Mechanism of antitumor effect on mouse hepatocellular carcinoma by intratumoral injection of OK-432, a streptococcal preparation.

Intratumoral (i.t.) injection of OK-432, a streptococcal preparation, into implanted tumors of mouse hepatocellular carcinoma (MIH-2) showed antitumor effect including tumor eradication. Intraperitoneal administration of same dose OK-432 did not exhibit tumor suppressive effect. In vitro cytotoxic test suggested that direct cytotoxic effect of OK-432 was not associated with antitumor activity by i.t.-OK-432 treatment. It was also found that Toll-like receptor 4 signaling was not involved in i.t.-OK-432 treatment. Three mice out of five, which had shown tumor eradication by i.t.-OK-432 treatment did not reject re-challenge of MIH-2 cells. Splenocytes from i.t.-OK-432 treated mice did not produce IFN-gamma by stimulation with MIH-2 cells in vitro, but produced abundant IFN-gamma by stimulation with OK-432. Immunofluorescence microscopy demonstrated that CD4+T cells, but not CD8+T cells, infiltrated to i.t.-OK-432 treated tumor tissue produced IFN-gamma. Tumor-infiltrating CD4+T cells from i.t.-OK-432 treated tumor tissue produced IFN-gamma by in vitro stimulation with OK-432 higher than those from untreated tumor tissue. IFN-gamma directly induced apoptosis of MIH-2 cells in vitro. Collectively, i.t.-OK-432 treatment induced priming of CD4+T cells to antigenecity of OK-432, and repetitive i.t.-OK-432 treatment induced IFN-gamma production from OK-432-sensitized CD4+T cells in tumor site, leading to apoptosis of MIH-2 cells susceptible to IFN-gamma.

Randomized phase II trial of OK-432 in patients with malignant pleural effusion due to non-small cell lung cancer.

To determine the optimum dose of OK-432 for intrathoracic administration, a multicenter randomized phase II trial was conducted in patients with malignant pleural effusion due to non-small cell lung cancer. Patients with histologically- or cytologically-proven malignant pleural effusions were randomized to arm A (10 Klinische Einheit (KE) of OK-432) or arm B (1 KE of OK-432). OK-432 was injected intrapleurally over 30 min on days 1 and 3 and the chest tube was clamped for 6 h. If control was inadequate on day 8, 10 KE was administered on days 8 and 10 in each treatment arm. Forty patients were enrolled and 38 patients were eligible (19 in arm A and 19 in arm B). The effusion control rate on day 8 was 79% in arm A and 53% in arm B, while control rates on day 28 were 74% and 84%, respectively. The median drainage time after administration was significantly shorter in arm A (4.0 +/- 1.2 days) than in arm B (7.0 +/- 1.7 days). The total drainage volume was also significantly less in arm A than in arm B. No grade 4 toxicities or treatment-related deaths were observed in either treatment arm. Intrathoracic injection of OK-432 is a feasible treatment for malignant pleural effusion. Although the malignant pleural effusion control rate was equivalent in each treatment arm, faster control and less drainage were achieved in arm A. A dose of OK-432 10 KE/body is, therefore, recommended for further trial.

Involvement of nitric oxide in anti-tumor effects of OK-432, a streptococcal anti-tumor immunotherapeutic agent.

We examined the role of nitric oxide (NO) induced by OK-432, a streptococcal immunotherapeutic agent, in anti-tumor effects of the OK-432 by in vitro and in vivo experiments using an NO synthase inhibitor, N-monomethyl-l-arginine acetate (NMA). The in vitro treatment of mouse splenocytes with OK-432 increased the expression of inducible NO synthase (iNOS) gene and NO production in a dose-dependent manner. Although it is well known that OK-432 induces cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, both of which are known to be potent NO inducers, we observed only a partial reduction of OK-432-induced NO production with the addition of anti-IFN-gamma and/or anti-TNF-alpha neutralizing antibodies. The cytotoxicity of the splenocytes increased by in vitro OK-432 stimulation was almost completely inhibited by the treatment with NMA. OK-432 administration resulted in a marked prolongation of survival and a significant inhibition of tumor growth in syngeneic tumor-bearing mice, whereas NMA significantly inhibited the anti-tumor effects of OK-432. Although the increased cytotoxicity of adherent splenocytes derived from OK-432-treated tumor-bearing mice was almost completely inhibited by NMA, only partial inhibition by NMA was observed in the cytotoxicity of the nonadherent splenocytes. These findings strongly suggest that the iNOS/NO induced by OK-432 is intimately involved in the anti-tumor effects of OK-432.

Antitumor effect of OK-432-derived DNA: one of the active constituents of OK-432, a streptococcal immunotherapeutic agent.

OK-432 is a Streptococcus-derived immunotherapeutic agent for malignancies. Our group has tried to identify the effective components of OK-432 and has succeeded in isolating a lipoteichoic acid-related preparation designated as OK-PSA, which is a strong inducer of T helper 1 (T(H)1) cells, and elicits an anticancer effect via Toll-like receptor (TLR) 4. Conversely, bacterial DNA with unmethylated CpG motifs can stimulate a T(H)1-type host response via TLR9. The unmethylated CpG DNA contained in OK-432 may play a role in its anticancer effect. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting the anticancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and MspI revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced TH1-type cytokines such as interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-12, and IL-18 and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. The antitumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced compared with that in wild-type mice, whereas the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG DNA in OK-432 functions as an active component in OK-432-induced anticancer immunity via TLR9.

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)-kappaB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-kappaB 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.

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.

Biological effect of OK-432 (picibanil) and possible application to dendritic cell therapy.

OK-432 (Picibanil), a streptococcal preparation with potent biological response modifying activities, was approved in Japan as an anticancer agent in 1975. In the ensuing 30 years, since then, a significant amount of data, including clinical as well as experimental studies, has been accumulated. OK-432 has been reported to induce various cytokines, activate immunological cells and thus augment anticancer immunity. Recently, the interrelation between innate immunity and adaptive immunity has become clear and it was reported that OK-432 acts, at least in part, via Toll-like receptor (TLR) 4-MD2 signaling pathway. In addition, dendritic cells (DCs) are considered to play a pivotal role in immunological response and it is reported that OK-432 induced maturation of DCs both in vitro and in vivo. These results suggest that OK-432 is a useful adjuvant in DC-based anticancer immunotherapy. Clinical studies of DC therapy with OK-432 are under way.

A randomized controlled study of immunochemotherapy with OK-432 after curative surgery for gastric cancer.

The effect of adjuvant immunochemotherapy including OK-432 (Picibanil) on survival was assessed in patients who underwent curative resection of gastric cancer. Patients enrolled in this randomized controlled study were randomly assigned to group A or group B. Group A patients received 800 mg/d 5'-DFUR (Furtulon) for 2 years from 2 weeks after the operation. Group B patients received OK-432 plus 5'-DFUR by the same regimen as in group A. This study enrolled 288 patients, and 1 patient with malignant lymphoma was excluded. Among the remaining 287 patients, 143 and 144 were allocated to group A and group B, respectively, and their data were included in statistical analysis. The 5-year survival rates for groups A and B were 62.9% and 63.8%, respectively, showing no significant difference (P = 0.7996).

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-gamma 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.

Anti-tumor immune response induced by the fractions derived from OK-432, a streptococcal preparation, by using a monoclonal antibody TS-2 that neutralizes the interferon-gamma-inducing activity of OK-432: comparison between the TS-2-binding and TS-2-unbinding fraction.

We have previously isolated a lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432, a streptococcal agent, by affinity chromatography on a CNBr-activated Sepharose 4B bound TS-2 monoclonal antibody (mAb) that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432. In the current study, we compared the cytokine-inducing and anti-tumor activities of OK-PSA, a TS-2-binding fraction, with those of OK-PTF, a TS-2-unbinding fraction, in order to determine the efficacy of OK-PSA for clinical use in affinity chromatography using TS-2. In the in vitro experiments using human peripheral blood mononuclear cells (PBMCs), OK-PSA markedly induced Th1-type cytokines, while interleukin (IL)-6 and IL-10, Th2-type cytokines, were induced by OK-PTF. Th1-cytokine induction by OK-PTF was not dose-dependent and was suppressed when PBMCs were treated with a high concentration of OK-PTF. In a mouse model, Th1 cytokines were also induced by OK-PSA and Th2 cytokines were induced by OK-PTF. Th2 cytokine-inducing activity of OK-PTF was accelerated in tumor-bearing mice relative to that in healthy mice. Although the anti-tumor effect of OK-PTF was statistically significant, it was much weaker than that of OK-PSA. A significant difference between the anti-tumor effect of OK-PSA and that of OK-PTF was observed (P<0.05). Finally, OK-PSA elicited its cytokine-inducing effect via Toll-like receptor (TLR) 4, whereas OK-PTF-induced signaling was mediated by both TLR2 and TLR4. These findings strongly suggested that the affinity chromatography using TS-2 is a useful strategy to separate the effective component for cancer therapy (OK-PSA) from other components.

Involvement of Toll-like receptor 4 signaling in interferon-gamma production and antitumor effect by streptococcal agent OK-432.

BACKGROUND: The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity. METHODS: Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acid-related molecule that is an active component of OK-432), and interferon-gamma (IFN-gamma) mRNA expression, an immune response measure, was analyzed by RT-PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN-gamma protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN-gamma levels were measured over time. All statistical tests were two-sided. RESULTS: Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN-gamma induction in 19 patients administered OK-432 (Fisher's exact test P<.001). Although both OK-432 and OK-PSA induced IFN-gamma expression from PBMCs in vitro, expression of TLR4 and MD-2 was associated only with IFN-gamma expression induced by OK-PSA (P<.001). In vivo intraperitoneal administration of OK-432 resulted in an increase of IFN-gamma in sera from wild-type mice but not in sera from TLR4-deficient mice. Tumors in wild-type mice treated with OK-432 were statistically significantly smaller than those in mice treated with saline (P =.007). By contrast, in TLR4-deficient mice, there was no difference in tumor volume between the two treatment groups. CONCLUSIONS: TLR4 and MD-2 may mediate OK-432-induced anticancer immunity.

Efficacy of adjuvant immunochemotherapy with OK-432 for patients with curatively resected gastric cancer: a meta-analysis of centrally randomized controlled clinical trials.

The benefit of immunochemotherapy employing a streptococcal preparation, OK-432 (Picibanil), in patients with curatively resected gastric cancer was reassessed by meta-analysis of data from 1,522 patients enrolled in six clinical trials with central randomization. All six trials began between 1985 and 1993, and patients were followed-up for at least 3 years after surgery and enrollment of the last patient. In these trials, standard chemotherapy was compared with the same chemotherapy plus OK-432. The endpoint was overall survival and intent-to-treat analysis was done without patient exclusion. Data were analyzed using the Mantel-Haenszel method. The 3-year survival rate for all eligible patients in the six trials was 67.5% in the immunochemotherapy group versus 62.6% in the chemotherapy group. The 3-year overall survival odds ratio was 0.81 (95% confidence interval: 0.65-0.99). The treatment effect was shown to be statistically significant (p = 0.044). The results of this meta-analysis suggest that immunochemotherapy after surgery with OK-432 can improve the survival of patients with curatively resected gastric cancer.

Enhancement of anti-tumor immunity by lipoteichoic acid-related molecule isolated from OK-432, a streptococcal agent, in athymic nude mice bearing human salivary adenocarcinoma: role of natural killer cells.

BACKGROUND: OK-PSA, a lipoteichoic acid (LTA)-related molecule isolated from a streptococcal agent OK-432, enhances anti-tumor immunity as a potent inducer of Th1-type cytokines. Recently, we obtained the data suggesting that natural killer (NK) cells may play a significant role for OK-PSA-induced cytokine production in vitro. MATERIALS AND METHODS: We conducted the animal experiments using athymic nude mice bearing human salivary adenocarcinoma to examine the role of NK cells in OK-PSA-induced anti-tumor immunity. OK-PSA was peritumorally injected into the mice. Cytokines in the sera were analyzed by ELISA. mRNAs for cytokines were detected by RT-PCR. 51Cr release test was performed to measure killer cell activities. RESULTS: OK-PSA markedly increased the amounts of IFN-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18 that are generally called "Th1-type cytokines" in the sera derived from tumor-bearing nude mice, and also accelerated the killing activities of tumor-infiltrating lymphocytes as well as of draining lymph node cells. Furthermore, OK-PSA administration resulted in significant inhibition of tumor growth, but the effect of OK-PSA was almost completely inhibited by the deletion of NK cells using anti-asialo GM1 antibody. CONCLUSION: These findings strongly suggested that NK cells are closely involved in OK-PSA-mediated anti-tumor immunity.