Potentiating CD20 monoclonal antibody therapy by targeting complement C3 fragments covalently deposited on lymphoma cells

• Complement-fixing CD20 mAbs deposit C3d on lymphoma cells; this neo-antigen can be selectively targeted to boost therapeutic efficacy.

• In B-cell lymphoma xenografts, C3d combined with CD20 mAbs extended survival over single-agent therapy, without apparent toxicity.

Monoclonal antibodies (mAbs) improve survival of patients with mature B-cell malignancies. Fcγ receptor–dependent effector mechanisms kill tumor cells but can promote antigen loss through trogocytosis, contributing to treatment failures. Cell-bound mAbs trigger the complement cascade to deposit C3 activation fragments and lyse cells. Within 24 hours after ofatumumab administration to patients with chronic lymphocytic leukemia (CLL), circulating tumor cells had lost CD20 and were opsonized with C3d, the terminal covalently bound form of complement protein C3. We hypothesized that C3d provides a target to eliminate residual CD20⁻ tumor cells. To test this hypothesis, we generated C8xi, a mouse/human chimeric immunoglobulin G1 (IgG1) that reacts with human but not mouse C3d. C8xi was effective in a patient-derived xenograft model against CD20⁻, C3d opsonized CLL cells from patients treated with ofatumumab. We also generated rabbit mAbs, 2 of which were chosen because they bound mouse and human C3d with low nanomolar affinity but were minimally cross-reactive with full-length C3. Anti-C3d rabbit/human chimeric IgG1 in combination with ofatumumab or rituximab prolonged survival of xenografted mice that model 3 different types of non-Hodgkin lymphoma (NHL). For example, in a diffuse large B-cell lymphoma model (SU-DHL-6), median survival with single-agent CD20 mAb was 114 days but was not reached for mAb combination treatment (P = .008). In another NHL model (SU-DHL-4), single-agent and combination mAb therapy eradicated lymphoma in most mice. In long-term survivors from both cohorts, there was no evidence of adverse effects. We propose that C3d mAbs combined with complement-fixing CD20 mAbs can overcome antigen-loss escape and increase efficacy of mAb-based therapy.