Introduction: Two anti-CD38 monoclonal antibodies (mAb) are approved for the treatment of multiple myeloma: isatuximab (Isa) and daratumumab (Dara); binding to CD38 on red blood cells (RBCs) results in false positive agglutination on indirect antiglobulin tests (IATs). Compared to Dara, which interferes with IATs in nearly 100% of patients, Isa interfered in 67/99 (67%) patients in the Phase 3 ICARIA-MM (NCT02990338) study and 95/150 (63%) patients in the Phase 3 IKEMA (NCT0327585) study. We sought to understand differences between the binding abilities of Isa and Dara to CD38 on RBCs.
Experimental Procedures: In vitro binding experiments in RBCs were performed following pretreatment of RBCs isolated from healthy donors +/- a mouse anti-human CD38 mAb (clone HB-7 or AT13/5) or a nicotinamide adenine dinucleotide (NAD) analog (irreversible CD38 inhibitor) before incubation with Isa, Dara, or control human IgG (hIgG). RBC surface bound CD38 antibodies were detected with fluorophore conjugated secondary antibodies and quantified by flow cytometry, imaging or mass spectrometry. Binding of Isa to recombinant human CD38 (rhCD38) pretreated with HB-7 or an NAD analog was quantified by Surface Plasmon Resonance (SPR). Ex vivo IAT was performed on plasma samples spiked with Isa or Dara.
Results: In vitro testing in the absence of human plasma showed that incubation of RBCs with Dara but not Isa resulted in a dose-dependent increase in RBC binding. The binding specificity of Dara was demonstrated by treatment with HB-7, an anti-CD38 mAb that recognizes epitopes that overlap with Dara but not Isa binding. Flow cytometry, confocal microscopy, SPR and mass spectrometry showed HB-7 had a competitive effect on Dara binding and induced Isa to bind to CD38 without competition. Pretreatment of RBCs with another anti-CD38 mAb AT13/5 or an NAD analog also induced Isa binding to RBCs. The NAD analog is an irreversible CD38 inhibitor that stabilizes CD38 dimer conformation. SPR demonstrated that pretreatment with HB-7 or the NAD analog may enhance dimer formation increasing Isa binding affinity to rhCD38. These results indicate that CD38 conformational changes caused by anti-CD38 antibodies or the NAD analog may facilitate Isa-binding to RBC-expressing CD38. In the ex vivo IAT, except for gel testing in which both Isa and Dara were panreactive, including all other IAT methods Dara reacted with many more cells, and reactions were stronger than Isa at all concentrations tested.
Conclusions: In this study, we confirmed that both Isa and Dara interfere with IATs but at different magnitudes. This reflects distinct mechanisms of CD38 binding. In the absence of human plasma, CD38 on RBCs can be directly recognized by Dara while Isa requires a cofactor, such as an anti-CD38 antibody or an NAD analog. Future studies are essential to characterize the impact of human plasma on Isa binding to RBCs-expressing CD38.
Citation Format: Zhili Song, Olivier Bedel, Bailin Zhang, Joern Hopke, Gejing Deng, Sandrine Macé, Marielle Chiron, Francisco Adrian, Taro Fukao, Frank Basile, France Pirenne, Makoto Okuda, Morvarid Moayeri, Btissam Chami, Yoko Hidaka, Ashok Nambiar, Thomas Martin, Chen Zhu. Anti-CD38 interference with blood compatibility testing: Differentiating isatuximab and daratumumab via functional epitope mapping [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1888.