OK-432 inhibited B16 melanoma growth and induced a TH<Subscript>1</Subscript> dominant state in tumor bearing mouse

Objective: To investigate the antitumor mechanisms of the streptococcal preparation OK-432. Methods: Using C57BL/6 mouse bearing B16 melanoma, we observed the antitumor activity of OK-432 and investigated the effect of OK-432 on multi-cytokine (IL-2, IL-4, IL-6, IL-10, IL-12, IFN-γ) production of mouse splenocyte both in vitro and in vivo. Results: As compared with control, OK-432 significantly inhibited B16 melanoma growth and lengthened mice survival time (P<0.05). In vitro OK-432 could stimulate splenocyte from tumor bearing mice to secrete IL-6, IL-12, IFN-γ and IL-10 remarkably (P<0.01). In vivo OK-432 led to the increased production of IL-2, IL-12 and IFN-γ but decreased production of IL-10 (P<0.05). When the splenocytes harvested from OK-432 treated mice were stimulated with OK-432 again in vitro, the production of IFN-γ increased and IL-10 decreased significantly (P<0.05). Conclusion: OK-432 could boost multiple cytokines production, especially IL-12 which skewed T cells in a Th₁ dominant state and enhanced the host antitumor activities. Foundation item: This work was supported in part by the Japan-China Sasakawa Medical Fellowship. Biography: WANG Xiang-hui (1963–), master of medicine, associate professor, Lanzhou General Hospital of PLA, majors in general surgery.     

Adoptive immunotherapy by pantropic killer cells recovered from OK-432-injected tumor sites in mice

A murine malignant ascites model with BAMC-1 tumors was established previously, which was cured completely by five consecutive i.p. injections of OK-432. We have found that peritoneal mononuclear cells from these animals contained antitumor effector cells which could destroy nonspecifically a variety of tumor cells in vitro. They were tentatively called pantropic killer cells (PKCs). The present study was essentially designed to show the antitumor effectiveness of the PKCs in vivo by the use of an adoptive immunotherapy model. The growth of BAMC-1 tumors transplanted s.c. 5 days earlier was significantly suppressed by passive transfer of 5 x 10(6) to 2 x 10(7) PKCs induced by injection of OK-432 into BAMC-1 bearing donor mice, while more than 1 x 10(8) immune spleen cells from the same donors treated with OK-432 were required to achieve the similar effects. Furthermore, if the tumor-bearing recipients were pretreated with 180 mg/kg of cyclophosphamide 1 h before the adoptive transfer, even 5 x 10(6) PKCs could induce complete regression of the tumors transplanted 5 days earlier. This protocol made it possible even to achieve the complete regression of larger tumors (9-10 mm in diameter) in recipients transplanted 12 days earlier. The PKCs were, as expected, able to cure not only BAMC-1-bearing animals but also Meth-A-bearing mice. As effector cells for adoptive immunotherapy, therefore, the PKCs induced by OK-432 seem to be as effective as, if not better than, lymphokine-activated killer cells expanded in vitro by culturing tumor infiltrating lymphocytes with interleukin-2. Although the study on surface markers of PKCs did not unequivocally discriminate these from lymphokine-activated killer cells, the present findings are considered significant indicating that a potent biological response modifier such as OK-432 can induce pantropic killer cells which are extremely effective in destroying various tumor cells in vivo. One of the advantages of OK-432 therapy over lymphokine-activated killer cell therapy, therefore, is that the former does not require the tedious and time-consuming in vitro procedures which are essential for the latter.     

Antitumor activity of streptococcal acid glycoprotein produced by Streptococcus pyogenes Su

Streptococcal acid glycoprotein (SAGP) was purified from the cultured cells of Streptococcus pyogenes Su, and its in vitro and in vivo antitumor activities were investigated in comparison with those of OK-432, a cell preparation of S. pyogenes Su which is used clinically as a potent antitumor agent. SAGP inhibited the growth of several tumor cell lines in vitro at less than 0.1 microgram/ml, while it did not affect the growth of the other tumor and normal cell lines even at 10 micrograms/ml. This selective cytotoxicity is a unique characteristic of SAGP. OK-432 did not show cytotoxicity in vitro. SAGP also showed a considerable life-span-prolonging effect on mice bearing Meth A tumor and inhibited the growth of sarcoma 180 tumor implanted im. The comparison of antitumor activities between SAGP and OK-432 definitely suggested a difference in the mechanisms of their actions, even though they were derived from the same bacterial strain.     

Inhibitory effects of B cells on antitumor immunity

B-cell functions in antitumor immunity are not well understood. In this study, we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag), D5 mouse melanoma, or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments, spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors), IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells.     

Combined effect of intraperitoneally administered OK-432 and recombinant interleukin (rIL-2) against mouse tumors

Combined effect of the streptococcal preparation, OK-432 and rIL-2, both administered intraperitoneally, was examined against Meth-A tumor, a syngeneic tumor of inbred BALB/c mice and an analysis of the effector cells also was performed. The combined treatment resulted in significant in vitro inhibition of tumor growth and increase in the length of survival of the Meth-A bearing mice. The spleen cells obtained from Meth-A bearing mice treated with both OK-432 and rIL-2 on the 12th day after the tumor transplantation were capable of lysing syngeneic Meth-A tumor cells in addition to NK-sensitive allogenic YAC-1 cells and LAK-sensitive EL-4 cells in vitro in a 4 hour 51Cr-release assay. Neither spleen cells obtained from the tumor bearing mice treated with OK-432 or rIL-2 alone, nor those obtained from normal mice treated with both agents, showed cytotoxic activity against Meth-A cells. The in vitro growth of NK-resistant allogenic P-388 was not inhibited by the spleen cells obtained from any treated mouse, with or without Meth-A tumor. In order to determine the effector subpopulation against Meth-A cells, the spleen cells obtained from the tumor-bearing mice treated with OK-432 and rIL-2 on the 12th day after tumor transplantation, were treated with either anti-Thy-1, 2 antibody plus complement, anti-asialo GM1 serum plus complement, or by adherence to a plastic plate or nylon wool column.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of factor(s) that render polymorphonuclear leukocytes cytostatic from spleen cells stimulated with a streptococcal preparation, OK-432

We previously reported the augmentation of tumor cytotoxicity of polymorphonuclear leukocytes (PMN) by in vivo administration of a streptococcal preparation, OK-432 (S. Watabe et al., J. Natl. Cancer Inst., 72: 1365-1370, 1984). The present study was undertaken to elucidate the mechanisms of the phenomena. Mouse and rat spleen cells were stimulated in vitro with OK-432. The culture supernatants from the stimulated spleen cells (OK sup) contained factor(s) that rendered mouse and rat PMN cytostatic [neutrophil activating factor (NAF)]. The stimulation of spleen cells with a small dose of OK-432 (0.05 micrograms/ml) resulted in the production of maximum NAF, and NAF was produced soon (12 h) after OK-432 stimulation. NAF was partially inactivated with 60 degrees C 30-min treatment, and completely inactivated with 100 degrees C 10 min. NAF was sensitive to pH 2 treatment. The treatment of PMN with OK sup for 5 min at 37 degrees C was sufficient to induce cytostatic activity of PMN. That OK sup contained gamma-interferon and recombinant gamma-interferon showed NAF activity indicate that gamma-interferon is a NAF in OK sup.     

Antitumor efficacy of activated macrophages against murine glioma cells

A crucial manifestation of malignant gliomas is the regrowth of already-invaded neoplastic cells after surgical intervention. One possible approach for inhibiting such tumor growth is to utilize the tumoricidal potential of macrophages. In order to investigate the clinical application of this concept, peritoneal exudate cells (PEC) activated in vitro and in vivo by immunomodulating agents were tested for cytotoxic activity against murine glioma (203-glioma) cells. As immunomodulating agents, heat-killed Propionibacterium acnes (P. acnes), OK-432 and Concanavilin A supernatant (Con A sup) were used in these experiments. P. acnes was provided by Kowa Pharmaceutical Co., Tokyo, and OK-432 by Chugai Pharmaceutical Co., Ltd., Tokyo. Klinische Einheit (KE) units were used to express the strength of the preparation, with 1 KE equal to 0.1 mg of dried streptococci. Con A sup was produced by Con A pulsing of BALB/c splenocytes resuspended in complete medium. PEC harvested from mice to which 5% glycogen in saline had been inoculated intraperitoneally 6 d previously were activated in vitro by P. acnes (P. acnes-PEC), OK-432 (OK-432-PEC) and Con A sup (Con A-PEC). The cytotoxic activities of P. acnes-PEC, OK-432-PEC and Con A-PEC were approximately 25%, 65% and 60%, respectively. PEC were then collected from mice into which either 100 micrograms of P. acnes or 1 KE of OK-432 had been injected intraperitoneally several times. The antitumor effects of P. acnes-PEC and OK-432-PEC were about 35% and 50%, respectively. These activated PEC demonstrated cytotoxic activity against murine glioma in the tumor neutralization assay (Winn assay). Also, the antitumor efficacy of OK-432-PEC belonged mainly to adherent cells. Meningeal gliomatosis (MG) models were prepared for clinical studies. Viable 203-glioma cells (5 X 10(6) were injected percutaneously into the cisterna magna of C57BL/6 mice. The median survival time (MST) of the untreated group was 8.5 days. The MST of the groups treated by intraperitoneal and intracisternal administration of P. acnes were 26 and 33 days. This therapy significantly prolonged the survival time of these models, particularly by the intracisternal treatment. The differential cell count by Giemsa staining and latexphagocytic cell findings revealed that macrophages accounted for more than 90% of the P. acnes-PEC. These results may indicate that activated (PEC) macrophages were induced intracisternally by P. acnes and that activated macrophages induced intraperitoneally exerted antitumor effects in MG models.     

Locoregional immunotherapy of malignant ascites from gastric cancer using DTH-oriented doses of the streptococcal preparation OK-432: Treatment of Th1 dysfunction in the ascites microenvironment

Locoregional administration of the streptococcal preparation OK-432 is effective in treating malignant ascites from gastric cancer. In order to enhance the efficacy, we conducted a pilot study of locoregional immunotherapy for malignant ascites using host-oriented doses of OK-432. Moreover, action mechanisms of OK-432 were further explored in view of the T-helper type 1 (Th1)-Th2 concept. Gastric cancer patients with cytologically determined malignant ascites were locoregionally administered with OK-432. The dose of OK-432 was selected according to the delayed-type hypersensitivity (DTH) reaction levels to OK-432. Cytokine production profiles of ascites cells were determined using whole ascites assay by stimulation with OK-432. IL-10 mRNA expression was analyzed using RT-PCR. It was found that a positive clinical response was observed in 37 of the 51 (73%) patients with the DTH-oriented approach, showing a significantly higher efficacy than traditional dosage methods using empirical doses (31/58, 53%) (p=0.0487). The DTH-oriented administration of OK-432 produced adverse effects such as fever elevation (p<0.0001) and abdominal pain (p=0.0013) to a significantly lesser extent compared with the traditional treatment. Analysis of the action mechanism of OK-432 revealed that the DTH reaction in responders (19+/-6 mm) was stronger than that in non-responders (6+/-4 mm) (p<0.0001). Tumor necrosis factor (TNF)-alpha production of ascites cells was also higher in responders (3943+/-1247 pg/ml) than in non-responders (1217+/-939 pg/ml) (p=0.0002). There was a significant positive correlation (p=0.0085) between the levels of DTH reaction and TNF-alpha production of ascites cells, but not of blood cells. Responders appeared to polarize on the Th1 axis when clinical responses were plotted on Th1-Th2 dimensions according to the cytokine production profiles of TNF-alpha, IFN-gamma, IL-4 and IL-6 of ascites cells. In vitro culture with IL-2 of ascites cells after OK-432 administration demonstrated an almost clonal expansion of CD4+ lymphocytes, which produced TNF-alpha and IFN-gamma, but did not produce IL-4 or IL-6. IL-10 mRNA expression was detectable in ascites cells from non-responders before treatment. These results suggest that the DTH-oriented locoregional administration of OK-432 may be both effective and less toxic in treating malignant ascites from gastric cancer, showing a possibility of the tailored immunotherapy for malignant ascites. Th1 dysfunction exists in the microenvironment of malignant ascites from gastric cancer, in which IL-10 may, in part, play a role. The up-regulation of Th1 responses by OK-432 may result in positive clinical responses. The DTH reaction to OK-432 may be a useful tool not only for predicting clinical response but also for selecting the optimal dose of OK-432.     

OK-432联合IL-2对Lewis肺癌及c-fos蛋白表达的抑制作用

 目的　研究OK 4 32与IL 2抑制近交系小鼠C57BL/6Lewis肺癌 (LLC)的生长及c fos蛋白的表达。方法　以C57BL/6荷瘤LLC小鼠为模型 ,观察OK 4 32联合IL 2对肿瘤发生、生长转移的影响 ,及用抗鼠c fos单克隆抗体进行SABC免疫组化技术半定量测定c fos在Lewis肺癌原发灶中的表达。结果　二者联用对肿瘤体积和重量的抑制作用明显增强 (P <0 0 1) ,增强抑瘤率 (P <0 0 1) ,肺转移灶数目明显减少。光镜下显示肿瘤灶中出现许多凋亡细胞 ;肿瘤组织电镜观察 ,瘤细胞核染色质浓集成块 ,在核膜内呈新月形或环形排列 ,核膜清楚 ,瘤细胞内质网扩张 ,线粒体肿胀。c fos在联合用药组与对照组比较明显减少 (P <0 0 1)。结论　OK 4 32与IL 2有明显的协同抗肿瘤活性 ,部分原因可能阻抑c fos基因的表达 ;提示OK 4 32联合IL 2可望为临床治疗癌症患者提供一种有效的生物治疗方法。     

Anti-tumor immunity induced by OK-432-derived DNA

We have tried to identify the effective components of OK-432, a Streptococcus-derived anti-cancer immunotherapeutic agent. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting anti-cancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and Msp I revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced Th1-type cytokines, such as IFN-gamma and IL-12, and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, a peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. Anti-tumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced as compared with that in wild-type mice, while the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG-DNA in OK-432 functions as an active component in OK-432-induced anti-cancer immunity via TLR9, at least in part.     

Radiation, 5-FU and OK-432: inhibitory effect of IL-10 and TGF-beta

We investigated the effect of 5-FU and radiation in OK-432-induced cytokine production. Stimulation of peripheral blood mononuclear cells (PBMCs) with OK-432 (1 micro/ml) for 24 h induced Th1-type cytokines (IFN-gamma, TNF-alpha, IL-12, IL-18) as well as IL-10 and TGF-beta. When the PBMCs were stimulated by 5-FU (5 microg/ml) or X-ray (2 Gy) simultaneously with OK-432, production of IL-10 and TGF-beta was significantly inhibited, while no significant change in Th1 cytokine production was observed. Although OK-432 also enhanced the expression of the genes encoding SOCS-1 and SOCS-3, which are negative regulators for cytokine signaling, this was reduced by 5-FU or X-irradiation. Induction of IL-10 and TGF-beta by OK-432 was significantly decreased by adding antisense ODN for SOCS-1 or that for SOCS-3. Radiation and 5-FU induce Th1-dominant state by inhibiting the OK-432-induced production of IL-10 and TGF-beta mediated by regulation of SOCS-1 and SOCS-3 expression, and are suggested to increase anti-cancer immunity.     

白细胞介素-18通过诱导肿瘤细胞凋亡抑制人肺腺癌细胞皮下移植瘤的生长

目的:探讨白细胞介素-18(IL-18)对人肺癌裸鼠皮下移植瘤生长的影响及抗肿瘤机制。方法:采用荷人肺腺癌A549细胞的裸鼠皮下移植瘤模型,不同剂量(5μg/100μl、50μg/100μl)的IL-18进行腹腔内注射,对照组为磷酸盐缓冲液(PBS溶液),观察瘤细胞生长能力和裸鼠生存期,应用光学显微镜观察肿瘤组织形态学变化,TUNEL法观察肿瘤细胞凋亡情况。结果:与对照组比较,IL-18高剂量组和IL-18常规剂量组明显抑制了皮下移植瘤生长(P<0.05)。IL-18常规剂量组生存期(63±8天)明显延长(P<0.05),IL-18高剂量组(44±5天)与空白对照组(42±6天)生存期无明显差异(P>0.05)。IL-18常规剂量治疗组和IL-18高剂量治疗组肿瘤组织内见大量淋巴细胞及炎细胞浸润,可见肿瘤细胞点、片状坏死;IL-18高剂量组和IL-18常规剂量组的凋亡细胞数均明显高于空白对照组(P<0.01)。结论:IL-18在裸鼠体内具有明显的抗肿瘤作用,其机制可能是刺激淋巴细胞、NK细胞和吞噬细胞等炎细胞分泌各种细胞因子,诱导肿瘤细胞凋亡,发挥抗肿瘤作用。     

Usefulness of immunomodulators for maturation of dendritic cells

Biological response modifiers (BRMs) augment the cytotoxic activity of various effector cells by the induction of multiple cytokines and suppression of immunosuppressive factors. BRMs are used extensively in adjuvant therapy for gastric cancer in Japan. In dendritic cell (DC)-based vaccine therapy, the quality of DCs is important in inducing strong antitumor immunity. A good manufacturing practice (GMP) grade agent for DCs maturation is desirable for safety. Here we report the effects of two BRMs, OK432 and PSK, which are GMP grade agents for the functional maturation of DCs. OK432 and PSK were examined in vitro, and compared with lipopolysaccharide (LPS) and a cytokine cocktail (IL-1beta, TNF-alpha, IL-6 and PGE2). In the immunophenotypical analysis, the expression of CD80 and CD83 of DCs stimulated with OK-432 increased significantly compared with PSK and medium, and this up-regulation was the same as levels of DCs stimulated with cytokine cocktail. DCs stimulated with OK-432 showed significantly higher production of IL-12 and Th1-type cytokines (IL-2 and IFN-gamma) compared with DCs stimulated with LPS or cytokine cocktail. OK-432 stimulated DCs could induce the significantly high level of cytotoxic T cell activity compared with PSK-stimulated or unstimulated DCs. These results suggest that OK432 is a GMP-grade reagent that promotes functional maturation of DCs and could be applied in DC-based vaccinations.     

Immunosuppressive effects of interleukin-12 coexpression in melanoma antigen gene-modified dendritic cell vaccines

Genetic immunotherapy with tumor antigen gene-modified dendritic cells (DC) generates robust immunity, although antitumor protection is not complete in all models. Previous experience in a model in which C57BL/6 mice immunized with DC transduced with adenoviral vectors expressing MART-1 demonstrated a 20-40% complete protection to a tumor challenge with B16 melanoma cells. Tumors that did develop in immunized mice had slower growth kinetics compared to tumors implanted in na?ve mice. In the present study, we wished to determine if the supraphysiological production of the Th1-skewing cytokine interleukin-12 (IL-12) could enhance immune activation and antitumor protection in this model. In a series of experiments immunizing mice with DC cotransduced with MART-1 and IL-12, antitumor protection and antigen-specific splenocyte cytotoxicity and interferon gamma production inversely correlated with the amount of IL-12 produced by DC. This adverse effect of IL-12 could not be explained by a direct cytotoxic effect of natural killer cells directed towards DC, nor the production of nitric oxide leading to down-regulation of the immune response - the two mechanisms previously recognized to explain immune-suppressive effects of IL-12-based vaccine therapy. In conclusion, in this animal model, IL-12 production by gene-modified DC leads to a cytokine-induced dose-dependent inhibition of antigen-specific antitumor protection.     

Comparative studies between liposomes containing muramyl dipeptide and various immunomodulators on activation of mouse peritoneal macrophages and NK cells

We compared the effects among muramyl dipeptide (MDP), liposome-encapsulated MDP (liposome MDP), bacillus Calmette-Guérin (BCG) and OK-432 on cytotoxic activity of mouse peritoneal macrophages (PM) and natural killer (NK) cells in vitro and in vivo, and their tumor-inhibitory effects against MH134 ascitic tumors in C3H/He mice. The cytotoxicity of PM induced by free MDP was lower than that induced by BCG, but a significantly higher cytotoxicity was induced by liposomes containing MDP and OK-432. The peritoneal NK cells were not activated by MDP, liposome MDP or BCG, but OK-432 profoundly augmented peritoneal NK activity. Growth inhibition of ascitic tumor was not observed in free MDP and BCG intraperitoneally treated mice, but moderate growth inhibition was noted in liposome-MDP-treated mice; and in OK-432-treated mice, marked tumor growth inhibition and prolongation of survival time were observed. These results suggested that OK-432 is more advantageous in controlling malignant tumor growth in vivo than free MDP, liposome MDP or BCG because of its ability to activate both macrophages and NK cells.     

Immunobiological studies of interferon-alpha A/D in comparison with a streptococcal preparation, OK-432

In order to clarify the characteristics of interferon-alpha A/D (IFN-alpha) as a biological response modifier (BRM), several immunobiological activities were compared with OK-432. 1) Both IFN-alpha and OK-432 inhibited the multiplication of Meth-A cells in vitro. 2) IFN-alpha (2 X 10(5) IU, ip) augmented the NK activity of peritoneal exudate cells (PEC) and spleen cells, and the peak of NK activity was observed 1 day after injection. OK-432 (1 KE, ip) augmented the NK activity of PEC, but not of spleen cells, and the peak was 3 days after. 3) Macrophage activating activity was more potent with OK-432 (1 KE) than IFN-alpha (2 X 10(5) IU). 4) Induction of CTL against alloantigens was augmented by IFN-alpha and OK-432. 5) By the combination of IFN-alpha with OK-432, a synergistic antitumor effect was obtained against Meth-A ascites tumor. Immunobiological effects of IFN-alpha seemed to be somewhat different from those of OK-432. Therefore, the combination of the two agents might cause a synergistic antitumor effect.     

Priming effect of interferons and interleukin 2 on endogenous production of tumor necrosis factor in mice

The effects of interferons (IFNs) and interleukin 2 (IL 2) on endogenous production of tumor necrosis factor (TNF) were investigated in mice. Production of serum TNF was triggered by iv injection of OK-432 and tested by in vitro cytotoxicity assay. Injection of recombinant IFN-gamma with OK-432 and tested by in vitro cytotoxicity assay. Injection of recombinant IFN-gamma with OK-432 or of IFN-alpha/beta, recombinant IFN-beta, recombinant IFN-alpha A/D or recombinant IL 2 six hours before OK-432 enhanced TNF production about 10-fold, which indicated priming actions of these compounds in TNF production. These findings suggest that these compounds could also be used as priming agents for endogenous production of TNF in cancer patients.     

Chemoimmunotherapy of L1210 leukemia with adriamycin, cyclophosphamide, and OK-432, and their effects on the generation of antitumor immunity

The synergistic effects of combined chemotherapy with adriamycin (ADR) and cyclophosphamide (CY) on L1210 tumors in mice were potentiated by use of a streptococcal preparation, OK-432, in a time- and dose-dependent way. Some mice were cured by treatment with the three agents, and resisted a later challenge by L1210 but not P388 leukemia cells. This immunity was blocked by administration of antimacrophage agents or CY. The effects of OK-432 were also studied with mice sensitized by L1210 cells attenuated with mitomycin C. OK-432 potentiated syngeneic and semi-syngeneic transplantation resistance in vivo and augmented primary and secondary cytotoxicity mediated by spleen cells in vitro. In vivo administration of ADR and CY together enhanced in vivo tumor transplantation resistance and in vitro cytotoxicity was blocked, but this inhibition was reversed by injection of OK-432. The results suggested that OK-432 acted by increasing the activity of cytotoxic spleen cells against L1210 cells, which are fast-growing and poorly immunogenic, and that this cytotoxicity killed tumor cells that survived the chemotherapy.