Characterization of notch1 antibodies that inhibit signaling of both normal and mutated notch1 receptors

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 RotiKenneth 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

Research output: Contribution to journalArticlepeer-review

150 Scopus citations

Abstract

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 EC50 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.

Original languageEnglish (US)
Article numbere9094
JournalPloS one
Volume5
Issue number2
DOIs
StatePublished - Feb 8 2010

Bibliographical note

Funding Information:
MAA, FW, NZ, AJB, JCA, and SCB are co-inventors of a patent application related to the antibodies reported in the study. The patent is entitled: “Generation and Characterization of anti-Notch Monoclonal Antibodies” (Application Serial No 61/199,753, filed on 11/20/2008). MAA, NZ, JEL, BAA, TJT, MG, LH, SV, KTV, PH, JZZ, ET, KTV, LLF, HW, FW, MC, AJB, LPA, and HEH are employees of Merck & Co., Inc. None of the authors from Merck & Co., Inc. received funding from any of the organizations (National Institutes of Health, Leukemia and Lymphoma Society, and the William Lawrence and Blanche Hughes Foundation) listed in the Financial Disclosure.

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