Chimeric Ad5/35 oncolytic adenovirus overcome preexisting neutralizing antibodies and enhance tumor targeting efficiency.

KD01, a third-generation conditionally replicating adenovirus serotype 5 developed by our team, has approved by the China Center for Drug Evaluation (CDE) for Phase I clinical trials (NCT06552598). However, 60% seroprevalence of anti-Ad5 neutralizing antibodies is a major hurdle for Ad5-based oncolytic viruses. To address this issue, we developed oAd5/35-HF, a fourth-generation oncolytic adenovirus vector designed to enhance infection efficiency and evade pre-existing neutralizing antibodies (NABs). To achieve this, we introduced targeted capsid modifications, replacing hexon hypervariable regions (HVRs) 1 and 5 with those from adenovirus serotype 35 (Ad35), along with alterations to the fiber region. These combined modifications significantly improved infection efficiency, maintained high viral titers, and enabled the virus to resist NABs. This is the first report of replacing both the Ad5 hexon HVRs and fiber regions with those from Ad35 in an oncolytic adenovirus, resulting in potent antitumor activity across multiple cancer types, even in the presence of high NAB levels. The oAd5/35-HF backbone provides a versatile platform for developing new chimera oncolytic adenovirus and adenovirus vector-based vaccine.