Recombinant human monoclonal HLA antibodies of different IgG subclasses with the same epitope specificity: excellent tools to study the differential effect of donor specific antibodies in transplantation.

C.S.M. Kramer, M.E.I. Franke-van Dijk, A.J. Priddey, H. Car, E. Gnudi, G.E. Karahan, E. van Beelen, C.C.C. Zilvold-van den Oever, H.J. Rademaker, P.W.H.I. Parren, V. Kosmoliaptsis, A. Mulder, D.L. Roelen, F.H.J. Claas, S. Heidt

Wednesday 13 march 2019

14:08 - 14:10h at Koningin Máximazaal

Categories: Basaal, Parallelsessie

Parallel session: Parallelsessie III - Basaal I

Background: The humoral immune response against mismatched HLA antigens in renal transplantation is associated with inferior graft survival, but only in a subpopulation of patients. The various clinical effects may be caused by different IgG subclasses of donor-specific HLA antibodies. IgG subclass antibodies have been associated with either complement dependent or independent graft damage. The mechanism of action of IgG subclass antibodies can be studied in experimental systems using HLA monoclonal antibodies (mAbs). Since currently available human HLA-specific mAbs are mainly of the IgG1 subclass, our aim was to generate human HLA class I and II mAbs of all four IgG subclasses.

Methods: Recombinant HLA class I and II-specific mAbs were generated from established human B cell hybridomas by cloning genes encoding the antibody variable domains into vectors. The antibodies were expressed by transient co-transfection of heavy and light chain vectors and purified using protein G column. IgG subclass cloning was confirmed with modified luminex screening assay using detection antibodies specific for each IgG subclass. Antibody specificity was determined by luminex single antigen bead (SAB) technology, affinity by bio-layer interferometry, and the effector functions were determined by complement dependent cytotoxicity (CDC), as well as antibody dependent cellular cytotoxicity (ADCC) assays.

Results: The IgG subclass cloning was verified with the modified luminex screening. Screening of the recombinant mAbs with luminex SAB assays showed that all four IgG subclasses have identical HLA specificities. Importantly, the four IgG subclasses have similar binding affinity for the immunizing HLA allele. Next, the four IgG subclass mAbs were tested with CDC and cell lysis of target cells was only observed for IgG1 and IgG3 mAbs in a dose-dependent manner. Thus, both IgG1 and IgG3 mAbs are highly cytotoxic and the most cell lysis was observed for IgG1 mAbs. No cytotoxicity was observed for IgG2 and IgG4 mAbs. In addition, both IgG1 and IgG3 mAbs can trigger effector cells to lyse target cells in ADCC. As expected, IgG2 and IgG4 mAbs did not induced specific cell lysis.

Conclusions: It is feasible to generate human HLA class I and II IgG subclass mAbs recognizing the same HLA epitope with the same affinity and with the appropriate functional properties. These mAbs can be used to understand the mechanism of HLA IgG subclass antibodies in renal transplantation and other clinical settings.