A multivalent peptide library approach to identify functional TRAIL mimetics – ApoMULTI-LIB

1-Scientific background and objectives : The modulation of cell signaling by small molecules, designed to interfere with pathological processes to induce a therapeutically beneficial effect is a major challenge which necessitates a multidisciplinary effort. Current cancer therapy follows treatment paradigms that attack general features of neoplastic growth (chemotherapy) and only in a few cases it has been possible to target directly the tumorigenic cause (APL, ATRA; CML, imatinib). Recently a novel therapeutic option has been recognized, which is based on the cancer-selective death inducing member of the TNF family, termed TNF related apoptosis-inducing ligand (TRAIL). TRAIL is a type II membrane bound ligand that engages with its membrane bound signaling receptors DR4 and DR5 which trimerizes and activates the extrinsic death signaling pathway. Evidence provided by one partner laboratory indicates that the two non-signaling ( decoy ) receptors DcR1 and DcR2 have functions in attenuating the TRAIL response. There is proof-of-principle confirming the tumor-selectivity of the TRAIL signaling pathway based on preliminary clinical results, mouse knock out data, xenograft experiments and cellular tumorigenesis models, some of these studies are actively pursued in the partner laboratories. The present therapeutic exploitation of the TRAIL pathway is based on the use of recombinant TRAIL (Genentech/Immunogen, phase I clinical trial) and the use of DR4 and DR5 activating antibodies (HGS, phase II clinical trial). We propose to establish an entirely novel procedure to activate the TRAIL system using an approach that has been originally developed and patented by one of the partners and has the promise to lead to a superior drug. 2-Description of the project, methodology : One of the partners has pioneered the development of 3D structure-based trimeric ligand mimetics for the CD40 member of the TNF receptor family. We will use a plethora of complementary design and screening approaches to generate a panel of both agonistic and antagonistic mimetics for the 4 TRAIL receptors. We will characterize and validate these mimetics in a variety of in vitro and in vivo settings and define their tumor-selective death potential in mono and combo-therapeutic settings. Two approaches will be used for mimetics synthesis (i) Rational design. Based on the known 3D structure data of the DR5 trimer and phage display results we will predict, synthesize and validate mimetics for all TRAIL receptors; (ii) Multivalent peptide library. To facilitate large scale screening, we will generate one bead/one compound trivalent tetra (and subsequently penta)peptide libraries using solid phase combi-chem technology. The characterization, screening and validation will be done as follows: (iii) Small scale validation will be done by testing existing systems to define TRAIL receptor binding selectivity, DISC induction, caspase activation and apoptosis. We will use an established panel of stepwise cellular tumorigenesis models to define the tumor-selectivity and potency of the mimetics relative to soluble recombinant TRAIL. (iv) HTS of trivalent peptide libraries. Subsequent to assay validation done with positive control mimetics (CD40, DR5) to quantify robustness, selectivity and sensitivity several in vitro and in vivo screening systems will be used. Autofluorescence will be limited by quantum dots. (a) Affinity capture. Recombinant extracellular domains of TRAIL receptors will be used for affinity capture of bead dispenser-fractionated sub-libraries. (b) Robotized HTS. Libraries will be dispensed in 96/384 well plates (1bead/well) and apoptosis induction assayed with engineered sensitized vs. TRAIL resistant cells, either directly on beads or after cleavage. (c) Manual picking. Fluorescence-tagged recombinant TRAIL receptors will be added to batches of 1000 beads and labeled beads will be removed (µmanipulator). In all cases identified mimetics will be ident...