Recent studies in mice, nonhuman primates, and clinical trials in human patients have emphasized the importance of the persistence of the vaccine-induced immune response, immunological memory, in mediating protective immunity against infectious diseases and cancer. Multiple extrinsic pathways control the differentiation of CD8+ cytotoxic T lymphocytes (CTL) into effector or memory cells by regulating the balanced expression of intracellular mediators in the antigen-activated T cells. Notably, inhibition of mediators of effector differentiation like mTOR, GSK3β or T-bet using genetic means or whenever available pharmacological agents, not only prevented the accumulation of the short-lived effectors but also redirected the activated T cells to differentiate along the memory pathway. For example, pharmacological inhibition of mTOR or GSK3β in mice with rapamycin or TSW119, respectively, led to the differentiation of antigen activated CD8+ T cells into long lasting memory cells that exhibited enhanced antiviral and antitumor immunity. Nevertheless, pharmacological agents often exhibit undesirable effects reflecting the broad distribution of their targets. For example, rapamycin inhibition of mTOR promotes the development of immune suppressive regulatory CD4+ T cells and GSK3β; inhibition polarizes dendritic cells (DC) to a tolerogenic state, arguably counterproductive in the setting of vaccination. In addition, development of pharmacological agents to modulate the function of intracellular targets that are not accessible to antibodies (“undruggable targets”) is highly challenging, and their availability especially for clinical use is limited.
Here we describe a versatile, broadly applicable, and clinically feasible approach to promote the generation of memory T cell responses that addresses the main limitations of pharmacological agents. RNAi was used to downregulate intracellular mediators of effector differentiation that were targeted to CD8+ T cells by conjugation to aptamer ligands. We have shown that a CD8+ T cell targeted 4-1BB aptamer-raptor siRNA conjugate administered to mice by tail vein injection downregulated mTORC1, while preserving mTORC2, activity in at least 60% of adoptively transferred OVA-specific transgenic OT-I cells while sparing host cells. Both rapamycin and aptamer-siRNA conjugate led to the development of an enhanced memory response in mice as judged by the ability of the memory cells to proliferate in response to re-exposure to antigen at a latter time point. However, the rapamycin generated CTL were defective in their cytotoxic effector functions and failed to elicit protective immunity against a tumor challenge. In contrast, the aptamer-raptor siRNA generated CTL exhibited normal cytotoxic effector functions and enhanced vaccine-induce protective antitumor immunity in both prophylactic and therapeutic tumor models. Reflecting the lack of cell specificity of mTOR inhibition by rapamycin, and providing a potential mechanisms underlying the effector defect of rapamycin generated CTL, dendritic cells from mice treated with rapamycin, but not with aptamer-raptor siRNA conjugate, exhibited reduced MLR activity, consistent with the known suppressive effects of rapamycin induced mTOR inhibition on DC.
These studies demonstrate the feasibility and efficiency of aptamer targeted delivery of siRNAs to immune cells, and underscore the potential advantages of aptamer-targeted siRNA delivery over nontargeted administration of pharmacological agents.
Citation Format: Alexey Berezhnoy, Iris Castro, Agata Levay, Thomas Malek, Eli Gilboa. Aptamer-targeted siRNA-mediated inhibition of mTORC1 in antigen-primed CD8+ cytotoxic T lymphocytes enhances immunological memory and antitumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B68.