Background: The long-term complete remission experienced by some cancer patients after receiving immunotherapies, such as adoptively transferred tumor-infiltrating lymphocytes (ACT-TIL), represent the ideal outcome to pursue for the development of therapies for oncology. At the same time, those responses are limited to a minority of treated patients, and adverse events resulting from preconditioning chemotherapy and postconditioning IL2 are a rather common scenario. TILT-123 is an oncolytic adenovirus (Ad5/3-E2F-D24-TNFa-IRES-IL2) designed to enable T-cell therapies and checkpoint inhibition against cancer. Ultimately, the aim of this approach is to expand the proportion of patients benefiting from immunotherapies. Extensive preclinical studies with this technology showed that the virus repolarizes the tumor’s immune microenvironment in a way that favors T-cell presence and their activity against tumor cells. When TILT-123 was used together with TILs in preclinical in vivo models, animals had a higher chance to display curative results while showing reduced toxicity as the use of TILT-123 replaces the pre and post conditioning (Havunen R. et al Mol Ther Oncolytics 2016, Santos J.M. Mol Ther 2018). A Phase I clinical trial (NCT04217473) is ongoing to evaluate safety of the approach in advanced melanoma patients. Extensive biological assays of patient aim to characterize viral transduction of tumors through the intravenous and intratumoral routes, and the recruitment of activated lymphocytes to tumors in order to elucidate the immunological impact of the drug. Methods: The primary aim of NCT04217473 is to evaluate safety of TILT-123 in advanced melanoma patients. Refractory or recurrent stage III/IV patients, which cannot be treated with curative intent with available therapies, and are eligible for ACT-TIL therapy, can potentially participate in the study. TILT-123 administration begins while the manufacturing of ACT-TILs takes place and continues after the TIL-therapy is administered to the patient. In contrast with standard ACT-TIL therapy, patients are not conditioned with lymphodepletion or IL-2 in this approach. Patients must present at least one biopsiable/operable tumor for the generation of TILs and another injectable lesion for intratumoral administration of TILT-123. The open label, dose escalation trial has the main endpoint of establishing TILT-123 safety by day 36 (prior to TIL administration), which is based on the incidence of adverse events, severe adverse events, vital signs, ECG, and safety laboratory results. Secondary endpoints include safety and tolerability after TIL therapy has been administered, evaluation of antitumor responses and studies of tumor immune repolarization. Cohorts 1-3 have been completed without DLTs. Enrollment in cohort 4 was initiated in December 2021. Clinical trial information: NCT04217473.