ABSTRACT: The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialised immune cells, such as regulatory T cells (TReg), are key components of active intratumoral immunosuppression. Inducible Co-Stimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive TReg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor bearing mice and cancer patients to demonstrate differential expression of ICOS in immune T cell subsets in different tissues. ICOS expression was higher on intratumoral TReg than on effector CD8 T cells. In addition, by immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOShigh T cells but was also associated with increased secretion of pro-inflammatory cytokines from ICOSlow TEFF cells. In syngeneic mouse tumor models, KY1044 depleted ICOShigh TReg and increased the intratumoral TEFF:TReg ratio, resulting in increased secretion of IFN_ and TNF_ by TEFF cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response.