Molecular studies of anti-HLA-A2 using light-chain shuffling: a structural model for HLA antibody binding.
Human leukocyte antigen (HLA) A2 is one of the most immunodominant HLA antigens. Through a process of light-chain variable domain (VL) shuffling, we analyzed the VL domains' role in anti-HLA-A2/A28-binding site diversity. This was achieved by combining a VH3-30-encoded HLA-A2/A28-specific heavy-chain variable domain with 10(4) non-immune VL domains. Twelve HLA-A2/A28-specific antibodies were subsequently identified. VL gene analysis demonstrated an absence of Vlambda domains and that all have VkappaI-encoded light chains. The affinities correlated with the VkappaI gene present, with the seven highest affinity antibodies using Vkappa domains encoded by the O18 gene segment. A 300-fold difference in affinity was observed between the 12 antibodies, and homology modeling demonstrated a correlation between electrostatic surface potential of the antigen-binding site and affinity for HLA. Overlap between the T-cell receptor-binding site and that of the antibodies was indicated by inhibition of cytotoxic T-lymphocyte killing of peptide-pulsed target cells. A model of antibody binding to HLA-A2 suggested contact with both alpha helices of the HLA molecule, such that the antigen-binding site spans the peptide-binding groove. These data increase the understanding of antibody recognition of HLA and may facilitate the production of clonotypic antibodies with peptide-specific binding.
Department of Hematology, Division of Transfusion Medicine, University of Cambridge, Long Road, Cambridge CB2 2PT, UK. naw23@cam.ac.uk
N A
NA
Watkins
T R
TR
Dafforn
M
M
Kuijpers
C
C
Brown
B
B
Javid
P J
PJ
Lehner
C
C
Navarrete
W H
WH
Ouwehand
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity / Q000235:genetics; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D003601:Cytotoxicity Tests, Immunologic; D000939:Epitopes / Q000276:immunology*; D015723:Gene Library; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015789:HLA-A2 Antigen; D007128:Immunoglobulin Fragments