Immunological Compatibility of Novel Enzyme-Converted Universal Blood Donor Cells

Introduction: In transfusion, unintentional mismatching of blood groups remains one of the most common causes of serious and sometimes fatal adverse reactions. Scarcity of certain blood types is another major transfusion challenge. The ABO blood group specificity is determined by the terminal monosaccharides, N-acetylgalactosamine on A and galactose on B RBCs. To create a universal blood supply, cell surface engineering has been studied as a method to minimize the immunological rejection of donor cells. Enzymatic removal of immunodominant sugars would uncover universally acceptable H-antigen (O group RBC). However, enzymatic conversion of A-type RBCs was lagging due to a lack of very active and specific glycosidases and the complex nature of the A blood group antigen. In this research, the performance of two novel enzymes that act together extremely efficiently to convert A-type RBCs to universal donor O RBCs, with high specificity for A antigen, is evaluated. Optimal conversion conditions are explored, as well as the immunological compatibility of the converted RBCs.

Materials & Methods: Whole blood samples are collected from consenting A donors (with approval from the University of British Columbia clinical research ethics board), washed and diluted to 10 % hematocrit in PBS buffer (pH7.4). The washed RBCs were treated with the newly developed glycosidases for 1 h at 37 ^oC. The efficiency of the newly developed glycosidases and the characterization of enzyme converted O (ECO)-RBCs were evaluated by gel-column based micro typing system (MTS) cards against A antigen and flow cytometry using anti A and H antibodies. Safety and compatibility of ECO-RBCs were assessed by blood group serology studies. In brief, ECO-RBCs were incubated in Rh-matched sera collected from diverse donors and any serum reactivity against ECO-RBCs was assessed using anti IgG MTS cards and flow cytometry using anti C3d (complement protein) antibody. To further confirm the cross-matching of ECO-RBCs, the monocyte monolayer assay (MMA), a clinically used method, was performed using monocytes collected from B and O blood donors to assess the phagocytic activity against ECO-RBCs pre-incubated with serum samples. Phagocytic activity against ECO-RBCs was calculated using the monocyte index (MI).

Results: Anti A MTS cards and flow cytometry analysis of antigen A and H expression, after enzymatic treatment, confirmed successful conversion of A blood group to universal O group. Our studies showed no significant hemolysis and no complement protein (C3d) deposition on ECO-RBCs pre-incubated with diverse sera, demonstrating the compatibility. Serology studies showed that ECO-RBCs are compatible with most sera (~50 donors) studied under transfusion conditions, though serum reactivity was shown by some O and B sera. A MI value of ≤ 6 % was obtained for all ECO-RBCs indicating minimal risk of an acute hemolytic reaction associated with ECO-RBC transfusion.

Conclusions: We have developed novel glycosidases that efficiently remove immunodominant A antigen from RBC surfaces with high specificity to create universal blood donor cells at neutral pH and at enzyme concentrations that are much lower than those previously required. Based on preliminary results, ECO-RBCs have the potential to increase donor blood supply and thereby greatly advance the field of transfusion.

Devine:Canadian Blood Services: Current Employment; Macopharma: Membership on an entity's Board of Directors or advisory committees; STRM.Bio: Membership on an entity's Board of Directors or advisory committees; Univ. of British Columbia: Current Employment.