Self-assembled cGAMP-STINGΔTM signaling complex as a bioinspired platform for cGAMP delivery

Abstract

The stimulator of interferon (IFN) genes (STING) pathway constitutes a highly important part of immune responses against various cancers and infections. Consequently, administration of STING agonists such as cyclic GMP-AMP (cGAMP) has been identified as a promising approach to target these diseases. In cancer cells, STING signaling is frequently impaired by epigenetic silencing of STING; hence, conventional delivery of only its agonist cGAMP may be insufficient to trigger STING signaling. In this work, while expression of STING lacking the transmembrane (TM) domain is known to be unresponsive to STING agonists and is dominant negative when coexpressed with the full-length STING inside cells, we observed that the recombinant TM-deficient STING protein complexed with cGAMP could effectively trigger STING signaling when delivered in vitro and in vivo, including in STING-deficient cell lines. Thus, this bioinspired method using TM-deficient STING may present a universally applicable platform for cGAMP delivery.

Fig. 1. Overview of state-of-the-art approaches of cGAMP delivery and schematics of recombinant STINGΔTM structure and therapeutic strategy.

(A) State-of-the-art approaches through directly encapsulating cGAMP into liposomes or polymersomes for cell transfection. (B) Current strategy of delivering cGAMP with a recombinant, transmembrane-deficient STING as carrier in the form of a ribonucleoprotein complex.

Fig. 2. cGAMP binding induces near-complete self-assembly of cGAMP-STINGΔTM tetramer.

FPLC analyses of (A) mouse STINGΔTM and (B) R237A/Y239A mutant in PBS, titrated with various molar ratios of cGAMP and schematics of cGAMP-STINGΔTM tetramer self-assembly with (C) mouse STINGΔTM and (D) R237A/Y239A mutant, which is not capable of binding cGAMP.

Fig. 3. cGAMP-STINGΔTM tetramer effectively triggers IFN expression in vitro, including in STING-deficient and STING-defective cell lines.

(A) Immunoblotting of endogenous expression of STING, TBK1, and IRF3 in HEK293T and RAW264.7 cell line. (B) RAW264.7 cells (n = 3) and (C) HEK293T cells (n = 4) treated with different combinations/mutations of cGAMP-STINGΔTM tetramer (10 μg of STINGΔTM with 0.25 μg of cGAMP per milliliter). Luciferase and single enzyme activity–based protein profiling (SEAP) activity were determined 24 hours after treatment. (D) Immunoblotting of HEK293T cells transiently transfected with plasmid DNA overexpressing full-length human STING (WT, HAQ, S366A, and L374A) and hSTINGΔTM. (E) Transfected HEK293T cells (n = 4) in (D) treated with cGAMP-STINGΔTM tetramer (plus R238A/Y240A mutant), cGAMP only, and 10 μg of STINGΔTM with 0.25 μg of cGAMP per milliliter. Luciferase activity were determined 24 hours after treatment. (F) Confocal micrograph of HEK293T cells (upper) transfected with plasmid DNA encoding for STINGΔTM expression and then stimulated with cGAMP and (lower) with cGAMP-STINGΔTM tetramer delivered as ribonucleoprotein complex. (G) HEK293T cells (n = 4) pretreated with TBK1 inhibitor MRT67307 (MRT) and then treated with different combinations/mutations of cGAMP-STINGΔTM tetramer. (H) Confocal micrograph of HEK293T cells treated with cGAMP-STINGΔTM tetramer showing colocalization of STINGΔTM and TBK1. (I) HEK293T cells (n = 4) pretreated with BFA, which blocks ER-Golgi trafficking and then treated with different combinations/mutations of cGAMP-STINGΔTM tetramer. (J) Confocal micrograph of HEK293T cells (n = 4) treated with cGAMP-STINGΔTM tetramer showing no colocalization of STINGΔTM with Golgi apparatus, in the presence or absence of BFA. Values are reported as means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, as analyzed by one-way analysis of variance (ANOVA). Scale bars, 50 μm. ns, not significant.

Fig. 4. cGAMP-STINGΔTM tetramer activates dendritic cells and promotes potent humoral response.

(A) Groups of C57BL/6 mice (n = 4) were tail base injected with 40 μg of STINGΔTM, with or without 1 μg of cGAMP, or 1 μg of cGAMP alone on day 0, and then on day 1.5, draining (inguinal) lymph node lymphocytes were collected for analysis by flow cytometry. (B) Dendritic cell activation in draining (inguinal lymph node) gated by % MHC-II⁺ cells in CD11c⁺ cells. (C) C57BL/6 mice (n = 4) were immunized with 10 μg of ovalbumin (OVA) alone or OVA mixed with 2.5 μg of cGAMP or 100 μg of STINGΔTM or both via tail base injection on days 0 and 7. On days 14 (D), 28 (E), and 42 (F), OVA-specific total immunoglobulin G (IgG) antibody level in mouse serum was measured via enzyme-linked immunosorbent assay (ELISA). In (E) and (F), five mice were lost because of accidental cage flooding. Values are reported as means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, as analyzed by one-way ANOVA.

Fig. 5. cGAMP-STINGΔTM tetramer promotes robust antigen-specific T cell responses.

(A) Groups of C57BL/6 mice (n = 7) were immunized with 50 μg of OVA alone or OVA mixed with 1 μg of cGAMP or 40 μg of STINGΔTM (or 40 μg of S365A STINGΔTM) on days 0 and 7. On day 14, PBMCs were collected and CD8⁺ T cells were analyzed by CD8 OVA epitope SIINFEKL tetramer staining (B) or stimulated ex vivo with CD8 OVA epitope SIINFEKL and analyzed by intracellular cytokine staining of IFN-γ (C) and TNF-α (D). (E) Groups of C57BL/6 mice (n = 5) were immunized with 50 μg of OVA alone or OVA mixed with 1 μg of cGAMP or 40 μg of STINGΔTM (or 40 μg of S365A STINGΔTM) on days 0 and 14. On day 21, PBMCs and lymphocytes in dLN and splenocytes were collected and CD8⁺ T cells were analyzed by CD8 OVA epitope SIINFEKL tetramer staining. Among CD8⁺ SIINFEKL tetramer⁺ T cells, effector memory precursors T_EMP were gated by CD27⁺ CD62L⁻ and KLRG1⁻ [(F) in dLN lymphocytes, (H) in PBMCs, and (I) in splenocytes], and central memory precursors T_CMP were gated by CD27⁺ CD62L⁺ and KLRG1⁻ [(G) in dLN lymphocytes and (J) in splenocytes, T_CMP was generally not found in PBMCs]. Values are reported as means ± SEM. ***P < 0.001, **P < 0.01, and *P < 0.05, as analyzed by one-way ANOVA.

Fig. 6. cGAMP-STINGΔTM tetramer promotes potent antitumor immunity in B16 melanoma model.

(A) Groups of C57BL/6 (n = 7) mice were immunized with 50 μg of OVA alone or OVA mixed with 1 μg of cGAMP or 40 μg of STINGΔTM (or 40 μg of S365ASTINGΔTM) on days 0 and 7. On day 21, mice were challenged with 1 million B16-OVA cells subcutaneously. Plots of overall (B) and individual (D) tumor growth curves, with numbers of surviving mice at the end of study (day 100) denoted. (C) Survival curves of mice. (E) Groups of C57BL/6 (n = 7) mice were first inoculated with 1 million MC38 cells and then treated with 100 μg of STINGΔTM (or 100 μg of S365A, R237A/Y239A STINGΔTM) mixed with 2.5 μg of cGAMP starting on day 7 for five times, 7 days apart via intratumoral injection. Plots of (F) overall and (H) individual tumor growth curves, with numbers of surviving mice at the end of study (day 60) denoted. (G) Survival curves of mice.