178 H-antigen re-emergence in FucOB enzyme-treated HUVEC cells

Abstract Introduction Enzymatic blood group antigen conversion is a promising strategy to enable ABO-incompatible kidney transplantation. An enzyme called FucOB can cleave the H-antigen, a precursor to all blood group antigen structures. When applied during ex vivo machine perfusion it could provide an ABO compatible option for rare Bombay blood group kidney recipients. However, this provides only phenotype alteration of the antigens on the vascular endothelial cells. As these cells remain genetically unmodified and metabolically active, it is believed that these antigens will re-express on the cell surface, but the timeline of this re-expression is unknown. Methods A re-emergence assay was carried out using human umbilical vein endothelial cells (HUVEC), which express the H antigen. Cells were treated with FucOB enzyme in vitro at different predefined time-points and cultured for up to 24hrs. The cells were stained for H-antigen expression and examined using the NXT attune flow cytometer. Results The results showed that immediately after enzyme treatment, H antigen expression decreased to 0.78-2.3%. After 24 hours 40-47% emergence was observed, relative to control cells without enzyme treatment. Results were similar with the variation the cultured media. Conclusion Our in vitro model demonstrated that over 40% re-emergence of H-antigen expression after 24 hrs. This possible antigen re-emergence kinetic period will help future treatment options for patients after an ABO-incompatible kidney transplantation.