OR1 Mapping anti-HLA antibody epitopes by painting the HLA blue

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Aim Mapping anti-HLA antibody epitopes has commonly been done by comparing antibody reactivity patterns to a panel of naturally occurring HLA allelic variants such as the solid phase HLA antibody screening panels. Information including the donor’s immunizing alleles and the recipient-derived HLA further helps to refine HLA antibody epitopes. While these methods are inexpensive and widely available, they represent an indirect means of mapping those amino acids on HLA that are critical to antibody recognition and binding. Here we test a complementary direct approach to measure the ‘foot print’ of an antibody at it interacts with HLA. A recently developed method called protein painting was used to map the interaction of mouse monoclonal antibody BB7.2 with HLA-A*02:01. Methods Briefly, purified soluble HLA-A*02:01 was incubated with and without BB7.2. Protein mixtures were incubated with the trypsin inhibitor Remazol Brilliant Blue (RBB), trypsin digested, and tryptic peptide fragments of HLA-A*02:01 were analyzed using LCMS. In this manner BBB protects solvent accessible Arg and Lys residues from trypsin digestion. When HLA-A*02:01 is first incubated with BB7.2, Arg and Lys residues covered by the antibody are not painted by BBB and therefore they remain trypsin susceptible. Results A mass spectrometric analysis of the resulting trypic peptides revealed that BB7.2 protects R44, R97, R108, R111, R131, and K144 from painting. These residues are primarily located on lateral edge of the alpha-2 alpha helix as shown on the three dimensional structure of HLA-A*02:01. In a solid phase assay, BB7.2 reacts exclusively with A2 and A28 allomorphs, data that identifies the well defined 144TKH epitope. The protein painting data shown here are consistent with the 144TKH epitope as K144 was protected from RBB binding by BB7.2. Conclusion We demonstrate a new method for direct mapping of anti-HLA antibody epitopes that is complementary to solid phase assays. Furthermore, protein painting reveals not only the critical residues for antibody binding, but the surrounding amino acids covered by the antibody. R. Buchli : Employee; Company/Organization; Pure Protein LLC. W. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure Protein LLC.