title: Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.
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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Biologics Research, Merck Research Laboratories, West Point, Pennsylvania, United States of America.
authors.forename: Miguel
authors.initials: M
authors.lastname: Aste-Amézaga
authors.forename: Ningyan
authors.initials: N
authors.lastname: Zhang
authors.forename: Janet E
authors.initials: JE
authors.lastname: Lineberger
authors.forename: Beth A
authors.initials: BA
authors.lastname: Arnold
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Toner
authors.forename: Mingcheng
authors.initials: M
authors.lastname: Gu
authors.forename: Lingyi
authors.initials: L
authors.lastname: Huang
authors.forename: Salvatore
authors.initials: S
authors.lastname: Vitelli
authors.forename: Kim T
authors.initials: KT
authors.lastname: Vo
authors.forename: Peter
authors.initials: P
authors.lastname: Haytko
authors.forename: Jing Zhang
authors.initials: JZ
authors.lastname: Zhao
authors.forename: Frederic
authors.initials: F
authors.lastname: Baleydier
authors.forename: Sarah
authors.initials: S
authors.lastname: L'Heureux
authors.forename: Hongfang
authors.initials: H
authors.lastname: Wang
authors.forename: Wendy R
authors.initials: WR
authors.lastname: Gordon
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Thoryk
authors.forename: Marie Blanke
authors.initials: MB
authors.lastname: Andrawes
authors.forename: Kittichoat
authors.initials: K
authors.lastname: Tiyanont
authors.forename: Kimberly
authors.initials: K
authors.lastname: Stegmaier
authors.forename: Giovanni
authors.initials: G
authors.lastname: Roti
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Ross
authors.forename: Laura L
authors.initials: LL
authors.lastname: Franlin
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Fubao
authors.initials: F
authors.lastname: Wang
authors.forename: Michael
authors.initials: M
authors.lastname: Chastain
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bett
authors.forename: Laurent P
authors.initials: LP
authors.lastname: Audoly
authors.forename: Jon C
authors.initials: JC
authors.lastname: Aster
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Blacklow
authors.forename: Hans E
authors.initials: HE
authors.lastname: Huber
mesh_terms: D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000918:Antibody Specificity / Q000276:immunology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D001667:Binding, Competitive; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000072101:Jagged-2 Protein; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051881:Receptor, Notch1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000911:Antibodies, Monoclonal; D036341:Intercellular Signaling Peptides and Proteins; C490504:JAG2 protein, human; D000072101:Jagged-2 Protein; D008024:Ligands; D008565:Membrane Proteins; D051881:Receptor, Notch1