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Plants and plant genes for the development of cancer vaccines
S470
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
properties validating the active role of the optimal nanostructure dimensions on the hBM-MSCs responses
[P-M.114]
Acknowledgments
S Massa 1,2,3,∗ , A Venuti 1,2,3 , L Spanò 1,2,3 , R Franconi 1,2,3
This study was supported by the Italian Ministero della Salute (grant no. RF-UMB-2006-339457 to A.O.), the Italian Fondazione Cassa di Risparmio di Perugia (grant no. 2009.020.0050 to A.O.), the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (grants: FIRB Idea Progettuale no. RBIP06FH7J 002 and PRIN no. 20084XRSBS 001 to A.O.), as well as the Istituto Nazionale Biostrutture e Biosistemi. doi:10.1016/j.jbiotec.2010.09.700 [P-M.113] A computational approach to kinome-wide binding affinity profiling of small-molecular inhibitors using dynamic conformations of kinases Q. Huang ∗ , L. Yu, X. Feng, M. Xu, B. Wan, L. Yu Fudan University, China Keywords: kinase profiling; kinase inhibitor selectivity; smallmolecular binding affinity; molecular modelling Selectivity is important for small-molecular inhibitors of protein kinases. Recently kinome-wide profiling of small-molecular binding affinity has become one of main experimental approaches to discovery of selective kinase inhibitors. To conduct the profiling in a relatively rapid and cheap way, it is very interesting to develop computational method to accurately predict the binding affinity profiles of inhibitors to all human kinases. Here we present a computational approach for accurately predicting such kinome-wide binding affinities of small molecules. To the end, structural models of catalytic domains of about 500 human protein kinases were first built by homology modelling and refined by high-resolution modelling protocols with Rosetta. Then, nanosecond-scale molecular dynamics (MD) simulations with explicit solvent representation were performed to simulate the kinases in complex with ATP in active sites, in order to mimic the active forms of kinases. To evaluate the binding affinities of small molecules, for each kinase 25 conformational snapshots were extracted from the MD trajectories to form a dynamic conformational ensemble for molecular docking of small-molecular ligands. With this conformational ensemble, binding free energies of a small molecule to all 25 conformations of a given kinase were calculated with the molecular docking program AutoDock Vina, and the average value was used to quantitatively describe the binding affinity of the small molecule to the given kinase. To test this approach, we calculated the kinome-wide binding affinity profiles for clinical kinase inhibitors. The comparison of calculated values with experimental data showed that our approach is a very promising tool to predict the kinase inhibitor selectivity.
Plants and plant genes for the development of cancer vaccines
1
ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, C.R. Casaccia, Plant Genetics and Genomics Section, Italy 2 Regina Elena Cancer Institute, Laboratory of Virology, Italy 3 University of L’Aquila, Basic and Applied Biology Department, Italy Keywords: Cancer Immunotherapy; plant proteins; plant expression systems; launch vector Introduction: 1) Immunotherapy against Human Papillomavirus (HPV) cancers is seeking for safe/effective vaccines. Linking tumor-specific antigens (HPV16 E7) to molecules increasing their ‘visibility’ represents a strategy to force the immune system to fight cancer. We focused on plant proteins as innovative immunemodulators with few clinical use constraints. 2) the E7 antigen can be expressed in numerous systems: it is essential to determine which system offers the most advantages for production. We have exploited an accelerated production platform applicable for expressing vaccine antigens that is based on a ‘launch vector’ enabling the use of non-genetically modified plants for target production and, so, creating a highly competitive platform that brings a new concept to biomanufacturing. Methods: 1) Ribosome-inhibiting proteins have features (immunogenicity, inflammation and apoptosis induction, detrimental in their former use as immunotoxins) that might be advantageous in tumor therapy. A saporin non toxic mutant was used as a carrier for the E7GGG gene in the context of a genetic vaccine (Italian Patent RM2009A000383). 2) We engineered the HPV16 E7 sequence as a fusion to -1,31,4-glucanase (LicKM) of Clostridium thermocellum and produced it in Nicotiana benthamiana plants using this launch vector (leaves and root cultures). Results and discussion: 1) Preliminary results show that fusion constructs are able to induce E7-specific IgGs, CTLs and Th1 cytokine-mediated inflammation affecting the growth of E7expressing tumours in mice, demonstrating that (mutant) plant genes hold promise to obtain humoral/cell-mediated specific immune responses. 2) The purified target antigen induced E7-specific IgG and cytotoxic T-cell responses inhibiting tumor development following challenge with an E7-expressing tumor cell line, even in a novel orthotopic mouse model. These data demonstrate the potential of this plant-based platform for producing human therapeutic vaccines. Especially root bioreactor culture is the future key step towards commercial production of bioactive therapeutics by plant biotechnology being a fast-growing, contained biotechnology. doi:10.1016/j.jbiotec.2010.09.702 [P-M.115]
doi:10.1016/j.jbiotec.2010.09.701
AA Sequencing, Glycosylation and Phosphorylation Site Characterization of Human Serum Clusterin using Liquid Chromatography-High Resolution Mass Spectrometry S. Sidoli ∗ , L. Elviri, M. Careri, A. Mangia, F. Rizzi, S. Bettuzzi Università degli Studi di Parma, Italy Introduction: An advanced method for peptide mixture analysis was developed and applied to characterization of human serum clusterin (sCLU), a glycoprotein secreted in all biological fluids
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
properties validating the active role of the optimal nanostructure dimensions on the hBM-MSCs responses
[P-M.114]
Acknowledgments
S Massa 1,2,3,∗ , A Venuti 1,2,3 , L Spanò 1,2,3 , R Franconi 1,2,3
This study was supported by the Italian Ministero della Salute (grant no. RF-UMB-2006-339457 to A.O.), the Italian Fondazione Cassa di Risparmio di Perugia (grant no. 2009.020.0050 to A.O.), the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (grants: FIRB Idea Progettuale no. RBIP06FH7J 002 and PRIN no. 20084XRSBS 001 to A.O.), as well as the Istituto Nazionale Biostrutture e Biosistemi. doi:10.1016/j.jbiotec.2010.09.700 [P-M.113] A computational approach to kinome-wide binding affinity profiling of small-molecular inhibitors using dynamic conformations of kinases Q. Huang ∗ , L. Yu, X. Feng, M. Xu, B. Wan, L. Yu Fudan University, China Keywords: kinase profiling; kinase inhibitor selectivity; smallmolecular binding affinity; molecular modelling Selectivity is important for small-molecular inhibitors of protein kinases. Recently kinome-wide profiling of small-molecular binding affinity has become one of main experimental approaches to discovery of selective kinase inhibitors. To conduct the profiling in a relatively rapid and cheap way, it is very interesting to develop computational method to accurately predict the binding affinity profiles of inhibitors to all human kinases. Here we present a computational approach for accurately predicting such kinome-wide binding affinities of small molecules. To the end, structural models of catalytic domains of about 500 human protein kinases were first built by homology modelling and refined by high-resolution modelling protocols with Rosetta. Then, nanosecond-scale molecular dynamics (MD) simulations with explicit solvent representation were performed to simulate the kinases in complex with ATP in active sites, in order to mimic the active forms of kinases. To evaluate the binding affinities of small molecules, for each kinase 25 conformational snapshots were extracted from the MD trajectories to form a dynamic conformational ensemble for molecular docking of small-molecular ligands. With this conformational ensemble, binding free energies of a small molecule to all 25 conformations of a given kinase were calculated with the molecular docking program AutoDock Vina, and the average value was used to quantitatively describe the binding affinity of the small molecule to the given kinase. To test this approach, we calculated the kinome-wide binding affinity profiles for clinical kinase inhibitors. The comparison of calculated values with experimental data showed that our approach is a very promising tool to predict the kinase inhibitor selectivity.
Plants and plant genes for the development of cancer vaccines
1
ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, C.R. Casaccia, Plant Genetics and Genomics Section, Italy 2 Regina Elena Cancer Institute, Laboratory of Virology, Italy 3 University of L’Aquila, Basic and Applied Biology Department, Italy Keywords: Cancer Immunotherapy; plant proteins; plant expression systems; launch vector Introduction: 1) Immunotherapy against Human Papillomavirus (HPV) cancers is seeking for safe/effective vaccines. Linking tumor-specific antigens (HPV16 E7) to molecules increasing their ‘visibility’ represents a strategy to force the immune system to fight cancer. We focused on plant proteins as innovative immunemodulators with few clinical use constraints. 2) the E7 antigen can be expressed in numerous systems: it is essential to determine which system offers the most advantages for production. We have exploited an accelerated production platform applicable for expressing vaccine antigens that is based on a ‘launch vector’ enabling the use of non-genetically modified plants for target production and, so, creating a highly competitive platform that brings a new concept to biomanufacturing. Methods: 1) Ribosome-inhibiting proteins have features (immunogenicity, inflammation and apoptosis induction, detrimental in their former use as immunotoxins) that might be advantageous in tumor therapy. A saporin non toxic mutant was used as a carrier for the E7GGG gene in the context of a genetic vaccine (Italian Patent RM2009A000383). 2) We engineered the HPV16 E7 sequence as a fusion to -1,31,4-glucanase (LicKM) of Clostridium thermocellum and produced it in Nicotiana benthamiana plants using this launch vector (leaves and root cultures). Results and discussion: 1) Preliminary results show that fusion constructs are able to induce E7-specific IgGs, CTLs and Th1 cytokine-mediated inflammation affecting the growth of E7expressing tumours in mice, demonstrating that (mutant) plant genes hold promise to obtain humoral/cell-mediated specific immune responses. 2) The purified target antigen induced E7-specific IgG and cytotoxic T-cell responses inhibiting tumor development following challenge with an E7-expressing tumor cell line, even in a novel orthotopic mouse model. These data demonstrate the potential of this plant-based platform for producing human therapeutic vaccines. Especially root bioreactor culture is the future key step towards commercial production of bioactive therapeutics by plant biotechnology being a fast-growing, contained biotechnology. doi:10.1016/j.jbiotec.2010.09.702 [P-M.115]
doi:10.1016/j.jbiotec.2010.09.701
AA Sequencing, Glycosylation and Phosphorylation Site Characterization of Human Serum Clusterin using Liquid Chromatography-High Resolution Mass Spectrometry S. Sidoli ∗ , L. Elviri, M. Careri, A. Mangia, F. Rizzi, S. Bettuzzi Università degli Studi di Parma, Italy Introduction: An advanced method for peptide mixture analysis was developed and applied to characterization of human serum clusterin (sCLU), a glycoprotein secreted in all biological fluids