anchorage-dependent growth of both GM3- and SM3-reconstituted cells was similar. However, anchorage-independent growth, as measured by colony forming ability in soft agar, of the SM3-reconstituted cells was almost completely lost, which supports our previous observatio
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dissociation of the insulin receptor and caveolin 1 complex by Ganglioside GM3 in the state of insulin resistance
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Kazuya Kabayama, Takashige Sato, Yasuyuki Igarashi, Alessandro Prinetti, Sandro Sonnino, Kumiko Saito, Nicoletta Loberto, Masataka Kinjo, Jin-ichi InokuchiAbstract:Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNFα-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the Ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR–Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR β-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR–Cav1 complex by the interactions of IR with GM3 in microdomains.
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Dissociation of the insulin receptor and caveolin-1 complex by Ganglioside GM3 in the state of insulin resistance.
Proceedings of the National Academy of Sciences, 2007Co-Authors: Kazuya Kabayama, Takashige Sato, Yasuyuki Igarashi, Alessandro Prinetti, Sandro Sonnino, Kumiko Saito, Nicoletta Loberto, Masataka Kinjo, Jin-ichi InokuchiAbstract:Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNFalpha-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the Ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR-Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR beta-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR-Cav1 complex by the interactions of IR with GM3 in microdomains.
Amy S. Paller - One of the best experts on this subject based on the ideXlab platform.
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Ganglioside GM3 mediates glucose induced suppression of igf 1 receptor rac1 activation to inhibit keratinocyte motility
Journal of Investigative Dermatology, 2017Co-Authors: Duncan Hieu M Dam, Xiaoqi Wang, Sarah L Sheu, Mahima Vijay, Desmond Shipp, Luke Miller, Amy S. PallerAbstract:Activation of insulin-like growth factor-1 (IGF-1) receptor (IGF1R) signaling induces keratinocyte migration, but little is known about its regulation, including in diabetic wounds. GM3, a lipid raft Ganglioside synthesized by GM3 synthase (GM3S), regulates receptor signaling. In diabetic mice, knockout or topically applied nanoconstruct-mediated knockdown of GM3S promotes wound edge IGF1R phosphorylation and re-epithelialization. Through modulating GM3 expression, we explored the role of GM3 in regulating human keratinocyte IGF1R signaling. Increases in GM3 and GM3S expression, including by exposure to high glucose, inhibit keratinocyte migration and IGF-1–induced chemotaxis in association with inhibition of IGF1R phosphorylation, suppression of Rac1 signaling, and activation of RhoA signaling. In contrast, GM3 depletion accelerates cell migration; increases cell velocity, displacement, and persistence; and activates IGF1R-Rac1 signaling. These data implicate GM3 in mediating glucose-induced suppression of IGF1R-Rac1 signaling. Furthermore, our findings provide evidence of a pivotal role for GM3-induced insulin resistance in impairing keratinocyte migration and reinforce the previously published studies in diabetic mice supporting GM3-depleting strategies as an approach for accelerating the healing of human diabetic wounds.
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Ganglioside GM3 synthase depletion reverses neuropathic pain and small fiber neuropathy in diet-induced diabetic mice
Molecular Pain, 2016Co-Authors: Daniela Menichella, Nirupa D. Jayaraj, Heather M. Wilson, Dongjun Ren, Kelsey Flood, Xiaoqi Wang, Andrew Shum, Richard J. Miller, Amy S. PallerAbstract:BackgroundSmall fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that Ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown.PurposeDetermine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal.ResultsWe demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro.ConclusionsThese studies establish Ganglioside GM3 as a new candidate responsible for neuropathic pain an...
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sirna based spherical nucleic acids reverse impaired wound healing in diabetic mice by Ganglioside GM3 synthase knockdown
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Pratik S Randeria, Heather M. Wilson, Xiaoqi Wang, Mark A. Seeger, Desmond Shipp, Chad A Mirkin, Amy S. PallerAbstract:Spherical nucleic acid (SNA) gold nanoparticle conjugates (13-nm-diameter gold cores functionalized with densely packed and highly oriented nucleic acids) dispersed in Aquaphor have been shown to penetrate the epidermal barrier of both intact mouse and human skin, enter keratinocytes, and efficiently down-regulate gene targets. Ganglioside-monosialic acid 3 synthase (GM3S) is a known target that is overexpressed in diabetic mice and responsible for causing insulin resistance and impeding wound healing. GM3S SNAs increase keratinocyte migration and proliferation as well as insulin and insulin-like growth factor-1 (IGF1) receptor activation under both normo- and hyperglycemic conditions. The topical application of GM3S SNAs (50 nM) to splinted 6-mm-diameter full-thickness wounds in diet-induced obese diabetic mice decreases local GM3S expression by >80% at the wound edge through an siRNA pathway and fully heals wounds clinically and histologically within 12 d, whereas control-treated wounds are only 50% closed. Granulation tissue area, vascularity, and IGF1 and EGF receptor phosphorylation are increased in GM3S SNA-treated wounds. These data capitalize on the unique ability of SNAs to naturally penetrate the skin and enter keratinocytes without the need for transfection agents. Moreover, the data further validate GM3 as a mediator of the delayed wound healing in type 2 diabetes and support regional GM3 depletion as a promising therapeutic direction.
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Ganglioside GM3 Depletion Reverses Impaired Wound Healing in Diabetic Mice by Activating IGF-1 and Insulin Receptors
Journal of Investigative Dermatology, 2014Co-Authors: Xiaoqi Wang, Heather M. Wilson, Sarah Lee, Mark A. Seeger, Hristo Iordanov, Nandita Gatla, Adam Whittington, Daniel Q. Bach, Amy S. PallerAbstract:Ganglioside GM3 mediates adipocyte insulin resistance, but the role of GM3 in diabetic wound healing, a major cause of morbidity, is unclear. The purpose of this study was to determine whether GM3 depletion promotes diabetic wound healing and directly activates keratinocyte (KC) insulin pathway signaling. GM3 synthase (GM3S) expression is increased in human diabetic foot skin, ob/ob and diet-induced obese diabetic mouse skin, and in mouse KCs exposed to increased glucose. GM3S knockout in diet-induced obese mice prevents the diabetic wound-healing defect. KC proliferation, migration, and activation of insulin receptor (IR) and insulin growth factor-1 receptor (IGF-1R) are suppressed by excess glucose in wild-type cells, but increased in GM3S -/- KCs with supplemental glucose. Co-immunoprecipitation of IR, IR substrate 1 (IRS-1), and IGF-1R, and increased IRS-1 and Akt phosphorylation accompany receptor activation. GM3 supplementation or inhibition of IGF-1R or PI3K reverses the increased migration of GM3S -/- KCs, whereas IR knockdown only partially suppresses migration.
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Cutaneous dyspigmentation in patients with Ganglioside GM3 synthase deficiency.
American Journal of Medical Genetics Part A, 2013Co-Authors: Heng Wang, Alicia Bright, Baozhong Xin, J.r. Bockoven, Amy S. PallerAbstract:Ganglioside GM3 synthase deficiency is a rare autosomal recessive metabolic disorder characterized by infantile onset of severe irritability and epilepsy, failure to thrive, developmental stagnation, and cortical blindness. Because of the lack of easily recognizable dysmorphism and specific neurologic manifestations, identification of patients with this condition is extremely challenging. Here we report on previously undescribed pigmentary abnormalities in 20 of 38 patients with GM3 synthase deficiency. All 20 of the patients showed freckle-like hyperpigmented macules, ranging in size from 2 to 5 mm in diameter and usually found bilaterally on the extremities, especially the dorsal aspects of the hands and feet. Seven of these patients also had depigmented macules and patches, especially on the face and extremities. These cutaneous changes were asymptomatic, and were not associated with the severity or particular phenotype of the neurologic disease. They became visible only after the first years of life with an increased incidence with advancing age. These distinct pigmentary features are not identified in 54 normal siblings, and may provide a useful clue in identifying patients with Ganglioside metabolic disorders. © 2013 Wiley Periodicals, Inc.
Frederic Ronzon - One of the best experts on this subject based on the ideXlab platform.
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effect of the β propiolactone treatment on the adsorption and fusion of influenza a brisbane 59 2007 and a new caledonia 20 1999 virus h1n1 on a dimyristoylphosphatidylcholine Ganglioside GM3 mixed phospholipids monolayer at the air water interface
Langmuir, 2011Co-Authors: Bernard Desbat, Eloise Lancelot, Tino Krell, Marieclaire Nicolai, Fred Vogel, Michel Chevalier, Frederic RonzonAbstract:The production protocol of many whole cell/virion vaccines involves an inactivation step with β-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL alkylates nucleotide bases, but its interaction with proteins has not been studied in depth. In the present study we use ellipsometry to analyze the influence of BPL treatment of two H1N1 influenza strains, A/Brisbane/59/2007 and A/New Caledonia/20/1999, which are used for vaccine production on an industrial scale. Analyses were conducted using a mixed lipid monolayer containing Ganglioside GM3, which functions as the viral receptor. Our results show that BPL treatment of both strains reduces viral affinity for the mixed monolayer and also diminishes the capacity of viral domains to self-assemble. In another series of experiments, the pH of the subphase was reduced from 7.4 to 5 to provoke the pH-induced conformational change of hemagglutinin, which occurs following endocytosi...
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effect of the β propiolactone treatment on the adsorption and fusion of influenza a brisbane 59 2007 and a new caledonia 20 1999 virus h1n1 on a dimyristoylphosphatidylcholine Ganglioside GM3 mixed phospholipids monolayer at the air water interface
Langmuir, 2011Co-Authors: Bernard Desbat, Eloise Lancelot, Tino Krell, Marieclaire Nicolai, Fred Vogel, Michel Chevalier, Frederic RonzonAbstract:The production protocol of many whole cell/virion vaccines involves an inactivation step with β-propiolactone (BPL). Despite the widespread use of BPL, its mechanism of action is poorly understood. Earlier work demonstrated that BPL alkylates nucleotide bases, but its interaction with proteins has not been studied in depth. In the present study we use ellipsometry to analyze the influence of BPL treatment of two H1N1 influenza strains, A/Brisbane/59/2007 and A/New Caledonia/20/1999, which are used for vaccine production on an industrial scale. Analyses were conducted using a mixed lipid monolayer containing Ganglioside GM3, which functions as the viral receptor. Our results show that BPL treatment of both strains reduces viral affinity for the mixed monolayer and also diminishes the capacity of viral domains to self-assemble. In another series of experiments, the pH of the subphase was reduced from 7.4 to 5 to provoke the pH-induced conformational change of hemagglutinin, which occurs following endocytosis into the endosome. In the presence of the native virus the pH decrease caused a reduction in domain size, whereas lipid layer thickness and surface pressure were increased. These observations are consistent with a fusion of the viral membrane with the lipid monolayer. Importantly, this fusion was not observed with adsorbed inactivated virus, which indicates that BPL treatment inhibits the first step of virus-membrane fusion. Our data also indicate that BPL chemically modifies hemagglutinin, which mediates the interaction with GM3.
Masami Suzuki - One of the best experts on this subject based on the ideXlab platform.
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recombinant human hexamer dominant igm monoclonal antibody to Ganglioside GM3 for treatment of melanoma
Cancer Research, 2008Co-Authors: Yumiko Azuma, Yuji Ishikawa, Shigeto Kawai, Toshiaki Tsunenari, Hiroyuki Tsunoda, Tomoyuki Igawa, Shinichiro Iida, Masahiko Nanami, Masami Suzuki, Reiko F IrieAbstract:2406 Ganglioside GM3 is the most common Ganglioside found on human melanoma cells. It is expressed at much higher densities and frequencies on melanoma cells than on normal cells. A human B-lymphoblastoid cell line that was established by immortalization of human B-cells by Epstein-Barr virus produces a human IgM antibody to GM3 (L612). L612 binds to GM3 and kills GM3-positive human melanoma cells in the presence of complement. L612 has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (~20%), pentameric IgM (~74%), and other minor IgM molecules. Because hexameric IgMs have been shown to activate complement more effectively than pentameric IgMs, we tested whether hexameric L612 plays a dominant role in the antitumor activity. Chinese hamster ovary (CHO) cells were transfected with L612 heavy (H)- and light (L)-chain genes, with or without the joining (J)-chain gene, that resulted in generation of pentamer dominant- and hexmaner dominant-IgM, respectively. Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was ~80% hexameric, whereas recombinant IgM secreted from CHO cells transfected with joining-chain genes (designated CJ45) was ~90% pentameric. CA19 and CJ45 were compared for their anti-melanoma activities by in vitro and in vivo experiments. Both CA19 and CJ45 IgMs caused complement-dependent cytotoxicity (CDC) against GM3 rich M-1 human and B16F10 mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller as compared to that of CJ45. Neither L612 nor its two recombinant forms (CA19 and CJ45) induced CDC against a GM3-negative A549 lung cancer cells, showing an antigen-specific binding and killing by these antibodies. The in vivo antitumor effects of the recombinant IgMs were evaluated using B16F10 cell line in a nude rat xenograft model. The nude rat’s complement had been shown to be as effective as human complement with respect to induction of CDC by these human IgM antibodies against the mouse melanoma. Intravenous injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing B16F10 xenograft. Compared to vehicle, CA19, CJ45, and L612 prolonged survival by 238%, 162%, and 155%, respectively. These results suggest that a hexamer-dominant human IgM against GM3 may provide a more potent treatment option for GM3-positive melanoma.
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recombinant human hexamer dominant igm monoclonal antibody to Ganglioside GM3 for treatment of melanoma
Clinical Cancer Research, 2007Co-Authors: Yumiko Azuma, Yuji Ishikawa, Shigeto Kawai, Toshiaki Tsunenari, Hiroyuki Tsunoda, Tomoyuki Igawa, Shinichiro Iida, Masahiko Nanami, Masami SuzukiAbstract:Purpose: L612, a human IgM monoclonal antibody produced by an EBV-transformed human B-cell line, binds to Ganglioside GM3 and kills GM3-positive human melanoma cells in the presence of complement. It has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (about 20%), pentameric IgM (about 74%), and other minor IgM molecules. Because hexameric IgM activates complement more effectively than pentameric IgM, we developed and evaluated a hexamer-dominant recombinant IgM for clinical applications. Experimental Design: Chinese hamster ovary (CHO) cells were transfected with heavy- and light-chain genes of L612, with or without the joining-chain gene. Antitumor effects of the recombinant IgM secreted from CHO cells were evaluated in vitro and in vivo . Results: Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was ∼80% hexameric, whereas recombinant IgM from CHO cells transfected with heavy-, light-, and joining-chain genes (designated CJ45) was about 90% pentameric. Both CA19 and CJ45 recombinant IgMs caused complement-dependent cytotoxicity against human and mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller. I.v. injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing a GM3-positive mouse melanoma xenograft. Conclusions: A hexamer-dominant human IgM against GM3 may provide a more potent treatment option for patients with GM3-positive melanoma.
Yumiko Azuma - One of the best experts on this subject based on the ideXlab platform.
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recombinant human hexamer dominant igm monoclonal antibody to Ganglioside GM3 for treatment of melanoma
Cancer Research, 2008Co-Authors: Yumiko Azuma, Yuji Ishikawa, Shigeto Kawai, Toshiaki Tsunenari, Hiroyuki Tsunoda, Tomoyuki Igawa, Shinichiro Iida, Masahiko Nanami, Masami Suzuki, Reiko F IrieAbstract:2406 Ganglioside GM3 is the most common Ganglioside found on human melanoma cells. It is expressed at much higher densities and frequencies on melanoma cells than on normal cells. A human B-lymphoblastoid cell line that was established by immortalization of human B-cells by Epstein-Barr virus produces a human IgM antibody to GM3 (L612). L612 binds to GM3 and kills GM3-positive human melanoma cells in the presence of complement. L612 has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (~20%), pentameric IgM (~74%), and other minor IgM molecules. Because hexameric IgMs have been shown to activate complement more effectively than pentameric IgMs, we tested whether hexameric L612 plays a dominant role in the antitumor activity. Chinese hamster ovary (CHO) cells were transfected with L612 heavy (H)- and light (L)-chain genes, with or without the joining (J)-chain gene, that resulted in generation of pentamer dominant- and hexmaner dominant-IgM, respectively. Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was ~80% hexameric, whereas recombinant IgM secreted from CHO cells transfected with joining-chain genes (designated CJ45) was ~90% pentameric. CA19 and CJ45 were compared for their anti-melanoma activities by in vitro and in vivo experiments. Both CA19 and CJ45 IgMs caused complement-dependent cytotoxicity (CDC) against GM3 rich M-1 human and B16F10 mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller as compared to that of CJ45. Neither L612 nor its two recombinant forms (CA19 and CJ45) induced CDC against a GM3-negative A549 lung cancer cells, showing an antigen-specific binding and killing by these antibodies. The in vivo antitumor effects of the recombinant IgMs were evaluated using B16F10 cell line in a nude rat xenograft model. The nude rat’s complement had been shown to be as effective as human complement with respect to induction of CDC by these human IgM antibodies against the mouse melanoma. Intravenous injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing B16F10 xenograft. Compared to vehicle, CA19, CJ45, and L612 prolonged survival by 238%, 162%, and 155%, respectively. These results suggest that a hexamer-dominant human IgM against GM3 may provide a more potent treatment option for GM3-positive melanoma.
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recombinant human hexamer dominant igm monoclonal antibody to Ganglioside GM3 for treatment of melanoma
Clinical Cancer Research, 2007Co-Authors: Yumiko Azuma, Yuji Ishikawa, Shigeto Kawai, Toshiaki Tsunenari, Hiroyuki Tsunoda, Tomoyuki Igawa, Shinichiro Iida, Masahiko Nanami, Masami SuzukiAbstract:Purpose: L612, a human IgM monoclonal antibody produced by an EBV-transformed human B-cell line, binds to Ganglioside GM3 and kills GM3-positive human melanoma cells in the presence of complement. It has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (about 20%), pentameric IgM (about 74%), and other minor IgM molecules. Because hexameric IgM activates complement more effectively than pentameric IgM, we developed and evaluated a hexamer-dominant recombinant IgM for clinical applications. Experimental Design: Chinese hamster ovary (CHO) cells were transfected with heavy- and light-chain genes of L612, with or without the joining-chain gene. Antitumor effects of the recombinant IgM secreted from CHO cells were evaluated in vitro and in vivo . Results: Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was ∼80% hexameric, whereas recombinant IgM from CHO cells transfected with heavy-, light-, and joining-chain genes (designated CJ45) was about 90% pentameric. Both CA19 and CJ45 recombinant IgMs caused complement-dependent cytotoxicity against human and mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller. I.v. injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing a GM3-positive mouse melanoma xenograft. Conclusions: A hexamer-dominant human IgM against GM3 may provide a more potent treatment option for patients with GM3-positive melanoma.
