Induced DC Maturation Research Articles

Light-Controlled Bioorthogonal Chemistry Altered Natural Killer Cell Activity for Boosted Adoptive Immunotherapy.

Natural killer (NK) cell-based immunotherapy has received much attention in recent years. However, its practical application is still suffering from the decreased function and inadequate infiltration of NK cells in the immunosuppressive microenvironment of solid tumors. Herein, we construct light-responsive porphyrin Fe array-armed NK cells (denoted as NK@p-Fe) for cell behavior modulation via bioorthogonal catalysis. By installing cholesterol-modified porphyrin Fe molecules on the NK cell surface, a catalytic array with light-harvesting capabilities is formed. This functionality transforms NK cells into cellular factories capable of catalyzing the production of active agents in a light-controlled manner. NK@p-Fe can generate the active antineoplastic drug doxorubicin through bioorthogonal reactions to enhance the cytotoxic function of NK cells. Beyond drug synthesis, NK@p-Fe can also bioorthogonally catalyze the production of the FDA-approved immune agonist imiquimod (IMQ). The activated immune agonist plays a dual role, inducing dendritic cell maturation for NK cell activation and reshaping the tumor immunosuppressive microenvironment for NK cell infiltration. This work represents a paradigm for the modulation of adoptive cell behaviors to boost cancer immunotherapy by bioorthogonal catalysis.

Innate immune cell activation by adjuvant AS01 in human lymph node explants is age independent.

Vaccine adjuvants are thought to work by stimulating innate immunity in the draining lymph node (LN), although this has not been proven in humans. To bridge the data obtained in animals to humans, we have developed an in situ human LN explant model to investigate how adjuvants initiate immunity. Slices of explanted LNs were exposed to vaccine adjuvants and revealed responses that were not detectable in LN cell suspensions. We used this model to compare the liposome-based AS01 with its components, monophosphoryl lipid A (MPL) and QS-21, and TLR ligands. Liposomes were predominantly taken up by subcapsular sinus-lining macrophages, monocytes, and DCs. AS01 induced DC maturation and a strong proinflammatory cytokine response in intact LN slices but not in dissociated cell cultures, in contrast to R848. This suggests that the onset of the immune response to AS01 required a coordinated activation of LN cells in time and space. Consistent with the robust immune response observed in older adults with AS01-adjuvanted vaccines, the AS01 response in human LNs was independent of age, unlike the response to R848. This human LN explant model is a valuable tool for studying the mechanism of action of adjuvants in humans and for screening new formulations to streamline vaccine development.

Vaccination against neoantigens induced in cross-priming cDC1 in vivo

The conventional type 1 dendritic cells (cDC1) play a pivotal role in protective immunity against pathogens and cancer. However, their low frequency in the blood and tissues limits their use in immune therapy. We have recently described a method to vaccinate against neoantigens that are induced in tumor cells by targeted delivery of a TAP siRNA to dendritic cells using a TLR9 binding CpG oligonucleotide. Since TLR9 is also expressed in immune suppressive myeloid populations TLR9 targeting could reduce the effectiveness of this approach. Here, we describe a modular multivalent antibody platform to target the TAP siRNA to resident Clec9a expressing cDC1 and show that it leads to selective and sustained TAP downregulation in cDC1 and inhibits tumor growth in mice more effectively than CpG targeted siRNA. To induce DC maturation an agonistic CD40 antibody was administered to the siRNA treated mice. To obviate the need for a second drug formulation and reduce the risk of toxicity, we exploited the multivalent nature of this targeting platform to co-deliver the TAP siRNA and a DC maturation agent, a CpG containing oligonucleotide, to cDC1 in vivo and show that it was more effective than Clec9a targeting of TAP siRNA in combination with CD40 antibody. This study describes a way to manipulate the function of cDC1 cells in vivo using a broadly applicable antibody-based targeting platform to deliver multiple biological agents to specific cells in vivo to potentiate (immune) therapy and to probe the biology of specific cell types in their natural settings.

Monophosphoryl lipid A-assembled nanovaccines enhance tumor immunotherapy.

Therapeutic cancer nanovaccines can induce strong antitumor immunity and establish long-term immune memory and have shown potential for curing tumors in some clinical trials. However, weak immunogenicity and safety concerns of nanocarriers limit the clinical translation of some therapeutic nanovaccines. Here, we developed minimal-component cancer nanovaccines, monophosphoryl lipid A (MPLA)-assembled nanovaccines (MANs), that could facilitate the clinical application of nanovaccines. The MANs were formed by protein antigens extracted from chemotherapy-induced tumor cell cultures and the amphiphilic immune adjuvant MPLA. Compared with free chemotherapy-induced antigens, MANs can activate the Toll-like receptor 4 (TLR4)-mediated signalling pathway and promote adaptive immunity against tumor antigens. Mechanistic analysis indicated that MANs induced antigen capture of DCs and promoted the activation of DCs and T cells, thereby optimizing the ratio of CD8+ T/Tregs in tumors and facilitating the transformation of the tumor immune microenvironment (TIME) from "cold" to "hot". In a CT26 colorectal cancer model, MANs+αPD-1 significantly improved the efficacy of αPD-1 treatment. Our work offers a strategy for designing minimal-component cancer nanovaccines with potential clinical benefits. STATEMENT OF SIGNIFICANCE: To address the weak immunogenicity of cancer vaccines and the safety concerns of nanocarriers, we prepared MPLA-assembled nanovaccines (MANs) using chemotherapy induced antigens and the immune adjuvant MPLA to promote cancer vaccines to clinical practice. MANs effectively internalized tumor antigens and induced DC maturation, indicating that the initial anti-tumor response had been activated. MANs+αPD-1 induced APCs, CD8+ T cells and memory T cells with positive anti-tumor effects to migrate to tumor tissue, thus leading to the transformation of the tumor immune microenvironment from "cold" to "hot". At the animal level, the combination of MANs and αPD-1 exerted synergistic effects and significantly enhanced tumor immunotherapy. Therefore, the treatment regimen of MANs+αPD-1 has potential clinical benefits.

NSAID-Au(I) Complexes Induce ROS-Driven DAMPs and Interpose Inflammation to Stimulate the Immune Response against Ovarian Cancer.

Inflammation contributes to the development of ovarian cancer, and chemoresistance is a principal obstacle in ovarian cancer treatment. Herein, we designed and synthesized a series of gold(I) complexes derived from NSAIDs or their analogues. Among them, complex B3 (Npx-Au) displayed higher antitumor activity than cisplatin and other gold(I) complexes. Npx-Au could induce oxidative stress and the damage-associated molecular patterns (DAMPs) process by the inhibition of TrxR activity. Mechanistic studies revealed that simultaneous downregulation of COX-2 and PD-L1 was observed after Npx-Au treatment. Interestingly, in vivo experiments demonstrated that Npx-Au treatment could stimulate the immune response via reducing the expression of PD-L1, inducing DC maturation and increasing the infiltration of T (CD4+ and CD8+) cells. Collectively, our studies found that the gold(I) complex Npx-Au could elicit immunogenic cell death (ICD) and provide a promising strategy for chemotherapy combined with immunotherapy in the treatment of ovarian cancer.

Nanoscale metal-organic framework delivers rapamycin to induce tissue immunogenic cell death and potentiates cancer immunotherapy

Rapamycin has great potential in the antitumor application, but its therapeutic effect is seriously affected by poor water solubility, targeting ability, and low bioavailability. Here, we constructed a novel composite nanomaterial with PCN-224 as a drug carrier and loaded rapamycin, named R@BP@HA. The nanoplate not only improves targeting, but also synergizes rapamycin with PCN-224 to effectively promote tumor cell apoptosis, which subsequently causes immunogenic cell death (ICD), and shows strong therapeutic effect in 4T1 breast cancer model. The treatment effect depends on three main points:(i)Proapoptotic effect of rapamycin on tumor cells;(ii)ROS production by PCN-224-mediated photodynamic therapy;(iii)ICD induced DC maturation, increased immune response and promoted T cell proliferation and differentiation. This nanoplate offers potential antitumor efficacy in combination with chemotherapy, photodynamic therapy, and immunotherapy.

Atorvastatin-induced tolerogenic dendritic cells improve cardiac remodeling by suppressing TLR-4/NF-κB activation after myocardial infarction.

Myocardial infarction (MI) caused by ischemic cardiomyocyte necrosis induces inflammatory responses that strongly affect ventricular remodeling. Tolerogenic dendritic cells (tDCs) can suppress this effect on inflammatory responses. However, the precise role of atorvastatin-induced tDCs in ventricular remodeling after MI remains unclear. To explore the effect of necrotic cardiomyocytes (SNC) and/or atorvastatin on DC function, the expression of CD40, CD80, CD86, and MHC-II was determined using flow cytometry. The protein levels of TLR-4/NF-κB-related molecules were evaluated using western blotting. The infarct area after MI was determined via 2,3,5-triphenyltetrazolium chloride staining. The TUNEL assay was employed to evaluate the apoptosis of cardiomyocytes in heart sections. Masson's trichrome method was used to determine the extent of fibrosis. Compared to the DCs co-cultured with PBS (control), cells co-cultured with Supernatant-IM or Supernatant-NH produced higher levels of inflammatory cytokines, including TNF-α, IL-1, IL-6, IL-12P40, and IL-8. This cytokine production was impaired by atorvastatin treatment. SNC treatment induced DC maturation and enhanced inflammatory cytokine secretion and oxidative stress through TLR-4/NF-κB pathway activation. Compared to that in the PBS-treated group, the left ventricular ejection fraction was significantly improved after tDC treatment. Additionally, compared to that in the PBS-treated group, tDC treatment reduced the left ventricular end-diastolic and end-systolic diameters in mice. Furthermore, treatment with tDCs improved the left ventricular systolic function, attenuated inflammatory cell infiltration, and reduced cardiomyocyte apoptosis, myocardial fibrosis, and infarct size compared to those in the control group. Adoptive transfer of atorvastatin-induced tDCs alleviated post-infarction cardiomyocyte apoptosis and myocardial fibrosis in association with decreased inflammatory cell infiltration and inhibited oxidative stress, likely by suppressing TLR-4/NF-κB activation after myocardial infarction.

Tfh Exosomes Derived from Allergic Rhinitis Promote DC Maturation Through miR-142-5p/CDK5/STAT3 Pathway.

BackgroundDendritic cells (DCs) play an important role in allergen signal presentation. Many studies showed that follicular helper T cells (Tfhs) are related to allergic rhinitis (AR). However, the relationship between Tfhs and DCs and the mechanism of their interaction with AR remain unclear.PurposeTo explore the mechanism of Tfhs on DC maturation in AR.MethodsTfhs were isolated from OVA-sensitized mice and co-cultured with DCs derived from mouse bone marrow. DCs maturity was monitored using flow cytometry and immunofluorescence staining. Exosomes of Tfhs were extracted, and miRNAs inside exosomes were analyzed using RNA-seq to identify differentially expressed genes. Using the TargetScan algorithm, it was predicted that CDK5 is a direct target gene, which is validated in a dual luciferase assay. DCs were treated with miR-142-5p mimics or inhibitors or transfected with CDK5 small interfering RNAs to verify the regulatory effects of miR-142-5p and CDK5 on DC maturation. How CDK5 regulates STAT3 signaling pathway was investigated to elucidate the molecular mechanism of DC maturation. Finally, in an in vivo experiment, the exosomes of AR-derived Tfhs were injected intravenously to detect their promotion of AR.ResultsTfh exosomes derived from AR mice contributed to DC maturation. RNA-seq results showed that miR-142-5p was the differentially decreased gene. Using the TargetScan algorithm, it was predicted that CDK5 was the target gene for the direct action of miR-142-5p. By detecting the effects of changes in the expression levels of miR-142-5p and CDK5 on DC maturation, it was demonstrated that miR-142-5p inhibits DC maturation by inhibiting CDK5 expression. CDK5-regulated STAT3 signaling pathway during DC maturation, and inhibition of the STAT3 signaling pathway can reverse the regulation of miR-142-5p/CDK5 on DC maturation. Finally, in vivo experiment indicated that the injection of AR-derived Tfhs promoted AR in mice.ConclusionTfh-derived exosomes induce DC maturation by regulating miR-142-5p/CDK5/STAT3 signaling pathway, thereby promoting the occurrence of AR.

Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses.

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo, while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses.

SARS-CoV-2 infection activates dendritic cells via cytosolic receptors rather than extracellular TLRs.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes coronavirus disease 2019 (COVID‐19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation, and potentially multiorgan dysfunction. It remains unclear how SARS‐CoV‐2 infection leads to immune activation. The Spike (S) protein of SARS‐CoV‐2 has been suggested to trigger TLR4 and thereby activate immunity. Here, we have investigated the role of TLR4 in SARS‐CoV‐2 infection and immunity. Neither exposure of isolated S protein, SARS‐CoV‐2 pseudovirus nor primary SARS‐CoV‐2 isolate induced TLR4 activation in a TLR4‐expressing cell line. Human monocyte‐derived DCs express TLR4 but not angiotensin converting enzyme 2 (ACE2), and DCs were not infected by SARS‐CoV‐2. Notably, neither S protein nor SARS‐CoV‐2 induced DC maturation or cytokines, indicating that both S protein and SARS‐CoV‐2 virus particles do not trigger extracellular TLRs including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS‐CoV‐2 and, strikingly, efficient type I IFN and cytokine responses. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS‐CoV‐2. These data imply that SARS‐CoV‐2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS‐CoV‐2 early during infection.

Oral administration of Lactobacillus delbrueckii enhances intestinal immunity through inducing dendritic cell activation in suckling piglets.

Lactobacillus delbrueckii (LAB) has been demonstrated to exert versatile beneficial effects on modulating intestinal immunity, increasing gut microbial diversity, promoting growth performance, and even preventing disease onset in pigs. However, the underlying mechanism of LAB-mediated gut immunity regulation in piglets remains unclear. In this study, we found that supplementation of LAB significantly increases serum TNF-α, ileum IL-4, and IL-10 levels compared with the control group. Meanwhile, oral supplementation of LAB-modified gut microbial communities was evidenced by the increased abundance of the Lactobacillus genus in the colon. Mechanistically, LAB induced dendritic cell (DC) maturation and activation, which may be relevant to the activation of NF-κB and MAPK signaling pathways. Moreover, we found that oral administration of LAB during the suckling period shows long-lasting immunomodulatory impacts on intestinal immunity after weaning. Collectively, this study uncovers the mechanism of LAB in regulating the intestinal immunity of piglets, suggesting that LAB can be developed as an immunoenhancing biological agent during the suckling period.

Combination of epigenetic regulation with gene therapy-mediated immune checkpoint blockade induces anti-tumour effects and immune response in vivo

Immunotherapy has become a powerful cancer treatment, but only a small fraction of patients have achieved durable benefits due to the immune escape mechanism. In this study, epigenetic regulation is combined with gene therapy-mediated immune checkpoint blockade to relieve this immune escape mechanism. PPD (i.e., mPEG-b-PLG/PEI-RT3/DNA) is developed to mediate plasmid-encoding shPD-L1 delivery by introducing multiple interactions (i.e., electrostatic, hydrogen bonding, and hydrophobic interactions) and polyproline II (PPII)-helix conformation, which downregulates PD-L1 expression on tumour cells to relieve the immunosuppression of T cells. Zebularine (abbreviated as Zeb), a DNA methyltransferase inhibitor (DNMTi), is used for the epigenetic regulation of the tumour immune microenvironment, thus inducing DC maturation and MHC I molecule expression to enhance antigen presentation. PPD plus Zeb combination therapy initiates a systemic anti-tumour immune response and effectively prevents tumour relapse and metastasis by generating durable immune memory. This strategy provides a scheme for tumour treatment and the inhibition of relapse and metastasis.

Activation of GM-CSF and TLR2 signaling synergistically enhances antigen-specific antitumor immunity and modulates the tumor microenvironment

BackgroundThe major challenge of antitumor immunotherapy is dealing with the immunosuppressive tumor microenvironment, which involves immature myeloid cell accumulation that results in T cell dysfunction. Myeloid cell activation is induced...

Trp2 Peptide-Assembled Nanoparticles with Intrinsically Self-Chelating 64Cu Properties for PET Imaging Tracking and Dendritic Cell-Based Immunotherapy against Melanoma.

Dendritic cell-based immunotherapy, in which the antigen is effectively delivered to dendritic cells and then the dendritic cells stimulated by the antigen migrate to draining lymph nodes (DLNs) to induce the CD8+ T-cell immune response, shows great promise for tumor immunotherapy. In this study, we used coassembled nanoparticles formed by Trp2 antigen and the conjugates of short-chain poly(ethylene glycol) (PEG) and pyropheophorbide-A (PPa) (Trp2/PPa-PEGm) to deliver Trp2 to DCs. Intrinsically self-chelating 64Cu of coassemblies could be used to sensitively image the migration of DCs in vivo by positron emission tomography (PET) imaging. The coassemblies of the Trp2 antigen were efficiently engulfed by DCs without causing DC cytotoxicity in vitro and induced DC maturation. After injection of DCs labeled by coassemblies of the Trp2 antigen, the homing of DCs to DLNs in vivo could be sensitively observed by PET imaging. The C57BL/6 mice injected with DCs containing the Trp2/PPa-PEGm NP showed antigen-specific immune responses including enhanced interferon-γ (IFN-γ) production, splenocyte proliferation, and percentage of IFN-γ-secreting CD8+ T cells. In addition, C57BL/6 mice inoculated with B16-F10 tumor cells showed delayed tumor growth after immunization with the Trp2/PPa-PEGm NP-labeled DC vaccine and enhanced infiltration of CD8+ T cells in tumors.

Modulation of lactate-lysosome axis in dendritic cells by clotrimazole potentiates antitumor immunity

BackgroundDendritic cells (DCs) play a critical role in antitumor immunity, but the therapeutic efficacy of DC-mediated cancer vaccine remains low, partly due to unsustainable DC function in tumor antigen presentation....

The bovine dialysable leukocyte extract IMMUNEPOTENT CRP induces immunogenic cell death in breast cancer cells leading to long-term antitumour memory

BackgroundCancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. IMMUNEPOTENT CRP (ICRP) acts as an immunomodulator and can be cytotoxic to cancer cells. Thus, we evaluated if ICRP induces ICD in BC cells.MethodsImmunogenicity of ICRP-induced cell death was evaluated in vitro, analysing the principal biochemical characteristics of ICD in MCF-7, MDA-MB-231 and 4T1 cells. Ex vivo, we assessed the ability of killed cancer cells (KCC) obtained from ICRP-treated 4T1 cells (ICRP-KCC) to induce DC maturation, T-cell priming and T-cell-mediated cancer cytotoxicity. In vivo, we evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice.ResultsICRP induced caspase-independent, ROS-dependent cell death, autophagosome formation, P-eIF2α, chaperone protein exposure, CD47 loss, ATP and HMBG1 release in BC cells. Additionally, ICRP-KCC promoted DC maturation, which triggered T-cell priming and cancer cytotoxicity. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes.ConclusionsICRP induces ICD in BC cells, leading to long-term antitumour memory.

Portulaca oleracea L. polysaccharides enhance the immune efficacy of dendritic cell vaccine for breast cancer.

Previous studies have reported that Portulaca oleracea L. polysaccharides (POL-P3b) is an immunoregulatory agent. However, few studies exist on POL-P3b as a novel immune adjuvant in combination with the DC vaccine for breast cancer treatment. In this work, a DC vaccine loaded with mouse 4T1 tumor cell antigen was prepared to evaluate the properties of POL-P3b in inducing the maturation and function of DC derived from mouse bone marrow, and then to investigate the effect of the DC vaccine combined with POL-P3b on breast cancer in vivo and in vitro. Morphological changes of DC were observed using scanning electron microscopy. Phenotypic and functional analyses of DC were detected by flow cytometry and allogeneic lymphocyte reaction. Cytokine levels in the DC culture supernatant were detected by ELISA. Western blotting analysis was used for the protein expression of TLR4, MyD88 and NF-κB. Apoptosis detection and protein expression of the tumor tissue were analyzed by TUNEL staining and immunohistochemistry, respectively. The security of POL-P3b was evaluated by the detection of hematological and blood biochemical indicators and pathological analysis for tissues. POL-P3b can induce DC activation and maturation, which is attributed to increasing the specific anti-tumor immune response, and the mechanism of action involved in the TLR4/MyD88/NF-κB signaling pathway. Experimental results in vivo further suggested that the administration of POL-P3b-treated antigen-primed DC achieved remarkable tumor growth inhibition through inducing apoptosis and enhancing immune responses. Moreover, the POL-P3b-treated DC vaccine was able to inhibit lung metastases. The results proved the feasibility of POL-P3b as an edible adjuvant of the DC vaccine for anti-breast cancer therapy.

Modular formation of hyaluronic acid/β-glucan hybrid nanogels for topical dermal delivery targeting skin dendritic cells

Transdermal immunomodulation is of increasing interest as an efficient drug delivery method. It non-invasively delivers drugs directly to skin-resident immune cells, thereby avoiding the first-pass metabolism. Herein, we prepared ovalbumin-conjugated hyaluronic acid-methacrylate (HAMA-OVA) and schizophyllan-methacrylate (SPGMA) hybrid nanogels and investigated their suitability for topical delivery. The particle size was controlled to between 100 and 300 nm using ultrasonication and filtering processes. The nanogels penetrated the porcine stratum corneum layer and were deposited in the dermis via hybridization with HAMA. In addition, the hybridized SPGMA promoted the internalization of the nanogels into dendritic cells (DCs; JAWSII), which resulted in an improvement in the ovalbumin delivery efficiency. In molecular biological assessments, the hybrid nanogels upregulated the DC activation marker interleukin-6 and induced DC maturation, indicating antigen-presenting behavior. These results suggest that HAMA/SPGMA hybrid nanogels are a promising topical delivery carrier for immunomodulation and vaccinations.

Induction of Robust Immune Responses by CpG-ODN-Loaded Hollow Polymeric Nanoparticles for Antiviral and Vaccine Applications in Chickens.

BackgroundPoultry vaccine has limited choices of adjuvants and is facing severe threat of infectious diseases due to ineffective of widely used commercial vaccines. Thus, development of novel adjuvant that offers safe and effective immunity is of urgent need.Materials and MethodsThe present research engineers a novel chicken adjuvant with potent immune-potentiating capability by incorporating avian toll-like receptor 21 (TLR21) agonist CpG ODN 2007 with a poly(lactic-co-glycolic acid) (PLGA)-based hollow nanoparticle platform (CpG-NP), which subsequently assessed ex vivo and in vivo.ResultsCpG-NPs with an average diameter of 164 nm capable of sustained release of CpG for up to 96 hours were successfully prepared. With the ex vivo model of chicken bone marrow-derived dendritic cells (chBMDCs), CpG-NP was engulfed effectively and found to induce DC maturation, promoting dendrite formation and upregulation of CD40, CD80 and CCR7. In addition to enhanced expression of IL-1β, IL-6, IL-12 and IFN-γ, 53/84 immune-related genes were found to be stimulated in CpG-NP-treated chBMDCs, whereas only 39 of such genes were stimulated in free CpG-treated cells. These upregulated genes suggest immune skewing toward T helper cell 1 bias and evidence of improved mucosal immunity upon vaccination with the CpG-NP. The CpG-NP-treated chBMDCs showed protective effects to DF-1 cells against avian influenza virus H6N1 infection. Upon subsequent coupling with infectious bronchitis virus subunit antigen administration, chickens were immunostimulated to acquire higher humoral immune response and protective response against viral challenge.ConclustionThis work presents a novel hollow CpG-NP formulation, demonstrating effective and long-lasting immunostimulatory ability ex vivo and in vivo for chickens, as systemically compared to free CpG. This enhanced immune stimulation benefits from high stability and controlled release of internal component of nanoparticles that improve cellular delivery, lymphoid organ targeting and sustainable DC activation. CpG-NP has broad application potential in antiviral and vaccine development.

Abstract B75: Nanoparticle formulation of the STING agonist SB 11285

Abstract Background: Immunotherapy has emerged as a transformative approach for the treatment of cancer. Recent evidence suggests that the activation of Stimulator of Interferon Genes (STING) pathway in immune calls within the tumor microenvironment (TME) can induce the production of Interferons and cytokines that would lead to the induction of innate and adaptive immunity. We have previously disclosed the discovery and development of SB 11285 as a first-in-class synthetic cyclic dinucleotide STING agonist, which has demonstrated potent antitumor activity, when used alone or combined with other antitumor agents, in several syngeneic mouse and rat tumor models when administered by intratumoral, intravenous, or intraperitoneal routes. Presented here is the development of a controlled-release nanoparticle formulation of SB 11285 for subcutaneous administration. Methods: (a) Nanoparticle formulations of SB 11285. SB 11285-NPs were generated using Polylactic-glycolic acid copolymer (PLGA) using double emulsion and nano-precipitation methods (b) Imaging of nanoparticles: Scanning electron microscopy was performed to visualize and measure size of the nanoparticles. (c) Induction of IRF3 and NF-KB. THP-1 cells and RAW macrophages carrying dual reporter constructs were treated with either SB 11285-NPs or naked SB 11285 for 22hrs and induction of IRF3 and NF-KB was calculated as % fold-change in luminescence compared to vehicle-treated cells. (d) Induction of cytokines: PBMCs were treated with different concentrations of 11285-NPs or naked SB 11285, and cell supernatants were analyzed for IFN-β, TNF-α, and RANTES by ELISA. (e) Dendritic cell maturation assay: PBMCs were differentiated into immature dendritic cells by addition of IL4 and GM-CSF followed by treatment with either SB 11285-NP or naked SB 11285. The maturation of dendritic cells was then evaluated by analyzing expression of CD83 and CD86 by flow cytometry. Results: PLGA formulations of SB 11285 prepared by double-emulsion method with an entrapment efficiency of 30 to 40% were spherical particles with an average diameter of 800nm. The nanoparticles were stable in aqueous media with minimal release of the entrapped SB 11285. In cell-culture studies, SB 11285-NPs showed potent induction of: (a) STING-dependent IRF3 induction in THP-1 cells (EC50: 3nM) as well as RAW macrophages (EC50: 3nM); (b) secretion of IFN-β (35 pg/ml), TNF-α (800 pg/ml), and RANTES (200 pg/ml), when tested at a low concentration of 112nM; and (c) SB 11285-NPs effectively induced DC maturation that was evident by increase in CD83 and CD86 expression. Conclusion: PLGA nanoparticles of SB 11285 were successfully prepared with submicron particle size that demonstrated potent expression of STING-dependent Type I IFN and cytokines in immune cells. The nanoparticles also induced maturation of dendritic cells. In vivo evaluation is in progress. Citation Format: Sreerupa Challa, Leena Suppiah, Dillon Cleary, Anjaneyulu Sheri, Rayomand Gimi, Geeta Meher, Seetharamaiyer Padmanabhan, Sumit Shah, Pranav Bhatt, Jovita Tauro, Radhakrishnan Iyer. Nanoparticle formulation of the STING agonist SB 11285 [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B75.