Construction of a bispecific antibody reacting with the alpha- and beta-chains of the human IL-2 receptor. High affinity cross-linking and high anti-proliferative efficiency.

Abstract

A bispecific antibody recognizing both the alpha- and beta-chains of the IL-2R was generated by sulfhydryl-directed chemical reassociation of monovalent Fab' fragments prepared from the anti-alpha mAb 33B3.1 (rat IgG2a) and from the anti-beta mAb A41 (mouse IgG1). Whereas the 33B3.1/A41 bispecific mAb (bi-mAb) binds to isolated alpha- and beta-chains with low affinity (Kd = 4 nM), its binding to cells co-expressing the two chains shows both low and high affinity components. The high affinity-binding sites (Kd = 100 pM) most probably correspond to the cross-linking by the bi-mAb of alpha- and beta-chains, whereas the low affinity component corresponds to the excess of alpha-chains. High affinity binding of bi-mAb on activated T cells is observed at 37 degrees C and not at 4 degrees C, suggesting that i) the two chains are dissociated at 4 degrees C in the absence of ligand and ii) the mechanism of bi-mAb catalyzed cross-linking of these two chains is temperature dependent. In contrast to parental 33B3.1 and A41 IgG, which recognize single positive (alpha + and beta +, respectively) and double positive alpha +/beta + cells with similar affinities, the 33B3.1/A41 bi-mAb is specific for activated alpha +/beta + cells with respect to its high affinity binding. In contrast to A41, which does not affect IL-2-induced proliferation of 4AS cells or anti-CD3-activated PBL, and to 33B3.1, which do inhibit proliferation but only partially and at high doses, the bi-mAb showed full blocking efficiencies at low concentrations (IC50 of 300 to 400pM) corresponding to the formation of high affinity alpha/bi-mAb/beta complexes. These half-maximal effects were observed at 10-fold lower concentrations than when using a combination of equimolar concentrations of parental 33B3.1 and A41 IgG. Because of their specificity and high blocking efficiencies, anti-alpha/anti-beta bi-mAb may constitute a better alternative for IL-2R-directed immunosuppression.