Extra domain B splice variant of fibronectin (EDB+FN) is an extracellular matrix protein (ECM) deposited by tumor associated fibroblasts, and is associated with tumor growth, angiogenesis and invasion. We hypothesized that EDB+FN is a safe and abundant target for therapeutic intervention with an antibody drug conjugate (ADC). We describe the generation, pharmacology, mechanism of action and safety profile of an ADC specific for EDB+FN (EDB-ADC). EDB+FN is broadly expressed in the stroma of pancreatic, non-small cell lung (NSCLC), breast, ovarian and head and neck cancers, while restricted in normal tissues. In patient derived xenograft (PDX), cell line xenograft (CLX), and mouse syngeneic tumor models, EDB-ADC, conjugated to auristatin Aur0101 through site-specific technology, demonstrated potent anti-tumor growth inhibition. Increased phospho-histone H3, a pharmacodynamic biomarker of response, was observed in tumor cells distal to the target site of tumor ECM after EDB-ADC treatment. EDB-ADC potentiated infiltration of immune cells, including CD3+ T lymphocytes into the tumor, providing rationale for the combination of EDB-ADC with immune checkpoint therapy. EDB-ADC and anti-PD-L1 combination in a syngeneic breast tumor model led to enhanced anti-tumor activity with sustained tumor regressions. In nonclinical safety studies in non-human primates, EDB-ADC had a well-tolerated safety profile without signs of either on-target toxicity or the off-target effects typically observed with ADCs that are conjugated through conventional conjugation methods. These data highlight the potential for EDB-ADC to specifically target the tumor microenvironment, provide robust therapeutic benefit against multiple tumor types and enhanced activity anti-tumor in combination with checkpoint blockade.