Mapping and characterization of the epitope(s) of Sch 55700, a humanized mAb, that inhibits human IL-5.
mAb against human IL-5 inhibit pulmonary eosinophilia, tissue damage and airway hyper-reactivity in allergic animal models. Sch 55700 is a humanized, neutralizing anti-IL-5 antibody. To better understand the molecular mechanism by which Sch 55700 blocks IL-5 bioactivity, we have mapped its epitope by scanning IL-5 with synthetic peptides. Those peptides containing a region, ERRRV, corresponding to amino acids 89-93 of IL-5 specifically interact with both Sch 55700 and its parental rat IgG, 39D10. Among the five residues of this region, all three arginine residues were particularly critical for interaction of these peptides with Sch 55700. We further characterized this region by alanine scanning using site-directed mutagenesis. Examination of COS-expressed IL-5 mutants by Western blot showed that single mutations of E(89), R(90), R(91) or R(92) to alanine caused a loss of IL-5 binding to both Sch 55700 and 39D10. We further demonstrated in surface plasmon resonance studies using a BIAcore biosenosor that E(89), R(90) or R(91) are involved in the interaction between IL-5 and its receptor alpha subunit. Based upon the findings here and previously reported structures of the IL-5 and 39D10 variable region, we propose a model suggesting that the molecular interactions between the IL-5 and Sch 55700 mainly involve several ion pair interactions. We conclude that Sch 55700 occupies a region, ERRR, on IL-5 that is essential for its interaction with the receptor and thereby blocks IL-5 bioactivity.
Zhang
J
J
Schering-Plough Research Institute, K-15C113/1600, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA.
Kuvelkar
R
R
Murgolo
N J
NJ
Taremi
S S
SS
Chou
C C
CC
Wang
P
P
Billah
M M
MM
Egan
R W
RW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D018604:Epitope Mapping; D006801:Humans; D015848:Interleukin-5 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D018123:Receptors, Interleukin / Q000378:metabolism; D053649:Receptors, Interleukin-5
D000911:Antibodies, Monoclonal; D015848:Interleukin-5; D018123:Receptors, Interleukin; D053649:Receptors, Interleukin-5