Aim: Analysis of monoclonal antibodies (mAbs) using HLA single antigen beads (SABs) reveals that some specificity profiles are concentration dependent. The inclusion or exclusion of weaker mean fluorescence intensities (MFIs) could impact the prediction outcome of a functional epitope which was termed based on assumption that it interacts with Ab heavy chain CDR3 and determines specificity, but such a definition still awaits confirmation based on structural information. Weak MFI from SAB assay was often interpreted to be high background, false positive and/or against denatured antigen (Ag). To elucidate whether the weaker MFIs also reflect specific Ag-Ab interactions, mutagenesis study comparing signal shifts of engineered variants (EVs) vs. wild types was performed.
Methods: Twelve HLA-DQ mAbs developed by Leiden University Medical Center were serially diluted and tested on LABScreenTM SAB panels to reveal if and which MFIs can be differentially diluted out. Functional epitope analyses were performed using both HLAMatchmaker and AminoAcid modules in HLA Fusion software. The predicted residues were further examined in sequence alignments and pHLA3D molecular models. Residues likely involved in binding were mutated and the Ags expressed on cell surface. Flow cytometry of these cells was used to discern which residues are critical. To quantify, mutated residue(s) that causes > 95% loss of function while maintaining expression is defined as a “functional epitope”.
Results: Concentration-dependent specificity profiles were observed for 9 out of the 12 mAbs. The weaker MFIs had to be included in some cases but excluded in others to reach satisfactory predictions consistent with mutagenesis results. Surprisingly, in one case, 2 separate functional epitopes were predicted based on 2 sets of specificity profiles and both matched the experimental results. Confirmation of these observations are on-going.
Conclusion: To better predict functional epitopes, our data so far suggest weaker signals should be excluded if there are strong signals from the same or a closely related evolutionary group, but included if the strong signals are from a distinct evolutionary group. Site-directed mutagenesis provides a spot view on whether the predicted residues are critical in Ab binding. These experimental data in turn provide clues on how to better the predictions based on specificity profiles.
