Mechanisms and Barriers in Nanomedicine: Progress in the Field and Future Directions.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

Apolipoprotein-mimetic nanodiscs reduce lipid accumulation and improve liver function in acid sphingomyelinase deficiency.

Acid sphingomyelinase deficiency (ASMD) is a severe lipid storage disorder caused by the diminished activity of the acid sphingomyelinase enzyme. ASMD is characterized by the accumulation of sphingomyelin in late endosomes and lysosomes leading to progressive neurological dysfunction and hepatosplenomegaly. Our objective was to investigate the utility of synthetic apolipoprotein A-I (ApoA-I) mimetics designed to act as lipid scavengers for the treatment of ASMD. We determined the lead peptide, 22A, could reduce sphingomyelin accumulation in ASMD patient skin fibroblasts in a dose dependent manner. Intraperitoneal administration of 22A formulated as a synthetic high-density lipoprotein (sHDL) nanodisc mobilized sphingomyelin from peripheral tissues into circulation and improved liver function in a mouse model of ASMD. Together, our data demonstrates that apolipoprotein mimetics could serve as a novel therapeutic strategy for modulating the pathology observed in ASMD.

Clinical Efficacy of ONC201 in H3K27M-Mutant Diffuse Midline Gliomas Is Driven by Disruption of Integrated Metabolic and Epigenetic Pathways.

Patients with H3K27M-mutant diffuse midline glioma (DMG) have no proven effective therapies. ONC201 has recently demonstrated efficacy in these patients, but the mechanism behind this finding remains unknown. We assessed clinical outcomes, tumor sequencing, and tissue/cerebrospinal fluid (CSF) correlate samples from patients treated in two completed multisite clinical studies. Patients treated with ONC201 following initial radiation but prior to recurrence demonstrated a median overall survival of 21.7 months, whereas those treated after recurrence had a median overall survival of 9.3 months. Radiographic response was associated with increased expression of key tricarboxylic acid cycle-related genes in baseline tumor sequencing. ONC201 treatment increased 2-hydroxyglutarate levels in cultured H3K27M-DMG cells and patient CSF samples. This corresponded with increases in repressive H3K27me3 in vitro and in human tumors accompanied by epigenetic downregulation of cell cycle regulation and neuroglial differentiation genes. Overall, ONC201 demonstrates efficacy in H3K27M-DMG by disrupting integrated metabolic and epigenetic pathways and reversing pathognomonic H3K27me3 reduction. SIGNIFICANCE: The clinical, radiographic, and molecular analyses included in this study demonstrate the efficacy of ONC201 in H3K27M-mutant DMG and support ONC201 as the first monotherapy to improve outcomes in H3K27M-mutant DMG beyond radiation. Mechanistically, ONC201 disrupts integrated metabolic and epigenetic pathways and reverses pathognomonic H3K27me3 reduction. This article is featured in Selected Articles from This Issue, p. 2293.

Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance.

PURPOSE: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). EXPERIMENTAL DESIGN: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. RESULTS: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. CONCLUSIONS: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.

Systematic evaluation of the effect of different apolipoprotein A-I mimetic peptides on the performance of synthetic high-density lipoproteins in vitro and in vivo.

Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.

Cancer stem cell antigen nanodisc cocktail elicits anti-tumor immune responses in melanoma.

One of the major reasons for poor cancer outcomes is the existence of cancer stem cells (CSCs). CSCs are a small subpopulation of tumor cells that can self-renew, differentiate into the majority of tumor cells, and maintain tumorigenicity. As CSCs are resistant to traditional chemotherapy and radiation, they contribute to metastasis and relapse. Thus, new approaches are needed to target and eliminate CSCs. Here, we sought to target and reduce the frequency of CSCs in melanoma by therapeutic vaccination against CSC-associated transcription factors, such as Sox2 and Nanog, and aldehyde dehydrogenase (ALDH). Toward this goal, we have identified novel immunogenic peptide epitopes derived from CSC-associated Sox2 and Nanog and synthesized synthetic high-density lipoprotein (sHDL) nanodisc vaccine formulated with Sox2, Nanog, and ALDH antigen peptides together with CpG, a Toll-like receptor 9 agonist. Vaccination with nanodiscs containing six CSC antigen peptides elicited robust T cell responses against CSC-associated antigens and promoted intratumoral infiltration of CD8+ T cells, while reducing the frequency of CSCs and CD4+ regulatory T cells within melanoma tumors. Nanodisc vaccination effectively reduced tumor growth and significantly extended animal survival without toxicity toward normal stem cells. Overall, our therapeutic strategy against CSCs represents a cost-effective, safe, and versatile approach that may be applied to melanoma and other cancer types, as well as serve as a critical component in combined therapies to target and eliminate CSCs.

Efficient aqueous remote loading of peptides in poly(lactic-co-glycolic acid).

Poly(lactic-co-glycolic acid) (PLGA) long-acting release depots are effective for extending the duration of action of peptide drugs. We describe efficient organic-solvent-free remote encapsulation based on the capacity of common uncapped PLGA to bind and absorb into the polymer phase net positively charged peptides from aqueous solution after short exposure at modest temperature. Leuprolide encapsulated by this approach in low-molecular-weight PLGA 75/25 microspheres slowly and continuously released peptide for over 56 days in vitro and suppressed testosterone production in rats in an equivalent manner as the 1-month Lupron Depot®. The technique is generalizable to encapsulate a number of net cationic peptides of various size, including octreotide, with competitive loading and encapsulation efficiencies to traditional methods. In certain cases, in vitro and in vivo performance of remote-loaded PLGA microspheres exceeded that relative to marketed products. Remote absorption encapsulation further removes the need for a critical organic solvent removal step after encapsulation, allowing for simple and cost-effective sterilization of the drug-free microspheres before encapsulation of the peptide.

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

Replenishing HDL with synthetic HDL has multiple protective effects against sepsis in mice.

Sepsis is a major health issue with mortality exceeding 30% and few treatment options. We found that high-density lipoprotein cholesterol (HDL-C) abundance was reduced by 45% in septic patients compared to that in nonseptic patients. Furthermore, HDL-C abundance in nonsurviving septic patients was substantially lower than in those patients who survived. We therefore hypothesized that replenishing HDL might be a therapeutic approach for treating sepsis and found that supplementing HDL with synthetic HDL (sHDL) provided protection against sepsis in mice. In mice subjected to cecal ligation and puncture (CLP), infusing the sHDL ETC-642 increased plasma HDL-C amounts and improved the 7-day survival rate. Septic mice treated with sHDL showed improved kidney function and reduced inflammation, as indicated by marked decreases in the plasma concentrations of blood urea nitrogen (BUN) and the cytokines interleukin-6 (IL-6) and IL-10, respectively. We found that sHDL inhibited the ability of the endotoxins LPS and LPA to activate inflammatory pathways in RAW264.7 cells and HEK-Blue cells expressing the receptors TLR4 or TLR2 and NF-κB reporters. In addition, sHDL inhibited the activation of HUVECs by LPS, LTA, and TNF-α. Together, these data indicate that sHDL treatment protects mice from sepsis in multiple ways and that it might be an effective therapy for patients with sepsis.

Krüppel-like factor 14 deletion in myeloid cells accelerates atherosclerotic lesion development.

AIMS: Atherosclerosis is the dominant pathologic basis of many cardiovascular diseases. Large genome-wide association studies have identified that single-nucleotide polymorphisms proximal to Krüppel-like factor 14 (KLF14), a member of the zinc finger family of transcription factors, are associated with higher cardiovascular risks. Macrophage dysfunction contributes to atherosclerosis development and has been recognized as a potential therapeutic target for treating many cardiovascular diseases. Herein, we address the biologic function of KLF14 in macrophages and its role during the development of atherosclerosis. METHODS AND RESULTS: KLF14 expression was markedly decreased in cholesterol loaded foam cells, and overexpression of KLF14 significantly increased cholesterol efflux and inhibited the inflammatory response in macrophages. We generated myeloid cell-selective Klf14 knockout (Klf14LysM) mice in the ApoE-/- background for the atherosclerosis study. Klf14LysMApoE-/- and litter-mate control mice (Klf14fl/flApoE-/-) were placed on the Western Diet for 12 weeks to induce atherosclerosis. Macrophage Klf14 deficiency resulted in increased atherosclerosis development without affecting the plasma lipid profiles. Klf14-deficient peritoneal macrophages showed significantly reduced cholesterol efflux resulting in increased lipid accumulation and exacerbated inflammatory response. Mechanistically, KLF14 upregulates the expression of a key cholesterol efflux transporter, ABCA1 (ATP-binding cassette transporter A1), while it suppresses the expression of several critical components of the inflammatory cascade. In macrophages, activation of KLF14 by its activator, perhexiline, a drug clinically used to treat angina, significantly inhibited the inflammatory response and increased cholesterol efflux in a KLF14-dependent manner in macrophages without triggering hepatic lipogenesis. CONCLUSIONS: This study provides insights into the anti-atherosclerotic effects of myeloid KLF14 through promoting cholesterol efflux and suppressing the inflammatory response. Activation of KLF14 may represent a potential new therapeutic approach to prevent or treat atherosclerosis.

Phospholipid nanoparticles: Therapeutic potentials against atherosclerosis via reducing cholesterol crystals and inhibiting inflammation.

BACKGROUND: Atherosclerosis-related cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. Cholesterol crystals (CCs) induce inflammation in atherosclerosis and are associated with unstable plaques and poor prognosis, but no drug can remove CCs in the clinic currently. METHODS: We generated a phospholipid-based and high-density lipoprotein (HDL)-like nanoparticle, miNano, and determined CC-dissolving capacity, cholesterol efflux property, and anti-inflammation effects of miNano in vitro. Both normal C57BL/6J and Apoe-deficient mice were used to explore the accumulation of miNano in atherosclerotic plaques. The efficacy and safety of miNano administration to treat atherosclerosis were evaluated in the Ldlr-deficient atherosclerosis model. The CC-dissolving capacity of miNano was also detected using human atherosclerotic plaques ex vivo. FINDINGS: We found that miNano bound to and dissolved CCs efficiently in vitro, and miNano accumulated in atherosclerotic plaques, co-localized with CCs and macrophages in vivo. Administration of miNano inhibited atherosclerosis and improved plaque stability by reducing CCs and macrophages in Ldlr-deficient mice with favorable safety profiles. In macrophages, miNano prevented foam cell formation by enhancing cholesterol efflux and suppressed inflammatory responses via inhibiting TLR4-NF-κB pathway. Finally, in an ex vivo experiment, miNano effectively dissolved CCs in human aortic atherosclerotic plaques. INTERPRETATION: Together, our work finds that phospholipid-based and HDL-like nanoparticle, miNano, has the potential to treat atherosclerosis by targeting CCs and stabilizing plaques. FUNDING: This work was supported by the National Institutes of Health HL134569, HL109916, HL136231, and HL137214 to Y.E.C, HL138139 to J.Z., R21NS111191 to A.S., by the American Heart Association 15SDG24470155, Grant Awards (U068144 from Bio-interfaces and G024404 from M-BRISC) at the University of Michigan to Y.G., by the American Heart Association 19PRE34400017 and Rackham Helen Wu award to M.Y., NIH T32 GM07767 to K. H., Barbour Fellowship to D.L.

Research and Education Needs for Complex Generics.

Complex generics are generic versions of drug products that generally have complex active ingredients, complex formulations, complex routes of delivery, complex dosage forms, are complex drug-device combination products, or have other characteristics that can make it complex to demonstrate bioequivalence or to develop as generics. These complex products (i.e. complex generics) are an important element of the United States (U.S.) Food and Drug Administration's (FDA's) Generic Drug User Fee Amendments (GDUFA) II Commitment Letter. The Center for Research on Complex Generics (CRCG) was formed by a grant from the FDA to address challenges associated with the development of complex generics. To understand these challenges, the CRCG conducted a "Survey of Scientific Challenges in the Development of Complex Generics". The three main areas of questioning were directed toward which (types of) complex products, which methods of analysis to support a demonstration of bioequivalence, and which educational topics the CRCG should prioritize. The survey was open to the public on a website maintained by the CRCG. Regarding complex products, the top three selections were complex injectables, formulations, and nanomaterials; drug-device combination products; and inhalation and nasal products. Regarding methods of analysis, the top three selections were locally-acting physiologically-based pharmacokinetic modeling; oral absorption models and bioequivalence; and data analytics and machine learning. Regarding educational topics, the top three selections were complex injectables, formulations, and nanomaterials; drug-device combination products; and data analytics, including quantitative methods and modeling & simulation. These survey results will help prioritize the CRCG's initial research and educational initiatives.

Vaccine nanodiscs plus polyICLC elicit robust CD8+ T cell responses in mice and non-human primates.

Conventional cancer vaccines based on soluble vaccines and traditional adjuvants have produced suboptimal therapeutic efficacy in clinical trials. Thus, there is an urgent need for vaccine technologies that can generate potent T cell responses with strong anti-tumor efficacy. We have previously reported the development of synthetic high-density protein (sHDL) nanodiscs for efficient lymph node (LN)-targeted co-delivery of antigen peptides and CpG oligonucleotides (a Toll-like receptor-9 agonist). Here, we performed a comparative study in mice and non-human primates (NHPs) to identify an ideal vaccine platform for induction of CD8+ T cell responses. In particular, we compared the efficacy of CpG class B, CpG class C, and polyICLC (a synthetic double-stranded RNA analog, a TLR-3 agonist), each formulated with antigen-carrying sHDL nanodiscs. Here, we report that sHDL-Ag admixed with polyICLC elicited robust Ag-specific CD8+ T cell responses in mice, and when used in combination with α-PD-1 immune checkpoint inhibitor, sHDL-Ag + polyICLC eliminated large established (~100 mm3) MC-38 tumors in mice. Moreover, sHDL-Gag + polyICLC induced robust Simian immunodeficiency virus Gag-specific, polyfunctional CD8+ T cell responses in rhesus macaques and could further amplify the efficacy of recombinant adenovirus-based vaccine. Notably, while both sHDL-Ag-CpG-B and sHDL-Ag-CpG-C generated strong Ag-specific CD8+ T cell responses in mice, their results were mixed in NHPs. Overall, sHDL combined with polyICLC offers a strong platform to induce CD8+ T cells for vaccine applications.

Targeting Neuroinflammation in Brain Cancer: Uncovering Mechanisms, Pharmacological Targets, and Neuropharmaceutical Developments.

Gliomas are one of the most lethal types of cancers accounting for ∼80% of all central nervous system (CNS) primary malignancies. Among gliomas, glioblastomas (GBM) are the most aggressive, characterized by a median patient survival of fewer than 15 months. Recent molecular characterization studies uncovered the genetic signatures and methylation status of gliomas and correlate these with clinical prognosis. The most relevant molecular characteristics for the new glioma classification are IDH mutation, chromosome 1p/19q deletion, histone mutations, and other genetic parameters such as ATRX loss, TP53, and TERT mutations, as well as DNA methylation levels. Similar to other solid tumors, glioma progression is impacted by the complex interactions between the tumor cells and immune cells within the tumor microenvironment. The immune system's response to cancer can impact the glioma's survival, proliferation, and invasiveness. Salient characteristics of gliomas include enhanced vascularization, stimulation of a hypoxic tumor microenvironment, increased oxidative stress, and an immune suppressive milieu. These processes promote the neuro-inflammatory tumor microenvironment which can lead to the loss of blood-brain barrier (BBB) integrity. The consequences of a compromised BBB are deleteriously exposing the brain to potentially harmful concentrations of substances from the peripheral circulation, adversely affecting neuronal signaling, and abnormal immune cell infiltration; all of which can lead to disruption of brain homeostasis. In this review, we first describe the unique features of inflammation in CNS tumors. We then discuss the mechanisms of tumor-initiating neuro-inflammatory microenvironment and its impact on tumor invasion and progression. Finally, we also discuss potential pharmacological interventions that can be used to target neuro-inflammation in gliomas.

Genetic Alterations in Gliomas Remodel the Tumor Immune Microenvironment and Impact Immune-Mediated Therapies.

High grade gliomas are malignant brain tumors that arise in the central nervous system, in patients of all ages. Currently, the standard of care, entailing surgery and chemo radiation, exhibits a survival rate of 14-17 months. Thus, there is an urgent need to develop new therapeutic strategies for these malignant brain tumors. Currently, immunotherapies represent an appealing approach to treat malignant gliomas, as the pre-clinical data has been encouraging. However, the translation of the discoveries from the bench to the bedside has not been as successful as with other types of cancer, and no long-lasting clinical benefits have been observed for glioma patients treated with immune-mediated therapies so far. This review aims to discuss our current knowledge about gliomas, their molecular particularities and the impact on the tumor immune microenvironment. Also, we discuss several murine models used to study these therapies pre-clinically and how the model selection can impact the outcomes of the approaches to be tested. Finally, we present different immunotherapy strategies being employed in clinical trials for glioma and the newest developments intended to harness the immune system against these incurable brain tumors.

Inhibition of 2-hydroxyglutarate elicits metabolic reprogramming and mutant IDH1 glioma immunity in mice.

Mutant isocitrate dehydrogenase 1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into 2 molecular subgroups: 1p/19q codeletion/TERT-promoter mutations or inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work focuses on glioma subtypes harboring mIDH1, TP53, and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of D-2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma-bearing mice. Also, D-2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to D-2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in WT-IDH gliomas. Thus, we combined D-2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint blockade and observed complete tumor regression in 60% of mIDH1 glioma-bearing mice. This combination strategy reduced T cell exhaustion and favored the generation of memory CD8+ T cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data support the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.

Cancer Immunotherapy via Targeting Cancer Stem Cells Using Vaccine Nanodiscs.

Cancer stem cells (CSCs) proliferate extensively and drive tumor metastasis and recurrence. CSCs have been identified in over 20 cancer types to date, but it remains unknown how to target and eliminate CSCs in vivo. Aldehyde dehydrogenase (ALDH) is a marker that has been used extensively for isolating CSCs. Here we present a novel approach to target and reduce the frequency of ALDHhigh CSCs by vaccination against ALDH. We have identified ALDH1-A1 and ALDH1-A3 epitopes from CSCs and developed synthetic high-density lipoprotein nanodiscs for vaccination against ALDHhigh CSCs. Nanodiscs increased antigen trafficking to lymph nodes and generated robust ALDH-specific T cell responses. Nanodisc vaccination against ALDHhigh CSCs combined with anti-PD-L1 therapy exerted potent antitumor efficacy and prolonged animal survival in multiple murine models. Overall, this is the first demonstration of a simple nanovaccine strategy against CSCs and may lead to new avenues for cancer immunotherapy against CSCs.

Immunotherapy for gliomas: shedding light on progress in preclinical and clinical development.

INTRODUCTION: Gliomas are infiltrating brain tumors associated with high morbidity and mortality. Current standard of care includes radiation, chemotherapy, and surgical resection. Today, survival rates for malignant glioma patients remain dismal and unchanged for decades. The glioma microenvironment is highly immunosuppressive and consequently this has motivated the development of immunotherapies for counteracting this condition, enabling the immune cells within the tumor microenvironment to react against this tumor. AREAS COVERED: The authors discuss immunotherapeutic strategies for glioma in phase-I/II clinical trials and illuminate their mechanisms of action, limitations, and key challenges. They also examine promising approaches under preclinical development. EXPERT OPINION: In the last decade there has been an expansion in immune-mediated anti-cancer therapies. In the glioma field, sophisticated strategies have been successfully implemented in preclinical models. Unfortunately, clinical trials have not yet yielded consistent results for glioma patients. This could be attributed to our limited understanding of the complex immune cell infiltration and its interaction with the tumor cells, the selected time for treatment, the combination with other therapies and the route of administration of the agent. Applying these modalities to treat malignant glioma is challenging, but many new alternatives are emerging to by-pass these hurdles.

Synthetic High-density Lipoprotein Nanodiscs for Personalized Immunotherapy Against Gliomas.

PURPOSE: Gliomas are brain tumors with dismal prognoses. The standard-of-care treatments for gliomas include surgical resection, radiation, and temozolomide administration; however, they have been ineffective in providing significant increases in median survival. Antigen-specific cancer vaccines and immune checkpoint blockade may provide promising immunotherapeutic approaches for gliomas. EXPERIMENTAL DESIGN: We have developed immunotherapy delivery vehicles based on synthetic high-density lipoprotein (sHDL) loaded with CpG, a Toll-like receptor-9 agonist, and tumor-specific neoantigens to target gliomas and elicit immune-mediated tumor regression. RESULTS: We demonstrate that vaccination with neoantigen peptide-sHDL/CpG cocktail in combination with anti-PD-L1 immune checkpoint blocker elicits robust neoantigen-specific T-cell responses against GL261 cells and eliminated established orthotopic GL261 glioma in 33% of mice. Mice remained tumor free upon tumor cell rechallenge in the contralateral hemisphere, indicating the development of immunologic memory. Moreover, in a genetically engineered murine model of orthotopic mutant IDH1 (mIDH1) glioma, sHDL vaccination with mIDH1 neoantigen eliminated glioma in 30% of animals and significantly extended the animal survival, demonstrating the versatility of our approach in multiple glioma models. CONCLUSIONS: Overall, our strategy provides a general roadmap for combination immunotherapy against gliomas and other cancer types.

Synthetic HDL Nanoparticles Delivering Docetaxel and CpG for Chemoimmunotherapy of Colon Adenocarcinoma.

Colon carcinomas comprise over two-thirds of all colorectal cancers with an overall 5-year survival rate of 64%, which rapidly decreases to 14% when the cancer becomes metastatic. Depending on the stage of colon carcinoma at diagnosis, patients can undergo surgery to attempt complete tumor resection or move directly to chemotherapy with one or a combination of drugs. As with most cancers, colon carcinomas do not always respond to chemotherapies, so targeted therapies and immunotherapies have been developed to aid chemotherapy. We report the development of a local combination therapy for colon carcinoma whereby chemo- and immunotherapeutic entities are delivered intratumorally to maximize efficacy and minimize off-target side effects. A hydrophobic chemotherapeutic agent, docetaxel (DTX), and cholesterol-modified Toll-like receptor 9 (TLR9) agonist CpG (cho-CpG) oligonucleotide are co-loaded in synthetic HDL (sHDL) nanodiscs. In vivo survival analysis of MC-38 tumor-bearing mice treated intratumorally with DTX-sHDL/CpG (median survival; MS = 43 days) showed significant improvement in overall survival compared to mice treated with single agents, free DTX (MS = 23 days, p < 0.0001) or DTX-sHDL (MS = 28 days, p < 0.0001). Two of seven mice treated with DTX-sHDL/CpG experienced complete tumor regression. None of the mice experienced any systemic toxicity as indicated by body weight maintenance and normal serum enzyme and protein levels. In summary, we have demonstrated that chemo- and immunotherapies can be co-loaded into sHDLs, delivered locally to the tumor, and can be used to improve survival outcomes significantly compared to chemotherapy alone.