Abstract B127: Pharmacodynamics and functionality of RheoSwitch® regulated immunomodulatory proteins, expressed from a multigenic embedded cellular bioreactor following intramuscular electroporation in mice.

Abstract

Abstract Over the past decade, immunotherapies have emerged as prominent means to fight cancer. It is currently well accepted that combining multiple immunomodulatory therapeutic modalities will likely have a deeper impact in promoting cancer remission than monotherapies. Toward the development of a tri-immuno-therapeutic approach, we evaluated the feasibility and expression potency of multigenic plasmid constructs that simultaneously expressed three immunomodulators - human IL-12, human IFNα, and a CTLA4 decoy - in single, dual or triple combinations. In all constructs, the three effectors were expressed under the control of Intrexon's RheoSwitch Therapeutic System® (RTS®) activated by an orally available small molecule activator ligand (AL), veledimex (also known as INXN-1001). Expression of the three genes of interest (GOI) was driven by distinct RTS® inducible promoters, which allow for conditional gene expression following oral treatment with veledimex. Seven plasmids were constructed; three encoding for each GOI alone, three containing a combination of two GOIs; and one plasmid expressing all three GOIs. Expression of each GOI from each plasmid was evaluated in vitro in HT1080 and HEK293T cells for expression and function, and in vivo following administration through a single IM injection and electroporation into mice pre-exposed to the AL. Three days post IM/EP and daily oral treatment with veledimex, animals were bled and sera were evaluated for the single or concomitant expression and function of the encoded GOIs. Transient transfection and in vivo electroporation of the single and multi-effector plasmids yielded increased levels of hIL-12, hIFNα, and the CTLA4 decoy, when combined with veledimex. In contrast, no expression was seen in cell culture supernatants or sera in the absence of the activator ligand. Importantly, all three effectors, expressed from single plasmids were functionally active in cell based assays - hIL-12 increased IFNγ secretion from NK92 cells, hIFNα enhanced STAT1 activation, and the CTLA4 decoy blocked CD80 binding to CTLA4. Taken together, these results show for the first time the feasibility of systemic expression of three distinct immune effectors from a single RTS® regulated multigenic construct in mice. The in vivo studies also highlight the potential use of an ECB to generate therapeutics for tumor-targeted delivery of single or multiple RTS® regulated cancer immunotherapies. Altogether, use of these novel regulated immunotherapeutic approaches could potentially be translated into an effective clinical regimen for a variety of cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B127. Citation Format: Pooja Agarwal, Stephen Schauer, Xiaohong Ma, Jacques Plummer, Tim Chan, Lindsay Williams, Michele Kaloss, John A. Barrett, Richard Einstein, Laurent M. Humeau, Thomas R. Reed. Pharmacodynamics and functionality of RheoSwitch® regulated immunomodulatory proteins, expressed from a multigenic embedded cellular bioreactor following intramuscular electroporation in mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B127.