Human interleukin 4 receptor complex: neutralization effect of two monoclonal antibodies.
The interaction of human interleukin 4 with the extracellular domain of its receptor alpha-subunit (shuIL-4R alpha) 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 angstroms). Using surface plasmon resonance, (SPR) technology, we established the high-affinity of human interleukin 4 (huIL-4) to shuIL-4R alpha which was immobilized on a BIAcore sensor chip (K(d) = 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-4R alpha. SPR studies showed that Mab 25D2 binds to huIL-4 and reduces its affinity for shuIL-4R alpha by 54-fold. Formation of a ternary complex between Mab 25D2 and the huIL-4/shuIL-4R alpha 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-4 alpha during size exclusion chromatography. SPR studies supported this finding and showed that the interactions of Mab 35F2 and shuIL-4R alpha 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-4R alpha and potentially blocks interaction with a secondary receptor subunit such as the IL-2R gamma [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-4R alpha.
Taremi
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SS
Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
Prosise
W W
WW
Rajan
N
N
O'Donnell
R A
RA
Le
H V
HV
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D006801:Humans; D015847:Interleukin-4 / Q000378:metabolism; D007700:Kinetics; D009500:Neutralization Tests; D011485:Protein Binding; D018123:Receptors, Interleukin / Q000276:immunology / Q000378:metabolism*; D019948:Receptors, Interleukin-4
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018123:Receptors, Interleukin; D019948:Receptors, Interleukin-4; D015847:Interleukin-4