Targeting of Adenovirus Serotype 5 Pseudotyped with Short Fiber from Serotype 41 to c-erbB2-Positive Cells using Bispecific Single-Chain Diabody

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Abstract

The purpose of the current study was to alter the broad native tropism of human adenovirus for virus targeting to c-erbB2-positive cancer cells. First, we engineered a single-chain antibody (scFv) against the c-erbB2 oncoprotein into minor capsid protein IX (pIX) of adenovirus serotype 5 (Ad5) in a manner commensurate with virion integrity and binding to the soluble extracellular c-erbB2 domain. To ablate native viral tropism and facilitate binding of the pIX-incorporated scFv to cellular c-erbB2, we replaced the Ad5 fiber with the Ad41 short (41s) fiber devoid of all known cell-binding determinants. The resultant Ad5F41sIX6.5 vector demonstrated increased cell binding and gene transfer as compared to the Ad5F41s control; however, this augmentation of virus infectivity was not c-erbB2 specific. Incorporation of a six-histidine (His6) peptide into the C-terminus of the 41s fiber protein resulted in markedly increased Ad5F41s6H infectivity in 293AR cells, which express a membrane-anchored scFv against the C-terminal oligohistidine tag, as compared to the Ad5F41s vector and the parental 293 cells. These data suggested that a 41s-fiber-incorporated His6 tag could serve for attachment of an adapter protein designed to guide Ad5F41s6H infection in a c-erbB2-specific manner. We therefore engineered a bispecific scFv diabody (scDb) combining affinities for both c-erbB2 and the His6 tag and showed its ability to provide up to 25-fold increase of Ad5F41s6H infectivity in c-erbB2-positive cells. Thus, Ad5 fiber replacement by a His6-tagged 41s fiber coupled with virus targeting mediated by an scDb adapter represents a promising strategy to confer Ad5 vector tropism for c-erbB2-positive cancer cells.

Introduction

The human adenovirus (Ad) family includes 51 serotypes divided into six species (A–F) that represent a large family of nonenveloped viruses containing a linear double-stranded DNA genome of approximately 36 kb1 packaged into an icosahedral capsid.2, 3 The major capsid components are the trimeric hexon protein and the penton that is a noncovalent complex between the pentameric penton base and the trimeric fiber protein. The interactions between Ad capsid proteins and host cell have been extensively characterized in vitro, revealing two distinct sequential steps critical for initiation of Ad infection. Ad attachment to the cell is mediated by fiber binding with its C-terminal knob domain to a primary cellular receptor.4 Subsequent interaction of the αvβ3/5 integrins, the secondary cellular receptor, with an Arg-Gly-Asp (RGD) sequence within a protein loop extended from the penton base is required to trigger endocytosis resulting in virus internalization.5, 6 Most Ads of species B have been shown to utilize human membrane cofactor CD46 as the predominant binding site7, 8 for infection of dendritic cells, hematopoietic stem cells, and some malignant cell types. The primary high-affinity cellular attachment receptor for many representatives of species A, C, D, E, and F has been identified9 as the coxsackievirus group B and Ad receptor,10 called CAR. The presence of CAR on human airway epithelia determines the susceptibility of epithelial cells to Ad5 infection both in vitro and in vivo.11, 12 Other receptors including heparan sulfate proteoglycans (HSPGs)13, 14 and class I histocompatibility complex15 have also been described for Ad serotype 2 (Ad2) and 5 (Ad5) from species C that are most extensively used for vector development.

The delineation of key aspects of the Ad cellular entry pathway in vitro, wherein cell attachment is distinct from subsequent virus internalization, suggested that binding of Ad to the primary receptor determines the host cell range and native viral tropism.16, 17, 18 The modification of residues critical for Ad binding to CAR19, 20, 21 and/or integrins22, 23, 24 has resulted in a dramatic decrease of Ad5 infectivity in vitro. However, these mutations were shown to have only a marginal effect on the biodistribution pattern of the virus in vivo, including its predominant accumulation in hepatic tissue after intravascular delivery in mice,24, 25, 26, 27, 28, 29 an important route of administration for many clinical applications. On the other hand, a significant impact on vector-mediated liver transduction has been demonstrated in several animal species by mutating the KKTK motif, a putative HSPG-binding site found in the Ad5 fiber shaft,30, 31 or by pseudotyping the Ad5 capsid with short-shafted fibers from Ad serotype 35,30, 32, 33, 34 40,35 or 4136, 37 that lack both CAR-binding and the KKTK motif. Furthermore, a number of plasma proteins including coagulation factors VII, IX, X, protein C, and complement component C4-binding protein have been shown to facilitate CAR-independent infection of hepatic cells by virtue of binding to either trimeric fiber knob38 or hexon39, 40 and bridging Ad particles to the cell surface in vitro and in vivo. Therefore, improving the therapeutic potential of Ad vectors requires the elimination of the natural viral tropism and introduction of a novel mechanism of selective cell recognition that would allow directed virus localization to the target tissue.

Several strategies including the use of bispecific adapter molecules and the genetic incorporation of targeting ligands into capsid proteins have been developed to redirect Ad5 infection via nonnative pathways (reviewed in Ref. 41). Originally, Ad targeting was demonstrated using a bispecific protein complex containing chemically conjugated virus- and receptor-binding moieties to achieve indirect virus linkage with the cells of interest via the folate receptor.42 This approach was further translated to confer Ad5 vector targeting capabilities toward receptors upregulated in the cells of interest with the use of a variety of targeting ligands that include Fab fragments of monoclonal antibodies,43, 44 engineered single-chain antibody (scFv) molecules,45, 46 and growth factors.47, 48, 49 Evaluation of adapter-mediated Ad targeting in vivo provided compelling evidence of a substantial reduction of virus uptake in the liver and a significant increase in Ad vector delivery to the target organ44, 50, 51 or tumors52, 53 in a receptor-specific manner.

Since single-component vector systems are favored for application in clinical settings, genetic ligand incorporation into the viral capsid has been extensively explored to achieve Ad targeting. While engineering of the Ad5 fiber knob domain to incorporate peptide ligands derived from phage display libraries has been shown to be feasible,54, 55, 56 subsequent attempts to employ various natural ligands with augmented affinity have been challenged by the limited tolerance of the fiber for genetic modifications57. In contrast, the C-terminus of the minor capsid protein IX (pIX) has been identified58, 59 as a locale amenable to incorporation of various heterologous polypeptides including fluorescent proteins,60, 61, 62 enzymes,63, 64, 65 an scFv,66 and single-chain T-cell receptor (scTCR)67 with minimal effect on viral function.

Herein, we explored the utility of the pIX ectodomain to display ligands with augmented size/complexity for retargeting of Ad infection via cellular markers associated with malignant cell transformation. This study was designed to restrict the broad native tropism of Ad5 to HER-2-positive cancer cells by means of genetic incorporation of anti-c-erbB2 scFv into pIX and replacing the Ad5 fiber with the 41s fiber, which is devoid of all known cell-binding determinants. We showed that the pIX-incorporated scFv failed to provide receptor-specific virus infection, while modification of the 41s fiber to contain a six-histidine (His6) peptide allowed efficient c-erbB2 targeting using a bispecific single-chain diabody (scDb) adapter, an scFv-derived diabody molecule that was engineered with affinities for both c-erbB2 and the His6 tag.

Section snippets

Genetic scFv incorporation into Ad5 capsid

To incorporate the scFv ligand into the viral capsid, we engineered the pIX gene in the Ad5 genome to encode a C-terminal Flag peptide followed by anti-c-erbB2 scFv C6.568 fused to the pIX ectodomain. The replication-competent Ad5pIXC6.5 vector was rescued and the presence of protein with molecular mass of approximately 45 kDa, as predicted for the pIXflagC6.5 polypeptide, was confirmed by Western blotting (Fig. 1a), suggesting that the full-length scFv fused to the pIX ectodomain had been

Discussion

Targeted Ad vectors hold the promise to expand the types of diseases that can be treated by gene therapy and to make the therapeutic applications of Ad vectors more effective. The increased specificity achieved by targeting virus infection to cells of interest will ultimately allow lower and safer doses of Ad vectors to be provided when regional or systemic delivery is contemplated in the future. The purpose of this study was to design an Ad vector that embodies genetic modifications to allow

Cells

The 293 human kidney cell line transformed with Ad5 DNA was purchased from Microbix (Toronto, Ontario, Canada). 293AR cells were generated previously70 to express an artificial receptor derived from 3D5 scFv75 specific for a C-terminal oligohistidine tag. The SKOV3 human ovarian cancer, MDA-MB-468, AU-565, and SKBR-3 human breast cancer, and HepG2 human hepatocellular carcinoma cell lines were obtained from the American Type Culture Collection (Manassas, VA). MDA-MB-435 breast adenocarcinoma

Acknowledgements

This work was supported by grants R21CA116525 (to I.P.D.), 5R01CA083821 (to D.T.C.), and R01CA108585 (to J.T.D.) from the National Cancer Institute and DAMD17-03-1-0629 (to I.P.D.) from the Department of Defense Breast Cancer Research Program.

We are grateful to Leigh Millican from the High Resolution Imaging Facility at University of Alabama Birmingham for her help with electron microscopy. We would like to disclose that D.T.C. and J.T.D. hold equity in VectorLogics, Inc. and I.P.D. has

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