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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., (© 2018 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.)

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.

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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2011 UICC.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In order to enhance the antitumor vaccination effect of dendritic cells (DC) pulsed with class I tumor peptide, we tried to utilize the local cytokine help of CD4+ T cells reactive to a streptococcal preparation OK432. DC were prepared from murine bone marrow cells by culture with both granulocyte/macrophage-colony-stimulating factor and interleukin(IL)-4. The peritumoral injections of OK432 induced OK432-reactive CD4+ T cells in the draining lymph nodes, and their in vitro production of interferon gamma was thus significantly enhanced by restimulation with OK432-pulsed DC. In addition, anti-P815 mastocytoma cytotoxic T lymphocytes were generated from the in vivo OK432-treated P815-draining lymph node cells only when the lymph node cells were restimulated in vitro with the DC pulsed with both P1A peptide and OK432. Moreover, the peritumoral injections of OK432 and the subsequent vaccination of the DC, pulsed with both OK432 and P1A peptide, significantly suppressed the growth of s.c. inoculated P815. Interestingly, a significant level of IL-12 was detected in the coculture supernatant of both OK432-pulsed DC and OK432-reactive CD4+ T cells. Collectively, our results suggest that the antitumor vaccination effect of DC pulsed with class I tumor peptide could thus be effectively augmented by locally utilizing the Th1-type cytokines from OK432-reactive CD4+ T cells.

Adoptive immunotherapy (AIT) for non-hematological malignancies, using HLA-matched donor lymphocytes, has been rarely reported. For a 35-year-old male patient with peritoneal disseminated advanced gastric cancer, we performed AIT using lymphocytes from his HLA-matched 37-year-old brother and a streptococcal preparation, OK-432, as an antigen. After the donor had been immunized by intradermal administration of OK-432, OK-432-reactive lymphocytes were induced in vitro and transferred to the patient intravenously with OK-432. Low-dose systemic immunochemotherapy, using interleukin-2, 5-fluorouracil and cyclophosphamide, was concurrently administered with AIT. As a result, the Schnitzler metastasis in the patient reduced in size without any significant graft-versus-host-related complications. One of the effector mechanisms of therapeutic benefit was suggested to be cytokine release from the transferred OK-432-reactive lymphocytes. Our findings suggest the safety and efficacy of AIT using lymphocytes from an HLA-matched sibling and OK-432 as an antigen. Further studies to investigate the use of tumor-associated antigen and an HLA-matched sibling's lymphocytes for AIT of advanced cancer are warranted.

Vaccinations with tumor cells engineered to express certain cytokines have been demonstrated to induce potent and specific antitumor immunity. In our previous report, we carried out a comparative study on the ability of cytokine-gene-modified tumor vaccines to induce host immune responses, and found that irradiated tumor cells, genetically modified to secrete granulocyte/macrophage-colony-stimulating factor (GM-CSF tumor vaccine), were the most potent stimulators of systemic antitumor immunity. In this report, using the experimental tumor models in which the GM-CSF tumor vaccine was less effective in immunopotentiation, we found that the combined use of a biological response modifier (BRM) OK-432 remarkably enhanced the antitumor activity induced by the GM-CSF tumor vaccine. These data indicate the possible role of a BRM such as OK-432 to intensify further the specific tumor vaccination therapy.

Low concentrations of exogenously added recombinant interleukin 2 (rIL-2) were able to augment OK-432-induced natural killer (NK) cell activity. This kind of augmenting effect depended on the dose of rIL-2 and manifested itself only in PBMC stimulated with OK-432 (OK-MC) followed by rIL-2; augmentation did not happen in the reverse order. The existence of CD16+/CD25+ (IL-2 receptor positive; IL-2R+) and CD57+/CD25+ double positive cells which possess NK cell surface markers in OK-MC markedly increased in a long-term culture (12 days). A strong positive correlation was observed between the IL-2-dependent augmentation of NK activity and the quantitative changes in cell populations that possessed NK cell phenotypes. Treatment of the day-12-OK-MC with monoclonal anti-CD56 antibody plus complement could almost completely abrogate the augmented NK cytotoxicity. Furthermore, this augmenting effect was detectable within 4 hr after addition of rIL-2 at single cell level, suggesting that the effect did not require NK cell's DNA synthesis. Thus it was suggested that OK-432 could promote and upregulate the expression of IL-2 receptor (CD25) on CD56+ NK cell populations. Moreover, it was considered that the interaction of low concentration rIL-2 with IL-2 receptors on OK-432-activated NK cells could augment their lytic function.

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.

Twenty two surgical specimens of gastric cancer resected after administration of OK-432 for the skin reaction test were examined to determine whether the cancer cells had the same antigens as OK-432, a product of hemolytic streptococcus cells. When the tissues were stained by the PAP method with anti-Su streptococcus antibody used as the primary antibodies, the common antigens were demonstrated in 10 (45.5%) of the 22. The presence or absence of the common antigens was independent of the degree of skin reaction to OK-432, and the relations of the common antigens to other host responses were not clear in this study. This is the first report for the presence of such common antigens between human gastric cancer and OK-432.

We established a method of in vitro immunization of human lymphocytes against human cancer cells and efficiently obtained human-human hybridomas producing cancer-specific antibodies using the in vitro immunized lymphocytes. Human lymphocytes were cocultured with human lung cancer cell line A549 for four days in medium containing various immunoactive reagents. In vitro immunization was effectively caused by the addition of OK-432 or muramyl peptides, IL-2, and IL-6, to the coculture of A549 cells and lymphocytes. Although specific antibodies of both IgM and IgG classes were produced by this method, the ratio of IgG to IgM was greater than or equal to 2. The efficiencies of in vitro immunization fluctuated about threefold in lymphocytes derived from several donors. The in vitro immunized lymphocytes were fused with human fusion partner A4H12 cells. Hybridomas producing specific antibodies reactive to A549 cells were efficiently obtained by sequential cell fusion. The efficiency of acquisition of hybridomas producing specific antibodies with the in vitro immunized lymphocytes was about 10-fold higher than that with lymphocytes spontaneously immunized in bodies of lung cancer patients.

Polyclonal activation of human peripheral blood lymphocytes (PBLs) in vitro by preparations of Streptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4+ helper type of T cells, and CD4-CD8- double-negative (DN) lymphocytes, including both CD3+ TcR gamma + T cells and CD2+CD3- immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3+ and CD3- types of CD4-CD8- DN lymphocytes, but not CD4+ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4-CD8- T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLs in vitro to induce the proliferation/activation of CD4+ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.

Peritoneal polymorphonuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 micrograms)/head of OK-432 or 200 micrograms/head of Nocardia rubra cell wall skeleton (N-CWS). Without in vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in 51Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 micrograms/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H2O2 production of PSK-induced PMNs was increased by the in vitro addition of 25 micrograms/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.

OK-432, a streptococcal preparation, has been used in the treatment of malignant diseases. We have found that OK-432 can act as an antigen and have established an OK-432-specific L3T4+ Lyt2- T-cell line (OK2) and a clone (OK2.21) from OK-432-immunized BALB/c mice (Iad) as antitumor effector cells. OK2 proliferated and secreted interleukin 2, but only when OK-432 was presented by Iad-positive antigen-presenting cells. Despite its helper phenotype and function, OK2 could kill OK-432-pulsed Iad-positive B-lymphoma cells. This killing was inhibited only by cold specific target cells (cold-target inhibition). OK-432 induced the cytotoxicity of OK2 as a specific antigen, not as a nonspecific immunostimulator. OK2 and OK2.21 also killed Ia-negative bystander target cells only in the presence of OK-432-pulsed Iad-positive cells (bystander killing). Double-chamber experiments suggested that the bystander killing was mediated by a short-acting soluble cytolytic factor. Finally, the OK-432-specific T-cells selectively killed tumor cells, suggesting that these T-cells play an important role in the immune surveillance against malignancy.

Antibody-dependent cell-mediated cytotoxicity (ADCC) through serum, which was obtained from B6D2F1 mice pretreated with heated group A streptococci, and spleen cells of normal B6D2F1 mice was examined for different kinds of mouse tumor cell line by using methods to estimate specific 51Cr-release from each 51Cr-labeled target cells. Serum obtained from mice 14 days after the injection with heated streptococci was evidently potent to cause ADCC for all tumor cells tested. When the experiments were carried out under the condition which E/T ratio is 50:1, specific 51Cr-release from each target cells were about 32% for L1210 cells, about 12% for MOPC 31C cells, and about 23% for MH 134 and about 10% for P 388 cells, respectively. Meanwhile, serum obtained from either BCG-injected mice or normal mice was not so much potent for these tumor cells. Experiments to identify immunoglobulin subclasses participating in antibody-dependent cell-mediated cytotoxic reactions clearly showed that only IgG2a immunoglobulin was effective to induce cytotoxic reaction. All classes or subclasses of serum immunoglobulins from mice pretreated with group A streptococci were estimated using single radial immunodiffusion plates which were incorporated with monospecific rabbit antiserum for each classes or subclasses. Results showed that either IgM or IgG2a levels were significantly increased 7 days and 14 days after the injection, but other immunoglobulin classes or subclasses did not. A possible consideration on the production of immunoglobulin to cause ADCC in mice treated with heated streptococci was discussed.

Delayed hypersensitivity (DH) against OK-432 was induced in BALB/c mice by injecting 2 KE of OK-432 with Freund's incomplete adjuvant into foot pads. One week after presensitization with OK-432, 2 X 10(5) cells of Meth A tumor were implanted subcutaneously in all mice, followed by intratumoral injection of 1 KE of OK-432 five times every other day starting at 7th day in experimental group. Tumor growth was significantly inhibited in the OK-432 presensitized group comparing to non-sensitized group. In experimental groups marked inhibition was observed in mice which were injected with OK-432 intratumorally 2 to 3 weeks after presensitization. This effect correlated well with delayed hypersensitivity against OK-432. Histological changes after intratumoral injection of OK-432 were examined in order to analyse the mechanism of this effect. The main finding of OK-432 injected specimen by H.E. staining were degeneration of tumor cells and infiltration of inflammatory cells. These changes were stronger in OK-432 presensitized mice. By beta-D-galactosidase staining accumulation of macrophages was found both inside the tumor and the surrounding tissue, and these macrophages increased in OK-432 presensitized mice. Immunoperoxidase staining with antiasialo GM1 anti-serum was also performed. Greater number of activate macrophages were observed to accumulate in the specimen of OK-432 presensitized mice than that of control mice. Some T cells were observed only around tumor tissues and not in the tumor of both presensitized and unsensitized mice. These results suggest that the activated macrophages play a major role in the augmentation of antitumor effect by presensitization with OK-432.

The action mechanism of antibody-dependent macrophage-mediated cytotoxicity (ADMC) induced by OK-432 against MM2 carcinoma cells was examined. Adherent peritoneal exudate cells (adherent PEC) harvested from mice 4 days after intraperitoneal injection of OK-432 exhibited potent cytotoxic activity against MM2 cells in the presence of anti-serum obtained from tumor-free mice which had survived over 60 days following treatment with OK-432 and resisted rechallenge with MM2 cells. The cytotoxic activity of the adherent PEC was not abolished by treatment with anti-Thy-1.2 and complement, and there were no differences in ADMC between adherent PEC from BALB/c mice and those from athymic BALB/c (nu/nu) mice. Further, ADMC activity was shown not only against MM2 cells, but also against other allogeneic tumor cells, such as MOPC-11 plasmacytoma cells. On the other hand, the effective factor(s) in anti-serum to MM2 cells was eliminated by passage through a protein A-Sepharose CL-4B affinity column. The action mechanism of ADMC caused by the adherent PEC and anti-serum to MM2 cells is discussed.

Cord blood mononuclear cells (MNC) were defective in their ability to produce interferon-gamma (IFN-gamma) on stimulation with phytohemagglutinin (PHA) or recombinant interleukin 2, whereas cord MNC could induce comparable amounts of IFN-gamma with adult controls on stimulation with a streptococcal preparation, OK-432. Moreover, irradiation of cord MNC with 1,500 rad before PHA stimulation could restore the IFN-gamma production. Kinetic studies indicated that such augmentation of IFN-gamma production by irradiation was evident when cord MNC were irradiated before or by 12 hr of PHA-stimulated culture. But irradiation after 18 hr or more of PHA stimulation did not exert any significant augmentation on IFN-gamma production by cord MNC. It seemed most likely that the ability of IFN-gamma production is already mature at birth, but radiosensitive suppressor effectors on IFN-gamma production are activated within cord MNC at an early stage of PHA stimulation, resulting in poor IFN-gamma production by cord MNC. PHA-induced IFN-gamma production by OKT3+, OKT4+, and OKT8- cord cells were markedly enhanced by irradiation with 1,500 rad before the culture. Coculture experiments disclosed that cord OKT4+ cells, but not OKT4- cells, when prestimulated with PHA for 24 hr, exerted active suppression on PHA-induced IFN-gamma production by adult MNC in a dose-dependent manner. These results suggested that radiosensitive suppressor effectors on IFN-gamma production were induced within the OKT4+ T cell subset of cord MNC on PHA stimulation.

The streptococcal preparation OK432 augments natural cytotoxicity of human blood lymphocytes and monocytes. It also enhanced the production of natural killer soluble cytotoxic factors (NKCF) when the effector cells interact with K562 cells. There was a good correlation between the OK432-induced enhancement of NK cell-mediated cytotoxicity and the released NKCF activity. OK432-pretreated monocytes secreted higher amounts of monocyte cytotoxic factors (MCF) than the untreated monocytes. With the monocytes the enhanced generation of MCF was not always accompanied by the increase in direct cell-mediated lysis of K562. OK432 treatment alone did not induce NKCF release from lymphocytes, and the presence of K562 in the culture was necessary. In contrast, monocytes generated MCF when exposed to OK432. In the supernatants of cocultures of OK432-activated effectors and K562 the NKCF and MCF activity was elevated two- to ten-fold. The OK432-induced augmentation of natural cytotoxicity exerted by lymphocytes and monocytes may be mediated through an increase in the synthesis, activation and/or release of NKCF and MCF.

Injection ip of a streptococcal preparation, OK-432, into WKA and DONRYU rats induced in vitro cytotoxicity of polymorphonuclear leukocytes (PMN) against tumor cells in terms of their cytostasis and cytolysis. PMN were obtained from peritoneal exudates of WKA and DONRYU rats. Cytotoxicity of activated PMN was immunologically nonspecific, although a certain target selectivity was observed in PMN cytotoxicity. The titer of cytotoxicity of PMN was dependent on the dose of injected OK-432 and on the number of PMN in the cytotoxicity assay. Activated PMN appeared very early (6 hr) after OK-432 injection, and the cytotoxic titer of PMN decreased from 24 hours and disappeared at 96 hours after injection of the reagent. In vitro culture of PMN with OK-432 also evoked cytotoxicity of PMN.

The early cellular responses to antitumor immunomodulators and conventional inducers, especially the polymorphonuclear leukocyte (PMN) responses, were examined in the peritoneal cavity of mice to investigate their effect on primary defense mechanisms. Immunomodulators were classified into 5 groups in terms of PMN response on the basis of its duration (declining or persistent) and extent (high or low induction): 1) TAK (beta-1,3-glucan)-type (high, persistent), 2) lentinan-type (high, declining), 3) yeast mannan-type (low, declining), 4) LPS (lipopolysaccharide)-type (low, persistent), 5) others (no effect). Since the general PMN response is of the declining type, the persistence of PMN with TAK- and LPS-type immunomodulators is a characteristic of the PMN-inducing activity. With respect to the extent, TAK- and lentinan-type immunomodulators induced larger numbers of PMN and macrophages than conventional inducers. These results suggest that some types of immunomodulators have effects on the early host-defense mechanism. From the viewpoint of the general self-defense mechanism we also compared these PMN responses with those to bacteria and to tumor inoculation, and the properties of substances inducing high PMN response, i.e., those with the quality of "foreignness," are discussed.

Effect of mitomycin C (MMC) administration on the generation of cytotoxic cells induced by in vitro activation of peripheral blood mononuclear cells (PBM) with OK-432, a bacterial immunopotentiator, was studied in patients with various carcinomas. Following i.v. injection of a single dose of 12 mg/m2 MMC, the ability of PBM to generate OK-432 activated killer cells was markedly increased. Thus, the cytotoxic activity observed 7 days after MMC administration was significantly augmented as compared to that before treatment. Therefore, the ability to generate lymphokine activated killer (LAK) cells was examined, and significantly increased capacity was observed 5 and 7 days after MMC injection. Then, the OK-432 activated killer cell activity significantly correlated with the LAK activity. After treatment, the distribution of lymphocyte subsets exhibited a significant decrease in the percentage of OKT8+ cells. Leu-11+ cells were also reduced. The results appear to indicate that the imbalance in T-cell subsets and the increase in the ability to induce LAK cells may be related to the augmenting effect of MMC administration on the generation of OK-432 activated killer cells in cancer patients.

In 38 patients with superficial bladder, local immunotherapy with streptococcal preparation OK-432 has been performed. We investigated whether OK-432 was an effective biological response modifier (BRM) against bladder tumors or not, and the relationship between the common antigens which OK-432 shared with the tumors, and antitumor effects of OK-432. In six out of 28 patients treated by intravesical instillation, and three out of 10 cases treated by intratumor injection, tumors were eliminated endoscopically. In the other patients, the tumors did not change. The PAP study using an anti-OK-432 antibody, showed a positive reaction in 66.7% of the instillation cases and in 40.0% in the injection cases. In 66.7% of the patients with the common antigens treated by the instillation and in 75.0% of patients with the antigens treated by the injection, tumors were eliminated. However, the PAP study showed a positive reaction in 9.1% of the no-change cases treated by the instillation and in 14.3% out of the no-change cases treated by the injection. We concluded that OK-432 was a favorable BRM for topical immunotherapy against bladder tumor and the presence of the common antigens between OK-432 and tumor may enhance the immune response of patients and promote tumor regression.

The pretreatment of B6D2F1 mice with OK-432 alone was not so effective to retard the growth of L1210 leukemia, and the leukemic cell was poorly immunogenic in the mice. However, when OK-432 was given by ip injection in combination with mitomycin-C-treated L1210 cells, the growth of L1210 leukemia was significantly retarded and some mice did not "take" the leukemia. Meanwhile, BCG was also effective in this respect. Differences in properties as an immunopotentiator between OK-432 and BCG were suggested by the experiments of in vitro cytotoxicity test of spleen cells.

The inactivate streptococcal preparation OK432 activates the cytotoxic function of natural killer (NK) cells. Moreover, it induces cytotoxic activity against freshly isolated tumor cells. The present study was aimed at assessing whether OK432-activated effector cells expressed cytotoxicity against tumor cells pleiotropically resistant to cancer chemotherapy agents. OK432-treated lymphoid cells killed the multidrug resistant (MDR) LOVO DX line as efficiently as drug sensitive parental LOVO N carcinoma line. Effector cells involved in killing MDR cells were low density large granular lymphocytes with NK functions. Activation of effector cells has the potential to complement conventional cytoreductive therapy by eliminating residual-tumor cells surviving and resistant to chemotherapy.

Interleukin-1 (IL-1) inducing activity of a streptococcal preparation OK-432 and its fractions by monocytes were investigated in patients with lung cancer and healthy subjects. The results showed that cell free extracts and cell wall fractions of OK-432 were the strong IL-1 inducing fractions. IL-1 activity released by OK-432-stimulated monocytes of patients with lung cancer fell within normal range. OK-432 stimulated intracellular IL-1 synthesis as well as extracellular release by monocytes. These results, therefore, suggested that OK-432 immunotherapy in patients with malignant diseases might be effective by increasing IL-1 production of monocytes.

Immunobiological activities of OK-432 and N-CWS were studied in BALB/c-Meth-A and C3H-MH 134 systems. Peritoneal exudate cells obtained from mice injected i.p. with OK-432 or N-CWS showed stronger in vitro cytotoxicity against Meth-A and YAC-1 cells than did those from non-treated mice. Spleen cells from OK-432 or N-CWS-treated mice exhibited lower Con A mitogenic response in comparison with those from non-treated mice. When Meth-A or MH-134 tumor cells were admixed with OK-432 or N-CWS, the intradermally inoculated tumor cells showed no or slower growth than did those admixed with saline only. There were no significant differences in all the above mentioned activities between OK-432 and N-CWS, and there was no difference in the direct tumor inhibitory activity of the two agents in vitro. However, the administration of N-CWS had no therapeutic effect against i.p.-inoculated tumor cells while OK-432 showed a strong therapeutic effect by the same treatment schedule. Further studies are required to clarify the mechanisms responsible for the difference in the therapeutic effect of the two agents.

The cross-antigenicity between streptococcus, Su strain (OK-432), and rat bladder cancer BC-47 was studied by the immunocytochemistry. Both anti-Su (OK-432) and anti-BC-47 reacted with both homo- and heterogenous antigens. The specificity of the PAP reactions using these sera was confirmed by absorption study of the first antibodies. But, OK-432 did not share with normal rat tissues. Furthermore, the presence of common antigens between OK-432 and animal tumors where OK-432 was effective was indicated by the PAP studies, but OK-432 did not share with tumors where OK-432 was ineffective. By the PAP studies using antigroup A streptococcus-specific C-polysaccharide and antigroup-specific sera, it was suggested that the determinant of the cross-antigenicity was group A-specific C-polysaccharide. The common antigens between OK-432 and human urogenital cancers were also identified by the PAP study. Then, local immunotherapy with OK-432 was carried out in 38 patients with superficial bladder tumor and the tumors were eliminated in 23.7%. In 77.8% of the complete response and in 10.3% of the no change, the tumor tissues demonstrated antigens common between OK-432 and tissues. From these results, it was concluded that common antigens which OK-432 shared with tumors could favorably augment host defense against human bladder tumors, the same as animal tumors.

The reaction of skin to 10-micrograms and 20-micrograms quantities of Su-polysaccharide (Su-Ps) were examined in 117 patients with cancer, and the results obtained were as follows. Reaction was a little greater to the 20-micrograms dose than to the 10-micrograms dose, but the two parameters were much correlated with each other. The rates of patients showing positive reactions to each dose were 30.8% for 10-micrograms and 34.2% for 20-micrograms. The reactions were augmented by administration of OK-432. The augmentation of skin reaction by OK-432 was more prominent at 20-micrograms doses than at 10-micrograms doses, therefore appearing to be Su-polysaccharide dose-dependent. The ideal Su-polysaccharide dose for Su-Ps skin reaction thus seemed to be 20-micrograms.

Murine spleen cells cultured for 3 or more days in medium with streptococcal preparation OK-432 became cytotoxic in vitro against several allogeneic and syngeneic tumor cells. These cytotoxic cells were designated OK-432-induced killer (OIK) cells. This study examined the in vivo antitumor efficacy of OIK cells in adoptive immuno- and immunochemo-therapy in mice bearing syngeneic tumors, such as EL-4 lymphoma, Meth-A fibrosarcoma, and MOPC-31C plasmacytoma. OIK cells neutralized these tumor cells, as shown by Winn-type tests, and the cell transfer prolonged the survival of mice inoculated intraperitoneally (ip) with EL-4 or Meth-A cells. Concomitant administration of OK-432 plus recombinant interleukin 2 (rIL-2) significantly improved the therapeutic efficacy of the transferred OIK cells. In mice inoculated with 1 x 10(4) EL-4 cells, chemoimmunotherapy consisting of ip administration of 200 mg/kg cyclophosphamide on day 3 followed by treatment with OIK cell (1 x 10(7)) transfer and with OK-432 (50 KE/kg) plus rIL-2 (50 units/mouse) 6 hr later and on day 6, prolonged the survival. Therefore, the immunotherapy with OIK-cell transfer followed by administration of OK-432 and rIL-2 may be clinically useful as an adjunct of cytoreductive chemotherapy for cancer.