Abstract Integrins αvβ6 and αvβ8 in the tumor microenvironment (TME) have been shown to activate immunosuppressive TGF-β, which serves as an important mechanism for immune checkpoint inhibitor resistance in a range of tumors. In this study, we demonstrate the utility of lasso peptides as versatile scaffolds for designing new therapeutics. A series of highly potent and selective dual αvβ6/8 inhibitors were engineered through a combination of epitope scanning, computational design, and directed evolution. Several analogs, such as lassotides 36 and 47 , were fully characterized and physicochemical, in vitro pharmacological, and in vivo data are reported. Lassotide 47 , a half-life extended derivative of 36 , was shown to strongly sensitize anti-mPD-1-resistant tumors in mice when dosed in combination with the checkpoint inhibitor. The 47 /anti-mPD-1 combination was shown to halt tumor growth and regress tumors in mouse models of triple negative breast and ovarian cancers. Dual inhibition of αvβ6/8 integrins expressed in the TME thus represents a promising tumor-specific strategy to overcome TGF-β-driven resistance and enhance the anti-tumor efficacy of immune checkpoint inhibitors.