CONSTRUCTION OF A BISPECIFIC ANTIBODY REACTING WITH THE ALPHA-CHAIN AND BETA-CHAIN OF THE HUMAN IL-2 RECEPTOR - HIGH-AFFINITY CROSS-LINKINGAND HIGH ANTIPROLIFERATIVE EFFICIENCY

A bispecific antibody recognizing both the alpha- and beta-chains of t he IL-2R was generated by sulfhydryl-directed chemical reassociation o f monovalent Fab' fragments prepared from the anti-alpha mAb 33B3.1 (r at IgG2a) and from the anti-beta mAb A41 (mouse IgG1). Whereas the 33B 3.1/A41 bispecific mAb (bi-mAb) binds to isolated alpha- and beta-chai ns with low affinity (K(d) = 4 nM), its binding to cells co-expressing the two chains shows both low and high affinity components. The high affinity-binding sites (K(d) = 100 pM) most probably correspond to the cross-linking by the bi-mab of alpha- and beta-chains, whereas the lo w affinity component corresponds to the excess of alpha-chains. High a ffinity binding of bi-mAb on activated T cells is observed at 37-degre es-C and not at 4-degrees-C, suggesting that i) the two chains are dis sociated 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 s pecific for activated alpha+/beta+ cells with respect to its high affi nity binding. In contrast to A41, which does not affect IL-2-induced p roliferation of 4AS cells or anti-CD3-activated PBL, and to 33B3.1, wh ich do inhibit proliferation but only partially and at high doses, the bi-mAb showed full blocking efficiencies at low concentrations (IC50 of 300 to 400 pM) corresponding to the formation of high affinity alph a/bi-mAb/beta complexes. These half-maximal effects were observed at 1 0-fold lower concentrations than when using a combination of equimolar concentrations of parental 33B3.1 and A41 IgG. Because of their speci ficity and high blocking efficiencies, anti-alpha/anti-beta bi-mAb may constitute a better alternative for IL-2R-directed immunosuppression.