Programable Albumin-Hitchhiking Nanobodies Enhance the Delivery of STING Agonists to Potentiate Cancer Immunotherapy.

Stimulator of interferon genes (STING) is a promising target for potentiating antitumor immunity, but multiple pharmacological barriers limit the clinical utility, efficacy, and/or safety of STING agonists. Here we describe a modular platform for systemic administration of STING agonists based on nanobodies engineered for in situ hitchhiking of agonist cargo on serum albumin. Using site-selective bioconjugation chemistries to produce molecularly defined products, we found that covalent conjugation of a STING agonist to anti-albumin nanobodies improved pharmacokinetics and increased cargo accumulation in tumor tissue, stimulating innate immune programs that increased the infiltration of activated natural killer cells and T cells, which potently inhibited tumor growth in multiple mouse tumor models. We also demonstrated the programmability of the platform through the recombinant integration of a second nanobody domain that targeted programmed cell death ligand-1 (PD-L1), which further increased cargo delivery to tumor sites while also blocking immunosuppressive PD-1/PD-L1 interactions. This bivalent nanobody carrier for covalently conjugated STING agonists stimulated robust antigen-specific T cell responses and long-lasting immunological memory, conferred enhanced therapeutic efficacy, and was effective as a neoadjuvant treatment for improving responses to adoptive T cell transfer therapy. Albumin-hitchhiking nanobodies thus offer an enabling, multimodal, and programmable platform for systemic delivery of STING agonists with potential to augment responses to multiple immunotherapeutic modalities.

STING-Pathway Inhibiting Nanoparticles (SPINs) as a Platform for Treatment of Inflammatory Diseases.

Aberrant activation of the cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) pathway has been implicated in the development and progression of a myriad of inflammatory diseases including colitis, nonalcoholic steatohepatitis, amyotrophic lateral sclerosis (ALS), and age-related macular degeneration. Thus, STING pathway inhibitors could have therapeutic application in many of these inflammatory conditions. The cGAS inhibitor RU.521 and the STING inhibitor H-151 have shown promise as therapeutics in mouse models of colitis, ALS, and more. However, these agents require frequent high-dose intraperitoneal injections, which may limit translatability. Furthermore, long-term use of systemically administered cGAS/STING inhibitors may leave patients vulnerable to viral infections and cancer. Thus, localized or targeted inhibition of the cGAS/STING pathway may be an attractive, broadly applicable treatment for a variety of STING pathway-driven ailments. Here we describe STING-Pathway Inhibiting Nanoparticles (SPINS)-poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with RU.521 and H-151-as a platform for enhanced and sustained inhibition of cGAS/STING signaling. We demonstrate that SPINs are equally or more effective at inhibiting type-I interferon responses induced by cytosolic DNA than free H-151 or RU.521. Additionally, we describe a SPIN formulation in which PLGA is coemulsified with poly(benzoyloxypropyl methacrylamide) (P(HPMA-Bz)), which significantly improves drug loading and allows for tunable release of H-151 over a period of days to over a week by varying P(HPMA-Bz) content. Finally, we find that all SPIN formulations were as potent or more potent in inhibiting cGAS/STING signaling in primary murine macrophages, resulting in decreased expression of inflammatory M1-like macrophage markers. Therefore, our study provides an in vitro proof-of-concept for nanoparticle delivery of STING pathway inhibitors and positions SPINs as a potential platform for slowing or reversing the onset or progression of cGAS/STING-driven inflammatory conditions.

Covalent Polymer-RNA Conjugates for Potent Activation of the RIG-I Pathway.

RNA ligands of retinoic acid-inducible gene I (RIG-I) are a promising class of oligonucleotide therapeutics with broad potential as antiviral agents, vaccine adjuvants, and cancer immunotherapies. However, their translation has been limited by major drug delivery barriers, including poor cellular uptake, nuclease degradation, and an inability to access the cytosol where RIG-I is localized. Here this challenge is addressed by engineering nanoparticles that harness covalent conjugation of 5'-triphospate RNA (3pRNA) to endosome-destabilizing polymers. Compared to 3pRNA loaded into analogous nanoparticles via electrostatic interactions, it is found that covalent conjugation of 3pRNA improves loading efficiency, enhances immunostimulatory activity, protects against nuclease degradation, and improves serum stability. Additionally, it is found that 3pRNA could be conjugated via either a disulfide or thioether linkage, but that the latter is only permissible if conjugated distal to the 5'-triphosphate group. Finally, administration of 3pRNA-polymer conjugates to mice significantly increases type-I interferon levels relative to analogous carriers that use electrostatic 3pRNA loading. Collectively, these studies have yielded a next-generation polymeric carrier for in vivo delivery of 3pRNA, while also elucidating new chemical design principles for covalent conjugation of 3pRNA with potential to inform the further development of therapeutics and delivery technologies for pharmacological activation of RIG-I.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

Engineering endosomolytic nanocarriers of diverse morphologies using confined impingement jet mixing.

The clinical translation of many biomolecular therapeutics has been hindered by undesirable pharmacokinetic (PK) properties, inadequate membrane permeability, poor endosomal escape and cytosolic delivery, and/or susceptibility to degradation. Overcoming these challenges merits the development of nanoscale drug carriers (nanocarriers) to improve the delivery of therapeutic cargo. Herein, we implement a flash nanoprecipitation (FNP) approach to produce nanocarriers of diverse vesicular morphologies by using various molecular weight PEG-bl-DEAEMA-co-BMA (PEG-DB) polymers. We demonstrated that FNP can produce uniform (PDI < 0.1) particles after 5 impingements, and that by varying the copolymer hydrophilic mass fraction, FNP enables access to a diverse variety of nanoarchitectures including micelles, unilamellar vesicles (polymersomes), and multi-compartment vesicles (MCVs). We synthesized a library of 2 kDa PEG block copolymers, with DEAEMA-co-BMA second block molecular weights of 3, 6, 12, 15, 20, and 30 kDa. All formulations were both pH responsive, endosomolytic, and capable of loading and cytosolically delivering small negatively charged molecules - albeit to different degrees. Using a B16.F10 melanoma model, we showcased the therapeutic potential of a lead FNP formulated PEG-DB nanocarrier, encapsulating the cyclic dinucleotide (CDN) cGAMP to activate the stimulator of interferon genes (STING) pathway in a therapeutically relevant context. Collectively, these data demonstrate that an FNP process can be used to formulate pH-responsive nanocarriers of diverse morphologies using a PEG-DB polymer system. As FNP is an industrially scalable process, these data address the critical translational challenge of producing PEG-DB nanoparticles at scale. Furthermore, the diverse morphologies produced may specialize in the delivery of distinct biomolecular cargos for other therapeutic applications, implicating the therapeutic potential of this platform in an array of disease applications.

STING-activating nanoparticles normalize the vascular-immune interface to potentiate cancer immunotherapy.

The tumor-associated vasculature imposes major structural and biochemical barriers to the infiltration of effector T cells and effective tumor control. Correlations between stimulator of interferon genes (STING) pathway activation and spontaneous T cell infiltration in human cancers led us to evaluate the effect of STING-activating nanoparticles (STANs), which are a polymersome-based platform for the delivery of a cyclic dinucleotide STING agonist, on the tumor vasculature and attendant effects on T cell infiltration and antitumor function. In multiple mouse tumor models, intravenous administration of STANs promoted vascular normalization, evidenced by improved vascular integrity, reduced tumor hypoxia, and increased endothelial cell expression of T cell adhesion molecules. STAN-mediated vascular reprogramming enhanced the infiltration, proliferation, and function of antitumor T cells and potentiated the response to immune checkpoint inhibitors and adoptive T cell therapy. We present STANs as a multimodal platform that activates and normalizes the tumor microenvironment to enhance T cell infiltration and function and augments responses to immunotherapy.

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

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.

Structural Optimization of Polymeric Carriers to Enhance the Immunostimulatory Activity of Molecularly Defined RIG-I Agonists.

RNA ligands of retinoic acid-inducible gene I (RIG-I) hold significant promise as antiviral agents, vaccine adjuvants, and cancer immunotherapeutics, but their efficacy is hindered by inefficient intracellular delivery to the cytosol where RIG-I is localized. Here, we address this challenge through the synthesis and evaluation of a library of polymeric carriers rationally designed to promote the endosomal escape of 5'-triphosphate RNA (3pRNA) RIG-I agonists. We synthesized a series of PEG-block-(DMAEMA-co-A n MA) polymers, where A n MA is an alkyl methacrylate monomer ranging from n = 2-12 carbons, of variable composition, and examined effects of polymer structure on the intracellular delivery of 3pRNA. Through in vitro screening of 30 polymers, we identified four lead carriers (4-50, 6-40, 8-40, and 10-40, where the first number refers to the alkyl chain length and the second number refers to the percentage of hydrophobic monomer) that packaged 3pRNA into ∼100-nm-diameter particles and significantly enhanced its immunostimulatory activity in multiple cell types. In doing so, these studies also revealed an interplay between alkyl chain length and monomer composition in balancing RNA loading, pH-responsive properties, and endosomal escape, studies that establish new structure-activity relationships for polymeric delivery of 3pRNA and other nucleic acid therapeutics. Importantly, lead carriers enabled intravenous administration of 3pRNA in mice, resulting in increased RIG-I activation as measured by increased levels of IFN-α in serum and elevated expression of Ifnb1 and Cxcl10 in major clearance organs, effects that were dependent on polymer composition. Collectively, these studies have yielded novel polymeric carriers designed and optimized specifically to enhance the delivery and activity of 3pRNA with potential to advance the clinical development of RIG-I agonists.

Synergistic formation and stabilization of oil-in-water emulsions by a weakly interacting mixture of zwitterionic surfactant and silica nanoparticles.

Oil-in-water emulsions were formed and stabilized at low amphiphile concentrations by combining hydrophilic nanoparticles (NPs) (i.e., bare colloidal silica) with a weakly interacting zwitterionic surfactant, caprylamidopropyl betaine, to generate a high hydrophilic-lipophilic balance. The weak interaction of the NPs with surfactant was quantified with contact angle measurements. Emulsions were characterized by static light scattering to determine the droplet size distributions, optical photography to quantify phase separation due to creaming, and both optical and electron microscopy to determine emulsion microstructure. The NPs and surfactant acted synergistically to produce finer emulsions with a greater stability to coalescence relative to the behavior with either NPs or surfactant alone. As a consequence of the weak adsorption of the highly hydrophilic surfactant on the anionic NPs along with the high critical micelle concentration, an unusually large surfactant concentration was available to adsorb at the oil-water interface and lower the interfacial tension. The synergy for emulsion formation and stabilization for the two amphiphiles was even greater in the case of a high-salinity synthetic seawater aqueous phase. Here, higher NP adsorption at the oil-water interface was caused by electrostatic screening of interactions between (1) NPs and the anionic oil-water interface and (2) between the NPs. This greater adsorption as well as partial flocculation of the NPs provided a more efficient barrier to droplet coalescence.