Engineering better immunotherapies via RNA interference

Abstract

The therapeutic potential of dendritic cell (DC) cancer vaccines has gained momentum in recent years. However, clinical data indicate that antitumor immune responses generally fail to translate into measurable tumor regression. This has been ascribed to a variety of tolerance mechanisms, one of which is the expression of immunosuppressive factors by DCs and T cells. With respect to cancer immunotherapies, these factors antagonise the ability to induce robust and sustained immunity required for tumor cell eradication. Gene silencing of immunosuppressive factors in either DCs or adoptive transferred T cells enhanced anti-tumor immune responses and significantly inhibited tumor growth. Therefore, engineered next generation of DC vaccines or adoptive T-cell therapy should include immunomodulatory siRNAs to release the "brakes" imposed by the immune system. Moreover, the combination of gene silencing, antigen targeting to DCs and cytoplasmic cargo delivery will improve clinical benefits.

Keywords: AML, acute myeloid leukemia; CMV, human cytomegalovirus; CTLA4, T-lymphocyte-associated antigen 4; DC, Dendritic cells; Gal, galectin hTERT, human telomerase reverse transcriptase; IDO, indoleamine 2,3-dioxygenase; IL, interleukin; INF, interferon; NK, natural killer; PD1, programmed cell death; RNA interference; RNAi, RNA interference; SOCS1, suppressor of cytokine signaling; STAT, Signal transducer and activator of transcription; T-cell therapy; TCR, T cell receptor; TLR, toll like receptor; Treg, Regulatory T; cancer vaccine; gene silencing; immunotherapy; siRNA, small interfering RNA; targeted therapies.

Figure 1.

Various co-stimulatory and co-inhibitory molecules regulate T cell activation. Indicated are multiple molecules that are involved in the regulation of T-cell responses under physiological conditions. One important family of membrane-bound molecules that bind co-stimulatory and co-inhibitory receptors is the B7 family (e.g., CD86, DC80). Although B7-CD28-specific signaling is a critical component of T cell priming, signaling through others receptors, including OX40 and ICOS is often required to further enhance CD4 and CD8 T cell priming and generation of memory cells. Strong signaling through the TCR and CD28 upregulates both co-inhibitory (e,g. CTLA4 and PD1) and co-stimulatory molecules (e.g. OX40, ICOS) on T cells. Inhibition via CTLA4 and PD1 in the context TCR signaling is likely of central importance in controlling immunity (see main text).

Figure 2.

Conversion of IDO negative DCs to IDO positive DCs. Subsequent to T-cell activation, IFN-γ produced by T cells induces the expression of IDO in DCs resulting in their conversion into tolerogenic DCs. This counter-regulatory mechanism is expected to control the magnitude and duration of adaptive immune responses. Activation of naïve T cells by IDO positive DCs leads to the generation of adaptive Treg cells, a population of CD4+ T cells that inhibit, rather than promotes, immune responses. Moreover, IDO positive DCs convert tryptophan into several metabolites with general immunosuppressive activity on lymphocytes.

Figure 3.

Timeline for the treatment and clinical development. The figure illustrates the treatment schedule for a patient with metastatic ovarian cancer. After surgery, the patient has received 4 combinations of chemotherapy prior to IDO-silenced DC vaccine. Chemo 1: Carboplatin, Taxol, Avastin; Chemo 2: Carboplatin, Caleyx; Chemo 3: Taxol; Chemo 4: Taxol, Avastin; SD, stable disease; PD, progressive disease; PR, partial remission.

Figure 4.

Simultaneous suppression of inhibitory signals enhanced T-cell proliferation. (A) Untransfected DCs or DCs transfected with IDO, PD-L1, or the combination IDO/PD-L1 siRNA for 24 h were co-cultured with allogeneic CD4+ T cells for 6 d at DC:T cell ratio 1:10. T cell proliferation was measured by thymidine incorporation as described previously. (B) As in A, except that allogeneic CD4+ T cells were transfected with PD1 siRNA. The results are represented as means of triplicate samples from the same experiment.

Figure 5.

Galectins 1 and 2 control T cell receptor (TCR) activation threshold. (A) Under steady-state conditions, DCs maintain an immature state (iDCs). Upon activation through inflammatory cytokines or pathogen-derived products, they mature (mDCs) and upregulate the expression of co-stimulatory molecules, such as CD80, CD86, and CD40. The lower expression of costimulatory molecules has been proposed to account for poor capacity of iDCs to stimulate T cells. (B) Gal1 and Gal3 gene silencing in iDCs enhanced T cell activation, despite low expression of costimulatory molecules. Also, gene silencing in mDCs enhanced T-cell activation. This enhancement is more likely due to a drop in TCR activation threshold. Endogenous or secreted Gal1 and Gal3 might interact with immunological synapses indicated by the circles. MHC = major histocompatibility complex.

Figure 6.

Dual siRNA can function as TLR activator and gene silencer. A dual siRNA can activate innate immunity via either endosomal TLRs or cytoplasmic RIG1 leading to cytokine production and eventually DC maturation. In addition, it is able to silence the expression of immunosuppressive factors (e.g., IL10, IDO, and SOCS1). Combination of gene silencing and induction of DC-maturation should enhance anti-tumor or anti-viral immunity.