OA4-AM24-MN-23 - Treatment-Emergent Antibodies to Amustaline/Glutathione Pathogen-Reduced Red Blood Cells in the ReCePI Phase III Clinical Trial

The ReCePI Phase III randomized, controlled clinical study compared amustaline (S-303)/glutathione pathogen-reduced (PR)RBCs (Test) with conventional RBCs (Control). The incidence and clinical significance of treatment emergent antibodies with specificity for PRRBCs was assessed.

Study

Design/Methods: Subjects received study RBCs during and for 7 days after complex cardiac surgery and were screened using an indirect antiglobulin test (IAT) for PRRBC-specific antibodies at baseline, when a routine IAT test was performed, and at 28- and 75-days post-surgery. Subjects with positive antibody screens were followed at ~2-week intervals and investigated for clinical evidence of increased RBC clearance. Serological confirmation was performed at Versiti Blood Center (Milwaukee, WI) and monocyte monolayer (MMA) assays at American Red Cross (Pomona, CA).

Results/Findings: One screened subject was excluded with a natural antibody specific for PRRBCs. 159 Test and 162 Control subjects received 456 Test and 524 Control (P=0.049) study RBCs. Four Test and 4 Control subjects developed RBC alloantibodies. Five Test (3.1%,) and 0 Control subjects (P=0.015) developed PRRBC-specific antibodies, first detected on Days 26-80 after surgery after receipt of 1-3 study units. Antibody titers were low (titer ≤8) and decreased over time (Table).  A MMA for clinically relevant antibodies was either non-reactive (3/5) or indeterminate (2/5) and the DAT was negative in 4 of 5 subjects. One subject had a weak positive DAT and an eluate specific for PRRBCs. Of four tested, three antibodies could be inhibited by free acridine (a component of S-303) and one could not, suggesting two epitope specificities. The protocol required the DSMB to advise a clinical stop if one subject showed evidence of hemolysis; none was observed and the study proceeded. Flow cytometry of RBC samples frozen during the investigations revealed circulating acridine-positive PRRBCs at levels consistent with the expected post transfusion levels (Table) considering RBC dose, hemorrhagic loss, RBC survival and hematopoietic recovery after surgery. RBC acridine surface density on transfused PRRBCs in vivo was substantially lower (200-300 acridine/cell) than on PRRBCs before transfusion (~8,500 acridine/cell) suggesting acridine loss in the presence of antibodies.

Conclusions:

PRRBC specific antibodies occurred in 3.1% of Test subjects. There was no evidence of hemolysis or in vitro properties indicative of clinical significance. Circulating PRRBCs could be enumerated by flow cytometry with a loss of RBC surface acridine  expression, which is a potential escape mechanism from destruction. (funded by the Biomedical Advanced Research and Development Authority, DHHS; ClinicalTrials.gov, NCT03459287).