874 Design and Characterization of Her2 Targeted Adenovirus Vectors coagulation factor IX (FIX) and the fiber knob domain derived from human adenovirus serotype 9 (Ad9) Tandem mass spectrometry data r[.]
coagulation factor IX (FIX) and the fiber knob domain derived from human adenovirus serotype (Ad9) Tandem mass spectrometry data revealed that interaction site involves Glu248 from the Ad9 fiber knob domain and Tyr47 from FIX, which is in the N-terminus of the FIX Gla-domain Thermodynamic and kinetic characterization of the complex formation using BIACORE technology will be also presented The obtained data can be used for the design of mutated forms offibcr knob domains with decreased affinity towards coagulation factor IX 872 Chimeric Adenoviral Vectors Displaying Fibers of Subgroup B Effectively Mediate Transgene Expression into Specific Cancer Cells Miho Murakami,'> I-1ideyo Ugai,' Natalya Belousova.' Masato Yamamoto, I Maaike Everts.P David T Curiel.' Therapy Departments 0/ Medicine Obstetrics and Gynecology, Pathology Surgery and the Gene Therapy Center; University ofAlabama at Birmingham Birmingham, AL; ]Division ofMolecular and Cellular Pathology; Department a/Pathology, University 0/Alabama at Birmingham Birmingham AL; 'Department ofExperimental Diagnostic Imaging University a/Texas Houston TX IDivision 0/ Human Gene Subgroup C Adenovirus (Ad) vectors arc being exploited for cancer gcnc therapy because of their ability to provide high levels oftransgene expression in infected cells However, since many types of cancer cells lack or express reduced levels ofthe primary receptor for subgroup C viruses [the coxsackie and adenovirus receptor (CAR)], the application for gene delivery into cancer cells is limited To circumvent this issue, chimeric subgroup C vectors displaying a fiber from other subgroups have been developed and demonstrated a CAR independent pathway to infect target cells It has been reported that the subgroup B virus receptor is highly expressed in cancer cells In this regard, we sought to evaluate that a chimeric subgroup C vector displaying a subgroup B fiber will effectively infect distinct tumors derived from different organs For this purpose, the fibers ofAd5 vectors (subgroup C) were genetically replaced with fibers of various subgroup B (Ad3, Ad II, or Ad35) viruses The resulting chimeric viral vectors were compared with the original Ad5 vector for transductional infectivity in a variety of cancer cell lines, using luciferase as a reporter gene The luciferase activities of these chimeric viral vectors were higher than that of the original Ad5 vector in CAR-negative glioma (30-fold) and chronic myelogenous leukemia (12 to 17-fold) cell lines, as well as in a CAR-positive lung cancer cell line (2 to 3-fold) Moreover, in prostate cancer cell lines, the chimeric viral vector displaying an Ad3 fiber on viral particles expressed luciferase 60-70 times higher than the original Ad5 vector and the other chimeric viral vectors Our results suggest that the chimeric vectors allow tumor targeting of cancer cells that not express CAR Moreover, the chimeric viral vector displaying an Ad3 fiber on viral particles may be effectively used for prostate cancer targeting Adenoviral receptors for particular serotypes will be differentially expressed on cancer cells The variability on cancer cells in susceptibility to infection by the different chimeric vectors is probably due to differential expression oftheir respective receptors This issue will need to be addressed in future studies Achievement of effective gene transfer with Ad vectors into specific cancer cells should be optimized by vectorology of fiber chimerism 873 Circumventing Induction of Neutralizing Antibodies to Adenovirus by Manipulating Vector Dose Marlene S Strayer, Kristin Blauvelt, David S Strayer, I Pathology, Thomas Jefferson University; Philadelphia, PA Antibodies that bind and inactivate vectors are a major limitation to the effectiveness of gene transfer for therapeutics or irnrnunization At the same time, strategies that allow repeat dosing have become increasingly important Recombinant human Adenovirus type (rAd) has not realized its potential as a gene therapy or vaccine vector in part because of pre-existing and induced anti-vector immunity Attempts to avoid this include using chimeric rAds with a different hexon protein , different serotypcs of human rAd or rAd from other species These approaches may address the issue of pre-existing anti-vector immunity, but repeat administration will likely still be a problem Therefore, in the context of using rAd vectors repeatedly to immunize against a transgene product, we studied the effects of man ipulating rAd vector doses on the induction ofneutralizing antibodies (NAbs) and anti-transgene antibodies (Abs) by repeat administration of different doses of 1st generation EIIE3 -deleted humanAd5 Outbred Swiss Webster mice were given 10(6), 10(7) or 10(8) IFU (infectious units) rAd.EGFP intranasally monthly for months , and bled weeks after each dose Serum anti-rAd NAbs and anti-EGFP Abs were measured Serum NAbs vs rAd were measured by in vitro neutralization assay rAd.EGFP was incubated with serum from control or immunized mice The transducing ability ofthe resulting mixture was tested in 293A cells using fluorescence microscopy EGFP expression was quantitated after one round of transduction/infection and total fluorescence was compared Higher immunizing doses of rAd.EGFP, c1icited NAbs after significantly fewer immunizations than did lower vector inocula Thus sera collected after I immunization with 10(8) infectious units (IFU) substantially neutralized the vector (P=.O15, 37% neutralization) while immunizations with 10(7) IFU were required to achieve significant levels of neutralization (P=.007, 43.9% neutralization) Interestingly, lowering the immunizing dose still farther, to 10(6) IFU, did not generate detectable NAbs, even after doses Titers ofanti-EGFPAbs were measured by ELISA and reflect transgene expression as well as immune response A single immunization with rAd.EGFP produced serum Abs vs EGFP at all IFU tested The second immunization at all doses increased serum titers approximately IO-fold Anti-EGFP titers were similar for 10(7) and 10(8) IFU, and significantly less for 10(6) IFU (P=.025 and 011, respectively) More than immunizations boosted titers only 2-fold at best Maximum titers were achieved with different numbers of immunizations, depending upon dose The highest titers were reached in some mice after only doses at 10(8) IFU, after doses at 10(7) IFU and after doses at 10(6) In fact 10(6) IFU produced little or no Ab in out mice Therefore anti-rAd-neutralizing Abs need not be as limiting as previously thought NAbs are dose-dependent: more vector elicits more NAbs with fewer administrations Inducing anti-transgene Abs does not necessarily require using rAd doses that elicit rapid NAbs An optimal dose of rAd may be the lowest dose that elicits adequate transgene expression or anti-transgene Abs after two or more doses 874 Design and Characterization of Her2Targeted Adenovirus Vectors Natalya Bclousova, Marine Kojanyan, Galina Mikhecva, Victor Krasnykh I Experimental Diagnostic Imaging University a/Texas M.D Anderson Cancer Center; Houston TX Earlier efforts to develop adenovirus(Ad)-based vectors suitab le for therapeutic gene delivery have identified the lack oftarget tissue Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © The American Soci ety ot G ene "1l1f:r:lpy S333 specificity as a major shortcoming ofthese agents One ofthe major strategies used to overcome this deficiency is termed transductional targeting It uses the concept of redirecting the virus from its native receptor, whose expression has not been linked to any known pathology, to those molecules that are preferentially expressed by the target tissue Direct genetic incorporation into the capsid ofAd vector of ligands that specifically bind to the molecular markers ofthe disease allows the virus to exploit these cell-associated markers as surrogate receptors for cell entry While the feasibility ofthis concept has been proven, the overall progress with developing genetically targeted Ad vectors has been slow for two reasons First, the receptor-binding component ofAd virion, the fiber protein , has proven to have a limited capacity in accommodating targeting ligands Second , the repertoire ofligand candidates is very limited by both the structural constrains imposed by the fiber architecture, and the requirements imposed by the fiber biosynthesis and intracellular trafficking In our recent studies, these two problems have been addressed by combining the previously developed fiber modification strategy with the use of protein ligands that have been designed to satisfy both the structural and biosynthetic requirement of an ideal targeting ligand for Ad vectors SpecificalIy, successful targeting of Ad to a major molecular marker of cancer, the Her2 receptor, has been achieved by replacing the viral fiber protein with a tripartite protein chimera containing a Her2-specific affibody ligand By improving on the previously designed fiber-flbritin protein backbone and taking advantage of the unique structural and biosynthetic characteristics ofaffibodies, the efficacy of incorporation ofthe resultant targeting chimeras intoAd virions has been vastly improved These improvements yielded an Ad vector whose infectivity in the Her2- and CAR-expressing cells rivals that orthe virus that contains unmodified wild-type fibers The specificity of this vector for Her2 and its high infectivity on Her2-expressing tumor cells make it an excellent prototype for therapeutic and imaging gene vectors that peritoneal macrophages produce chemokines and activate IFN cascade by the infection ofAd vectors Next, we analyzed the profile of in vivo gene expression in mice after systemic administration of Ad vectors Our previous studies showed that, after the systemic administration of Ad vectors, liver is a major transduction tissue, whereas spleen is the origin of IL-6 production Therefore, DNA microarray analysis in hepatic and splenic tissues was performed hours after intravenous injection of the conventional Ad vector Genes of many inflammatory cytokines and chemokines, such as (L-6, CXCL2, CXCL5 and CCL II , were up-regulated only in spleen, whereas CXCL9 was up-regulated only in liver These results indicate that the major origin of the inflammatory eytokines! ehemokines production is spleen, as expected The expression of toll-like receptors, including TLR2 and TLR3, and IFN-indueible genes, including lfit I and [fiG, were also augmented by Ad vectors These results indicate that Ad vectors could stimul ate various genes for the activation of innate immune responses 875 Comprehensive Search for Factors Involved in the Innate Immune Response Induced by Adenovirus Vectors Adenoviral vectors have been successfully used for gene therapy purposes as well as vaccines for antigen delivery; they have been proven particularly efficacious to generate a CTL response against selected antigens More recently, adenoviral vectors have also been used as "cancer vaccine" for colon cancer Most of the studies are based on vectors derived from the human adenovirus type 5; however, more than 50% ofthe adult human population has been already naturally infected and exhibits neutralizing antibodies against vector capsid proteins with consequent reduction of vector uptake and efficacy of transduction In order to circumvent this problem , it is possible to generate vectors from either alternative human serotypes or non-human adenoviruses We decided to follow the latter approach and tested several first generation adenoviral vectors expressing the E-GFP protein , based on different virus serotypes isolated from chimpanzee These vector serotypes were classified on the basis ofexon iper-variable region sequence In order to apply these vectors for transduction of leukemic cells, we analyzed their tropism on several leukemia cell lines determining E-GFP using a cytofluorimetric method We infected NB -4 (acute promyelocityc leukemia), HL60 and Kasumi I (acute leukemia myeloid) , RS4; II (acute Iinfoblastic leukemia), K562 (erythromyeloblastoid leukemia) cell lines with twelve chimp adenoviral serotypes; we used human adenoviral serotype and uninfected 293 cells as controls The adenoviraJ vector derived from the CI serotype, a subgroup B adenovirus, exhibited the best transduction efficacy in all infected cell lines when compared to other chimp adenovirus-derived vectors and the human serotype control vector In vivo infection pattern and stem cells transduction are currently being tested in order to obtain a more detailed assessment of the tropism of these vectors and, therefore, evaluate possible applications Haruna Sakurai.P Katsuhide Igarashi,' Katsuhisa Tashiro.t-' Kenji Kawabata, I Fuminori Sakurai,' Shinnosuke Kurachi,'> Shinsaku Nakagawa.' Ken-ichi Aisaki,' Jun Kanno," Hiroyuki Mizuguchi.P I Laboratory ofGene Transfer and Regulation, National Institute ofBiomedical Innovation, Ibaraki, Osaka, Japan; 2Gradllate School ofPharmaceutical Sciences, Osaka University, Suita Osaka, Japan; ' Dtvision ofToxicology, National Institute of Health Sciences, Biological Safety Research Center; Setagaya, TOkyo, Japan Adenovirus (Ad) vectors are highly efficient in transducing cells, however, they are thought to be immunogenic Intravenous injection of conventional Ad vectors induces innate immune response, such as the production oflL-6 and IL-12 Thus , elarification ofthe mechanism of innate immune response induced by Ad vector is essential for the establishment of the safe gene therapy Using DNA mieroarray, we searched factors which are involved in the innate immune response by Ad vectors in vitro and in vivo We normalized data from DNA mieroarray analysis by thc "Percellome" method, which represent the copy number of mRNA per template DNA At first, we analyzed the alteration of gene expressions in mouse peritoneal macrophages after infection of Ad vectors Since the conventional Ad vector has difficulty in infecting macrophages, AdRGD vectors (the fiber-mutant Ad vector with RGD peptide in the HI loop of fiber) as well as conventional Ad vectors were used for the infection Although only few genes were up-regulated at I hour after the Ad vector infection , 26 genes were up-regulated at hours after the infection ofAd vectors, including chemokines (CXCL 10, CCL4 etc) and interferon-inducible genes This result indicates S334 876 Tropism Analysis of Several PrimateDerived Adenoviral Vectors in Leukemia Cell Lines Barbara Lombardo.P Agostino Cirillo ," Carmine Cozzolino.F Barbara Izzo,' Paola Rinaldi,' Nicola Esposito," Paolo Morabito," Luigi Del Vecchio.t-' Fabrizio Pane.t-' Francesco Salvatore,'> Alfredo Nicosia,' Riccardo Cortese," Lucio Pastore, I.l Stefano Colloca.' 'Lab Terapia Genica e Cellulare, CEINGE-Biotecnologia Avanzate , Napoli, Nil, Italy; 'Dipantmemo di Biochimica e Biotecnologie Mediche, Universita degli Studi di Napoli Federico II, Napoli, NA, Italy; 'Molecular and Cellular Biology, IRBM, Pomezia, Rill, Italy; "Istituto Biostruttura e Bioimmagini, CNR, Napoli, NA, Italy; sDivisione di Immunoematologia, Ospedale A Cardarelli, Napoli, NA, Italy; 611• a., Okairos, Pomezia, RAI, Italy Molecular Therapy Yofume 15 Supplement I, \by 2007 Copyright © '111C AmericanSocietyo f Gene TIICr.lpr ... use of protein ligands that have been designed to satisfy both the structural and biosynthetic requirement of an ideal targeting ligand for Ad vectors SpecificalIy, successful targeting of Ad... the previously designed fiber-flbritin protein backbone and taking advantage of the unique structural and biosynthetic characteristics ofaffibodies, the efficacy of incorporation ofthe resultant... prototype for therapeutic and imaging gene vectors that peritoneal macrophages produce chemokines and activate IFN cascade by the infection ofAd vectors Next, we analyzed the profile of in vivo gene expression