A HLA-Cw6 specific single-chain antibody fragment (scFv) recognizing a natural killer cell receptor epitope.
Major histocompatibility complex (MHC) class I molecules induce inhibitory signals on natural killer (NK) cells via killer cell immunoglobulin-like receptors (KIR). We recently reported a human single-chain antibody (scFv#1), which recognizes an epitope on HLA-Cw6 (genotype: *0602). Flow cytometry showed scFv#1 binding to HLA-Cw6 (strong) and also to HLA-Cw2, 4, 5 (very weak) but not to HLA-Cw1, 3, 7, 8. The presumptive epitope of the antibody fragment, which includes residues Asn77 and Lys80 was verified by introducing point mutations into HLA-Cw6 encoding cDNAs. Asn77 --> Ser77 (N77S) and Lys80- -> Asn80 (K80N) mutants of Cw6 lost scFv#1 binding capacity whereas an additional mutation at aa position 90 (Asp-->Ala, D90A) did not influence scFv#1 binding characteristics. Since residues 77 and 80 of HLA-C are directly involved in KIR/MHC interaction, we expected the induction of target cell lysis upon addition of scFv#1 when bringing NK and HLA-Cw6 positive cells together. To prove this interference, we performed Cr-release assays, using Cw*0602 and mock-transfected K562 erythroleukemia cells as targets and freshly prepared peripheral blood NK cells as effector cells. scFv#1 appeared to influence KIR on ligand binding and restored lysis at low effector to target (E/T) ratios. Pan HLA class I antibody W6/32 did not show such effects. Taken together scFv#1 binding patterns with mutagenized HLA-Cw6 and Cr-release assays are strong evidence that the scFv#1 epitope on HLA-Cw6 is at or close to the binding site of CD158a.
Marget
Matthias
M
Institute of Immunology, Universitaetsklinikum Schleswig-Holstein, Campus Kiel, Michaelisstrasse 5, Kiel 24105, Germany. marget@immunologie.uni.kiel.de
Bohlsen
Dennis
D
Davarnia
Parvin
P
Yoo-Ott
Kyoung
K
Kabelitz
Dieter
D
Skerra
Arne
A
Steinmann
Jörg
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes; D015236:HLA-C Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000276:immunology*; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000276:immunology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000276:immunology*; D054340:Receptors, KIR; D054342:Receptors, KIR2DL1; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014162:Transfection
D018076:DNA, Complementary; D000939:Epitopes; D015236:HLA-C Antigens; C528994:HLA-C*06 antigen; D007135:Immunoglobulin Variable Region; D011971:Receptors, Immunologic; D054340:Receptors, KIR; D054342:Receptors, KIR2DL1; D011994:Recombinant Proteins