Patent Application: US-64644903-A

Abstract:
adenoviral vectors can be used in vaccines to cause antigen - presenting cells to display desired antigens . disclosed is a vector and associated methods that transduce antigen - presenting cells better than currently available vectors , enabling the vector to be delivered in lower doses , and thus improves the efficiency of adenoviral vaccine technology .

Description:
a gene delivery vehicle according to the invention preferably has at least one of the protein fragments comprising a tissue tropism determining fragment of a fiber protein derived from a subgroup b adenovirus . preferably , at least one of the protein fragments comprises a tissue tropism determining fragment of a fiber protein derived from a subgroup b adenovirus . a still more preferred gene delivery vehicle has at least one of the protein fragments comprising a tissue tropism determining fragment of a fiber protein derived from a subgroup b adenovirus , such as adenovirus 16 . in one embodiment , however , a gene delivery vehicle according to the invention further includes protein fragments derived from an adenovirus of subgroup c . also , the gene delivery vehicle can include a nucleic acid derived from one or more adenovirus . in one embodiment , the gene delivery vehicle according to the invention has a nucleic acid comprising at least one sequence encoding a fiber protein comprising at least a tissue tropism determining fragment of a subgroup b adenovirus fiber protein , preferably of adenovirus 16 . furthermore , the adenovirus nucleic acid can be modified such that the capacity of the adenoviral nucleic acid to replicate in a target cell has been reduced or disabled or the adenoviral nucleic acid can be modified so that the capacity of a host immune system to mount an immune response against adenoviral proteins encoded by the adenovirus nucleic acid has been reduced or disabled . a gene delivery vehicle according to the invention can comprise a minimal adenovirus vector or an ad / aav chimeric vector and can comprise at least one non - adenovirus nucleic acid . to load dcs with immunostimulatory proteins or peptides to become therapeutically feasible at least two distinct criteria have to be met . first , the isolation of large numbers of dcs that can be isolated , manipulated , and re - infused into a patient , making the procedure autologous . to date , it is possible to obtain such large quantities of immature dcs from cultured peripheral blood monocytes from any given donor . second , a vector that can transduce dcs efficiently such that the dna encoding for an immunostimulatory protein can be delivered . the latter is extremely important since it has become clear that the time required for dcs to travel to the lymphoid organs is such that most proteins or peptides are already released from the dcs , resulting in incomplete immune priming . because dcs are terminally differentiated and thus non - dividing cells , recombinant adenoviral vectors are being considered for delivering the dna encoding for antigens to dcs . ideally , this adenovirus should have a high affinity for dendritic cells , but should also not be recognized by neutralizing antibodies of the host such that in vivo transduction of dcs can be accomplished . the latter would obviate the need for ex vivo manipulations of dcs but would result in a medical procedure identical to the vaccination programs that are currently in place , i . e ., intramuscular or subcutaneous injection predominantly . thus , dendritic cells transduced by adenoviral vectors encoding an immunogenic protein may be ideally suited to serve as natural adjuvants for immunotherapy and vaccination . efficient gene delivery to dcs is a major interest in the field of gene therapy . therefore , alteration of the ad5 host cell range to be able to target dcs in vitro as well as in vivo is a major interest of the invention . to identify a chimeric adenovirus with preferred infection characteristics for human dcs , we generated a library of ad5 based viruses carrying the fiber molecule from alternative serotypes ( serotypes 8 , 9 , 13 , 16 , 17 , 32 , 35 , 45 , 40 - l , 51 ). ad5 was included as a reference . as more thoroughly herein , the susceptibility of human monocyte derived immature and mature dendritic cells to recombinant chimeric adenoviruses expressing different fibers was evaluated . the invention is further explained by the use of the following illustrative examples : an ad5 / fiber35 chimeric vector with cell type specificity for dendritic cells . human pbmc from healthy donors were isolated through ficoll - hypaque density centrifugation . monocytes were isolated from pbmc by enrichment for cd14 + cells using staining with fitc labeled anti - human cd 14 monoclonal antibody ( becton dickinson ), anti - fitc microbeads , and macs separation columns ( miltenyi biotec ). this procedure usually results in a population of cells that are & lt ; 90 % cd14 + as analyzed by facs . cells were placed in culture using rpmi - 1640 medium ( gibco ) containing 10 % foetal bovine serum (“ fbs ”) ( gibco ), 200 ng / ml rhu gm - csf ( r & amp ; d / itk diagnostics , 100 ng / ml rhu il - 4 ( r & amp ; d / itk diagnostics ) and cultured for 7 days with feeding of the cultures with fresh medium containing cytokines on alternate days . after 7 days , the immature dendritic cells resulting from this procedure express a phenotype cd83 − , cd14 low or cd14 − , hla - dr + , as was demonstrated by facs analysis . immature dcs were matured by culturing the cells in a medium containing 100 ng / ml tnf - a for 3 days , after which , they expressed cd83 on their cell surface . 5 × 10 5 immature dcs were seeded in wells of 24 - well plates and exposed for 24 hours to 100 and 1000 virus particles per cell of each fiber recombinant virus . virus tested was ad5 , and the fiber chimeric viruses based on ad5 : ad5 . fib12 , ad5 . fib16 , ad5 . fib28 , ad5 . fib32 , ad5 . fib40 - l ( long fiber of serotype 40 ), ad5 . fib49 , and ad5 . fib51 ( where fibxx stands for the serotype from which the fiber molecule is derived ). these viruses are derived from subgroup c , a , b , d , d , f , d , and b respectively . after 24 - hours , cells were lysed ( 1 % triton x - 100 / pbs ) and luciferase activity was determined using a protocol supplied by the manufacturer ( promega , madison , wis ., usa ). the results of this example , shown in fig1 demonstrate that ad5 poorly infects immature dcs as evidenced by the low level of transgene expression . in contrast , ad5 . fib16 and ad5 . fib51 ( both a b - group fiber chimeric virus ) and also ad5 . fib40 - l ( subgroup f ) show efficient infection of immature dcs based on luciferase transgene expression . in a second experiment , 5 × 10 5 immature and mature dendritic cells were infected with 10 , 000 virus particles per cell of ad5 , ad5 . fib16 , ad5 . fib40 - l , and ad5 . fib51 all carrying the lacz gene as a marker . lacz expression was monitored by flow cytometric analysis using a cm - fdg kit system and the instructions supplied by the manufacturer ( molecular probes , leiden , nl ). the results of this experiment , shown in fig2 correlate with the previous experiment in that ad5 . fib16 and ad5 . fib51 are superior to ad5 in transducing mature and immature human dcs . also , this example shows that ad5 . fib40 - l is not as good as ad5 . fib16 and ad5 . fib51 , but is better than ad5 . based on the earlier examples , we tested other chimeric adenoviruses containing fibers of b group viruses , for example , ad5 . fib11 and ad5 . fib35 for their capacity to infect dcs . we focused on immature dcs , since these are the cells that process an expressed transgene product into mhc class i and ii presentable peptides . immature dc &# 39 ; s were seeded at a cell density of 5 × 10 5 cells / well in 24 well plates ( costar ) and infected with 1 , 000 and 5 , 000 virus particles per cell after which the cells were cultured for 48 hours under conditions for immature dcs prior to cell lysis and luciferase activity measurements . the results of this example , shown in fig3 demonstrate that ad5 based chimeric adenoviruses containing fibers of group - b viruses efficiently infect immature dcs . in a fourth experiment , we again infected immature dcs identically as described in the former experiments but this time ad5 , ad5 . fib16 , and ad5 . fib35 were used carrying gfp as a marker gene . the results on gfp expression measured with a flow cytometer 48 hours after virus exposure are shown in fig4 and correlate with the data obtained so far . thus , the results so far are consistent in that ad5 based vectors carrying a fiber from an alternative adenovirus derived from subgroup b predominantly fiber of 35 , 51 , 16 , and 11 are superior to ad5 for transducing human dcs . the adenoviruses disclosed herein are also very suitable for vaccinating animals . to illustrate this , we tested dcs derived from mice and chimpanzees to identify whether these viruses could be used in these animal models . chimpanzees in particular , since the receptor for human adenovirus derived from subgroup b is unknown to date and therefore it is unknown whether this protein is conserved among species . for both species , immature dcs were seeded at a density of 10 5 cells per well of 24 - well plates . cells were subsequently exposed for 48 hours to 1000 virus particles per cell of ad5 , ad5fib16 , and ad5 . fib51 in case of mouse dendritic cells and ad5 , and ad . fib35 in case of chimpanzee dcs ( see , fig5 ). the mouse experiment was performed with viruses carrying luciferase as a marker , and demonstrated approximately 10 - 50 fold increased luciferase activity as compared to ad5 . the chimpanzee dcs were infected with the gfp viruses , and were analyzed using a flow cytometer . these results ( also shown in fig5 ) demonstrate that ad5 ( 3 %) transduces chimpanzee dcs very poorly in comparison to ad5 . fib35 ( 66 . 5 %). immature dcs were incubated with ad5 . luc or with the fiber - modified vectors at a virus dose of 10 5 virus particles per dc . luciferase transgene expression was determined 24 hours after virus exposure . results are a representative of two independent experiments performed with dc derived from 2 different individuals , and depicted graphically in fig6 . results are expressed in relative light units (“ rlu ”) per 10 4 dc versus recombinant fiber modified vector . following - up on example v , immature dcs were incubated with ad5 . gfp or ad5fib16 . gfp , ad5fib35 . gfp , ad5fib40 - l . gfp , or ad4 . fib51 . gfp . different dosages were used : 10 3 , 10 4 , or 10 5 virus particles per dc ( white bar , grey bar , or black bar respectively in fig7 ). gfp expression was determined 24 hours after virus exposure . fig7 expresses the results in graph a as percentage gfp positive cells and , in graph b , as median fluorescence intensity . results shown are derived from 3 independent experiments performed with dc derived from 3 different individuals . immature dc were treated for 48 hours with lps to allow maturation of the dc . matured dcs were incubated with ad5 . gfp or ad5fib16 . gfp , ad5fib35 . gfp , ad5fib40 - l . gfp , or ad4 . fib51 . gfp . dosages used were 10 3 , 10 4 , or 10 5 virus particles per dc ( depicted as white , grey , and black bars respectively in fig8 ). gfp expression was determined 24 hours after virus exposure . in fig8 results are expressed as ( a ) percentage gfp positive cells and ( b ) median fluorescence intensity ( b ). results shown are derived from 3 independent experiments performed with dc derived from 3 different individuals . immature dendritic cells were exposed for 48 hours to different maturation agents before being exposed to various viruses . the virus dosage used was 10 4 virus particles per dc . gfp expression was determined 24 hours after virus exposure . the results are expressed as percentage gfp positive cells . results shown are representative of 2 independent experiments . the results are graphically depicted in fig9 . the maturation agents used were lps ( black bars ), tnf - a ( white with black dots ), mcm ( diagonal downward ), poly i : c ( black with white dots ), and anti - cd40 antibodies ( diagonal upwards ). as a negative control for a maturation marker , ifn - a ( grey bars ) was used , while immature dendritic cells were used as a general control ( white bars ). dendritic cells were exposed to 10 4 virus particles per cell where various vectors ( f5 , f16 , f35 , and f51 ) were used . the results are graphically depicted in fig1 , wherein , in ( a ), the percentage gfp positive cells detected is depicted , in ( b ) the median fluorescence intensity is depicted and , in ( c ), cells that were frozen and genomic dna extracted to quantify the number of adenoviral genomes using real - time pcr . the dc types were immature dc ( white bar ), mature dc ( black bar ), or immature dc transduced and subsequently matured using lps . cells were analyzed for gfp expression 48 hours after virus exposure . the data are representative for two independent experiments . as graphically depicted in fig1 , immature dendritic cells were transduced with 10 5 , 10 4 or 10 3 virus particles ( top , middle , and bottom graphs respectively ) of ad5 . gp100 or ad5 . fib35 . gp100 ( white squares and circles , respectively ). likewise , matured dc were transduced with 10 5 , 10 4 or 10 3 virus particles of ad5 . gp100 or ad5 . fib35 . gp100 ( black squares and circles respectively ). transduced dc ( 10 4 cells ) were cultured with the hla - 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