The immunosuppressive effects of CD4+ CD25 high regulatory T cells (Tregs) 
interfere with antitumor immune responses in cancer patients. Here, we present a 
novel class of engineered human interleukin (IL)-2 analogs that antagonizes the 
IL-2 receptor, for inhibiting regulatory T cell suppression. These antagonists 
have been engineered for high affinity to the alpha subunit of the IL-2 receptor 
and very low affinity to either the beta or gamma subunit, resulting in a 
signaling-deficient IL-2 analog that sequesters the IL-2 receptor alpha subunit 
from wild type IL-2. Two variants, "V91R" and "Q126T" with residue substitutions 
that disrupt the beta and gamma subunit binding interfaces, respectively, have 
been characterized in both a T cell line and in human primary Tregs. These 
mutants retain their high affinity binding to IL-2 receptor alpha subunit, but 
do not activate STAT5 phosphorylation or stimulate T cell growth. The 2 mutants 
competitively antagonize wild-type IL-2 signaling through the IL-2 receptor with 
similar efficacy, with inhibition constants of 183 pM for V91R and 216 pM for 
Q126T. Here, we present a novel approach to CD25-mediated Treg inhibition, with 
the use of an engineered human IL-2 analog that antagonizes the IL-2 receptor.