Abstract 4747: Novel T-cell redirecting trivalent bispecific antibodies.
Authors: Rossi Diane L. , Rossi Edmund A. , Cardillo Thomas M. , Goldenberg David M. , Chang Chien-Hsing 
Published: August 2014

Abstract
Background: Various formats of bispecific antibodies (bsAbs) to redirect effector T cells for killing of targeted tumor cells have shown considerable promise both pre-clinically and clinically. Notable examples are BiTE and DART, which comprise scFv-based anti-CD3 and anti-tumor antibodies. Herein, we describe, for the first time, the generation of a panel of novel T-cell redirecting trivalent bsAbs, each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of tumor-targeting Fab by the DOCK-AND-LOCKTM (DNLTM) method, as potential therapeutics for hematological or solid malignancies. We provide a detailed characterization of a CD19-specific prototype designated (19)-3s. DNL is a modular platform that combines recombinant engineering with site-specific chemical conjugation to generate bsAbs whose conceivable advantages include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance, and potent T-cell mediated cytotoxicity. Methods: Seven DNL complexes, designated (19)-3s, (14)-3s, (15)-3s, (E1)-3s, (1R)-3s, (C2)-3s, and (M1)-3s, were prepared by linking anti-CD3 scFv to a stabilized dimer of humanized Fab with specificity for CD19, CEACAM5, CEACAM6, Trop-2, IGF-1R, HLA-DR, and PAM4-antigen, respectively. Each complex was affinity purified and showed more than 85% of the desired product in a single peak by size-exclusion HPLC. Extensive in vitro characterizations were performed on (19)-3s using a variety of cell-based assays and CD19-expressing human non-Hodgkin lymphoma cell lines (Daudi, Raji and Ramos). The in vivo antitumor activity of (19)-3s was evaluated in NOD/SCID mice injected SC with a mixture of Raji cells and human peripheral blood mononuclear cells (hPBMCs). Results: (19)-3s at 1 μg/mL induced synapse formation between freshly isolated human T cells and Daudi cells, resulting in 45.5% CD20/CD7 dual-positive events with &gt;90% of Daudi cells associated with T cells, compared with 2% for untreated cells. At an E:T ratio of 12.5:1, (19)-3s-mediated T-cell killing of Daudi cells at &lt;1 pM, with maximal activity at 10 pM. Similar results were seen with both Ramos and Raji cells. In the presence of Daudi cells, (19)-3s specifically induced the activation of T cells, as evidenced by a substantial (&gt;50-fold) upregulation of CD69 expression and T cell proliferation. The (19)-3s was highly effective in vivo. In the Raji/hPBMC model of NOD/SCID mice, three of the 5 animals treated every other day with 43 μg of (19)-3s for three doses remained alive after 84 days and had significantly (P = 0.0018) improved median survival time (&gt; 84 days) compared to the untreated control group (28 days). Conclusions: (19)-3s effectively induce T-cell-mediated killing of CD19-expressing cells in vitro and in vivo. We are currently evaluating the in vitro and in vivo properties of other DNL complexes in cognate solid tumors.
Citation Format: Diane L. Rossi, Edmund A. Rossi, Thomas M. Cardillo, David M. Goldenberg, Chien-Hsing Chang. Novel T-cell redirecting trivalent bispecific antibodies. [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 4747. doi:10.1158/1538-7445.AM2013-4747