874 design and characterization of her2 targeted adenovirus vectors

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 9 (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

characteriza-tion of the complex formacharacteriza-tion 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,' NatalyaBelousova.'Masato

Yamamoto,IMaaikeEverts.P David T Curiel.'

IDivision0/Human Gene Therapy Departments0/Medicine.

Obstetrics and Gynecology, Pathology Surgery and the Gene

TherapyCenter;University ofAlabama at Birmingham

Bir-mingham, AL; ]Division ofMolecular and Cellular Pathology;

Department a/Pathology, University0/Alabama at Birmingham.

Birmingham AL; 'Department ofExperimental Diagnostic

Imag-ing University a/Texas Houston TX.

Subgroup C Adenovirus (Ad) vectors arc being exploited for

cancer gcnc therapy because oftheir ability to provide high levels

oftransgene expression in infected cells However,since many types

of cancer cells lack or express reduced levels ofthe primary

recep-tor for subgroup C viruses [the coxsackie and adenovirus receprecep-tor

(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,

us-ing luciferase as a reporter gene The luciferase activities of these

chimeric viral vectors were higher than that ofthe 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 60-70times 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

do 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

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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 irnrnuniza-tion At the same time, strategies that allow repeat dosing have become increasingly important Recombinant human Adenovirus type 5 (rAd) has not realized its potential as a gene therapy or vac-cine vector in part because of pre-existing andinduced 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 manipulatingrAd vector doses on the induc-tion ofneutralizing antibodies (NAbs) and anti-transgene antibodies (Abs) by repeat administration of different doses of 1st generation EIIE3-deletedhumanAd5 Outbred Swiss Webster mice were given 10(6),10(7) or 10(8) IFU (infectious units) rAd.EGFP intranasally monthly for 4 months,and bled 2 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 vec-tor inocula Thus sera collected after I immunization with 10(8) infectious units (IFU) substantiallyneutralized the vector (P=.O15, 37% neutralization) while 3immunizations 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 4 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 2 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 2 doses at 10(8) IFU, after

3 doses at 10(7) IFU and after 4 doses at 10(6) In fact 10(6) IFU produced little or no Ab in 2 out 5 mice.Therefore anti-rAd-neu-tralizing 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 expres-sion or anti-transgene Abs after two or more doses

874 Design and Characterization of Her2-Targeted 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 suitable for therapeutic gene delivery have identified the lack oftarget tissue

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specificity as a major shortcoming ofthese agents One ofthe major

strategies used to overcome this deficiency is termed transductional

targeting Ituses the concept of redirecting the virus from its native

receptor, whose expression has not been linked to any known

pathol-ogy,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-bind-ing 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 intracellulartrafficking. 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

target-ing ligand for Ad vectors SpecificalIy, successful targettarget-ing 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 improve

-ments yielded an Ad vector whose infectivity in the Her2- and

CAR-expressing cells rivals that orthe virus that contains

unmodi-fied 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

875 Comprehensive Search for Factors

Involved in the Innate Immune Response Induced

by Adenovirus Vectors

Haruna Sakurai.P Katsuhide Igarashi,' Katsuhisa Tashiro.t-'Kenji

Kawabata,IFuminori Sakurai,' Shinnosuke Kurachi,'> Shinsaku

Nakagawa.' Ken-ichi Aisaki,' Jun Kanno," Hiroyuki Mizuguchi.P

ILaboratory ofGene Transfer and Regulation, National Institute

ofBiomedical Innovation, Ibaraki , Osaka, Japan; 2 Gradllate

School ofPharmaceutical Sciences , Osaka University, Suita.

Osaka, Japan; ' Dtvision ofToxicology, National Institute of

Health Sciences, Biological Safety ResearchCenter;Setagaya,

TOkyo , Japan.

Adenovirus (Ad) vectors are highly efficient in transducing cells,

however,they are thought to be immunogenic Intravenous

injec-tion of conveninjec-tional 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

es-sential 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 3 hours

after the infection ofAd vectors, including chemokines (CXCL 10,

CCL4 etc) and interferon-inducible genes This result indicates

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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

3 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 re-sults 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 stimulatevarious genes for the activation of innate immune responses

876 Tropism Analysis of Several Primate-Derived Adenoviral Vectors in Leukemia Cell Lines

Barbara Lombardo.PAgostino 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.lStefano Colloca.'

'Lab Terapia Genica e Cellulare, CEINGE -Biotecnologia

Avanzate , Napoli , Nil, Italy;'Dipantmemodi 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.

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 al-ready 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 leuke-mia) cell lines with twelve chimp adenoviral serotypes;we used human adenoviral serotype 5 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 5 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

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