SITE-DIRECTED C3A RECEPTOR ANTIBODIES FROM PHAGE DISPLAY LIBRARIES

Recent cloning of the human C3a receptor (C3aR) revealed that this rec eptor belongs to the large family of rhodopsin-type receptors, A uniqu e feature of the C3aR is the large second extracellular loop comprisin g about 175 amino acid residues, We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized w ith the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Pv fr agments (scFvs) was selected after four rounds of panning using the se cond extracellular loop of the C3aR, fused to the maltose binding prot ein as Ag, Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct reg ions within the loop, i,e,, positions 185 to 193 and 218 to 226, repre senting the immunodominant domains of the loop, By flow cyotmetric ana lyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 c ells, In addition, the scFvs bound to the human mast cell line HMC-1. Immunofluorescence studies showed C3aR expression on polymorphonuclear granulocytes and monocytes, but not on lymphocytes, In addition, no C 3aR expression was observed on human erythrocytes or platelets, Surpri singly, none of the scFvs alone or in combination inhibited C3a-induce d Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR, In a ddition, C3a did not displace binding of the scFvs to the receptor, st rongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.