Masato Okamoto

Expression of Toll-Like Receptor 4 on Dendritic Cells Is Significant for Anticancer Effect of Dendritic Cell-Based Immunotherapy in Combination with an Active Component of OK-432, a Streptococcal Preparation

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432, a Streptococcus-derived anticancer immunotherapeutic agent. In the present study, we first examined the effect of OK-PSA on the maturation of dendritic cells (DCs) in vitro by using the DCs derived from 5 healthy donors and 10 patients with head and neck cancer with or without expression of toll-like receptor 4 (TLR4) or MD-2 mRNA. OK-PSA treatment effectively increased the surface expression of MHC class II, CD80, CD83, and CD86. OK-PSA-stimulated DCs secreted the cytokines that can induce helper T-cell 1 (Th1)-type T-cell response, and stimulated allogeneic T cells to produce IFN-γ and to elicit an allogeneic antigen-specific cytotoxicity. These activities almost depended on expression of TLR4 and MD-2 genes. We next investigated the in vivo anticancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. C57BL/6 mice, which express wild-type TLR4, and C57BL/6-derived TLR4-knockout (TLR4−/−) mice were used. Although OK-PSA accelerated the antitumor effect of intratumoral DC administration in wild-type mice bearing syngeneic tumors, the antitumor effect of OK-PSA as well as of the combination therapy with DCs and OK-PSA was not significant in TLR4−/− mice. Interestingly, an administration of wild-type-mouse-derived DCs followed by OK-PSA exhibited a marked antitumor effect even in the TLR4−/− mice. These findings suggest that OK-PSA may be a potent adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anticancer activity even in a TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.

Clinical and immunologic evaluation of dendritic cell-based immunotherapy in combination with gemcitabine and/or S-1 in patients with advanced pancreatic carcinoma

Objectives In the current study, we have evaluated the clinical and immunological responses in patients with advanced pancreatic carcinoma who received dendritic cell (DC)–based immunotherapy in combination with gemcitabine and/or S-1. Methods Dendritic cell–based immunotherapy (DC vaccine alone or DC vaccine plus lymphokine-activated killer [LAK] cell therapy) in combination with gemcitabine and/or S-1 has been carried out in 49 patients with inoperable pancreatic carcinoma refractory to standard treatment. Results Of 49 patients, 2 patients had complete remission, 5 had partial remission, and 10 had stable disease. Prolongation of survival in this cohort was highly likely (median survival, 360 days). Survival of patients receiving DC vaccine and chemotherapy plus LAK cell therapy was longer than those receiving DC vaccine in combination with chemotherapy but no LAK cells. Increased numbers of cancer antigen-specific cytotoxic T cells and decreased regulatory T cells were observed in several patients on immunotherapy, but increased overall survival time tended to be associated only with the latter. None of the patients experienced grade 3 or worse adverse events during the treatment period. Conclusions Dendritic cell vaccine–based immunotherapy combined with chemotherapy was shown to be safe and possibly effective in patients with advanced pancreatic cancer refractory to standard treatment.

Mechanism of anticancer host response induced by OK-432, a streptococcal preparation, mediated by phagocytosis and Toll-like receptor 4 signaling

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-κB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-κB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.

Immunotherapy for colorectal cancer

The incidence of colorectal cancer (CRC) is on the rise, and the prognosis for patients with recurrent or metastatic disease is extremely poor. Although chemotherapy and radiation therapy can improve survival rates, it is imperative to integrate alternative strategies such as immunotherapy to improve outcomes for patients with advanced CRC. In this review, we will discuss the effect of immunotherapy for inducing cytotoxic T lymphocytes and the major immunotherapeutic approaches for CRC that are currently in clinical trials, including peptide vaccines, dendritic cell-based cancer vaccines, whole tumor cell vaccines, viral vector-based cancer vaccines, adoptive cell transfer therapy, antibody-based cancer immunotherapy, and cytokine therapy. The possibility of combination therapies will also be discussed along with the challenges presented by tumor escape mechanisms.

Purification and characterization of interferon-gamma-inducing molecule of OK-432, a penicillin-killed streptococcal preparation, by monoclonal antibody neutralizing interferon-gamma-inducing activity of OK-432.

An immunoglobulin M mouse monoclonal antibody (MAb) to streptococcal preparation OK-432, TS-2, was generated. The TS-2 MAb showed positive reaction with butanol extract of OK-432 and fungal, branched (1-->3)-gamma-glucans (lentinan and schizophyllan) as well as lipoteichoic acids. Moreover, the interferon (IFN)-gamma-inducing activity of OK-432 was neutralized by TS-2 MAb. The affinity column of butanol extract of OK-432 on CNBr-activated Sepharose 4B-bound TS-2 antibody was prepared and the fractions containing IFN-gamma-inducing activity were eluted. The polysaccharide sample carrying the IFN-gamma-inducing activity with a molecular weight of 700,000 was destroyed by treatment with acid or sodium metaperiodate, but was stable to treatment with heat, alkali, pronase, or neuraminidase. Survival time of human salivary adenocarcinoma-bearing animals given a combination of the polysaccharide sample purified from butanol extract of OK-432 and TS-2 MAb was significantly shorter compared with that of the tumor-bearing animals given only the purified polysaccharide sample of OK-432. Moreover, a high level of effector cell activity in natural killer (NK) and lymphokine-activated killer (LAK) assays and significant increase of IFN-gamma-positive cells or asialo-GM1-positive cells were detected in the spleen cells from the animals given the polysaccharide sample purified from butanol extract of OK-432. These findings indicate that the polysaccharide sample purified by the affinity chromatography of butanol extract of OK-432 on CNBr-activated Sepharose 4B-bound TS-2 MAb carries the IFN-gamma-inducing activity of OK-432 and marked antitumor activity.

Effect of YM529 on a Model of Mandibular Invasion by Oral Squamous Cell Carcinoma in Mice

Purpose: This study examined the mechanisms of osteoclast-mediated bone invasion in a model of oral squamous cell carcinoma (OSCC). C3H/HeN mice were inoculated with SCC VII cells into the masseter region to establish an animal model of mandibular invasion by OSCC. Experimental Design: The mice were divided into three groups: a control group, given daily s.c. injections of saline; group 1, given 2 μg per mouse per day of the bisphosphonate YM529; and group 2, given 10 μg per mouse per day of YM529. After 3 weeks of treatment, the lesions were studied by micro-computed tomography. After tartrate-resistant acid phosphatase (TRAP) staining, the osteoclasts were easily identified, and the percentages of the area occupied by osteoclasts were calculated by computer for each sample. The tumors were analyzed by RT-PCR to determine the mRNA expression of interleukin-6 (IL-6), parathyroid hormone–related protein (PTHrP), tumor necrosis factor-α (TNF-α), receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin. Results: SCC VII cells rapidly multiplied in the masseter muscle of the mice. Bone invasion was evident only in the control group on micro-computed tomography. On TRAP-stained slices, the percentages of osteoclasts in groups 1 and 2 were significantly lower than that in the control group. The mRNA expressions of IL-6, PTHrP, THF-α, and RANK decreased as the concentration of YM529 increased. Conclusions: We conclude that various cancer-derived cytokines play important roles in the invasion of bone by OSCC. YM529, a third-generation bisphosphonate, can suppress osteoclast-mediated bone invasion by OSCC. The mechanism of this effect might involve inhibition of cytokines such as IL-6, PTHrP, TNF-α, and RANK by YM529.

Oral squamous cell carcinoma cells induce osteoclast differentiation by suppression of osteoprotegerin expression in osteoblasts

The invasion of oral squamous cell carcinoma (SCC) cells into the mandibular bone is a common clinical problem. It has been reported that BHY cells, a human oral SCC cell line, are capable of invading mandibular bone of nude mice. These results led us to examine possible mechanisms of osteoclastogenesis induced by BHY cells using in vitro culture systems. When BHY cells were cocultured with mouse bone marrow cells (BMCs), only few osteoclasts were formed, even though BHY cells express the receptor activator of NF‐κB ligand (RANKL). However, adding BHY cells to a coculture of mouse primary osteoblasts (POBs) and BMCs markedly induced osteoclastogenesis in the absence of osteotropic factors. Furthermore, another oral SCC cell line, HSC‐2, which does not express RANKL, also induced osteoclastogenesis in our cocultures. These effects were significantly, but not completely, inhibited by adding osteoprotegerin (OPG). In addition, we also found that TNFα released from these cells partially contributes to osteoclastogenesis via a RANKL‐independent mechanism. Adding BHY or HSC‐2 cells suppressed mouse OPG mRNA expression and protein production by POBs in cocultures of POBs and human oral SCC cells. This finding is consistent with the result that BHY cells and HSC‐2 cells did not enhance osteoclastogenesis in cocultures of BMCs and POBs from OPG‐deficient mice. Immunohistochemical analysis showed a reduction of OPG expression in osteolytic lesions as compared to normal lesions from oral SCC patients. Therefore, oral SCC‐induced suppression of OPG expression in POBs appears critical for osteoclastogenesis, rather than expression of RANKL in SCC cells.

Anti-tumor effect of an intratumoral administration of dendritic cells in combination with TS-1, an oral fluoropyrimidine anti-cancer drug, and OK-432, a streptococcal immunopotentiator: Involvement of toll-like receptor 4

The authors investigated the in vivo anti-tumor effect of intratumoral administration of bone marrow-derived dendritic cells (DCs) after chemotherapy using an oral fluoropyrimidine anti-cancer drug TS-1, and followed by immunotherapeutic agent OK-432, in two syngeneic tumor-bearing mouse models. Both in Meth-A fibrosarcoma-bearing BALB/c mice and in SCCVII-bearing C3H/HeN mice, 1 week of oral administration of TS-1 effected partial eradication of established tumors. Intratumoral injection of DCs and OK-432 caused only slight inhibition of the tumor growth. However, TS-1 administration followed by DCs and OK-432 resulted in a marked inhibition in the tumor growth and also contributed to a greater prolongation of survival. By the injection of DCs and OK-432 after TS-1 administration, a significant infiltration of immune cells, especially CD8+ T cells, was observed. Furthermore, the cytotoxic activities of tumor-infiltrating lymphocytes and draining lymph node cells against inoculated tumor cells were significantly increased by the therapy, while activities against nonspecific target cells were not. Cytotoxic memory T cells were also induced; the main effectors were MHC class I-restricted, CD8+ T cells. The same therapy was also applied to SCCVII-bearing C3H/HeJ mice in which the Toll-like receptor (TLR) 4 is mutated and its function impaired; no immunotherapeutic effect was observed in the TLR4-deficient mouse model. These findings suggest that the local DC therapy in combination with TS-1 and OK-432 may be a useful strategy for the treatment of solid tumors, and that TLR4 signaling is involved in the success of this therapy.

Phase I pilot study of Wilms tumor gene 1 peptide‐pulsed dendritic cell vaccination combined with gemcitabine in pancreatic cancer

This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide‐pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first‐line therapy among patients with advanced pancreatic cancer. Ten HLA‐A*2402 patients were treated with WT1 peptide‐pulsed DC vaccination (1 × 107 cells) on days 8 and 22 and gemcitabine (1000 mg/m2) on days 1, 8 and 15. Induction of a WT1‐specific immune response was evaluated using the delayed‐type hypersensitivity (DTH) skin test, interferon‐γ enzyme‐linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well‐tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C‐reactive protein and interleukin‐8 (IL‐8) showed poor survival even though a WT1‐specific immune response was induced in them. WT1 peptide‐pulsed DCGEM is feasible and effective for inducing anti‐tumor T‐cell responses. Our results support future investigations for pancreatic cancer patients with non‐liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry (http://www.umin.ac.jp/ctr/ number: UMIN‐000004855).

Fusions between dendritic cells and whole tumor cells as anticancer vaccines

Various strategies have been developed to deliver tumor-associated antigens (TAAs) to dendritic cells (DCs). Among these, the fusion of DCs and whole cancer cells can process a broad array of TAAs, including hitherto unidentified molecules, and present them in complex with MHC Class I and II molecules and in the context of co-stimulatory signals. DC-cancer cell fusions have been shown to stimulate potent antitumor immune responses in animal models. In early clinical trials, however, the antitumor effects of DC-cancer cell fusions are not as vigorous as in preclinical settings. This mini-review summarizes recent advances in anticancer vaccines based on DC-cancer cell fusions.