Substitution of the Ad3 hexon onto the Ad5 capsid confers ablation of binding to FX and thereby decreases liver tropism and enhances the anti-tumor therapeutic window. Herein, we have demonstrated that these hexon substituted vectors do not bind FX which translates into decreased liver transduction and improved tumor transduction. Furthermore, by ablating FX binding and decreasing liver sequestration the effects of infectivity enhancement are improved as demonstrated by improved tumor transduction with the Ad3 fiber knob modified vectors in conjunction with hexon modification.
It was surprising that the Ad5HVR5-6H vector bound to FX and didn’t exhibit any decreased liver tranduction based on previously published findings of the role of HVR5 in binding to FX. However, after further investigation it is likely the lack of ablation of binding was due to a combination of factors. Some of these factors are that enough of the residues in HVR5 responsible for binding to FX were preserved (8
), HVR7 remained intact, and the peptide insertion was insufficient to confer a conformational change in the hexon protein. Previously published HVR5 mutants didn’t preserve the threonine amino acid at position 269 (which was preserved in our vectors) and included insertions of at least 8 amino acids (6
The treatment of animals with warfarin during the administration of Ad vectors has been previously shown to result in improved anti-tumor efficacies (11
). The lack of a lasting effect of the warfarin treated animals with the CRAd agent containing the wild-type Ad5 hexon is likely because the progeny virus contain the wild-type Ad5 hexon and can bind to FX, which is repleted after dosing with warfarin wears off and therefore the circulating FX would help clear the progeny virus. In our experiments therapeutic doses of warfarin were only administered immediately prior to the initial Ad injection.
In this study we substituted the Ad3 hexon onto the Ad5 capsid, therefore, it remains to be clearly determined if the ablation to binding FX of this vector results from an intrinsic lack of binding by the Ad3 hexon or if it is the conformational change from incorporation of this foreign hexon into the Ad5 capsid that ablates any binding to FX. Although previous experiences have noted a decrease in viral titer production with this modification, we were able to generate adequate viral titers (1 × 1012 vp/ml) for our experimental purposes with comparable TCID50 (data not shown).
Overcoming the obstacles to safe and effective systemic administration of Ad vectors is essential in advancing an effective anti-cancer Ad based therapy. Several key components of achieving this have been recently elucidated, notably, the liver tropism mediated by Ad5 hexon binding to coagulation FX. There are now several published Ad hexon mutants that demonstrate ablation of FX binding with resultant decreased liver tropism. Which one of these mutants is the “best” will be dependent on further comparison analysis and the result may differ for the particular application. Beyond its role in binding to FX, hexon serves as one of the major capsid proteins and modification of it may lead to vector instability. However, modifications may also serve multiple purposes beyond ablating FX binding, such as re-targeting through peptide insertions or changing the immunogenicity of the vector.
Three major approaches will likely be advanced for FX-ablating hexon modifications. Two strategies retain the majority of the Ad5 hexon: 1) minimal point mutations of the required residues in HVR5 and HVR7 to minimally affect the vector capsid yet ablate FX binding. 2) Insertion of peptides into HVR5 and/or HVR7 that ablate FX binding and allow further benefits such as re-targeting, epitope display, or avoidance of immune recognition. 3) hexon pseudotyping with Ad serotypes that don’t bind FX. The first approach has been successfully achieved by substitution of 3 residues (2 in HVR5 and 1 in HVR7) with corresponding residues from Ad26 (8
). The second approach has been initiated by some groups but the full potential of such hasn’t been completely demonstrated. The third approach involves substitution of the majority of the hexon protein with that of an alternate serotype that ablates FX binding and potentially provides additional benefits such as decreased immunogenicity for repeat vector administration.
The findings presented herein, offer another strategy for Ad hexon modification for ablation of FX binding and decreasing liver tropism. Furthermore, the synergistic effect of combining infectivity enhancement strategies with decreased liver sequestration provide promise that effective targeting and therapeutic effects following IV administration can be achieved. To apply Ad vectors for several systemic disease targets, including metastatic cancer, will necessitate effective IV administration. Several obstacles will be necessary to overcome to achieve this end, these include decreasing liver toxicity and sequestration of injected vector, improving target cell transduction, and decreasing vector-immune system interactions. Adenovirus pseudotyping of the hexon and fiber proteins with alternate serotypes, as presented here, may offer the opportunity to overcome several of these obstacles.