PROJECT SUMMARY/ABSTRACT Approximately half of all patients diagnosed with colorectal cancer develop liver metastases (stage 4 disease) and have a poor prognosis. Surgical resection remains the standard of care for 10-30% of patients with colorectal liver metastasis, but the recurrence rate for these patients is high (60-80%). Oncolytic virus immunotherapy represents a novel therapeutic approach for liver metastasis, combining cell killing by oncolysis and immune-mediated effects that are crucial for response durability. DNX-2440 is a tumor-selective, conditionally-replicative oncolytic adenovirus encoding human OX40 ligand (OX40L), developed to treat cancer. DNX-2440 combines in a single agent the oncolytic activity and anti-tumor immune activity of the predicate adenovirus, DNX-2401 (tasadenoturev) which has completed Phase 2 testing in recurrent glioblastoma, with additional immune modulation mediated by OX40 pathway engagement. The OX40L genetic modification in DNX-2440 enhances immune activation within the tumors, as engagement of the OX40 receptor by OX40L enhances memory T-cell survival and suppresses the differentiation and activity of regulatory T-cells. Preclinical studies demonstrate that DNX-2440 leads to high expression levels of OX40L, tumor-specific anti-tumor immune memory, and effective cell killing in both injected and non-injected tumors. In this application, a Phase 1 open-label, window-of-opportunity study is proposed to assess the clinical safety and anti-tumor immune response to neoadjuvant DNX-2440 in patients with resectable liver metastasis. The study intervention includes two sequential intratumoral injections of DNX-2440, two weeks apart, to a single lesion of liver metastasis (target lesion) in patients presenting with two or more lesions of liver metastasis and scheduled to have surgery for resection of all lesions. This study will evaluate the safety and maximum tolerated dose of intratumoral administration of DNX-2440 and will provide extensive information about the biological and immunotherapeutic effects of DNX-2440 in the studied patient population. Correlative analyses will be performed on collected pre-treatment tumor biopsies from the target lesion and post-treatment tissue (injected lesion, non-injected lesions, normal tissue) collected during the surgical resection.