Impact of heme on the therapeutic efficacy of anti-CD20 antibodies

Polyreactivity, the capacity of antibodies to recognize unrelated antigens, is often an undesirable trait in therapeutic antibodies, as it can impair pharmacokinetics and elevate the risk of adverse effects. A growing body of evidence indicates that antibodies can acquire polyreactivity and autoreactivity after transient contact with heme, a byproduct of hemolysis. In our earlier work, we found that nearly 14% of the clinical-stage antibodies are sensitive to heme exposure. Here, we investigated how heme-induced polyreactivity influences the function of anti-CD20 antibodies, focusing on rituximab and CAT 13 (the parental antibody of ublituximab). Using a broad array of approaches, including ELISA, protein microarrays, spectroscopy, surface plasmon resonance, flow cytometry, molecular modeling, cytotoxicity assays, and murine lymphoma models, we demonstrate that rituximab readily acquires polyreactivity upon heme binding. This modification enabled promiscuous interactions with diverse human proteins while leaving recognition of the intended target, CD20, intact. In vitro, complement-dependent cytotoxicity remained unaffected by heme treatment. Mechanistic analyses revealed that heme associates with rituximab at a moderate-affinity site distinct from its CD20 binding interface. Strikingly, however, in vivo experiments showed that systemic heme exposure reduces the therapeutic efficacy of anti-CD20 antibodies. Our findings uncover a context-dependent vulnerability of rituximab and related CD20-targeting antibodies to heme, suggesting that hemolysis and extracellular heme levels may represent overlooked determinants of clinical response in lymphoma treatment.