Therapeutic antibody targeting of individual Notch receptors

Abstract

The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.

Notch1‐Snail1‐E‐cadherin pathway in metastatic hepatocellular carcinoma

“…41 Other new agents that targeting Notch pathways have also primarily shown effects in inhibiting tumor growth and reducing angiogenesis. 43 Our results demonstrated that knockdown of Notch1 was able to significantly inhibit tumor metastasis from the liver to the lung in a mouse model (Figs. 2a and 2f) and thereby established the potential for targeting Notch signaling as an approach to inhibit tumor metastasis.…”

“…Gamma-secretase inhibitors (GSIs) as inhibitors of key mediators of Notch signaling 41 inhibition of the Notch transcription complex 42 and the development of antibodies targeting individual Notch receptors 43 have shown great potential as new targeted therapeutic agents. GSIs can suppress tumor growth in cell lines and in xenografts in mice 41 through the inhibition of cell proliferation and induction of apoptosis; thus, some GSIs have progressed into Phase I clinical trials.…”

A Monoclonal Antibody against Human Notch1 Ligand–Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem–like Cells

“…for targeting the Notch-associated pathobiology. Effectiveness of anti-NRR antibodies in targeting oncogenic Notch signaling in T-acute lymphoblastic leukemia (T-ALL) cell lines has already been reported (32,34). Here, we show effectiveness of the EGF repeats 11 to 12 specific mAbs in selectively affecting ligand-dependent Notch function in breast cancer cell lines.…”

“…Individual receptor-specific antibodies against Notch1 and Notch3 NRR domains have been shown to stabilize the receptors in an autoinhibited state and are allosteric in nature (32)(33)(34). However, antibodies specific for the ligand-binding domain of the receptor can serve dual functions of being the potential therapeutic tools while providing insights into the mechanism of ligand binding and subsequent receptor activation.…”

Notch1 controls development of the extravillous trophoblast lineage in the human placenta

“…For manipulation of Notch1 activity in first trimester villous explant cultures, a specific Notch1-blocking antibody (Notch1-IgG1), inhibiting ADAM-mediated cleavage (25), was used (Fig. 2).…”