Background S-303 was developed to inactivate viruses, bacteria, protozoa, and leukocytes in red blood cell concentrates (RBC). S-303 is a modular FRALE compound (FRangible Anchor Linker Effector) designed to bind to nucleic acids with its Anchor, to react through its Effector, and to form cross-links. S-303 spontaneously decomposes to the non-reactive compound S-300 by hydrolysis of the Linker, to minimize protein adducts. Pathogen inactivation (PI) treatment utilized the co-addition of S-303 and unbuffered GSH to quench non-specific S-303 reactions. The treatment process was optimized to maintain RBC function and maximize PI. Pre-clinical dog and rabbit chronic transfusion studies with allogeneic S-303 RBC showed no detectable antibodies to S-303 RBC. In Phase 1 studies, transfusion of healthy subjects with autologous human S-303 RBC demonstrated acceptable post-transfusion recovery and life span. Repeated transfusion (n=5) of 28 healthy subjects with autologous S-303 RBC showed no detectable antibodies to S-303 RBC. In a Phase 3 trial evaluating chronic transfusion of allogeneic S-303 RBC to patients with thalassemia or sickle cell anemia, 2 of 26 patients developed low titer positive Indirect Antiglobulin Tests (IAT) to S-303 RBC (one of the 2 patients also had a direct reacting IgM agglutinin). For both patients Direct Antiglobulin Tests (DAT) were negative. However, pretreatment RBC from the same unit remained compatible. S-303-related Anchor derivatives inhibited the positive IAT. Following this observation, clinical trials of S-303 RBC were stopped, studies were initiated to define the immunologic response to S-303 RBC, and an improved S-303 treatment process was developed.

Methods The original PI process utilized 200 μM S-303 and 2 mM unbuffered GSH. The process was modified to use 10-fold more neutralized GSH (20 mM) added to RBC 10 minutes prior to addition of S-303 (200μM). High titer anti-Anchor sera (RaS) were elicited by immunizing rabbits with a stable Anchor-KLH construct. A FACS assay to detect decoration of RBC with S-303 was developed using RaS and FITC goat anti-rabbit (GAR) IgG. IAT assays were performed with two methods: High titer RaS were tested with buffer gel cards (MTS), S-303 RBC suspended in low ionic strength solution (LISS) and GAR IgG. Reactive patient sera were tested with S-303 RBC and anti-IgG gel cards (MTS)

Results S-303 RBC prepared with the original clinical process were positive for IAT by gel card for both the RaS (1:100) and for the patient sera (1:3). FACS analysis using RaS (1:100) with FITC GAR IgG (1:64) demonstrated a high level of labeling. Under the modified conditions (S-303m), S-303m RBC exhibited minimal labeling above background by FACS with RaS. Sera from the 2 patients with positive IAT against S-303 RBC were negative against S-303m RBC. In addition, high titer RaS were negative against S-303m RBC in IAT by gel card. Potent inactivation of bacteria in S303m RBC (S. epidermis, S. marcescens) and viruses (Vesicular stomatitis virus) was retained. Storage of S-303m RBC for 42 days exhibited hemolysis and K+ levels comparable to S303 RBC and higher ATP levels than S-303 RBC.

Conclusions An improved PI process has been developed that significantly reduces RBC decoration by S-303 while maintaining PI and RBC in vitro function. The new process eliminated the positive IAT reactivity with sera from patients previously alloimmunized to S-303 RBC.

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