Mapping the epitopes of neutralizing anti-human IL-3 monoclonal antibodies. Implications for structure-activity relationship.
The epitopes of neutralizing mAb were mapped in order to identify a receptor binding site on human IL-3 (huIL-3). To initiate this structure and activity analysis, four neutralizing mAb were selected on the basis of preventing rhuIL-3 stimulated proliferation of peripheral blood cells from a patient with chronic myelogenous leukemia (CML). In order to identify continuous epitopes, the neutralizing mAb were assayed in a solid-phase ELISA for their reactivity with either denatured rhuIL-3 or with the peptides generated by digestion of rhuIL-3 by using two different proteinases. Two of the neutralizing mAb recognized single fragments from both digestions. Amino acid (aa) sequence determination showed that these peptides overlap, defining a region of 22 aa (aa 29 to 50 of the mature rhuIL-3 protein). In a competition ELISA, the two continuous epitopes were shown to be linked to one another and to the two discontinuous epitopes, suggesting that all four neutralizing mAb bind to a discrete region of the IL-3 molecule, which might be involved in binding to the IL-3R.
Lokker
N A
NA
Preclinical Research, Sandoz Pharma Ltd., Basle, Switzerland.
Strittmatter
U
U
Steiner
C
C
Fagg
B
B
Graff
P
P
Kocher
H P
HP
Zenke
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D007377:Interleukin-3 / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007377:Interleukin-3