Phage selection of bicyclic peptides binding Her2

Aberrant expression of the epidermal growth factor receptor Her2 has been implicated in various malignancies including breast cancer. Monoclonal antibodies and an antibody drug conjugate targeting Her2 have found wide clinical application. Herein, we aimed at developing Her2-specifc ligands based on peptides that have a 100-fold smaller molecular weight than antibodies. Such peptides could potentially offer advantages in the development of ligand drug conjugates, such as ease of synthesis and conjugation, higher molecule-per-mass ratios, and better tumor penetration. Panning of large bicyclic peptide phage display libraries against Her2 yielded a range of Her2-specific ligands having different formats and binding motifs. Strong sequence similarities among several of the isolated peptides indicated that they interact with Her2 in a specific manner. The best bicyclic peptide obtained after affinity maturation bound Her2 with a K-D of 304 nM. The diverse peptide ligands may offer valuable starting points for the development of high-affinity Her2 binders with potential application for tumor imaging and therapy. (C) 2014 Elsevier Ltd. All rights reserved.

Development of bicyclic peptide Her2 binders and phage selection of alpha-helical peptide ligands

Philippe Diderich

Conformationally constrained peptide ligands offer an attractive format for the development of therapeutics. They can bind with high affinity and selectivity to protein targets, they are small enough to diffuse into tissues, they can be synthesized chemically, and their degradation products are not toxic. In recent years, a number of novel techniques were innovated to develop peptide-based ligands. One such technique is the generation of bicyclic peptides by phage display. This technique is well established in our laboratory and has led to the isolation of high-affinity antagonist for a range of important therapeutic targets. In a first project I aimed at developing bicyclic peptide ligands of the epidermal growth factor receptor Her2. Aberrant expression of Her2 has been implicated in various malignancies including breast cancer. In the treatment of this cancer type several Her2 specific antibodies were proved to be efficient. Due to their small size, bicyclic peptides could potentially offer advantages over antibodies such as ease of synthesis and conjugation, higher molecule-permass ratios, and better tumor penetration. I panned a large bicyclic peptide phage library against the extra-cellular domain of Her2 and obtained a range of peptide sequences binding to Her2 in a specific manner. After affinity maturation, a bicyclic peptide that bound Her2 with a Kd of 304 nM could be obtained. This peptide ligand offers a valuable starting point for further improvement and the development of high-affinity Her2 binders with potential application for tumor imaging and therapy. A second project was triggered by my observation that it is relatively difficult to generate high-affinity bicyclic peptide ligands to proteins with flat and featureless surfaces such as Her2. The major reason for the limited binding affinity of bicyclic peptides was supposed to lie in the lack of a defined secondary structure in solution and the resulting entropic penalty upon binding. To overcome this problem, I proposed to evolve ligands based on a-helices. Chemically stabilized a-helices, also named stapled peptides, were previously developed by rational design. I proposed to evolve a-helical peptides by phage display wherein the helix, is stabilized in a chemical reaction prior to phage panning. I tested this strategy by affinity maturing an alpha-helical peptide binding to beta-catenin. The project resulted with a 250-fold improved ligand of beta-catenin. In a third project, I developed a novel constrained peptide format that I dubbed âhelix-loopâ motif. A stabilized alpha-helix was expanded with a peptide loop in order to increase the number of amino acids that can formcontacts with a target. The loop was linked to either the N- or C-terminal end of the helix and via a cysteine residue to the chemical linker stabilizing the alpha-helix. Libraries were created adding randomized loops with different length to either side of the peptide. Ligands with improved affinity were isolated against the cancer target beta-catenin. In summary, I have exploited the existing bicyclic peptide format to evolve bicyclic peptide ligands of Her2. In addition, I have developed a new approach to affinity mature stabilized helical peptides by phage display as well as a new constrained peptide format. This new approach should be applicable for affinity maturation of any stabilized alpha-helix. The new peptide format promises the generation of high affinity ligands to any protein target, including Her2.

EPFL2014

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

Middle-down approaches for mass spectrometry-based protein identification and characterization

Kristina Srzentic

Mass spectrometry (MS) has emerged over the last two decades as the analytical technique of choice in systems-level protein studies, known as proteomics. Two are the MS-based approaches generally applied to proteomics: bottom-up (BU), which relies on the proteolytic digestion of proteins into short (~10 amino acids) peptides, and top-down (TD), where proteolysis is omitted, intact proteins are detected and fragmented in gas phase. Both methods present advantages as well as drawbacks. Here, we sought to establish a complete platform to put forward a third MS-based proteomic approach; middle-down (MD). It implies protein digestion as in BU, but aims to generate large peptides which size approaches the one of small intact proteins that are readily analyzed in TD. This novel domain aims to account for the shortcoming of both classical approaches. Until now, the main reasons behind the limited use of MD proteomics have been the lack of easy-to-use cleaving agents capable of producing peptides in the desired 3-15 kDa mass range with high specificity, limitations in MS and allied instrumentation, and the absence of dedicated bioinformatics tools for processing of acquired data. The latter greatly impedes the next milestone in MD proteomics â large-scale analysis. MD can potentially combine the analysis of large portions of proteins carrying set of biologically-relevant modifications â allowing exploring proteins of molecular weight or complexity incompatible with current TD capabilities â with the high-throughput hallmark of BU proteomics. Here, we first in silico evaluated the potential target amino acid residues to produce peptides in the MD mass range within the proteomes of different organisms. This bioinformatics work was followed by an experimental study based on synthetic MD-sized peptides, aimed at determining the optimal MS and tandem MS parameters for large peptide characterization. Next, we pursued two distinct ways of performing MD proteolysis: i) the use of an enzyme and ii) the use of a chemical reagent. We selected the protease Sap9 as a target enzyme for MD, which we fully characterized and successfully applied to the study of a mixture of monoclonal antibodies, where it showed an advantage over traditional BU in terms of reduced introduction of artifacts to the sample, allowing the post-translational modification investigation and unambiguous antibody identification. The chemical cleavage way we addressed via judicious protocol optimization for hydrolysis at the N-terminal side of cysteine with NTCB reagent. We also advanced MD protocols by generation of large (~50 kDa) subunits of monoclonal antibodies through the use of papain and another more specific novel protease, GingisKHAN, combined with new MS signal processing and data analysis capabilities. The developed workflow improved mapping of the connectivity of cysteines involved in inter- and intra-molecular disulfide bridges in antibodies. To summarize, we demonstrated that MD approach to mass spectrometry and proteomics is a powerful, yet underdeveloped, complement to BU and TD. This work has benchmarked MD for targeted protein analysis. In the near future, with advancements of the field, we envision its growing use for large-scale complex proteome analysis.