Targeted delivery of immune-stimulating bispecific RNA, inducing apoptosis and anti-tumor immunity in cancer cells

Abstract

Double-stranded RNAs (dsRNA)-based strategies appeared as promising therapies to induce an inflammation in the tumor microenvironment. However, currently described systems generally lack active targeting of tissues, and their clinical translation is thus limited to intratumoral injection. Herein, we developed an antibody-siRNA-5'triphosphate conjugate with multiple modes of action, combining cell surface EphA2-specific internalization, leading to a simultaneous gene silencing and activation of the receptor retinoic acid-inducible gene I (RIG-I). Recognition of cytosolic siRNA-5'triphosphate by RIG-I triggers the expression of interferons and pro-inflammatory cytokines, inducing an inflammation of the tumor environment and activating neighboring immune cells. In addition, these RIG-I-specific effects synergized with siRNA-mediated PLK1 silencing to promote cancer cell death by apoptosis. Altogether, such immune-stimulating antibody-RNA conjugate opens a novel modality to overcome some limitations encountered by dsRNA molecules currently in clinical trials.

Keywords: Cancer; Cell biology; Conjugate; Drug delivery system.

Graphical abstract

Figure 1

Schematic dual mode of action of antibody-siRNA-5′ppp conjugate If delivered in the cytosol, the siRNA-5′ppp can be recognized by RIG-I, leading to the activation of both IRF 3/7 (1) and NF-kB (2) signaling pathways, respectively inducing cancer-selective apoptosis, and activating anti-tumor immunity. Concomitantly, the same construct can be loaded in RISC, guiding it to a sequence-complementary mRNA, triggering its degradation (3). In our postulate, silencing PLK1 could lead to the apoptosis by cell-cycle arrest while enhancing MAVS-mediated production of interferon (4).

Figure 2

Synthesis scheme and representative analysis of antibody-siRNA conjugate by native SEC-MS (A) Synthesis of mAb-siRNA-5′ppp 1 and (B) deconvoluted spectra of mAb-siPLK1-5′ppp 1 by native SEC-MS, showing an average degree of conjugation of 1.3. D1 and D2 stand for one and two dsRNA conjugated to the antibody, respectively, with expected D1 being 160,467 Da and expected D2 being 173,769, the naked antibody was discarded during the SEC purification.

Figure 3

Antibody-siRNA conjugate first colocalize with lysosomes, then siRNA is released in the cytosol Immunofluorescence pictures obtained from a dual-labeled ARC after 1 h of incubation (A–D, upper line) and 4 h of incubation (E–H, bottom line). Cy5 (red) was conjugated to the antibody’s lysines, Cy3 (yellow) was placed at the 5′-end of the siRNA, Lysoview 488 (green, white for the merged pictures) was used to label endo/lysosomes. Pearson’s correlation coefficient graphs are provided in the supplemental information in Figure S5.

Figure 4

Simultaneous activation of RIG-I and silencing of PLK1 mediated by mAb-siPLK1-5′ppp 1 on A549-Dual siPLK1-5′ppp was delivered by either RNAiMAX or EphA2-antibody. (A) RIG-I-dependent activation of the IRF pathway (N = 6, triplicates). (B) RIG-I-dependent activation of the NF-κB pathway (N = 6, triplicates). Dual activity of the siPLK1-5′ppp delivered by either RNAiMAX or EphA2-antibody regarding RIG-I/PLK1 expression levels or cell-cycle arrest. (C) Western-blot on RIG-I, PLK1, and GAPDH, showing a simultaneous PLK1 knockdown and RIG-I activation, 72 h after incubation at 40 nM. (D) Proportion of cells in G2/M phase analyzed by FACS after 24 h of incubation at 40 nM (N = 2, triplicates). Data are presented as mean ± SD. Significancy was assessed by comparing treated cells with non-treated cells, ns = non-significant, ∗∗∗ = p < 0.0005, ∗∗∗∗ = p < 0.00005).

Figure 5

mAb-siPLK1-5′ppp induces A549-Dual cell death by apoptosis and activates PBMCs to promote anti-tumor inflammation A549-Dual confluence over time after treatments with transfected siRNAs (A) or ARCs (B for A549 cells alone, C for a mixture of A549/PBMC: 1/5). One picture of A549 cells was taken every 2 h for 4 days, PBMC were excluded by the software because of the difference of size between A549 and PBMC, N = 2 with two different donors of PBMCs—(D) Cytokine quantification after mAb-siRNA-5′ppp 1 treatment for 24 h at 100 nM to the A549/PBMC mixture. Data are presented as mean ± SD. Significancy was assessed by comparing the secreted cytokine obtained from mAb-siRNA-5′ppp 1 compared to the non-treated mixture of cells (ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005, ∗∗∗∗ = p < 0.00005), N = 1, triplicates.

Figure 6

mAb-siPLK1-5′ppp can induce immunogenic cell death CALR exposition after 48 h of incubation at 100 nM, indicating a possible release of damage-associated molecular patterns (N = 2, triplicates). Data are presented as mean ± SD. Significancy was assessed by comparing treated cells with non-treated cells (ns = non-significant, ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005).