Duobody-CD3xCD20 Shows Unique and Potent Preclinical Anti-Tumor Activity in Vitro and In Vivo, and Is Being Evaluated Clinically in Patients with B-Cell Malignancies
Authors: Hiemstra Ida H. , Engelberts Patrick J. , de Jong Bart , Schuurhuis Danita H , Salcedo Theodora W. , Verploegen Sandra , van der Zee Marten , Gerritsen Arnout F. , Losic Nedjad , Horbach Sjeng , Oliveri Roberto , Lammerts van Bueren Jeroen J. , Autzen Usher Pernille , Schuurman Janine , Parren Paul W. H. I. , Breij Esther C. W. 
Published: October 2019

DuoBody®-CD3xCD20 (GEN3013) is a bispecific antibody (bsAb), recognizing the T-cell antigen CD3 and the B-cell antigen CD20, that triggers potent T-cell-mediated lysis of CD20-expressing cells. DuoBody-CD3xCD20 is a full-length bispecific IgG1 generated by controlled Fab-arm exchange (cFAE) [1, 2] and contains an effector function-silenced Fc region.

In vitro, DuoBody-CD3xCD20 induced potent activation, proliferation and cytotoxic activity of both CD4+ and CD8+ T cells in the presence of CD20-expressing cells, as measured by flow cytometry and bromodeoxyuridine (BrdU) incorporation assays. DuoBody-CD3xCD20 induced T-cell-mediated cytotoxicity towards a diverse panel of cell lines derived from various B-cell malignancies and endogenous B cells, with EC50 values in the low picomolar range (EC50: 0.2-5.0 pM).

The CD20-specific antibody 7D8 [3-5] forms the basis for the CD20-specific Fab arm of DuoBody-CD3xCD20. To study the contribution of this specific Fab arm to the observed potency of DuoBody-CD3xCD20, we compared the target binding characteristics and the capacity to induce T-cell-mediated cytotoxicity of a CD3 bsAb based on 7D8, with CD3 bsAbs using B-cell targeting arms derived from alternative CD20 antibodies or from antibodies against other well-known B-cell membrane molecules CD22, CD24, CD37, CD70, CD79b, CD138 and HLA-DR. In addition, target expression levels of the B-cell targets were assessed in a panel of B-cell lines. Using a classic chromium release assay, the 7D8-based CD3 bsAb displayed cytotoxic activity superior to all other B-cell-targeting CD3 bsAbs tested, including alternative CD20-targeting CD3 bsAbs. This unique cytotoxic activity could not be explained by expression levels of the target antigen, nor by the binding affinity or epitope of the B-cell specific Fab arm. This illustrates the complexity of factors that determine the potency of CD3 bsAbs.

The anti-tumor activity of DuoBody-CD3xCD20 was confirmed in vivo in humanized mouse models using three different B-cell lymphoma xenograft models, in prophylactic and therapeutic settings.

Non-clinical safety studies with DuoBody-CD3xCD20 in cynomolgus monkeys demonstrated profound and long-lasting B-cell depletion (at least 70 days, at dose levels > 0.1 mg/kg) from both peripheral blood and lymphoid organs. B-cell depletion was reversible, with time to B-cell recovery correlating with the treatment dose. Notably, at the same dose level, B-cell depletion was comparable between subcutaneous and intravenous administration. Pharmacokinetic (PK) analysis demonstrated comparable bioavailability for the two administration routes, although peak plasma levels were lower and delayed after subcutaneous administration. Moreover, lower plasma cytokine levels were observed after subcutaneous administration.

Based on these data, Genmab has initiated a First-in-Human clinical trial to evaluate the safety and preliminary efficacy of DuoBody-CD3xCD20 by subcutaneous administration in patients with B-cell malignancies. The study is currently enrolling (EudraCT No: 2017-001748-36).

References Labrijn, A.F., et al., Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange. Proc Natl Acad Sci U S A, 2013. 110(13): p. 5145-50. Labrijn, A.F., et al., Controlled Fab-arm exchange for the generation of stable bispecific IgG1. Nat Protoc, 2014. 9(10): p. 2450-63. Teeling, J.L., et al., Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood, 2004. 104(6): p. 1793-800. Teeling, J.L., et al., The Biological Activity of Human CD20 Monoclonal Antibodies Is Linked to Unique Epitopes on CD20. Journal of Immunology, 2006. 177(1): p. 362-71. van Meerten, T., et al., HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis. Haematologica, 2010. 95(12): p. 2063-71.

Disclosures

Hiemstra: Genmab: Employment, Other: Warrants. Engelberts:Genmab: Employment, Other: Warrants. de Jong:Genmab: Employment, Other: Warrants. Schuurhuis:Genmab: Employment, Other: Warrants. Salcedo:Genmab: Employment, Other: Warrants. Verploegen:Genmab: Employment, Equity Ownership. van der Zee:Genmab: Employment, Other: Warrants. Gerritsen:Genmab: Employment, Other: Warrants. Losic:Genmab: Employment, Other: Warrants. Horbach:Genmab: Employment, Other: Warrants. Oliveri:Genmab: Employment, Other: Warrants. Lammerts van Bueren:Genmab: Employment, Other: Warrants. Autzen Usher:Genmab: Employment, Other: Warrants. Schuurman:Genmab: Employment, Other: Warrants. Parren:Genmab: Equity Ownership; Lava Therapeutics: Employment. Breij:Genmab: Employment, Equity Ownership.