Assessing and mitigating the interference of ALX148, a novel CD47 blocking agent, in pretransfusion compatibility testing

Abstract

Funding information ALX148, soluble CD47, and high-affinity SIRP monomers were provided by ALX Oncology Background ALX148, a novel CD47 blocking agent, is in clinical development for the treatment of advanced solid tumors and lymphoma. Because CD47 is highly expressed on red blood cells (RBCs), its therapeutic blockade can potentially interfere with pretransfusion compatibility testing. This study describes the interference of ALX148 in pretransfusion compatibility testing and evaluates the methods used for mitigating such interference. Study Design and Methods: Routine serologic tests were performed on six samples from four patients treated with ALX148. Antibody screening tests were performed on ALX148-spiked plasma, and RBC testing including antigen typing was performed on ALX148-coated RBCs. Soluble CD47 or high-affinity signal regulatory protein α (SIRPα) monomers were used to remove the falsepositive reactivity of ALX148-spiked plasma with or without anti-E. Results: ALX148 caused false-positive reactivity in antibody screening using indirect antiglobulin testing (IAT) and two-stage papain testing. However, falsepositive reactivity was not observed at the immediate spin (IS), room temperature (RT), and 37°C phases. Direct antiglobulin testing, autologous controls, and eluates showed positive results. ALX148 did not affect blood group antigen typing performed at the IS or RT phases. The use of 50-to 100-fold molar excess of soluble CD47 or 300-fold molar excess of high-affinity SIRPα monomers removed false-positive reactivity in IAT without affecting anti-E detection. Conclusion: ALX148 generates false-positive reactivity in IAT, interfering with pretransfusion compatibility testing. The use of soluble CD47 or highaffinity SIRPα monomers can resolve the interference without possibly missing clinically significant alloantibodies.

Application of Blood Group Genotyping by Next-Generation Sequencing in Various Immunohaematology Cases

“…Anti-CD47 drugs such as ALX148 and Hu5F9-G4 interfere with pretransfusion compatibility testing, thus complicating the safe and timely provision of RBC units [42][43][44]. One possible option to resolve the anti-CD47 interference is to provide phenotypically or genotypically matched RBC units.…”

Evorpacept-Induced Interference and Application of a Novel Mitigation Agent, Evo-NR, in Pretransfusion Testing

“…The following three mixtures were prepared: 25 μL evorpacept-spiked plasma (0.1, 1, 10, 100, 1,000, and 2,000 μg/mL) was mixed with 50 μL 0.8% rr (ce/ce) RBCs (ID-DiaCell III, Bio-Rad); 25 μL evorpacept-spiked plasma (2,000 μg/mL) preincubated with 1-, 3-, 6-, and 9-fold molar excess of Evo-NR was mixed with 50 μL 0.8% rr (ce/ce) RBCs (ID-DiaCell III); and one volume of 3% rr (ce/ce) RBCs (Panoscreen, Immucor), preincubated with two volumes of 10 μg/mL evorpacept-spiked plasma followed by two washes with normal saline, was mixed with 10 volumes of EGA or PBS, or 500-, 1,000-, 1,500-, and 2,000-fold molar excess of Evo-NR. FITC-conjugated F(ab’)2 goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used for staining, as described in a previous study [ 18 ]. The test results were compared to those obtained using a blank plasma sample as a negative control.…”

“…Previously reported pattern of interference by evorpacept was considerably different from that of magrolimab. In contrast to magrolimab, evorpacept did not cause spontaneous agglutination, DAT was positive, and strong interference was primarily observed in the IAT phase and not in the IS or RT phases during antibody testing [ 18 , 19 ]. For patients receiving evorpacept, blood type and baseline unexpected antibody status are determined before starting treatment, often with extended RBC antigen genotyping and/or phenotyping.…”

Transfusion management in the era of magrolimab (Hu5F9‐G4), an anti‐CD47 monoclonal antibody therapy

“…The anti‐CD47 therapy magrolimab binds CD47 on RBCs, 12 resulting in transient anemia 21,35 and interference with pretransfusion testing in clinical trials 11,12,36 . Interference with antibody detection has been reported with investigational CD47‐blocking agents, although the degree of interference depends on the IgG subclass, the design of the drug molecule, plasma drug levels, and the reagents and methods used for blood typing 37–40 . This review focuses specifically on the magrolimab interference with pretransfusion testing.…”