Background: Microsatellite instability-high (MSI-H) tumors are characterized by deficient mismatch repair, high tumor mutation burden, and exceptional anti-tumor responses to immunotherapy. While durable disease control is often observed for advanced MSI-H cancers treated with immune checkpoint blockade (ICB), some patients (pts) experience treatment resistance. In melanoma and urothelial cancer, acquired resistance to ICB has been associated with increased signaling of the immunosuppressive TGF-β pathway. Bintrafusp alfa (BA) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 monoclonal antibody blocking PD-L1. Methods: In this phase II study (NCT03436563), pts with MSI-H metastatic solid tumors who had progressed on prior ICB were eligible. Pts received 1200 mg BA intravenously every 14 days. Paired tumor biopsies were collected for biomarker analysis. The primary objective was to estimate response rate (RR) per iRECIST. Median progression-free survival (PFS) and overall survival (OS) were calculated according to the Kaplan-Meier method. Adverse events (AEs) were assessed according to CTCAE v5.0. Results: 15 pts [arising from the colon/rectum (12), pancreas (1), duodenum (1), appendix (1)] were enrolled. Median duration of prior ICB before progression was 7.2 months (range, 1.8-24). All pts were evaluable for toxicity, and 14 for response. There was 1 grade 3 treatment-related AE (adrenal insufficiency) and 1 grade 5 treatment-related immune AE (hepatic failure). Median number of doses with BA was 4 (interquartile range, 2-4). There were 3 pts with stable disease and 11 with progressive disease as best response. RR was 0% (95% confidence interval (CI), 0-24%), and disease control rate was 21% (95% CI, 5-51%). Median PFS and OS were 1.8 months (95% CI, 1.5-5.7) and 9.1 months (95% CI, 5.3-not estimable), respectively. One pt with colorectal cancer demonstrated sustained stable disease (20.5 months) with BA. Conclusions: Dual inhibition of TGF-β and PD-L1 by BA did not demonstrate significant anti-tumor activity in the majority of pts with MSI-H metastatic cancer who had progressed on prior ICB. One pt did experience prolonged clinical benefit from BA. Ongoing correlative studies may inform on the effect of TGF-β and PD-L1 modulation by BA within the tumor microenvironment. Clinical trial information: NCT03436563.