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[Cancer Research 62, 609-616, January 15, 2002]
© 2002 American Association for Cancer Research

Virology

Adenovirus Targeting to c-erbB-2 Oncoprotein by Single-Chain Antibody Fused to Trimeric Form of Adenovirus Receptor Ectodomain1

Elena A. Kashentseva, Toshiro Seki, David T. Curiel and Igor P. Dmitriev2

Division of Human Gene Therapy [E. A. K., T. S., D. T. C., I. P. D.], Departments of Medicine, Pathology, and Surgery [D. T. C.], and Gene Therapy Center [D. T. C.], University of Alabama at Birmingham, Birmingham, Alabama 35294-3300


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of adenovirus (Ad) vectors for cancer gene therapy applications is currently limited by several factors, including broad Ad tropism associated with the widespread expression of coxsackievirus and adenovirus receptor (CAR) in normal human tissues, as well as limited levels of CAR in tumor cells. To target Ad to relevant cell types, we have proposed using soluble CAR (sCAR) ectodomain fused with a ligand to block CAR-dependent native tropism and to simultaneously achieve infection through a novel receptor overexpressed in target cells. To confer Ad targeting capability on cancer cells expressing the c-erbB-2/HER-2/neu oncogene, we engineered a bispecific adapter protein, sCARfC6.5, that consisted of sCAR, phage T4 fibritin polypeptide, and C6.5 single-chain fragment variable (scFv) against c-erbB-2 oncoprotein. Incorporation of fibritin polypeptide provided trimerization of sCAR fusion proteins that, compared with monomeric sCAR protein, resulted in augmented affinity to Ad fiber knob domain and in increased ability to block CAR-dependent Ad infection. We demonstrated that sCARfC6.5 protein binds to cellular c-erbB-2 oncoprotein and mediates efficient Ad targeting via a CAR-independent pathway. As illustrated in cancer cell lines that overexpress c-erbB-2, targeted Ad, complexed with sCARfC6.5 adapter protein, provided from 1.5- to 17-fold enhancement of gene transfer compared with Ad alone and up to 130-fold increase in comparison with untargeted Ad complexed with sCARf control protein. The use of recombinant trimeric sCAR-scFv adapter proteins may augment Ad vector potency for targeting cancer cell types.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ad3 represents a large family of nonenveloped viruses (1) . Human Ad includes 47 known viral serotypes grouped into six distinct subgroups, A to F. Most of the studies on the mechanism of Ad infection have concluded that receptor recognition is one of the key factors that determines cell tropism (2 , 3) . In this regard, the initial steps of Ad infection involve at least two sequential virus-cell interactions, each mediated by a specific viral capsid protein. Ad infection is initiated by the binding of globular knob domain of trimeric fiber protein to a host cell primary receptor (4 , 5) . Subsequent interaction of the penton base with {alpha}v integrins mediates virion internalization via receptor-mediated endocytosis (6) . Fiber receptor for Ad subgroups A, C, D, E, and F has been identified as the CAR (7, 8, 9) . CAR is an integral membrane protein consisting of two extracellular immunoglobulin-like D1 and D2 domains, a transmembrane region, and a COOH-terminal cytoplasmic domain (8 , 10) . The extracellular domain of CAR is sufficient for virus attachment and infection (11 , 12) , whereas both transmembrane and intracellular regions appear to be dispensable for these functions (13) . Both structural analysis of fiber knob complexed with CAR D1 domain (14) and knob mutagenesis studies (15) revealed that amino acid residues responsible for CAR binding are located on lateral surfaces formed by the interface of two adjacent knob monomers. These data suggest an avidity mechanism when three CAR molecules could simultaneously bind per one fiber knob trimer, which was recently supported by kinetic analysis of Ad2 knob binding to the CAR D1 domain (16) .

Well-characterized Ad serotypes 2 and 5 from subgroup C are predominantly used as vectors for in vitro and in vivo gene delivery (17) , because of high infection efficiency in a variety of human cell types and tissues. However, this broad viral tropism is disadvantageous for gene delivery to cancer cell types refractory to Ad infection because of the absence or low levels of CAR expression (18, 19, 20, 21) . This limitation could be solved by Ad targeting via a nonnative viral receptor (22 , 23) . Several strategies have been tested in an effort to target Ad via CAR-independent pathways (24) including chemical conjugation or genetic modification of viral capsid proteins to incorporate targeting ligands and the use of bispecific adapter molecules to provide indirect virus linkage with the cell-surface receptors. The technical achievement of Ad targeting via adapter molecules has been approached by a variety of methods. Bispecific conjugates of antibodies or their Fab fragments were used to achieve linkage between target receptor and v.p. by means of specific recognition through either a fiber knob domain or penton base (reviewed in Refs. 17 , 22 , 23 ). Further refinement of this strategy has been accomplished by the engineering of recombinant proteins consisting of an anti-knob scFv fused with human EGF (25) or a scFv against EGFR (26) . The original concept of employment of fusion proteins comprising a soluble viral receptor and targeting ligand was proposed for retrovirus targeting to specific cell types (27) . Applying this strategy to Ad targeting, we have developed an approach based on the use of sCAR ectodomain fused with EGF, achieving simultaneously the blocking of virus-CAR interaction and the redirection of Ad to cells overexpressing EGFR (28 , 29) . A similar approach was successfully applied to target Ad to high-affinity Fc{gamma} receptor I-positive human monocytic cells (30) . The use of recombinant adapter molecules eliminates chemical conjugation and provides a high degree of flexibility for ligand substitution and, consequently, expands the targeting capabilities of Ad vectors.

We hypothesized that the predicted 3:1 stoichiometry of CAR-knob binding could provide high-affinity linkage of trimeric sCAR-ligand proteins to v.p. and, thereby, promote the ligand-mediated binding to target receptors. In this study, we describe a novel approach of Ad transductional targeting to cancer cell types expressing c-erbB-2 oncoprotein by means of a recombinant protein adapter. The gene known as c-erbB-2/HER-2/neu, encoding a member of the erbB family of growth factor receptors, is most frequently altered in human cancer and was shown overexpressed in a number of malignancies including tumors that arise in the breast and ovary (31 , 32) . We engineered a bispecific protein, sCARfC6.5, featuring a unique trimeric design and consisting of sCAR fused with phage T4 fibritin polypeptide and C6.5 scFv against c-erbB-2. We have demonstrated that the sCARfC6.5 protein efficiently blocks Ad native tropism while simultaneously mediating virus infection via an alternative CAR-independent pathway, which markedly enhances gene transfer efficiency to cell lines that overexpress c-erbB-2. Our data suggest that the use of this original approach may augment the potency of Ad vectors for cancer gene therapy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Media.
The 293 human kidney cell line, transformed with Ad5 DNA, was purchased from Microbix (Toronto, Ontario, Canada). The human breast cancer cell lines MDA-MB-468, AU-565, SK-BR-3, BT-474, and MCF-7 and the ovarian cancer cell line SK-OV-3, established from adenocarcinomas of mammary gland and ovary, respectively, were from the American Type Culture Collection (Manassas, VA). All of the cell lines were maintained in recommended growth media supplied by Mediatech (Herndon, Va.) containing 10% FBS (HighClone, Logan, UT) and 2 mM glutamine at 37°C in a humidified atmosphere of 5% CO2. Infection of the cells with Ad was carried out in the infection medium containing 2% FBS.

Enzymes.
Restriction endonucleases, Klenow enzyme, T4 DNA ligase, and proteinase K were from either New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN).

Antibodies.
Murine serum to baculovirus-produced human sCAR protein was generated at the University of Alabama at Birmingham Hybridoma Core Facility. The MAb RmcB (33) to human CAR were produced using hybridoma purchased from American Type Culture Collection and kindly provided by J. T. Douglas (University of Alabama at Birmingham). Penta·His MAbs were from Qiagen Inc. (Valencia, CA). Rabbit serum against phage T4 fibritin protein was kindly provided by V. Mesyanzhinov (Shemykin and Ovchinnicov Institute of Bioorganic Chemistry, Moscow, Russia). Mouse MAbs to the human c-erbB-2/HER-2/neu oncoprotein, Ab-2 (Clone9G6.10), were purchased from NeoMarkers Inc., (Fremont, CA). Normal mouse IgG1 were from OEM Concepts (Toms River, NJ). Goat antimouse and antirabbit IgG conjugated with alkaline phosphatase were from Sigma Chemical Co. (St Louis, MO) and Pierce (Rockford, IL), respectively. Streptavidin-alkaline phosphatase conjugate was from Bio-Rad Laboratories (Hercules, CA). Alexa 488-labeled goat antimouse IgG were from Molecular Probes (Eugene, OR).

Viruses.
A recombinant Ad5 vector, AdLucGFP, containing double expression cassette consisting of firefly luciferase gene and GFP gene under the control of cytomegalovirus immediate early promoter in place of the E1 region of the Ad genome, was constructed as described by Seki et al. (34) . Ad was propagated on 293 cells and purified by centrifugation in CsCl gradients by a standard protocol. The titers of physical v.p. and infectious v.p. were determined by using the methods of Maizel et al. (35) and Mittereder et al. (36) , respectively.

Construction of Recombinant Plasmids.
To generate the recombinant gene encoding the extracellular domain of human CAR followed by polypeptide sequence derived from bacteriophage T4 fibritin protein (37) , PCR was used. Sense primer 5'-GTT GAA AGA TCT GGA TTA ACC AAT AAA ATA AAA GCT ATC GAA ACT GAT ATT GCA TCA G complementary to position 1240 of the fibritin gene was designed to introduce BglII restriction site into the amplified DNA sequence, and antisense primer 5'-TTG CGG CCC CAG CGG CCG CTG GTG ATA AAA AGG TAG complementary to position 16 of untranslated 3'-region was designed both to introduce NotI restriction site and to substitute a stop codon for an alanine (GCC) codon. The PCR fragment (238 bp) was digested with BglII and NotI, and a 214-bp DNA fragment encoding 71 COOH-terminal amino acids of fibritin M polypeptide (38) was purified. A BglII-NotI-fragment was ligated with BamHI- and NotI-digested plasmid pFBshCAR-EGF (29) that contained the recombinant gene for the CAR ectodomain, His6, short linker, and human EGF to substitute EGF for the fibritin sequence. This plasmid, designated pFBsCARfibritin, was then cleaved with NotI and ligated with oligonucleotide duplex 5'-GGC CCA ACC GCA GCC AAA ACC TCA ACC CCA GCC ACA ACC TCA GCC CAA ACC TCA GCC TAA ACC GGT TTA AAC GGC C coding for a proline-rich hinge region that was derived from camel immunoglobulins and containing an AgeI site followed by a stop codon. A plasmid clone that contained the DNA duplex in the correct orientation was selected by sequencing and was designated pFBsCARfCh. The resultant plasmid was then used as a vector to generate the recombinant baculovirus using the Bac-to-Bac baculovirus construction system (Life Technologies, Inc., Grand Island, N.Y.) to express sCARf protein. Then, oligonucleotides 5'-CCG GGA GCT CTG CGC TAG CT and 5'-CCG GAG CTA GCG CAG AGC TC, designed to contain SacI and NheI restriction sites and AgeI-compatible cohesive 5'-ends, were annealed to form duplex DNA and ligated to AgeI-digested pFBsCARfCh. Plasmid clones were sequenced, and the plasmid containing the DNA duplex in the correct orientation was designated pFBsCARfChSN. DNA sequence coding for C6.5 scFv against c-erbB-2 was PCR amplified from cDNA (provided by J. D. Marks, Department of Anesthesia and Pharmaceutical Chemistry, University of California, San Francisco, CA) using primers 5'-AGG AAA CCG GTG GTC TAG ATC AGG TGC AGC and 5'-AGT ATC TAG AGG GAA CTA GTA CGG TCA GCT TGG TCC CTC, which were designed to introduce XbaI and SpeI restriction sites, respectively. The PCR product was digested with XbaI and SpeI, and a purified 769-bp DNA fragment was cloned into SpeI-cleaved pFastBacHTa (Life Technologies, Inc.), which resulted in plasmid pFB6hC6.5. Then, plasmid pFBsCARfChSN was digested with SacI and NheI and ligated with 775-bp SacI-SpeI-fragment DNA, coding for C6.5 scFv isolated from pFB6hC6.5. The constructed plasmid, containing recombinant gene encoding sCAR, His6, short linker, fibritin polypeptide, hinge region, and C6.5 scFv, was sequenced to confirm the correct DNA structure. The resultant plasmid, designated pFBsCARfC6.5, was then used to generate the recombinant baculovirus using the Bac-to-Bac system.

Expression, Purification, and Biotinylation of the Fusion Proteins.
The fusion proteins, sCARf and sCARfC6.5, comprised of sCAR-His6-fibritin and sCAR-His6-fibritin-hinge-C6.5scFv polypeptide sequences, respectively, were expressed in High Five cells (Invitrogen, Carlsbad, CA) that were infected with recombinant baculoviruses. Recombinant His6-tagged proteins were purified from dialyzed culture medium by immobilized metal-ion-affinity chromatography on Ni-nitrilotriacetic acid (Ni-NTA)-Sepharose (Qiagen Inc.) as described previously (29) . Protein concentrations were determined by the BCA-200 protein assay kit using bovine gamma globulin as the standard (Pierce). Purified sCARf and previously produced sCAR-His6 (29) proteins were biotinylated using EZ-Link SulfoNHS-LS-Biotinylation kit (Pierce). The degree of biotin-protein incorporation [determined using HABA method (Pierce)] was 0.6 biotin per molecule of sCAR-His6 monomeric protein and 0.5 biotin per trimeric molecule of sCARf protein.

Protein Electrophoresis and Western Blot.
To determine whether the recombinant sCARf and sCARfC6.5 fusion proteins could form trimers, they were analyzed by SDS-PAGE. Purified proteins were either boiled in Laemmli loading buffer prior to electrophoresis to denature proteins to monomers or loaded on the gel without denaturation. The trimeric or monomeric configurations of protein molecules were determined based on their mobilities in the gel. To analyze the composition of sCAR fusion proteins, we used Western blot. Samples of boiled sCARfC6.5 and sCARf proteins separated on 4–15% gradient SDS-PAGE were transferred to polyvinylidene difluoride membrane and probed with murine anti-sCAR serum, Penta·His MAb, or rabbit antifibritin serum. Bound IgG were detected with secondary alkaline phosphatase-conjugated antibodies.

ELISA.
Solid-phase binding ELISA was performed as follows. Recombinant Ad5 knob protein, expressed in Escherichia coli as described previously (39) , was diluted in 50 mM NaHCO3 (pH 9.6) at a concentration of 1 µg/ml and was immobilized on Nunc-Maxisorp ELISA plate overnight. The wells were blocked with PBS [10 mM NaH2PO4, 10 mM KH2PO4 (pH 7.4), and 136 mM NaCl] containing 0.05% Tween 20 and 2% BSA and then were washed with PBS containing 0.05% Tween 20. Biotinylated sCAR-His6 and sCARf proteins, diluted in blocking buffer to concentrations ranging from 0.01 to 25 pmol/ml, were added to the wells in 100-µl aliquots. After a 1-h incubation at room temperature, the wells were washed, and bound biotinylated proteins were detected by 45-min incubation with 1:1000 dilution of streptavidin-alkaline phosphatase conjugate (Bio-Rad). The plates were then developed using signal-producing reagent p-nitrophenyl phosphate (Sigma Chemical Co.). Plates were read in a microtiter plate reader, set at 405 nm; results are presented as mean absorbance ± SD.

Indirect Immunofluorescence.
The analysis of cell lines for expression levels of CAR and c-erbB-2 oncoprotein was performed by indirect immunofluorescence assay using flow cytometry as follows. Aliquots (100 µl) of cells, resuspended in FACS buffer [10 mM NaH2PO4, 10 mM KH2PO4 (pH 7.4), 136 mM NaCl, 1% BSA, and 0.1% NaN3] at a concentration of 2 x 106 cells/ml were incubated with either RmcB (anti-CAR) or Ab-2 (anti-c-erbB-2) MAb at a concentration of 5 µg/ml for 1 h at 4°C. An isotype-matched normal mouse IgG1 was used as a negative control. Cells were washed with FACS buffer by centrifugation and then were incubated with secondary Alexa 488-labeled goat antimouse antibody (Molecular Probes) at a concentration of 5 µg/ml for 1 h at 4°C. Cells were washed with FACS buffer prior to flow cytometry analysis. To validate that C6.5 scFv, incorporated in the context of sCARfC6.5 protein, are able to bind to cellular c-erbB-2, cells were incubated first with sCARfC6.5 or with sCARf protein as a negative control at a concentration of 10 µg/ml. After a 1-h incubation, cells were washed and incubated with primary RmcB MAb and then with secondary Alexa 488-labeled antibody as described above. Cell samples (104 cells/sample) were analyzed by flow cytometry performed at the University of Alabama at Birmingham FACS Core Facility. Data were expressed as the geometric mean fluorescence intensity of the entire gated population. The positive cell population was determined by gating the right-hand tail of the distribution of the negative control sample for each cell line at 1%.

Gene Transfer Assay.
The assay of Ad-mediated gene transfer to the cells was performed as follows. Aliquots (3 µl) of AdLucGFP vector were mixed with 6-µl aliquots of sCARf or sCARfC6.5 protein dilutions ranging from 0.2 to 53 pmol or of PBS [10 mM NaH2PO4, 10 mM KH2PO4 (pH 7.4), and 136 mM NaCl] for 15 min at room temperature. The virus-sCARprotein-containing complexes were diluted to 1 ml with infection medium containing 2% FBS, and 200-µl aliquots were then added to the cell monolayers [grown in a 24-well plate (3–5 x 105 cells/well) at MOI of 100 v.p./cell] and were incubated for 30 min at room temperature to allow virus internalization. Then, infection medium was aspirated, the cells were washed with PBS, and the cells were incubated in a growth medium containing 10% FBS at 37°C to allow expression of the reporter genes. Forty-six h postinfection, cells were lysed and luciferase activity was analyzed by using the Promega (Madison, WI) luciferase assay system and a Berthold (Gaithersburg, MD) luminometer. For inhibition of the Ad infection of 293 cells AdLucGFP vector was mixed with sCARf or sCARfC6.5 protein dilutions (1.1–30 pmol), or with sCAR-His6 protein dilutions (3–230 pmol), or with PBS for 15 min at room temperature. Monolayers of 293 cells were exposed to the virus-sCARprotein-containing complexes at MOI of 13 v.p./cell for 30 min and then were incubated for an additional 20 h at 37°C to allow expression of luciferase gene prior to analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Design and Generation of sCAR Fusion Proteins.
To exploit the trivalent nature of CAR-knob interaction for the purposes of Ad targeting, we engineered a recombinant adapter protein consisting of soluble CAR in fusion with a trimerization sequence and a targeting ligand (Fig. 1A)Citation . We hypothesized that the predicted 3:1 stoichiometry of CAR-knob binding could provide high-affinity linkage of trimeric sCAR-ligand adapter proteins to virus and thereby block CAR-dependent Ad infection. Our goal was to generate a trimeric sCAR-ligand protein capable of efficiently blocking Ad native tropism while providing a novel target-selective tropism to c-erbB-2-positive cells (Fig. 1B)Citation . Because of the absence of a specific cognate ligand for c-erbB-2 oncoprotein, we chose C6.5 scFv as a targeting moiety that binds to the extracellular domain of this tumor antigen (40) .



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  		Fig. 1. A, design of sCARfC6.5 fusion protein. The recombinant fusion gene encoding the human CAR ectodomain, His6, polypeptide derived from phage T4 fibritin protein (Fibritin); proline-rich hinge region (Hinge), and C6.5 scFv to c-erbB-2 oncoprotein was constructed in a baculovirus expression vector. The sequence encoding fibritin polypeptide was introduced into the design to achieve sCARfC6.5 protein trimerization. Recombinant gene was expressed in baculovirus-infected insect cells and secreted sCARfC6.5 protein was purified. B, use of sCARfC6.5 protein for Ad targeting. Engineered bispecific sCARfC6.5 fusion protein serves as an adapter between Ad and cells expressing c-erbB-2 oncoprotein. Presence of three sCAR domains in the context of trimerized adapter molecule potentially provides high-affinity viral linkage because of the trivalent stoichiometry of CAR-knob binding. The use of trimeric sCARfC6.5 adapter protein might, therefore, allow efficient blocking of Ad infection of CAR-bearing cells. C6.5 scFv targeting moiety of virus-bound adapter protein mediates recognition of c-erbB-2-positive cells, thereby providing novel target-specific Ad tropism.

 
The gene encoding the extracellular part of human CAR including secretion signal, six-histidine tag (His6), flexible linker, trimerization domain, hinge region, and C6.5 scFv was designed to produce sCARfC6.5 targeting protein. To achieve trimerization of the sCAR fusion molecule, we used a polypeptide derived from bacteriophage T4 fibritin protein, which is known to form highly stable homotrimers (37) . Genetically engineered fibritin M polypeptide (38) , containing 71 amino acids corresponding to the last {alpha}-helical coiled-coil segment and the complete COOH-terminal domain of phage T4 fibritin protein, was used. A proline-rich hinge region (20 amino acids) derived from camel antibodies served as a junction between the fibritin polypeptide and downstream scFv. The gene encoding sCAR, His6, linker, fibritin M, and hinge region was used to produce sCARf control protein. The constructed genes for sCARf and sCARfC6.5 proteins were expressed in a baculovirus expression system that has already proved its utility for the expression of functional sCAR (9) and chimeric sCAR-EGF (29) . Both of the fusion proteins were produced in a secreted soluble form after infection of High Five insect cells with generated recombinant baculoviruses. Secreted sCARf and sCARfC6.5 His6-tagged proteins were purified and were analyzed for the presence of encoded polypeptide sequences and trimerization.

Characterization of Recombinant sCAR Fusion Proteins.
The polypeptide composition of produced fusion proteins was characterized by Western blot analysis. Detection of denatured electrophoretically resolved sCARf and sCARfC6.5 proteins using specific antibodies revealed the presence of the CAR ectodomain, His6 tag, and fibritin sequences in the context of both of the fusion proteins (Fig. 2A)Citation . The incorporation of scFv sequence in sCARfC6.5 protein was confirmed by the shift of its electrophoretic mobility compared with sCARf, indicating the predicted 27 kDa increase of molecular mass. The presence of additional minor bands in the sample of sCARfC6.5 protein was likely the result of incomplete translation of sCARfC6.5 mRNA. To determine whether the recombinant secreted sCARf and sCARfC6.5 proteins form trimers, these proteins were analyzed by SDS-PAGE. Electrophoresis of denatured protein samples showed the presence of major bands with molecular masses close to 36 and 63 kDa as expected for monomeric forms of sCARf and sCARfC6.5 molecules, respectively (Fig. 2B)Citation . Electrophoretic mobility of nondenatured protein samples was greatly decreased compared with denatured proteins as was predicted for trimeric forms of sCARf and sCARfC6.5 molecules. This demonstrated that incorporation of fibritin polypeptide in the context of these recombinant fusion proteins results in efficient trimerization of both sCARfC6.5 and sCARf control protein. Thus, the analysis of sCARf and sCARfC6.5 proteins indicates that generated sCAR fusion proteins maintain both designed composition and stable trimeric conformation.



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  		Fig. 2. Characterization of sCAR fusion proteins. A, Western blot of sCAR fusion proteins. Samples of purified sCARfC6.5 (Lanes 2, 4, and 6) and sCARf (Lanes 3, 5, and 7) proteins were boiled in Laemmli loading sample buffer and separated on 4–15% gradient SDS-PAGE. Electrophoretically resolved proteins were transferred to polyvinylidene difluoride membrane and probed with murine anti-sCAR serum (Lanes 2 and 3), Penta·His MAb (Lanes 4 and 5), or rabbit anti-fibritin serum (Lanes 6 and 7). Bound murine or rabbit IgG were detected with secondary alkaline phosphatase-conjugated goat antimouse or antirabbit antibodies, respectively. Numbers on the left, molecular masses of marker proteins (Lane 1) in kilodaltons. B, trimerization analysis of sCAR fusion proteins. Samples of sCARfC6.5 (Lanes 1 and 2) and sCARf (Lanes 3 and 4) proteins and molecular mass marker (Lane 5) were separated on 4–15% gradient SDS-PAGE. The samples in Lanes 2 and 4 were boiled in Laemmli loading buffer to denature proteins to monomers, whereas proteins in Lanes 1 and 3 were not denatured by boiling. Protein bands were visualized by GELCODE blue stain reagent. Numbers on the right, molecular masses of marker proteins in kilodaltons.

 
Analysis of sCAR Fusion Proteins Binding to Ad Fiber Knob.
We characterized trimeric sCARf protein with respect to its ability to bind Ad fiber knob compared with monomeric sCAR-His6 protein generated previously (29) . The knob-binding affinities of sCARf and sCAR-His6 proteins were compared by ELISA using immobilized Ad5 knob expressed in E. coli (39) . Compared with monomeric sCAR-His6 protein, the knob-binding affinity of trimeric sCARf protein was increased at least 20-fold in a range of tested concentrations from 0.1 to 5 pmol/ml (Fig. 3A)Citation . This result suggests that, compared with sCAR-His6 monomer, trimeric sCARf protein possesses augmented ability to provide viral linkage by means of high-affinity binding to Ad fiber knob domain. Therefore, trimeric sCAR fusion proteins might offer improved blocking capability of CAR-dependent virus-cell attachment and viral infection.



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  		Fig. 3. Analysis of sCAR fusion proteins binding of to Ad fiber knob. A, comparison of monomeric and trimeric sCAR protein knob-binding by ELISA. Biotinylated trimeric sCARf and monomeric sCAR-His6 fusion proteins were incubated at various concentrations with immobilized recombinant Ad5 fiber knob protein. Biotinylated sCAR fusion proteins bound to fiber knob were detected with alkaline phosphatase conjugated with streptavidin. Each point, the cumulative mean ± SD of triplicate determinations. Error bars (some are smaller than the symbols), SDs. B, inhibition of Ad infection of CAR-positive cells by sCAR fusion proteins. AdLucGFP vector containing luciferase expression cassette was incubated with either PBS or with increasing amounts of monomeric sCAR-His6, or trimeric sCARf, or sCARfC6.5 fusion proteins. Viral mixtures were added to monolayers of 293 cells at MOI of 100 v.p./cell. After 30-min incubation to allow virus internalization, the medium was changed and cells were incubated for an additional 20 h at 37°C to allow luciferase expression. Then, cells were lysed, and relative luciferase activity was analyzed. Luciferase activities, detected in cells infected in the presence of sCAR fusion proteins, are shown as percentages of luciferase activity registered in control cells infected with AdLucGFP incubated with PBS. Each point represents the cumulative mean ± SD of triplicate determinations. Error bars (some are smaller than the symbols), SDs.

 
To determine whether augmented binding to Ad fiber knob results in increased ability of trimeric sCAR proteins to block Ad infection, we performed an infection inhibition assay. AdLucGFP vector, expressing both luciferase and GFP reporter genes, was preincubated with either PBS or one of the sCAR-His6, sCARf, or sCARfC6.5 proteins at varying concentrations and was used to infect 293 cells, which are known to express a high level of CAR. The ability of sCAR fusion proteins to block viral infection was assessed by sCAR protein dose-dependent impairment of Ad-mediated gene transfer as measured by luciferase activity in infected cells (Fig. 3B)Citation . It was shown that sCARfC6.5 as well as sCARf displayed an increased ability to inhibit CAR-dependent Ad infection compared with monomeric sCAR-His6 protein. The concentrations of sCARfC6.5, sCARf, and sCAR-His6 needed to block Ad infection by 50% were 3, 6, and 54 nM, respectively. Interestingly, sCARfC6.5 protein displayed Ad infection inhibition efficiency somewhat higher than did sCARf control protein. This experiment validated the utility of trimeric sCAR fusion proteins to block CAR-dependent Ad tropism and, therefore, provided a rationale for additional c-erbB-2 targeting studies.

Bispicific sCARfC6.5 Protein Binds to Cellular c-erbB-2.
Flow cytometry analysis was performed to validate that C6.5 scFv incorporated into recombinant sCARfC6.5 fusion protein retained its ability to bind c-erbB-2 oncoprotein at the cell surface. The sCARfC6.5 protein was used to bind to c-erbB-2 that was overexpressed on AU-565 breast cancer cells. The MDA-MB-468 breast cancer cell line, previously shown to be c-erbB-2-negative, was used as a control. The sCARfC6.5 protein, bound to c-erbB-2 displayed at the cell surface, presented the CAR ectodomain for antibody detection with primary anti-CAR RmcB MAb (33) followed by a secondary antimouse fluorochrome-conjugated antibody. As shown in Fig. 4Citation , incubation of AU-565 cells, naturally low in CAR (Fig. 5)Citation , with sCARfC6.5 protein increased cell binding of anti-CAR antibody. In contrast, neither the incubation of AU-565 cells with sCARf control protein nor the incubation of sCARfC6.5 with MDA-MB-468 c-erbB-2-negative cells revealed any increase of RmcB MAb antibody binding compared with MAb alone (Fig. 4)Citation . Thus, we demonstrated that C6.5 scFv that was incorporated in the context of fusion protein retained its functional ability to recognize cellular c-erbB-2 oncoprotein, which enabled sCARfC6.5 protein binding to c-erbB-2-positive cells.



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  		Fig. 4. Confirmation of sCARfC6.5 protein binding to cellular c-erbB-2. Trimeric sCARfC6.5 and sCARf fusion proteins were incubated with either c-erbB-2-positive AU-565 or c-erbB-2-nagative MB-468 cells. The sCAR fusion proteins bound to cells were probed with anti-CAR RmcB MAb and then detected with secondary Alexa 488-labeled goat antimouse antibodies. Binding of sCARfC6.5 protein (black line) to c-erbB-2-positive AU-565 cells is seen because of the positive staining relative to sCARf control protein (gray line) or anti-CAR MAb alone (spike filled in black). Representative data from two independent experiments are shown.

 


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  		Fig. 5. Expression of CAR and c-erbB-2 in cancer cell lines. The breast cancer cell lines MCF-7, BT-474, SK-BR-3, AU-565, and MB-468 and ovarian cancer cell line SK-OV-3 were analyzed for CAR and c-erbB-2 expression ( by indirect immunofluorescence assay using anti-CAR RmcB and anti-c-erbB-2/HER-2/neu Ab-2 MAb, respectively. Positive staining for CAR (thin black line) and c-erbB-2 (bold black line) is seen relative to an isotype control IgG (spike filled in gray). Representative data from two independent experiments are shown.

 
sCARfC6.5 Adapter Protein Mediates c-erbB-2-specific Ad Infection.
To test the utility of sCARfC6.5 adapter protein for Ad targeting, we evaluated its ability to improve Ad-mediated gene transfer to c-erbB-2-positive cells. The established human breast cancer cell lines AU-565, SK-BR-3, BT-474, MCF-7, and MDA-MB-468 and ovarian cancer cell line SK-OV-3 were used to validate the ability of c-erbB-2 oncoprotein to mediate Ad infection. Our previous study showed that these cells are relatively refractory to Ad infection.4 The data were corroborated by flow cytometry analysis that showed either absence or low level of CAR on their cell surface (Fig. 5)Citation . Importantly, high levels of c-erbB-2 were detected in these cell lines (Fig. 5)Citation , which suggested that Ad targeting to c-erbB-2 may overcome poor vector susceptibility attributable to the lack of CAR. To determine the optimal adapter protein to virus ratio, sCARfC6.5 protein was titered against a constant dose of AdLucGFP vector (100 v.p./cell) as measured by improvements in gene transfer efficiency. The magnitude of gene transfer augmentation by targeted Ad complexed with sCARfC6.5 adapter was illustrated on selected c-erbB-2-positive cell lines and c-erbB-2-negative MDA-MB-468 cells compared with untargeted Ad preincubated with sCARf control protein or Ad alone (Fig. 6)Citation . Fig. 6ACitation shows sCARfC6.5 protein dose-dependent enhancement of gene transfer that was achieved by targeted Ad compared with that achieved by untargeted Ad, as measured by luciferase activity that was detected in infected cells. The sCARfC6.5/Ad ratio providing maximal gene-transfer increase ranged from 1 x 10-7 to 3 x 10-7 pmol/v.p. depending on the cell line tested. As shown in Fig. 6ACitation , the sCARfC6.5-targeting protein mediated a 3.4-, 11-, 32-, 47-, and 135-fold enhancement of gene transfer to MCF-7, SK-OV-3, BT-474, SK-BR-3, and AU-565 cells, respectively. The sCARfC6.5 adapter protein provided 1.5- to 17-fold increase of Ad gene transfer compared with Ad alone in most cell lines; however, the levels of improvement in cell transduction were highly variable (Fig. 6B)Citation . Although c-erbB-2-targeted Ad showed a 6-fold enhancement of gene transfer to SK-OV-3 cells and no transduction improvement of MCF-7 cells compared with Ad alone, both of the cell lines demonstrated similar levels of gene transfer and remained relatively Ad refractory. Consistent with the augmentation of the Ad gene transfer to c-erbB-2-positive cell lines achieved by sCARfC6.5 targeting protein, untargeted Ad that was complexed with sCARf control protein showed a marked decrease in gene transfer. Importantly, the use of both targeting and control protein to mediate the Ad infection of c-erbB-2-negative MDA-MB-468 cells that expressed moderate levels of CAR resulted in an 8-fold decrease of gene transfer. These data strongly indicate that the sCARfC6.5-targeting adapter promoted the Ad infection of CAR-deficient cells specifically via a c-erbB-2-dependent pathway.



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  		Fig. 6. sCARfC6.5 targeting adapter protein promotes Ad gene transfer to c-erbB-2-positive cells. A, determination of optimal sCARfC6.5 adapter protein:Ad ratio. AdLucGFP vector expressing luciferase reporter gene was preincubated with either sCARfC6.5 targeting protein or sCARf control protein at varying concentrations to form c-erbB-2-targeted or untargeted viral complexes, respectively. The monolayers of MCF-7, BT-474, SK-BR-3, AU-565, SK-OV-3, and MB-468 cells were infected with targeted or untargeted viral complexes at MOI of 100 v.p./cell. Cells were incubated for 46 h to allow expression of reporter gene, then were lysed, and the luciferase activity was analyzed. Results are presented as logarithm of ratio of luciferase activities detected in the cells infected with targeted Ad to luciferase activities detected in the cells infected with untargeted Ad complexes formed at the same concentration of each sCAR protein (Targeted Ad:Untargeted Ad). Each point represents the cumulative mean ± SD of triplicate determinations. Error bars (some are smaller than the symbols), SDs. B, enhancement of Ad gene transfer by sCARfC6.5 targeting protein. AdLucGFP vector was preincubated with either PBS (Ad alone) or one of sCARfC6.5 (c-erbB-2-targeted Ad) or sCARf (untargeted Ad) proteins at the concentration providing maximal gene transfer augmentation as determined in A. The cell monolayers were infected with Ad alone ({blacksquare}), c-erbB-2-targeted Ad (), or untargeted Ad () viral complexes at MOI of 100 v.p./cell and analyzed for luciferase expression 46 h postinfection. Luciferase activities detected in cell lysates are shown as the cumulative mean of triplicate determinations of relative light units (RLU) ± SD.

 
Augmentation of c-erbB-2-targeted Ad Infection Efficiency.
The Ad infection of the cells overexpressing c-erbB-2 oncoprotein mediated by the sCARfC6.5-targeting protein resulted in the enhancement of luciferase reporter gene expression. This increase in reporter activity could result from an increased number of infected cells or, alternatively, from an elevated level of transgene expression caused by the augmented infection of a limited population of cells. To address this issue, cells were infected with AdLucGFP vector, preincubated with PBS (Ad alone), sCARf (untargeted Ad), or sCARfC6.5 (c-erbB-2-targeted Ad) proteins at a sCAR protein:Ad ratio of 2 x 10-7 pmol/v.p. Ad infection efficiency was monitored by direct visualization of GFP expression by fluorescence microscopy. Fig. 7Citation shows the results of Ad-mediated GFP reporter gene delivery to three representative cell lines: AU-565, SK-BR-3, and BT-474. Infection with AdLucGFP vector alone resulted in a low percentage of GFP-expressing cells, whereas highly increased numbers of infected cells were detected in the case of c-erbB-2-targeted Ad. In contrast, infection with untargeted Ad showed a decreased infection efficiency compared with that of Ad alone, which resulted in few or no fluorescent cells. Infection of c-erbB-2-negative MDA-MB-468 cells with targeted Ad did not show any improvement of infection efficiency compared with that achieved by untargeted Ad or by Ad alone (data not shown). These results were consistent with data obtained for Ad-mediated luciferase gene delivery (Fig. 6)Citation .



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  		Fig. 7. The use of sCARfC6.5 targeting protein improves efficiency of Ad infection of c-erbB-2-positive cells. AdLucGFP vector expressing GFP was preincubated with PBS (Ad alone), sCARfC6.5 adapter protein, or sCARf control protein at the concentration of 2 x 10-7 pmol/v.p to form c-erbB-2-targeted or untargeted viral complexes, respectively. The monolayers of SK-BR-3, BT-474, and AU-565 cells were infected with Ad alone, untargeted Ad, or c-erbB-2-targeted Ad complexes at MOI of 100 v.p./cell. Infected cells expressing GFP were detected 24 h postinfection by fluorescence microscopy.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
One of the major challenges facing Ad gene delivery systems is the modification of viral native tropism to confer targeting capability on selected cell types. The limitation of Ad vectors associated with broad CAR-dependent tropism and inefficient infection of CAR-deficient cells could be solved by Ad targeting to a novel receptor overexpressed in the cells of interest. In this study, we explored the utility of a recombinant adapter protein to achieve modification of Ad tropism. The use of adapter molecules to mediate cell-specific Ad infection relies on the following: (a) the ability to be produced and purified at preparative amounts and retain stable structure; (b) the ability to provide efficient linkage to the v.p. while blocking Ad native tropism; and (c) the ability to achieve binding to cell-specific receptors, thus generating a novel tropism. We previously developed a targeting approach based on the use of the sCAR ectodomain fused with EGF. which allowed both the blocking of CAR-dependent tropism and the Ad targeting to the cells overexpressing EGFR (29) . To extend this approach, we engineered an adapter protein, sCARfC6.5, comprised of sCAR fused with a trimerization domain and a scFv against c-erbB-2 oncoprotein to target cancer cell types, and a sCARf control protein that lacked the anti-c-erbB-2 scFv targeting moiety. A trimerization domain derived from phage T4 fibritin protein was incorporated into the design of the sCAR fusion proteins to achieve tight viral linkage by virtue of trivalent binding to the Ad fiber knob. Both the sCARfC6.5 targeting protein and the control sCARf protein were expressed in insect cells and purified at preparative amounts by affinity chromatography. An analysis of purified fusion proteins showed that sCARf and sCARfC6.5 retain their designed composition and maintain stable trimeric structure. As expected, trimerization of sCAR proteins resulted in the augmentation of knob-binding efficiency by at least 20-fold compared with that achieved by the monomeric sCAR-His6 protein. This result is consistent with a kinetic analysis showing that, in contrast to the high on/off interaction rates between CAR D1 domain and Ad2 fiber knob, the binding of the knob domain to three D1 molecules simultaneously leads to a low overall off rate and Kd of ~1 nM (16) . We then evaluated whether trimerization would result in an improved ability of sCAR proteins to block Ad infection. An infection inhibition assay demonstrated that the concentrations (3 and 6 nM, respectively) of trimeric sCARfC6.5 and sCARf proteins that were needed to inhibit Ad-mediated gene transfer by 50% were, respectively, 18- and 9-fold lower than the concentration (54 nM) of monomeric sCAR-His6 protein that was needed. These data proved the utility of trimeric sCAR adapter proteins in blocking CAR-dependent Ad infection. The fact that T4 fibritin forms homotrimers that are resistant to dissociation by SDS and digestion by trypsin (37 , 38) suggests that the incorporation of a fibritin polypeptide in the context of sCAR fusion might provide a highly stable trimeric structure compatible with in vitro, and likely in vivo, Ad targeting schemes. We showed that, in addition to its ability to block Ad infection, the sCARfC6.5 adapter protein binds to cellular c-erbB-2 oncoprotein and, therefore, enables Ad targeting via a CAR-independent pathway. In ovarian and breast cancer cell lines overexpressing c-erbB-2, the ability of the c-erbB-2 oncoprotein to mediate Ad infection was illustrated by markedly increased levels of gene transfer and numbers of infected cells. Thus, the use of the sCARfC6.5 adapter protein overcomes the barrier of CAR deficiency by retargeting the Ad infection via the c-erbB-2 oncoprotein and provides a <=2-fold enhancement of gene transfer efficiency in comparison with that provided by the sCARf control protein. Our observation that the use of the sCARfC6.5 adapter decreased the Ad infection efficiency in c-erbB-2-negative MDA-MB-468 cells that expressed moderate levels of CAR suggests the high specificity of this Ad-targeting approach. Importantly, the presence of three scFvs in each trimeric sCARfC6.5 molecule likely contributes to the increase in apparent receptor affinity attributable to polyvalent binding and contributes, therefore, to the efficiency of Ad targeting. In fact, the presence of additional binding sites prolongs the association of scFv-based molecules with tumor cells in vitro and in vivo. Studies of bivalent diabody molecules that were constructed from C6.5 scFv demonstrated a more highly prolonged association (60-fold) with c-erbB-2 on the surface of SK-OV-3 cells and more tumor-retained diabodies (6.5-fold) when compared with scFv monomer (41) .

The use of trimeric sCAR-ligand fusion proteins or recombinant adapter molecules sharing a trimeric design to confer Ad targeting to specific cell types may augment the utility of current Ad vectors. In addition, the availability of scFvs with defined specificities to tumor antigens offers the flexibility of ligand substitution to expand the targeting capabilities of Ad vectors. Of note, Ad targeting by means of bispecific antibody conjugates was shown to achieve direct therapeutic goals in in vivo models relevant to human clinical cancer gene therapy schemes (42, 43, 44, 45) . Thus, modification of Ad tropism based on the use of recombinant adapters could facilitate target-directed infection, which will reduce the effective therapeutic viral dose, thereby decreasing the immediate toxicity and increasing the safety and efficiency of Ad vectors.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. James D. Marks for the provision of the cDNA for C6.5 scFv, Dr. Robert Finberg (Dana Farber Cancer Institute, Boston, MA) for cDNA for CAR, and Dr. V. Mesyanzhinov for anti-fibritin serum. We thank Dr. Victor Krasnykh (Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL) for the plasmid encoding recombinant Ad5 fiber knob protein and Dr. Joanne T. Douglas (Division of Human Gene Therapy, University of Alabama-Birmingham) for making RmcB antibody available to us. We thank the DNA Sequencing Core and the FACS Core Facility at the University of Alabama at Birmingham for providing assistance. Thanks to Dr. Joel N. Glasgow for fruitful discussions and proofreading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Idea Award DAMD17-00-1-0115 from United States Army Department of Defense (I. P. D.), National Cancer Institute Grant N01 C0–97110, and NIH Grants P50 CA89019 and RO1 CA86881. Back

2 To whom requests for reprints should be addressed, at the University of Alabama at Birmingham, WTI-620, 1824 Sixth Avenue South, Birmingham, AL 5294-3300. Phone: (205) 934-2326; Fax: (205) 975-7949; E-mail: idmitriev{at}gtp.ccc.uab.edu Back

3 The abbreviations used are: Ad, adenovirus; CAR, coxsackievirus and Ad receptor; FACS, fluorescent-activated cell sorting; sCAR, soluble CAR ectodomain; scFv, single-chain fragment variable; EGF, epidermal growth factor; EGFR, EGF receptor; MAb, monoclonal antibody; GFP, green fluorescent protein; v.p., viral particle(s); MOI, multiplicity/multiplicities of infection; FBS, fetal bovine serum. Back

4 Unpublished observations. Back

Received 8/17/01. Accepted 11/14/01.


   REFERENCES
Top
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online