DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing

TLDR

DuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody engineered by controlled Fab‑arm exchange to simultaneously engage CD3 on T cells and CD20 on B cells. The Fc domain was silenced by L234F/L235E/D265A mutations, and the bispecific’s ability to activate T cells and mediate cytotoxicity was quantified by flow cytometry in co‑culture assays, while its anti‑tumor activity and safety were evaluated in humanized mouse models and cynomolgus monkeys, and pharmacokinetics and cytokine responses were compared after subcutaneous versus intravenous administration. DuoBody-CD3xCD20 induced highly potent, T‑cell‑mediated killing of malignant B cells in vitro, outperformed alternative CD3‑targeting bispecifics, demonstrated robust anti‑tumor activity in vivo even with competing CD20 antibodies, and achieved profound, long‑lasting B‑cell depletion in monkeys with comparable efficacy after subcutaneous and intravenous dosing while lowering cytokine release.

Abstract

BackgroundDuoBody®-CD3xCD20 (GEN3013) is a full-length human IgG1 bispecific antibody (bsAb) recognizing CD3 and CD20, generated by controlled Fab-arm exchange. Its Fc domain was silenced by introduction of mutations L234F L235E D265A.MethodsT-cell activation and T-cell-mediated cytotoxicity were measured by flow cytometry following co-culture with tumour cells. Anti-tumour activity of DuoBody-CD3xCD20 was assessed in humanized mouse models in vivo. Non-clinical safety studies were performed in cynomolgus monkeys.FindingsDuoBody-CD3xCD20 induced highly potent T-cell activation and T-cell-mediated cytotoxicity towards malignant B cells in vitro. Comparison of DuoBody-CD3xCD20 to CD3 bsAb targeting alternative B-cell antigens, or to CD3xCD20 bsAb generated using alternative CD20 Ab, emphasized its exceptional potency. In vitro comparison with other CD3xCD20 bsAb in clinical development showed that DuoBody-CD3xCD20 was significantly more potent than three other bsAb with single CD3 and CD20 binding regions and equally potent as a bsAb with a single CD3 and two CD20 binding regions. DuoBody-CD3xCD20 showed promising anti-tumour activity in vivo, also in the presence of excess levels of a CD20 Ab that competes for binding. In cynomolgus monkeys, DuoBody-CD3xCD20 demonstrated profound and long-lasting B-cell depletion from peripheral blood and lymphoid organs, which was comparable after subcutaneous and intravenous administration. Peak plasma levels of DuoBody-CD3xCD20 were lower and delayed after subcutaneous administration, which was associated with a reduction in plasma cytokine levels compared to intravenous administration, while bioavailability was comparable.InterpretationBased on these preclinical studies, a clinical trial was initiated to assess the clinical safety of subcutaneous DuoBody-CD3xCD20 in patients with B-cell malignancies.FundingGenmab