Abstract 1243: Bispecific TandAbs: a safe and potent platform for T cell-mediated killing of CD19+ cells.
Authors: Zhukovsky Eugene A. , Reusch Uwe , Burkhardt Carmen , Knackmuss Stefan , Fucek Ivica , Eser Markus , McAleese Fionnuala , Ellwanger Kristina 
Published: August 2014

Abstract
CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for therapeutic antibodies that treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The TandAb technology enables the development of bispecific antibody therapeutics for oncology indications that harness the cytotoxic nature of T cells. The CD3 RECRUIT-TandAb AFM11, a human bispecific tetravalent antibody with two binding sites for both CD3 and CD19, exhibits highly potent cytotoxic activity and possesses pharmacokinetic properties that are considerably improved relative to alternative bispecific formats. In vitro assays demonstrate high affinity binding of AFM11 to CD3+ and CD19+ cells leading to higher potency and efficacy of T cell recruitment and target cell lysis relative to alternative bispecific antibody formats(tandem scFv, diabody). Cytotoxicity assays show that AFM11 mediates target cell lysis by CD4+ and CD8+ T cells with EC50 values in the low to sub-picomolar range that is independent of the target cell CD19 density. AFM11 exhibits nearly identical cytotoxicity across a broad range of effector-to-target ratios, from 5:1 to 1:5, and facilitates serial target cell killing by T cells. Binding of antibodies to CD3 on T cells may elicit devastating cytokine release if it results in systemic rather than target-specific activation of T cells. Indeed, the anti-CD3 antibody OKT3 exhibits high toxicity in humans due to non-specific activation of T cells. Thus, in vitro assays were used to assess AFM11 off-target activity. AFM11 activates T cells inly in the presence of CD19+ cells, resulting in induction of CD69 and CD25 expression and the on-target release of IFN-γ, TNF-α, IL-2, and IL-6. However, depletion of CD19+ cells from PBMC cultures completely abrogates AFM11-mediated activation marker expression, T cell proliferation, and cytokine release, and supports strict CD19+ target-dependent T cell activation. Conversely, OKT3, produced cytokine release and T cell proliferation in all cultures except purified T cells. AFM11-mediated target cell lysis is specific for and restricted to CD19+ target cells since CD19− bystanders were not lysed in assays with mixed target populations. Binding of AFM11 to CD3 also did not induce anergy since its incubation with T cells for several days did not inhibit their potent cytotoxicity. AFM11 demonstrated a robust dose-dependent inhibition of growth of Raji tumor xenografts in NOD/scid mice reconstituted with human PBMC. Moreover, a single dose of AFM11 resulted in tumor growth inhibition similar to that produced by 5 daily injections. Altogether, these data suggest that AFM11 is a highly efficacious novel drug candidate for the treatment of hematological malignancies with advantages in both safety profile and anticipated dosing regimen.
Citation Format: Eugene A. Zhukovsky, Uwe Reusch, Carmen Burkhardt, Stefan Knackmuss, Ivica Fucek, Markus Eser, Fionnuala McAleese, Kristina Ellwanger. Bispecific TandAbs: a safe and potent platform for T cell-mediated killing of CD19+ cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1243. doi:10.1158/1538-7445.AM2013-1243