IgG4 anti-CD47: Erythrophagocytosis and potential transfusion complications

IgG4 monoclonal anti-CD47 for cancer immunotherapy resulted in anemia necessitating red cell (RBC) transfusions. Although the antibody readily binds to CD47 on RBCs, the mechanism underlying the anemia associated with IgG4 is poorly understood. We investigated samples from patients receiving Hu5F9-G4 (magrolimab) with positive reactivity in compatibility tests using a monocyte-monolayer assay (MMA).

For RBC opsonization, patient plasma was diluted to avoid direct agglutination and to give a 4+ indirect antiglobulin test (IAT) and >10³ mean fluorescent intensity (MFI) by flow cytometry. Fcγ receptor blocking used F(ab')₂ antibodies. Antibody-dependent cellular cytotoxicity (ADCC) used purified natural killer (NK) cells. Recombinant IgG4 anti-K and polyclonal anti-D were tested by MMA concurrent with blocking of CD47 with a deglycosylated antibody.

Five of six samples showed significant erythrophagocytosis (phagocytosis index [PI] = 20-60; PI > 5 clinically significant) with the exception having 30-fold lower MFI. Phagocytosis was not increased in the presence of complement. Fcγ receptor blocking showed FcγRI and FcγRIIa were involved but not FcγRIIIa, supported by negative results when tested for NK ADCC. IgG4 anti-K also showed significant erythrophagocytosis (PI = 30). Blocking of CD47 increased phagocytosis, 1.7 to 3.9-fold, respectively, of RBCs opsonized with IgG4 anti-K or polyclonal anti-D.

IgG4 RBC antibodies, generally not considered clinically significant for transfusion, can result in erythrophagocytosis providing an explanation for anemia related to anti-CD47 mediated by FcγRI and FcγRIIa. Enhanced phagocytosis of alloantibody-coated RBCs with concurrent blocking CD47 suggests potential for enhanced complications for patients who develop RBC alloantibodies while receiving cancer therapies targeting CD47.