Background: The TLR-9 agonist lefitolimod (MGN1703) significantly increases the Th1 response in pre-clinical models and has demonstrated antitumor activity in early phase clinical trials. This trial assessed the safety and preliminary efficacy of the rational combination of lefitolimod with ipilimumab in patients (pts) with advanced solid tumors. Methods: This was a single-center, open-label, investigator-initiated, dose-finding, and expansion Phase I trial (ClinicalTrials.gov no. NCT0266877). Key inclusion criteria were histologically confirmed advanced solid tumor, ECOG performance status ≤ 2; age ≥ 18 years. Patients (pts) were enrolled at escalating doses of lefitolimod (15-120 mg/kg), subcutaneously (SC) administered weekly and a fixed dose of ipilimumab 3 mg/kg in a 3-week cycle. For the expansion cohort, lefitolimod was administered intratumorally (IT) at 15 mg weekly. The primary endpoint was safety and tolerability according to CTCAE v4.0, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD)/recommended Phase II dose (RP2D) for the lefitolimod in this combination. The secondary endpoint was efficacy, evaluated according to Immune-Related Response Criteria (irRC). Exploratory endpoints included changes in the intra-tumoral T-cell microenvironment in pre- and post- treatment tumor biopsies and the correlation of immune biomarkers with response. Results: From March 2017 to February 2020, 28 pts were enrolled. Median age was 56 years (range: 19-92). Median prior lines of therapy was 4 (range: 0-12). Pts 1-4 received lefitolimod 15 mg SC, pts 5-8 received lefitolimod 30 mg SC, pts 9-12 received lefitolimod 60 mg SC, pts 13-19 received lefitolimod 120 mg SC, and pts 20-28 received lefitolimod 15 mg IT. Eleven pts had at least one treatment-related AE (TRAE). The most common TRAE was skin rash (n=4, 14.2%), followed by fatigue (n=3, 10.7%) and pruritis (n=2, 7.1%). There were no grade 4 or 5 AEs, none of the pts required dose reduction, and there was no discontinuation of treatment due to AEs. DLT and MTD were not reached and the RP2D was established as lefitolimod SC 120 mg weekly with ipilimumab 3mg/Kg every 3 weeks. Of the 28 pts, 8 pts had stable disease as best response. 7 pts could not be evaluated by irRC due to clinical progression before restaging, non-measurable lesions and withdrawal of consent. Paired biopsy samples were analyzed by flow cytometry. Pro-inflammatory immune conditioning of the tumor microenvironment by the combination of the TLR9 agonist MGN1703 in combination with ipilimumab showed increases in intra-tumoral CD8 T cell frequency, memory CD8 phenotype (CD45RO+) and proliferation (Ki67+). Conclusions: The combination of high subcutaneous doses or intra-tumoral administration of lefitolimod in combination with ipilimumab is safe and well tolerated in patients with advanced cancers, with preliminary antitumor activity observed. Clinical trial information: NCT0266877.