Monoclonal anti‐CD47 interference in red cell and platelet testing

Abstract

BACKGROUND Anti‐CD47 (Hu5F9‐G4) is a human monoclonal immunoglobulin G (IgG)4 antibody that is in clinical trials to treat hematologic or solid malignancies. CD47, a glycoprotein expressed on all cells, binds to signal‐regulatory protein α on macrophages and regulates phagocytosis. Blocking CD47 is thought to enhance phagocytosis and promote antitumor responses. Here, we evaluate drug interference in pretransfusion testing, determine mitigation strategies, and compare interference with anti‐CD38 (Daratumumab). STUDY DESIGN AND METHODS Samples from four patients were tested by standard methods. Anti‐IgG (Immucor monoclonal Gamma‐clone and Ortho BioClone) were used, and dithiothreitol and enzyme‐treated RBCs were tested. Allo‐adsorption was performed with papain treated RBCs, pooled platelets, or with commercial human platelet concentrate. Platelet antibody testing was performed according to manufacturer's instructions. RESULTS All plasma samples reacted 3+ to 4+ in all phases with all red blood cells (RBCs) by all methods including immediate spin. Stronger reactivity was observed with D– RBCs with titers as high as 16,384 at indirect antiglobulin testing. Reactivity at indirect antiglobulin testing using Gamma‐clone anti‐IgG (which does not detect IgG4) was only weakly positive and confirmed to be carryover agglutination. Plasma reacted with dithiothreitol, trypsin, papain, α‐chymotrypsin, or warm autoantibody removal medium (W.A.R.M., Immucor) treated RBCs. Direct antiglobulin testing and autocontrol were negative or weak with 3+ reactive eluates. Reactivity was removed by multiple alloadsorptions with papain‐treated cells or pooled platelets. Polyethylene glycol adsorption was invalid due to precipitation of antibody. CONCLUSION Anti‐CD47 (Hu5F9‐G4) interferes with all phases of pretransfusion testing, including ABO reverse typing. To remove interference requires multiple RBC alloadsorptions and/or the use of monoclonal Gamma‐clone anti‐IgG in the indirect antiglobulin testing.

Reliability of labeling red cell units with minor antigen historical results and process considerations

“…Donor units with extended antigen profiles are increasingly requested for patients receiving chronic transfusion to prevent alloimmunization, particularly to C, E, and K as common causes of sensitization . Extended antigen typed blood is also used for antigen matching (providing donor units negative for clinically significant antigens the recipient lacks) for patients with warm autoantibodies when compatibility cannot be demonstrated and underlying alloantibodies cannot, or have not, been ruled out, and is also an option for patients receiving monoclonal antibody therapies that interfere in pre‐transfusion testing …”

“…[1][2][3][4] Extended antigen typed blood is also used for antigen matching (providing donor units negative for clinically significant antigens the recipient lacks) for patients with warm autoantibodies when compatibility cannot be demonstrated and underlying alloantibodies cannot, or have not, been ruled out, 5,6 and is also an option for patients receiving monoclonal antibody therapies that interfere in pre-transfusion testing. [7][8][9][10] Recent regulatory changes in the industry have the potential to positively impact the availability of extended typed units to meet growing demand. Following a 2012 Blood Product Advisory Committee (BPAC) meeting, 11 which focused on the lack of regulatory standards for testing and labeling of donor units with minor blood group antigens, an AABB/FDA workgroup was organized.…”

The effects of monoclonal anti‐CD47 on RBCs, compatibility testing, and transfusion requirements in refractory acute myeloid leukemia

“…14,15 For anti-CD47, use of multiple alloadsorptions and monoclonal gamma-clone anti-IgG, which does not detect IgG4, have been proposed to help mitigate antibody interference. 16 In the Phase 1b study of Hu5F9-G4 in relapsed/ refractory B-NHL, anemia occurred in approximately 42% of patients. This was mitigated by the use of a priming dose of 1 mg/kg to enable clearance of older RBCs and a compensatory reticulocytosis to occur before commencing a maintenance dose.…”

“…Multiple RBC alloadsorptions and/or the use of monoclonal gamma-clone anti-IgG (which does not detect IgG4) may help limit interference with compatibility testing. 16 In due course, use of an anti-idiotype against Hu5F9-G4 on patient serum samples could further reduce this interference.…”

Two case reports involving therapeutic monoclonal anti‐CD47 (Hu5F9‐G4), it's effect on compatibility testing and subsequent selection of components for transfusion

“…CD47 surface glycoprotein (integrin-associated protein) ligates signal-regulatory protein alpha on macrophages, initiating selfrecognition and inhibiting phagocytosis. 5,6 Malignant cells show high expression of CD47 and are resistant to phagocytosis. Anti-CD47 aims to prompt the destruction of malignant cells by blocking CD47 positive cells allowing for phagocytosis.…”