Wild-Type Human Adenovirus (Serotype 35)

Oncolytic adenovirus (OAd) is one of the most promising oncolytic viruses. In this study, a novel OAd composed entirely of oncolytic adenovirus serotype 35 (Ad5) (OAd35) was developed. OAd35 recognizes CD46 as an infection receptor, which is ubiquitously expressed in almost all human cells and is often upregulated in malignant tumor cells. In addition, 20% or less of adults have neutralizing antibodies against Ad35. In CAR-positive tumor cells, OAd35-mediated cytolytic activity was similar to that of OAd5, while in CAR-negative tumor cells, OAd35 exhibited higher cytolytic activity than OAd5. Anti-Ad5 serum significantly inhibited the in vitro tumor cell lytic activity of OAd5, while OAd35 exhibited comparable levels of in vitro tumor cell lytic activity in the presence of anti-Ad5 and naive serum. After intratumoral injection, OAd35 significantly inhibited the growth of subcutaneous CAR-positive and CAR-negative tumors. These results indicate that OAd35 is a promising oncolytic virus to replace OAd5.

To examine the tumor cell lytic activity of OAds, crystal violet staining was performed 5 days after virus infection (Figure 1A). Both OAds exhibited potent and comparable tumor cell lytic activity on HepG2 and A549 cells, both of which are CAR-positive. OAd35 killed H1299 cells less efficiently than OAd5. OAd35 achieved 100% H1299 cell lysis at 300 viral particles (VP)/cell, whereas 100% H1299 cell lysis was observed with OAd5 at 100 VP/cell. On the other hand, in CAR-negative T24 and MCF-7 cells, OAd5 did not exhibit significant tumor cell lytic activity at less than 1,000 VP/cell, whereas OAd35 mediated potent tumor cell lytic activity in these CAR-negative tumor cells. In addition, OAd5 mediated lower tumor cell lytic activity than wild-type Ad5 in A549, H1299, and MCF-7 cells. On the other hand, OAd35 showed comparable or higher levels of tumor cell lytic activity than wild-type human adenovirus (Serotype 35) in all tumor cells except A549 cells. These data indicate that E1A gene expression using the hTERT promoter reduced the tumor cell lytic activity of OAd5, whereas insertion of the hTERT promoter enhanced the tumor cell lytic activity of OAd35. The efficiency of OAd5 and OAd35 in inducing cell lysis in NHLF and MRC5 cells was lower than that of the corresponding wild-type Ads (Figure 1B), indicating that insertion of the hTERT promoter significantly enhanced the safety of OAd5 and OAd35.

Figure 1. Tumor cell lysis activities and safety profiles of OAd35. (Ono R, et al., 2021)