Abstract

Interleukin-2 (IL-2) is an immunoregulatory cytokine whose biological effects are mediated through interaction with specific receptors on the surface of target cells. Due to its presumed role in generating a normal immune response, IL-2 is being evaluated for the treatment of a variety of tumors, in addition to infectious diseases. During the study of the structure-activity relationships for IL-2 and its receptors, one analog in which threonines at positions 41 and 51 were replaced by prolines (T41/51P) was found to possess apparent signaling abnormalities. Bioassays and receptor binding assays with human peripheral blood lymphocytes revealed the EC50 and Kd values of this analog to be 200 pM and 5.9 nM, respectively. Although the EC50 is greater and the receptor affinity of T41/51P is much weaker than that of wild-type IL-2, receptor occupancy versus biological response comparisons indicated that a much lower receptor occupancy was required to generate an equivalent biological response. Competitive receptor binding analyses with both intermediate affinity (beta/gamma subunit complex) and low affinity (alpha subunit) receptors were carried out to assess the origin of this phenomenon. Similar analyses of the singly substituted T41P and T51P analogs were carried out. From these studies, it was apparent that facilitated signaling was mainly attributable to position 51, whereas mutations at position 41 primarily influenced low affinity binding. The observation that the T51P analog facilitates response, compared with wild-type IL-2, may indicate a signaling-dependent conformational change in IL-2 upon receptor binding.