Background: Glioblastoma (GBM) is an aggressive brain tumor and despite efforts in developing new effective therapies, patient survival remains low. There is increasing interest in using immune checkpoint inhibitors (ICIs) for GBM, however the immunosuppressive (cold tumor) characteristics of GBM limit the efficacy of ICIs. Consequently, there is a need to identify patients with active tumor-infiltrating lymphocytes (hot tumor). Several studies have shown that brain extracellular matrix such as type IV collagen has a dynamic composition with protease-induced alterations. In this study, we evaluated the clinical utility of a non-invasive biomarker of granzyme B degraded type IV collagen (C4G) reflecting tumor-infiltrating lymphocyte activity and of matrix metalloproteinase (MMP) degraded type IV collagen (C4M) in GBM patients treated with nivolumab (anti-PD-1) and bevacizumab (anti-VEGF) in the recurrent setting. Methods: C4G and C4M were measured in serum from 22 controls and 39 GBM patients previously treated with surgery, radiotherapy and chemotherapy in the primary setting. After GBM recurrence, 18 patients underwent salvage resection (arm A) however 21 patients had no possibility for resection (arm B). All patients were treated with nivolumab and bevacizumab (NCT03890952 phase II study). Baseline GBM samples were taken before the second-line treatment. The association between C4G levels and outcome was evaluated by Cox regression analysis for overall survival (OS) and odds ratio (OR) calculations for complete response (CR) rate after dichotomizing patients into low vs high levels of C4G (median cutpoint). Results: C4G (p = 0.004), but not C4M (p = 0.166), was significantly elevated in serum from GBM patients compared to controls. Moreover, patients with high C4G levels had a significantly increased likelihood of experiencing CR (OR=6.68, p<0.0001). Furthermore, patients with high C4G experienced improved OS compared to low C4G (HR=0.39), and this remained significant after adjusting for other significant risk factors (treatment arm and MGMT methylation) by multivariate analysis (HR=0.44) (table). Conclusions: A non-invasive biomarker reflecting tumor-infiltrating lymphocyte activity (C4G) has the potential to identify GBM patients responding to nivolumab and bevacizumab in the recurrent setting. In the future, this may provide a non-invasive biomarker tool for stratifying patients with GBM for ICI trials. Clinical trial information: NCT03890952.