Development of a Nanocarrier-Based Splenic B Cell-Targeting System for Loading Antigens in Vitro.

B cells are types of lymphocytes that are involved in the production of antibodies against pathogens. They also deliver and present antigens for the priming of T cells. Recently, we developed an in vivo splenic marginal zone (MZ) B cell-targeting liposomes decorated with polyethylene glycol (PEG) containing a hydroxyl-terminus group (HO-PEG-Lip). In an expansion of a previous study, we used HO-PEG-Lip as an in vitro antigen delivery tool to splenic B cells to test the ability of this formulation to overcome the limitations of the poor antigen uptake ability of B cells for implantation. To achieve our purpose, various factors were optimized. These factors include cell number, liposome concentration, pre-opsonization of liposomes, fresh serum concentration, and incubation time, all of which affect the extent of interaction between liposomes and B cells. As a result, we confirmed that the HO-PEG-Lip required incubation at 37 °C for at least 20 min with 50% mouse fresh serum followed by a subsequent incubation at 37 °C for at least another 30 min with splenic B cells. By using such a loading system, fluorescein isothiocyanate (FITC)-labeled ovalbumin (OVA), a model antigen, encapsulated in HO-PEG-Lip could be efficiently loaded into splenic B cells. In addition, HO-PEG-Lip and FITC-labeled OVA encapsulated in HO-PEG-Lip were efficiently associated with MZ-B cells with high levels of complement receptors (CRs) rather than follicular B cells with low levels of CRs. These results propose a novel and useful system to efficiently load antigens into B cells in vitro by taking advantage of complement systems.

Development of an Antigen Delivery System for a B Cell-Targeted Vaccine as an Alternative to Dendritic Cell-Targeted Vaccines.

Vaccines have contributed to the prevention of infectious diseases for a long time. Pathogen-derived antigens and adjuvants in vaccine formulations stimulate immune cells to elicit humoral and cellular immune responses against pathogens. Achieving highly immune responses with decreased adverse effects requires the development of a system that can deliver antigens to specific immune cells. Dendritic cells (DCs) are well-known professional antigen presenting cells (APCs) that initiate acquired immune responses by presenting antigens to T cells. Accordingly, DC-targeted vaccines have been investigated and applied in clinical trials for the treatment of infectious diseases and for chronic diseases such as cancers. In addition to DCs, B lymphocytes are regarded as professional APCs despite their primary role in humoral immunity. Therefore, B cell-targeted vaccines are also expected to elicit both humoral and cellular immune responses. In this review we summarize the basic functions of DCs and B cells as APCs. We also provide information on DC and B cell targeted vaccines in preclinical and clinical settings. Finally, we introduce our novel antigen delivery system that targets splenic marginal zone B cells and the ability of this system to act as a novel vaccine that elicits both humoral and cellular immune responses.

Increasing Tumor Extracellular pH by an Oral Alkalinizing Agent Improves Antitumor Responses of Anti-PD-1 Antibody: Implication of Relationships between Serum Bicarbonate Concentrations, Urinary pH, and Therapeutic Outcomes.

Acidic extracellular pH (pHe) is characteristic of the tumor microenvironment. Several reports suggest that increasing pHe improves the response of immune checkpoint inhibitors in murine models. To increase pHe, either sodium bicarbonate (NaHCO3) or citric acid/potassium-sodium citrate (KNa-cit) was chronically administered to mice. It is hypothesized that bicarbonate ions (HCO3-), produced from these alkalinizing agents in vivo, increased pHe in the tumor, and excess HCO3- eliminated into urine increased urinary pH values. However, there is little published information on the effect of changing serum HCO3- concentrations, urinary HCO3- concentrations and urinary pH values on the therapeutic outcomes of immunotherapy. In this study, we report that oral administration of either NaHCO3 or KNa-cit increased responses to anti-programmed cell death-1 (PD-1) antibody, an immune checkpoint inhibitor, in a murine B16 melanoma model. In addition, we report that daily oral administration of an alkalinizing agent increased blood HCO3- concentrations, corresponding to increasing the tumor pHe. Serum HCO3- concentrations also correlated with urinary HCO3- concentrations and urinary pH values. There was a clear relationship between urinary pH values and the antitumor effects of immunotherapy with anti-PD-1 antibody. Our results imply that blood HCO3- concentrations, corresponding to tumor pHe and urinary pH values, may be important factors that predict the clinical outcomes of an immunotherapeutic agent, when combined with alkalinizing agents such as NaHCO3 and KNa-cit.

A Novel Platform for Cancer Vaccines: Antigen-Selective Delivery to Splenic Marginal Zone B Cells via Repeated Injections of PEGylated Liposomes.

Treating cancer with vaccines has been a challenge. In this study, we introduce a novel Ag delivery platform for cancer vaccines that delivers an encapsulated Ag to splenic marginal zone B (MZ-B) cells via the aid of a PEGylated liposome (PL) system. Splenic MZ-B cells have recently attracted interest as alternative APCs. In mice, preimmunization with empty (no Ag encapsulation) PLs triggered the efficient delivery of a subsequent dose of Ag-containing PLs, injected 3 d later, to the spleen compared with a single dose of Ag-containing PLs. In addition, immunization with empty PLs allowed three subsequent sequential injections of OVA-PLs to efficiently induce a CTL response against OVA-expressing murine thymoma (EG7-OVA) cells and resulted in in vivo growth inhibition of subsequently inoculated EG7-OVA cells. However, these sequential treatments require repeated immunizations to achieve their antitumor effect. Therefore, to improve the antitumor effect of our novel vaccine system, an adjuvant, α-galactosylceramide (αGC), was incorporated into the OVA-PLs (αGC/OVA-PLs). As expected, the incorporation of αGC reduced the required number of immunizations with OVA-PLs to the point that a single immunization treatment with empty PLs and an injection of αGC/OVA-PL efficiently triggered a potent CTL induction, resulting in a rejection of the development and a suppression of the growth of tumors that had already developed s.c. Results of this study indicate that a novel Ag delivery platform that grants efficient Ag delivery to splenic MZ-B cells shows promise as a therapeutic modality for conquering tumor growth and/or progression.

Case-based similar image retrieval for weakly annotated large histopathological images of malignant lymphoma using deep metric learning.

In the present study, we propose a novel case-based similar image retrieval (SIR) method for hematoxylin and eosin (H&E) stained histopathological images of malignant lymphoma. When a whole slide image (WSI) is used as an input query, it is desirable to be able to retrieve similar cases by focusing on image patches in pathologically important regions such as tumor cells. To address this problem, we employ attention-based multiple instance learning, which enables us to focus on tumor-specific regions when the similarity between cases is computed. Moreover, we employ contrastive distance metric learning to incorporate immunohistochemical (IHC) staining patterns as useful supervised information for defining appropriate similarity between heterogeneous malignant lymphoma cases. In the experiment with 249 malignant lymphoma patients, we confirmed that the proposed method exhibited higher evaluation measures than the baseline case-based SIR methods. Furthermore, the subjective evaluation by pathologists revealed that our similarity measure using IHC staining patterns is appropriate for representing the similarity of H&E stained tissue images for malignant lymphoma.

Nucleic acids delivered by PEGylated cationic liposomes in systemic lupus erythematosus-prone mice: A possible exacerbation of lupus nephritis in the presence of pre-existing anti-nucleic acid antibodies.

Nucleic acid-based therapy with plasmid DNA (pDNA) and small interfering RNA (siRNA) have received recent attention for their ability to modulate the cellular expression of genes and proteins. Polyethylene glycol-modified (PEGylated) cationic nanoparticles have been used as non-viral vectors for the in vivo delivery of these nucleic acids. We have reported that PEGylated cationic liposomes (PCL) including pDNA or siRNA induce anti-PEG antibodies upon repeated intravenous injection, leading to the formation of immune complexes and enhanced clearance from the blood of subsequent doses. However, the issue surrounding the association of nucleic acids with PCL whether induces anti-nucleic acid antibodies has not been studied. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with the character of end-organ damage and the presence of anti-nuclear antibodies. We used a healthy mouse and an SLE mouse model to test the hypothesis that nucleic acids associated with PCL induce anti-nuclear antibodies and then induce SLE and exacerbate SLE symptoms. We report here that pDNA or siRNA associated with PCL (pDNA/PCL or siRNA/PCL) induced anti-DNA or RNA antibodies, respectively, in healthy mice. Repeated injections did not, however, cause SLE-like symptoms in the healthy mice. In addition, in SLE-prone mice with pre-existing anti-nuclear antibodies, pDNA/PCL were deposited on the kidneys and exacerbated lupus nephritis subsequent to the formation of immune complexes. These results may imply that nucleic acids associated with PCL do not contribute to the onset of SLE in healthy individuals who lack anti-nuclear antibodies, but nucleic acids may exacerbate the symptoms in SLE patients who have pre-existing anti-nuclear antibodies.

Anti-PEG IgM production and accelerated blood clearance phenomenon after the administration of PEGylated exosomes in mice.

Recently, there is an increasing interest in exosomes or extracellular vesicles as potential candidates for delivering RNAs, proteins, genes, and anticancer agents. Engineering of exosome properties is rapidly evolving as a means of expanding exosome applications. PEGylation of exosomes is a technique used to improve their in vivo stability, circulation half-lives, and sometimes to allow the binding targeting ligands to the exosome exterior. According to FDA guidelines for the development of PEGylated proteins, immunological responses to PEGylated molecules and particles should be examined. In this study, we prepared PEGylated exosomes and investigated the production of anti-PEG IgM antibodies after single i.v. injections in mice. In addition, we monitored blood concentrations and tumor accumulation of a second dose of PEGylated exosomes administered after the initial dose. Single injections of PEGylated exosomes in mice induced anti-PEG IgM production in a T cell-dependent manner. The anti-PEG IgM production decreased when the injection dose of PEGylated exosomes was further increased. Anti-PEG IgM induced by injection of PEGylated exosomes decreased blood concentrations of a second dose of PEGylated exosomes and suppressed their tumor accumulation in a C26 murine colorectal cancer model. Initial injection doses of either PEGylated liposomes or PEGylated ovalbumin (PEG-OVA), both of them induced anti-PEG IgM production, also decreased the blood concentration of PEGylated exosomes. Interestingly, anti-PEG IgM induced by injection of PEGylated exosomes did not affect the blood concentration of PEG-OVA. These results imply the importance of monitoring anti-PEG IgM when repeat PEGylated exosome doses are required and/or when PEGylated exosomes are used together with other PEGylated therapeutics.

Complement activation induced by PEG enhances humoral immune responses against antigens encapsulated in PEG-modified liposomes.

Splenic marginal zone B (MZ-B) cells have attracted attention as alternative antigen-presenting cells. We recently developed an original delivery system, using PEGylated liposomes (PEG-Lip) to deliver antigens to MZ-B cells. In this system, to induce antigen-specific immunity, empty PEG-Lip and antigen-containing PEG-Lip were intravenously (i.v.) injected sequentially at 3 day intervals. Since complement activation by the second dose is required for the delivery of antigen-containing PEG-Lip to splenic MZ-B cells, we investigated the ability of liposomes, modified with various PEG derivatives having different functional terminal groups (methoxy PEG (CH3O-PEG), hydroxy PEG (HO-PEG) or polyglycerol (PG), to activate the complement system and deliver a model antigen, ovalbumin (OVA), to splenic MZ-B cells in vitro and in vivo. Hydroxy PEG-modified liposomes (HO-PEG-Lip) both activated the complement system in vitro, and facilitated the preferential association of HO-PEG-lip with MZ-B cells in vitro. Manipulating HO-PEG density, in particular a density of 2 mol% in total lipids, significantly enhanced the association of HO-PEG-Lip with splenic MZ-B cells in vivo. Consequently, a single i.v. injection of HO-PEG-Lip (2 mol%) containing OVA induced OVA-specific IgG response. Our immunization system with HO-PEG-Lip, achieved efficient antigen delivery to MZ-B cells after a single i.v. injection, improving on our previous immunization system. This new delivery technique may be an improved, simple, antigen delivery system to MZ-B cells that induces meaningful levels of humoral immune response.

Hepatosplenic phagocytic cells indirectly contribute to anti-PEG IgM production in the accelerated blood clearance (ABC) phenomenon against PEGylated liposomes: Appearance of an unexplained mechanism in the ABC phenomenon.

The accelerated blood clearance (ABC) phenomenon, caused in large degree via in vivo anti-PEG IgM production, is one of obstacles for development of PEGylated liposome and protein formulations, due to decreased efficiency and/or side effects such as anaphylaxis upon repeat administrations. We have shown in murine ABC models that splenectomy suppressed the level of anti-PEG IgM production induced by PEGylated liposomes, indicating that murine splenic B cells play an important role in its production. However, splenectomy did not completely inhibit production of anti-PEG IgM, suggesting that other cells may contribute to its production in the ABC phenomenon. In this study, we examined the contribution of hepatosplenic phagocytic cells to anti-PEG IgM production and clearance of PEGylated liposomes during the ABC phenomenon. Depletion of hepatosplenic phagocytic cells by pretreatment of mice with clodronate-containing non-PEGylated liposomes suppressed anti-PEG IgM production to a considerable degree, without a change in the number of splenic B cells, and attenuated the enhanced clearance of second dose of PEGylated liposomes. These results suggest that hepatosplenic phagocytic cells, in addition to splenic B cells, contribute to the production of anti-PEG IgM and the ABC phenomenon against PEGylated liposomes. The mechanism whereby splenic B cells interact with hepatosplenic phagocytic cells to produce anti-PEG IgM, upon administration of an initial dose of PEGylated liposomes remains to be elucidated.