Enhanced Adenovirus Infection of Melanoma Cells by Fiber-Modification: Incorporation of RGD Peptide or Ad5/3 Chimerism

Abstract

The incidence of malignant melanoma has been increasing. Unfortunately, advanced melanomas are rarely curable with standard therapy; therefore, new forms of treatment such as gene therapy are needed. The success of gene delivery or oncolysis depends on the nature of the vector. Adenoviral vectors are advantageous for several reasons; however, they are dependent on CAR (coxsackie and adenovirus receptor) which is deficient or heterogeneously expressed on melanoma cells in situ. Correspondingly, transduction of freshly purified melanoma cells has been shown to be minimal or variable. In order to overcome this shortcoming, it is necessary to construct tropism modified adenoviral vectors. With this goal in mind, we generated two tropism modified vectors, Ad5lucRGD which has an RGD motif incorporated into the HI loop of the fiber knob and Ad5/3luc1 which contains the tail and shaft domain of the Ad5 fiber and the knob domain of the Ad3 fiber. Herein we demonstrate that Ad5/3luc1 infects CAR-negative primary melanoma cells 1128 times better than Ad5luc1 and 34 times better than Ad5lucRGD. Furthermore we show that Ad5/3luc1 and Ad5lucRGD infect via a CAR independent route by blocking the CAR receptor. In addition, we show that the infectivity of the cells correlates with the expression of CAR and Ad3 receptors determined by FACS analysis. Therefore, Ad5/3 is very attractive as a potential therapeutic vector for malignant melanoma.

Complex mosaicism is a novel approach to infectivity enhancement of adenovirus type 5-based vectors

“…5,9,11,12,27,28 In fact, F.5/3 provided consistently stronger augmentation of infectivity than the HI loop-modified F.5 knob in all cancer cell lines analyzed in this study. A complete resistance of all the F.5/3 chimerabased vectors to recombinant Ad5 knob blocking, on the one hand, but clear sensitivity to the recombinant Ad3 knob, on the other, as illustrated by Figure 5, demonstrates ablation of CAR-tropism and strongly suggests that the major interaction of the F.5/3 viruses with cancer cells involves the Ad3 receptor.…”

“…12,15,[24][25][26] Thus our data indicate that due to some structural features the Ad3 knob is more refractory to genetic incorporation of the RGD-4C peptide than the Ad5 fiber knob domain.…”

“…In addition, serotype chimerism has been demonstrated to accomplish CAR-independent entry with infectivity enhancement. 5,9,11,12 The most recent additional approach has employed a combination of two different functional ligands genetically incorporated in two distinct locales of a single-fiber knob. This approach has proved to be useful as it has allowed an additional infectivity enhancement of the Ad5 vector due to the additive effect of dual-receptor targeting via both ligands within a single fiber molecule.…”

Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways

“…Earlier studies which utilized these vectors have demonstrated that the employed MOI elicits optimum gene transfer of target cells based on available cellular receptors. 17 We would also note that the MOIs we employed do not differ substantially from recent microarray studies of adenoviral entry biology, as noted above. 19,20 Changes in gene expression were considered to be significant when P-values were less than 0.05.…”

“…The rationale for using the human melanoma cell line, M21, was linked to our previous findings which demonstrate effective adenoviral vector-mediated infection of these melanoma cells by tropism-modified vectors, Ad5lucRGD and Ad5/ 3luc1. 17 Herein, we utilize microarray technology to investigate the resultant changes in gene expression after infection of melanoma cells with recombinant adenoviral vectors containing a wild-type capsid, Ad5luc1, or with tropism-modified adenoviral vectors Ad5lucRGD and Ad5/3luc1.…”