Phage display

L'exposition sur phage (ou présentation sur phage, termes traduisant l'anglais phage display) est une technique in vitro permettant d'étudier les interactions entre protéines, peptides et ADN grâce à des bactériophages. L'exposition sur phage a d'abord été décrite par George P. Smith en 1985, quand il a fait la démonstration de l'exposition de peptides à la surface d'un phage filamenteux, grâce à la fusion du gène codant un peptide d'intérêt avec le gène III du phage. Un brevet appartenant à George Pieczenik, antérieur à 1985, décrit également l'obtention de bibliothèques de phages. Cette technologie a par la suite été perfectionnée par des équipes du MRC Laboratory of Molecular Biology de Cambridge, comprenant Winter et McCafferty, ainsi que par le Scripps Research Institute, ce qui a rendu possible l'exposition de protéines telles que des anticorps à usage thérapeutique. En faisant la connexion entre le génotype et le phénotype, cette technique permet de cr

Exploring the potency of normal and pathological human thymic epithelial cells

Tiphaine Mélodie Chantal Arlabosse

Thymic epithelial cells (TECs) are organised in a unique 3D network that is critical for the development of efficient and self-tolerant T-cells. We report for the first time that the human thymus of all ages, even old and involuted, contains a significant number (up to 30%) of clonogenic TECs with extensive growth potential. All clonogenic TECs derive from a common EpCAM+ precursor, which within 48h in culture undergoes a molecular switch and gives rise to refringent EpCAM- colonies and stratified EpCAM+ colonies. The latter two subpopulations can be distinguished based on their morphology, their expression of genes implicated in epidermal stratification and their expression of genes implicated in epithelia-mesenchymal transition. Clonal analysis revealed that clonogenic cells are hierarchically organised, refringent cells being higher in the hierarchy than stratified cells. Although the thymus normally involutes with age, it can be the site of uncontrolled proliferation, resulting in the formation of thymic tumours including thymomas. Thymomas are classified by the WHO based on histological criteria as type A, B and AB and are frequently associated with Myasthenia Gravis, an autoimmune disease affecting the neuromuscular junction. The biology of thymomas is poorly understood, because of the rarity of the tumours and because of the lack of a reliable in vitro model. We report for the first time, that clonogenic cells can be isolated from all thymoma subtypes as well as from thymic hyperplasia and mediastinal teratoma. The latter cells displayed many similarities with normal TECs but also differences in terms of morphology, gene expression and genetic profile. Importantly, all thymic tumours studied contained refringent-like EpCAM- cells and stratified EpCAM+ cells, supporting the hypothesis that thymic tumour-initiating cells arise from clonogenic TECs. We have not succeeded yet in developing an appropriate assay to support the growth of clonogenic epithelial cells from thymic tumours in vivo. Finally, we report that the SY5 monoclonal antibody raised against human involucrin reproducibly cross-reacts with a molecule present in the Golgi apparatus of refringent cells derived from normal and pathological thymi. Immunocytochemistry and knock-down experiments revealed that the latter molecule is not involucrin. Epitope mapping by phage-display experiments and data base search identified putative candidate proteins, including nesprin-1, a player of the neuromuscular junction, which could be the key to understand the frequent association of thymomas with Myasthenia Gravis.

EPFL2017

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

Cyclic peptide ligands are a promising molecular format for the development of therapeutics. They combine some of the advantages of large protein therapeutics (high affinity and specificity) and of small molecule drugs (accessibility to chemical synthesis and low immunogenicity). With phage display, large combinatorial libraries of more than one billion mono- and polycyclic peptides can be developed and screened against virtually any target. During phage affinity selections, the physical link between phenotype (encoded peptide) and genotype (phage DNA) allows the identification of enriched peptide binders by sequencing of the phage vector DNA. Based on these concepts, our laboratory has established a robust chemistry in which phage displayed peptides containing two or three cysteines are cyclized or bi-cyclized with thiol-reactive linkers. This procedure has been generally applied for the isolation of potent and selective (bi)cyclic peptide ligands with therapeutic potential. The isolation of binders with optimal properties is highly dependent on the possibility to generate (bi)cyclic peptide libraries with high structural diversity and on the ability to thoroughly decode the phage selection output. In my PhD work, I developed new methods to generate large and structurally diverse libraries and to decode more efficiently phage selected peptides. In my first project, I explore the cyclization of peptides with two chemical bridges as a method to provide rapid access to phage-encoded libraries with high scaffold diversity. To this end, a range of twelve structurally diverse chemicals were tested for thiol-reactivity and phage compatibility. Subsequently, peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) were cyclized with six of these chemical linkers and panned against the protease plasma kallikrein. The selection yielded inhibitors with remarkable affinity (sub-nM KI), target selectivity (>1000-fold) and proteolytic stability (t1/2 in plasma > 3 days) despite a relatively small molecular mass (~ 1200 Da). In the second project, I developed phage-encoded libraries of small cyclic peptides in order to identify ligands that have the potential to be orally absorbed. A major challenge for the generation of such library is the usually low diversity of compounds due to the limited number of amino acids. In this work, we addressed this limitation by taking advantage of a recently discovered peptide cyclization reaction in which the thiol of a cysteine is ligated with the N-terminal amino group using a chemical reagent. We applied this strategies to two phage-encoded peptide libraries of four and five amino acids (XXXC and XXXXC) that were cyclized with eight chemical linkers yielding a collection of more than 1.3 million compounds. Panning the libraries against the two disease targets plasma kallikrein and coagulation factor XIa yielded ligands with single-digit micromolar affinity. In a third project, I developed a strategy to bypass the bacterial infection step in phage display. Bacterial infection is usually crucial for the propagation and identification of enriched clones by DNA sequencing. However, mutations or chemical modifications of the phage coat proteins, sometimes lead to decreased infection rates and therefore compromise the entire procedure. Consequently, there is an interest for developing methods in which this step could be completely omitted.

EPFL2017

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

EPFL2017

Exploring the potency of normal and pathological human thymic epithelial cells

Tiphaine Mélodie Chantal Arlabosse

EPFL2017

Selection of antibodies against a Muc1 peptide by phage display using a novel antigen presentation procedure

Exploring the potency of normal and pathological human thymic epithelial cells

New methods for developing (bi)cyclic peptides by phage display

New methods for developing (bi)cyclic peptides by phage display

Selection of antibodies against a Muc1 peptide by phage display using a novel antigen presentation procedure

Exploring the potency of normal and pathological human thymic epithelial cells