Site-directed C3a receptor antibodies from phage display libraries.
Recent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellular loop comprising about 175 amino acid residues. We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized with the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Fv fragments (scFvs) was selected after four rounds of panning using the second extracellular loop of the C3aR, fused to the maltose binding protein as Ag. Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct regions within the loop, i.e., positions 185 to 193 and 218 to 226, representing the immunodominant domains of the loop. By flow cyotmetric analyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 cells. 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 C3aR expression was observed on human erythrocytes or platelets. Surprisingly, none of the scFvs alone or in combination inhibited C3a-induced Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR. In addition, C3a did not displace binding of the scFvs to the receptor, strongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.
Hawlisch
H
H
Institute of Medical Microbiology, Hannover Medical School, Germany.
Frank
R
R
Hennecke
M
M
Baensch
M
M
Sohns
B
B
Arseniev
L
L
Bautsch
W
W
Kola
A
A
Klos
A
A
Köhl
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D015926:Complement C3a / Q000276:immunology*; D018604:Epitope Mapping; D015723:Gene Library; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011951:Receptors, Complement / Q000032:analysis / Q000276:immunology*; D013329:Structure-Activity Relationship
D007128:Immunoglobulin Fragments; D011951:Receptors, Complement; C055966:immunoglobulin Fv; D015926:Complement C3a