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.     

In vivo immunological antitumor effect of OK-432-stimulated dendritic cell transfer after radiofrequency ablation

Radiofrequency ablation therapy (RFA) is a radical treatment for liver cancers and induces tumor antigen-specific immune responses. In the present study, we examined the antitumor effects of focal OK-432-stimulated dendritic cell (DC) transfer combined with RFA and analyzed the functional mechanisms involved using a murine model. C57BL/6 mice were injected subcutaneously with colon cancer cells (MC38) in their bilateral flanks. After the establishment of tumors, the subcutaneous tumor on one flank was treated using RFA, and then OK-432-stimulated DCs were injected locally. The antitumor effect of the treatment was evaluated by measuring the size of the tumor on the opposite flank, and the immunological responses were assessed using tumor-infiltrating lymphocytes, splenocytes and draining lymph nodes. Tumor growth was strongly inhibited in mice that exhibited efficient DC migration after RFA and OK-432-stimulated DC transfer, as compared to mice treated with RFA alone or treatment involving immature DC transfer. We also demonstrated that the antitumor effect of this treatment depended on both CD8-positive and CD4-positive cells. On the basis of our findings, we believe that combination therapy for metastatic liver cancer consisting of OK-432-stimulated DCs in combination with RFA can proceed to clinical trials, and it is anticipated to be markedly superior to RFA single therapy.     

Combined effects of cis-DDP and OK-432 on ascitic mastocytoma in mice: optimal period for the administration of OK-432

Combined effects of cis-DDP and OK-432 on the ascites mastocytoma was studied in mice laying emphasis on the best timing for the administration of the OK-432. Mice were transplanted with FMA3 mastocytoma cells into the abdominal cavity at a dose of 10(5) cells per mouse. They were injected with cis-DDP next day at a single shot of 8 mg/kg in the abdominal cavity. A streptococcal preparation, OK-432, was i.p. injected two times at intervals of one week at a dose of 50 KE/kg per injection. The pair of injections started 1, 2, 3, 4 or 5 weeks after the transplantation. Mean survival time (M. S. T.) of mice was 16.8, 16.2 and 52.3 in the groups of mice nontreated, given OK-432 alone, and given cis-DDP alone, respectively. In comparison of M. S. T. within the groups of mice treated with the combination of cis-DDP and OK-432, the highest value was observed in the group which was given OK-432 between 3 and 4 weeks after the transplantation.     

Orally administered streptococcal preparation,OK-432 augments the antitumor immunity of patients with gastric or colorectal cancer

A streptococcal preparation, OK-432, was orally administered at a dose of 5 KE to patients with gastric or colorectal cancer for 7–14 days before their operations, and its immunomodulatory effects on peripheral blood lymphocytes (PBL), regional node lymphocytes (RNL) and tumor infiltrating lymphocytes (TIL) were assessed. The group treated with OK-432 included 8 gastric and 6 colorectal cancer patients, and the control group included 8 gastric and 8 colorectal cancer patients. The NK cell activity of PBL was significantly augmented by the oral administration of OK-432, and the proportions of Leu 7+ and Leu 11+ cells in PBL also increased. The responses of PBL and TIL to autologous tumor extracts in the presence of interleukin-2 were enhanced after the oral administration of OK-432. The proportion of OKT8+ cells in PBL increased after treatment with oral OK-432, whereas the proportion in RNL significantly decreased. These results indicate that oral OK-432 affects NK and T cells and may augment the antitumor immunity of patients with gastrointestinal cancer.     

Augumentation of splenic antitumor immunity by local immunotherapy in gastric cancer patients

We previously reported that the antitumor effect of OK-432, a streptococcal preparation, was markedly augmented when this agent was injected into tumors together with fibrinogen. In order to elucidate the effect of this treatment on the spleen, we assessed splenic function in gastric cancer patients receiving preoperative local immunotherapy with OK-432 and fibrinogen. Immunohistochemical studies of the spleen at 7 days after intratumoral injection therapy revealed numerous macrophages phagocytizing OK-432 in the splenic sinuses. Phenotypic analysis of splenocytes by flow cytometry revealed an increase in the CD4/CD8 ratio and in the expression of HLA-DR, CD25, and Leu M3 by splenic T cells of the patients treated with OK-432 plus fibrinogen when compared to patients treated with OK-432 alone or untreated patients. Splenic T cells from patients treated with OK-432 plus fibrinogen showed significantly higher cytotoxicity against Daudi and K562 cells than T cells from control patients (p<0.05), and culture of these splenic T cells with recombinant IL-2 induced the expansion of lymphokine-activated killer cells. These results demonstrate that local immunotherapy with a mixture of OK-432 and fibrinogen effectively augumented splenic antitumor immunity in gastric cancer patients.     

Therapeutic and life-prolonging effect of intrapleural injection with a streptococcal preparation,OK-432, and IL2-cultured effusion lymphocytes to breast cancer patients with malignant pleural effusion

We developed a local AIT using PEL cultured with TCGF combined with preadministration of OK-432. Twenty-six patients of breast cancer with pleural effusion have been treated with this therapy since 1983. PEL expanded and tumor cells collapsed by day 9 in culture with TCGF. Cultured PEL possessed significantly higher cytotoxic activity against autologous tumor cells than PBL cultured in the same condition (p < 0.05), but there was no difference between their cytotoxic activities against K562. The proliferation rate of PEL obtained after intrapleural administration of OK-432 was higher than that obtained before OK-432 (p < 0.01). Moreover, the cytotoxic activities against both autologous tumor and K562 of cultured PEL obtained after OK-432 administration was significantly (p < 0.05) higher than those cultured PEL obtained before.Cultured PEL (1 x 10⁸ - 6 x 10⁹) were transferred into the pleural cavity after the intrapleural administration of OK-432 (1–5 KE). The volume of pleural effusion increased temporarily after the administration of OK-432 but significantly (p < 0.01) decreased after AIT. Tumor cells disappeared cytologically in 22 patients at the last puncture of pleural effusion. Pleural effusion disappeared completely in 19 of 26 patients and decreased by more than 50% in volume in 6 patients. Performance status improved in 22 patients. The response rate for OK-432-combined AIT in the present study was 96%. The survival period of the patients treated by OK-432-combined AIT in this trial was significantly (p < 0.002) prolonged compared to that of the patients receiving chemotherapy alone. The side effects were fever and general malaise after OK-432 administration but no critical toxicity was observed.     

Mechanism of immunotherapeutic activity of OK-432 in the treatment of peritoneal carcinomatosis

Summary In this report the mechanism of therapeutic activity of OK-432 for the treatment of peritoneal carcinomatosis was investigated by correlating effector-cell augmentation with therapeutic activity in rats bearing MADB-106 carcinomatosis. Tumor cells were injected i.p. and the treatment with OK-432 was initiated 5 days later with 0.5, 1, 5 or 10 KE/animal of OK-432 injected i.p. semiweekly. Significant therapeutic activity was observed at all doses examined with greater prolongation of survival noted at the higher doses of OK-432. Animals treated with 0.5 KE/animal had a prolongation of the median survival time from 14 days for saline-treated animals to 17 days for the OK-432 treated animals (P<0.0008), while animals treated with higher doses had much longer periods of survival, some animals being tumor-free at 185 days. In the same studies, natural killer (NK) cell, lymphokine-activated killer cell, cytotoxic T lymphocyte, and macrophage tumoricidal/cytostatic activities were measured 7 days and 14 days following tumor injection (2 days and 9 days after initiation of immunotherapy). OK-432 had immunostimulatory activity in most of the assays of immune function examined and this correlated with host survival, including augmentation of peritoneal and peripheral blood cytotoxic T lymphocyte activity on day 14, peritoneal and alveolar macrophage activity on day 7 and day 14, as well as natural killer cell activity on day 14. These results suggest that the therapeutic doses are also immunomodulatory doses for the effector cells mentioned above. We suggest, therefore, that immunological monitoring may help to optimize treatment protocols for the treatment of peritoneal and perhaps pleural effusions with OK-432.By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. This research was sponsored by the National Cancer Institute, DHHS, under contract no. N01-23910 with Program Resources Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government     

Prevention of lymph node metastases by adoptive transfer of CD4+ T lymphocytes admixed with irradiated tumor cells

CD4⁺8^– T lymphocytes with potent antitumor activity in vivo were obtained in peritoneal exudate cells by immunizing mice with irradiated MM48 tumor cells admixed with OK-432. These immune CD4⁺ T cells were used in adoptive immunotherapy for prevention of lymph node metastases after removal of the primary tumor. Complete cure of metastases was obtained by adoptive transfer of CD4⁺ T cells admixed with irradiated MM48 tumor cells, but not by CD4⁺ T cells alone. To analyze the curative effect of admixing tumor cells on the prevention of metastases, a model of 1-day tumor inoculated with macrophages was used. Administration of immune CD4⁺ T cells alone resulted in the regression of local tumor in more than half of the mice, although all of them eventually died of lymph node metastases. On the other hand, adoptive transfer of immune CD4⁺ T cells plus irradiated tumor cells resulted in the complete regression of local tumors in all the mice, which survived without any sign of metastasis. The curative effect of the immune CD4⁺ T cells obtained by admixing irradiated tumor cells was tumor-specific. Macrophages induced by OK-432 (tumoricidal), implanted together with tumor, assisted tumor regression more than did macrophages elicited by proteose peptone (nontumoricidal) in the same adoptive transfer system. Administration of recombinant interleukin-2 instead of stimulant tumor cells did not enhance, but rather eliminated the constitutive antitumor activity of CD4⁺ T cells. On the other hand, exogenous recombinant interleukin-1 was more effective in the enhancement of antitumor activity of the CD4⁺ T cells as compared with stimulant tumor cell administration. In this case, the activating states of macrophages at the implanted tumor site had no influence on the therapeutic efficacy. A possible role of macrophages for induction of tumor-specific cytotoxic T cells that were mediated by tumor-specific CD4⁺ T cells is discussed.     

Search for immunobiological parameters predictive of clinical effects of OK-432 in patients with malignant ascites

Although OK-432, a potent BRM, has been known to induce the remarkable improvement of clinical conditions in cancer patients through its strong effects on their immune capabilities, no specific immune parameters have been identified to best predict the clinical outcome after the OK-432 treatment. In an attempt to identify early parameters indicative of the clinical effects, we have administered 0.1 mg of OK-432 intraperitoneally to a total of 12 patients with malignant ascites and examined peritoneal fluid and peripheral blood obtained on 4 days before, 1, 3, and 7 days after the OK-432 injection using various immunobiological assays. Four weeks later, clinical improvements were evaluated by the disappearance of malignant cells from and/or substantial decrease in ascites. Four patients (responders) showed the improvements while 8 patients (nonresponders) showed no clinical evidence for improvement. In a few parameters among the many examined, significantly different patterns of changes were noted between responders and nonresponders. Thus, in nonresponder patients MØ and T cell population returned to an initial low level after early increases (on days 1 and/or 3), while they remained increased day 1 through 7 in responders. In responder patients, the cytotoxicity of peritoneal mononuclear cells against K562 and Daudi cells were augmented on day 7, but not in nonresponder patients. Thein vitro stimulation of the mononuclear cells with OK-432 enhanced the cytotoxic activity and induced the interferon (IFN) production in the responders but not in nonresponders. These parameters will be useful for the early prediction of the expected clinical effects of OK-432.     

Induction of antitumor L3T4-positive T cells by OK-432 at tumor sites in mice

We have previously reported the development of antitumor effector cells by day 12 after tumor implantation using a murine malignant ascites model with BAMC-1 tumor, which could be cured completely by five consecutive i.p. injections of OK-432 starting on day 2. In contrast, the OK-432 treatment with the same protocol failed to cure the tumor-bearing athymic mice, though it could suppress tumor growth temporarily. The results suggest that T cells may play a critical role in achieving a therapeutic effect. The present study was designed to clarify the nature of the antitumor effector cells induced by OK-432 in euthymic mice. The number of tumor cells in the pertioneal cavity of OK-432-treated euthymic mice increased gradually up to day 12 and dropped suddenly on day 14, while in the athymic mice the tumor cells transiently decreased in the first 7 days then started to expand drastically on day 8. The timing of the appearance of the effector cells was examined by adoptive-transfer experiments. The peritoneal exudate cells (PEC) obtained from BAMC-1 bearing euthymic mice on various days during the treatments with OK-432 were passively transferred intraperitoneally on the respective days (synchronous transfer) or on day 7 (convergent transfer) to BAMC-1-bearing athymic mice, which were treated similarly with OK-432. More than 85% of the recipient athymic mice survived when an adoptive transfer was made on and after day 7. These results indicated that the effector cells developed before day 8 in euthymic mice. The effector cells detectable on day 7 in the PEC represent plastic- or nylon-wool-column-nonadherent cells, which could cure the tumor-bearing athymic mice. Furthermore, the effector cells were destroyed when the nylon-wool-column-nonadherent cells were treated with an anti-L3T4 antibody and complement whereas the same treatment with anti-Lyt2 antibody had no effect. These L3T4⁺ cells did not possess asialo-GM1 antigen. Although the exact mechanism of action of the effector cells is yet to be clarified, the induction of human equivalents of this type of effector cell would be a good parameter indicative of clinical effects induced by OK-432 or other biological response modifiers in an individual cancer patient.     

Induction of tumor growth inhibitory factor (TGIF) in human mononuclear cells by OK-432, a streptococcal preparation

Summary A tumor growth inhibitory factor (TGIF) was induced in the culture supernatant from mixed culture of human peripheral blood mononuclear cells (PBMC) and a streptococcal preparation, OK-432, in vitro. The activity generated in the supernatant increased in a time-dependent fashion and first appeared 6 h after the initiation of culture, reaching its maximum around 48 h. The TGIF was cytostatic against seven of ten human tumor targets, but not against three murine tumor targets. Tumor cell growth was inhibited by a transient contact, i.e., 1 h, with TGIF. The TGIF was produced by lymphocytes but not by monocytes, because the activity was usually enhanced by elimination of plastic-adherent cells from the original PBMC fraction. The TGIF was relatively stable against heating at 56° C for 30 min, but the activity was totally destroyed after heating at 70° C for 5 min. The molecular weight of TGIF was estimated to be about 43×10³ daltons by gel filtration. No interferon (IFN) activity was detected in the TGIF-positive fractions obtained by gel filtration, and the TGIF-positive fractions did not inhibit the growth of tumor necrosis factor (TNF)-sensitive mouse L929 cells. The TGIF activity was not significantly affected in neutralizing tests using specific antibodies against human IFN and TNF. The OK-432 was administered i.p. for management of cancer patients with malignant ascites. Ascites-derived mononuclear cells (ASMC) were obtained before and 3 to 5 days after OK-432 injection. The ASMC obtained after the injection produced TGIF in vitro in the absence of OK-432; the preinjection ASMC showed no such production. A positive correlation was found between TGIF-producing activity by ASMC and the effect of OK-432 injection on ascites volume. These results indicate that TGIF is induced in mononuclear cells by OK-432 not only in vitro but also in vivo and plays an important role in inhibition of tumor growth in cancer patients.     

The therapeutic effect of OK-432-combined adoptive immunotherapy against liver metastases from gastric or colorectal cancers

Twenty-four patients with liver metastases from gastric or colorectal cancer were treated with OK-432-combined adoptive immunotherapy (AIT). Lymphocytes isolated from regional lymph nodes or peripheral blood were cultured with medium containing T cell growth factor and sonicated tumor extract antigen (SE-Ag) for 9–13 days. The cultured lymphocytes were transferred mainly through the hepatic artery after the administration of OK-432, a streptococcal preparation. Sixteen of the 24 patients received a low dose of anti-cancer agents between the OK-432 injection and cell transfer. When cultured without SE-Ag, regional lymph node lymphocytes (RLNL) showed significantly (P<0.05) higher cytotoxic activity against autologous tumor cells and, on the contrary, lower cytotoxic activity against K562 than peripheral blood lymphocytes (PBL). When cultured with SE-Ag, cytotoxicity of RLNL against autologous tumor cells was nearly equivalent to that of PBL. The blastogenesis of fresh PBL to SE-Ag was significantly (P<0.05) augmented after the OK-432-combined AIT. Two patients showed complete response and 4 patients showed partial response among 19 patients who had evaluable lesions. Five patients whose liver metastases were resected were treated with OK-432-combined AIT as an adjuvant therapy. To date they are alive without recurrence in the liver.Abbreviations AIT adoptive immunotherapy - RLNL regional lymph node lymphocytes - SE-Ag sonicated tumor extract antigen     

Phase IB trial of picibanil (OK-432) as an immunomodulator in patients with resected high-risk melanoma

 The microbial immunostimulant OK-432 has been studied intensively in preclinical systems and has shown promise as an anticancer agent in trials that have been conducted over the past 20 years in Japan. To date, no systematic dose response evaluation of this agent has defined its dose-limiting toxicity or immunobiological activity. A phase IA study has been conducted in 25 patients with metastatic cancer at the University of Pittsburgh Cancer Institute Melanoma Center, establishing 30 KE as the maximal tolerable dosage, on the basis of cutaneous reactions. Subsequently, 48 patients with resected high-risk melanoma participated in a phase IB study of OK-432. This study has evaluated the immunomodulatory activity of OK-432 at five dosages ranging from 1 KE to 20 KE, administered ID twice weekly for 3 months. A formal analysis of the treated population in comparison to the randomized control group has been conducted, and profound immunological effects have been defined in the group of patients treated with OK-432. Patients who participated in this trial had a significant depression of OK-432- inducible cytokine production (interleukin-1β, interferon γ, and tumor necrosis factor α) at baseline. Treatment with OK-432 reversed this deficit for interferon γ (IFNγ) production in a dose-dependent manner, and mitigated the inhibition for interleukin-1 (IL-1) across all dosage groups. The impact of OK-432 upon other immunological functions of the treated cohorts is more variable, with durable suppression of mononuclear cell superoxide production, and in vitro cytotoxicity to tumor. Immunological characteristics of the entire cohort demonstrate a strong and significant correlation of elevated blood CD16⁺ cell counts and natural killer activity with early tumor progression and death due to melanoma. Favorable prognosis is associated with monocyte capacity to produce tumor necrosis factor (TNF), and polymorphonuclear leukocyte formylmethionyl-leucylphenylalanine-inducible superoxide release. This study reveals several new immunological correlates of tumor progression and lethal outcome in resected high-risk melanoma. It demonstrates that the depressed IL-1, TNF, and IFNγ release associated with melanoma may be mitigated by treatment with OK-432. This study has defined treatment and dose response patterns of immunomodulation associated with one of the most complex immunological agents yet evaluated in phase IB trials, in a well-defined population of high-risk patients with resected melanoma. Received: 2 October 1996 / Accepted: 28 January 1997     

Dual effects of OK-432 on mitogenic response of splenocytes to concanavalin A

OK-432, a streptococcal preparation, was studied for its effect on the concanavalin A (Con A)-induced mitogenesis of the host spleen cells. When mice were given a single intraperitoneal injection of OK-432, there was a substantial increase in the mitogenic response of splenocytes, whereas multiple injections conversely resulted in a marked reduction of the mitogenic response, when the spleen cells were cultured at high cell densities of over than 5 X 10(5) cells/well. The reduced Con A-responsiveness in the latter was not restored by mixing spleen cells from mice given multiple OK-432 injections with those from normal mice. Moreover, splenic macrophages from OK-432-injected mice exhibited marked inhibitory activity against Con A-mitogenesis of normal splenocytes, while normal splenic macrophages failed to show such an effect. Splenic T cells from OK-432-injected mice also showed an inhibitory activity against Con A-mitogenesis of normal splenocytes and similar activity was also noted in normal splenic T cells. Therefore, the OK-432-spleen cells contain two types of suppressor cells; one is a newly elicited suppressor macrophage and the other is a suppressor T cell supposedly resident also in normal spleen cells. In the OK-432-injected spleen cells, accessory cell function for T cell Con A-mitogenesis was markedly reduced. On the other hand, it was noted that the interleukin 2-producing ability of the OK-432-splenocytes was augmented more than that of normal splenocytes, indicating that multiple OK-432 injections also cause an increase in the helper T cell activity of the host spleen cells.     

Combination immunotherapy with OK-432, recombinant granulocyte-colony-stimulating factor and recombinant interleukin-2 for human hepatocellular carcinoma

 The antitumor effects of immunotherapy using streptococcal preparations (OK-432), recombinant granulocyte-colony-stimulating factor (rG-CSF) and recombinant interleukin-2 (rIL-2) were examined for human hepatocellular carcinoma (HCC). Following subcutaneous injection of OK-432 (2 KE) and rG-CSF (50 – 60 μg), low-dose intratumoral administration of OK-432 (3 – 12 KE) was performed. Thereafter, 2×10⁵ JRU of rIL-2 was subcutaneously injected. This therapeutic regimen was repeated twice. Serum α-fetoprotein levels were markedly decreased in three of seven patients with HCC by this treatment. Post-therapeutic histological examination revealed that trabecular cords or pseudoglandular arrangements of tumor cells were completely disordered in all cases and that extensive infiltration of lymphocytes into the tumor stroma was present in five cases. The number of CD4- and CD57-positive cells among tumor-infiltrating lymphocytes after immunotherapy was significantly higher than that in patients without immunotherapy (P <0.01). These findings suggest that even a small intratumoral injection of OK-432 can induce extensive infiltration of helper/inducer and natural killer cells into the tumor stroma when combined with subcutaneous injection of OK-432, rG-CSF and rIL-2 and that these cells might play important roles in tumor cytotoxicity. Received: 30 December 1994 / Accepted: 6 November 1995     

Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

Activation of human peripheral blood-derived monocytes by OK-432 (Streptococcus pyogenes): augmented cytotoxicity and secretion of TNF and synergy with rIFN-gamma

The biological response modifier OK-432 has been shown both to exert the enhancement of several immunological activities and to have a direct anti-tumor effect. The present study examines the immunopotentiating effect of OK-432 on peripheral blood monocytes (PBM) derived from normal humans. Monocyte activation was assessed by examining direct cell-mediated cytotoxic activity (CMC) and secretion of cytotoxic factors in the supernatant by the 51Cr release assay and secretion of tumor necrosis factor (TNF)-alpha detected by a sensitive radioimmunoassay. The OK-432-augmented activity was compared to that achieved by recombinant interferon-gamma (rIFN-gamma). Coculture of PBM with OK-432 overnight resulted in significant augmentation of CMC and secretion of cytotoxic factors and TNF in the supernatant. The effects observed were dose dependent and the resulting activity was much more pronounced than that achieved with an optimal concentration of IFN-gamma. The monocyte- and supernatant-mediated cytotoxic activities were in a large part attributed to TNF as both activities were inhibited by anti-TNF antibody. Several parameters of monocyte activation by OK-432 were examined. The kinetics of monocyte activation revealed that a short time exposure (2-6 hr) was sufficient for activation but maximal activation was detected after 18 hr. However, the kinetics of the cytotoxic assay were not shortened and 16-20 hr was necessary for optimal cytotoxic activity. Significant synergy was obtained when suboptimal concentrations of OK-432 and IFN-gamma were used. The synergy was noted in CMC, supernatant activity, and TNF concentration. These results demonstrate that OK-432 is a potent activator of monocyte cytotoxicity and also activates secretion of TNF. Also, OK-432 is a much more potent activator than rIFN-gamma. The synergy with OK-432 and IFN-gamma suggests that OK-432-mediated activation of monocytes takes place by a different mechanism than that mediated by rIFN-gamma. Thus, monocytes and products thereof may actively participate in the in vivo anti-tumor effect mediated by OK-432.     

Therapeutic efficacy of sequential therapy with OK-432, cyclophosphamide,IL2-cultured lymphocytes andin vivo IL2 against advanced murine plasmacytoma

BALB/c mice inoculated IP with a syngeneic plasmacytoma MOPC104E were treated with a combination of a streptococcal preparation, OK-432 (1 KE, 0.1 mg/mouse), low-dose of cyclophosphamide (CPA, 1 mg/kg) and adoptive transfer of tumor-bearer-spleen cells (2 x 10(7) cells) cultured with IL2 and sonicated tumor extract (adoptive immunotherapy; AIT). The consecutive protocol of OK-432 (day 8, 9 post inoculation) - CPA (day 10) - AIT (day 11) was the most effective. Rate of complete remission was highest when recombinant (r-) IL2 was injected to the mice after AIT. Moreover, another bacterial preparation, Nocardia rubra cell wall skeleton and another low-dose chemotherapy, Mitomycin C could be used successfully instead of OK-432 or CPA. Transfer test of intraperitoneal cells (tumor cells plus host cells) of mice on day 11 post inoculation (on the day of AIT) revealed that OK-432 augmented the susceptibility of peritoneal cells to cultured lymphocytes in inhibition of transplantability, and that CPA after OK-432 augmented the anti-tumor effect of tumor-bearer-spleen cells which act synergistically with cultured lymphocytes. This therapy schedule seems to be the best model to augment the effect of AIT with minimal side effect.     

Pronounced antitumor effect of LAK-like cells induced in the peritoneal cavity of mice after intraperitoneal injection of OK-432, a killed Streptococcal preparation

Summary More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i. p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i. p. injection of the admixture of the nonadherent PEC (1×10⁷ cells) with BAMC-1 cells (1×10⁵) survived for more than 60 days. When the same nonadherent PEC (1×10⁷ cells) were i. p. transferred adoptively 1 day after the inoculation of 1×10⁵ BAMC-1 tumor cells, again all mice survived.When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations.These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.