In this study, we evaluated a targeting approach, utilizing a genetically modified Ad fiber and adapter-mediated retargeting, both combined in the context of a fiber-mosaic viral capsid. We applied this approach for EGFR targeting, to demonstrate the functionality of our strategy in vitro and to establish the potential of this system for in vivo targeting applications.
Bifunctional adapter molecules have been engineered with specificity for both the Ad capsid protein (mainly Ad fiber) and to alternative cellular receptors distinct from CAR. The main advantage of this system is in its flexibility for using high affinity binders, regardless of their nature and size. However, the necessity for producing complex adapter molecules, such as whole antibodies, Ab fragments or recombinant fusion proteins, has always been regarded as an inherent drawback of this approach. Nevertheless, efficient adenovirus targeting in vivo
has only been reported using adapter-based approaches.14–16
Genetically modified vectors have a theoretical advantage in that they do not require additional soluble components for infectivity. A number of viruses with fibers constructed in this manner have been reported ranging from minor modifications, including single amino-acid substitutions17
and addition of small peptides18,19
to more substantial alterations, such as switching entire protein functional domains (knob, shaft)20–22
and generation of artificial fiber-like scaffolds that retain the essential functional properties of the whole Ad fiber.23,24
These vectors generally fulfill the basic targeting requirements on cells in vitro
, but the expected targeting efficacy has not yet been translated to in vivo
experiments. In fact, the genetically modified single component vectors most often require careful optimization of vector design by trial and error. In view of these obstacles, we hypothesized that the fiber mosaic platform, where two fibers are included in the viral genome, presents a more flexible mode for Ad vector modification and may simplify optimization of targeting efforts.
Here, we engineered a novel fiber-mosaic Ad vector, which comprises two fiber types: the wt fiber and FF chimera fused to the BAP. This strategy has previously been used to metabolically conjugate biotin to viral proteins in mammalian cells, which allows them to be coupled to retargeting adapter molecules that contain streptavidin.4,5,25
However, the utility of biotin incorporation for virus retargeting has thus far only been demonstrated in vitro
and, among all of the structural proteins tested, only fiber-incorporated biotin was capable of efficient retargeting.5
These studies established that biotin attachment is determined by the locale for BAP incorporation and provided the rationale for our fiber-based retargeting strategy.
Although our fiber-mosaic vector does provide an alternative tropism, the native tropism is not completely ablated due to the retention of the wt fiber in the viral capsid. The targeting function is delegated to a recombinant fiber, while keeping the structural and functional integrity of the wt fiber during all stages of the viral life cycle. The presence of wt fiber served to amplify the fiber-mosaic Ad to titers as high as 1012
vp/ml in regular laboratory-scale preparations. The presence of wt fiber may undermine the efficiency of retargeting, as just a fraction of fibers present on the viral capsid will serve for retargeting purposes. However, efficient redirection of our vector towards cells expressing EGFR was validated in vitro
. The adapter-driven mBfMAd increased transduction of EGFR-positive cell lines 10- to 23-fold, compared to transduction without adapter. This level of enhancement is comparable with previously reported values obtained utilizing a complex of the wt virus with different bispecific molecular adapters.12,26
Similarly, adenovirus retargeted through an ‘adenobody’ strategy demonstrated a 10-fold enhancement of infectivity on A431 cell line.27
Efficient utilization of the retargeting fiber was demonstrated using blocking experiments. The mBfMAd vector complexed with EGF-Streptavidin maintained a high level of gene transfer in the presence of Ad5 fiber knob concentrations that blocked infectivity of the Ad5luc control virus in the presence or absence of the adapter. These data additionally support the ability of mBfMAd to redirect infection through the EGFR pathway. Thus, we believe that the proposed mosaic virus can efficiently utilize one of the mosaic fibers to redirect virus to alternative receptor in vitro
Retargeting of different viruses through cell growth factor receptors, such as EGFR, which is highly expressed on tumors of different origin, has been validated in several adapter-based studies.12,26–29
All reported adapter constructs were effective at coupling Ad to EGFR and resulted in increased gene transfer to EGFR expressing cell lines. This proved that EGFR pathway is compatible with the adenoviral infection cycle. EGF exhibits high affinity binding to EGFR, which leads to rapid internalization via the receptor-mediated endocytic pathway but no recycling of the receptor-ligand complex.30
Thus, the EGFR pathway is one of the best studied and proven pathways for adenoviral retargeting in vitro
. However, in vivo
studies involving such experiments are scarce and primarily utilize intratumoral administration of retargeted viruses.28,31
There is often a disconnect between the virus targeting efficacy in vitro
and that in vivo
, whereby the retargeting results obtained in cell culture does not translated into in vivo
gains. Thus, we next wanted to test if our retargeting strategy using fiber-mosaic virus can be effective in vivo
and retarget mBFmAd to EGFR expressing cells.
Initial in vivo
studies for retargeting adenoviral vectors were carried out using adapter-mediated approaches in i.p. models of human cancer in mice.32,33
This approach allows evaluation of targeting without the major hurdle of systemic virus administration, sequestration of the injected virus by the liver. The SKOV3ip ovarian cancer cell line expresses a very high level of EGFR and thus presents a good model to test EGFR targeting. Depending on the experimental conditions, mBfmAd demonstrated a 5-to 20–fold increase in gene transfer on these cells in vitro
. Thus, it seemed logical to test whether the targeting gains would be paralleled in vivo
. When injected in mice with pre-established i.p. SKOV3ip xenografts, EGFR-retargeted mBfmAd increased tumor luciferase expression sevenfold, whereas gene expression in the liver was not affected. Gene transfer efficiency with the Ad5luc vector was also slightly enhanced by the presence of the adapter. We have noted the similar effect of adapter on Ad5luc infectivity in vitro
, but the adapter-based gene transfer enhancement of Ad5luc was lower than that of the mBFmAd. Moreover, the addition of the adapter to Ad5luc increased gene transfer in both tumor and liver to the same extent, which finally resulted in similar tumor-to-liver ratios for Ad5luc with or without adapter (9.4 vs 8.9), whereas the mean tumor-to-liver ratio calculated for the group receiving mBfMAd increased from 39 to 69 for virus without adapter and EGFR-retargeted virus, respectively. Of note, previous publications on adapter-mediated Ad retargeting, under similar experimental conditions, report only qualitative data on targeting gains32,34
or targeting gains of at most two- to fivefold. Thus, the retargeting strategy applied to mBFmAd allowed to achieve comparable increase in tumor gene transfer in the context of ovarian cancer xenografts.
Another approach to test retargeting properties of adenoviral vectors in an efficient manner was recently developed in our group.2
As systemically introduced vectors retargeted to tumors are to overcome multiple physiological barriers before reaching their targets, it has been proposed that the display of targeting molecules at accessible sites would facilitate testing of targeting gains of systemically administered adenoviral vectors. An hCAR-transgenic mouse model that is sensitized to Ad infection and is used to transiently express tumor antigens in the lung vasculature was recently reported to be efficient for evaluating vectors targeted to CD40 and carcinoembryonic antigen (CEA).2,35
Thus, the hCAR mice were used to confirm whether our targeting strategy using the combination of two targeting modes in a single vector could efficiently target hEGFR expressed in the lungs. Expression of hEGFR was transiently induced in the mice pulmonary endothelium by systemic injection of recombinant adenovirus AdfltEGFR. In this model, mBFmAd retargeted to EGFR expressed in mouse lungs showed a fivefold enhancement in the lung gene transfer, which resulted in increased the lung-to-liver retargeting ratio from 1.3 for the virus without adapter to 6.3 for the retargeted virus. The lung-to-liver ratios calculated in our experiment for the retargeted fiber-mosaic vector correlated well with the values obtained in a previous study utilizing this model for CD40 retargeting (lung-to-liver ratio of 5.2 for CD40-retargeted virus and 1.2 for irrelevant virus),35
thus providing a good estimate of the level of transductional retargeting gains achievable in this in vivo
model. Overall, this transient transgenic model system allowed our targeting strategy to be evaluated.
Despite the fact that statistically significant differences in gene transfer was obtained by targeted vs untargeted virus in both models tested, a considerable variation of gene transfer values were obtained for individual animals, particularly for the calculated tumor-to-liver or lung-to-liver ratios. Specifically, this was observed in the groups receiving mBFmAd with adapter. These variations could be associated with the individual in vivo conditions, experimental errors or factors related to the retargeting system itself in its current design, such as uniformity of virus prep and virus-adapter formulation.
All experimental work included in this study was carried out using single mBFmAd and Ad5luc preps, which were characterized for vp and PFU content, as well as the degree of fiber incorporation and biotinylation. However, we would like to stress that the uniformity of viral preparation in terms of the fiber content of each individual vp remains unknown. It is likely that the preparation of fiber-mosaic virus could contain (i) truly fiber-mosaic particles having both fibers in one viral capsid, (ii) a mixture of viral capsids displaying just one of the fibers and (iii) a combination of both variants. This factor may affect the overall performance of the fiber-mosaic virus. Of note, native fiber-mosaic viruses of serotype 40 and 41 have equal presentation of both fibers and apparently display both fibers in a single vp.
Another possible cause for inconsistence of viral preps is potential recombination. Although we were trying to minimize homologous sequences in mosaic genome, the rearrangement at low level still exists. DNA isolated from several viral preps of mBFmAd was tested for rearrangement and reversion to the single fiber genome by PCR. A low level of PCR product with that size corresponded to the recombination event was detected in viral preps, and in plasmid preps of mBFmAd Shuttle vector and Ad genome, which are the standard steps of designing adenoviral vector. We believe that recombination at some low level occurred in the plasmid DNA, whereas it is being propagated in bacteria, thus mBFmAd viral prep also may carry the low level of contamination with viruses with one fiber. To minimize recombination in improved generations of fiber-mosaics, silent point mutations can be introduced into fiber tail sequences. In addition, we also cannot exclude possible batch to batch variation of mBFmAd preps in terms of biotinylated fiber incorporation. For this experiment virus was preincubated with the adapter without any additional purification. However, further optimization for obtaining the virus-adapter complex can be considered. The crude viral prep obtained after the first virus CsCl banding can be further purified on avidin columns to eliminate virions lacking the recombinant fibers fibers. We are currently testing the consistency of fiber-mosaic virus preparations. Several lots of virus preparation did show a similar ratio of wt fiber to biotinylated fiber incorporation as described previously. However, different experimental and viral amplification conditions may bias or favor incorporation of the retargeting fiber and influence the overall efficiency of the retargeting strategy.
In this study, we have demonstrated enhanced gene transfer based on transductional retargeting of our vector. It would be of a considerable interest to investigate the extent to which it will translate to therapeutic end points. Several publications indicated the feasibility of conversion of the vector targeting gains to the treatment benefits in experimental animal models.28,33
Thus, our future goal will include the introduction of an anticancer therapeutic gene in the context of the proposed fiber-mosaic platform to test therapeutic efficacy of our retargeting strategy.
In summary, we have confirmed that mBfMAd complexed with EGF-Streptavidin could successfully retarget virus to EGFR-expressing cancer cell lines. Most importantly, the evidence of utility of this strategy was demonstrated in vivo. The targeting potential of mBfMAd complexed with EGF-Streptavidin was tested on two in vivo models that overexpress EGFR in different tissues. First, we utilized a mouse model of locoregional EGFR-positive ovarian xenografts. Secondly, the retargeting capacity of mBfMAd/EGF was tested on a ‘transient transgenic’ mouse model with transient expression of hEGFR in the mouse lung vasculature. mBfMAd/EGF achieved a higher transduction level in the lung compared to mBfMAd without adapter. Moreover, tumor-to-liver or lung-to-liver gene expression ratios for retargeted mBfMAd increased in both models tested. Thus, we demonstrated that an additional fiber in fiber-mosaic virions could be used for biotinylation and this modification, in combination with appropriate adapters, can be successfully exploited for adenoviral retargeting strategies. Importantly, our study demonstrated the proof of preclinical utility of our targeting strategy in animal models.