Amphiphilic Polyelectrolyte Graft Copolymers Enhance the Activity of Cyclic Dinucleotide STING Agonists

Abstract

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) hold great therapeutic potential, but their activity is hindered by poor drug-like properties that restrict cytosolic bioavailability. Here, this challenge is addressed through the synthesis and evaluation of a novel series of PEGMA-co-DEAEMA-co-BMA copolymers with pH-responsive, membrane-destabilizing activity to enhance intracellular delivery of the CDN, cGAMP. Copolymers are synthesized with PEGMA of two different molecular weights (300 and 950 Da) and over a range of PEG mass fraction and polymer molecular weight, and relationships between copolymer structure, self-assembly, endosomal escape, and cGAMP activity are elucidated. A subset of polymers that self-assembled into 50-800 nm nanoparticles is identified, which can be loaded with cGAMP via a simple mixing strategy, resulting in significantly enhanced immunostimulatory activity. Increased cGAMP activity is found to be highly correlated with the capacity of carriers to enhance intracellular CDN uptake and to promote endosomal destabilization, findings that establish efficient cytosolic delivery as a criterion for CDN carriers. Additionally, it is demonstrated that a lead CDN carrier formulation can enhance STING activation in vivo in a model of intratumoral immunotherapy. Collectively, these investigations demonstrate the utility of PEGMA-co-DEAEMA-co-BMA copolymers as carriers for CDNs and potentially other cytosolically-acting drug cargo.

Keywords: endosomal escape; graft copolymers; polymer nanoparticles; self-assembling nanoparticles; stimulator of interferon genes (STING).

Figure 1.. Synthesis and evaluation of PEGMA-co-DEAMEA-co-BMA copolymers for cGAMP delivery.

(a) RAFT polymerization scheme used for synthesis of a series of PEGMA-co-DEAMEA-co-BMA of variable PEGMA molecular weight (MW_PEGMA), PEG weight fraction (wt%_PEG), and molecular weight of the DEAMEA and BMA component of the copolymer (MW_DB). (b) Schematic representation of approach used to formulate and evaluate the self-assembly properties of copolymers and to (c) screen the capacity of copolymers to enhancing the intracellular uptake and cytosolic delivery of cGAMP to activate the STING pathway. Image created with Biorender.com.

Figure 2.. Effect of PEGMA-co-DEAEMA-co-BMA properties on colloidal self-assembly properties.

(a) Number average diameter and polydispersity index (PDI) of PEGMA-co-DEAEMA-co-BMA polymers diluted in PBS as measured by dynamic light scattering analysis (n=3 independently-prepared samples). (b) Representative electron micrographs of copolymers of synthesized with 300 Da PEGMA of variable PEG weight percent (wt% PEG) and molecular weight of the endosomolytic DEAEMA and BMA component. Micrographs of the 950 Da PEGMA-containing library can be found in Figure S3 of the SI.

Figure 3.. Effect of PEGMA-co-DEAEMA-co-BMA properties on membrane-destabilizing and endosomal escape properties.

(a) Relative degree of erythrocyte hemolysis mediated by PEGMA-co-DEAEMA-co-BMA copolymers at different pH values (n=3-4). (b) Schematic illustrating Galectin 8 (Gal8) recruitment assay used to investigate endosomal escape of polymers. (c) Representative fluorescent images of cells expressing Gal8-YFP fusion protein upon treatment with polymers synthesized with 300 Da PEGMA with 15 kDa of DEAEMA-co-BMA (DB) with 25 wt% PEG (highest Gal8 recruitment), 30 wt% (medium), and 20 wt% (lowest), or PBS (NT). (d) Mean intensity of Gal8-YFP vesicles normalized to cell area for indicated polymers (n=6).

Figure 4.. Effect of PEGMA-co-DEAEMA-co-BMA structure on cGAMP delivery.

(a) Relative uptake (MFI: median fluorescence intensity) of a fluorescently-labeled CDN (cdGMP-Dy547) formulated with indicated polymer by THP-1 monocytes (n=1). (b) Dose-response curves for cGAMP formulated with indicated copolymer in THP-1 ISG cells with an IFN regulatory factor (IRF)-inducible reporter construct (n=4 for each dose). The horizontal line represents the half-maximal activity of free cGAMP at a concentration of 125 μg/mL; the dose response curve for free cGAMP is shown in Figure S6. (c) Summary of the cGAMP concentration at which the half-maximal maximal activity of cGAMP was achieved for each polymer formulation. Note that for carriers that did not induce a response between 0.1-500 ng/mL cGAMP that this value was set at 500 ng/mL.

Figure 5.. Heat map summarizing relationships between formulation properties, endosomal escape, and cGAMP activity.

Maximal level of cGAMP activity (MaxAct), the cGAMP concentration at half-maximal activity (CHMA), and the ratio of MaxAct and CMHA are representative of carrier activity, which tends to correlate with CDN uptake, particle size, hemolysis at pH 6.6, and level of Gal8 recruitment. Note that copolymers are identified using the following convention: the first number of the PEGMA molecular weight (3 for 300 Da and 9 for 950 Da)- the weight (w) percent PEGMA-the molecular weight in kilodaltons of the DEAEMA and BMA (DB) component (e.g., 9-w20-DB15k). All properties were normalized to the lowest and highest values on a 0.0-1.0 scale.

Figure 6.. In vivo evaluation of PEGMA-co-DEAEMA-co-BMA as cGAMP carriers.

(a) qPCR analysis of Ifnb1, Cxcl10, and Tnf expression in B16.F10 tumors 4 h after intratumoral administration of indicated copolymers formulated with cGAMP, free cGAMP or PBS as a vehicle control (n=7-10 biologically independent samples, one-way ANOVA with Tukey’s post-hoc test; *P<0.05, ** P<0.01). (b) Mice were treated intratumorally with cGAMP formulated with indicated carrier, free cGAMP, or vehicle control three times, three days apart. Mean tumor volume in response to intratumoral administration of free cGAMP or cGAMP formulated with indicated copolymer (n=6-7 biologically independent samples, one-way ANOVA with Tukey’s post-hoc test on days 15 and 18; *P<0.05). cGAMP dose in all studies was 10 μg per injection.