Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity

Abstract

Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8 + T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8 + T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8 + T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8 + T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8 + T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.

Aptamer-Targeted Attenuation of IL-2 Signaling in CD8 + T Cells Enhances Antitumor Immunity

“…11 Conjugation of siRNA cargos to aptamers often compromises activity of conjugated siRNA. Here, we used an algorithm whereby siRNAs with reduced melting temperature (TM) are chosen for conjugation to aptamers, which was shown to significantly increase the success of identifying siRNAs that retain bioactivity upon conjugation to aptamer.…”

“…11 As shown in Figure S4A, targeted inhibition of Axin-1 was more effective than that of raptor, and inhibition of Axin-1 or CD25 exhibited a comparable antitumor effect ( Figure S4B). …”

Reducing Toxicity of Immune Therapy Using Aptamer-Targeted Drug Delivery

“…Systemic administration of the 4-1BB aptamer-RPTOR siRNA conjugates to mice via the tail vein downregulated mTORC1, but not mTORC2, activity in circulating 4-1BB-expressing CD8 þ T cells, led to the generation of potent memory CD8 þ T-cell responses, and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, although nontargeted administration of the general mTOR inhibitor rapamycin also enhanced antigen-activated CD8 þ T-cell memory, both mTORC1 and mTORC2 were downregulated in the CD8 þ T cells, the cytotoxic effector functions of the reactivated memory cells were reduced, alloreactivity of splenic DCs was diminished, and mice failed to reject a tumor challenge (21). Enhancing CD8 þ T-cell memory and protective immunity in vivo is predicated on the efficient delivery of siRNA to a sizable proportion of the circulating CD8 þ T cells.…”

“…Yet, unlike delivery to hepatocytes or tumors, delivery of siRNA to normal or malignant hematopoietic cells has been notoriously difficult and inefficient (22). Systemic administration of the 4-1BB aptamer-targeted RPTOR siRNA conjugates via the tail vein was remarkably efficient, leading to the downregulation of RPTOR RNA and mTORC1 activity in more than 60% of the circulating 4-1BB þ CD8 þ T cells (21). Preliminary studies suggest that siRNA knockdown of CD25, Axin-1, Smad3, or Smad4 in CD8 þ T cells is equally efficient (E. Gilboa, unpublished data).…”

“…The preclinical murine studies to promote immune memory discussed above and in ref. (21) suggest that aptamer-targeted siRNA delivery is uniquely capable of doing that.…”