Direct quantitative measurement of the kinetics of HLA-specific antibody interactions with isolated HLA proteins

Abstract

HLA specific antibodies vary in their pathogenicity and this is likely to be the net effect of constant chain usage, quantity, specificity, and affinity. Here we have measured the affinity of human monoclonal antibodies for a range of HLA proteins. Purified antibodies and ligands allowed dynamic interactions to be measured directly by surface plasmon resonance. Physiochemical differences between pairs of ligands were quantified using electrostatic mismatch and hydrophobic mismatch scores. All antibodies were characterized by fast on-rates and slow off rates but with a wide range of association rates (k 3.63-24.25 × 10 per mol per second) and dissociation rates (k, 0.99-10.93 × 10 per second). Dissociation constants (K) ranged from 5.9 × 10 M to 3.0 × 10 M. SN320G6 has approximately a twenty-fold greater affinity for HLA A2 compared with SN607D8, but has a similar affinity for HLA-A2 and B57. In contrast, SN607D8 has greater than a twofold greater affinity for HLA-A2 compared with A68. Similarly, WK1D12 has about a threefold greater affinity for HLA-B27 compared with B7. The higher affinity interactions correlate with the specificity of stimulating antigen. This is the first study to directly measure the binding kinetics and affinity constants for human alloantibodies against HLA.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR

“…AA substitutions within the functional epitope that do not affect binding of mAbs to an HLA allele in SAB assay may affect the ability to induce complement‐dependent cytotoxicity, 17 as was the case for LB_DR4_A. In addition, MFI values can reflect differential affinity of the mAbs for specific HLA alleles 57 and AA substations within the structural epitope can lead to lower affinity, which can be reflected in the MFI values 58 . Mutation studies have been informative on determining the involvement of single AAs in the interaction between the HLA molecule and antibody 48,49,59 .…”

A subset of anti-HLA antibodies induces FcγRIIa-dependent platelet activation

“…Similarly, whereas SN607D8 and SN230G6 both bind to HLA-A2, only SN607D8 induces platelet activation. Recently the affinities of SN607D8 and SN230G6 for HLA-A2 were reported: SN607D8 has a K D of 1.2×10 −8 M and SN230G6 has a K D of 5.9×10 −10 M. 33 It is likely that the affinity of HLA antibodies affects their ability to activate platelets to some extent. However, as the non-activating antibody SN230G6 has a significantly higher affinity than that of the activating antibody SN607D8, the propensity of HLA monoclonal antibodies to induce FcγRIIa-dependent platelet activation is not exclusively dependent on their affinity.…”

Caveats of HLA antibody detection by solid‐phase assays

“…It is important to realize that MFI reflects just a proportion of all HLA antibodies present in the serum, namely the rate of antibodies capable of binding to SAB in a stable manner under the applied assay conditions (i.e., short incubation time). The assessed proportion is, therefore, not only triggered by the antibodies’ concentration but also by their affinity, a characteristic that greatly differs between monoclonal HLA antibodies, as accurately shown by surface plasmon resonance . Furthermore, the measured MFI represents only the fraction of isotypes that are targeted by the applied detection antibody conjugate.…”