Abstract
Abstract 2762
Background:
The use of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells is a very active area of antibody engineering. Various formats of such agents made recombinantly have shown considerable promise both pre-clinically and clinically. For example, one design termed Bispecific T-cell engager (BiTE) employs a single polypeptide containing 2 antigen-binding specificities (each contributed by a cognate VH and VL) linked in tandem via a flexible linker, and another design termed DART (Dual-Affinity Re-Targeting) utilizes a disulfide-stabilized diabody. Both BiTE and DART, however, exhibit fast blood clearance due to their small size (∼55 kDa). Herein, we describe, for the first time, the generation of a novel T-cell redirecting bsAb, (19)-3s, comprising an anti-CD3 scFv covalently conjugated to a stabilized anti-CD19 F(ab)2. The potential advantages of (19)-3s include bivalent binding to tumor cells, a larger size (∼130 kDa) to preclude rapid renal clearance, and potent T-cell mediated cytotoxicity.
Methods and Results:
The Dock-and-Lock (DNL) method was used to generate (19)-3s by combining a stabilized anti-CD19 F(ab)2 with an anti-CD3-scFv, resulting in a homogeneous covalent structure of the designed composition, as shown by SE-HPLC, ELISA, SDS-PAGE, and immunoblot analyses. Functionally, (19)-3s induced synapse formation between effector and target cells using freshly isolated human T cells mixed with Daudi Burkitt lymphoma cells. Using an E:T ratio of 2.5:1 and 1 μg/mL of (19)-3s, the cell mixture was stained with anti-CD20-APC (for Daudi) and anti-CD7-FITC (for T cells), and cobinding was measured by flow cytometry as the % of CD20+/CD7+ events. After treatment with (19)-3s, 45.5% of events were found to be CD20/CD7 dual-positive, indicating synapsed Daudi and T cells, compared with 2% measured for untreated cells. Gating of the Daudi cell population showed that >90% of Daudi cells were associated with T cells. To access the targeted T-cell killing of Daudi, isolated T cells and Daudi were mixed at an E:T ratio of 12.5:1 and treated with serial dilutions of (19)-3s. After 18-h incubation at 37°C, cytotoxicity was measured using a LDH-release assay. Potent (19)-3s-mediated T-cell killing of Daudi cells was observed at <1 pM, with maximal activity at 10 pM. Similar results were seen with both Ramos and Raji NHL cell lines. In vivo studies to determine Pk and efficacy are underway. Based on DNL constructs of similar design, we expect (19)-3s to have an elimination rate longer than that of MT103, a BiTE comprising scFvs derived from anti-CD19 and anti-CD3, thus perhaps avoiding continuous infusions with this new construct.
Conclusions:
(19)-3s can bind T cells and NHL cells simultaneously and induce T-cell-mediated killing at pM concentrations in an ex vivo setting. The modular nature of the DNL method will allow the rapid production of a large number of related conjugates for redirected T-cell killing of various malignancies, without the need for additional recombinant engineering and protein production. We are currently evaluating the in vivo activity of (19)-3s, as a prototype, to determine if this novel bsAb format offers additional advantages.
Disclosures:
Rossi: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Cardillo:Immunomedics, Inc: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership. Chang:Immunomedics, Inc.: Employment.
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