Session topic


Title: Properties of Adenovirus vectors with increased affinity to DSG2 and its potential benefits for oncolytic approaches and gene therapy
ID: PS 26
Type: Poster session
Talk time: 3 + 2 min
Session: Poster session 1
Receptors and entry

Speaker: Nora Bahlmann (Witten/DE)

Abstract - Text

Abstract text (incl. references and figure legends)

Carcinomas are known to have a widespread upregulation of intercellular junctions as barriers to immune responses and drug therapy. To address this, the junction opener 4 (JO4), a small recombinant protein, derived from the human adenovirus 3 (Ad3)-fiber knob was previously developed. Surface plasmon resonance analysis revealed an 885-fold higher affinity of JO4 to the Ad receptor Desmoglein 2 (DSG2) compared to the parental fiber knob. Binding of JO4 to DSG2 leads to shedding of the domain followed by less junction protein expression and transient tight junction opening. JO4 has proven effective to enhance antibody and chemotherapy and is now explored in clinical trials.

We investigated different JO4-containing adenoviral vectors in their ability of cell entry, transgene expression and oncolytic effect in different cancer cell lines to potentially find a therapeutic benefit. Based on the parental Ad3 and two types of chimeric Ad5/3 (Ad5-long fiber shaft with Ad3 fiber knob and Ad5 with short fiber shaft from Ad3 and Ad3 fiber knob), we constructed individual viruses containing the JO4-mutation. Virus derived GFP-expression was measured by FACS and luciferase expression was quantified by reporter assays. Viral vector cell entry was quantified using qPCR and to measure cell lysis efficiencies oncolytic assays were performed. For receptor expression analyses we applied DSG2/CAR and CD46 immunostaining in flow cytometry.

In all our experimental settings and investigated cell lines we could not find a benefit from the JO4-containg viruses compared to the parental viruses. Instead,  Ad3 and Ad5/3 with JO4-mutation presented a rather weakened effect in cellular entry, transgene expression, oncolysis assay, spheroid culture infection assay and TEER-measurement in multilayer transwell culture. We hypothesized that the JO4-mutation might influence CD46 and CAR binding of the viruses but even in CD46 and CAR- double-knock out-cells no enhancement was observed. Also, there was no distinct advantage of infecting cells with higher DSG2 expression levels. Comparing the viral vectors without JO4-mutation among themselves Ad5/3-long fiber seems to be a promising candidate for infecting Hs578t and K562, showing a more than 20% higher GFP-Expression level in FACS in the latter than wild type Ad3 and even an 8-fold higher level than wild type Ad5.