Cholesteryl Pullulan Research Articles

Induction of Mucosal IgA-Mediated Protective Immunity Against Nontypeable Haemophilus influenzae Infection by a Cationic Nanogel-Based P6 Nasal Vaccine.

Nontypeable Haemophilus influenzae (NTHi) strains form a major group of pathogenic bacteria that colonizes the nasopharynx and causes otitis media in young children. At present, there is no licensed vaccine for NTHi. Because NTHi colonizes the upper respiratory tract and forms biofilms that cause subsequent infectious events, a nasal vaccine that induces NTHi-specific secretory IgA capable of preventing biofilm formation in the respiratory tract is desirable. Here, we developed a cationic cholesteryl pullulan–based (cCHP nanogel) nasal vaccine containing the NTHi surface antigen P6 (cCHP-P6) as a universal vaccine antigen, because P6 expression is conserved among 90% of NTHi strains. Nasal immunization of mice with cCHP-P6 effectively induced P6-specific IgA in mucosal fluids, including nasal and middle ear washes. The vaccine-induced P6-specific IgA showed direct binding to the NTHi via the surface P6 proteins, resulting in the inhibition of NTHi biofilm formation. cCHP-P6 nasal vaccine thus protected mice from intranasal NTHi challenge by reducing NTHi colonization of nasal tissues and eventually eliminated the bacteria. In addition, the vaccine-induced IgA bound to different NTHi clinical isolates from patients with otitis media and inhibited NTHi attachment in a three-dimensional in vitro model of the human nasal epithelial surface. Therefore, the cCHP-P6 nanogel nasal vaccine induced effective protection in the airway mucosa, making it a strong vaccine candidate for preventing NTHi-induced infectious diseases, such as otitis media, sinusitis, and pneumonia.

Protein antigen conjugated with cholesteryl amino-pullulan nanogel shows delayed degradation in dendritic cells and augmented immunogenicity

Carriers that augment delivery, immunogenicity or both are crucial in the development of vaccines especially component vaccines as components of pathogens are often poorly immunogenic. Cholesteryl pullulan (CHP) that forms nano-sized hydrogel (nanogel) and encapsulates proteins was shown to be useful in the delivery of vaccines. Here we demonstrate that subcutaneous immunization of mice with bovine serum albumin (BSA) chemically conjugated to NH2-CHP nanogel induces strong antibody production. This augmented antibody production requires covalent conjugation between BSA and CHP, but does not require nanogel formation. Conjugation of NH2-CHP nanogel induces persistence of BSA in dendritic cells (DCs) in vivo. As resistance to lysosomal degradation was previously shown to augment antigen presentation by DCs, conjugation of antigens with CHP nanogel may enhance antibody production to antigens by delaying lysosomal degradation. Therefore, delayed degradation of antigens by covalent conjugation with nanoparticles may be a good strategy for the development of effective vaccines.

IMMU-08. NOVEL NANOPARTICLE-BASED DELIVERY OF H3.3K27M PEPTIDE TO TUMOR-ASSOCIATED MACROPHAGES ENHANCES THE TUMOR HOMING OF H3.3K27M-TCR TRANSDUCED T-CELLS IN HLA-A2/DR1 TRANSGENIC MICE WITH H3.3K27M+

Abstract We have identified a novel HLA-A*02:01-restricted CD8 T-cell epitope encompassing the H3.3K27M mutation and a corresponding high-affinity T-cell receptor (TCR) that recognizes the epitope. While the development of adoptive cell transfer therapy using TCR-transduced T-cells holds a promise, we still need to overcome multiple challenges, such as suboptimal T-cell trafficking and the immunosuppressive environment of malignant glioma. For example, tumor-associated macrophages (TAMs) mediate immunosuppression but do not function as effective antigen-presenting cells. We have developed a novel cholesteryl pullulan (CHP) nanogel as a highly biocompatible and efficient vaccine delivery system targeting TAMs. In this study, we investigated whether the CHP nanogel loaded with the H3.3K27M peptide would deliver the peptide to TAMs and convert TAMs to better antigen-presenting cells that enhance the anti- H3.3K27M+ glioma activity of the TCR-transduced T-cells. As a clinically relevant mouse model, we used HLA-A2/HLA-DR1-transgenic mice and generated a syngeneic glioma cell line that expresses H3.3K27M from their astrocytes. We also generated a retroviral vector encoding the H3.3K27M-specific TCR for transduction of mouse T cells. HLA-A2/HLA-DR1-transgenic mice bearing day 16 intracerebral H3.3K27M+ glioma received an intravenous administration of the CHP nanogel along with poly-ICLC, a Toll-like receptor 3 agonist. The mice then received an intravenous infusion of TCR-transduced or control, non-transduced T-cells on the following day. The triple combination regimen with the CHP, poly-ICLC and TCR-transduced T-cells significantly suppressed the tumor growth, associated with increased levels of T-cell infiltration into the tumors compared with the dual-therapy with poly-ICLC and TCR-T-cells without the CHP. Furthermore, TAMs isolated from CHP-treated mice showed evidence of CHP-uptake, abilities to stimulate proliferation of TCR-transduced T-cells, and higher levels of HLA.A2 expression. These results suggest that the antigen-loaded CHP nanogel can promote the local antigen-presentation to T-cells and represent a promising approach for improving the efficacy of adoptive T-cell therapy for gliomas.

A nanogel-based trivalent PspA nasal vaccine protects macaques from intratracheal challenge with pneumococci

Current polysaccharide-based pneumococcal vaccines are effective but not compatible with all serotypes of Streptococcus pneumoniae. We previously developed an adjuvant-free cationic nanogel nasal vaccine containing pneumococcal surface protein A (PspA), which is expressed on the surfaces of all pneumococcal serotypes. Here, to address the sequence diversity of PspA proteins, we formulated a cationic nanogel-based trivalent pneumococcal nasal vaccine and demonstrated the vaccine’s immunogenicity and protective efficacy in macaques by using a newly developed nasal spray device applicable to humans. Nasal vaccination of macaques with cationic cholesteryl pullulan nanogel (cCHP)-trivalent PspA vaccine effectively induced PspA-specific IgGs that bound to pneumococcal surfaces and triggered complement C3 deposition. The immunized macaques were protected from pneumococcal intratracheal challenge through both inhibition of lung inflammation and a dramatic reduction in the numbers of bacteria in the lungs. These results demonstrated that the cCHP-trivalent PspA vaccine is an effective candidate vaccine against pneumococcal infections.

Characterization and Specification of a Trivalent Protein-Based Pneumococcal Vaccine Formulation Using an Adjuvant-Free Nanogel Nasal Delivery System.

We previously developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan. Here, we generated three pneumococcal surface protein A (PspA) fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel and mixed the three resulting monovalent formulations into a trivalent nanogel-PspA formulation. First, to characterize the nanogel-PspA formulations, we used native polyacrylamide gel electrophoresis (PAGE) to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, we adopted two methods-a densitometric method based on lithium dodecyl sulfate (LDS)-PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)-to determine the PspA content in the nanogel formulations. Third, treatment of nanogel-PspA formulations by adding methyl-β-cyclodextrin released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. However, when nanogel-PspA formulations were heat-treated at 80 °C for 16 h, CD spectroscopy showed that each PspA was released in a denatured form. Fourth, we confirmed that the nanogel-PspA formulations were internalized into nasal mucosa effectively and that each PspA was gradually released from the nanogel in epithelial cells in mice. Fifth, LDS-PAGE densitometry and ELISA both indicated that the amount of trivalent PspA was dramatically decreased in the heat-treated nanogel compared with that before heating. When mice were immunized nasally using the heat-treated formulation, the immunologic activity of each PspA was dramatically reduced compared with that of the untreated formulation; in both cases, the immunologic activity correlated well with the content of each PspA as determined by LDS-PAGE densitometry and ELISA. Finally, we confirmed that the trivalent nanogel-PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent PspA nasal vaccine formulation.

First-in-human phase I clinical trial of the NY-ESO-1 protein cancer vaccine with NOD2 and TLR9 stimulants in patients with NY-ESO-1-expressing refractory solid tumors

Cholesteryl pullulan (CHP) is a novel antigen delivery system. CHP and New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen complexes (CHP-NY-ESO-1) present multiple epitope peptides to the MHC class I and II pathways. Adjuvants are essential for cancer vaccines. MIS416 is a non-toxic microparticle that activates immunity via the nucleotide-binding oligomerization domain 2 (NOD2) and TLR9 pathways. However, no reports have explored MIS416 as a cancer vaccine adjuvant. We conducted a first-in-human clinical trial of CHP-NY-ESO-1 with MIS416 in patients with NY-ESO-1-expressing refractory solid tumors. CHP-NY-ESO-1/MIS416 (μg/μg) was administered at 100/200, 200/200, 200/400 or 200/600 (cohorts 1, 2, 3 and 4, respectively) every 2 weeks for a total of 6 doses (treatment phase) followed by one vaccination every 4 weeks until disease progression or unacceptable toxicity (maintenance phase). The primary endpoints were safety and tolerability, and the secondary endpoint was the immune response. In total, 26 patients were enrolled. Seven patients (38%) continued vaccination in the maintenance phase. Grade 3 drug-related adverse events (AEs) were observed in six patients (23%): anorexia and hypertension were observed in one and five patients, respectively. No grade 4–5 drug-related AEs were observed. Eight patients (31%) had stable disease (SD). Neither augmentation of the NY-ESO-1-specific IFN-γ-secreting CD8+ T cell response nor an increase in the level of anti-NY-ESO-1 IgG1 was observed as the dose of MIS416 was increased. In a preclinical study, adding anti-PD-1 monoclonal antibody to CHP-NY-ESO-1 and MIS416 induced significant tumor suppression. This combination therapy is a promising next step.

1214P - Randomized phase II clinical trial of NY-ESO-1 protein vaccine combined with cholesteryl pullulan (CHP-NY-ESO-1) in resected esophageal cancer patients

BackgroundSince PD-1/PD-L1 blockade has displayed clinical efficacy in esophageal cancer patients, an immunological therapeutic approach such as a cancer vaccine will be realistic in clinics. NY-ESO-1, one of cancer-testis antigens, is expressed in approximately 30% esophageal squamous cell carcinoma (ESCC). Cholesteryl pullulan (CHP) is an antigen delivery system to antigen-presenting cells including macrophages. The complex of CHP and NY-ESO-1 protein (CHP-NY-ESO-1) is a vaccine that activates CD4+ and CD8+ T cells. We aimed to evaluate clinical efficacy of the CHP-NY-ESO-1 for esophageal cancer patients after radical surgery in a randomized phase II trial. Methods54 NY-ESO-1-expressing ESCC patients who underwent radical surgery following neoadjuvant chemotherapy of cisplatin/5-FU were randomized to two arms, CHP-NY-ESO-1 vaccine and observation as a control arm. The vaccine was composed of 200mg full-length NY-ESO-1 protein, which was subcutaneously given 15 doses with 2 or 4-week interval for 12 months. Primary endpoints were disease-free survival (DFS) and safety. Secondary endpoints were immune-responses and overall survival (OS). 49 patients were evaluated for DFS and OS. ResultsDFS in 2 years are 56.0% and 58.3% in the vaccine arm and in the control. OS in 2 years are 76.0% and 79.2%, respectively. No differences were seen between the vaccine and the control group. Subgroup analysis demonstrated that T-bet+ CD8+ T cell infiltration was significantly correlated to DFS and that PD-L1-expression in tumors showed unfavorable tendency for the vaccine group. Exploratory analysis of intra-cohort correlations among the vaccinated patients revealed that 5% or more expression of NY-ESO-1 and high polymeric immunoglobulin receptor (PIGR)-gene expression in tumors were favorable factors. ConclusionsThe clinical trial revealed that CHP-NY-ESO-1 vaccine alone did not display clinical efficacy compared to the control. It suggested that CHP-NY-ESO-1 vaccine would be indicated to>5% NY-ESO-1 and/or high PIGR gene-expressing esophageal tumors that are infiltrated with activated T cells. Clinical trial identificationUMIN000007905. Legal entity responsible for the studyThe authors. FundingJapan Agency for Medical Research and Development. DisclosureAll authors have declared no conflicts of interest.

Phase I/II clinical trial of NY-ESO-1-specific TCR-engineered T-cell transfer combined with a novel T-cell stimulator CHP:NE1 for patients with refractory soft tissue sarcoma.

TPS11074 Background: Combination therapy to enhance the efficacy of T cell receptor (TCR)-engineered T cells (TCR-T) has received increasing attention. We found that a combination therapy of TCR-T and a long peptide vaccine with CpG adjuvant without lymphodepletion regimen caused regression of immune checkpoint inhibitor-resistant sarcoma in a preclinical mouse model. Based on this finding, we initiated a clinical trial of TBI-1301 combined with CHP:NE1 without lymphodepletion for the patients with NY-ESO-1-expressing advanced soft tissue sarcoma. TBI-1301 is an NY-ESO-1157–165/HLA-A*02:01- or -A*02:06-specific TCR-T engineered to reduce endogenous TCR mRNA expression. CHP:NE1 is a novel T cell stimulator consisting of NY-ESO-1 long peptide antigen, cholesteryl pullulan (CHP) nanogel, and CpG oligoDNA. CHP nanogel is used for efficient delivery of long peptide antigen into the lymph nodes. CpG oligoDNA is a TLR9 agonist and used as an adjuvant. CHP:NE1 is expected to reinforce TBI-1301 T cells in the lymph nodes. Methods: This is an investigator-initiated multi-institutional first-in-human phase I/II clinical trial. TBI-1301 is infused at 5×109 cells one day after subcutaneous injection of CHP:NE1. CHP:NE1 is injected again 7 days after TBI-1301 infusion. This cycle is repeated once more. Lymphodepletion using cyclophosphamide and/or fludarabine is not performed. Key inclusion criteria include: refractory soft tissue sarcoma with NY-ESO-1 antigen expression, HLA-A*02:01- or HLA-A*02:06- positive, ECOG Performance Status 0 or 1, and adequate organ function. The primary objective is to assess the safety and efficacy in the phase 1 and 2 parts, respectively. The secondary objective is to assess the efficacy and immune response (blood concentration, immunophenotype and activity of TBI-1301) in the phase 1 part and the safety and immune response in the phase 2 part. Enrollment in this study is currently ongoing. Clinical trial information: JMA-IIA00346.

NY-ESO-1 antigen expression and immune response are associated with poor prognosis in MAGE-A4-vaccinated patients with esophageal or head/neck squamous cell carcinoma.

MAGE-A4 antigen is a cancer-testis antigen that is frequently expressed in tumor tissues. Cholesteryl pullulan (CHP) is a novel antigen delivery system for cancer vaccines. This study evaluated the safety, immune responses and clinical outcomes of patients who received a CHP-MAGE-A4 vaccine. Twenty-two patients with advanced or metastatic cancer were enrolled, and were subcutaneously vaccinated with either 100 μg or 300 μg of CHP-MAGE-A4. Seven and 15 patients, respectively, were repeatedly vaccinated with 100 μg or 300 μg of CHP-MAGE-A4; patients in both groups received a median of 7 doses. No serious adverse events related to the vaccine were observed. Of 7 patients receiving the 100 μg dose, 2 (29%) showed immune responses, compared with 3 of the 14 (21%) patients who received the 300 μg dose. In total, MAGE-A4-specific antibody responses were induced in 5 of 21 (24%) patients. No differences in survival were seen between patients receiving the 100 μg and 300 μg doses, or between immune responders and non-responders. Eleven (50%) patients had pre-existing antibodies to NY-ESO-1. In 16 patients with esophageal or head/neck squamous cell carcinoma, the survival time was significantly shorter in those who had NY-ESO-1-co-expressing tumors. Patients with high pre-existing antibody responses to NY-ESO-1 displayed worse prognosis than those with no pre-existing response. Therefore, in planning clinical trials of MAGE-A4 vaccine, enrolling NY-ESO-1-expressing tumor or not would be a critical issue to be discussed. Combination vaccines of MAGE-A4 and NY-ESO-1 antigens would be one of the strategies to overcome the poor prognosis.

First-in-human phase I clinical trial of NY-ESO-1 protein cancer vaccine with a novel adjuvant MIS416, NOD2 and TLR9 stimulant, for patients with NY-ESO-1 expressing solid tumors.

e15176Background: Complexes of Cholesteryl pullulan and NY-ESO-1 antigen (CHP-NY-ESO-1) present multiple epitope peptides to both the MHC class I and class II pathways. MIS416 is a non-toxic microp...

Time-dependent transition of the immunoglobulin G subclass and immunoglobulin E response in cancer patients vaccinated with cholesteryl pullulan-melanoma antigen gene-A4 nanogel.

A phase I+II clinical trial of vaccination with MAGE-A4 protein complexed with cholesteryl pullulan melanoma antigen gene-A4 nanogel (CHP-MAGE-A4) is currently underway in patients with MAGE-A4-expressing cancer. In the present study, the primary phase I endpoint was to test the safety of the administration of 300 µg CHP-MAGE-A4 with and without OK-432. Another aim of the study was to clarify the details of the specific humoral immune response to vaccination. The 9 patients enrolled for phase I were vaccinated 6 times, once every 2 weeks: 3 patients with 100 µg and 3 patients with 300 µg CHP-MAGE-A4, and 3 patients with 300 µg CHP-MAGE-A4 plus 0.5 clinical units of OK-432. Toxicities were assessed using Common Terminology Criteria for Adverse Events v3.0. Clinical response was evaluated by modified Response Evaluation Criteria in Solid Tumours. Immunological monitoring of anti-MAGE-A4-specific antibodies was performed by ELISA of pre- and post-vaccination patient sera. The 6 vaccinations produced no severe adverse events. Stable disease was assessed in 4/9 patients. Anti-MAGE-A4 total immunoglobulin (Ig)G titers increased in 7/9 patients. Efficacious anti-MAGE-A4 IgG1, 2 and 3 antibody responses were observed in 7/9 patients. Among them, positive conversions to T helper 2 (Th2)-type antibody responses (IgG4 and IgE) were observed after frequent vaccination in 4/7 patients. The Th2 conversion was possibly associated with undesirable clinical observations, including progressive disease and the appearance of a new relapse lesion. The present study suggested that frequent vaccinations activated a Th2-dominant status in the cancer patients. The identification of a time-dependent IgG subclass and IgE antibody production during vaccination protocols may be a useful surrogate marker indicating a potentially undesirable change of the immunological environment for an effective antitumor immune response in cancer patients.

PD32-05 PHASE I CLINICAL STUDY ON THE COMBINATION THERAPY OF CHP-NY-ESO-1 CANCER VACCINE AND MIS416 FOR THE TREATMENT OF PATIENTS WITH NY-ESO-1 EXPRESSING REFRACTORY UROTHELIAL CANCER OR CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Advanced (including Drug Therapy) III1 Apr 2016PD32-05 PHASE I CLINICAL STUDY ON THE COMBINATION THERAPY OF CHP-NY-ESO-1 CANCER VACCINE AND MIS416 FOR THE TREATMENT OF PATIENTS WITH NY-ESO-1 EXPRESSING REFRACTORY UROTHELIAL CANCER OR CASTRATION-RESISTANT PROSTATE CANCER Takeshi Sasaki, Mikiya Ishihara, Hideki Kanda, Yasuhide Hori, Norihito Soga, Naozumi Harada, Yoshihiro Miyahara, Shinichi Kageyama, Hiroshi Shiku, and Yoshiki Sugimura Takeshi SasakiTakeshi Sasaki More articles by this author , Mikiya IshiharaMikiya Ishihara More articles by this author , Hideki KandaHideki Kanda More articles by this author , Yasuhide HoriYasuhide Hori More articles by this author , Norihito SogaNorihito Soga More articles by this author , Naozumi HaradaNaozumi Harada More articles by this author , Yoshihiro MiyaharaYoshihiro Miyahara More articles by this author , Shinichi KageyamaShinichi Kageyama More articles by this author , Hiroshi ShikuHiroshi Shiku More articles by this author , and Yoshiki SugimuraYoshiki Sugimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.681AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES NY-ESO-1 antigen is a cancer-testis antigen that is exclusively expressed in several types of cancer, aside from expression in the normal testis and placenta. This antigen is considered an ideal target for cancer immunotherapy. Cholesteryl pullulan (CHP) is a novel antigen delivery system. Complexes of CHP and NY-ESO-1 antigen (CHP-NY-ESO-1) present multiple epitope peptides to both the MHC class I and class II pathways. MIS416 is a non-toxic microparticle adjuvant that activates immune responses via NOD-2 and TLR9 pathways. Vaccination studies in mice had demonstrated that CHP-NY-ESO-1 with MIS416 enhanced anti-tumor efficacy. This study evaluated the safety, tolerability, and clinical efficacy of CHP-NY-ESO-1 with adjuvant MIS416 in NY-ESO-1-expressing patients (pts) with refractory urothelial cancer (UC) or castration-resistant prostate cancer (CRPC) (Clinical trial registration number, UMIN000005246). METHODS Archival or newly obtained tumor samples from pts were screened for NY-ESO-1 expression using immunohistochemistry and RT-PCR. Eligible pts were those with NY-ESO-1 expression in ≥1% of tumor cells and/or ≥NY-ESO-1 1 copy/GADPH 104 copy. Pts received CHP-NY-ESO-1/MIS416 100 μg/200 μg, 200 μg/200 μg, 200 μg/400 μg, or 200 μg/600 μg every 2 wk 6 doses followed by every 4 wks vaccination until disease progression or unacceptable toxicity was observed. Response was assessed at 12 wks by CT and bone scan per RECIST v1.1 and serum PSA levels. The primary endpoints were safety and tolerability, and the secondary endpoints were clinical efficacy and quality of life. RESULTS A total of 16 pts were enrolled (4 pts with UC and 12 pts with CRPC). Median age was 74 y (range, 48-84). All pts had prior therapies (chemotherapy, radiation, and/or surgery). Five were repeatedly vaccinated with 100 µg (median 4.5 [1-7] doses) and 10 pts with 200 μg (6 [3-37] doses) of CHP-NY-ESO-1. Ten pts (63%) received ≥6 doses. One pt remains on therapy. G1-2 drug-related adverse event (AE) was observed in all pts, most commonly injection site reaction (n = 16), malaise (n = 3), sinus tachycardia (n = 3), and hyperkalemia (n = 3). Grade 3 drug-related AEs were observed in 5 pts (31%), hypertension (n = 3) and anorexia (n = 1). No G4 drug-related AEs were observed. All pts with refractory UC had PD <6 doses. In CRPC pts, 4 pts (48%) had SD, 7 pts (58%) had PD, and one pt had N/A. No significant difference was seen in QLQ-C30 symptom score during treatment. CONCLUSIONS CHP-NY-ESO-1 with adjuvant MIS416 demonstrates acceptable safety and tolerability in refractory UC and CRPC pts with promising anti-tumor efficacy in CRPC pts. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e762 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takeshi Sasaki More articles by this author Mikiya Ishihara More articles by this author Hideki Kanda More articles by this author Yasuhide Hori More articles by this author Norihito Soga More articles by this author Naozumi Harada More articles by this author Yoshihiro Miyahara More articles by this author Shinichi Kageyama More articles by this author Hiroshi Shiku More articles by this author Yoshiki Sugimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Highlights from the latest research in nanomedicine.

2015 A nanoparticulate hydrogel anticancer vaccine Evaluation of: Muraoka D, Harada N, Hayashi T et al. Nanogel-based immunologically stealth vaccine targets macrophages in the medulla of lymph node and induces potent antitumor immunity. ACS Nano 8(9), 9209–9218 (2014). Therapeutic cancer vaccines are designed to stimulate highly specific immune responses against cancers by targeting tumor-associated antigens. Various vaccines have utilized a wide range of immunogenic agents, including peptides, recombinant viruses, tumor cells and primed antigen-presenting dendritic cells, in combination with immunogenicityboosting adjuvants, immune suppression inhibitors or traditional chemotherapeutic agents [1]. However, cancer vaccines have been of varying effectiveness in clinical trials due to tumor characteristics such as antigenic heterogeneity and immunosuppressive mechanisms. Antigenic heterogeneity may allow the tumors to develop resistance against single-target immunogenic agents, while immunosuppressive mechanisms reduce the effectiveness of the immune response and alter the self-recognition mechanisms, causing cancerous cells to be ignored. Furthermore, tumor-associated antigen-targeting agents show varying immunogenic properties, with tradeoffs between specificity and effectiveness [1–3]. Nanoparticles have been of interest as efficacy-enhancing vaccine delivery agents due to their size and customizability. They are small enough to move into lymphatic systems and enter antigen-presenting cells through engulfment, and can be designed for specific parameters in terms of shape, surface properties and composition in order to enable antigen delivery specificity and presentation longevity [4,5]. Muraoka et al. developed hydrogel nanoparticles (nanogels) of approximately 50 nm in diameter using cholesteryl pullulan (CHP) polysaccharides, which self-assemble in water. These nanogels stably complex with polypeptides through hydrophobic interactions. Stability was shown for over 40 h in serum using nanogels complexed with a long-peptide antigen (LPA,) which either included the murine tumor-specific antigen mERK2 or the human tumor antigen MAGE-A4. Due to their small particle size, lack of charge and potential ligand sites, the CHP–LPA nanogel complexes travel efficiently in the lymphatic system to the draining lymph node, escaping uptake by lymph node dendritic cells until they reach the central medulla portion of the node. There, they are largely engulfed by medullary macrophages. Cellular uptake was evaluated through flow cytometry assessments of rhodamine-labeled CHP nanogels upon immune cell populations isolated from the draining lymph node. LPA that was complexed with other formulations did not show similar uptake patterns. Medullary macrophage antigen cross-presentation was assessed with CHP–mERK2 LPA, a Toll-like receptor 9 agonist and a CpG oligodeoxynucleotide adjuvant. The results showed successful T-cell recognition of mERK2-derived tumor epitopes mainly with the macrophage cells purified from the lymph nodes, with weak T-cell responses from purified dendritic cell populations. These outcomes were confirmed by impaired Highlights from the latest research in nanomedicine

Cholesteryl Pullulan Encapsulated TNF-α Nanoparticles Are an Effective Mucosal Vaccine Adjuvant against Influenza Virus.

We encapsulated tumor necrosis factor-α (TNF-α), a major proinflammatory cytokine, into cholesteryl pullulan (CHP) to prepare TNF/CHP nanoparticles. In this report, we describe the immune-enhancing capability of the nanoparticles to act as a vaccine adjuvant. TNF/CHP nanoparticles showed excellent storage stability and enhanced host immune responses to external immunogens. The nanoparticles were effective via the nasal route of administration for inducing systemic IgG1 as well as mucosal IgA. We applied the nanoparticles in a model experimental influenza virus infection to investigate their adjuvant ability. TNF/CHP nanoparticles combined with a conventional split vaccine protected mice via nasal administration against a lethal challenge of A/PR/8/34 (H1N1) influenza virus. Mechanistic studies showed that the nanoparticles enhanced antigen uptake by dendritic cells (DCs) and moderately induced the expression of inflammation-related genes in nasopharynx lymphoid tissue (NALT), leading to the activation of both B and T cells. Preliminary safety study revealed no severe toxicity to TNF/CHP nanoparticles. Slight-to-moderate influences in nasal mucosa were observed only in the repeated administration and they seemed to be reversible. Our data show that TNF/CHP nanoparticles effectively enhance both humoral and cellular immunity and could be a potential adjuvant for vaccines against infectious diseases, especially in the mucosa.

Nanogel-based immunologically stealth vaccine targets macrophages in the medulla of lymph node and induces potent antitumor immunity.

Because existing therapeutic cancer vaccines provide only a limited clinical benefit, a different vaccination strategy is necessary to improve vaccine efficacy. We developed a nanoparticulate cancer vaccine by encapsulating a synthetic long peptide antigen within an immunologically inert nanoparticulate hydrogel (nanogel) of cholesteryl pullulan (CHP). After subcutaneous injection to mice, the nanogel-based vaccine was efficiently transported to the draining lymph node, and was preferentially engulfed by medullary macrophages but was not sensed by other macrophages and dendritic cells (so-called "immunologically stealth mode"). Although the function of medullary macrophages in T cell immunity has been unexplored so far, these macrophages effectively cross-primed the vaccine-specific CD8(+) T cells in the presence of a Toll-like receptor (TLR) agonist as an adjuvant. The nanogel-based vaccine significantly inhibited in vivo tumor growth in the prophylactic and therapeutic settings, compared to another vaccine formulation using a conventional delivery system, incomplete Freund's adjuvant. We also revealed that lymph node macrophages were highly responsive to TLR stimulation, which may underlie the potency of the macrophage-oriented, nanogel-based vaccine. These results indicate that targeting medullary macrophages using the immunologically stealth nanoparticulate delivery system is an effective vaccine strategy.

P56. A novel cancer vaccine with nanogel-based antigen transporter and sequence-optimised long peptide antigen

Background The limited success of cancer vaccines in clinical could be owing to their low efficacy that can be largely affected by their formulation. Vaccine formulation usually consists of three components, antigen, delivery system, and adjuvant. Recently, long peptide antigen (LPA) has been attracting interest, because (1) one LPA can provide antigen presenting cells (APCs) with multiple T cell epitopes and (2) it avoids undesired presentation by non-professional APCs that can lead to the exhaustion of antigen-specific T cells. While a LPA can include multiple epitopes, the prerequisite design of junction of epitopes (inter-epitope sequence, IES) for robust immunogenicity is not yet established, and we explored it in this study. On the other hand, accumulating data has been suggesting the importance of delivery system for cancer vaccines. By employing a novel nanogelbased delivery system, we tried to improve the immunogenicity of LPA vaccine. Materials and methods LPA was designed to include three mouse cytotoxic T cell (CTL) epitopes that were linked with the IES of oligotyrosine, oligothreonine, oligoglycine or oligoproline. The complex of LPA and cholesteryl pullulan (CHP) nanogel was fabricated, and subcutaneously injected to mice with TLR agonist such as CpG oligoDNA or polyIC RNA as an adjuvant. Frequency of vaccine-induced CTLs specific to each epitope was then determined by intracellular IFN-g staining assay. Using similar mouse model, the effect of CHP nanogel delivery system on transportation of LPA to APCs in the draining lymph node (DLN) as well as T cell response to vaccination was also examined, comparing with a conventional delivery system, incomplete freund adjuvant (IFA). Results The IES candidates were selected based on in silico prediction of the sensitivity to proteasomal cleavage. The sensitivity of oligotyrosine and oligothreonine was predicted high, while that of oligoglycine and oligoproline was low. In the vaccinated mice, the LPA including the oligotyrosine IES most robustly stimulated specific CTLs towards all three epitopes included in LPA. The LPA including the oligothreonine IES was second best. In contrast, LPA including the oligoglycine or oligoproline IES often induced very limited CTL response. In the evaluation of delivery system, the CHP nanogel transported LPA to APCs in the DLN much more efficiently than IFA did. The kinetics of the CHP nanogel-mediated LPA transportation was closely similar to that of APC activation by TLR agonists, suggesting that the CHP nanogel harmonises the action of LPA and adjuvant. Indeed, in the presence of TLR agonists, the CHP nanogel greatly augmented the immunogenicity and anti-tumour effect of LPA vaccine. Conclusions We succeeded in the enhancement of immunogenicity of LPA vaccine by the rational design of IES in LPA and the CHP nanogel-mediated efficient antigen transportation to the DLN. These technologies will bring a remarkable improvement of vaccine efficacy.

Reduction breakable cholesteryl pullulan nanoparticles for targeted hepatocellular carcinoma chemotherapy.

In this study, a novel nanoparticulate drug delivery platform with inherent targeting ability to hepatic carcinoma cells and reduction-triggered drug release property was developed based on reducible cholesterol-modified pullulan (rCHP). The nanoparticle characteristics and antitumor effects were investigated in vitro and in vivo. The results revealed that drug-loaded rCHP nanoparticles were spherical and their diameter ranged between 80 and 160 nm with a change of molecular structure and drug loading content. rCHP nanoparticles with pullulan shells could anchor to human hepatocellular carcinoma cells (HepG2) due to specific recognition of asialoglycoprotein receptors (ASGPRs) overexpressing at the cytomembrane, reduction-sensitively release doxorubicin (DOX) in tumor cells, and effectively suppress the growth of HepG2 in vitro. In a hepatoma-bearing nude mouse model, attributed to their uncharged pullulan surface layer, the nanoparticles efficiently accumulated in the tumor site and were internalized by tumor cells. After cellular uptake, DOX release triggered by the reductive circumstance of tumor cells was achieved benefiting from the reduction-sensitive DOX release property. These nanoparticles showed significantly better antitumor effect and biosafety than DOX·HCl in the nude mice bearing hepatocellular carcinoma tumor.

Synthesis and characterization of biotin modified cholesteryl pullulan as a novel anticancer drug carrier

A series of biotin modified cholesteryl pullulan (Bio-CHSP) conjugates with different degrees of substitution (DS) of biotin moiety were synthesized and characterized by Fourier transform infrared (FT-IR), proton nuclear magnetic resonance (1H NMR) and X-ray diffraction (XRD). Bio-CHSP conjugates were amphiphilic in nature and their self-aggregation behavior in aqueous media was evaluated by the fluorescence probe technique. Bio-CHSP self-aggregated nanoparticles (Bio-CHSP NPs) were prepared and analyzed by dynamic light scattering (DLS), zeta potential and transmission electron microscopy (TEM) technologies. These novel nanoparticles were almost spherical in shape, and their size, ranging from 178.8 to 100.0nm. The safety of Bio-CHSP NPs was studied through single dose toxicity test in mice, and the result showed that Bio-CHSP NPs were well tolerated at the intravenous dose of 200mg/kg in mice. Moreover, as a model anticancer drug, mitoxantrone loaded Bio-CHSP NPs were also prepared and characterized in this study.

Dose-dependent effects of NY-ESO-1 protein vaccine complexed with cholesteryl pullulan (CHP-NY-ESO-1) on immune responses and survival benefits of esophageal cancer patients

BackgroundCholesteryl pullulan (CHP) is a novel antigen delivery system for cancer vaccines. This study evaluated the safety, immune responses and clinical outcomes of patients who received the CHP-NY-ESO-1 complex vaccine, Drug code: IMF-001.MethodsPatients with advanced/metastatic esophageal cancer were enrolled and subcutaneously vaccinated with either 100 μg or 200 μg of NY-ESO-1 protein complexed with CHP. The primary endpoints were safety and humoral immune responses, and the secondary endpoint was clinical efficacy.ResultsA total of 25 patients were enrolled. Thirteen and twelve patients were repeatedly vaccinated with 100 μg or 200 μg of CHP-NY-ESO-1 with a median of 8 or 9.5 doses, respectively. No serious adverse events related to the vaccine were observed. Three out of 13 patients in the 100-μg cohort and 7 out of 12 patients in the 200-μg cohort were positive for anti-NY-ESO-1 antibodies at baseline. In the 100-μg cohort, an antibody response was observed in 5 out of 10 pre-antibody-negatives patients, and the antibody levels were augmented in 2 pre-antibody-positive patients after vaccination. In the 200-μg cohort, all 5 pre-antibody-negative patients became seropositive, and the antibody level was amplified in all 7 pre-antibody-positive patients. No tumor shrinkage was observed. The patients who received 200 μg of CHP-NY-ESO-1 survived longer than patients receiving 100 μg of CHP-NY-ESO-1, even those who exhibited unresponsiveness to previous therapies or had higher tumor burdens.ConclusionsThe safety and immunogenicity of CHP-NY-ESO-1 vaccine were confirmed. The 200 μg dose more efficiently induced immune responses and suggested better survival benefits. (Clinical trial registration number NCT01003808).

Self-assembled pH-sensitive cholesteryl pullulan nanogel as a protein delivery vehicle.

A self-assembled nanogel, derived from an acid-labile cholesteryl-modified pullulan (acL-CHP), was prepared by grafting vinyl ether-cholesterol substituents onto a 100 kD pullulan main chain polymer backbone. Stable nanogels are formed by acL-CHP self-assemblies at neutral pH. The hydrodynamic radius of the nanogels, observed to be 26.5 ± 5.1 nm at pH 7.0, increased by ~135% upon acidification of the solution to pH 4.0. SEC analysis of the acL-CHP nanogel at pH 4.0 showed that the grafts were nearly 80% degraded after 24 h, whereas little or no degradation was observed over the same time period for a pH stable analog (acS-CHP) at pH 4.0 or the acL-CHP at pH 7.0. Complexation of BSA with the acL-CHP nanogel was observed at pH 7.0 with subsequent release of the protein upon acidification. These findings suggest that stimuli-responsive, self-assembled nanogels can release protein cargo in a manner that is controlled by the degradation rate of the cholesterol-pullulan grafting moiety.