Aptamer-targeted inhibition of mTOR in T cells enhances

antitumor immunityAlexey Berezhnoy, Iris Castro, Agata Levay, Thomas R. Malek, and Eli

Gilboa

J Clin Invest. 2014;124(1):188–197

Background (siRNA)• siRNA (small interfering RNA) - ability to inhibit

target genes

• Aptamer - oligonucleic acid molecules that bind to a target molecule.

• Aptamer-siRNA conjugate - delivery vehicle composed of a targeting part (aptamer) and a therapeutic part (siRNA).

Background (T Cells)

•Memory T cells – T cells that have encountered and responded to their specific antigen.

• They can recognize invaders and at a 2nd encounter can reproduce to mount a faster and stronger immune response than the 1st time.

Background• Elevated levels of mTOR promote the accumulation of short-lived

effectors rather than memory cells.

• Pharmacological agents can have undesirable effects due to their broad range

Aim of the study

Aptamer-targeted siRNA inhibition of mTOR in CD8+ T

cells enhances a vaccine-induced memory and an

antitumor immunity that is superior to pharmacological

treatment.

Methods• siRNA specific to raptor (a component of mTORC1) • Aptamer that binds to 4-1BB, a molecule that is expressed on CD8+ T

cells

Test if siRNA inhibit

s mTOR

C1 activity

.

Test if mTOR

inhibition causes formation of

memory CD8+ T

cell

Test if siRNA

can enhance vaccine-induced memory respons

es

Methods• Goal: 4-1BB-raptor is capable of inhibiting mTOR activity

• OT-I cells transferred into mice, and mice vaccinated with OVA peptide and then treated with:• rapamycin • 4-1BB aptamer-GFP siRNA (4-1BB-GFP) • 4-1BB aptamer-raptor siRNA (4-1BB-raptor)

•mTORC activity measured

Results

• The 4-1BB-raptor conjugate inhibited the activity of mTORC1, but not mTORC2,

• Rapamycin inhibited both mTOR complexes.

• The target specificity of raptor siRNA inhibition is shown

Methods• Goal: mTOR inhibition leads to the generation of enhanced memory

CD8+ T cell responses

• OT-I cells transferred into mice, mice vaccinated with OVA peptide and then treated with:• rapamycin • 4-1BB aptamer-GFP siRNA (4-1BB-GFP) • 4-1BB aptamer-raptor siRNA (4-1BB-raptor)

• Cells were analyzed by flow cytometry.

• Cells treated with rapamycin or 4-1BB-raptor have greater CD62Lhi CD44hi or CD62LhiCD127hi phenotype.

• Phenotypes show an enhanced potential to develop into memory cells.

Methods• Goal: 4-1BB-raptor can enhance vaccine-induced memory responses

• Mice were vaccinated with B16 melanoma cells (GVAX) and then treated with: • Rapamycin• 4-1BB aptamer-GFP siRNA conjugate (4-1BB-GFP)• 4-1BB aptamer-raptor siRNA conjugate (4-1BB-raptor)

• Then, on day 50, the mice were challenged with parental B16 tumor cells and survival was determined

Results• Treatment with 4-1BB-raptor

enhanced GVAX-induced antitumor immunity.

• Rapamycin is less effective at promoting memory responses and has less antitumor immunity

Author’s Interpretation• siRNA inhibition of mTORC1 enhances vaccine-induced immunity.

• Aptamer-targeted siRNA superior to rapamycin in terms of protective antitumor immunity in mice.

• Rapamycin inhibits mTORC1 and mTORC2. Since mTORC2 is in cell survival, and glucose homeostasis, inhibition could be harmful

• Cell targeting reduces dose of siRNA needed, reducing the risk of nonspecific immune activation

Critique• Side-effects of inhibition of mTOR on other cellular processes was not

examined

• Enhancement of memory T-cells not directly measured

• Mechanism of the immune response in the enhancement of antitumor immunity still needs to be investigated

• 4-11B expression is upregulated on cells other than CD8+ so more specific targeting receptors will improve the specificity

• Currently, only in mouse model, no human studies conducted so therapeutic applications not completely confirmed

Questions?