ABSTRACT: Despite advances in cancer immunotherapy, treatment response is still highly variable. One contributing factor is the tumor microenvironment and specifically the presence of suppressive immune cells such as regulatory T (Treg) cells. Being able to target these specifically, whilst leaving effector T cell populations untouched is an attractive strategy that may overcome some of these issues, improving responses. To generate antibodies specific for tumor-associated Tregs, lymphocytes were isolated from tumor-bearing mice and panned against the n-CoDeR phage antibody library. Using the target agnostic F.I.R.S.T. discovery platform, they were evaluated in ex vivo and in vivo models to determine tissue and cell selectivity and specificity, ability to deplete Tregs and elicit tumor control in subcutaneous tumor models. 24 antibodies were identified and explored, representing a range of specificities from pan-T cell to Treg and tumor Treg specific. Relative expression/binding of these mAbs on tumor Tregs was not a predictor of subsequent Treg deletion efficacy or tumor control, whereas tumor Treg selectivity was. One mAb in particular demonstrated tumor specific depletion of Tregs, leaving those in the spleen and blood untouched. This Fc:Fc_R mediated tumor specific Treg depletion was important for antitumor effects. Target deconvolution showed this mAb binds a distinct epitope within ICAM-1, that is hypothesized to mediate its selectivity towards tumor Tregs. These data validate the target agnostic discovery approach as a viable means to identify new therapeutic antibodies.