Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.
<h4>Background</h4>Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor an...
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2010-02-01
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| author | Miguel Aste-Amézaga Ningyan Zhang Janet E Lineberger Beth A Arnold Timothy J Toner Mingcheng Gu Lingyi Huang Salvatore Vitelli Kim T Vo Peter Haytko Jing Zhang Zhao Frederic Baleydier Sarah L'Heureux Hongfang Wang Wendy R Gordon Elizabeth Thoryk Marie Blanke Andrawes Kittichoat Tiyanont Kimberly Stegmaier Giovanni Roti Kenneth N Ross Laura L Franlin Hui Wang Fubao Wang Michael Chastain Andrew J Bett Laurent P Audoly Jon C Aster Stephen C Blacklow Hans E Huber |
| author_facet | Miguel Aste-Amézaga Ningyan Zhang Janet E Lineberger Beth A Arnold Timothy J Toner Mingcheng Gu Lingyi Huang Salvatore Vitelli Kim T Vo Peter Haytko Jing Zhang Zhao Frederic Baleydier Sarah L'Heureux Hongfang Wang Wendy R Gordon Elizabeth Thoryk Marie Blanke Andrawes Kittichoat Tiyanont Kimberly Stegmaier Giovanni Roti Kenneth N Ross Laura L Franlin Hui Wang Fubao Wang Michael Chastain Andrew J Bett Laurent P Audoly Jon C Aster Stephen C Blacklow Hans E Huber |
| author_sort | Miguel Aste-Amézaga |
| collection | DOAJ |
| description | <h4>Background</h4>Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.<h4>Principal findings</h4>Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.<h4>Conclusions/significance</h4>Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation. |
| format | Article |
| id | doaj-art-b4ac98e727c04ff3ad08212b68b47d08 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2010-02-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-b4ac98e727c04ff3ad08212b68b47d082025-08-20T02:31:50ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-02-0152e909410.1371/journal.pone.0009094Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.Miguel Aste-AmézagaNingyan ZhangJanet E LinebergerBeth A ArnoldTimothy J TonerMingcheng GuLingyi HuangSalvatore VitelliKim T VoPeter HaytkoJing Zhang ZhaoFrederic BaleydierSarah L'HeureuxHongfang WangWendy R GordonElizabeth ThorykMarie Blanke AndrawesKittichoat TiyanontKimberly StegmaierGiovanni RotiKenneth N RossLaura L FranlinHui WangFubao WangMichael ChastainAndrew J BettLaurent P AudolyJon C AsterStephen C BlacklowHans E Huber<h4>Background</h4>Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.<h4>Principal findings</h4>Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.<h4>Conclusions/significance</h4>Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0009094&type=printable |
| spellingShingle | Miguel Aste-Amézaga Ningyan Zhang Janet E Lineberger Beth A Arnold Timothy J Toner Mingcheng Gu Lingyi Huang Salvatore Vitelli Kim T Vo Peter Haytko Jing Zhang Zhao Frederic Baleydier Sarah L'Heureux Hongfang Wang Wendy R Gordon Elizabeth Thoryk Marie Blanke Andrawes Kittichoat Tiyanont Kimberly Stegmaier Giovanni Roti Kenneth N Ross Laura L Franlin Hui Wang Fubao Wang Michael Chastain Andrew J Bett Laurent P Audoly Jon C Aster Stephen C Blacklow Hans E Huber Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. PLoS ONE |
| title | Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. |
| title_full | Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. |
| title_fullStr | Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. |
| title_full_unstemmed | Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. |
| title_short | Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. |
| title_sort | characterization of notch1 antibodies that inhibit signaling of both normal and mutated notch1 receptors |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0009094&type=printable |
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