Bispecific antibodies and their derivatives can induce potent cytolytic activity through activation of T cells which target tumor cells. Cell linking moieties (CLM) represent engineered, bispecific antibodies consisting of two scFv. Previous studies have shown that the systemic distribution and pharmacokinetic profile of these agents has limited their utility for many target and effector combinations. We hypothesize that a genetically-modified, allogeneic cellular product could be developed to express such molecules leading to the inducible release at the tumor site following systemic delivery and thereby providing spatial and temporal control of the CLM distribution to increase the number of target-effector combinations. The RheoSwitch Therapeutic System® (RTS®) is a gene switch activated by a small molecule ligand that has been demonstrated clinically to control the timing and extent of gene expression. The RTS® platform has three essential elements: (1) switch components (co-activation partner and a ligand-controllable transcription factor); (2) activator ligand (veledimex); and (3) inducible promoter. Sans ligand, the switch complex is inactive and inhibits gene expression. By adding ligand, the switch components form an active complex, prompting gene expression in a dose-dependent manner. Endometrial regenerative cells (ERC) are a mesenchymal-like stem cell with a high replicative potential. We demonstrate that genetically modified ERC (GM-ERC) can be generated with high efficiency via nucleofection, lipofection or transduction with adenovirus, adeno-associated virus (AAV) or lentivirus. GM-ERC expressing a CLM construct comprised of anti-CD3-anti-EGFR bispecific antibody (CLM) under constitutive expression secretes up to 300 ng/ml of CLM as evaluated by ELISA. Addition of GM-ERC cells, supernatants from GM-ERC, or purified CLM to co-cultures of unstimulated peripheral blood mononuclear cells and EGFR+ A549 lung carcinoma cells led to T cell activation as measured by cytokine secretion (IL-2 and IFN-γ) and upregulation of CD69, CD25, and EGFR-specific cell-mediated cytotoxicity of A549 cells in vitro. CLM-expressing ERC were effective in coculture killing assays at doses as low as 1% of A549 target cells. Expression of CLM under the inducible promoter (RTS-CLM) yielded effective control of CLM secretion and killing activity, with greater than 80% cytotoxicity against A549 at 48-72 hours of cocultures with ERC-RTS-CLM, A549 cells and T cells when in the presence of veledimex (ligand), but not vehicle control. The data demonstrate that GM-ERC can secrete a bispecific antibody that induces T cell mediated killing in vitro, and that this activity can be controlled using an RTS® expression vector in combination with an activator ligand. This provides evidence supporting the feasibility of the cytolytic activity of CLM-secreting GM-ERC as an allogeneic cell therapy product, a novel and promising approach for the therapy of lung carcinoma and other EGFR+ malignancies.
Citation Format: Amy Wesa, Yumei Xiong, Tim Chan, Jeff Rosenbloom, Srinivas Rengarajan, Paul Szymanski, John A. Barrett, Francois Lebel, Farzad Haerizadeh, Richard Einstein. Controlled production of a bispecific antibody by a genetically modified stem cell triggers T cell activation and cytolysis in non-small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B25.
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