ABSTRACT
BACKGROUND: Advanced clear cell renal cell carcinoma (ccRCC) is a prevalent kidney cancer for which long-term survival rates are abysmal, though immunotherapies are showing potential. Not yet clinically vetted are bispecific T cell engagers (BTEs) that activate T cell-mediated cancer killing through intercellular synapsing. Multiple BTE formats exist, however, with limited cross-characterizations to help optimize new drug design. Here, we developed BTEs to treat ccRCC by targeting carbonic anhydrase 9 (CA9) while characterizing the persistent BTE (PBTE) format and comparing it to a new format, the persistent multivalent T cell engager (PMTE). These antibody therapies against ccRCC are developed as both recombinant and synthetic DNA (synDNA) medicines. METHODS: Antibody formatting effects on binding kinetics were assessed by flow cytometry and intercellular synaptic strength assays while potency was tested using T-cell activation and cytotoxicity assays. Mouse models were used to study antibody plasma and tumor pharmacokinetics, as well as antitumor efficacy as both recombinant and synDNA medicines. Specifically, three models using ccRCC cell line xenografts and human donor T cells in immunodeficient mice were used to support this study. RESULTS: Compared with a first-generation BTE, we show that the PBTE reduced avidity, intercellular synaptic strength, cytotoxic potency by as much as 33-fold, and ultimately efficacy against ccRCC tumors in vivo. However, compared with the PBTE, we demonstrate that the PMTE improved cell avidity, restored intercellular synapses, augmented cytotoxic potency by 40-fold, improved tumor distribution pharmacokinetics by 2-fold, and recovered synDNA efficacy in mouse tumor models by 20-fold. All the while, the PMTE displayed a desirable half-life of 4 days in mice compared with the conventional BTE's 2 hours. CONCLUSIONS: With impressive efficacy, the CA9-targeted PMTE is a promising new therapy for advanced ccRCC, which can be effectively delivered through synDNA. The highly potent PMTE format itself is a promising new tool for future applications in the multispecific antibody space.
Subject(s)
Antibodies, Bispecific , Carcinoma, Renal Cell , Kidney Neoplasms , T-Lymphocytes , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/immunology , Humans , Animals , Mice , Kidney Neoplasms/drug therapy , Kidney Neoplasms/immunology , T-Lymphocytes/immunology , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Cell Line, Tumor , Immunotherapy/methods , Carbonic Anhydrase IX/metabolism , Female , Xenograft Model Antitumor AssaysABSTRACT
Cancer immunotherapy has demonstrated great promise with several checkpoint inhibitors being approved as the first-line therapy for some types of cancer, and new engineered cytokines such as Neo2/15 now being evaluated in many studies. In this work, we designed antibody-cytokine chimera (ACC) scaffolding cytokine mimetics on a full-length tumor-specific antibody. We characterized the pharmacokinetic (PK) and pharmacodynamic (PD) properties of first-generation ACC TA99-Neo2/15, which synergized with DLnano-vaccines to suppress in vivo melanoma proliferation and induced significant systemic cytokine activation. A novel second-generation ACC TA99-HL2-KOA1, with retained IL-2Rß/γ binding and attenuated but preserved IL-2Rα binding, induced lower systemic cytokine activation with non-inferior protection in murine tumor studies. Transcriptomic analyses demonstrated an upregulation of Type I interferon responsive genes, particularly ISG15, in dendritic cells, macrophages and monocytes following TA99-HL2-KOA1 treatment. Characterization of additional ACCs in combination with cancer vaccines will likely be an important area of research for treating melanoma and other types of cancer.
Subject(s)
Melanoma , Nanoparticles , Vaccines, DNA , Mice , Animals , Cytokines , Antibodies , DNAABSTRACT
Glioblastoma multiforme (GBM) is among the most difficult cancers to treat with a 5-year survival rate less than 5%. An immunotherapeutic vaccine approach targeting GBM-specific antigen, EGFRvIII, previously demonstrated important clinical impact. However, immune escape variants were reported in the trial, suggesting that multivalent approaches targeting GBM-associated antigens may be of importance. Here we focused on multivalent in vivo delivery of synthetic DNA-encoded bispecific T cell engagers (DBTEs) targeting two GBM-associated antigens, EGFRvIII and HER2. We designed and optimized an EGFRvIII-DBTE that induced T cell-mediated cytotoxicity against EGFRvIII-expressing tumor cells. In vivo delivery in a single administration of EGFRvIII-DBTE resulted in durable expression over several months in NSG mice and potent tumor control and clearance in both peripheral and orthotopic animal models of GBM. Next, we combined delivery of EGFRvIII-DBTEs with an HER2-targeting DBTE to treat heterogeneous GBM tumors. In vivo delivery of dual DBTEs targeting these two GBM-associated antigens exhibited enhanced tumor control and clearance in a heterogeneous orthotopic GBM challenge, while treatment with single-target DBTE ultimately allowed for tumor escape. These studies support that combined delivery of DBTEs, targeting both EGFRvIII and HER2, can potentially improve outcomes of GBM immunotherapy, and such multivalent approaches deserve additional study.
ABSTRACT
Despite advances in ovarian cancer (OC) therapy, recurrent OC remains a poor-prognosis disease. Because of the close interaction between OC cells and the tumor microenvironment (TME), it is important to develop strategies that target tumor cells and engage components of the TME. A major obstacle in the development of OC therapies is the identification of targets with expression limited to tumor surface to avoid off-target interactions. The follicle-stimulating hormone receptor (FSHR) has selective expression on ovarian granulosa cells and is expressed on 50%-70% of serous OCs. We generated mAbs targeting the external domain of FSHR using in vivo-expressed FSHR vector. By high-throughput flow analysis, we identified multiple clones and downselected D2AP11, a potent FSHR surface-targeted mAb. D2AP11 identifies important OC cell lines derived from tumors with different mutations, including BRCA1/2, and lines resistant to a wide range of therapies. We used D2AP11 to develop a bispecific T cell engager. In vitro addition of PBMCs and T cells to D2AP11-TCE induced specific and potent killing of different genetic and immune escape OC lines, with EC50s in the ng/ml range, and attenuated tumor burden in OC-challenged mouse models. These studies demonstrate the potential utility of biologics targeting FSHR for OC and perhaps other FSHR-positive cancers.
Subject(s)
Ovarian Neoplasms , Receptors, FSH , Humans , Animals , Mice , Female , Receptors, FSH/genetics , Receptors, FSH/metabolism , Neoplasm Recurrence, Local , Immunotherapy , Ovarian Neoplasms/genetics , Ovarian Neoplasms/therapy , Ovarian Neoplasms/pathology , Antibodies, Monoclonal/therapeutic use , Adaptive Immunity , Tumor MicroenvironmentABSTRACT
Glioblastoma is an aggressive tumor with poor survival rates. Bispecific T cell engagers (BTEs) against different cancers are in various stages of clinical development. Toxicity resulting from cytokine release syndrome and the short half-life of BTEs, which necessitates continuous infusion, complicating delivery and increasing costs, are major challenges in the field. Here we describe the development of in vivo DNA-launched BTEs (dBTEs) with highly focused targeting of interleukin-13 receptor α2 (IL-13Rα2), a glioblastoma cell-surface target. We developed 4 BTEs targeting 2 epitopes of IL-13Rα2 and studied how heavy-light chain orientation affects BTE function. The dBTEs induced T cell activation, cytokine production, and tumor cytolysis in the presence of IL-13Rα2+ tumor cells, but we observed unique patterns of immune activation. We found a strong correlation between granzyme B secretion and dBTE-induced cytolysis of specific and nonspecific tumors. We down-selected dBTE PB01-forward based on lower cytokine induction profile and highest activation specificity. In vivo, dBTE PB01-forward demonstrated an improved half-life versus intravenous recombinant BTE delivery. In an orthotopic glioblastoma model, dBTE PB01-forward controlled tumor growth, improving animal survival, supporting the hypothesis that the blood-brain barrier does not affect the function of systemically delivered dBTE. Further study of PB01-forward for targeting glioblastoma and other IL-13Rα2+ cancers is warranted.
ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease where muscle weakness and neuromuscular junction (NMJ) denervation precede motor neuron cell death. Although acetylcholine is the canonical neurotransmitter at the mammalian NMJ synapse, glutamate has recently been identified as a critical neurotransmitter for NMJ development and maintenance. One source of glutamate is through the catabolism of N-acetyl-aspartyl-glutamate (NAAG), which is found in mM concentrations in mammalian motoneurons, where it is released upon stimulation and hydrolyzed to glutamate by the glial enzyme glutamate carboxypeptidase II (GCPII). Using the SOD1G93A model of ALS, we found an almost fourfold elevation of GCPII enzymatic activity in SOD1G93A versus WT muscle and a robust increase in GCPII expression which was specifically associated with activated macrophages infiltrating the muscle. 2-(Phosphonomethyl)pentanedioic acid (2PMPA) is a potent GCPII inhibitor which robustly blocks glutamate release from NAAG but is highly polar with limited tissue penetration. To improve this, we covalently attached 2PMPA to a hydroxyl polyamidoamine (PAMAM-G4-OH) dendrimer delivery system (D-2PMPA) which is known to target activated macrophages in affected tissues. Systemic D-2PMPA therapy (20 mg/kg 2PMPA equivalent; IP 2 × /week) was found to localize in muscle macrophages in SOD1G93A mice and completely normalize the enhanced GCPII activity. Although no changes in body weight or survival were observed, D-2PMPA significantly improved grip strength and inhibited the loss of NMJ innervation in the gastrocnemius muscles. Our finding that inhibiting elevated GCPII activity in SOD1G93A muscle can prolong muscle function and delay NMJ denervation may have early therapeutic implications for ALS patients.
Subject(s)
Amyotrophic Lateral Sclerosis , Dendrimers , Neurodegenerative Diseases , Amyotrophic Lateral Sclerosis/metabolism , Animals , Dendrimers/pharmacology , Denervation , Disease Models, Animal , Glutamates , Humans , Mammals , Mice , Mice, Transgenic , Muscle, Skeletal , Superoxide Dismutase , Superoxide Dismutase-1/geneticsABSTRACT
Cognitive impairment is a common aspect of multiple sclerosis (MS) for which there are no treatments. Reduced brain N-acetylaspartylglutamate (NAAG) levels are linked to impaired cognition in various neurological diseases, including MS. NAAG levels are regulated by glutamate carboxypeptidase II (GCPII), which hydrolyzes the neuropeptide to N-acetyl-aspartate and glutamate. GCPII activity is upregulated multifold in microglia following neuroinflammation. Although several GCPII inhibitors, such as 2-PMPA, elevate brain NAAG levels and restore cognitive function in preclinical studies when given at high systemic doses or via direct brain injection, none are clinically available due to poor bioavailability and limited brain penetration. Hydroxyl-dendrimers have been successfully used to selectively deliver drugs to activated glia. Methods: We attached 2-PMPA to hydroxyl polyamidoamine (PAMAM) dendrimers (D-2PMPA) using a click chemistry approach. Cy5-labelled-D-2PMPA was used to visualize selective glial uptake in vitro and in vivo. D-2PMPA was evaluated for anti-inflammatory effects in LPS-treated glial cultures. In experimental autoimmune encephalomyelitis (EAE)-immunized mice, D-2PMPA was dosed biweekly starting at disease onset and cognition was assessed using the Barnes maze, and GCPII activity was measured in CD11b+ hippocampal cells. Results: D-2PMPA showed preferential uptake into microglia and robust anti-inflammatory activity, including elevations in NAAG, TGFß, and mGluR3 in glial cultures. D-2PMPA significantly improved cognition in EAE mice, even though physical severity was unaffected. GCPII activity increased >20-fold in CD11b+ cells from EAE mice, which was significantly mitigated by D-2PMPA treatment. Conclusions: Hydroxyl dendrimers facilitate targeted drug delivery to activated microglia. These data support further development of D-2PMPA to attenuate elevated microglial GCPII activity and treat cognitive impairment in MS.
Subject(s)
Dendrimers , Multiple Sclerosis , Animals , Cognition , Dendrimers/pharmacology , Disease Models, Animal , Mice , Microglia , Multiple Sclerosis/drug therapyABSTRACT
BACKGROUND: Additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines that are safe and effective as primary vaccines and boosters remain urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic. We describe safety and durability of immune responses following 2 primary doses and a homologous booster dose of an investigational DNA vaccine (INO-4800) targeting full-length spike antigen. METHODS: Three dosage strengths of INO-4800 (0.5 mg, 1.0 mg, and 2.0 mg) were evaluated in 120 age-stratified healthy adults. Intradermal injection of INO-4800 followed by electroporation at 0 and 4 weeks preceded an optional booster 6-10.5 months after the second dose. RESULTS: INO-4800 appeared well tolerated with no treatment-related serious adverse events. Most adverse events were mild and did not increase in frequency with age and subsequent dosing. A durable antibody response was observed 6 months following the second dose; a homologous booster dose significantly increased immune responses. Cytokine-producing T cells and activated CD8+ T cells with lytic potential were significantly increased in the 2.0-mg dose group. CONCLUSIONS: INO-4800 was well tolerated in a 2-dose primary series and homologous booster in all adults, including elderly participants. These results support further development of INO-4800 for use as primary vaccine and booster. CLINICAL TRIALS REGISTRATION: NCT04336410.
Subject(s)
COVID-19 , Vaccines, DNA , Adult , Aged , Antibodies, Viral , Antibody Formation , COVID-19/prevention & control , COVID-19 Vaccines , Humans , Immunogenicity, Vaccine , SARS-CoV-2 , Vaccination/adverse effects , Vaccines, DNA/adverse effectsABSTRACT
Latent Epstein-Barr virus (EBV) infection is associated with several types of cancer. Several clinical studies have targeted EBV antigens as immune therapeutic targets with limited efficacy of EBV malignancies, suggesting that additional targets might be important. BamHI-A rightward frame 1 (BARF1) is an EBV antigen that is highly expressed in EBV+ nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC). BARF1 antigen can transform human epithelial cells in vivo. BARF1-specific antibodies and cytotoxic T cells were detected in some EBV+ NPC patients. However, BARF1 has not been evaluated as an antigen in the context of therapeutic immunization. Its possible importance in this context is unclear. Here, we developed a synthetic-DNA-based expression cassette as immunotherapy targeting BARF1 (pBARF1). Immunization with pBARF1 induced potent antigen-specific humoral and T cell responses in vivo. Immunization with pBARF1 plasmid impacted tumor progression through the induction of CD8+ T cells in novel BARF1+ carcinoma models. Using an in vivo imaging system, we observed that pBARF1-immunized animals rapidly cleared cancer cells. We demonstrated that pBARF1 can induce antigen-specific immune responses that can impact cancer progression. Further study of this immune target is likely important as part of therapeutic approaches for EBV+ malignancies.
ABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.
Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Nanoparticles/administration & dosage , SARS-CoV-2/immunology , Animals , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , Cricetinae , Epitopes , Guinea Pigs , Immunogenicity, Vaccine , Mice , Nanoparticles/chemistry , Nucleic Acid-Based Vaccines/administration & dosage , Nucleic Acid-Based Vaccines/chemistry , Nucleic Acid-Based Vaccines/genetics , Nucleic Acid-Based Vaccines/immunology , Polysaccharides/chemistry , Polysaccharides/genetics , Polysaccharides/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccine PotencyABSTRACT
Effective treatment of glioblastoma remains a daunting challenge. One of the major hurdles in the development of therapeutics is their inability to cross the blood-brain tumor barrier (BBTB). Local delivery is an alternative approach that can still suffer from toxicity in the absence of target selectivity. Here, we show that nanotubes formed from self-assembly of ssDNA-amphiphiles are stable in serum and nucleases. After bilateral brain injections, nanotubes show preferential retention by tumors compared to normal brain and are taken up by glioblastoma cells through scavenger receptor binding and macropinocytosis. After intravenous injection, they cross the BBTB and internalize in glioblastoma cells. In a minimal residual disease model, local delivery of doxorubicin showed signs of toxicity in the spleen and liver. In contrast, delivery of doxorubicin by the nanotubes resulted in no systemic toxicity and enhanced mouse survival. Our results demonstrate that ssDNA nanotubes are a promising drug delivery vehicle to glioblastoma.
ABSTRACT
Glioblastoma is among the most aggressive forms of cancers, with a median survival of just 15-20 months for patients despite maximum clinical intervention. The majority of conventional anti-cancer therapies fail due to associated off-site toxicities which can be addressed by developing target-specific drug delivery systems. Advances in nanotechnology have provided targeted systems to overcome drug delivery barriers associated with brain and other types of cancers. Dendrimers have emerged as promising vehicles for targeted drug and gene delivery. Dendrimer-mediated targeting strategies can be further enhanced through the addition of targeting ligands to enable receptor-specific interactions. Here, we explore the sugar moieties as ligands conjugated to hydroxyl-terminated polyamidoamine dendrimers to leverage altered metabolism in cancer and immune targeting. Using a highly facile click chemistry approach, we modified the surface of dendrimers with glucose, mannose, or galactose moieties in a well-defined manner, to target upregulated sugar transporters in the context of glioblastoma. We show that glucose modification significantly enhanced targeting of tumor-associated macrophages (TAMs) and microglia by increasing brain penetration and cellular internalization, while galactose modification shifts targeting away from TAMs towards galectins on glioblastoma tumor cells. Mannose modification did not alter TAMs and microglia targeting of these dendrimers, but did alter their kinetics of accumulation within the GBM tumor. The whole body biodistribution was largely similar between the systems. These results demonstrate that dendrimers are versatile delivery vehicles that can be modified to tailor their targeting for the treatment of glioblastoma and other cancers.
Subject(s)
Dendrimers , Glioblastoma , Dendrimers/therapeutic use , Drug Delivery Systems , Glioblastoma/drug therapy , Glycosylation , Humans , Macrophages , Tissue DistributionABSTRACT
More than 100 million people have been infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Common laboratory mice are not susceptible to wild-type SARS-CoV-2 infection, challenging the development and testing of effective interventions. Here, we describe the development and testing of a mouse model for SARS-CoV-2 infection based on transduction of the respiratory tract of laboratory mice with an adeno-associated virus vector (AAV6) expressing human ACE-2 (AAV6.2FF-hACE2). We validated this model using a previously described synthetic DNA vaccine plasmid, INO-4800 (pS). Intranasal instillation of AAV6.2FF-hACE2 resulted in robust hACE2 expression in the respiratory tract. pS induced robust cellular and humoral responses. Vaccinated animals were challenged with 105 TCID50 SARS-CoV-2 (hCoV-19/Canada/ON-VIDO-01/2020) and euthanized four days post-challenge to assess viral load. One immunization resulted in 50% protection and two immunizations were completely protective. Overall, the AAV6.2FF-hACE2 mouse transduction model represents an easily accessible, genetically diverse mouse model for wild-type SARS-CoV-2 infection and preclinical evaluation of potential interventions.
ABSTRACT
Glioblastoma is the most common and aggressive form of primary brain cancer, with median survival of 16-20 months and a 5-year survival rates of <5%. Recent advances in immunotherapies have shown that addressing the tumor immune profile by targeting the colony-stimulating factor 1 (CSF-1) signaling pathway of tumor-associated macrophages (TAMs) has the potential to improve glioblastoma therapy. However, such therapies have shown limited successes in clinical translation partially due to lack of specific cell targeting in solid tumors and systemic toxicity. In this study, we present a novel hydroxyl dendrimer-mediated immunotherapy to deliver CSF-1R inhibitor BLZ945 (D-BLZ) from systemic administration selectively to TAMs in glioblastoma brain tumors to repolarize the tumor immune environment in a localized manner. We show that conjugation of BLZ945 to dendrimers enables sustained release in intracellular and intratumor conditions. We demonstrate that a single systemic dose of D-BLZ targeted to TAMs decreases pro-tumor expression in TAMs and promotes cytotoxic T cell infiltration, resulting in prolonged survival and ameliorated disease burden compared to free BLZ945. Our results demonstrate that dendrimer-drug conjugates can facilitate specific, localized manipulation of tumor immune responses from systemic administration by delivering immunotherapies selectively to TAMs, thereby improving therapeutic efficacy while reducing off-target effects.
ABSTRACT
Switching microglia from a disease exacerbating, 'pro-inflammatory' state into a neuroprotective, 'anti-inflammatory' phenotype is a promising strategy for addressing multiple neurodegenerative diseases. Pro-inflammatory microglia contribute to disease progression by releasing neurotoxic substances and accelerating pathogenic protein accumulation. PPARα and PPARγ agonists have both been shown to shift microglia from a pro-inflammatory ('M1-like') to an alternatively activated ('M2-like') phenotype. Such strategies have been explored in clinical trials for neurological diseases, such as Alzheimer's and Parkinson's disease, but have likely failed due to their poor blood-brain barrier (BBB) penetration. Hydroxyl-terminated polyamidoamine dendrimers (without the attachment of any targeting ligands) have been shown to cross the impaired BBB at the site of neuroinflammation and accumulate in activated microglia. Therefore, dendrimer conjugation of a PPARα/γ dual agonist may enable targeted phenotype switching of activated microglia. Here we present the synthesis and characterization of a novel dendrimer-PPARα/γ dual agonist conjugate (D-tesaglitazar). In vitro, D-tesaglitazar induces an 'M1 to M2' phenotype shift, decreases secretion of reactive oxygen species, increases expression of genes for phagocytosis and enzymatic degradation of pathogenic proteins (e.g. ß-amyloid, α-synuclein), and increases ß-amyloid phagocytosis. These results support further development of D-tesaglitazar towards translation for multiple neurodegenerative diseases, especially Alzheimer's and Parkinson's Disease.
Subject(s)
Dendrimers , Microglia , Alkanesulfonates , Amyloid beta-Peptides , Phagocytosis , Phenotype , PhenylpropionatesABSTRACT
Novel delivery strategies are necessary to effectively address glioblastoma without systemic toxicities. Triptolide is a therapy derived from the thunder god vine that has shown potent anti-proliferative and immunosuppressive properties but exhibits significant adverse systemic effects. Dendrimer-based nanomedicines have shown great potential for clinical translation of systemic therapies targeting neuroinflammation and brain tumors. Here we present a novel dendrimer-triptolide conjugate that specifically targets tumor-associated macrophages (TAMs) in glioblastoma from systemic administration and exhibits triggered release under intracellular and intratumor conditions. This targeted delivery improves phenotype switching of TAMs from pro- towards anti-tumor expression in vitro. In an orthotopic model of glioblastoma, dendrimer-triptolide achieved significantly improved amelioration of tumor burden compared to free triptolide. Notably, the triggered release mechanism of dendrimer-mediated triptolide delivery significantly reduced triptolide-associated hepatic and cardiac toxicities. These results demonstrate that dendrimers are a promising targeted delivery platform to achieve effective glioblastoma treatment by improving efficacy while reducing systemic toxicities.
Subject(s)
Dendrimers , Glioblastoma , Phenanthrenes , Dendrimers/therapeutic use , Diterpenes , Epoxy Compounds , Glioblastoma/drug therapy , Humans , Tumor-Associated MacrophagesABSTRACT
Glioblastoma exhibits high mortality rates due to challenges with drug delivery to the brain and into solid tumors. This two-pronged barrier necessitates high doses of systemic therapies, resulting in significant off-target toxicities. Recently, dendrimer-nanomedicines (without ligands) have shown promise for targeting specific cells in brain tumors from systemic circulation, for improved efficacy and amelioration of systemic toxicities. A dendrimer-rapamycin conjugate (D-Rapa) is presented here that specifically targets tumor-associated macrophages (TAMs) in glioblastoma from systemic administration. D-Rapa improves suppression of pro-tumor expression in activated TAMs and antiproliferative properties of rapamycin in glioma cells in vitro. In vivo, D-Rapa localizes specifically within TAMs, acting as depots to release rapamycin into the tumor microenvironment. This targeted delivery strategy yields improved reduction in tumor burden and systemic toxicities in a challenging, clinically relevant orthotopic syngeneic model of glioblastoma, demonstrating the significant potential of dendrimers as targeted immunotherapies for improving glioblastoma treatment, still an unmet need.
Subject(s)
Brain Neoplasms , Dendrimers , Glioblastoma , Brain Neoplasms/drug therapy , Dendrimers/therapeutic use , Drug Delivery Systems , Glioblastoma/drug therapy , Humans , Sirolimus/pharmacology , Tumor Microenvironment , Tumor-Associated MacrophagesABSTRACT
Mitochondria mediate critical cellular processes, including proliferation, apoptosis, and immune responses; as such, their dysfunction is pathogenic in many neurodegenerative disorders and cancers. In glioblastoma, targeted delivery of mitochondria-focused anticancer therapies has failed to translate into clinical success due to the nonspecific cellular localization, heterogeneity of receptor expression across patients, poor transport across biological barriers to reach the brain, tumor, and mitochondria, and systemic side effects. Strategies that can overcome brain and solid tumor barriers and selectively target mitochondria within specific cell types may lead to improvements in glioblastoma treatment. Developments in dendrimer-mediated nanomedicines have shown promise targeting tumor-associated macrophages (TAMs) in glioblastoma, following systemic administration. Here, we present a novel dendrimer conjugated to the translocator protein (18 kDa) (TSPO) ligand 5,7-dimethylpyrazolo[1,5-α]pyrimidin-3-ylacetamide (DPA). We developed a clickable DPA for conjugation on the dendrimer surface and demonstrated in vitro that the dendrimer-DPA conjugate (D-DPA) significantly increases dendrimer colocalization with mitochondria. Compared to free TSPO ligand PK11195, D-DPA stimulates greater antitumor immune signaling. In vivo, we show that D-DPA targets mitochondria specifically within TAMs following systemic administration. Our results demonstrate that dendrimers can achieve TAM-specific targeting in glioblastoma and can be further modified to target specific intracellular compartments for organelle-specific drug delivery.
Subject(s)
Dendrimers , Glioblastoma , Cell Line, Tumor , Dendrimers/pharmacology , Dendrimers/therapeutic use , Glioblastoma/drug therapy , Humans , Ligands , Mitochondria , Receptors, GABA/therapeutic use , Tumor-Associated MacrophagesABSTRACT
BACKGROUND: Given the emergent aging population, the identification of effective treatments for Alzheimer's disease (AD) is critical. OBJECTIVE: We investigated the therapeutic efficacy of JHU-083, a brain-penetrable glutamine antagonist, in treating AD using the humanized APOE4 knock-in mouse model. METHODS: Cell culture studies were performed using BV2 cells and primary microglia isolated from hippocampi of adult APOE4 knock-in mice to evaluate the effect of JHU-083 treatment on LPS-induced glutaminase (GLS) activity and inflammatory markers. Six-month-old APOE4 knock-in mice were administered JHU-083 or vehicle via oral gavage 3x/week for 4-5 months and cognitive performance was assessed using the Barnes maze. Target engagement in the brain was confirmed using a radiolabeled GLS enzymatic activity assay, and electrophysiology, gastrointestinal histology, blood chemistry, and CBC analyses were conducted to evaluate the tolerability of JHU-083. RESULTS: JHU-083 inhibited the LPS-mediated increases in GLS activity, nitic oxide release, and pro-inflammatory cytokine production in cultured BV2 cells and primary microglia isolated from APOE4 knock-in AD mice. Chronic treatment with JHU-083 in APOE4 mice improved hippocampal-dependent Barnes maze performance. Consistent with the cell culture findings,postmortem analyses of APOE4 mice showed increased GLS activity in hippocampal CD11b+ enriched cells versus age-matched controls, which was completely normalized by JHU-083 treatment. JHU-083 was well-tolerated, showing no weight loss effect or overt behavioral changes. Peripheral nerve function, gastrointestinal histopathology, and CBC/clinical chemistry parameters were all unaffected by chronic JHU-083 treatment. CONCLUSION: These results suggest that the attenuation of upregulated hippocampal glutaminase by JHU-083 represents a new therapeutic strategy for AD.
Subject(s)
Apolipoprotein E4 , Azo Compounds/pharmacology , Caproates/pharmacology , Cognition/drug effects , Glutaminase/antagonists & inhibitors , Glutamine/antagonists & inhibitors , Hippocampus/drug effects , Alzheimer Disease/drug therapy , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Animals , Apolipoprotein E4/genetics , Azo Compounds/therapeutic use , Caproates/therapeutic use , Cell Line , Cells, Cultured , Cognition/physiology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Glutaminase/metabolism , Glutamine/metabolism , Hippocampus/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/drug effects , Microglia/metabolism , Microglia/pathologyABSTRACT
Malignant gliomas are the most common and aggressive form of primary brain tumors, with a median survival of 15-20 months for patients receiving maximal interventions. Advances in nanomedicine have provided tumor-specific delivery of chemotherapeutics to potentially overcome their off-target toxicities. Recent advances in dendrimer-based nanomedicines have established that hydroxyl-terminated poly(amidoamine) dendrimers can intrinsically target neuroinflammation and brain tumors from systemic administration without the need for targeting moieties. The size of nanocarriers is a critical parameter that determines their tumor-targeting efficiency, intratumor distribution, and clearance mechanism. In this study, we explore the dendrimer size effects on brain tumor targeting capability in two clinically relevant orthotopic brain tumor models, the 9L rat and GL261 mouse models, which capture differing aspects of gliomas. We show that increasing dendrimers from Generation 4 to Generation 6 significantly enhances their tumor accumulation (~10-fold greater at 24 hr), tumor specificity (~2-3 fold higher), and tumor retention. The superior tumor targeting effect of G6 dendrimers is associated with its reduced renal clearance rate, resulting in longer circulation time compared to G4 dendrimers. Additionally, the increase in dendrimer generation does not compromise its homogeneous tumor distribution and intrinsic targeting of tumor-associated macrophages. These results validate the potential for these dendrimers as an effective, clinically translatable platform for effectively targeting tumor-associated macrophages in malignant gliomas.