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

Upon kidney transplantation, de novo donor-specific antibodies (dnDSA) can be triggered by immunogenic polymorphic amino acids (AA) configurations on mismatched donor HLA. These configurations, called eplets, are theoretically defined and require experimental verification to establish whether allo-antibodies can actually bind to these specific polymorphic residues. Human HLA-specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we aimed to generate recombinant human HLA-DR specific mAbs for the verification of HLA-DR eplets. We isolated single HLA-DR-specific memory B cells from peripheral blood of immunised individuals (n = 3) using HLA class II tetramers. Upon cell sorting, an average of 5 HLA-antibody positive B cell clones (range 2-11) were isolated per individual. The clones obtained from a single individual differed in clonality and specificity. From the HLA-specific memory B cells we generated recombinant human HLA-DR-specific mAbs (n = 5). The mAbs were screened with Luminex single antigen bead assay. The AA sequences of the reactive HLA antigens were compared using the software program HLA-EMMA to identify uniquely shared AA within 3-3.5Å, which are referred to as functional epitopes. This reactivity analysis led to identification of three configurations i.e. 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognised respectively by HLA-DRB1*01:01​, HLA-DRB1*04:01​, and HLA-DRB1*07:01​ antigen-reactive mAbs. The former two correspond to eplets 70QA and 31FYY and can now be considered as antibody-verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody-verified. The verification of these eplets will contribute to the implementation of eplet matching in allocation systems aiming to prevent the development of dnDSA. The generated recombinant human HLA-DR-specific mAbs can also be used in functional studies to provide insight in the respective roles of HLA-specific IgG antibodies in causing graft damage.