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. Inhibition of mediators of effector differentiation like mTOR, Blimp-1, CD25, glycolysis, or GSK3β, 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 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 Treg, and GSK3β inhibition polarizes dendritic cells 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.
We are developing 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 is used to downregulate intracellular mediators of effector differentiation, that are targeted to CD8+ T cells by conjugation to an oligonucleotide aptamer ligand. 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. Whereas 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, the rapamycin generated CTL were defective in their cytotoxic effector functions and failed to elicit protective immunity against a tumor challenge. Underscoring the lack of cell specificity of rapamycin action, and providing a potential mechanisms underlying the effector defect of rapamycin generated memory CD8+ T cells, dendritic cells from mice treated with rapamycin, but not with aptamer-raptor siRNA conjugate, exhibited reduced alloMLR activity, consistent with the known suppressive effects of rapamycin induced mTOR inhibition on DC.
Given that inhibition of mTORC1 may not be the optimal way of enhancing memory, we have used the aptamer targeting platform to inhibit other mediators of effector differentiation in activated CD8+ T cells. In preliminary studies 4-1BB aptamer-targeted inhibition of Axin-1, to promote wnt signaling, or reducing IL-2 signaling by downregulating CD25 expression, enhanced vaccine-induced protective antitumor immunity in tumor-bearing mice. Using the OT-I adoptive transfer model, in preliminary experiments we have shown that 4-1BB aptamer targeted co-inhibition of both raptor and PD-1 increased the formation of memory OT-I cells; while not superior to that of inhibiting raptor alone, the OT-I cells expressed significantly less PD-1 on cell surface, and were, therefore, resistant to PD-1-mediated immune suppression.
Overall, 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.
This abstract is also presented as Poster A23.
Citation Format: Alexey Berezhnoy, Rajagopalan Anugraha, Thomas Malek, Eli Gilboa. Potentiating immunological memory in mice using aptamer targeted siRNA delivery to inhibit mediators of effector differentiation in CD8+ cytotoxic T lymphocytes. [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 PR11.
- ©2015 American Association for Cancer Research.