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Inhibiting Individual Notch Receptors Improves Treatment Print

Notch receptors constitute a family of evolutionarily conserved transmembrane proteins that function as conduits for cell communication, regulating cell fate and growth. Aberrant activation of any of the four human Notch receptors has been linked to diseases, particularly cancer, making the Notch pathway a compelling target for new drug studies. A research team from Bay Area companies Genentech and Exelixis has synthesized highly specialized antibody inhibitors that specifically inhibit only Notch1 or Notch2, acting through a potent and novel mechanism of Notch inhibition. These antibodies, characterized at ALS Beamline 5.0.2, could provide the tools to therapeutically target individual Notch receptors, avoiding the intestinal toxicity ascribed to treatment with more than one, or a pan-Notch, inhibitor.

Two Are Not Better Than One

A hurdle to the therapeutic application of pan-Notch inhibitors, such as GSIs, in the treatment of Notch defect disorders has been toxicity in the intestinal crypt. Genetic disruption of Notch1 or Notch2 suggests that when combined, the two receptors together control a classic Notch cell fate switch that relies on proper Notch signaling to maintain a healthy balance between secretory and absorptive cell fates. To test the success of the new Notch-specific inhibitors, mice were treated with α-NRR1, α-NRR2, or both. Mice treated with α-NRR1 or α-NRR2 maintained weight, but mice treated with α-NRR1 plus α-NRR2 rapidly lost weight.

Consistent with previously reported effects of GSIs, intestinal crypts from mice treated with the antibody combination showed a preponderance of secretory cells at the expense of absorptive cells, a state referred to as severe goblet cell metaplasia. In contrast, individual dosing with α-NRR1 or α-NRR2 generated few or no changes to intestinal morphology. This finding shows advanced therapeutic potential for treating certain cancers and other disorders caused by defects in individual Notch receptors because using a Notch-specific inhibitor circumvents intestinal toxicity, while still preventing unwanted activation.

Ligand binding triggers a conformational change in the Notch receptor’s negative regulatory region (NRR), enabling receptor activation via protease cleavage at a site that is otherwise protected within the quiescent NRR. This cleavage prompts a subsequent cleavage, catalyzed by the gamma-secretase complex, liberating the active Notch signaling fragment within the cell. Unfortunately, the application of gamma-secretase inhibitors (GSIs) appears to have limited therapeutic potential because they cause intestinal toxicity, attributed to dual inhibition of Notch1 and Notch2.

Researchers from Genentech and Exelixis used phage display to generate antibodies that specifically block only Notch1 or Notch2. By targeting the NRRs of that receptor specifically through a novel inhibitory mechanism, researchers hoped Notch could be stabilized in the “off” conformation, inhibiting signaling.


Side (top) and open-book (bottom) views of the high-resolution structure of the anti-NRR1 antibody (antibody binding fragment) binding across the face of the Notch regulatory region (NRR). Top: The heavy (yellow) and light (green) chains of the antibody closely contact amino acids across the LNR-A, LNR-B and HD domains of the NRR. Bottom: Notch1 signaling requires that the LNR-A and LNR-B domains peel away from the HD domain below (right). Antibody binding is expected to prohibit such a conformational change and thereby lock the NRR in a quiescent conformation.

This approach yielded α-NRR1 and α-NRR2: clinically relevant, fully human antibodies that potently inhibit Notch1 or Notch2 signaling by locking the NRR in the quiescent state. Receptor-specific inhibition was revealed not only in protein-binding and cell-based signaling assays in vitro, but also in rodent models of genetically defined Notch1- or Notch2-dependent processes. Furthermore, a high-resolution co-crystal structure of α-NRR1 in complex with NRR1 clearly illustrates the molecular basis of receptor antagonism: an antibody clamp that stabilizes the autoinhibited NRR1 and interferes with the conformational changes required for receptor activation.


Top: The growth rate in vivo (mouse xenograft model) of human HBP-ALL, a T cell leukemia line that expresses mutationally activated Notch1 and directly depends on Notch1 signaling for growth. Blocking Notch1 signaling using anti-NRR1 induces dramatic tumor regression, even in large tumors (approximately 2000 mm3). Bottom: Anti-NRR1 inhibits proper growth of blood vessels, resulting in a “hyper-vascularized” network of blood vessels in mouse neonate retinas. Such a dense and tangled network fails to efficiently conduct blood flow, an effect that can be used to inhibit tumor growth indirectly by choking off nutrient supplies.

A series of experiments studying cancer-cell growth and geared toward validating the therapeutic potential of α-NRR1 revealed that it holds promise as a “double whammy” weapon against certain tumor cells. First, α-NRR1 can dramatically decrease growth in cancer cells that depend directly on Notch1 signaling, which was illustrated in vitro and in preclinical xenograft models. In particular, α-NRR1 holds promise for treating T-cell acute lymphoblastic leukemia, a cancer for which over half of patients carry activating mutations in Notch1.

Second, since Notch1 inhibition disrupts proper tumor angiogenesis, α-NRR1 may provide a means to indirectly block growth of a wide variety of tumor types by inhibiting blood circulation to the tumor. In preclinical models of tumor growth using cells that do not directly depend on Notch signaling for growth or survival, researchers found that Notch1 antagonism dramatically inhibited tumor growth. By “flipping” a Notch-dependent cell fate switch in endothelial cells, tumor vasculature became compromised. Notch1 inhibition generated a tangled web of tumor vasculature rather than an organized one, blocking efficient blood circulation.


Top: Targeting either Notch1 or Notch2 had no effect on weight in treated mice, a stark contrast with the rapid weight loss caused by inhibiting both receptors simultaneously, a toxic effect previously observed in animal models and patients treated with gamma-secretase inhibitors (which block signaling from all Notch receptors). Bottom: This intestinal toxicity reflected a Notch1- and Notch2-controlled cell fate switch in microscopic images of the intestinal crypt. Treatment with either anti-NRR1 or anti-NRR2 had little or no effect, whereas treatment with both inhibitors caused goblet cell metaplasia, a shift away from the proper balance of absorptive and secretory cells to a dominance of secretory cells (revealed by Alcian Blue staining).

These receptor-specific inhibitors represent a clear breakthrough over existing pan-Notch inhibitors such as GSIs in reducing or avoiding the goblet cell metaplasia (a preponderance of secretory cells at the expense of absorptive cells) that is a hallmark of a general Notch blockade. These first studies of α-NRR1 and α-NRR2 show that individual dosing with α-NRR1 or α-NRR2 generated few or no changes to intestinal morphology and so make a compelling case to more deeply evaluate adding these Notch receptor-specific antibodies to the arsenal of cancer therapeutics. α-NRR1 and α-NRR2 may also find other uses in affecting Notch signaling, most notably with immune-based diseases and regenerative medicine.



Research conducted by C. Cain-Hom, C.A. Callahan, Y. Chen, L. Choy, G.J. Dow, D. Finkle, T.J. Hagenbeek, S.G. Hymowitz, G.P. de Leon, J. Ridgway, D. Schahin-Reed, A. Shelton, C.W. Siebel, S. Stawicki, R. Venook, R.J. Watts, X. Wu, Y. Wu, M. Yan, and J. Zha (Genentech) and R. Choy, P. Howard, L. Kadyk, and J. Zhang (Exelixis).

Research funding: Genentech, Inc., and Exelixis, Inc. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: Y. Wu, C. Cain-Hom, L. Choy, T.J. Hagenbeek, G.P. de Leon, Y. Chen, D. Finkle, R. Venook, X. Wu, J. Ridgway, D. Schahin-Reed, G.J. Dow, A. Shelton, S. Stawicki, R.J. Watts, J. Zhang, R. Choy, P. Howard, L. Kadyk, M. Yan, J. Zha, C.A. Callahan, S.G. Hymowitz, and C.W. Siebel, "Therapeutic antibody targeting of individual Notch receptors," Nature 464, 1052 (2009).


This research was featured on the cover of Nature:

Therapeutic Antibody Targeting of Individual Notch Receptors.



ALS Science Highlight #232


ALSNews Vol. 322