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840. Improved Tropisms of Adenoviral Vectors for EGF-R Positive Cells Using Different Bispecific Ligands
ADENOVIRUS VECTOR TARGETING needs to be altered. For these reasons we have designed Ad vectors expressing the EGFR binding Affibody molecule ZE. The ZE ligand has been inserted in tandem repeat in the HI-loop with flanking linker sequences. Fibres expressing both ZH-ZE and ZE-ZH have also been produced and found to bind both their targets. Current progress on these new vectors will be reported. The results presented here, together with previous Ad re-targeting using Affibody molecules, shows that this class of ligands will allow for re-targeting of Ad to new targets in a reproducible mode.
839. Identification of Novel Fiber-Knob Motif for Pancreatic Cancers from Adenoviral Library with Random HI-Loop Peptide Insertion
Yoshiaki Miura,1 Eric Brown,1 Julia Davydova,1 Kazunori Aoki,2 Masato Yamamoto.1 1 Department of Surgery, University of Minnesota, Minneapolis, MN; 2Section for Studies on Host-Immune Response, National Cancer Center Research Institute, Tokyo, Japan.
Adenoviral tropism is determined by the interaction between adenoviral capsid proteins and specific protein molecules on the cell surface. A human adenovirus type 5 (Ad5) initially binds to the cells via the interaction between the fiber knob and the cellular primary receptor, the coxsackie virus and adenovirus receptor (CAR). Thus, modification of the fiber knob region has been a major strategy for vector targeting at the level of cell binding/entry. We hypothesize that efficient presentation of peptide library in adenoviral fiber and high throughput screening of the resultant vectors would result in a new class fiber structure exploitable for gene therapy of cancers. First, we have developed a method to directly select the vectors with intended targeting ability from a random peptide library displayed in the HI-loop of CAR-binding ablated fiber. The vector was generated and amplified with fiber trans-complementing cell line to avoid the elimination of the clones which does not efficiently infect producer cells, and then the resultant adenoviral library was screened with pancreatic cancer cell line (AsPC-1). The convergence of HI-loop insert sequence to several particular sequences was observed. In order to examine the binding ability of selected adenoviruses for target cells, we compared wild-type adenovirus (Ad-WT) and the library derived viruses with selected motifs in HI-loop. AsPC-1 was infected with same copy number of each virus at 4°C for 3 hrs. After extensive washing, crude viral lysates (CVLs) were prepared, and the viral copy number was determined by Q-PCR. The copy number with the new HI-loop insertion was more than two times higher than that of Ad-WT. Thus, the binding ability for AsPC-1 cells was much higher than that of the native Ad5 although these adenoviruses with HI-loop insertion have AB-loop mutation ablating its CAR-binding. We then analyzed the viral replication in the target cells. AsPC-1 was infected at same condition as binding assay. After extensive washing, cells were cultured at 37°C for 48 hrs allowing viral replication and CVLs were prepared. Despite the absence of CAR binding in the library bearing vectors, the copy number with the library derived vector was slightly higher than that of Ad-WT. This data indicate that the insertion of the newly identified motif in the HI-loop of the fiber knob region did not hinder the adenoviral replication machinery in target cells. These results indicated that viral binding conferred by the novel peptide sequence in HI-loop beat the naturally selected native binding mediated by AB-loop binding to CAR and that the motif is usable in replication competent system. For the first time, we identified the peptide motif from random peptide library presented in HI-loop which is showing better binding compared to wild type Ad. We plan to analyze these adenoviruses with the novel binding peptides for specificity, efficacy, and in vivo function.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
840. Improved Tropisms of Adenoviral Vectors for EGF-R Positive Cells Using Different Bispecific Ligands
Maxime Pinard,1,2 Nasha Nassoury,1 Phuong Le,1 Birgit Kastberger,1 Ismaïla Ba,1 Thanh-Dung Nguyen,3 Maureen O’Connor,1 Roger MacKenzie,3 Rénald Gilbert,1,4 Bernard Massie.1,2 1 Genomics and Gene Therapy Vectors Group, Biotechnology Research Institute - CNRC, Montréal, QC, Canada; 2Microbiology and Immunology Department, Université de Montréal, Montréal, QC, Canada; 3Institute for Biological Sciences - CNRC, Ottawa, ON, Canada; 4Montreal Neurological Institute - McGill, Montréal, QC, Canada. Adenoviral vector (AdV) is being intensively investigated as a potential therapeutic agent through the use of suicide genes or through its oncolytic nature as a means to the kill tumor cells. These approaches are promising but cancer cells often express low level of the Coxsackie Adenovirus Receptor (CAR), the primary receptor for AdV. In order to compensate for the paucity of CAR, multiple strategies have been proposed. Some of them consist to alter the AdV fiber to enable recognition of new receptors at the cellular surface, such as PEGylation or using fibers derived from other serotypes. To target specifically tumor cells, our group has developed a bispecific ligand approach coupled with Ad5 fiber modification. The interaction between the virus and the ligand is generated by using an artificial interaction domain, the E.Coil/K.Coil. The K.Coil is inserted within the HI loop domain or at the C-terminus of the Ad5 fiber and its counter part, the E.Coil, is fused with a ligand of interest. An epidermal growth factor derived ligand (E5.Coil-EGF) obtained through refolding and purification from E.coli (Le et al., 2009) as well as a modified single domain antibody (AFAI-E.Coil) were used to evaluate our targeting system. The AFAI single domain antibody binds with high affinity to a cell surface receptor specifically expressed on lung carcinoma cells such an A549 (Zhang et al., 2004). The two fiber-modified AdVs were generated and they were infectious. Binding of the modified Ad5 fiber with the E5.Coil ligand was shown by ELISA and we demonstrated that the interaction strength of the K5.Coil modified fiber was threefold higher compared with the K4.Coil modified fiber. Using flow cytometry, we also demonstrated that the E5.Coil-EGF ligand could increase the infectivity of the C-terminus modified Ad5 for EGF-R expressing A549 cell by twofold (Zeng et al., 2008), whereas for the HI-loop modified AdV, this increase for U87 or A549 cells was 5- and 20-fold respectively. The AFAI-E.Coil ligand has shown an increase of 10 to 20-fold on A549 cell line.
841. Integrin-Binding RGD Insertion in Adenovirus-5 Fiber Shaft or Knob as a Selective Tumor-Targeting Strategy in Human Colorectal Cancer
Sergio Lavilla-Alonso,1,2 Neus Bayo-Puxan,3 Gerd Bauerschmitz,4 Sari Pesonen,1,2 Ramon Alemany,3 Akseli Hemminki.1,2 1 Cancer Gene Therapy Group, Molecular Cancer Biology Program and Transplantation Laboratory and Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2 Transplantation Laboratory, HUSLAB, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 3Translational Research Laboratory, IDIBELL-Institut Català d’Oncologia, Barcelona, Spain; 4Department of Obstetrics and Gynecology, Heinrich-Heine University, Duesseldorf, Germany. Colorectal cancer (CRC) is the third most common cancer worldwide and despite improved therapy regimes, metastatic disease usually remains incurable. Although not definitively shown heretofore, CRC cells might feature variable expression of CAR and hence fail to be effectively transfected by adenovirus 5. Focusing on S321
839. Identification of Novel Fiber-Knob Motif for Pancreatic Cancers from Adenoviral Library with Random HI-Loop Peptide Insertion
Yoshiaki Miura,1 Eric Brown,1 Julia Davydova,1 Kazunori Aoki,2 Masato Yamamoto.1 1 Department of Surgery, University of Minnesota, Minneapolis, MN; 2Section for Studies on Host-Immune Response, National Cancer Center Research Institute, Tokyo, Japan.
Adenoviral tropism is determined by the interaction between adenoviral capsid proteins and specific protein molecules on the cell surface. A human adenovirus type 5 (Ad5) initially binds to the cells via the interaction between the fiber knob and the cellular primary receptor, the coxsackie virus and adenovirus receptor (CAR). Thus, modification of the fiber knob region has been a major strategy for vector targeting at the level of cell binding/entry. We hypothesize that efficient presentation of peptide library in adenoviral fiber and high throughput screening of the resultant vectors would result in a new class fiber structure exploitable for gene therapy of cancers. First, we have developed a method to directly select the vectors with intended targeting ability from a random peptide library displayed in the HI-loop of CAR-binding ablated fiber. The vector was generated and amplified with fiber trans-complementing cell line to avoid the elimination of the clones which does not efficiently infect producer cells, and then the resultant adenoviral library was screened with pancreatic cancer cell line (AsPC-1). The convergence of HI-loop insert sequence to several particular sequences was observed. In order to examine the binding ability of selected adenoviruses for target cells, we compared wild-type adenovirus (Ad-WT) and the library derived viruses with selected motifs in HI-loop. AsPC-1 was infected with same copy number of each virus at 4°C for 3 hrs. After extensive washing, crude viral lysates (CVLs) were prepared, and the viral copy number was determined by Q-PCR. The copy number with the new HI-loop insertion was more than two times higher than that of Ad-WT. Thus, the binding ability for AsPC-1 cells was much higher than that of the native Ad5 although these adenoviruses with HI-loop insertion have AB-loop mutation ablating its CAR-binding. We then analyzed the viral replication in the target cells. AsPC-1 was infected at same condition as binding assay. After extensive washing, cells were cultured at 37°C for 48 hrs allowing viral replication and CVLs were prepared. Despite the absence of CAR binding in the library bearing vectors, the copy number with the library derived vector was slightly higher than that of Ad-WT. This data indicate that the insertion of the newly identified motif in the HI-loop of the fiber knob region did not hinder the adenoviral replication machinery in target cells. These results indicated that viral binding conferred by the novel peptide sequence in HI-loop beat the naturally selected native binding mediated by AB-loop binding to CAR and that the motif is usable in replication competent system. For the first time, we identified the peptide motif from random peptide library presented in HI-loop which is showing better binding compared to wild type Ad. We plan to analyze these adenoviruses with the novel binding peptides for specificity, efficacy, and in vivo function.
Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
840. Improved Tropisms of Adenoviral Vectors for EGF-R Positive Cells Using Different Bispecific Ligands
Maxime Pinard,1,2 Nasha Nassoury,1 Phuong Le,1 Birgit Kastberger,1 Ismaïla Ba,1 Thanh-Dung Nguyen,3 Maureen O’Connor,1 Roger MacKenzie,3 Rénald Gilbert,1,4 Bernard Massie.1,2 1 Genomics and Gene Therapy Vectors Group, Biotechnology Research Institute - CNRC, Montréal, QC, Canada; 2Microbiology and Immunology Department, Université de Montréal, Montréal, QC, Canada; 3Institute for Biological Sciences - CNRC, Ottawa, ON, Canada; 4Montreal Neurological Institute - McGill, Montréal, QC, Canada. Adenoviral vector (AdV) is being intensively investigated as a potential therapeutic agent through the use of suicide genes or through its oncolytic nature as a means to the kill tumor cells. These approaches are promising but cancer cells often express low level of the Coxsackie Adenovirus Receptor (CAR), the primary receptor for AdV. In order to compensate for the paucity of CAR, multiple strategies have been proposed. Some of them consist to alter the AdV fiber to enable recognition of new receptors at the cellular surface, such as PEGylation or using fibers derived from other serotypes. To target specifically tumor cells, our group has developed a bispecific ligand approach coupled with Ad5 fiber modification. The interaction between the virus and the ligand is generated by using an artificial interaction domain, the E.Coil/K.Coil. The K.Coil is inserted within the HI loop domain or at the C-terminus of the Ad5 fiber and its counter part, the E.Coil, is fused with a ligand of interest. An epidermal growth factor derived ligand (E5.Coil-EGF) obtained through refolding and purification from E.coli (Le et al., 2009) as well as a modified single domain antibody (AFAI-E.Coil) were used to evaluate our targeting system. The AFAI single domain antibody binds with high affinity to a cell surface receptor specifically expressed on lung carcinoma cells such an A549 (Zhang et al., 2004). The two fiber-modified AdVs were generated and they were infectious. Binding of the modified Ad5 fiber with the E5.Coil ligand was shown by ELISA and we demonstrated that the interaction strength of the K5.Coil modified fiber was threefold higher compared with the K4.Coil modified fiber. Using flow cytometry, we also demonstrated that the E5.Coil-EGF ligand could increase the infectivity of the C-terminus modified Ad5 for EGF-R expressing A549 cell by twofold (Zeng et al., 2008), whereas for the HI-loop modified AdV, this increase for U87 or A549 cells was 5- and 20-fold respectively. The AFAI-E.Coil ligand has shown an increase of 10 to 20-fold on A549 cell line.
841. Integrin-Binding RGD Insertion in Adenovirus-5 Fiber Shaft or Knob as a Selective Tumor-Targeting Strategy in Human Colorectal Cancer
Sergio Lavilla-Alonso,1,2 Neus Bayo-Puxan,3 Gerd Bauerschmitz,4 Sari Pesonen,1,2 Ramon Alemany,3 Akseli Hemminki.1,2 1 Cancer Gene Therapy Group, Molecular Cancer Biology Program and Transplantation Laboratory and Finnish Institute for Molecular Medicine, University of Helsinki, Helsinki, Finland; 2 Transplantation Laboratory, HUSLAB, Helsinki University Central Hospital (HUCH), Helsinki, Finland; 3Translational Research Laboratory, IDIBELL-Institut Català d’Oncologia, Barcelona, Spain; 4Department of Obstetrics and Gynecology, Heinrich-Heine University, Duesseldorf, Germany. Colorectal cancer (CRC) is the third most common cancer worldwide and despite improved therapy regimes, metastatic disease usually remains incurable. Although not definitively shown heretofore, CRC cells might feature variable expression of CAR and hence fail to be effectively transfected by adenovirus 5. Focusing on S321