Final Activity Report Summary - HUMPROTHER (New tools for delivering therapeutic proteins: application to human therapy)
The objectives of this program were to develop totally new and original strategies aimed at producing and delivering biotherapeutic proteins. Amongst the therapeutic proteins were included difficult to express proteins such as membrane proteins. During this project, we have developed an optimised cell-free expression system which is capable to overexpress membrane proteins either in a soluble form in the presence of detergents or embedded into a lipid vesicle to form proteoliposomes (patent WO2008152262).
The tremendous advantage of our system is that the production of the recombinant therapeutic soluble membrane proteins or proteoliposomes is a one-step reaction which can be easily scaled-up for industrial applications. We used these recombinant therapeutic proteoliposomes for analysing the structure and the function of the NADPH oxidase complex (which is responsible for the bactericidal activity). More importantly, these recombinant proteoliposomes have been used to restore an enzymatic deficiency in CGD-cells (Chronic Granulomatous Disease). Optimisation of these therapeutic Nox2 proteoliposomes is ongoing in order to quickly move to clinical trials. This latter project has been recently supported by an English private foundation (CGD Research Trust) demonstrating the European interest for the proposal.
Using this optimised cell-free expression system, we overexpressed a therapeutic proapoptotic Bak membrane protein which is capable to be specifically delivered to cancer cells including melanoma, colon carcinoma and brain cancer (glioblastoma). Using our Bak proteoliposome (LipoBak), we can propose for the first time, a highly potent therapeutic product for treating glioblastoma which is capable to increase the life expectation of patients bearing such tumours. A European industrial partner has been approached for testing this therapeutic product into phase I/II.
We used this expression system to produce VDAC mitochondrial membrane protein to get more insight into the function and the structure of this essential protein. We established national and international collaborations for resolving the structure of this important protein. Crystals and first structures (resolution at 7.5 A) of VDAC have been already obtained demonstrating the great potency of this system to produce fully active membrane proteins.
Finally, we used this expression technology to produce native antigens to develop vaccines against infectious diseases. One of these antigens is a bacterial porin which is capable to protect against Pseudomonas aeruginosa, a bacterial strain found in opportunist infections such cystic fibrosis. A development of this vaccine is on-going with a French specialised pharmaceutical company. Recombinant antigens from other bacterial species such as Haemophilus influenzae, Haemophilus ducreyi, Neisseria meningitidis or Yersinia pestis have been produced and tested in vaccine trials. All antigens stimulated an immune response in mice and vaccination experiments are on-going.
One of the actual challenges in drug delivery is to transport biologically active molecules in tumour cells without damaging surrounding normal tissues. During this proposal, we developed new polypeptide carriers able to transport and deliver soluble therapeutic proteins. Amongst these carriers, we optimised dodecahedra protein complex from the adenovirus type 5 fused to the WW domain of Nedd4. We used this complex to deliver native antigens and showed a total protection against bacterial challenges in mice vaccinated. This carrier was also used to specifically deliver the therapeutic p53 protein into tumour cells resulting in cellular death by apoptosis. During this project, we characterized a totally new protein transduction domain from the ZEBRA transcription factor from Epstein Barr virus. This domain is capable to deliver therapeutic proteins with high efficiency in tumour cells by a new transduction molecular mechanism. This transduction results specifically in cellular death of cancer cells.
The tremendous advantage of our system is that the production of the recombinant therapeutic soluble membrane proteins or proteoliposomes is a one-step reaction which can be easily scaled-up for industrial applications. We used these recombinant therapeutic proteoliposomes for analysing the structure and the function of the NADPH oxidase complex (which is responsible for the bactericidal activity). More importantly, these recombinant proteoliposomes have been used to restore an enzymatic deficiency in CGD-cells (Chronic Granulomatous Disease). Optimisation of these therapeutic Nox2 proteoliposomes is ongoing in order to quickly move to clinical trials. This latter project has been recently supported by an English private foundation (CGD Research Trust) demonstrating the European interest for the proposal.
Using this optimised cell-free expression system, we overexpressed a therapeutic proapoptotic Bak membrane protein which is capable to be specifically delivered to cancer cells including melanoma, colon carcinoma and brain cancer (glioblastoma). Using our Bak proteoliposome (LipoBak), we can propose for the first time, a highly potent therapeutic product for treating glioblastoma which is capable to increase the life expectation of patients bearing such tumours. A European industrial partner has been approached for testing this therapeutic product into phase I/II.
We used this expression system to produce VDAC mitochondrial membrane protein to get more insight into the function and the structure of this essential protein. We established national and international collaborations for resolving the structure of this important protein. Crystals and first structures (resolution at 7.5 A) of VDAC have been already obtained demonstrating the great potency of this system to produce fully active membrane proteins.
Finally, we used this expression technology to produce native antigens to develop vaccines against infectious diseases. One of these antigens is a bacterial porin which is capable to protect against Pseudomonas aeruginosa, a bacterial strain found in opportunist infections such cystic fibrosis. A development of this vaccine is on-going with a French specialised pharmaceutical company. Recombinant antigens from other bacterial species such as Haemophilus influenzae, Haemophilus ducreyi, Neisseria meningitidis or Yersinia pestis have been produced and tested in vaccine trials. All antigens stimulated an immune response in mice and vaccination experiments are on-going.
One of the actual challenges in drug delivery is to transport biologically active molecules in tumour cells without damaging surrounding normal tissues. During this proposal, we developed new polypeptide carriers able to transport and deliver soluble therapeutic proteins. Amongst these carriers, we optimised dodecahedra protein complex from the adenovirus type 5 fused to the WW domain of Nedd4. We used this complex to deliver native antigens and showed a total protection against bacterial challenges in mice vaccinated. This carrier was also used to specifically deliver the therapeutic p53 protein into tumour cells resulting in cellular death by apoptosis. During this project, we characterized a totally new protein transduction domain from the ZEBRA transcription factor from Epstein Barr virus. This domain is capable to deliver therapeutic proteins with high efficiency in tumour cells by a new transduction molecular mechanism. This transduction results specifically in cellular death of cancer cells.