ABSENCE OF IMMUNOGENICITY OF DIASPIRIN CROSS-LINKED HEMOGLOBIN IN HUMANS

Diaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly cross -linked hemoglobin-based oxygen carrier being developed as a therapy f or acute blood loss. We report here the absence of immunogenicity of D CLHb in patients enrolled in phase II and III clinical trials of DCLHb . Two very sensitive immunoassays, an enzyme-linked immunosorbent assa y (ELISA) and a Western blot assay, were developed and validated for t his assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for DCLHb and native human hemoglob in (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that these antibodies were elicited as a xenogenic response to the protein. In the ELISA, the optical densit y of a patient sample exposed to DCLHb-coated wells was compared with that of the patient sample exposed to carbonate buffer-coated wells; a n optical density ratio of 1.4 was established for discriminating betw een a positive (reactive) or negative DCLHb antibody response. To date , all of the more than 300 patient specimens (preinfusion and postinfu sion) from clinical trials have exhibited a ratio of less than 1.4, co nfirming the lack of preexisting antibodies to DCLHb and clearly showi ng the absence of DCLHb antibodies after exposure to this new biologic entity. There has been no requirement for use of the confirmatory Wes tern blot assay, Taken together, the results from this study indicate DCLHb is not immunogenic in humans at doses evaluated clinically. (C) 1998 by The American Society of Hematology.