Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma.

BACKGROUND: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. METHODS: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. RESULTS: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. CONCLUSION: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

Although a variety of urinary cancer markers are available for in vitro diagnosis, inherent problems of urine environment-containing various inorganic/organic ions/molecules that vary in concentration over a 20-fold range or more and significantly attenuate antibody avidity for markers-render conventional immunoassays unsuitable, remaining unresolved and a major challenge. Here we developed a 3D-plus-3D (3p3) immunoassay method, based on a single-step urinary marker detection by 3D-antibody probes, which are free of steric hindrance and capable of omnidirectional capture of markers in a 3D solution. The 3p3 immunoassay showed an excellent performance in the diagnosis of prostate cancer (PCa) through detecting PCa-specific urinary engrailed-2 protein, demonstrating 100% sensitivity and 100% specificity with the urine specimens of PCa-related and other related disease patients and healthy individuals. This innovative approach holds a great potential in opening up a novel clinical route for precise in vitro cancer diagnosis and also pushing urine immunoassay closer to more widespread adoption.

An Optimally Fabricated Platform Guides Cancer-Specific Activation of Chemotherapeutic Drugs and Toxicity-Free Cancer Treatment.

Cancer chemotherapeutic drugs such as doxorubicin, mitomycin C, and gemcitabine, which are mostly small synthetic molecules, are still clinically useful for cancer treatment. However, despite considerable therapeutic efficacy, severe toxicity-associated problems, which are mainly caused by the non-specific mode of action such as chromosomal DNA damage and interference in the DNA replication even in normal cells, remain unresolved and a major challenge for safer and thus more widespread adoption of chemotherapy. Herein, an innovative platform is developed through beneficially integrating core peptide units into highly-ordered, stable, and flexibly guest-adaptable structure of apoferritin, which simultaneously fulfills high-capacity loading of chemotherapeutic drugs compared with the case of FDA-approved antibody-drug conjugates, efficient drug targeting to cancer cells, and cancer cell-specific drug release and activation. This approach dramatically reduces drug toxicity to normal cells, significantly enhances efficacy in in vivo cancer treatment without toxicity to normal organs of mice, and thus is expected to open up a novel clinical route to break through the limits of current cancer chemotherapy.

Synthetic pro-peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae.

Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony-stimulating factor (hG-CSF) in S. cerevisiae, a secretion-enhancing peptide cassette including an hIL-1ß-derived pro-peptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters-N-glycosylation, net negative charge balance, and glycine-rich flexible linker-were investigated in batch cultures of S. cerevisiae. Using a three-stage design involving screening, selection, and optimization, the production and secretion of hG-CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG-CSF produced by the optimized pro-peptide after the final stage increased by 190% compared to that of the original pro-peptide. Although hG-CSF was used as the model protein in the current study, this strategy is applicable to the enhanced production of other heterologous proteins, using S. cerevisiae as the host.

Effects of anxiety on sedation among pediatric patients undergoing esophagogastroduodenoscopy.

We investigated whether the severity of anxiety among children undergoing endoscopy could affect sedation. We prospectively recruited patients under 18 years of age who were scheduled to undergo esophagogastroduodenoscopy (EGD). Baseline anxiety was evaluated using the Spence Children's Anxiety Scale (SCAS). Our analysis considered the type and dose of sedative drugs, degree of sedation, and complications associated with sedation according to the scale score. The mean total SCAS T-scores and each subscale score were significantly higher in the 6-12-year age group. The mean T-score among patients who exhibited irritability during EGD was higher than that among other patients, with significant differences in the Total, Obsessive Disorder, Panic Agoraphobia and General Anxiety subscales. The midazolam doses of children exhibiting irritability were more likely to need higher (p = 0.006). Other sedation-related complications were not associated with the T-scores in each subscale. Total sleep time was not associated with any T-score subscales. Anxiety levels were significantly higher among 6-12-year-olds and children exhibiting irritability. Anxiety was often associated with irritability, difficulties achieving adequate sedation during EGD, and additional sedative drug administration. We recommend higher doses or more potent drugs to facilitate endoscopy for children with high anxiety levels.

Structural and functional characterization of a monoclonal antibody blocking TIGIT.

TIGIT is an immune checkpoint receptor that is expressed on subsets of activated T cells and natural killer (NK) cells. Several ligands for TIGIT, including poliovirus receptor (PVR), are expressed on cancer cells and mediate inhibitory signaling to suppress antitumor activities of the immune cells. Many studies support that the TIGIT signaling is a potential target for cancer immunotherapy. We developed an IgG4-type monoclonal antibody against human TIGIT, designated as MG1131, using a phage display library of single-chain variable fragments (scFvs). MG1131 interacts with TIGIT much more tightly than PVR does. The crystal structure of a scFv version of MG1131 bound to TIGIT was determined, showing that MG1131 could block the PVR-TIGIT interaction and thus the immunosuppressive signaling of TIGIT. Consistently, MG1131 is bound to TIGIT-expressing cells and interferes with PVR binding to these cells. Moreover, MG1131 increased NK cell-mediated tumor killing activities, inhibited immunosuppressive activity of regulatory T (Treg) cells from healthy donors, and restored interferon-γ secretion from peripheral blood mononuclear cells derived from multiple myeloma patients. MG1131 also increased T cell infiltration to the tumor site and inhibited tumor growth in mice. Collectively, these data indicate that MG1131 modulates the effector functions of T cells and NK cells positively and Treg cells negatively.

Structural and Functional Characterizations of Cancer Targeting Nanoparticles Based on Hepatitis B Virus Capsid.

Cancer targeting nanoparticles have been extensively studied, but stable and applicable agents have yet to be developed. Here, we report stable nanoparticles based on hepatitis B core antigen (HBcAg) for cancer therapy. HBcAg monomers assemble into spherical capsids of 180 or 240 subunits. HBcAg was engineered to present an affibody for binding to human epidermal growth factor receptor 1 (EGFR) and to present histidine and tyrosine tags for binding to gold ions. The HBcAg engineered to present affibody and tags (HAF) bound specifically to EGFR and exterminated the EGFR-overexpressing adenocarcinomas under alternating magnetic field (AMF) after binding with gold ions. Using cryogenic electron microscopy (cryo-EM), we obtained the molecular structures of recombinant HAF and found that the overall structure of HAF was the same as that of HBcAg, except with the affibody on the spike. Therefore, HAF is viable for cancer therapy with the advantage of maintaining a stable capsid form. If the affibody in HAF is replaced with a specific sequence to bind to another targetable disease protein, the nanoparticles can be used for drug development over a wide spectrum.

Designed protein- and peptide-based hydrogels for biomedical sciences.

Proteins are fundamentally the most important macromolecules for biochemical, mechanical, and structural functions in living organisms. Therefore, they provide us with diverse structural building blocks for constructing various types of biomaterials, including an important class of such materials, hydrogels. Since natural peptides and proteins are biocompatible and biodegradable, they have features advantageous for their use as the building blocks of hydrogels for biomedical applications. They display constitutional and mechanical similarities with the native extracellular matrix (ECM), and can be easily bio-functionalized via genetic and chemical engineering with features such as bio-recognition, specific stimulus-reactivity, and controlled degradation. This review aims to give an overview of hydrogels made up of recombinant proteins or synthetic peptides as the structural elements building the polymer network. A wide variety of hydrogels composed of protein or peptide building blocks with different origins and compositions - including ß-hairpin peptides, α-helical coiled coil peptides, elastin-like peptides, silk fibroin, and resilin - have been designed to date. In this review, the structures and characteristics of these natural proteins and peptides, with each of their gelation mechanisms, and the physical, chemical, and mechanical properties as well as biocompatibility of the resulting hydrogels are described. In addition, this review discusses the potential of using protein- or peptide-based hydrogels in the field of biomedical sciences, especially tissue engineering.

Abrogation of HLA surface expression using CRISPR/Cas9 genome editing: a step toward universal T cell therapy.

As recent advancements in the chimeric antigen receptor-T cells have revolutionized the way blood cancers are handled, potential benefits from producing off-the-shelf, standardized immune cells entail the need for development of allogeneic immune cell therapy. However, host rejection driven by HLA disparity in adoptively transferred allogeneic T cells remains a key obstacle to the universal donor T cell therapy. To evade donor HLA-mediated immune rejection, we attempted to eliminate T cell's HLA through the CRISPR/Cas9 gene editing system. First, we screened 60 gRNAs targeting B2M and multiple sets of gRNA each targeting α chains of HLA-II (DPA, DQA and DRA, respectively) using web-based design tools, and identified specific gRNA sequences highly efficient for target deletion without carrying off-target effects. Multiplex genome editing of primary human T cells achieved by the newly discovered gRNAs yielded HLA-I- or HLA-I/II-deficient T cells that were phenotypically unaltered and functionally intact. The overnight mixed lymphocyte reactions demonstrated the HLA-I-negative cells induced decreased production of IFN-γ and TNF-α in alloreactive T cells, and deficiency of HLA-I/II in T cells further dampened the inflammatory responses. Taken together, our approach will provide an efficacious pathway toward the universal donor cell generation by manipulating HLA expression in therapeutic T cells.

Tumor-Infiltrating Regulatory T-cell Accumulation in the Tumor Microenvironment Is Mediated by IL33/ST2 Signaling.

Regulatory T cells (Treg) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity; however, the molecular mechanism underlying the accumulation of Tregs in the TME is poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in the TME and had significantly higher expression of immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression-related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared with tumor-infiltrating CD4+Foxp3- conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA-seq and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an IL33 receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor-infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.

Nonimmunogenetic Viral Capsid Carrier with Cancer Targeting Activity.

Although protein nanoparticles (PNPs) (e.g., viral capsids) capable of delivering a broad range of drug agents have shown distinctive advantages over synthetic nanomaterials, PNPs have an intrinsic drawback that hampers their clinical application, that is, potential immunogenicity. Here, a novel method for resolving the immunogenicity problem of PNPs, which is based on the genetic presentation of albumin-binding peptides (ABPs) on the surface of PNP, is reported. ABPs are inserted into the surface of a viral capsid (hepatitis B virus capsid/HBVC) while preserving the native self-assembly function of HBVC. The ABPs effectively gather human serum albumins around HBVC and significantly reduce both inflammatory response and immunoglobulin titer in live mice compared to ABP-free HBVC. Furthermore, ABP-conjugated HBVCs remain within tumors for a longer period than HBVCs conjugated to tumor cell receptor-bindingpeptides, indicating that the ABPs are also capable of enhancing tumor-targeting performance. Although applied to HBVC for proof of concept, this novel approach may provide a general platform for resolving immunogenicity and cancer-targeting problems of PNPs, which enables the development of a variety of PNP-based drug delivery carriers with high safety and efficacy.

Peptide ligand-mediated endocytosis of nanoparticles to cancer cells: Cell receptor-binding- versus cell membrane-penetrating peptides.

The endocytosis-mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands-human ferritin heavy chain (hFTH) nanoparticle. Twenty-four copies of a CMPP(human immunodeficiency virus-derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αv ß3 ) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye-labeled CRBP- and CMPP-presenting nanoparticles were estimated in the in vitro cultures of integrin- and EGFR-overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP- and CRBP-presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT-mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.

Tumor Necrosis Factor-producing T-regulatory Cells Are Associated With Severe Liver Injury in Patients With Acute Hepatitis A.

BACKGROUND AND AIMS: CD4+CD25+Foxp3+ T-regulatory (Treg) cells control immune responses and maintain immune homeostasis. However, under inflammatory conditions, Treg cells produce cytokines that promote inflammation. We investigated production of tumor necrosis factor (TNF) by Treg cells in patients with acute hepatitis A (AHA), and examined the characteristics of these cells and association with clinical factors. METHODS: We analyzed blood samples collected from 63 patients with AHA at the time of hospitalization (and some at later time points) and 19 healthy donors in South Korea. Liver tissues were collected from patients with fulminant AHA during liver transplantation. Peripheral blood mononuclear cells were isolated from whole blood and lymphocytes were isolated from liver tissues and analyzed by flow cytometry. Cytokine production from Treg cells (CD4+CD25+Foxp3+) was measured by immunofluorescence levels following stimulation with anti-CD3 and anti-CD28. Epigenetic stability of Treg cells was determined based on DNA methylation patterns. Phenotypes of Treg cells were analyzed by flow cytometry and an RORγt inhibitor, ML-209, was used to inhibit TNF production. Treg cell suppression assay was performed by co-culture of Treg-depleted peripheral blood mononuclear cells s and isolated Treg cells. RESULTS: A higher proportion of CD4+CD25+Foxp3+ Treg cells from patients with AHA compared with controls produced TNF upon stimulation with anti-CD3 and anti-CD28 (11.2% vs 2.8%). DNA methylation analysis confirmed the identity of the Treg cells. TNF-producing Treg cells had features of T-helper 17 cells, including up-regulation of RORγt, which was required for TNF production. The Treg cells had reduced suppressive functions compared with Treg cells from controls. The frequency of TNF-producing Treg cells in AHA patients' blood correlated with their serum level of alanine aminotransferase. CONCLUSIONS: Treg cells from patients with AHA have altered functions compared with Treg cells from healthy individuals. Treg cells from patients with AHA produce higher levels of TNF, gain features of T-helper 17 cells, and have reduced suppressive activity. The presence of these cells is associated with severe liver injury in patients with AHA.

Near-Infrared Plasmonic Assemblies of Gold Nanoparticles with Multimodal Function for Targeted Cancer Theragnosis.

Here we report a novel assembly structure of near-infrared plasmonic gold nanoparticles (AuNPs), possessing both photoacoustic (PA) and photothermal (PT) properties. The template for the plasmonic AuNP assembly is a bioconjugate between short double-strand DNA (sh-dsDNA) and human methyl binding domain protein 1 (MBD1). MBD1 binds to methylated cytosine-guanine dinucleotides (mCGs) within the sequence of sh-dsDNA. Hexahistidine peptides on the engineered MBD1 function as a nucleation site for AuNP synthesis, allowing the construction of hybrid conjugates, sh-dsDNA-MBD1-AuNPs (named DMAs). By varying the length of sh-dsDNA backbone and the spacer between two adjacent mCGs, we synthesized three different DMAs (DMA_5mCG, DMA_9mCG, and DMA_21mCG), among which DMA_21mCG exhibited a comparable photothermal and surprisingly a higher photoacoustic signals, compared to a plasmonic gold nanorod. Further, epidermal growth factor receptor I (EGFR)-binding peptides are genetically attached to the MBD1 of DMA_21mCG, enabling its efficient endocytosis into EGFR-overexpressing cancer cells. Notably, the denaturation of MBD1 disassembled the DMA and accordingly released the individual small AuNPs (<5 nm) that can be easily cleared from the body through renal excretion without causing accumulation/toxicity problems. This DMA-based novel approach offers a promising platform for targeted cancer theragnosis based on simultaneous PA imaging and PT therapy.

CXCL10 is produced in hepatitis A virus-infected cells in an IRF3-dependent but IFN-independent manner.

Acute hepatitis A caused by hepatitis A virus (HAV) infection is accompanied by severe liver injury in adult patients, and the liver injury is associated with the production of chemokines. Herein, we investigated the mechanism of how HAV infection induces the production of CXCR3 and CCR5 chemokines, such as CXCL10, CCL4 and CCL5. The production of CXCL10, CCL4 and CCL5 was markedly increased by HAV (HM-175/18f) infection in the culture of primary human hepatocytes and HepG2 cells. In particular, CXCL10 was produced in HAV-infected cells, not in neighboring uninfected cells. Moreover, these chemokines were significantly increased in the sera of acute hepatitis A patients. The production of IFN-λs was also robustly induced by HAV infection, and the blocking of secreted IFN-λs partially abrogated the production of CCL4 and CCL5 in HAV-infected cells. However, CXCL10 production was not decreased by the blocking of IFN-λs. Instead, CXCL10 production was reduced by silencing the expression of RIG-I-like receptor (RLR) signal molecules, such as mitochondrial antiviral signaling protein and interferon regulatory factor 3, in HAV-infected cells. In conclusion, HAV infection strongly induces the production of helper 1 T cell-associated chemokines, particularly CXCL10 via RLR signaling, even without secreted IFNs.

Superparamagnetic Gold Nanoparticles Synthesized on Protein Particle Scaffolds for Cancer Theragnosis.

Cancer theragnosis using a single multimodality agent is the next mainstay of modern cancer diagnosis, treatment, and management, but a clinically feasible agent with in vivo cancer targeting and theragnostic efficacy has not yet been developed. A new type of cancer theragnostic agent is reported, based on gold magnetism that is induced on a cancer-targeting protein particle carrier. Superparamagnetic gold-nanoparticle clusters (named SPAuNCs) are synthesized on a viral capsid particle that is engineered to present peptide ligands targeting a tumor cell receptor (TCR). The potent multimodality of the SPAuNCs is observed, which enables TCR-specific targeting, T2 -weighted magnetic resonance imaging, and magnetic hyperthermia therapy of both subcutaneous and deep-tissue tumors in live mice under an alternating magnetic field. Furthermore, it is analytically elucidated how the magnetism of the SPAuNCs is sufficiently induced between localized and delocalized spins of Au atoms. In particular, the SPAuNCs show excellent biocompatibility without the problem of in vivo accumulation and holds promising potential as a clinically effective agent for cancer theragnosis.

Development of large-scale and economic pH control system for outdoor cultivation of microalgae Haematococcus pluvialis using industrial flue gas.

The aim of this study was to develop the economic and effective buffer system for microalgae mass cultivation using industrial flue gas. Due to the continuous flue gas supplement, culture media acidified, therefore cell growth inhibited. Although buffering agent was added, this result increase in cost for overall culture process. Therefore combined buffer system of bicarbonate and phosphate (BP) for large-scale use was investigated. The bicarbonate buffer system generated from CO2 dissolution, additionally phosphate buffer system improves the buffer performance under the continuous CO2 supplementation from flue gas. The microalgae Haematococcus pluvialis was cultivated under autotrophic outdoor conditions using these buffer solutions. As a result, the autotrophic BP buffer system enhanced the biomass and astaxanthin productivity of H. pluvialis to 105% and 103%, respectively. The results confirm that the BP buffer system reduces the cost of microalgal CO2 conversion process, particularly for the outdoor mass cultivation.

Genetic Assembly of Double-Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging.

Hepatitis B virus capsid (HBVC), a self-assembled protein nanoparticle comprised of 180 or 240 subunit proteins, is used as a cage for genetic encapsulation of fluorescent proteins (FPs). The self-quenching of FPs is controlled by varying the spacing between FPs within the capsid structure. Double-layered FP nanoparticle possessing cancer cell-targeting capabilities is also produced by additionally attaching FPs and cancer cell receptor-binding peptides (affibodies) to the outer surface of the capsid. The generically modified HBVC with double layers of mCardinal FPs and affibodies (mC-DL-HBVC) exhibit a high fluorescence intensity and a strong photostability, and is efficiently internalized by cancer cells and significantly stable against intracellular degradation. The mC-DL-HBVC effectively detects tumor in live mice with enhanced tumor targeting and imaging efficiency with far less accumulation in the liver, compared to a conventional fluorescent dye, Cy5.5. This suggests the great potential of mC-DL-HBVC as a promising contrast agent for in vivo tumor fluorescence imaging.

Effects of gold nanoparticle-based vaccine size on lymph node delivery and cytotoxic T-lymphocyte responses.

Although it has been shown that the size of nanoparticle-based vaccines is a key determining factor for the induction of immune responses, few studies have provided detailed analyses of thresholds or critical sizes of nanoparticle vaccines. Here we report effects of the size of gold nanoparticle (GNP)-based vaccines on their efficiency of delivery to lymph nodes (LNs) and induction of CD8+ T-cell responses. We further propose a threshold size of GNPs for use as an effective vaccine. To examine the effects of GNP size, we synthesized GNPs with diameters of 7, 14 and 28nm, and then conjugated them with recombinant ovalbumin (OVA) as a model antigen. The resulting OVA-GNPs had hydrodynamic diameter (HD) of ~10, 22, and 33nm for 7, 14 and 28nm GNPs, respectively and exhibited a size-dependent increase in cellular uptake by dendritic cells (DCs) and subsequent T-cell cross-priming and activation. Upon injection into a mouse footpad, both 22- and 33-nm OVA-GNPs showed much higher delivery efficiency to draining LNs than did 10-nm OVA-GNPs. An ex vivo restimulation assay using OVA as an antigen revealed that frequencies of OVA-specific CD8+ T cells were higher in mice immunized with 22- and 33-nm OVA-GNPs than in those immunized with 10-nm OVA-GNPs; moreover, these cells were shown to be poly-functional. In a tumor-prevention study, 22-nm OVA-GNPs showed greater antitumor efficacy, and higher infiltration of CD8+ T-cells and greater tumor cell apoptosis and cell death than 10-nm OVA-GNPs. Taken together, our results suggest that the size threshold for induction of potent cellular responses and T-cell poly-functionality by GNPs lies between 10nm and 22nm, and highlight the importance of nanoparticle size as a critical parameter in designing and developing nanoparticle-based vaccines.

Engineered Human Ferritin Nanoparticles for Direct Delivery of Tumor Antigens to Lymph Node and Cancer Immunotherapy.

Efficient delivery of tumor-specific antigens (TSAs) to lymph nodes (LNs) is essential to eliciting robust immune response for cancer immunotherapy but still remains unsolved. Herein, we evaluated the direct LN-targeting performance of four different protein nanoparticles with different size, shape, and origin [Escherichia coli DNA binding protein (DPS), Thermoplasma acidophilum proteasome (PTS), hepatitis B virus capsid (HBVC), and human ferritin heavy chain (hFTN)] in live mice, using an optical fluorescence imaging system. Based on the imaging results, hFTN that shows rapid LN targeting and prolonged retention in LNs was chosen as a carrier of the model TSA [red fluorescence protein (RFP)], and the flexible surface architecture of hFTN was engineered to densely present RFPs on the hFTN surface through genetic modification of subunit protein of hFTN. The RFP-modified hFTN rapidly targeted LNs, sufficiently exposed RFPs to LN immune cells during prolonged period of retention in LNs, induced strong RFP-specific cytotoxic CD8+ T cell response, and notably inhibited RFP-expressing melanoma tumor growth in live mice. This suggests that the strategy using protein nanoparticles as both TSA-carrying scaffold and anti-cancer vaccine holds promise for clinically effective immunotherapy of cancer.