Generation of human monoclonal HLA-DR antibodies for verification of HLA-DR eplets

C.S.M. Kramer, M.E.I. Franke-van Dijk, K.H. Bakker, M. Uyar-Mercankaya, G.E. Karahan, D.L. Roelen, F.H.J. Claas, S. Heidt

Wednesday 4 march 2020

14:30 - 14:40h

Parallel session: Parallel sessie IV – Basale abstracts

Background: In renal transplantation, recipients can form de novo donor-specific antibodies (DSA) against the mismatched HLA antigens of the donor and especially DSA against HLA class II are formed. Recent studies have shown that the development of these de novo DSA correlates with the number of HLA-DR/DQ eplet mismatches between recipient and donor, but not every individual eplet mismatch will result in DSA response. HLA class II eplets are theoretically defined single or configurations of surface exposed polymorphic amino acids. Therefore, experimental verification of immunogenic eplets is required, but is currently lacking due to the limited number of available HLA class II monoclonal antibodies (mAbs). To this end, we aimed to develop recombinant human HLA-DR specific mAbs.

Methods: From peripheral blood mononculear cells of three pregnancy immunised individuals, single memory B cells positive for HLA-DR tetramers were sorted. After expansion, supernatants were screened for the presence of HLA antibodies. From the HLA antibody positive B cell clones, RNA was isolated to obtain the variable heavy and light chains for cloning into pcDNA3.3 expression vectors. Next, both vectors were co-transfected in Expi293 cells to produce recombinant human HLA mAbs, which were analysed with luminex single antigen beads (SAB).

Results: HLA-DRB1*07:01, -DRB1*01:01, and -DRB1*04:01 tetramers were used for cell sorting and an average of 5 (range 2-11) HLA positive clones were obtained from sorted memory B cells, which represented an average of 0.0002% of total B cells. From these clones, eight recombinant mAbs were generated with six different antibody reactivity patterns: DR7, DR7/DR9, DR7/DR12, DR1/DR4/DR15/DR51, DR1/DR9/DR10/DR51 and DR4/DR1303/DR8/DR11/DR15/DR16. By analysing the antibody reactivity patterns, single or configurations of amino acids uniquely shared by the reactive HLA alleles could be defined. This analysis led to antibody-verification of HLA-DR eplets, 78V, 70Q 73A, and 31F 32Y 37Y. In addition, these mAbs could bind their native HLA targets expressed on cells and led to complement dependent cytotoxicity.

Conclusions: In conclusion, recombinant human HLA-DR specific mAbs can be generated from single memory B cells, and antibody reactivity pattern analysis led to verification of HLA-DR eplets. Additionally, these mAbs can also be used for functional studies on the effect of HLA antibodies in transplantation.