Phage Selection of Bicyclic Peptide Ligands of the Notch1 Receptor

Precise regulation of the Notch signaling pathway is crucial, as increases or deficiencies in signaling are associated with diseases, including a wide range of cancers. Recent studies have demonstrated that monoclonal antibodies that bind and stabilize the structure of the negative regulatory region (NRR) in the extracellular domain of the Notch receptor can inhibit Notch signaling. In this work, we posed the question whether bicyclic peptides, being around 100-fold smaller than antibodies, can also stabilize the NRR and inhibit Notch signaling. Bicyclic peptides that bind the NRR of human Notch1 were isolated from combinatorial libraries by phage display. Affinity maturation yielded ligands with dissociation constant (K-d) values as low as 150nM. The bicyclic peptides increased the melting temperature of the NRR by up to 8 degrees C, thus substantially stabilizing the protein structure, but they did not inhibit Notch signaling in cellular assays. Although Notch signaling could not be inhibited, this work demonstrates that phage-selected bicyclic peptides can stabilize proteins; this capacity of bicyclic peptides may be exploited for modulating the conformation of other disease targets.

Peptide ligands stabilized by small molecules

Alessandro Angelini, Davide Bertoldo, Shiyu Chen, Christian Heinis, Florence Pojer

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle-free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

Wiley-V C H Verlag Gmbh2014

Kinetics of Antibody Binding to Membranes of Living Bacteria Measured by a Photonic Crystal-Based Biosensor

Carine Ben Adiba, Giovanni Dietler, Ekaterina Rostova

Optical biosensors based on photonic crystal surface waves (PC SWs) offer a possibility to study binding interactions with living cells, overcoming the limitation of rather small evanescent field penetration depth into a sample medium that is characteristic for typical optical biosensors. Besides this, simultaneous excitation of s-and p-polarized surface waves with different penetration depths is realized here, permitting unambiguous separation of surface and volume contributions to the measured signal. PC-based biosensors do not require a bulk signal correction, compared to widely used surface plasmon resonance-based devices. We developed a chitosan-based protocol of PC chip functionalization for bacterial attachment and performed experiments on antibody binding to living bacteria measured in real time by the PCSW-based biosensor. Data analysis reveals specific binding and gives the value of the dissociation constant for monoclonal antibodies (IgG2b) against bacterial lipopolysaccharides equal to K-D = 6.2 +/- 3.4 nM. To our knowledge, this is a first demonstration of antibody-binding kinetics to living bacteria by a label-free optical biosensor.

Mdpi Ag2016

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies

Floriane Auderset, Ute Koch, Freddy Radtke, Anne Wilson

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4–CD8– thymocytes. Notch1 is progressively down-regulated at the CD4+CD8+ and mature CD4+ or CD8+ thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1+CD25– cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117+ early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

2009