Portal de Periódicos da CAPES

Human Interleukin 4 Receptor Complex: Neutralization Effect of Two Monoclonal Antibodies

1996; American Chemical Society; Volume: 35; Issue: 7 Linguagem: Inglês

10.1021/bi951741t

1943-295X

S. Shane Taremi, W.W. Prosise, Nithya Rajan, Rosemary A. O'Donnel, Hung V. Le,

Advanced biosensing and bioanalysis techniques

The interaction of human interleukin 4 with the extracellular domain of its receptor α-subunit (shuIL-4Rα) was characterized in studies utilizing chemical cross-linking, size exclusion chromatography, and Western blot analysis. A 1:1 stoichiometric complex could be demonstrated over a wide range (0.04−2.7) of ligand−receptor concentration ratios. It could also be cross-linked with bifunctional reagents containing a minimum chain length of eight methylene residues or the equivalent (11.4 Å). Using surface plasmon resonance, (SPR) technology, we established the high-affinity of human interleukin 4 (huIL-4) to shuIL-4Rα which was immobilized on a BIAcore sensor chip (Kd = 46 pM). The mechanisms of action of neutralizing monoclonal antibodies (Mab) 25D2 and 35F2 [Abrams et al. (1991) U.S. Patent 5,041,381; Ramanathan et al. (1990) in Advances in Gene Technology: The Molecular Biology of Immune Diseases and the Immune Response (Streilein, J. W., et al., Eds.) p 163, IRL Press, Oxford; DeKruyff et al. (1989) J. Exp. Med. 170, 1477−1493] were subsequently evaluated on the basis of their interaction with huIL-4 in the presence of shuIL-4Rα. SPR studies showed that Mab 25D2 binds to huIL-4 and reduces its affinity for shuIL-4Rα by 54-fold. Formation of a ternary complex between Mab 25D2 and the huIL-4/shuIL-4Rα complex was demonstrated in size exclusion chromatography experiments. In contrast, Mab 35F2 which also binds huIL-4 failed to form a stable ternary complex with huIL-4 and shuIL-4Rα during size exclusion chromatography. SPR studies supported this finding and showed that the interactions of Mab 35F2 and shuIL-4Rα to huIL-4 are mutually exclusive. These data are consistent with results of previous epitope mapping studies showing that Mabs 25D2 and 35F2 bind to huIL-4 at two different sites [Ramanathan et al. (1993) Biochemistry 32, 3549−3556]. Together, the results suggest that Mab 25D2 binds to a domain in huIL-4 including helix D and exerts its inhibitory effect through a dual action. It decreases the affinity of huIL-4 for huIL-4Rα and potentially blocks interaction with a secondary receptor subunit such as the IL-2Rγ [Reusch et al. (1994) Eur. J. Biochem. 222, 491−499]. Mab 35F2 operates through a direct and simpler mechanism, binding to helix C and inhibiting huIL-4 activity by sterically excluding all interaction with huIL-4Rα.