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.

Nanoparticle delivery improves the pharmacokinetic properties of cyclic dinucleotide STING agonists to open a therapeutic window for intravenous administration.

The stimulator of interferon genes (STING) pathway plays an important role in the immune surveillance of cancer and, accordingly, agonists of STING signaling have recently emerged as promising therapeutics for remodeling of the immunosuppressive tumor microenvironment (TME) and enhancing response rates to immune checkpoint inhibitors. 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP) is the endogenous ligand for STING, but is rapidly metabolized and poorly membrane permeable, restricting its use to intratumoral administration. Nanoencapsulation has been shown to allow for systemic administration of cGAMP and other cyclic dinucleotides (CDN), but little is known about how nanocarriers affect important pharmacological properties that impact the efficacy and safety of CDNs. Using STING-activating nanoparticles (STING-NPs) - a polymersome platform designed to enhance cGAMP delivery - we investigate the pharmacokinetic (PK)-pharmacodynamic (PD) relationships that underlie the ability of intravenously (i.v.) administered STING-NPs to induce STING activation and inhibit tumor growth. First, we demonstrate that nanoencapsulation improves the half-life of encapsulated cGAMP by 40-fold, allowing for sufficient accumulation of cGAMP in tumors and activation of the STING pathway in the TME as assessed by western blot analysis and gene expression profiling. Nanoparticle delivery also changes the biodistribution profile, resulting in increased cGAMP accumulation and STING activation in the liver and spleen, which we identify as dose limiting organs. As a consequence of STING activation in tumors, i.v. administered STING-NPs reprogram the TME towards a more immunogenic antitumor milieu, characterized by an influx of >20-fold more CD4+ and CD8+ T-cells. Consequently, STING-NPs increased response rates to αPD-L1 antibodies, resulting in significant improvements in median survival time in a B16-F10 melanoma model. Additionally, we confirmed STING-NP monotherapy in an additional melanoma (YUMM1.7) and breast adenocarcinoma (E0771) models leading to >50% and 80% reduction in tumor burden, respectively, and significant increases in median survival time. Collectively, this work provides an examination of the PK-PD relationship governing STING activation upon systemic delivery using STING-NPs, providing insight for future optimization for nanoparticle-based STING agonists and other immunomodulating nanomedicines.

Co-delivery of Peptide Neoantigens and Stimulator of Interferon Genes Agonists Enhances Response to Cancer Vaccines.

Cancer vaccines targeting patient-specific neoantigens have emerged as a promising strategy for improving responses to immune checkpoint blockade. However, neoantigenic peptides are poorly immunogenic and inept at stimulating CD8+ T cell responses, motivating a need for new vaccine technologies that enhance their immunogenicity. The stimulator of interferon genes (STING) pathway is an endogenous mechanism by which the innate immune system generates an immunological context for priming and mobilizing neoantigen-specific T cells. Owing to this critical role in tumor immune surveillance, a synthetic cancer nanovaccine platform (nanoSTING-vax) was developed that mimics immunogenic cancer cells in its capacity to efficiently promote co-delivery of peptide antigens and the STING agonist, cGAMP. The co-loading of cGAMP and peptides into pH-responsive, endosomolytic polymersomes promoted the coordinated delivery of both cGAMP and peptide antigens to the cytosol, thereby eliciting inflammatory cytokine production, co-stimulatory marker expression, and antigen cross-presentation. Consequently, nanoSTING-vax significantly enhanced CD8+ T cell responses to a range of peptide antigens. Therapeutic immunization with nanoSTING-vax, in combination with immune checkpoint blockade, inhibited tumor growth in multiple murine tumor models, even leading to complete tumor rejection and generation of durable antitumor immune memory. Collectively, this work establishes nanoSTING-vax as a versatile platform for enhancing immune responses to neoantigen-targeted cancer vaccines.

Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a childhood cancer for which new treatment options are needed. The success of immune checkpoint blockade in the treatment of adult solid tumors has prompted the exploration of immunotherapy in NB; however, clinical evidence indicates that the vast majority of NB patients do not respond to single-agent checkpoint inhibitors. This motivates a need for therapeutic strategies to increase NB tumor immunogenicity. The goal of this study was to evaluate a new immunotherapeutic strategy for NB based on potent activation of the stimulator of interferon genes (STING) pathway. METHODS: To promote STING activation in NB cells and tumors, we utilized STING-activating nanoparticles (STING-NPs) that are designed to mediate efficient cytosolic delivery of the endogenous STING ligand, 2'3'-cGAMP. We investigated tumor-intrinsic responses to STING activation in both MYCN-amplified and non-amplified NB cell lines, evaluating effects on STING signaling, apoptosis, and the induction of immunogenic cell death. The effects of intratumoral administration of STING-NPs on CD8+ T cell infiltration, tumor growth, and response to response to PD-L1 checkpoint blockade were evaluated in syngeneic models of MYCN-amplified and non-amplified NB. RESULTS: The efficient cytosolic delivery of 2'3'-cGAMP enabled by STING-NPs triggered tumor-intrinsic STING signaling effects in both MYCN-amplified and non-amplified NB cell lines, resulting in increased expression of interferon-stimulated genes and pro-inflammatory cytokines as well as NB cell death at concentrations 2000-fold to 10000-fold lower than free 2'3'-cGAMP. STING-mediated cell death in NB was associated with release or expression of several danger associated molecular patterns that are hallmarks of immunogenic cell death, which was further validated via cell-based vaccination and tumor challenge studies. Intratumoral administration of STING-NPs enhanced STING activation relative to free 2'3'-cGAMP in NB tumor models, converting poorly immunogenic tumors into tumoricidal and T cell-inflamed microenvironments and resulting in inhibition of tumor growth, increased survival, and induction of immunological memory that protected against tumor re-challenge. In a model of MYCN-amplified NB, STING-NPs generated an abscopal response that inhibited distal tumor growth and improved response to PD-L1 immune checkpoint blockade. CONCLUSIONS: We have demonstrated that activation of the STING pathway, here enabled by a nanomedicine approach, stimulates immunogenic cell death and remodels the tumor immune microenvironment to inhibit NB tumor growth and improve responses to immune checkpoint blockade, providing a multifaceted immunotherapeutic approach with potential to enhance immunotherapy outcomes in NB.

At the bench: Engineering the next generation of cancer vaccines.

Cancer vaccines hold promise as an immunotherapeutic modality based on their potential to generate tumor antigen-specific T cell responses and long-lived antitumor responses capable of combating metastatic disease and recurrence. However, cancer vaccines have historically failed to deliver significant therapeutic benefit in the clinic, which we maintain is due in part to drug delivery challenges that have limited vaccine immunogenicity and efficacy. In this review, we examine some of the known and putative failure mechanisms of common first-generation clinical cancer vaccines, and describe how the rational design of materials engineered for vaccine delivery and immunomodulation can address these shortcomings. First, we outline vaccine design principles for augmenting cellular immunity to tumor antigens and describe how well-engineered materials can improve vaccine efficacy, highlighting recent innovations in vaccine delivery technology that are primed for integration into neoantigen vaccine development pipelines. We also discuss the importance of sequencing, timing, and kinetics in mounting effective immune responses to cancer vaccines, and highlight examples of materials that potentiate antitumor immunity through spatiotemporal control of immunomodulation. Furthermore, we describe several engineering strategies for improving outcomes of in situ cancer vaccines, which leverage local, intratumoral delivery to stimulate systemic immunity. Finally, we highlight recent innovations leveraging nanotechnology for increasing the immunogenicity of the tumor microenvironment (TME), which is critical to enhancing tumor infiltration and function of T cells elicited in response to cancer vaccines. These immunoengineering strategies and tools complement ongoing advances in cancer vaccines as they reemerge as an important component of the immunotherapeutic armamentarium.

Multimodal Multiplexed Immunoimaging with Nanostars to Detect Multiple Immunomarkers and Monitor Response to Immunotherapies.

The overexpression of immunomarker programmed cell death protein 1 (PD-1) and engagement of PD-1 to its ligand, PD-L1, are involved in the functional impairment of cluster of differentiation 8+ (CD8+) T cells, contributing to cancer progression. However, heterogeneities in PD-L1 expression and variabilities in biopsy-based assays render current approaches inaccurate in predicting PD-L1 status. Therefore, PD-L1 screening alone is not predictive of patient response to treatment, which motivates us to simultaneously detect multiple immunomarkers engaged in immune modulation. Here, we have developed multimodal probes, immunoactive gold nanostars (IGNs), that accurately detect PD-L1+ tumor cells and CD8+ T cells simultaneously in vivo, surpassing the limitations of current immunoimaging techniques. IGNs integrate the whole-body imaging of positron emission tomography with high sensitivity and multiplexing of Raman spectroscopy, enabling the dynamic tracking of both immunomarkers. IGNs also monitor response to immunotherapies in mice treated with combinatorial PD-L1 and CD137 agonists and distinguish responders from those nonresponsive to treatment. Our results showed a multifunctional nanoscale probe with capabilities that cannot be achieved with either modality alone, allowing multiplexed immunologic tumor profiling critical for predicting early response to immunotherapies.

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

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.

Poly(propylacrylic acid)-peptide nanoplexes as a platform for enhancing the immunogenicity of neoantigen cancer vaccines.

Cancer vaccines targeting patient-specific tumor neoantigens have recently emerged as a promising component of the rapidly expanding immunotherapeutic armamentarium. However, neoantigenic peptides typically elicit weak CD8+ T cell responses, and so there is a need for universally applicable vaccine delivery strategies to enhance the immunogenicity of these peptides. Ideally, such vaccines could also be rapidly fabricated using chemically synthesized peptide antigens customized to an individual patient. Here, we describe a strategy for simple and rapid packaging of peptide antigens into pH-responsive nanoparticles with endosomal escape activity. Electrostatically-stabilized polyplex nanoparticles (nanoplexes) can be assembled instantaneously by mixing decalysine-modified antigenic peptides and poly(propylacrylic acid) (pPAA), a polyanion with pH-dependent, membrane destabilizing activity. These nanoplexes increase and prolong antigen uptake and presentation on MHC-I (major histocompatibility complex class I) molecules expressed by dendritic cells, resulting in enhanced activation of CD8+ T cells. Using an intranasal immunization route, nanoplex vaccines inhibit formation of lung metastases in a murine melanoma model. Additionally, nanoplex vaccines strongly synergize with the adjuvant α-galactosylceramide (α-GalCer) in stimulating robust CD8+ T cell responses, significantly increasing survival time in mice with established melanoma tumors. Collectively, these findings demonstrate that peptide/pPAA nanoplexes offer a facile and versatile platform for enhancing CD8+ T cell responses to peptide antigens, with potential to complement ongoing advancements in the development of neoantigen-targeted cancer vaccines.

Gold Nanoantenna-Mediated Photothermal Drug Delivery from Thermosensitive Liposomes in Breast Cancer.

In this work, we demonstrate controlled drug delivery from low-temperature-sensitive liposomes (LTSLs) mediated by photothermal heating from multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. The unique geometry of MGNs enables the generation of mild hyperthermia (∼42 °C) by converting near-infrared light to heat and effectively delivering doxorubicin (DOX) from the LTSLs in breast cancer cells. We confirmed the cellular uptake of MGNs by using both fluorescence confocal Z-stack imaging and transmission electron microscopy (TEM) imaging. We performed a cellular viability assay and live/dead cell fluorescence imaging of the combined therapeutic effects of MGNs with DOX-loaded LTSLs (DOX-LTSLs) and compared them with free DOX and DOX-loaded non-temperature-sensitive liposomes (DOX-NTSLs). Imaging of fluorescent live/dead cell indicators and MTT assay outcomes both demonstrated significant decreases in cellular viability when cells were treated with the combination therapy. Because of the high phase-transition temperature of NTSLs, no drug delivery was observed from the DOX-NTSLs. Notably, even at a low DOX concentration of 0.5 µg/mL, the combination treatment resulted in a higher (33%) cell death relative to free DOX (17% cell death). The results of our work demonstrate that the synergistic therapeutic effect of photothermal hyperthermia of MGNs with drug delivery from the LTSLs can successfully eradicate aggressive breast cancer cells with higher efficacy than free DOX by providing a controlled light-activated approach and minimizing off-target toxicity.