BISPECIFIC HUMANIZED ANTI-IL-2 RECEPTOR-ALPHA-BETA ANTIBODIES INHIBITORY FOR BOTH IL-2-MEDIATED AND IL-15-MEDIATED PROLIFERATION

Humanized anti-Tac (HAT) and Mik beta 1 (HuMik beta 1) Abs directed at IL-2R alpha and IL-2R beta, respectively, inhibit IL-2 binding and bi ological activity and together act synergistically in vitro. The Abs h ave been used successfully in primate models of allograft rejection, g raft-vs-host disease, and autoimmunity. We produced bifunctional human ized anti-IL-2R alpha beta Abs (BF-IgG) to combine the specificity of the two Abs into one entity by fusing HAT-producing NSO cells and HuMi k beta 1-producing Sp2/0 cells. BF-IgG was purified using protein G-Se pharose affinity chromatography, followed by IL-2R alpha and IL-2R bet a affinity chromatography and hydrophobic interaction chromatography. BF-IgG exhibited both anti-IL-2R alpha and anti-IL-2R beta specificiti es in binding assays. While the Ab binds the IL-2R with intermediate a ffinity (K-d = 2.82 nM), it does not inhibit IL-15 binding to its high affinity IL-15R. In Kit225/K6 (IL-2R alpha beta gamma(+)) cells, BF-I gG was 10-fold more potent than a HAT/HuMik beta 1 equimolar mixture i n blocking IL-2-induced proliferation and, unexpectedly, was at least 65-fold more active than the mixture in blocking IL-15-induced prolife ration, This dual inhibitory activity may be due to cross-linking of t he IL-2R alpha and IL-2R beta, thus blocking IL-2 binding and possibly impeding the association of IL-2R beta with IL-15R. BF-IgG has potent immunosuppressant activities against both IL-2- and IL-15-mediated re sponses, and this antagonist could be more efficacious than HAT and/or HuMik beta 1 for the treatment of autoimmunity and the prevention of allograft rejection.