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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.

Using Charles RIver-SD 7-week-old rats, a study was carried out on the immunological effect of localized liver injections of OK-432 (Picibanil, Chugaipharma ceutical Co. Led., Tokyo, Japan). The subjects were categorized into groups injected with saline solution, PIC-0.5 KE/kg, PIC-1.0 KE/kg, and PIC-2.0 KE/kg. Measurements were undertaken prior to injection, on the first day and on the fifth day of injection through cardiac blood sampling to determine the absolute counts of T-cells, B-cells, helper T-cells and suppressor T-cells. On comparing these values in the saline group to the OK-432 groups prior to injection, no significant changes were observed on the first day. By the fifth day, however significant differences were evident between the changes which had occurred in the two groups. When changes in the saline group were studied prior to localized liver injection on the fifth day, there were no significant differences in the absolute counts of T-cells, B-cells, Th-cells and Ts-cells/lymphocytes. In the PIC-0.5 group the T-cells, B-cells, Th-cells and Ts-cells remained basically unchanged, but, a reduction in Ts-cells/lymphocytes (p less than 0.05) was observed. Although there were changes in the absolute counts of T-cells, B-cells and Th-cells in the PIC-1.0 group, reductions in the levels of Ts-cells (p less than 0.05) and Ts-cells lymphocytes (p less than 0.01) were observed. The PIC-2.0 group showed a decline in the Ts-cells (p less than 0.05) and Ts-cell/lymphocyte counts (p less than 0.01) with no changes in the T-cells, B-cells and Th-cells. As shown above, when the liver was injected with saline no immunological changes were recognized, while on the other hand, reductions in the Ts-cells and Ts/lymphocyte counts were ascertained when OK-432 was injected.

Heat-killed Lactobacillus casei YIT 9018 (LC 9018) cells enhanced the resistance to herpes simplex virus type 1 (HSV-1) in adult mice, but not significantly. The protection of mice against HSV-1 infection and the production of neutralizing antibodies were significantly enhanced by the administration of LC 9018 in combination with inactivated HSV-1 antigen. The optimal enhancement of resistance was seen in mice 14 days after the simultaneous administration of these substances. The resistance to HSV-1 infection in mice could be transferred with peritoneal exudate cells from syngeneic mice previously treated with LC 9018 alone and LC 9018 in combination with inactivated HSV-1 antigen or with thioglycollate broth, whereas the transfer of peritoneal exudate cells induced by thioglycollate broth alone and of spleen cells induced by LC 9018 in combination with thioglycollate broth or by thioglycollate broth alone was not effective. These results suggest that mouse peritoneal macrophages induced by the administration of LC 9018 in combination with inactivated HSV-1 antigen may play an important role in host defense mechanisms against HSV-1 infection.

An immunoglobulin M mouse monoclonal antibody (MAb) to the streptococcal preparation OK-432, TS-1, was generated. The TS-1 antigen is a carbohydrate epitope. This antigen is stable upon fixation and embedding in paraffin. The tissue and cellular OK-432 localization in human salivary adenocarcinoma-bearing nude mice given OK-432 intratumorally was examined by various methods according to the immunological procedures using the purified TS-1 MAb. The presence of OK-432 antigen recognized by TS-1 MAb was clearly observed in the tumor as well as the spleen and lung 24 or 48 h after OK-432 administration, whereas transfer of OK-432 from the site of injection to the organs, such as liver and kidney, was rarely seen. The presence of OK-432 antigen in some immunocompetent cells, as defined by Mac-1 antigen or asialo GM1 antigen, was observed by the double-antibody labeling technique in the tumor and spleen from tumor-bearing nude mice. Moreover, interaction between OK-432 and macrophages or natural killer (NK) cells in relation to expression of interferon (IFN) in tumor-bearing nude mice given OK-432 was observed. Consequently, significant increases of Ia-positive or Ia-negative macrophages, NK cells as well as IFN-alpha/beta- or IFN-gamma-positive cells in the tumor and/or spleen were found when compared with those without OK-432 administration.

The effects of the anti-tumour agent OK-432 on the immune response to hamster erythrocytes (HRBC) and nucleated chicken erythrocytes (CRBC) were studied in inbred SL mice. Mice were treated repeatedly with OK-432 before immunization with erythrocytes in saline. The cytotoxicity of CRBC-primed spleen cells, as demonstrated by 51Cr release from labelled CRBC, was markedly increased by treatment with OD-432. The delayed footpad reaction to CRBC was significantly augmented by treatment with OK-432. These results in mice indicate that OK-432 can enhance the cellular immune responses which require the contribution of T cells. Such an activation of T cells by OK-432 was observed in the humoral immune response to a trinitrophenyl group. Augmentation of anti-hapten antibody production, suggesting the enhancement of helper T cell activity by OK-432, was noticed after immunization with trinitrophenyl conjugated to erythrocytes. Furthermore, this enhancement of helper T cell activity by OK-432 was confirmed by utilizing an adoptive transfer system. These results support the possibility that T cell activation may be one of the important effects of OK-432 as an immunopotentiator.

To evaluate the efficacies of various administration routes of the streptococcal preparation, OK-432, we studied the lymphocyte proliferation responses to lectins in patients with malignancies. OK-432 was administered intravenously (I.V.), intramuscularly (I. M.) or intradermally (I.D.) for 2 weeks. Lymphocyte proliferation responses to concanavalin A, PHA and SuM (crude extract of OK-432) were studied before and after OK-432 administration. Enhanced responses were observed in 7 out of 9 patients (77.8%) in the I.V. group compared with 3 out of 7 (44.2%) in the I.M. group and 4 out of 9 (44.4%) in the I.D. group. Ratios of stimulation index (S.I.) after administration over S.I. before administration were highest in the I.V. group. These results suggest that I.V. administration of OK-432 is most effective for stimulating host immune systems.

Streptococcal immunopotentiator OK-432 (NSC-B116209) augmented the natural killer (NK) cell activity of peritoneal exudate cells (PEC) in inbred C57BL/6 mice given ip injections of 0.1 mg OK-432 per mouse. The cytotoxic activity of PEC increased as early as 1 day after inoculation, reached its peak on day 3, and gradually declined thereafter, YAC-1, K562, and MOLT-4 target cells were more sensitive to PEC than were EL 4 and P815 target cells. The elimination of adherent cells by a nylon wool column enriched the proportion of cytotoxic cells among PEC. Nylon wool column-passed PEC were resistant to treatment with anti-Thy 1.2 antibody plus complement and sensitive to anti-asialo GM1 serum plus complement. Because 1:40-diluted rabbit antiserum against glycosphingolipid asialo GM1 is capable of eliminating mouse NK cell activity and is not cytotoxic to killer T-cells, the above results strongly suggest that OK-432 augments the NK cell activity in mice.

OK-432, a killed preparation of Streptococcus pyogenes, as well as bacillus Calmette-Guérin (BCG) and Corynebacterium parvum are all known to induce immune interferon (IFN gamma) in mice. To examine the mechanisms of IFN gamma induction by OK-432, DDI mice were sensitized with various doses of OK-432, either by a single injection of a 1-mg dose or repeated injections of 0.1-mg doses given intraperitoneally. Spleen cells removed from the mice 7-9 days after the last injection produced high-titered IFN gamma (600-800 IU/ml) in vitro in the presence of 5 micrograms/ml of OK-432. In the absence of OK-432, however, in vitro IFN gamma production of sensitized spleen cells was quite limited. Moreover, when inducers of different antigenic entities such as serologically unrelated Streptococcus faecalis, Listeria monocytogenes, or Con A were added in vitro, instead of OK-432, to the OK-432-sensitized spleen cells, high-titered IFN gamma production also occurred. This may indicate that the signal required by T cells to produce IFN gamma in vitro need not necessarily be the same as that required to sensitize mouse macrophage in vivo. Once sensitized with OK-432, mice spleen cells continued to produce high-titered IFN gamma for more than 3 but less than 5 weeks.

In vivo animal studies support the concept that monocytes and macrophages are important in the immune surveillance of oncogenesis and that in vitro activated murine macrophages are cytocidal for tumour cells. In this study, the tumour cell cytotoxic activity of human peripheral blood monocytes was examined by measuring the inhibition of 3H-thymidine uptake in the human cancer cell line, established in our laboratory from human squamous cell lung cancer. The monocytes from 8 of the 31 lung cancer patients (26%) showed a percentage growth inhibition of less than 69.8%, which exceeded the 95% confidence limits of the percentage growth inhibition observed with healthy control monocytes. On the other hand, among the 16 sarcoidosis and the 8 tuberculosis cases no value was below 69.8%. However, there was no significant difference between the growth inhibition and the clinical stages or histological type. When OK-432, a Streptococcal agent, was administered in vivo to patients with lung cancer, an elevation of the growth inhibition was observed in 7 out of 8 patients. It was confirmed that the tumour cell cytostatic activity of the monocyte is suppressed in patients with lung cancer, and these monocyte deficits hinder the inhibition of tumour growth and metastasis.

The role of complement in the polymorphonuclear leukocyte (PMN)-mediated tumor cell destruction in cancer ascites was investigated in relation to a streptococcal preparation OK-432, a so-called biological response modifier. Incubation of OK-432 with fresh human serum at 37 degrees C for 60 min resulted in the generation of C3a and C5a chemotactic factors. Intraperitoneal (i.p.) injection of the mixture to a patient with cancer ascites revealed an accumulation of PMNs in the ascitic fluid for a longer period with a rapid reduction of the ascitic fluid, than an intraperitoneal injection of OK-432 alone examined in the same patient. PMNs were found to invade clusters of the tumor cells and then form rosettes followed by the destruction of tumor cells. These findings induced by OK-432 continued over 10 days in the presence of fresh serum, while diminished within 3-4 days when OK-432 alone was injected. When fresh human plasma or fresh frozen plasma was used instead of serum and i.p. injected with OK-432 avoiding preincubation, the same cytological and clinical changes were observed in other patients. These data strongly indicate that OK-432 activates human complement either in vitro or in the peritoneal cavity, and induces PMNs to accumulate in the ascitic fluid. Although the mechanism of killing of tumor cells by PMNs is obscure, addition of human serum or plasma to i.p. use of OK-432 seems to be valuable for the management of patients with malignant ascites.

The effects of inactivated streptococci (OK-432) on murine macrophage functions were investigated. In vivo treatment of peritoneal macrophages with OK-432 augmented the direct cytotoxic activity against TU5 tumor cells in a 48 h tritiated thymidine release assay. OK-432 also stimulated the rapid (6 h, 51Cr release) macrophage-mediated killing of Actinomycin D-sensitized WEHI 164 sarcoma cells. Moreover, the expression of la antigens on peritoneal macrophages was found to be greatly enhanced after in vivo treatment with OK-432. The immunomodulatory effects of OK-432 on macrophages functions may contribute to the antitumor activity of inactivated streptococci.

OK432, a Streptococcus preparation, is a biological response modifier that has been used in the treatment of malignant diseases. A cloned Thy 1.2+, L3T4+, Lyt 2.2- T cell (OK2.21), specific for OK432 and restricted to I-Ed, was isolated from BALB/c mice immunized with OK432. OK2.21 not only secreted interleukin 2 but also killed Iad-bearing B-lymphoma cells in an antigen-specific manner. The clone also killed Ia-negative bystander tumor targets, but only in the presence of both OK432 and antigen-presenting cells. Despite the cytotoxicity against tumor cells, OK2.21 did not kill OK432-pulsed normal spleen cells. Injection of this clone together with OK432 to tumor-bearing BALB/c mice prolonged their survival, suggesting that this clone acts as an anti-tumor effector (or effector-inducer) cell in the therapy with OK432.

This study was performed intending to demonstrate whether delayed hypersensitivity (DH) to OK-432 might augment the antitumor effect of intratumoral injection of OK-432. In this preliminary report, we showed that 1) DH to OK-432 was inducible in BALB/c mice by injecting 2KE of OK-432 with Freund's incomplete adjuvant into foot pads 2) DH was assayed by direct macrophage migration inhibition test (MIT) of Harrington's microdroplet method using 100 micrograms/ml of Su-M protein extracted from OK-432 as in vitro test antigen 3) the highest MI was observed in the second week after sensitization 4) strong and stable DH was induced after the second sensitization at the interval of two weeks.