3594 Background: The efficacy of checkpoint inhibitor immunotherapy in MSI-H mCRC has not been replicated in MSS mCRC. Therefore, additional interventions are needed to convert immunologically “cold” MSS CRC to “hot” tumors resembling MSI-H tumors. PolyPEPI1018 is an off-the-shelf, multi-peptide vaccine containing 12 immunogenic epitopes derived from 7 shared tumor antigens which demonstrated early evidence of clinical activity in first-line MSS mCRC. Methods: Patients with MSS mCRC who have progressed on 2-3 lines of prior chemotherapy regimen received PolyPEPI1018 (1.2 mg, sc) and atezolizumab (1,200 mg, iv) Q3W until disease progression or unacceptable toxicity. The primary endpoint was safety. A Simon 2-stage design was applied. Data on objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and correlation studies will be presented. Results: 18 patients (66 % female) were enrolled for stage 1 of the study. Median age was 54 years (range 38–79), 50% had liver metastases and 67% received at least 3 lines of prior therapies. PD-L1 expression was <1% for 85% of patients. The combination was well-tolerated; most common side effect related to treatment was Grade (Gr) 1-2 local skin reaction (n=22). Gr 2 events (n=2) at least possibly related to treatment were nausea, pyrexia and increased blood alkaline phosphatase. There were no Gr 3-5 events or study discontinuation due to treatment AE. The ORR was 0% and the DCR was 61%. The mPFS was 2.7 months (95%CI 1.4-4.0) and the mOS was not reached (95%CI 8.75 – NE months) at the data cut-off date. Ex-vivo FluoroSpot identified 13/16 subjects with robust, vaccine-specific CD8+ and/or CD4+ T cell responses detected against multiple vaccine antigens. For patients with available biopsy pairs (n=8), both the average PD-L1 expression (IC%, p=0.029) and the density of CD8+ tumor-infiltrating lymphocytes (TILs, p=0.019) were significantly increased, post-treatment. Patients with increased PFS (> 12 weeks) had both higher levels of post-treatment PD-L1 (p=0.007) and TILs (p=0.016) than patients with PFS ≤ 12 weeks. Conclusions: PolyPEPI1018 in combination with atezolizumab was well-tolerated with no Grade 3 AEs observed. Despite no objective tumor responses could be detected, correlative data suggest contribution of PolyPEPI1018-induced immune responses to the modulation of tumor microenvironment and to improved disease control. The study did not proceed to stage 2; it is on-going for the collection of survival data. Clinical trial information: NCT05243862 .