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Abstract
Background Viral entry into target cells through cell surface receptor-viral protein interactions is a crucial step in oncolytic virotherapy. The broad tropism and high transduction efficiency of adenoviruses (Ads) have contributed to their extensive employment in gene therapy and as oncolytic viruses (OVs). The commonly used Ad type-5 (Ad5) utilizes the coxsackie adenovirus receptor (CAR) for cell entry. However, CAR downregulation during cancer progression1,2 limits the therapeutic efficacy of OVs reliant on this surface protein. Group B Ads use CD46 (a ubiquitously expressed receptor frequently overexpressed in cancer)3,4 for cell entry, thereby presenting an opportunity to equip OVs with chimeric fibers to enhance tropism to malignant cells. We engineered ICVB-1042, a potent, selective, and systemically available OV, with an Ad5/Ad34 chimeric fiber to enable viral entry via CD46 instead of CAR proteins. Here, we demonstrate the cell entry requirements for ICVB-1042 compared to ICVB-421, an Ad5-derived virus with wild-type fiber and capsid.
Methods CD46 and CAR knockout (CD46- and CAR-) A549 human lung carcinoma cell lines were generated using CRISPR editing. The cell lines were exposed to ICVB-1042 or ICVB-421, and the yellow fluorescent reporter protein (YPET) expression was analyzed in viable CD46+ and CD46- cells. Viral cytotoxicity was measured using cell index, a cell viability surrogate, in CD46-, CAR-, and CD46+ cells. The percentage of cytolysis was quantified relative to controls (no virus). Mouse LL/2 cells expressing transgenic human CD46 were exposed to a replication-independent YPET-expressing vector with the capsid of ICVB-1042 to determine viral entry.
Results A dramatic increase in YPET expression in CD46+ cells was observed post-infection with ICVB-1042, suggesting that CD46 expression is a requisite for ICVB-1042 cell entry (figure 1A-B). ICVB-421 infection was not strongly associated with CD46 expression, indicating that the absence of CD46 did not impair ICVB-421 tumor cell entry. ICVB-1042 infection induced 100% cytolysis in all cell lines faster than ICVB-421 but with a lower rate in CD46- cells (figure 1C), demonstrating that CD46 deficiency induces resistance to ICVB-1042-induced tumor killing. High YPET fluorescence intensity in mouse LL/2 cells harboring human CD46 transgenes was observed, denoting that human CD46 expression by LL/2 cells enabled transduction by a vector version of ICVB-1042 (figure 2A-B).
Conclusions These results demonstrate that replacing wild-type fiber with the chimeric Ad5/Ad34 fiber of ICVB-1042 results in a potent OV that relies on CD46 expression for tumor entry.
References
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(A) Flow cytometry analysis of YPET expression in CD46 knockout cell lines; the A549 parental cell line with endogenous CD46 expression (CD46+) was used as a positive control. (B) Plots showing percentage of YPET-expressing CD46+ and CD46- cells (46.5% in CD46+ versus 1.9% in CD46- cells; p=0.0005, t-test). (C) Cell viability was measured at baseline and every 30 minutes for 144 hours following ICVB-1042 or ICVB-421infection. MOI: multiplicity of infection; 24hpi: 24 hours post infection.
(A) Representative images showing YPET fluorescence in mouse lung carcinoma LL/2 cells (generated via piggyBac transposition) following infection of a replication-independent YPET-expressing vector of ICVB-1042. (B) Plots showing average YPET fluorescence signal intensity (with background fluorescence subtracted). MOI: multiplicity of infection; 1085 and Clone 5 are constructs expressing CD46 isoforms in parental LL/2 cell lines.