Unresectable hepatic CRC metastases are one of the leading causes of cancer mortality. Adenovirus gene-delivery vectors provide substantial promise as therapeutic agents, and have proven effective in cases where local delivery is feasible (40
). Unfortunately, in the clinic it is often not possible to directly inject all metastases. Here we demonstrate that systemic adenovirus tumor targeting and therapy can be achieved by combined use of (i) transductional liver Ad untargeting with a trimeric bispecific adapter, to reduce complications of liver infection, (ii) transductional tumor retargeting with this bispecific adapter to increase efficacy and specificity of hepatic tumor transgene delivery and (iii) transcriptional restriction with a tumor-restricted promoter. This demonstration of enhanced adenovirus transgene tumor expression with therapeutic efficacy and reduced liver transgene expression and hepatic toxicity suggests a means to engineer practical, effective therapeutic agents, both for hepatic CRC metastases in particular and for hepatic metastases of other epithelial cancers.
Adenovirus gene transfer efficacy for most tumors, following systemic administration, has been limited. One reason is that many tumors lose CAR expression (41
). Moreover, over 90% of systemically administered Ad is taken up by the liver (44
). We previously demonstrated transductional Ad liver untargeting and CEA+
hepatic tumor reporter gene retargeting following systemic Ad administration, using monomeric sCARhMFE adapter (15
). In this report we demonstrate that combining transductional liver untargeting/tumor retargeting with COX-2 mediated transcriptional restriction increases hepatic tumor:liver targeting following systemic virus administration ( and ). We have now substantially improved the adapter, increasing its binding both to the virus knob and to CEA on target cells, by a trimerization protocol (). We demonstrate here that sCARfMFE trimerization results in (i) greater adapter affinity for the viral knob, (ii) increased untargeting of CAR-mediated infection in cultured cells, (iii) increased hepatic untargeting in vivo
and (iv) increased retargeting efficacy for CEA+
tumor cells, both in culture and in vivo
Although adenovirus-bispecific adapter complexes are prepared at high concentrations, the preparations are diluted substantially when injected intravenously. To determine whether association between the trimeric fiber knob and the trimeric sCARfMFE adapter renders the [Ad][sCARfMFE] complex less likely than the adenovirus-monomeric adapter [Ad][sCARhMFE] complex to dissociate following intravenous infection, we examined the effects of [Ad.CMVfLuc][sCARhMFE] and [Ad.CMVfLuc][sCARfMFE] dilution on Ad retargeting to cultured CEA+
cells (Supplementary Fig. S3
). Dilution decreased [Ad.CMVfLuc][sCARhMFE] CEA retargeting efficacy much more extensively than did dilution of [Ad.CMVfLuc][sCARfMFE].
Competition by soluble CEA (sCEA), often found in sera of colorectal cancer patients (46
), might also interfere with anti-CEA based Ad tumor retargeting for CEA+
tumors. To determine whether the [Ad][trimeric sCARfMFE] complex is more resistant than the [Ad][monomeric sCARhMFE] complex to sCEA competition for binding to CEA+
cells, infection of CEA+
cells by [Ad][adapter] complexes was competed with recombinant sCEA. Monomer sCARhMFE-mediated CEA retargeting is nearly eliminated by sCEA; only 6% of [Ad.CMVfLuc][sCARhMFE] binding is not blocked (Supplementary Fig. S4
). In contrast, nearly half the trimer sCARfMFE Ad retargeting ability cannot be competed by sCEA (Supplementary Fig. S4
), suggesting that the trimer binds to cell membrane CEA molecules much more avidly, presumably by multivalent “capping” of CEA molecules that are mobile in the cell membrane. Thus circulating sCEA is less likely to present a problem in retargeting adenovirus to CEA-shedding tumors when trimer sCARfMFE adapter, rather than monomer sCARhMFE adapter, is used.
These data support the potential use of trimerized adapters that untarget adenovirus liver infection and retarget the virus to hepatic tumors as a promising therapeutic strategy for hepatic metastases. In addition to reducing hepatic infection, enhancing CEA-dependent tumor targeting, minimizing dilution effects, and avoiding circulating CEA inhibition of CEA-dependent tumor targeting, sCARfMFE hepatic untargeting also decreases liver toxicity following systemic Ad vector administration both by reducing hepatic therapeutic gene expression and by reducing innate immune responses to Ad particles ().
Transgene expression can also be regulated by transcriptional restriction, using targeted, cell-specific promoters (47
). Even if Ad vectors infect hepatocytes, virally-delivered transgenes cannot be expressed if they are under the control of a promoter not expressed in liver cells. The COX-2
gene is ectopically activated in many epithelial cancers (22
) but is not expressed strongly in liver (50
). Here we used COX-2
promoter restriction to target reporter gene expression to CRC and NSCLC liver metastasis models ( and ) and demonstrated the efficacy of COX-2
promoter restriction in Ad-mediated HSV1-tk/GCV CRC liver metastasis therapy (). COX-2
promoter transcriptional restriction constrains Ad therapeutic transgene expression, following systemic Ad administration, to COX-2+
CRC liver metastases. The result is both to kill the cancer cells using a therapeutic transgene and, by preventing liver transgene expression, to reduce liver toxicity. These data demonstrate that transcriptional restriction can reduce liver toxicity by preventing hepatic therapeutic gene expression during systemic Ad gene therapy.
Imaging data suggest that combining the two technologies should prove more effective at therapeutic gene delivery than either transductional Ad untargeting/retargeting or transcriptional transgene restriction alone ( and ). As anticipated, combined transductional untargeting/retargeting and transcriptional restriction enhances therapeutic efficacy (, left). sCARfMFE transductional liver untargeting and CEA retargeting increased Ad.cox2NTP-mediated HSV1-tk/GCV killing of LS174T hepatic metastases; moreover the increased CRC hepatic tumor cell killing resulting from sCARfMFE transductional retargeting is accompanied by a decreased number of virus particles present in liver (, right).
Combining transductional untargeting/retargeting and tumor-restricted transcriptional expression offers substantial flexibility. Recombinant proteins can be created using alternative adenovirus untargeting components (e.g. sCAR, anti-knob Fab) and retargeting agents (e.g., receptor ligands, antibodies, lectins). Promoters for restricted expression can be chosen that utilize either tissue/cell specificity or tumor cell specificity. By varying retargeting moieties and transcriptionally restricted promoters, one can “tailor” untargeting/retargeting and transcriptional restriction combinations for specific tumors or for other cells. By utilizing alternative transgenes, different purposes can be achieved; cargo genes whose products can be imaged by bioluminescence, fluorescence, magnetic resonance imaging, positron emission tomography, single photon emission tomography, etc can be targeted. Alternatively, therapeutic genes that target tumor cells, tumor neovasculature, inflammatory cells and other cells that participate in tumor progression can be incorporated into these vectors and retargeted to alternative cells and/or tissues with appropriate bispecific adapters.