Hong Kong Workshop Lecture 6 Epitope Specificities of HLA Antibodies Tested in Ig- and C1q-Binding Assays and CDC.

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Hong Kong Workshop Lecture 6 Epitope Specificities of HLA Antibodies Tested in Ig- and C1q-Binding Assays and CDC

How do antibodies react with HLA epitopes? Epitopes are defined by eplets and other residue configurations Binding with antibody involves CDRs on heavy and light chains Binding energy (affinity) and biological activity of antibody

HLA Antibody Epitope Specificity in Different Assays (Duquesnoy et al HLA Antibody Epitope Specificity in Different Assays (Duquesnoy et al. Human Immunology. 74:1271-1279, 2013) Human monoclonal antibodies tested with Ig-binding (Luminex) with single alleles C1q-binding (Luminex) with single alleles CDC testing with a large HLA-typed cell panel (N>800; 13th International Workshop)

Human monoclonal antibodies generated by Arend Mulder Leiden University Medical Center The Netherlands

Monoclonal Antibody Reactivity Patterns of Eplet-Carrying Alleles Lum-Ig Lum-C1q CDC I Positive II Negative III Note: All monoclonals in this study had been selected for their lymphocytotoxic reactivity with the immunizing antigen

Complement-Binding Antibody

Antibody Binding Energy (Affinity) and Reactivity in Different Assays Interpretation Negative in all 3 assays No specific epitope recognition and binding Only Lum-Ig positive + Epitope is recognized but insufficient conformational change in antibody Lum-Ig positive Lum-C1q positive CDC negative ++ Epitope is recognized + conformational change exposes C1q-binding site on antibody but no Complement activation Positive in all 3 assays +++ Epitope is recognized + exposed C1q-binding site + conformational change in C1q to activate C1qrs complex, then Complement activation

Conclusion: all 62GE-carrying alleles are unacceptable mismatches Pattern I 62GE-Specific mAb ROU2D3 Conclusion: all 62GE-carrying alleles are unacceptable mismatches

Is A*01:01 an unacceptable mismatch? Pattern II 166DG-Specific mAb BVK5C4 Is A*01:01 an unacceptable mismatch?

Two lymphocytotoxic human monoclonal antibodies specific for 144TKR

144TKR-Specific mAb OK5A3 Pattern III Lum-Ig- and Lum-C1q reactivity is specific for 144TKR CDC reactivity is specific for 144TKR+151H Is A*80:01 an unacceptable mismatch?

Another 144TKR-Specific mAb: BRO11F6 Pattern III Another 144TKR-Specific mAb: BRO11F6 Lum-Ig and Lum-C1q reactivity is specific for 144TKR+151H

144TKR-Specific mAb BRO11F6 Pattern III Lum-Ig- and Lum-C1q reactivity is specific for 144TKR+151H CDC reactivity is specific for 144TKR+150AHA Are A*11:01 and A*11:02 the only unacceptable mismatches?

Binding energy with antibody Structural Model of HLA Epitope Reactivity of Complement-Fixing Antibodies Tested in Ig-Binding, C1q-Binding and CDC Assays Binding energy with antibody +++ Immunizing allele Lum-Ig positive Lum-C1q positive CDC positive

Binding energy with antibody Structural Model of HLA Epitope Reactivity of Complement-Fixing Antibodies Tested in Ig-Binding, C1q-Binding and CDC Assays Binding energy with antibody +++ + Immunizing allele Lum-Ig positive Lum-C1q positive CDC positive Allele 1 Lum-Ig positive Lum-C1q negative CDC negative

Binding energy with antibody Structural Model of HLA Epitope Reactivity of Complement-Fixing Antibodies Tested in Ig-Binding, C1q-Binding and CDC Assays Binding energy with antibody +++ + ++ Immunizing allele Lum-Ig positive Lum-C1q positive CDC positive Allele 1 Lum-Ig positive Lum-C1q negative CDC negative Allele 2 Lum-Ig positive Lum-C1q positive CDC negative

Binding energy with antibody Molecular Model of HLA Epitope Reactivity of Complement-Fixing Antibodies Tested in Ig-Binding, C1q-Binding and CDC Assays Binding energy with antibody +++ + ++ +++ Immunizing allele Lum-Ig positive Lum-C1q positive CDC positive Allele 1 Lum-Ig positive Lum-C1q negative CDC negative Allele 2 Lum-Ig positive Lum-C1q positive CDC negative Allele 3 Lum-Ig positive Lum-C1q positive CDC positive

Why Do Certain Antibodies React in Luminex but not in CDC? These antibodies are not Complement-fixing BUT (more likely) They can fix Complement but will react in CDC only with eplet-carrying alleles with proper structural epitope configurations

The determination of epitope-based mismatch acceptability might be technique dependent Which method is clinically relevant for determining mismatch acceptability?