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TRIDENT Sintesi della relazione

Project ID: 37686
Finanziato nell'ambito di: FP6-LIFESCIHEALTH
Paese: Ireland

Final Report Summary - TRIDENT (Therapeutic molecules for treatment of solid tumours by modulating death receptor-mediated apoptosis)

With almost 3 million new cases each year and 1.7 million deaths, cancer is an important public health problem in Europe. The ageing of the European population will cause these numbers to continue to increase. The most common cancers are lung, colorectal and breast cancers. New therapeutic strategies and novel tumour-selective anti-cancer agents are necessary in order to improve the treatment of these and other solid tumours. To address this challenge, the TRIDENT project aimed to define and develop novel therapeutic strategies and tumour-selective agents to treat solid tumours. These therapeutic strategies and agents will be based on modulation of signalling cascades induced by members of the TNF ligand (TNF-L) family and their cognate receptors of the TNF receptor (TNF-R) family. Numerous members of the TNF-L / TNF-R family are implicated in controlling cancer growth. TNF-ligands like TRAIL and FASL kill cancer cells by induction of apoptosis while other ligands, like APRIL enhance tumour proliferation. Therefore, signalling cascades modulated by the TNF-L / TNF-R family members provide attractive therapeutic targets.

The overall aim of this program was to define and develop novel therapeutic strategies and tumour-selective agents to treat solid tumours based on the modulation of signalling cascades induced by members of the TNF ligand (TNF-L) family and their cognate receptors of the TNF receptor (TNF-R) family.

Studies had demonstrated that in different tumours TRAIL induces apoptosis only via the DR4 or the DR5 receptor, despite both receptors being expressed and present on the cell surface. Therefore, a major objective of this program was to generate TRAIL variants with improved death inducing characteristics using rational structure assisted design. Receptor-selective variants of TRAIL promise more efficient tumour killing by only targeting the active receptor and therefore, we wanted to generate TRAIL variants with a high potency to activate apoptosis-inducing TNF receptors. Previous work by our network in the Fifth Framework Programme (FP5) had led to the generation of DR5-receptor selective TRAIL variants (a single mutant of WT TRAIL at position 269 from aspartate to histidine (D269H) and a double mutant, where the D269H mutation was combined with mutation E195R). In year 1 of TRIDENT, the computational design focused on identifying amino acid replacements predicted to increase selectivity to DR4. The mutant TRAIL variants were generated by site directed mutagenesis and purified as recombinant proteins. Two amino acid replacements (aspartate at position 218 to tyrosine or histidine) in wild type (WT) TRAIL were identified that significantly increased selectivity towards DR4. In year 2, the production, biophysical and biological characterisation of mutants identified in the first year was carried out.

Another important aim of this network in years 2 and 3 was to evaluate the biological activity / functionality of lead molecules (identified in year 1) in cancer cell lines. This was done by studying the mechanism and level of TRAIL receptor activation by WT TRAIL, versus the efficacy of receptor selective variants. These studies revealed that TRAIL variants which do not bind to the decoy receptors have higher pro-apoptotic activity. Furthermore, the DR5-selective TRAIL variant, due to its increased affinity towards one TRAIL receptor displayed enhanced receptor binding kinetics and reduced formation of heteromeric, non-functional receptor complexes (such as DR5-DR5-DcR2 heterotrimer).

These events appear to be important determinants of receptor activation, caspase-8 activation and resulting cell death. Thus, engineering TRAIL variants to possess receptor-selectivity and high agonistic activity are crucial to achieving effective tumour cell killing. A DR4 selective variant was found to be very active on a number of AML cell lines and, using death receptor selective variants and agonistic anti-DR4 and anti-DR5 antibodies, we demonstrated that TRAIL-induced apoptosis is primarily transmitted by DR4 in AML cells. The increased affinity towards DR4 and increased biological activity on DR4-responsive AML cell lines suggests potential use of rhTRAIL as an anticancer therapeutic agent on leukemic cells such as AML.

Finally, our aim was to exploit the reagents and protocols developed during this program by involving a start-up SME. A number of patents were filed protecting the intellectual content generated in this program and the production protocols for generating purified TRAIL variants suitable for preclinical and early clinical studies were completed. Pharmacokinetic and biodistribution properties of the patented lead molecules and pre-clinical animal studies were also conducted and are currently ongoing. These can now be used by a start-up SME to exploit and commercialise the knowledge generated over the course of this program.

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