Background: Microsatellite stable (MSS) or mismatch repair proficient (MMR-P) metastatic colorectal cancer is refractory to immune checkpoint inhibition. We hypothesized that a combination of intratumoral TLR9 agonist, radiosurgery and dual PD-1 and CTLA-4 blockade would induce a local focus of immune stimulation, evoking a systemic immune response. Methods: In this single institution investigator-initiated phase I study, patients with MSS / MMR-P metastatic colorectal cancer were treated with a priming dose of subcutaneous (s.c.) vidutolimod, three intratumoral injections of vidutolimod, and radiosurgery to a metastasis, combined with nivolumab 3mg/kg every 2 weeks and ipilimumab 1 mg/kg every 6 weeks. Following conclusion of intratumoral therapy, s.c. injections of vidutolimod commenced. Efficacy endpoints were based upon a non-irradiated and non-injected lesion. Cytokine levels were measured at baseline and at 7 (± 2) weeks. Patients were placed in 4 cohorts: (1) Safety run-in without radiosurgery (No RT); (2) Radiosurgery prior to intratumoral therapy (Early Liver RT Slow). An amendment was made due to early patient fall-out due to progressive disease; (3) Radiosurgery prior to intratumoral therapy with a condensed timeline (Early Liver RT Quick). An additional amendment was made due to unacceptable liver toxicity; (4) Radiosurgery to extrahepatic lesion following completion of intratumoral therapy (Late Extrahepatic RT). ClinicalTrials.gov Identifier NCT03507699. Results: A total of 19 patients were accrued (No RT, n=2; Early Liver RT Slow, n=7; Early Liver RT Quick, n=4; Late Extrahepatic RT, n=6). Median age was 59 years (range 40-71), 68% were male, median number of previous systemic treatments was 3 (range 2 – 5). None of the patients responded, aside from one patient in cohort #4 who achieved complete response, and was subsequently found to have a high tumor mutational burden (TMB) 79 mutations / Mb. Grade 3 liver toxicity, including elevated transaminases and hyperbilirubinemia, was reported in 0%, 0%, 75% and 17% in cohorts 1-4, respectively. Reactions following intratumoral injections, typically within 12 hours, included fever (68%), tachycardia (21%), chills (63%) and hypotension (47%). There was an increase in systemic CXCL10 levels at 7 (± 2) weeks compared to baseline, with a median of 407 versus 78 pg/ml, respectively, p<0.01. Conclusions: Intratumoral liver injection of vidutolimod was associated with acute systemic symptoms attributed to cytokine release. The juxtaposition of liver irradiation and intratumoral vidutolimod injection to the same lesion was associated with unacceptable hepatic toxicity. The combination of intratumoral vidutolimod, radiosurgery, nivolumab and ipilimumab was not found to be efficacious in MSS / MMR-P colon cancer. Clinical trial information: NCT03507699.