Final Report Summary - TRANSCOM (The Commercialisation of Transglutaminase.)
Summary
The TRANSCOM project was designed to facilitate the exchange of knowledge and technology between 3 leading European research teams and 4 SMEs each with an interest in the protein crosslinking enzymes Transglutaminases. Transglutaminases (TGs) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of stable isopeptide bonds or by deamidation of proteins. Such protein modifications have important roles under normal physiological conditions but under conditions where deregulation of enzyme activity may occur eg following chronic inflammation they can become pathological. A major objective of the TRANSCOM project was: to establish the molecular nature of the role of transglutaminases in the pathogenesis of diseases which are known to involve their cross-linking or deamidation activity such as coeliac disease and neurodegenerative diseases. Working between sectors our ultimate aim was to develop new point of care diagnostic assays for coeliac disease which reduce the invasive procedures used in diagnosis and to develop novel specific inhibitors that form the basis for new therapeutic approaches in the treatment of diseases and complications where transglutaminases are involved.
Societal Significance of the Project
The diseases chosen to study within this project need very little introduction with respect to their importance in Human Health. Celiac disease occurs, on average, once in every 1,000 live births, with a range from one in 250 to one in 4,000. Currently, the management of this disease is through diet alone and any therapy that would remove the need for a strict gluten-free diet would have a significant uptake. Moreover it is still one of most undiagnosed diseases and even though point of care kits are available their present accuracy still requires invasive surgery to confirm diagnosis. Hence there is a need for the introduction of more accurate point of care diagnostic tests. The celiac market is expected to be $8b by 2019 with no candidate drug therapies currently passing Phase II. Neurodegeneration is a group of diseases which hardly requires explanation of its competitive opportunities for European biotechnology – as life expectancy increases the Alzheimer’s market alone is expected to grow significantly, from 18 to 25 million patients by 2015. The total drug sales for all neurodegenerative diseases in 2008 was $18.5b and predicted to increase to over $35b by 2015. However there is still an urgent need for more effective drugs. Infection by MRSA (methicillin resistant Staphylococcus aureus) is frequently associated with blood clots from invasive surgery and via intravenous catheters. The most frequent complication associated with intravenous catheter implantation is microbial infection. Hence new catheter coatings are urgently required to prevent fouling and to alleviate related bloodstream infections which are estimated to occur at an annual rate of 80,000 to 250,000 cases in the US. The catheter market is forecast to be over $23b.
The work programme for TRANSCOM was divided into 4 major scientific and technological WPs each with its own set of objectives which utilise the expertise and skills of the TRANSCOM partnership.
WP1 Development of biomarkers for celiac disease diagnosis
WP2 Development/testing alternative treatment forms for celiac disease
WP3 Development of new models for testing potential therapeutics in neurodegenerative diseases associated with intracellullar inclusion formation. Engineering, characterization and production of "leakyTG2" and exploration of the biological effects of this enzyme in disease models.
WP4 Development of transglutaminase inhibitors and formulations suitable for therapeutic use and assays
Summary of the Project achievements
TRANSCOM has made a number of achievements during the life of the project facilitated by the sharing and transfer of knowledge between its academic and commercial partners which are in line with its original objectives. Of particular note has been the development of a new lateral flow test for the detection of both IgA and IgG deamidated gliadin peptide (DGP) antibodies for use in coeliac diagnosis. Bench marking of this new test has indicated that data from the lateral flow kit are in line with results produced by the standard ELISA test. Importantly unlike other point of care tests it detects 100% of celiac disease (CD) samples but also non-specific positives, especially in the case of IgA deficient celiac disease patients. The work has also led to the identification and publication of the major CD autoantibody-binding epitope in TG2, which is conformational and formed by three spatially close amino acids of adjacent domains which have been identified. In turn this has allowed the identification of monoclonal mouse anti-TG2 antibodies which bind near to the binding site of CD antibodies which from initial tests have the potential to be used therapeutically to interfere with the binding of celiac antibodies and modify their biological effects. The project has also given us further information on the pathogical implications resulting from celiac antibodies when targeted against the major autoantigen TG2 showing their inhibitory effects on angiogenesis which in turn affects development and wound healing. It has also highlighted the importance of TG2 in angiogenesis suggesting this enzyme may be a possible target in angiogenic pathologies since inhibitors developed in the project have been shown to be capable of blocking VEGF induced angiogenesis.The project has demonstrated that by using selected amino acid mutations in TG2 around the active transamidating site this can reverse the forward crosslinking activity of TG2 into a crosslink hydrolysing enzyme. Initial results have shown this reversible /leaky TG2 to have potential applications in a number of TG2 related diseases eg neurodegenerative inclusion body formation where breakdown of the aggregated proteins may be possible.
A further goal of the project was to develop specific small molecule inhibitors with high potency and low toxicity that do not inhibit cysteine proteases or other active members of the transglutaminase (TG) family. However to screen such inhibitors specific assay for the other members of the TG family must be available which have now been developed and optimised within this project. As a consequence small high potency (IC 50s in low nM level) peptidomimetic inhibitors have been developed that are highly specific for TG2 and which can be targeted to the inside or outside of the cell. TG2 inhibitors have been shown for their ability to prevent gliadin induced effects in celiac patient mucosal organ cultures. They were also able to prevent celiac IgA–induced increase of epithelial permeability of gliadin peptides and help prevent the pathological effects of CD antibodies. TG2 inhibitors have also been shown to block angiogenesis in cell, ex vivo and pre clinical models opening up the possibility that these TG2 specific inhibitors may be used in pathologies related to angiogenesis.
The project has also demonstrated that small compound inhibitors that block FXIIIa activity can be incorporated into silicone catheters with long release kinetics and with the ability to block FXIIIa activity giving proof of concept that this may be a very feasible way of preventing catheter fouling and associated MRSA infections.
Finally by incorporating TG2 inhibitors into liposomes that target to the liver the project has demonstrated that liposomes may be a specific way of targeting TG2 inhibitors to diseases like liver fibrosis thus limiting any toxic side effects that may result as a consequence of oral or other route of administration.
Technology Transfer
The work programme was designed to facilitate the exchange of knowledge and technology between partner institutions maximising on their strengths and expertise in order to undertake and achieve the interdisciplinary research programmes outlined. Four Experienced Researchers have been employed on the project and 121.5 months of secondments have taken place between partners involving a mixture of researcher experiences and a high degree of knowledge transfer. In order to increase the skill base of employed researchers and secondees, three training courses for ERs and secondees have taken place at each of the academic partner laboratories. A further course run by partner Debrecen in conjunction with FEBS has also taken place.
Research Impact
During the TRANSCOM project a total of 21 research papers and one patent were published in peer reviewed journals, with media press releases having taken place in the area of celiac disease. One patent on the new TG2 specific inhibitors has also been filed. In addition partners and ERs presented their work at a number of scientific conferences and all academic partners and one commercial partner (Covalab) were invited speakers at the Gordon Research Conference in Lucca, Italy on "Transglutaminases in Human Disease Processes”, June 29th- July 4th 2014. The project has also continually updated its own website www.mariecurie-transcom.org.
The TRANSCOM project was designed to facilitate the exchange of knowledge and technology between 3 leading European research teams and 4 SMEs each with an interest in the protein crosslinking enzymes Transglutaminases. Transglutaminases (TGs) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of stable isopeptide bonds or by deamidation of proteins. Such protein modifications have important roles under normal physiological conditions but under conditions where deregulation of enzyme activity may occur eg following chronic inflammation they can become pathological. A major objective of the TRANSCOM project was: to establish the molecular nature of the role of transglutaminases in the pathogenesis of diseases which are known to involve their cross-linking or deamidation activity such as coeliac disease and neurodegenerative diseases. Working between sectors our ultimate aim was to develop new point of care diagnostic assays for coeliac disease which reduce the invasive procedures used in diagnosis and to develop novel specific inhibitors that form the basis for new therapeutic approaches in the treatment of diseases and complications where transglutaminases are involved.
Societal Significance of the Project
The diseases chosen to study within this project need very little introduction with respect to their importance in Human Health. Celiac disease occurs, on average, once in every 1,000 live births, with a range from one in 250 to one in 4,000. Currently, the management of this disease is through diet alone and any therapy that would remove the need for a strict gluten-free diet would have a significant uptake. Moreover it is still one of most undiagnosed diseases and even though point of care kits are available their present accuracy still requires invasive surgery to confirm diagnosis. Hence there is a need for the introduction of more accurate point of care diagnostic tests. The celiac market is expected to be $8b by 2019 with no candidate drug therapies currently passing Phase II. Neurodegeneration is a group of diseases which hardly requires explanation of its competitive opportunities for European biotechnology – as life expectancy increases the Alzheimer’s market alone is expected to grow significantly, from 18 to 25 million patients by 2015. The total drug sales for all neurodegenerative diseases in 2008 was $18.5b and predicted to increase to over $35b by 2015. However there is still an urgent need for more effective drugs. Infection by MRSA (methicillin resistant Staphylococcus aureus) is frequently associated with blood clots from invasive surgery and via intravenous catheters. The most frequent complication associated with intravenous catheter implantation is microbial infection. Hence new catheter coatings are urgently required to prevent fouling and to alleviate related bloodstream infections which are estimated to occur at an annual rate of 80,000 to 250,000 cases in the US. The catheter market is forecast to be over $23b.
The work programme for TRANSCOM was divided into 4 major scientific and technological WPs each with its own set of objectives which utilise the expertise and skills of the TRANSCOM partnership.
WP1 Development of biomarkers for celiac disease diagnosis
WP2 Development/testing alternative treatment forms for celiac disease
WP3 Development of new models for testing potential therapeutics in neurodegenerative diseases associated with intracellullar inclusion formation. Engineering, characterization and production of "leakyTG2" and exploration of the biological effects of this enzyme in disease models.
WP4 Development of transglutaminase inhibitors and formulations suitable for therapeutic use and assays
Summary of the Project achievements
TRANSCOM has made a number of achievements during the life of the project facilitated by the sharing and transfer of knowledge between its academic and commercial partners which are in line with its original objectives. Of particular note has been the development of a new lateral flow test for the detection of both IgA and IgG deamidated gliadin peptide (DGP) antibodies for use in coeliac diagnosis. Bench marking of this new test has indicated that data from the lateral flow kit are in line with results produced by the standard ELISA test. Importantly unlike other point of care tests it detects 100% of celiac disease (CD) samples but also non-specific positives, especially in the case of IgA deficient celiac disease patients. The work has also led to the identification and publication of the major CD autoantibody-binding epitope in TG2, which is conformational and formed by three spatially close amino acids of adjacent domains which have been identified. In turn this has allowed the identification of monoclonal mouse anti-TG2 antibodies which bind near to the binding site of CD antibodies which from initial tests have the potential to be used therapeutically to interfere with the binding of celiac antibodies and modify their biological effects. The project has also given us further information on the pathogical implications resulting from celiac antibodies when targeted against the major autoantigen TG2 showing their inhibitory effects on angiogenesis which in turn affects development and wound healing. It has also highlighted the importance of TG2 in angiogenesis suggesting this enzyme may be a possible target in angiogenic pathologies since inhibitors developed in the project have been shown to be capable of blocking VEGF induced angiogenesis.The project has demonstrated that by using selected amino acid mutations in TG2 around the active transamidating site this can reverse the forward crosslinking activity of TG2 into a crosslink hydrolysing enzyme. Initial results have shown this reversible /leaky TG2 to have potential applications in a number of TG2 related diseases eg neurodegenerative inclusion body formation where breakdown of the aggregated proteins may be possible.
A further goal of the project was to develop specific small molecule inhibitors with high potency and low toxicity that do not inhibit cysteine proteases or other active members of the transglutaminase (TG) family. However to screen such inhibitors specific assay for the other members of the TG family must be available which have now been developed and optimised within this project. As a consequence small high potency (IC 50s in low nM level) peptidomimetic inhibitors have been developed that are highly specific for TG2 and which can be targeted to the inside or outside of the cell. TG2 inhibitors have been shown for their ability to prevent gliadin induced effects in celiac patient mucosal organ cultures. They were also able to prevent celiac IgA–induced increase of epithelial permeability of gliadin peptides and help prevent the pathological effects of CD antibodies. TG2 inhibitors have also been shown to block angiogenesis in cell, ex vivo and pre clinical models opening up the possibility that these TG2 specific inhibitors may be used in pathologies related to angiogenesis.
The project has also demonstrated that small compound inhibitors that block FXIIIa activity can be incorporated into silicone catheters with long release kinetics and with the ability to block FXIIIa activity giving proof of concept that this may be a very feasible way of preventing catheter fouling and associated MRSA infections.
Finally by incorporating TG2 inhibitors into liposomes that target to the liver the project has demonstrated that liposomes may be a specific way of targeting TG2 inhibitors to diseases like liver fibrosis thus limiting any toxic side effects that may result as a consequence of oral or other route of administration.
Technology Transfer
The work programme was designed to facilitate the exchange of knowledge and technology between partner institutions maximising on their strengths and expertise in order to undertake and achieve the interdisciplinary research programmes outlined. Four Experienced Researchers have been employed on the project and 121.5 months of secondments have taken place between partners involving a mixture of researcher experiences and a high degree of knowledge transfer. In order to increase the skill base of employed researchers and secondees, three training courses for ERs and secondees have taken place at each of the academic partner laboratories. A further course run by partner Debrecen in conjunction with FEBS has also taken place.
Research Impact
During the TRANSCOM project a total of 21 research papers and one patent were published in peer reviewed journals, with media press releases having taken place in the area of celiac disease. One patent on the new TG2 specific inhibitors has also been filed. In addition partners and ERs presented their work at a number of scientific conferences and all academic partners and one commercial partner (Covalab) were invited speakers at the Gordon Research Conference in Lucca, Italy on "Transglutaminases in Human Disease Processes”, June 29th- July 4th 2014. The project has also continually updated its own website www.mariecurie-transcom.org.