Background: Combination treatment with regorafenib (80–120 mg/day, PO, 3 wks on/1 wk off) plus nivolumab (480 mg IV Q4W) showed manageable safety but modest efficacy in a phase 2 study of 70 pts from North America with pMMR/MSS chemotherapy-resistant metastatic CRC (mCRC). Five pts had a partial response (PR; objective response rate [ORR]: 7%); all did not have liver metastases at baseline (n = 5/23; ORR: 22%). One pt had a confirmed complete response (CR) after the primary completion analysis of the study (ASCO 2021). This retrospective exploratory analysis investigated the potential association between specific biomarkers and anti-tumor activity, and how those biomarkers are modulated by treatment with regorafenib plus nivolumab. Methods: In formalin-fixed paraffin-embedded tumor samples obtained at baseline and Cycle (C) 2 Day (D) 8, immune-related biomarkers were assessed via immunohistochemistry (IHC), and RNA sequencing was used for gene expression profiling/gene signatures. Pre-/on-treatment blood samples were collected to measure circulating tumor DNA (ctDNA) and soluble biomarkers. Results: A total of 40 and 27 baseline tumor samples and 14 and 5 paired tumor samples at baseline/C2D8 were available for IHC and RNA sequencing, respectively. Higher baseline protein and mRNA expression of biomarkers for pre-existing immune sensitivity (eg, effector T cells) trended with anti-tumor activity. These biomarkers were expressed at lower levels in pts with liver metastases vs those without liver metastases at baseline. Cytotoxic T cell density was elevated on C2D8 but did not correlate with anti-tumor activity. Increased mean variant allelic frequency in ctDNA at C2D1 predominated in pts with progressive disease (PD), while clearance of ctDNA at C2D1 was only noted for the one pt with a CR. High clonal tumor mutational burden in ctDNA showed a numerical trend with anti-tumor activity (PD vs. SD/PR; P=0.058) and PFS (P = 0.072). Baseline serum levels of select markers related to angiogenesis (eg, vascular endothelial growth factor [VEGF] D) were associated with inferior anti-tumor activity (P = 0.002). Serum levels of immune-related soluble biomarkers (eg, tumor necrosis factor alpha) increased on treatment (P < 0.005), while levels of soluble VEGF receptor 2 decreased (P < 0.001). Conclusions: This study of pts with MSS mCRC treated with regorafenib plus nivolumab suggests that baseline tumor biomarkers for pre-existing immune sensitivity trended with anti-tumor activity, whereas select baseline peripheral biomarkers related to angiogenesis trended with inferior anti-tumor activity. Pharmacodynamics effects were observed, yet no significant correlation with anti-tumor activity was found. Due to the small sample size and retrospective nature, these analyses are hypothesis-generating. Clinical trial information: NCT04126733.