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Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors

BACKGROUND: 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...

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Autores principales: Aste-Amézaga, Miguel, Zhang, Ningyan, Lineberger, Janet E., Arnold, Beth A., Toner, Timothy J., Gu, Mingcheng, Huang, Lingyi, Vitelli, Salvatore, Vo, Kim T., Haytko, Peter, Zhao, Jing Zhang, Baleydier, Frederic, L'Heureux, Sarah, Wang, Hongfang, Gordon, Wendy R., Thoryk, Elizabeth, Andrawes, Marie Blanke, Tiyanont, Kittichoat, Stegmaier, Kimberly, Roti, Giovanni, Ross, Kenneth N., Franlin, Laura L., Wang, Hui, Wang, Fubao, Chastain, Michael, Bett, Andrew J., Audoly, Laurent P., Aster, Jon C., Blacklow, Stephen C., Huber, Hans E.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817004/
https://www.ncbi.nlm.nih.gov/pubmed/20161710
http://dx.doi.org/10.1371/journal.pone.0009094
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author Aste-Amézaga, Miguel
Zhang, Ningyan
Lineberger, Janet E.
Arnold, Beth A.
Toner, Timothy J.
Gu, Mingcheng
Huang, Lingyi
Vitelli, Salvatore
Vo, Kim T.
Haytko, Peter
Zhao, Jing Zhang
Baleydier, Frederic
L'Heureux, Sarah
Wang, Hongfang
Gordon, Wendy R.
Thoryk, Elizabeth
Andrawes, Marie Blanke
Tiyanont, Kittichoat
Stegmaier, Kimberly
Roti, Giovanni
Ross, Kenneth N.
Franlin, Laura L.
Wang, Hui
Wang, Fubao
Chastain, Michael
Bett, Andrew J.
Audoly, Laurent P.
Aster, Jon C.
Blacklow, Stephen C.
Huber, Hans E.
author_facet Aste-Amézaga, Miguel
Zhang, Ningyan
Lineberger, Janet E.
Arnold, Beth A.
Toner, Timothy J.
Gu, Mingcheng
Huang, Lingyi
Vitelli, Salvatore
Vo, Kim T.
Haytko, Peter
Zhao, Jing Zhang
Baleydier, Frederic
L'Heureux, Sarah
Wang, Hongfang
Gordon, Wendy R.
Thoryk, Elizabeth
Andrawes, Marie Blanke
Tiyanont, Kittichoat
Stegmaier, Kimberly
Roti, Giovanni
Ross, Kenneth N.
Franlin, Laura L.
Wang, Hui
Wang, Fubao
Chastain, Michael
Bett, Andrew J.
Audoly, Laurent P.
Aster, Jon C.
Blacklow, Stephen C.
Huber, Hans E.
author_sort Aste-Amézaga, Miguel
collection PubMed
description BACKGROUND: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.
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spelling pubmed-28170042010-02-17 Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors Aste-Amézaga, Miguel Zhang, Ningyan Lineberger, Janet E. Arnold, Beth A. Toner, Timothy J. Gu, Mingcheng Huang, Lingyi Vitelli, Salvatore Vo, Kim T. Haytko, Peter Zhao, Jing Zhang Baleydier, Frederic L'Heureux, Sarah Wang, Hongfang Gordon, Wendy R. Thoryk, Elizabeth Andrawes, Marie Blanke Tiyanont, Kittichoat Stegmaier, Kimberly Roti, Giovanni Ross, Kenneth N. Franlin, Laura L. Wang, Hui Wang, Fubao Chastain, Michael Bett, Andrew J. Audoly, Laurent P. Aster, Jon C. Blacklow, Stephen C. Huber, Hans E. PLoS One Research Article BACKGROUND: 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. PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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. Public Library of Science 2010-02-08 /pmc/articles/PMC2817004/ /pubmed/20161710 http://dx.doi.org/10.1371/journal.pone.0009094 Text en Aste-Amezaga et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Aste-Amézaga, Miguel
Zhang, Ningyan
Lineberger, Janet E.
Arnold, Beth A.
Toner, Timothy J.
Gu, Mingcheng
Huang, Lingyi
Vitelli, Salvatore
Vo, Kim T.
Haytko, Peter
Zhao, Jing Zhang
Baleydier, Frederic
L'Heureux, Sarah
Wang, Hongfang
Gordon, Wendy R.
Thoryk, Elizabeth
Andrawes, Marie Blanke
Tiyanont, Kittichoat
Stegmaier, Kimberly
Roti, Giovanni
Ross, Kenneth N.
Franlin, Laura L.
Wang, Hui
Wang, Fubao
Chastain, Michael
Bett, Andrew J.
Audoly, Laurent P.
Aster, Jon C.
Blacklow, Stephen C.
Huber, Hans E.
Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors
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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817004/
https://www.ncbi.nlm.nih.gov/pubmed/20161710
http://dx.doi.org/10.1371/journal.pone.0009094
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