Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters.

Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.

Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT.

The importance of the EGF receptor (EGFR) signaling pathway in the development and progression of nonsmall cell lung carcinomas (NSCLC) is widely recognized. Gene sequencing studies revealed that a majority of tumors responding to EGFR kinase inhibitors harbor activating mutations in the EGFR kinase domain. This underscores the need for novel biomarkers and diagnostic imaging approaches to identify patients who may benefit from particular therapeutic agents and approaches with improved efficacy and safety profiles. To this goal, we developed 4-[(3-iodophenyl)amino]-7-{2-[2-{2-(2-[2-{2-([(18)F]fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide ([(18)F]F-PEG6-IPQA), a radiotracer with increased selectivity and irreversible binding to the active mutant L858R EGFR kinase. We show that PET with [(18)F]F-PEG6-IPQA in tumor-bearing mice discriminates H3255 NSCLC xenografts expressing L858R mutant EGFR from H441 and PC14 xenografts expressing EGFR or H1975 xenografts with L858R/T790M dual mutation in EGFR kinase domain, which confers resistance to EGFR inhibitors (i.e., gefitinib). The T790M mutation precludes the [(18)F]F-PEG6-IPQA from irreversible binding to EGFR. These results suggest that PET with [(18)F]F-PEG6-IPQA could be used for the selection of NSCLC patients for individualized therapy with small molecular inhibitors of EGFR kinase that are currently used in the clinic and have a similar structure (i.e., iressa, gefitinib, and erlotinib).

Development of a targeted gene vector platform based on simian adenovirus serotype 24.

Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations--widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery--justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.

Modification of adenovirus capsid with a designed protein ligand yields a gene vector targeted to a major molecular marker of cancer.

The future of genetic interventions in humans critically depends on the selectivity and efficiency of gene transfer to target tissues. The viral gene vectors explored to date cannot selectively transduce the desired targets. While substantial progress has been made in developing targeting strategies for adenovirus (Ad) vectors, future advances in this direction are severely limited by the shortage of naturally existing molecules available for use as targeting ligands. This shortage is due to fundamental and irresolvable differences at the level of both posttranslational modifications and intracellular trafficking between the Ad structural proteins and those natural proteins that are involved in interactions with the cell surface and could otherwise be considered as potential targeting ligands. We hypothesized that this problem could be resolved by altering the natural tropism of Ad vector through incorporation into its capsid of a rationally designed protein ligand, an affibody, whose structural, functional, and biosynthetic properties make it compatible with the Ad assembly process. We tested this hypothesis by redesigning the receptor-binding Ad protein, the fiber, using affibodies specific for human epidermal growth factor receptor type 2 (Her2), a major molecular marker of human tumors. The biosynthesis and folding of these fiber chimeras were fully compatible with Ad virion formation, and the resultant viral vectors were capable of selective delivery of a dual-function transgene to Her2-expressing cancer cells. By establishing the feasibility of this affibody-based approach to Ad vector targeting, the present study lays the foundation for further development of Ad vector technology toward its clinical use.

Therapeutic Molecular Targetingof 15-Lipoxygenase-1 in Colon Cancer.

Molecular targeting for apoptosis induction is being developed for better treatment of cancer. Downregulation of 15-lipoxygenase-1 (15-LOX-1) is linked to colorectal tumorigenesis. Re-expression of 15-LOX-1 in cancer cells by pharmaceutical agents induces apoptosis. Antitumorigenic agents can also induce apoptosis via other molecular targets. Whether restoring 15-LOX-1 expression in cancer cells is therapeutically sufficient to inhibit colonic tumorigenesis remains unknown. We tested this question using an adenoviral delivery system to express 15-LOX-1 in in vitro and in vivo models of colon cancer. We found that (i) the adenoviral vector 5/3 fiber modification enhanced 15-LOX-1 gene transduction in various colorectal cancer cell lines, (ii) the adenoviral vector delivery restored 15-LOX-1 expression and enzymatic activity to therapeutic levels in colon cancer cell lines, and (iii) 15-LOX-1 expression downregulated the expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and BcL-XL, activated caspase-3, triggered apoptosis, and inhibited cancer cell survival in vitro and the growth of colon cancer xenografts in vivo. Thus, selective molecular targeting of 15-LOX-1 expression is sufficient to re-establish apoptosis in colon cancer cells and inhibit tumorigenesis. These data provide the rationale for further development of therapeutic strategies to target 15-LOX-1 molecularly for treating colonic tumorigenesis.

Therapeutic molecular targeting of 15-lipoxygenase-1 in colon cancer.

Molecular targeting for apoptosis induction is being developed for better treatment of cancer. Downregulation of 15-lipoxygenase-1 (15-LOX-1) is linked to colorectal tumorigenesis. Re-expression of 15-LOX-1 in cancer cells by pharmaceutical agents induces apoptosis. Antitumorigenic agents can also induce apoptosis via other molecular targets. Whether restoring 15-LOX-1 expression in cancer cells is therapeutically sufficient to inhibit colonic tumorigenesis remains unknown. We tested this question using an adenoviral delivery system to express 15-LOX-1 in in vitro and in vivo models of colon cancer. We found that (i) the adenoviral vector 5/3 fiber modification enhanced 15-LOX-1 gene transduction in various colorectal cancer cell lines, (ii) the adenoviral vector delivery restored 15-LOX-1 expression and enzymatic activity to therapeutic levels in colon cancer cell lines, and (iii) 15-LOX-1 expression downregulated the expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and BcL-XL, activated caspase-3, triggered apoptosis, and inhibited cancer cell survival in vitro and the growth of colon cancer xenografts in vivo. Thus, selective molecular targeting of 15-LOX-1 expression is sufficient to re-establish apoptosis in colon cancer cells and inhibit tumorigenesis. These data provide the rationale for further development of therapeutic strategies to target 15-LOX-1 molecularly for treating colonic tumorigenesis.

Infectivity-enhanced cyclooxygenase-2-based conditionally replicative adenoviruses for esophageal adenocarcinoma treatment.

The employment of conditionally replicative adenoviruses (CRAd) constitutes a promising alternative for cancer treatment; however, in the case of esophageal adenocarcinoma (EAC) the lack of an appropriate tumor-specific promoter and relative resistance to adenovirus infection have hampered the construction of CRAds with clinically applicable specificity and efficacy. By combining transcriptional targeting with infectivity enhancement for CRAds, we generated novel cyclooxygenase-2 (Cox-2) promoter-controlled replicative viral agents for the treatment of EAC. We used infectivity enhancement based on incorporation of an RGD-4C motif into the HI loop of the adenoviral (Ad) fiber knob domain as well as replacement of the Ad5 knob with the Ad3 knob. The Cox-2 promoter was highly active in EAC, whereas showing no significant activity in Cox-2-negative cell lines and primary cells isolated from normal mouse esophagus and stomach. Evaluation of infectivity-enhanced vectors revealed that the transduction and virus-cell binding ability of Ad5/Ad3-chimera were significantly more efficient than that of unmodified and Arg-Gly-Asp (RGD)-modified vectors. All of the Cox-2 CRAds demonstrated replication and subsequent oncolysis in EAC cells but not in Cox-2-negative cells in vitro, thus confirming the dependence of their replication on the Cox-2 promoter activity. Ad5/Ad3 CRAds exhibited significantly improved oncolysis and progeny production compared with unmodified and RGD-modified vectors without sacrificing tumor selectivity. Whereas unmodified and RGD-modified CRAds showed insignificant therapeutic effect in vivo, Ad5/Ad3 CRAds remarkably suppressed tumor growth of established xenografts in mice. Thus, our studies have demonstrated that Ad5/Ad3-chimeric Cox-2 promoter-driven CRAds are selective and potent agents for the treatment of EAC.

Substitution of the adenovirus serotype 5 knob with a serotype 3 knob enhances multiple steps in virus replication.

Adenovirus (Ad) serotype 5 (Ad5) continues to be the predominant vector used for cancer gene therapy. However, many tumor types are reported to be relatively refractory to Ad5 infection because of low surface expression of the native Ad5 receptor, CAR. The observation that many tumor cells are CAR deficient has necessitated the development of CAR-independent infection strategies, including the introduction of heterologous ligand sequences into the virus fiber gene and immunological or chemical modifications of the capsid proteins. Alternatively, native Ad5 tropism can be modified by substituting the knob region from other Ad serotypes such as Ad type 3 (Ad3) into the Ad5 knob region. To date, the effect(s) of tropism modification on the replication and oncolytic capacity of these chimeric Ad vectors has not been fully evaluated. To address this issue, Ad5 vectors and isogenically matched chimeric vectors with Ad3 tropism (Ad5/3) were compared in this study. Various parameters of virus infection were compared, including binding, nuclear translocation, E1A transcription, transgene expression, de novo virus production, and oncolysis. Overall, the chimeric Ad5/3 virus was progressively more efficient at each step of the replication cycle compared with its Ad5 counterpart. The higher replication efficiency of the chimeric Ad5/3 vector translated into improved therapeutic efficacy in a murine in vivo tumor rejection model. These findings suggest that in addition to the initial target cell interaction, multiple mechanisms contribute to the enhanced replication of the chimeric Ad5/3 vector. Furthermore, the data demonstrate that alternative Ad serotype receptors can be used to improve infection and subsequent oncolytic replication, which is particularly relevant in gene therapy applications for tumors that are inefficiently infected with Ad5.

Adenoviral gene therapy for renal cancer requires retargeting to alternative cellular receptors.

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies in humans. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced RCC. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new modality for cancer. However, a primary limiting factor for the use of Ad vectors for cancer gene therapy is their critical dependence on cellular expression of the primary Ad receptor, the coxsackie and adenovirus receptor (CAR), known to be down-regulated in many cancer types. Following the identification of CAR deficiency in RCC lines, we have found abundant membrane expression of alpha(v)beta 3 and alpha(v)beta 5 integrins and of the putative receptor to Ad serotype 3 (Ad3). As an alternative gene therapy approach for RCC that would circumvent CAR deficiency, we employed retargeting of replication-incompetent Ad vectors and replication-competent Ad viruses to alpha(v)beta 3 and alpha(v)beta 5 integrins and to the putative Ad3 receptor. These strategies to genetically alter Ad tropism were based on either the insertion of a cysteine-aspartate-cysteine-arginine-glycine-aspartate-cysteine-phenylalanine-cysteine (RGD) motif into the HI loop of the Ad fiber knob domain or on generation of a chimeric Ad fiber composed of adenovirus serotype 5 shaft/Ad3 knob. Both strategies proved highly efficient to circumvent CAR deficiency and enhance gene delivery into RCC cells. Furthermore, in the context of replication-competent Ad, tropism alteration resulted in distinct capacity of the retargeted viruses to infect, replicate, and lyse RCC models in vitro and in vivo. The retargeting strategies were particularly beneficial in the context of replication-competent Ad. These findings underscore the importance of CAR-independent cellular entry mechanisms in RCC and are highly consequential for the development of viral antitumor agents for RCC and other CAR-negative tumors.

Treatment of ovarian cancer with a tropism modified oncolytic adenovirus.

Ad5-Delta 24RGD is an adenovirus that is selectively replication competent in cells defective in the Rb/p16 pathway, such as ovarian cancer cells. The fiber of Ad5-Delta 24RGD contains an integrin binding RGD-4C motif, allowing Coxsackie adenovirus receptor-independent infection of cancer cells. Oncolysis of cell lines was similar to that of a wild-type control, and replication in primary tumor material was shown using a novel three-dimensional spheroid model. Finally, an orthotopic murine model of peritoneally disseminated ovarian cancer was used to test i.p. administration to tumor-bearing animals. Injection of the agent resulted in eradication of i.p. disease, whereas control animals expired (P < 0.0001). These results suggest that Ad5-Delta 24RGD could be useful for treatment of ovarian cancer in humans.

Native and engineered tropism of vectors derived from a rare species D adenovirus serotype 43.

Unique molecular properties of species D adenoviruses (Ads)-the most diverse yet underexplored group of Ads-have been used to develop improved gene vectors. The low seroprevalence in humans of adenovirus serotype 43 (Ad43), an otherwise unstudied species D Ad, identified this rare serotype as an attractive new human gene therapy vector platform. Thus, in this study we wished to assess biological properties of Ad43 essential to its vectorization. We found that (1) Ad43 virions do not bind blood coagulation factor X and cause low random transduction upon vascular delivery; (2) they clear host tissues more quickly than do traditionally used Ad5 vectors; (3) Ad43 uses CD46 as primary receptor; (4) Ad43 can use integrins as alternative primary receptors. As the first step toward vectorization of Ad43, we demonstrated that the primary receptor specificity of the Ad43 fiber can be altered to achieve infection via Her2, an established oncotarget. Whereas this modification required use of the Ad5 fiber shaft, the presence of this domain in chimeric virions did not make them susceptible for neutralization by anti-Ad5 antibodies.

Promoter-controlled infectivity-enhanced conditionally replicative adenoviral vectors for the treatment of gastric cancer.

BACKGROUND: Gastric cancer is the fourth most common malignancy worldwide. Adenoviral vectors (Ads) have been applied for gene therapy of various cancers because of their high transduction efficiency. However, the infectivity of gastrointestinal cancer cells is poor due to the limited expression of the Coxsackie-adenovirus receptor (CAR). In addition, few tumor-specific promoters (TSPs) have been characterized for this type of cancer. To overcome these problems, we proposed TSP-driven conditionally replicating adenoviruses (CRAds) with fiber modification for virotherapy of gastric cancer. METHODS: We assessed the expression profile of eight TSPs in gastric cancer cell lines and evaluated promising candidates in the context of CRAd cytocidal effect. Next, infectivity enhancement by fiber modifications was analyzed in the gastric cancer cell lines. Finally, we combined the TSP-driven CRAds of choice with the fiber modifications to augment the killing effect. RESULTS: Out of the eight TSPs, the midkine (MK) and cyclooxygenase-2 (Cox-2M and Cox-2L) promoters showed high transcriptional activity in gastric cancer cells. When these promoters were used in a CRAd context, Cox-2 CRAds elicited the strongest cytocidal effect. The greatest infectivity enhancement was observed with adenoviral vectors displaying 5/3 chimeric fibers. Likewise, Cox-2 CRAds with 5/3 chimeric fibers showed the strongest cytocidal effect in gastric cancer cell lines. Therefore, Cox-2 CRAds with 5/3 chimeric fiber modification showed good selectivity and infectivity in gastric cancer cells to yield enhanced oncolysis. CONCLUSIONS: Cox-2 CRAds with 5/3 chimeric fiber modification are promising for virotherapy of gastric cancer.

Cell cycle arrest and apoptosis induced by SART-1 gene transduction.

UNLABELLED: The biological function of the SART-1 gene product is demonstrated and its potential as a target for cancer gene therapy is discussed. MATERIALS AND METHODS: The SART-1 gene was transduced by a recombinant adenovirus vector and its expression was promoted by a CMV promoter. RESULTS: The transduction efficiency by recombinant adenoviruses in A549 and MCF-7 cells was determined using a vector expressing luciferase, which showed high expression in the cells. Cell count analysis using Trypan-Blue dye exclusion showed that SART-1 gene transduction inhibited cell growth. Flow cytometry analysis suggested that SART-1 gene transduction induced cell cycle arrest followed by apoptosis. Western blot analysis confirmed that the apoptosis pathway was activated by SART-1 gene transduction. CONCLUSION: These results show that SART-1 gene transduction induces cell cycle arrest leading to apoptosis and suggest the possibility of gene therapy against cancer. In addition, SART-1 is known to be a tumor antigen in a range of cancers recognized by T cells, thus a potential strategy would be the combination of suicide gene therapy with immuno-gene therapy.

Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells.

Gene delivery efficiency in clinical cancer gene therapy trials with recombinant adenoviruses (Ads) based on serotype 5 (Ad5) has been limited partly because of variable expression of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human primary cancer cells. As a means of circumventing CAR deficiency, Ad vectors have been retargeted by creating chimeric fibers possessing knob domains of alternate Ad serotypes. In this study, we have constructed an Ad5-based vector, Ad5/3luc1, with a chimeric fiber protein featuring a knob domain derived from Ad3. This virus is retargeted to the Ad3 receptor and, therefore, has different tissue tropism. A novel knob binding assay was used to measure expression of CAR and the Ad3 receptor. Further, to evaluate the correlation of receptor expression and infectivity by Ad, a panel of ovarian cancer cell lines and purified primary cancer cells were infected with Ad5luc1 and Ad5/3luc1 at 50, 200, and 1000 viral particles/cell. Our results confirm that Ad5/3luc1 is retargeted to the Ad3 receptor. Furthermore, the Ad3 receptor is present at higher levels than CAR on ovarian adenocarcinoma cells. Also, the amount of binding to primary receptor appears to be the major factor determining the efficiency of transgene expression. The Ad5/3 chimera displays enhanced infectivity for ovarian cancer cell lines and purified primary tumor cells, which could translate into increased efficacy in clinical trials.

Blood-based screening and light based imaging for the early detection and monitoring of ovarian cancer xenografts.

We report a novel technology for in vivo early detection, identification, and monitoring of ovarian cancer in live mice leading to better treatment outcome. A genetic dualistic reporter system that uses an adenoviral (Ad) vector to transfer the genetic reporters to the ovarian cancer is described. Infection of the cancer cells leads to expression of one reporter that is detected in blood, namely, secreted human placental alkaline phosphatase (SEAP). A second reporter, namely, enhanced green fluorescent protein (GFP) is also delivered by the Ad, leading to expression at the site of ovarian cancer. The SEAP gene under control of the cytomegalovirus (CMV) promoter element is linked to the GFP gene with an IRES element. A diagnostic adenoviral vector (Ad) encoding the SEAP and GFP (Ad5-SEAP-GFP) is produced. Efficacy of newly developed diagnostic vector is tested in cell culture and animal models. SKOV3ip.1 cells are infected with Ad5-SEAP-GFP. Over time the cells are monitored for fluorescence and SEAP is also measured in the growth media supernatant. For animal experiments, SKOV3ip.1 cells are implanted first in nude mice either subcutaneously (SC) or intraperitoneally (IP) separately. After 4-7 days, the Ad5-SEAP-GFP is administered. Control mice do not receive any Ad vector. All mice are imaged with a fluorescent stereomicroscope after 24 h, and blood is collected for SEAP analyses. Increasing green fluorescence is detected in all SKOV3ip.1 cells infected with Ad5-SEAP-GFP, while SEAP levels in growth media increase over monitoring period. Expression of GFP in both SC and IP tumors is detected by 24 h in the live mice. At this time, the SEAP blood levels are more than 2-3 fold greater than blood levels of control group. GFP fluorescence and SEAP levels continue to increase in all mice that are injected with Ad5-SEAP-GFP until termination. Control mice (both SC and IP) do not express GFP or SEAP throughout the experiment. GFP contrast is necessary to differentiate between micro-sized early stage non-palpable ovarian tumor nodules and surrounding normal tissue. While the studies are conducted in mice, it is envisioned that the dual-based approach will eventually be translated into human applications for routine diagnosis and monitoring of ovarian cancer when an ovarian cancer specific promoter will be available. Due to the thickness of the abdominal wall in human laparoscopy or laparotomy will be necessary. This system will provide gynecologic oncologists with a more effective tool for treating patients. The blood-based screening assay provides a quick test to determine the presence of the ovarian cancer at its earliest stage. The location of the ovarian cancer is afforded by the light-based imaging component, which represents a new and improving technology with tremendous advantages of sensitivity and spatial resolution to localize micro-sized tumor nodules. The novelty of the dualistic system is the linkage of blood-based reporter screening as a selection criteria for subsequent light-based imaging procedures. This combination will lead to an accurate and widely applicable method for the early detection and monitoring of ovarian cancer, especially in high-risk women

Development of a therapeutic adenoviral vector for cholangiocarcinoma combining tumor-restricted gene expression and infectivity enhancement.

Cholangiocarcinoma is an invasive malignancy that is most often unresectable upon diagnosis and unresponsive to chemotherapy and radiation. While adenoviral gene therapy has shown promise in treating many tumors, systemic toxicity and low tumor transduction efficiency have hampered its application in many gastrointestinal cancers. To overcome these difficulties, we have constructed an adenoviral vector utilizing a tumor-specific promoter (TSP) for selective transgene expression and a vector with an RGD-motif in the fiber-knob region for infectivity enhancement. In seeking a TSP for cholangiocarcinoma, Secretory Leukoprotease Inhibitor, Midkine, Gastrin Releasing Peptide, VEGF, Cox-2M, and Cox-2L promoters were configures in adenoviral vectors, and evaluated in cholangiocarcinoma cells lines (Oz and SkChA-1). Luciferase assays demonstrated that Cox-2 promoters (M and L) showed the highest promoter activity, with Cox-2M appearing slightly stronger than Cox-2L. Infectivity enhanced vectors with RGD-motif in the fiber-knob region were also constructed with the luciferase transgene driven by a CMV control and the Cox-2M and Cox-2L promoters. Subsequent luciferase assays comparing the unmodified vectors to the RGD-modified versions demonstrated higher levels of luciferase activity than the RGD-infected cells. This paradigm was then applied to a therapeutic HSV-TK/GCV model by constructing RGD-enhanced HSV-TK vectors driven by Cox-2M and Cox-2L promoters. In vitro cytocidal effect analysis confirmed that the RGD-modified, cox-2 (M and L) driven vectors showed a stronger cytocidal effect upon gancyclovir administration than the vectors with wild-type fiber. The Cox-2 promoter demonstrates a favorable selectivity profile for cholangiocarcinoma, and RGD-modification further enhances transduction efficiency. This combination has potential to overcome the obstacles to clinical application of adenoviral gene therapy in cholangiocarcinoma.

Adenovirus-mediated gene delivery to dendritic cells.

Dendritic cells (DCs) are "professional" antigen-presenting cells (APCs) that are uniquely capable of activating and instructing a naive immune system to mount a specific cellular and humoral response. Recognition of this crucial function makes the development of technologies for DC-based immuno-therapies a priority for the treatment of a wide variety of diseases. The most immediate impact of this emerging technology will be in the treatment of cancer and the development of third generation vaccines to protect against viral and intracellular pathogens. In addition to elicitation of immune responses, DCs also function to maintain tolerance to "self." Once the biological basis for this important function is understood, future applications of DC-based immuotherapies may be developed to ameliorate autoimmune diseases or enhance acceptance of transplanted organs. The feasibility of "engineering" the function of DCs has been realized by recent advances in ex vivo methodologies that allow selective DC propagation, antigen loading, and genetic modification in vitro for subsequent therapeutic transfer into the host. Ultimately, the ability to genetically modify these cells will allow us to design DC-mediated interventions that will direct predictable control of either immune activation or tolerance in vivo.

Her2-specific multivalent adapters confer designed tropism to adenovirus for gene targeting.

Adenoviruses (Ads) hold great promise as gene vectors for diagnostic or therapeutic applications. The native tropism of Ads must be modified to achieve disease site-specific gene delivery by Ad vectors and this should be done in a programmable way and with technology that can realistically be scaled up. To this end, we applied the technologies of designed ankyrin repeat proteins (DARPins) and ribosome display to develop a DARPin that binds the knob domain of the Ad fiber protein with low nanomolar affinity (K(D) 1.35 nM) and fused this protein with a DARPin specific for Her2, an established cell-surface biomarker of human cancers. The stability of the complex formed by this bispecific targeting adapter and the Ad virion resulted in insufficient gene transfer and was subsequently improved by increasing the valency of adapter-virus binding. In particular, we designed adapters that chelated the knob in a bivalent or trivalent fashion and showed that the efficacy of gene transfer by the adapter-Ad complex increased with the functional affinity of these molecules. This enabled efficient transduction at low stoichiometric adapter-to-fiber ratios. We confirmed the Her2 specificity of this transduction and its dependence on the Her2-binding DARPin component of the adapters. Even the adapter molecules with four fused DARPins could be produced and purified from Escherichia coli at very high levels. In principle, DARPins can be generated against any target and this adapter approach provides a versatile strategy for developing a broad range of disease-specific gene vectors.

A strategy for adenovirus vector targeting with a secreted single chain antibody.

BACKGROUND: Successful gene therapy will require targeted delivery vectors capable of self-directed localization. In this regard, the use of antibodies or single chain antibody fragments (scFv) in conjunction with adenovirus (Ad) vectors remains an attractive means to achieve cell-specific targeting. However, a longstanding barrier to the development of Ad vectors with genetically incorporated scFvs has been the biosynthetic incompatibility between Ad capsid proteins and antibody-derived species. Specifically, scFv require posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during protein synthesis and virion assembly. METHODOLOGY/PRINCIPAL FINDINGS: We have therefore sought to develop scFv-targeted Ad vectors using a secreted scFv that undergoes the requisite posttranslational modifications and is trafficked for secretion. Formation of the scFv-targeted Ad vector is achieved via highly specific association of the Ad virion and a targeting scFv employing synthetic leucine zipper-like dimerization domains (zippers) that have been optimized for structural compatibility with the Ad capsid and for association with the secreted scFv. Our results show that zipper-containing Ad fiber molecules trimerize and incorporate into mature virions and that zippers can be genetically fused to scFv without ablating target recognition. Most importantly, we show that zipper-tagged virions and scFv provide target-specific gene transfer. CONCLUSIONS/SIGNIFICANCE: This work describes a new approach to produce targeted Ad vectors using a secreted scFv molecule, thereby avoiding the problem of structural and biosynthetic incompatibility between Ad and a complex targeting ligand. This approach may facilitate Ad targeting using a wide variety of targeting ligands directed towards a variety of cellular receptors.

Adenoviruses with an RGD-4C modification of the fiber knob elicit a neutralizing antibody response but continue to allow enhanced gene delivery.

OBJECTIVE: The purpose of this study was to investigate the effect of preexisting neutralizing antibody (NAbs) in naive mice and the effect of induced NAbs in mice immunized with either an RGD or nonmodified Ad5 vector on the transduction efficiency of adenoviral vectors. METHODS: BALB/c mice were immunized with Ad5LucRGD, with the unmodified Ad5Luc1, or with Opti-MEM intraperitoneally (ip) from one to three times. Sera were collected on day 27 and serially diluted to block Ad5Luc1 or Ad5LucRGD prior to infection of SKOV3.ip1 human ovarian carcinoma cells with these same vectors. Forty-eight hours post Ad infection, a luciferase assay was performed to determine the titer of NAbs. RESULTS: Luciferase assay data showed that the gene transfer efficacy of Ad5LucRGD was 1.56-fold higher than Ad5Luc1 in the presence of serum from naive mice. In the presence of serum from Ad5Luc1-challenged mice, the transduction efficiency of Ad5LucRGD was 3.27-fold higher (single challenge) and 4.2-fold higher (triple challenge) than Ad5Luc1. In the presence of serum from Ad5LucRGD-challenged mice, the transduction efficiency of Ad5LucRGD was 2.24-fold higher (single challenge) and 2.53-fold higher (triple challenge) than Ad5Luc1. CONCLUSION: The RGD-modified human Ad vectors appear to be less recognizable than unmodified Ad to preexisting NAbs in mouse models. RGD-modified Ad vectors also appear to elicit a relatively lower level of NAbs that may also contribute to the higher gene transduction efficiency of these modified vectors. Therefore, RGD-modified Ad vectors may be reagents of clinical utility in the context of preformed anti-Ad immunity and in the setting of repetitive dosing.