NEUTRALIZING MONOCLONAL-ANTIBODIES TO HUMAN IL-8 RECEPTOR A MAP TO THE NH2-TERMINAL REGION OF THE RECEPTOR

mAbs against human IL-8 receptor A (IL-BR-A) were generated by immuniz ing mice with either: 1) peptides corresponding to various extracellul ar domains of IL-8R-A or 2) transfected 293 cells expressing IL-8R-A ( 293-71 cells). Among the seven peptides used for immunization, only th e peptide corresponding to residues 2-19 of IL-8R-A produced Abs capab le of recognizing native IL-8R-A on 293-71 cells. We screened for hybr idomas secreting mAbs capable of binding strongly to peptide 2-19 in a n ELISA and capable of recognizing native IL-8R-A in flow cytometry ex periments with 293-77 cells. Two clones secreting mAbs capable of reco gnizing native IL-8R-A were selected for further characterization. Non e of these mAbs were able to block the binding of I-125-IL-8 to 293-71 cells. We also screened hybridomas derived from mice immunized with t he intact receptor expressed on transfected 293-71 cells and identifie d several clones secreting mAbs capable of recognizing native IL-8R-A in flow cytometry experiments. Two of these mAbs were capable of block ing the binding of I-125-IL-8 to 293-71 cells. The epitopes, recognize d by these blocking mAbs and by the other nonblocking mAbs derived fro m the peptide immunization, mapped to the NH2-terminal region of IL-8R -A using an ELISA against synthetic peptides: the two blocking mAbs ma pped to residues 2-14 of IL-8R-A, whereas the nonblocking mAbs mapped to residues 2-11. Furthermore, flow cytometry analysis of IL-8 recepto r mutants showed that Asp(6) plays an important role in the binding of the blocking mAbs but not in the binding of the nonblocking mAbs. Con versely, a mutation of Asp(11) to Lys disrupts the binding of one of t he nonblocking mAbs (4C8) but has no effect on recognition by others. Analysis of the affinity of these mAbs for IL-8R-A demonstrated that b locking mAbs have affinities at least sevenfold higher than the nonblo cking mAbs.