We found that the initial delivery of systemically administered oHSV to distant tumors is impaired compared with normal organs, but that infectious virions are amplified rapidly in tumors and cleared from normal organs. Prior anti-angiogenic therapy did not enhance and in some cases was detrimental to tumor uptake of systemically administered oHSV, and combination therapy was more effective than single agent therapy only when virus was given first.
Systemic biodistribution has been a major stumbling block for virus vectors used in gene transfer and virotherapy. The best studied example is adenovirus, where the “first-pass effect” causes most particles given IV to be sequestered in the liver.18
Perhaps because HSV is a larger, enveloped particle there appears to be less of a first-pass effect, with only about one-third of detectable HSV in the liver at 12 hours post administration.19
In fact, antitumor efficacy has been demonstrated in animal models following intravenously administered HSV mutants for subcutaneous tumors20, 21
and pulmonary metastases,22-24
even in the presence of neutralizing antibodies.25
Uptake of oncolytic HSV following arterial infusion has also been demonstrated in human trials.26, 27
These data and studies of other viruses28, 29
suggest systemic intravenous virotherapy may be useful for the treatment of metastatic disease, though little is understood about the mechanisms mediating virus uptake in tumors.
Oncolytic HSV biodistribution has been examined in a genetically engineered spontaneous mouse model of prostate cancer using four virus injections over a 10 day period. HSV genomes were detected equally in prostate tumors and liver, with less in lymph node, lung, and blood.30
There was no evidence of virus amplification in tumor sites, but virus genomes were not quantified before day 11; importantly, virus genomes persisted in the prostate tumors whereas they were rapidly cleared from normal organs. The analyses were performed 11 days after the first virus injection and 1 day after the last, so the actual amount delivered to various sites, as opposed to that resulting from virus replication or migration, is unclear. Interestingly, oncolytic HSV given intraperitoneally was also found in distant tumor sites and induced an antitumor effect, though full biodistribution studies were not performed.
One limitation to systemic HSV delivery is innate immunity. Following infection with most strains of wild type HSV, mice mount vigorous innate and adaptive anti-viral immune responses. HSV has evolved mechanisms such as Fc receptor binding to subvert adaptive anti-viral immunity, but innate immune factors including complement have been shown in some cases to limit viral delivery to tumor sites when given regionally.31-33
One strategy to subvert inactivation of viruses given systemically that has been pioneered with other virus types is the use of tumor-homing cells, such as tumor cells or T cells, as delivery vehicles.34, 35
Induction of a transient vascular leak with vasoactive agents including IL-2, enhanced by inhibition of Tregs, has been shown to markedly enhance delivery of an oncolytic virus to distant tumor sites.36
It is well documented that small molecule drugs linked to or encapsulated in liposomes show preferential accumulation in tumors due to leaky neoangiogenic vessels (with intercellular gaps as large as 600-800 nm) and impaired lymphatic drainage, an effect known as enhanced permeability and retention.37
A similar phenomenon for HSV uptake was not borne out by our data, as we found decreased amounts of virus within the tumor relative to other organs shortly after virus administration. It may be that compared with small molecule drugs and their lipid conjugates, the larger size of HSV particles (~150nm diameter) prevents passive extravasation through inter-endothelial cell gaps regardless of a pressure differential. In addition, there may be fewer herpesvirus receptors in tumors compared with normal organs,38, 39
resulting in only low levels of active receptor-mediated uptake, which would be unaffected by pressure differentials. Whether or not tumors in different sites or organs other than the flanks and intramuscular tumors tested in this study differ in this regard is unknown.
VEGF blockade has been shown to decrease tumor interstitial pressure, transiently enabling increased delivery of systemic chemotherapeutic agents to xenograft tumors before the vasculature is fully remodeled.16, 40
In contrast, we found that VEGF blockade did not increase, and in fact in some cases decreased delivery of oHSV to tumor sites. The most likely explanation is the reduced tumor perfusion following antiangiogenic therapy, though we cannot rule out the formal possibility that HSV receptors were downregulated on endothelial or tumor cells by anti-VEGF antibody. This remains a possibility in light of data suggesting VEGF upregulates HSV receptors on endothelial cells,41
though we did not observe any effect of anti-VEGF on HSV-mediated cytotoxicity on tumor cells in culture (Supplementary Figure S1
). The effect of VEGF blockade on virus replication and spread after it has already reached the tumor is also unknown.
Anti-VEGF given after systemic oHSV administration led to better tumor control and animal survival than when given prior to virus. In the latter case, there was a trend toward additive effects of combination therapy compared with bevacizumab alone (p=0.13) but not compared to virus alone, suggesting that the lower antitumor effect of having less virus was offset by the additional antitumor effect of the bevacizumab. The reason DC101 appeared to have less of an effect than bevacizumab is not known, but may be related to the fact that we did not optimize the doses of each antibody or to variables inherent to binding soluble ligands versus membrane bound receptors. Also, administration of DC101 in mice results in a compensatory increase in native systemic mouse VEGF levels,42
which may have partially counteracted the effects of DC101. Whether such an effect occurs with bevacizumab has not been tested.
Whether or not our findings are broadly applicable to oncolytic viruses in general is not known. We did not investigate whether tumor uptake of smaller viruses, such as adenovirus or Sindbis virus, or those of a different shape (e.g. filamentous), which also require receptor-mediated uptake but may better fit through endothelial gaps, are also impeded by anti-angiogenic agents. Similarly, we do not yet know if the effect will be less pronounced in other tumor models that are less dependent on VEGF. Regardless, our data suggest that tumor vascular remodeling by anti-angiogenic agents fails to enhance and in some cases decreases tumor uptake of systemically administered oncolytic HSV particles and should therefore be used after, but not before, systemic virus administration.