Abstract Background: Non-invasive biomarkers for predicting response to immune checkpoint inhibitors (ICI) are urgently needed. Crucial for response to ICI's is a T-cell permissive tumor microenvironment (hot tumor), characterized by presence of activated T-cells and low fibrotic activity. Activated T-cells release the protease granzyme B (GzB), which can cleave type IV collagen during tumor infiltration. We evaluated the potential of measuring GzB generated type IV collagen degradation fragments in a liquid biopsy for identifying metastatic melanoma (MM) patients responding to ipilimumab. Methods: A monoclonal antibody was raised against a GzB generated neo-epitope on type IV collagen (C4G), and used to develop a technically robust competitive electro-chemiluminescence immunoassay (ECLIA). C4G was measured in serum from MM patients (n=54) before initiation of ipilimumab treatment. C4G was combined with the fibrosis biomarker PRO-C3, measuring type III collagen formation. Biomarker levels were associated with objective response rate (ORR) and overall survival (OS) outcomes. Results: The C4G assay was specific for a neo-epitope on type IV collagen degradation fragments generated by GzB. The ORR was 2.6 fold higher (18% vs 7%) in patients with high C4G levels (>25th percentile) vs low levels (≤25th percentile). Likewise, high C4G levels at baseline were associated with longer OS, with a median OS of 646 days vs 290 days for low C4G levels (HR=0.48, 95%CI: 0.24-0.98, p=0.045). When combining high C4G with low PRO-C3, the HR dropped to 0.30 (95%CI: 0.15-0.60, p=0.0006), and remained significant when adjusted for the covariates age, lactate dehydrogenase levels and prior treatment (HR: 0.35, 95%CI: 0.18-0.72, p=0.004). Conclusions: A liquid biopsy measuring granzyme B degraded type IV collagen (C4G) as a surrogate of active immune infiltration into the tumor microenvironment is associated with response to the ICI therapy ipilimumab. When combining C4G with the fibrosis biomarker PRO-C3, patients with this special phenotype - low fibrosis and high immune infiltration - have an even better chance of responding compared to high C4G levels alone. If validated, this suggests that specific collagen remodeling biomarkers (C4G+PRO-C3) have potential for predicting response to ICI's in clinical cancer trials. Citation Format: Christina Jensen, Dovile Sinkeviciute, Daniel H. Madsen, Patrik Önnerfjord, Morten Hansen, Henrik Schmidt, Inge Marie Svane, Morten A. Karsdal, Nicholas Willumsen. Liquid biopsy reflecting a T-cell permissive tumor microenvironment identifies metastatic melanoma patients responding to immune checkpoint inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3091.