Background: Despite recent FDA approval of immune checkpoint inhibitor pembrolizumab and drug-antibody conjugate in the treatment of metastatic triple negative breast cancer (mTNBC), the overall survival benefit of these therapies remains modest. Oncolytic virotherapy (OV) utilizes genetically modified viruses to infect and kill cancer cells while sparing healthy cells. CF33-hNIS-anti-PD-L1 (CHECKvacc) is a novel chimeric orthopoxvirus with robust anti-cancer activity in TNBC xenografts. Cells infected with CHECKvacc were shown to express functional human sodium-iodide symporter (hNIS) and anti-PD-L1 proteins. hNIS gene transfer allows tracking of virus by 99mTc single-photon emission computed tomography (SPECT). Our preliminary animal studies demonstrated that tumor cells infected with CHECKvacc successfully secrete functional hNIS and anti-PD-L1. CHECKvacc is safe and well-tolerated, and detects and effectively kills TNBC at doses several magnitudes lower than other OVs in xenograft models. Methods: This study is a first-in-human (FIH) phase I, single center, single arm clinical trial evaluating the safety and tolerability of CHECKvacc intratumoral injection in patients with mTNBC. Key eligibility criteria include patients with unresectable or metastatic TNBC; progressed on at least 2 prior chemotherapies including immune checkpoint inhibitor-containing regimen; ECOG 0-1; RECIST 1.1 measurable disease; and at least one tumor amenable to repeated intratumoral injections. Eligible patients receive CHECKvacc intratumorally at one of 8 assigned dose levels (1 × 10⁵ PFU, 3 × 10⁵ PFU, 1 × 10⁶ PFU, 3× 10⁶ PFU, 1 × 10⁷ PFU, 3 × 10⁷ PFU, 1 × 10⁸ PFU, 3 x 10⁸ PFU) on Days 1 and 15 of each 28-day cycle for a total of 3 cycles of treatment. Primary objectives are to evaluate the safety and tolerability of CHECKvacc (DLTs and other toxicities by CTCAE v5.0). Secondary objectives are to determine optimal biological dose (OBD), recommended phase II dose (RP2D), and response rate by RECIST1.1. Results: The first 3 subjects of dose level 1 will be enrolled sequentially for safety monitoring. Once the initial 3 subjects are treated sequentially, the study will follow the Phase I Queue 3+3 (IQ 3+3) design which expands a dose level up to 8 subjects after a single DLT has been observed. Enrollment to the final RP2D may be expanded to include up to 12 patients for efficacy assessment. The estimated targeted accrual is 33 patients (minimum) to 78 patients (maximum). Correlative aims include assessing viral kinetics, viral plaque assay, 99mTc SPECT imaging for virus tracking, peripheral blood and tumor tissue for antiviral immune activation, and tumor microenvironment changes in association with response to therapy. Conclusions: This FIH trial of CF33-hNIS-antiPD-L1 intratumoral injection in patients with mTNBC is currently underway. Clinical trial information: NCT05081492.