The binding pattern of a neutralizing monoclonal antibody to mutant oncostatin M molecules is correlated with functional activity.
Oncostatin M (OM) is a member of the cytokine family that includes leukaemia inhibitory factor (LIF), granulocyte colony stimulating factor (G-CSF), and interleukin 6 (IL-6). We previously reported the characterization of a monoclonal antibody (MAb) to OM, termed OM2, which neutralizes its functional activity. To gain information about the epitope detected by this MAb, we utilized a sandwich enzyme-linked immunoassay (EIA) to examine OM2 binding on a series of mutant recombinant OM molecules generated by site-directed mutagenesis, encompassing amino acid insertions, deletions, or alterations throughout the molecule. Carboxy-terminal deletions of a putative amphiphilic alpha helix past residue 185 abrogated binding of the OM2 MAb; alteration of a hydrophobic residue in the helix to a neutral one also prevented antibody binding. Analysis of mutants in which cysteines involved in intrachain disulfide binding were changed to serines revealed that one of two disulphide bonds was essential for OM2 binding. Two mutant molecules containing deletions in the amino-terminal one-fourth of the molecule were not bound by OM2, while a third mutant OM molecule with a C-terminal proximal internal deletion outside of the amphiphilic alpha helix was bound. The binding pattern of MAb OM2 to the OM mutant molecules correlated well with their functional activities. The data suggest that residues in both the C-terminal alpha helix and N-terminal one-fourth of the molecule are involved in neutralizing antibody binding and functional activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Radka
S F
SF
Regeneron Pharmaceuticals, Inc., Tarrytown, NY 10591.
Kallestad
J C
JC
Linsley
P S
PS
Shoyab
M
M
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001710:Biotin; D002460:Cell Line; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D053683:Oncostatin M; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D000906:Antibodies; D000911:Antibodies, Monoclonal; D016207:Cytokines; D010455:Peptides; D011994:Recombinant Proteins; D053683:Oncostatin M; D001710:Biotin