Retargeting of adenoviral infection to melanoma: Combining genetic ablation of native tropism with a recombinant bispecific single‐chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

Abstract

Gene therapy is an emerging and promising modality for the treatment of malignant melanoma and other neoplasms for which conventional therapies are inadequate. Various therapeutic genes have shown promise for tumor cell killing. However, successful gene therapy depends on the development of efficient and targeted gene transfer vectors. Here we describe a novel strategy for targeting of adenovirus-mediated gene transfer to melanoma cells. This strategy combines genetic ablation of native adenoviral tropism with redirected viral binding to melanoma cells via a bispecific adapter molecule, a bacterially expressed single-chain diabody, scDb MelAd, that binds to both the adenoviral fiber protein and to the high molecular weight melanoma-associated antigen (HMWMAA). This antigen is widely and specifically expressed on the surface of melanoma cells and its expression is associated with tumor development and progression. Our results showed specific and strong binding of the anti-HMWMAA scFv RAFT3 and the bispecific adapter scDb MelAd to melanoma cells. In adenoviral infection experiments, we demonstrated i) substantially (>50-fold) reduced infectivity of capsid mutant adenoviruses, ii) restored (up to 367-fold increase), CAR-independent and HMWMAAmediated infectivity of these mutant viruses by scDb MelAd specifically in melanoma cells, and iii) higher levels of transgene expression in melanoma cells by fiber mutant virus complexed with scDbMelAd, relative to a vector with wildtype fibers. We confirmed the utility of this targeting strategy with human primary melanoma cells that represent clinically relevant substrates. These experiments established that the retargeting strategy mediates up to 54-fold increased adenoviral gene transfer to CAR-negative melanoma cells compared to the vector with native tropism. Hence, the HMWMAA-targeted adenoviral vector lacking native tropism exhibits both enhanced specificity and augmented infectivity of gene transfer to melanoma cells, suggesting that it is feasible to use this vector to improve gene therapy for malignant melanoma.

Modulation of viability and maturation of human monocyte‐derived dendritic cells by oncolytic adenoviruses

“…Primary melanoma cells were obtained from surgically removed skin metastasis as described previously. 8 Cells were grown at 37°C in a humidified atmosphere of 5% CO 2 . Monocyte-derived dendritic cells (moDC) were generated from leukapheresis products of healthy donors in a standardized procedure as described previously.…”

“…First, previous studies have revealed a paucity of expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackievirus and adenovirus receptor) on cancer cells, which were freshly purified from tumor specimen of different tumor types. 7,8 These cells are therefore poorly infected and lysed by oncolytic adenoviruses derived from Ad5. 9,10 Secondly, animal experiments have shown that adenovirus lateralization is limited by anatomical barriers such as connective tissue, 11 which can constitute large fractions of human tumors.…”

Highly specific transgene expression mediated by a complex adenovirus vector incorporating a prostate-specific amplification feedback loop

“…Finally, the effective transcriptional regulation afforded by PFLPS could be combined with current methods of transductional regulation including manipulation of Ad fiber knob [41][42][43][44][45][46][47] and use of bi-specific antibodies 38,[48][49][50][51] to further improve the targeting of gene therapy vectors to specific cell types and therefore increase the specificity and the safety of the vectors.…”

A strategy for systemic delivery of the oncolytic herpes virus HSV1716: redirected tropism by antibody-binding sites incorporated on the virion surface as a glycoprotein D fusion protein

“…[14][15][16][17][18][19][20][21][22] Bridging, bispecific targeting molecules comprising either an anti-adenovirus scFv or the Coxsackievirus-adenovirus receptor linked to a targeting scFv, have been used for targeting oncolytic adenoviruses. [23][24][25][26][27][28][29] Results from a number of studies with HSV-1 have shown that it is possible to alter the tropism by incorporating ligands such as erythropoietin, interleukin (IL) 13, human hepatitis B virus preS1 peptide, the N-terminal fragment of urokinase-type plasminogen activator or 6-His into the viral envelope as glycoprotein fusion proteins. [30][31][32][33][34][35] Targeting has also been achieved using a soluble adapter molecule comprising an antiepidermal growth factor receptor (EGFR) scFv linked to the HSV-1 gD-binding domain of nectin-1 and more recently, a scFv against HER2/neu incorporated into the N terminus of gD has been shown to redirect the tropism of HSV-1 to this mammary tumour receptor.…”