Vaccination of Urological Cancer Patients With WT1 Peptide-Pulsed Dendritic Cells in Combination With Molecular Targeted Therapy or Conventional Chemotherapy Induces Immunological and Clinical Responses.

The prognosis of metastatic or relapsed renal cell carcinoma (RCC) or bladder cancer (BC) remains poor despite the introduction of immune checkpoint blockade agents. We aimed to investigate the safety and the feasibility of a vaccination with WT1 peptide-loaded dendritic cells (DCs) and OK-432 adjuvant combined with molecular targeted therapy or conventional chemotherapy. Five eligible patients with metastatic or relapsed RCC and five eligible patients with BC were enrolled. No severe adverse events related to a vaccination were observed. Seven patients with RCC or non-muscle invasive BC had durable stable disease and three other patients had disease progression after DC vaccination. DC vaccination augmented WT1 specific immunity and the reduction of regulatory T cells which might be related to clinical outcome. These results indicate that DC-based immunotherapy combined with a molecular targeted therapy or a conventional chemotherapy is safe and feasible for patients in advanced stage of RCC or BC.

TLR4 preconditioning is associated with low success of OK-432 treatment for lymphatic malformations in children.

PURPOSE: We have recently shown that the relative TLR4 expression on monocytes of low responding pediatric patients after OK-432 treatment is significantly reduced after stimulation with lipopolysaccharide (LPS) compared with high responding children. The aim of this study was to perform further analysis to explain this observation. METHODS: Monocytes from children with high (HR, n = 5) and low response (LR, n = 6) after previous OK-432 treatment were stimulated with LPS for 20 h and analyzed with fluorescence-activated cell sorting (mean fluorescence intensity, MFI; level of significance P ≤ 0.05). RESULTS: Mean MFI after LPS stimulation was comparable in both groups (HR 1142 ± 652 units, LR 839 ± 427 units, P = 0.85). Significant changes after LPS stimulation are explained by higher pre-stimulation values in the LR group compared with the HR group (950 ± 718 vs. 477 ± 341, P = 0.25) with considerable differences of the mean expression changes after LPS stimulation (HR 665 ± 683 vs. LR -111 ± 605, P = 0.08). CONCLUSION: The previously shown reduced TLR4 upregulation on monocytes after LPS stimulation in the LR group compared with the HR group can be primarily explained by TLR preconditioning. This observation implies the use of absolute values with definite thresholds.

Overcoming regulatory T-cell suppression by a lyophilized preparation of Streptococcus pyogenes.

Cancer vaccines have yet to yield clinical benefit, despite the measurable induction of humoral and cellular immune responses. As immunosuppression by CD4(+) CD25(+) regulatory T (Treg) cells has been linked to the failure of cancer immunotherapy, blocking suppression is therefore critical for successful clinical strategies. Here, we addressed whether a lyophilized preparation of Streptococcus pyogenes (OK-432), which stimulates Toll-like receptors, could overcome Treg-cell suppression of CD4(+) T-cell responses in vitro and in vivo. OK-432 significantly enhanced in vitro proliferation of CD4(+) effector T cells by blocking Treg-cell suppression and this blocking effect depended on IL-12 derived from antigen-presenting cells. Direct administration of OK-432 into tumor-associated exudate fluids resulted in a reduction of the frequency and suppressive function of CD4(+) CD25(+) Foxp3(+) Treg cells. Furthermore, when OK-432 was used as an adjuvant of vaccination with HER2 and NY-ESO-1 for esophageal cancer patients, NY-ESO-1-specific CD4(+) T-cell precursors were activated, and NY-ESO-1-specific CD4(+) T cells were detected within the effector/memory T-cell population. CD4(+) T-cell clones from these patients had high-affinity TCRs and recognized naturally processed NY-ESO-1 protein presented by dendritic cells. OK-432 therefore inhibits Treg-cell function and contributes to the activation of high-avidity tumor antigen-specific naive T-cell precursors.

Improved efficacy of therapeutic vaccination with dendritic cells pulsed with tumor cell lysate against hepatocellular carcinoma by introduction of 2 tandem repeats of microbial HSP70 peptide epitope 407-426 and OK-432.

Therapeutic vaccination with dendritic cells (DCs) pulsed with tumor cell lysate vaccine (H-D) represents an attractive approach for hepatocellular carcinoma (HCC) treatment. However, the efficacy of this approach is not most satisfactory for the low levels of T helper 1 (Th1)-type cytokines secretion and weak T cell responses. In this study, in order to increase the potency of H-D, two tandem repeats of microbial HSP70 peptide epitope 407-426 (2mHSP70(407-426), M2) which has been demonstrated to be effective in enhancing DC maturation were applied. The DC vaccine (HM-D) which was HCC tumor cell lysate pulsed with M2 was developed. Nevertheless, the immunotherapeutic effect was still not satisfactory enough even some promotion was obtained. Therefore, OK-432 (OK), which is a useful anti-cancer agent and effectively in stimulating DC maturation, was introduced to HM-D. Our results demonstrated that treatment with the improved DC vaccine which was tumor cell lysate pulsed with M2 and OK (HMO-D), compared with H-D and HM-D, significantly increased cell surface markers (MHC-I and II, CD40, CD80, CD86 and CD11c) expression on DCs, enhanced Th1-type cytokines (IL-12, TNF-α and IFN-γ) production but not Th2-type cytokine (IL-5) production, induced remarkable high levels of lymphocytes proliferation and CD8(+) cytotoxic T-lymphocyte (CTL). Furthermore, immunization with HMO-D effectively reduced tumor progression and enhanced the survival of mice with H22 tumors. Besides, we also found that the capability of M2 in inducing the Th1 cytokines was stronger than OK. In view of these results, HMO-D vaccination provided a novel immunotherapeutic approach for the treatment of HCC.

A phase I study of vaccination with NY-ESO-1f peptide mixed with Picibanil OK-432 and Montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.

We conducted a phase I clinical trial of a cancer vaccine using a 20-mer NY-ESO-1f peptide (NY-ESO-1 91-110) that includes multiple epitopes recognized by antibodies, and CD4 and CD8 T cells. Ten patients were immunized with 600 µg of NY-ESO-1f peptide mixed with 0.2 KE Picibanil OK-432 and 1.25 ml Montanide ISA-51. Primary end points of the study were safety and immune response. Subcutaneous injection of the NY-ESO-1f peptide vaccine was well tolerated. Vaccine-related adverse events observed were fever (Grade 1), injection-site reaction (Grade 1 or 2) and induration (Grade 2). Vaccination with the NY-ESO-1f peptide resulted in an increase or induction of NY-ESO-1 antibody responses in nine of ten patients. The sera reacted with recombinant NY-ESO-1 whole protein as well as the NY-ESO-1f peptide. An increase in CD4 and CD8 T cell responses was observed in nine of ten patients. Vaccine-induced CD4 and CD8 T cells responded to NY-ESO-1 91-108 in all patients with various HLA types with a less frequent response to neighboring peptides. The findings indicate that the 20-mer NY-ESO-1f peptide includes multiple epitopes recognized by CD4 and CD8 T cells with distinct specificity. Of ten patients, two with lung cancer and one with esophageal cancer showed stable disease. Our study shows that the NY-ESO-1f peptide vaccine was well tolerated and elicited humoral, CD4 and CD8 T cell responses in immunized patients.

Vaccination with OK-432 followed by TC-1 tumor lysate leads to significant antitumor effects.

Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancer. TC-1 cell is a cell line with high expression of E7 antigen of HPV type 16 and its cell lysate has been demonstrated as an ideal inducer of E7-specific, antitumor immunity. OK-432 (Picibanil), a penicillin-killed Streptococcus pyogenes, has been reported with potent immunomodulation properties in cancer treatment by stimulating the maturation of dendritic cells (DCs) and secretion of Th-1 type cytokines. The current study demonstrated that a protocol to immunize the C57BL/6 mice with OK-432 followed by treatment with TC-1 lysate can generate markedly increased immune responses of E7-specific CD4(+) T cells and a moderate increase of natural killer (NK) cell, as well as a satisfactorily protective and therapeutic antitumor effect by triggering the DCs to prime T cells. Depletion of lymphocyte subset in vivo suggested that the antitumor effects could be dominantly executed by CD8+ T cells and followed by NK cells, and both of these reactions were induced by the generation of robust E7-specific CD4(+) T helper cell response. These findings warrant OK-432 combination with tumor-lysate as an effective and safe vaccine in future clinical application of cervical cancer.

Cross-priming of CD8(+) T cells in vivo by dendritic cells pulsed with autologous apoptotic leukemic cells in immunotherapy for elderly patients with acute myeloid leukemia.

OBJECTIVE: The prognosis for elderly patients with acute myeloid leukemia (AML) remains dismal. To explore the potential of immunotherapy for improving clinical outcomes for these patients, we performed a phase I clinical trial of dendritic cell (DC)-based immunotherapy for elderly patients with AML. MATERIALS AND METHODS: Autologus monocytes were obtained after reducing tumor burden by chemotherapy. Immature DCs induced with granulocyte-macrophage colony-stimulating factor and interleukin-4 were pulsed with autologous apoptotic leukemic cells as antigens. DCs were administered intradermally to four patients five times at 2-week intervals. To facilitate DC migration to lymph nodes, injection sites were pretreated with killed Streptococcus pyogenes OK-432 one day before. DCs were coinjected with OK-432 to induce maturation and interleukin-12 production in vivo. RESULTS: Antileukemic responses were observed by an interferon-γ enzyme-linked immunospot assay or a tetramer assay in two of four patients. In a human leukocyte antigen-A∗2402-positive patient, induction of CD8(+) T-cell responses to WT1- and human telomerase reverse transcriptase - derived peptides were observed, indicating cross-priming in vivo. The two patients with antileukemic immunity showed longer periods of disease stabilization than the other two patients. CONCLUSIONS: This study demonstrates the immunogenicity of autologous DCs that cross-present leukemia-associated antigens from autologous apoptotic leukemic cells in vivo in elderly patients with AML.

Antibody responses against NY-ESO-1 and HER2 antigens in patients vaccinated with combinations of cholesteryl pullulan (CHP)-NY-ESO-1 and CHP-HER2 with OK-432.

Combination vaccines of the NY-ESO-1 protein complexed with cholesteryl pullulan (CHP), CHP-NY-ESO-1, and the truncated 146HER2 protein with CHP, CHP-HER2, were subcutaneously administered with the immuno-adjuvant OK-432 to eight esophageal cancer patients. Vaccination was well-tolerated. NY-ESO-1- and HER2-specific antibody responses were analyzed using the patients' sera and samples from previous single CHP-NY-ESO-1 or CHP-HER2 vaccine trial. The responses to NY-ESO-1 in the combination vaccine study were comparable to the single vaccine. For responses to HER2, there were fewer antibody responses in the combination vaccines. Although there were marked individual variations in the antibody responses to the NY-ESO-1 and HER2 antigens, the reaction patterns to these antigens were comparable within each patient. Antibodies to OK-432 were not augmented. Protein cancer vaccines targeting multiple antigens are feasible.

Chemokines are secreted by monocytes following OK-432 (lyophilized Streptococcus pyogenes) stimulation.

BACKGROUND: OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied in vitro the role of mononuclear phagocytes (MNPs), including purified monocytes (MOs), in the immune response to OK-432. MIP-1alpha/beta and MCP-1 secretions were assessed in whole blood (WB), peripheral blood mononuclear cells (PBMCs) and purified MOs, after in vitro stimulation with OK-432 with or without adherence for 24 hours. RESULTS: OK-432 stimulated MNPs to secrete MCP-1 and MIP-1alpha/beta in healthy individuals and in head and neck squamous cell carcinoma (HNSCC) patients, except for OK-432 stimulation of WB giving a minimal MIP-1alpha/beta response. Upon culture on low-attachment wells, a spontaneous chemokine secretion was observed, with an unchanged secretion following OK-432 stimulation. Inhibition of Syk kinase and/or PI-3 kinase did not significantly change the chemokine response to OK-432, except for MIP-1alpha production being increased upon Syk inhibitor addition and an increased MCP-1 response upon addition of both inhibitors. Adhesion may possibly involve beta1 and/or beta3 integrins, not beta2, whereas beta(1-3) integrins may act as co-stimulatory receptors for OK-432. Based on direct blockage of CD36 or CD18 by antibodies, MCP-1 production may be mediated by CD18 while MIP-1beta and MCP-1 production may occur upon binding to CD36. CONCLUSION: Adherent human MOs produce MCP-1 and MIP-1alpha/beta upon stimulation with OK-432. CD36 modulates MIP-1beta and MCP-1 response. Thus, to some extent OK-432 acts as a substance whereby only MOs adhered to surfaces secrete MCP-1 and MIP-1alpha/beta, in part explaining why OK-432 is suited as a biological response modifying drug.

Efficacy of adjuvant immunochemotherapy with polysaccharide K for patients with curative resections of gastric cancer.

Non-specific immunopotentiators, such as polysaccharide K (PSK), also known as OK-432, induce anti-tumor effects via immunological responses. The efficacy of combination immunochemotherapy using these immunopotentiators has been examined by multiple previous studies. The survival benefits of immunochemotherapy for patients with curative resections of gastric cancers are not widely accepted. To clarify this issue, we performed a meta-analysis to evaluate the effect of immunochemotherapy on survival in patients with curative resections of gastric cancer. For this study, we compared the results of chemotherapy and immunotherapy using the biological response modifier PSK as an immunopotentiator. The meta-analysis included 8,009 patients from eight randomized controlled trials after central randomization. The overall hazard ratio for eligible patients was 0.88 (95% confidence interval, 0.79-0.98; P = 0.018) with no significant heterogeneity [chi (2)(8) for heterogeneity = 11.7; P = 0.16]. The results of this meta-analysis suggest that adjuvant immunochemotherapy with PSK improves the survival of patients after curative gastric cancer resection.

Polyarginine-mediated protein delivery to dendritic cells presents antigen more efficiently onto MHC class I and class II and elicits superior antitumor immunity.

Protein transduction domains (PTDs) have been used increasingly to deliver reagents to a variety of cell types in vitro and in vivo. We have previously shown that HIV TAT-PTD-containing whole protein antigens (Ags)-transduced dendritic cells (DCs) stimulated Ag-specific CD8+ and CD4+ T cells. Although the cytotoxic T lymphocytes (CTL) activity generated was sufficient to prevent engraftment of mice with Ag-expressing tumors, treatment of tumor-bearing mice with TAT-PTD Ag-transduced DCs resulted in tumor regression in some animals. Recently, several other PTDs were reported to promote higher transduction efficiencies than TAT-PTD. To evaluate the role of individual PTDs in induction of immune responses in tumor vaccination studies, we engineered recombinant fusion Ovalbumin (OVA) that contained three differrent PTDs, including the most efficacious known PTD (polyarginine (R9)-PTD). Our results demonstrated that R9-PTD-containing OVA transduced DCs most efficiently, and that transduction efficacy was closely correlated with the extent of Ag-specific CD4+ and CD8+ T-cell activation in vitro and in vivo. Repeated vaccination with R9-PTD-OVA-transduced DC in (OVA-expressing) tumor-bearing mice induced enhanced antitumor immunity, and elicited complete rejection of tumors when DC was co-injected with adjuvants. This vaccination strategy may be clinically applicable, and offers theoretical and practical advantages to those that are in current use.

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.

Quick generation of fully mature dendritic cells from monocytes with OK432, low-dose prostanoid, and interferon-alpha as potent immune enhancers.

Dendritic cells (DCs) are one of the promising tools for enhancing antigen-specific immune responses in clinical settings. Many studies have been performed thus far to verify the efficacy of the DC vaccine in cancer patients; however, the responses have not always been satisfactory, partly because of DC incompetence. To obtain DCs potentially applicable for vaccination of cancer patients, our group sought to establish the strategy of DC generation mainly by modulating culture periods and maturation stimuli. Novel mature DCs that can be generated from monocytes within 3 days by using a combination of OK432 (Streptococcus pyogenes preparation), low-dose prostaglandin E2 (PGE2), and interferon-alpha (OPA-DCs) were developed. They strongly express CD83, CD86, and CCR7 and have potent ability to migrate to CCL21. In addition, they were able to activate natural killer and T helper 1 (TH1) cells and to induce peptide-antigen-specific cytotoxic T lymphocytes more significantly than monocyte-derived DCs stimulated with a conventional cytokine cocktail of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and PGE2 (monocyte-conditioned medium [MCM]-mimic DCs). The profound ability of OPA-DCs to stimulate these effectors is attributable to their higher expression of IL-12p70, IL-23, and IL-27 than MCM-mimic DCs, which was supported by the findings that the neutralization of IL-12p70 and IL-23 reduced the TH1 priming ability of OPA-DCs. Even when from advanced gastric or colonic cancer patients, OPA-DCs displayed abilities of migration and TH1 induction comparable to those from healthy subjects. Therefore, OPA-DCs may serve as a feasible vaccine with the potential to enhance TH1-dominant and cytolytic immune responses against cancers.

Understanding the response of dendritic cells to activation by streptococcal preparation OK-432.

BACKGROUND: The streptococcal preparation OK-432 induces maturation of T cells and dendritic cells (DCs). However, the mechanisms by which OK-432 induces DC maturation are not well understood. MATERIALS AND METHODS: The effects of OK-432 and TNF-alpha on peripheral blood mononuclear cells (PBMCs) were compared. Antibody-based approaches were used to detect proteins characteristic of antigen-presenting cells and cytokines. Cytotoxicity was evaluated by measuring the release of LDH after incubation of effector and target cells. The TLR-4 levels were measured by real-time quantitative PCR. RESULTS: Changes in cell surface marker levels were detected in both treatment groups but OK-432 had a greater effect on induction of Th-1-type cytokines. Furthermore, TLR-4 mRNA was up-regulated in cells from two out of five patients in response to OK-432, but not in response to TNF-alpha. CONCLUSION: OK-432 has a more profound effect on human DCs than TNF-alpha and may act through multiple signaling pathways.

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 role of cytokines in OK-432 injection therapy for cystic lymphangioma: an approach to the mechanism.

PURPOSE: This study examines cytokine levels of aspirates from cystic lymphangiomas after injection of OK-432 and over the course of several weeks to better understand the process of tumor regression. METHODS: Fluids aspirated from lymphangioma cysts of 3 patients were collected sequentially before and after OK-432 injection. Mononuclear cells (MNCs) were separated and cultured with or without OK-432. Vascular endothelial growth factor (VEGF), soluble VEGF receptor 1 (sVEGFR1), sVEGFR2, transforming growth factor beta-1 (TGF-beta1), interleukin (IL)-6, IL-8, IL-12+p40, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) levels in the supernatants of the aspirates and the culture supernatants of MNCs were then measured by ELISA. RESULTS: The aspirates exhibited a marked elevation in IL-6, IL-8, VEGF, and TGF-beta1 levels for a few weeks after the OK-432 injection. IL-6, IL-8, IL-12+p40, TNF-alpha, and IFN-gamma levels were elevated in the culture supernatants of the MNC cultured with OK-432 for up to 9 days. All the tumors regressed significantly, with sclerotic change, within 3 months after OK-432 injection. CONCLUSIONS: Cytokine production is maintained for a few weeks after OK-432 injection. Fibrotic changes may be another main mechanism in tumor regression in addition to cytotoxic effects on lymphangioma cells. A close relationship between cytokines from intracystic cells and lymphangioma cells is suggested.

Combined immunotherapy with intracavital injection of activated lymphocytes, monocyte-derived dendritic cells and low-dose OK-432 in patients with malignant effusion.

We have conducted a pilot study with combined immunotherapy using autologous lymphocytes activated ex vivo and monocyte-derived dendritic cells in combination with low-dose OK-432, a streptococcal preparation, in five patients with peritoneal or pleural carcinomatosis who were resistant to standard chemotherapy. All patients were given 3 to 10 courses of the combined immunotherapy. No severe adverse reactions occurred. Effusion production was decreased in all of the patients. Significant decreases in tumor markers of both effusions and sera as well as effusion volume occurred in all of the patients. Cytological examinations revealed a marked decrease or disappearance of cancer cells in those effusions. Three patients showed increase in IFN-gamma levels in the effusions. The overall prognosis of the patients was acceptable and the mean survival time was more than 9 months. The locoregional immunotherapy seems to be encouraging in view of therapeutic modality in patients who are resistant to standard chemotherapy. Our study provides a new protocol for immunotherapy and warrants further phase I/II clinical study for chemo-resistant patients with malignant effusion.

Locoregional immunotherapy of malignant effusion from colorectal cancer using the streptococcal preparation OK-432 plus interleukin-2: induction of autologous tumor-reactive CD4+ Th1 killer lymphocytes.

In total, 16 patients with cytologically proven malignant effusion from colorectal cancer were treated by locoregional administration of the streptococcal preparation OK-432 alone or OK-432 plus the T-cell growth factor interleukin (IL)-2, and the action mechanism of the treatment was studied. A positive clinical response, showing a cytologic disappearance of cancer cells and decrease of effusion, was observed in nine of 11 (82%) patients treated with OK-432 alone and in all five patients treated with OK-432 plus IL-2. Flow cytometric analysis revealed that OK-432 plus IL-2 locally induced acute inflammation-like responses, including serial cellular infiltrations of granulocyte migration within a matter of hours, and activation of macrophages and T lymphocyte involvement within the following days, and that a predominant expansion of CD3+CD4+ lymphocytes (CD: cluster of differentiation) was induced by in vitro stimulation with IL-2 of locoregional cells after the OK-432 administration (OK/IL-2AK cells). The OK/IL-2AK cells produced tumour necrosis factor-alpha and interferon-gamma, but these cells did not produce IL-4 and IL-6. The OK/IL-2AK cells expressed potent killing activity against autologous tumour cells. This activity was abrogated by treatment of the lymphocytes with anti-CD3, -CD4, -TCRalphabeta antibody, and by the treatment of target cells with anti-human leukocyte antigen (HLA)-DR antibody. The OK/IL-2AK cells expressed Fas-L gene, and flow cytometric analysis demonstrated HLA-DR expression in approximately 75% of CEA+ or cytokeratin+ effusion cells. TCRVbeta gene analysis of the OK/IL-2AK cells showed an oligoclonal usage of TCRbeta20, which was also involved in the cytotoxic mechanism of the OK/IL-2AK cells. Single-strand conformational polymorphism analysis demonstrated the clonotypes for the TCRVbeta20 gene, and the CDR3s of the gene were sequenced. The clonotypic PCR using the TCRVbeta20-CDR3 sequences could detect the CDR3-identical TCRs in effusion lymphocytes from the other patients. Taken together, it is suggested that locoregional administration of OK-432 plus IL-2 is highly effective for the management of malignant effusion from colorectal cancer. OK-432 plus IL-2 induces autologous tumour-reactive CD4+ Th1 killer lymphocytes, which recognise tumour antigen(s) presented with HLA class II molecules on effusion tumour cells by means of preferential usage of TCRVbeta20. The clonotypic PCR using the TCRVbeta20-CDR3 sequences may be informative for treating malignant effusion from colorectal cancer using OK-432 plus IL-2.

Novel OK-432-conjugated tumor vaccines induce tumor-specific immunity against murine tongue cancer.

Priming with tumor antigens is one of the most important strategies in cancer immunotherapy. To enhance tumor antigenicity, OK-432, a streptococcal preparation, was coupled to squamous cell carcinoma (KLN-205) by means of a 0.2% glutaraldehyde method. The purpose of this study was to investigate whether OK-432-conjugated tumor vaccines could induce tumor-specific immunity. Our originally developed mouse tongue cancer model was used throughout this work for the analysis of antitumor effects. Prepared OK-432-conjugated KLN-205 vaccines were immunized 3 times to DBA/2 mice. The results showed that the KLN-205 vaccines induced cytolytic activity and strongly suppressed both KLN-205 tumor incidence and growth, and survival of the mice was improved. Moreover, the histological results showed that a greater number of lymphocytes had infiltrated around tumor cells by 24 hours after tumor inoculation in the vaccine group. These results suggest that immunizations with KLN-205 vaccines increase the antitumor effects against tongue cancer.

Effects of streptococcal preparation OK-432 on cytokine induction in spleen and tumour tissues of mice bearing MH-134 tumour cells.

Streptococcal preparation OK-432 is a bacterial immunopotentiator extensively used in Japan for adjuvant cancer therapy. Using C3H/He mice bearing MH-134 tumour cells, cytokine inductions of tumour necrosis factor-alpha, interleukin 1 beta, interleukin 6 and interferon-gamma were determined in spleen and tumour tissues by reverse transcriptase-polymerase chain reaction analysis. No significant induction of cytokine mRNA was observed after subcutaneous administration of OK-432 (OK-432, s.c.) or after intratumoural injection of IFN-gamma (IFN-gamma, i.t.), compared with controls, either in spleen or tumour tissues. In contrast, subcutaneous administration of OK-432 followed by intratumoural OK-432 injection (OK-432, s.c. + i.t.) was found to induce some cytokine mRNAs significantly. The mRNA levels of tumour necrosis factor alpha and interferon-gamma in spleen tissue and those of interleukin 1 beta and interferon-gamma in tumour tissues were significantly elevated in mice with OK-432, s.c. + i.t. treatment compared with controls. These results suggest that treatment with OK-432, s.c. + i.t. effectively induced splenic antitumour immunity as well as local immunity against tumour cells.