Endosomolytic polymersomes increase the activity of cyclic dinucleotide STING agonists to enhance cancer immunotherapy

Abstract

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) are a promising class of immunotherapeutics that activate innate immunity to increase tumour immunogenicity. However, the efficacy of CDNs is limited by drug delivery barriers, including poor cellular targeting, rapid clearance and inefficient transport to the cytosol where STING is localized. Here, we describe STING-activating nanoparticles (STING-NPs)-rationally designed polymersomes for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). STING-NPs increase the biological potency of cGAMP, enhance STING signalling in the tumour microenvironment and sentinel lymph node, and convert immunosuppressive tumours to immunogenic, tumoricidal microenvironments. This leads to enhanced therapeutic efficacy of cGAMP, inhibition of tumour growth, increased rates of long-term survival, improved response to immune checkpoint blockade and induction of immunological memory that protects against tumour rechallenge. We validate STING-NPs in freshly isolated human melanoma tissue, highlighting their potential to improve clinical outcomes of immunotherapy.

Figure 1 |. Design, optimization, and characterization of STING-NPs.

a) Schematic of STING-NP structure and mechanism of enhanced intracellular delivery of 2’3’-cGAMP. cGAMP is encapsulated in endosomolytic polymersomes assembled from pH-responsive diblock copolymers. After polymersome self-assembly and cGAMP loading, polymer chains are crosslinked in situ via partial reduction of PDS groups with DTT resulting in formulation of disulfide crosslinks. 2PT: 2-pyridinethione. b) STING-NPs enhance intracellular uptake of cGAMP and in response to decreased pH within endosomal compartments disassemble and promote endosomal escape of cGAMP to the cytosol. Representative conventional (c) and cryo (d) transmission electron micrographs of polymersomes assembled using PEG_2kDa-DBP_4.5kDa polymers. Cryo-EM was performed once, while conventional EM was repeated independently twice with similar results. e) Zeta potential distribution of polymersomes at pH 7.4. Repeated twice independently with similar results. f) Dynamic light scattering analysis of number average particle size distribution of STING-NPs at extracellular and endosomal pH. Repeated twice independently with similar results. g) Gel permeation chromatograms of PEG_2kDa-DBP_4.5kDa copolymers before and after in situ crosslinking of polymersomes. Repeated twice independently with similar results. h) Effect of the degree of crosslinking, represented by equivalents of DTT to PDSMA, on pH-dependent membrane destabilizing activity as measured using an erythrocyte haemolysis assay (n=4 biologically independent samples, two-tailed Student’s t-test). i) Effect of second block molecular weight (MW) in PEG_2kDa-DB_MW copolymers on pH-dependent haemolysis (n=4 biologically independent samples). j) Dose-response curves of type-I IFN (IFN-I) response elicited by indicated cGAMP-containing formulations in THP-1, RAW264.7, and B16 interferon stimulated genes (ISG) cells with an IFN regulatory factor (IRF)-inducible reporter construct. STING-NP(NC): non-crosslinked; PEG-DB: cGAMP delivered with polymersomes assembled using non-crosslinkable PEG_2kDa-DB_5kDa chains; Mix: physical mixture of empty crosslinked PEG_2kDa-b-DBP_4.5kDa polymersomes and free cGAMP (n=4 biologically independent samples). k) Flow cytometric quantification of uptake of cdGMP-Dy547 co-delivered with cGAMP in indicated formulation by THP-1 and RAW264.7 cells (n=3 biologically independent samples, one-way ANOVA with Tukey test). All statistical data are presented as mean ± SD.

Figure 2 |. STING-NPs enhance the delivery and immunostimulatory activity of cGAMP in the tumour microenvironment.

a) qPCR analysis of Ifnb1, Cxcl9, and Cxcl10 expression in B16.F10 tumours 4h after intratumoural administration of STING-NP, free cGAMP, or PBS as vehicle control (for STING-NP, cGAMP and PBS, respectively, n=11, 10, and 12 biologically independent samples, one-way ANOVA with Tukey test) at a dose corresponding to 10 μg cGAMP. b) Luminescence of SC B16.F10 tumours expressing an ISRE luciferase reporter following intratumoural treatment with cGAMP formulations (mean ± SEM; for STING-NP, cGAMP and PBS, n=5, 4, and 5 biologically independent samples, two-way ANOVA with Tukey test). The values p=0.007 and p=0.002 denotes significance between STING-NP and PBS and cGAMP, respectively, at t=72h; p=0.018: STING-NP vs. PBS at 96h; p=0.006: STING-NP vs. cGAMP at 96h. c) Summary of selected differentially expressed genes (p <0.05, one-way ANOVA) in response to treatment with STING-NPs or cGAMP (n=4 biologically independent samples). d) Ranked analysis of differential gene expression between STING-NP and cGAMP administration (n=4 biologically independent samples). e) Unsupervised hierarchical clustering of relative gene expression. f) Percentage of cdGMP-Dy547⁺ cells amongst cell populations in the TME following intratumoural administration of Dy547-cdGMP formulations (n=4 biologically independent samples, one-way ANOVA with Tukey test). g) Flow cytometric quantification of the median fluorescent intensity (MFI) of cdGMP-Dy547⁺ cells among indicated cell populations in the TME (n=4 biologically independent samples, two-way ANOVA with Sidak’s multiple comparison test). h) Ifnb1 expression following in vitro incubation with STING-NP, cGAMP, or PBS for 4h at doses equivalent to 150 nM cGAMP in RAW264.7 macrophages, DC2.4 dendritic cells, primary NK cells, and B16.F10 melanoma cells (n=3 biologically independent samples, one-way ANOVA with Tukey test). Significance levels are shown for comparisons between cells treated with STING-NP formulations. i) Fluorescence spectrophotometric quantification of cdGMP-dy547 accumulation in the sentinel LN 2h following intratumoural administration (for cGAMP and STING-NP, respectively, n=3,4 biologically independent samples, two-tailed Student’s t-test). j) Ifnb1 expression in the sentinel (inguinal) LN following 4h following intratumoural administration (n=3 biologically independent samples, one-way ANOVA with Tukey test). Unless otherwise noted, statistical data are presented as mean ± SD.

Figure 3 |. STING-NPs shift the immunocellular composition of the tumor microenvironment.

a) Flow cytometric quantification of the number of monocytic (CD11b⁺Ly6c⁺Ly6g⁻) and granulocytic (CD11b⁺Ly6c⁺Ly6g⁺SSC^hi) myeloid derived suppressor cells, activated neutrophils (CD11b⁺Ly6c⁺Ly6g⁺SSC^lo), macrophages (mϕ; CD11b⁺F4/80⁺), natural kill (NK) cells (NK1.1⁺), and dendritic cells (DC; CD11c⁺MHC-II⁺) 48h following intratumoural injection (for m-MDSC, g-MDSC, neutrophil, macrophage, NK cells, and DCs, respectively n=6, 6, 6, 11, 11, and 11 biologically independent samples, one-way ANOVA with Tukey test). b) Representative flow cytometry histogram (left) and quantification (right) of CD86 expression by dendritic cells in the inguinal lymph node (n=6 biologically independent samples, one-way ANOVA with Tukey test). c) Representative flow cytometry histogram (left) and quantification (right) of CD206 expression by intratumoral macrophages (n=6 biologically independent samples, Kruskal-Wallace test with Dunn’s multiple comparison test) d) Representative flow cytometry dot plot and e) quantification of tumour infiltrating CD4⁺ and CD8⁺ T cells 48h following intratumoural injection (n=11 biologically independent samples, one-way ANOVA with Tukey test). f) Ratio of CD8⁺ to CD4⁺ T cells in the TME (n=11 biologically independent samples, one-way ANOVA with Tukey test). g) Intracellular cytokine staining and flow cytometry was used to evaluate TNF-α and IFN-γ production by tumour infiltrating CD4⁺ and CD8⁺ T cells in response to PMA/ionomycin stimulation (n=11 biologically independent samples, two-way ANOVA with Tukey test). Statistical data are represented as mean ± SD. All experiments were repeated once independently with similar results.

Figure 4 |. STING-NPs enhance the immunotherapeutic efficacy of cGAMP and synergize with immune checkpoint blockade.

a) Intratumoural administration and tumour rechallenge scheme for mice with a single established B16.F10 tumour. Mice with 100 mm³ subcutaneous (SC) tumours were administered STING-NPs, free cGAMP, empty nanoparticles (NP), a physical mixture of empty NPs and cGAMP (Mix), or PBS intratumourally (I.T.). b) Photographs of tumours 8 days after treatment. Repeated twice independently with similar results. c) Spider plots of individual tumour growth curves with number of complete responses (CRs) denoted. Repeated twice independently with similar results. (d) Mean tumour volume from 3 independent experiments (for PBS, NP, cGAMP, Mix, and STING-NP, n=7, 8, 8, 13, and 9 biologically independent samples; Kruskal-Wallis test with Dunn’s multiple comparisons test). (e) Kaplan-Meier survival curves of mice treated with indicated formulation using 1500 mm³ tumour volume as endpoint criteria (for PBS, NP, cGAMP, Mix, and STING-NP, n=7, 8, 8, 13, and 9 biologically independent samples, two-tailed Mantel-Cox test). f) Mice demonstrating complete responses to STING-NP treatment were rechallenged with B16.F10 cells on the contralateral flank 65 days after inoculation without any further treatment. (For treatment naïve and rechallenged CRs, n=4 and 7 biologically independent samples. ****: p<0.0001, two-tailed Student t test). g) Kaplan-Meier survival curves for treatment naïve and STING-NP treated CRs (n=7, two-tailed Mantel-Cox test). h) Treatment scheme for mice with two concurrently established contralateral B16.F10 tumours. Mice were treated with cGAMP containing formulations intratumourally in one tumour and administered a combination of anti-PD1 and anti-CTLA-4 antibodies (ICB) intraperitoneally (I.P.) three times, 4 days apart. i) Representative images of tumours 8 days after initiation of STING-NP or PBS administration (injected into left flank tumour). Repeated independently once with similar results. j) Average tumour volume of injected primary and untreated contralateral tumour (for PBS, cGAMP, ICB, cGAMP+ICB, STING-NP, and STING-NP+ICB, n=11, 7, 6, 10, 10, and 12 biologically independent samples, two-tailed Mann-Whitney test); p=0.069, p=0.001 denotes significance levels of STING-NP and STING-NP + ICB treated mice, respectively, vs. those treated with ICB alone. Kruskal-Wallis test with Dunn’s multiple comparison test. k) Treatment scheme for mice treated intravenously (I.V.) with cGAMP formulations and I.P. with ICB 3 times, 4 days apart. l) Representative images of tumours 8 days after initiation of treatment. Repeated twice independently with similar results. m) Spider plots of individual tumour growth curves of intravenously treated mice, n) average tumour volume (two-tailed Mann-Whitney test, p=0.003 denotes significance of STING-NP relative to mix+ICB group), and o) Kaplan-Meier survival analysis (two-tailed Mantel-Cox test). m-o: for PBS, cGAMP, ICB, cGAMP+ICB, Mix+ICB, STING-NP, and STING-NP+ICB, n= 10, 10, 10, 10, 10, 8, and 10 biologically independent samples. All statistical data are represented as mean ± SEM.

Figure 5 |. STING-NPs enhance cGAMP activity in human metastatic melanoma.

Surgically resected melanoma metastases were divided into nine sections (3 per treatment, one-way ANOVA with Tukey test), randomized, and injected intratumourally with STING-NPs or cGAMP at 150 nM and cultured for 24 h. qPCR analysis of Ifnb1, Tnf and Cxcl10 gene expression in tissue freshly isolated from two different melanoma patients after indicated treatment. All statistical data are presented as mean ± SD.