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.