Mechanism of anticancer host response induced by OK-432, a streptococcal preparation, mediated by phagocytosis and Toll-like receptor 4 signaling.

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-kappaB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-kappaB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.

Receptors for human plasminogen on the biological response modifier OK-432.

The biological response modifier OK-432, constituting cell wall fragments from a group A Streptococcus strain and used in anticancer therapy trials, was tested for its ability to interact with different plasma proteins. The uptake of 125I-labelled protein was measured using a panel of six different plasma proteins all known to react with receptors on a majority of streptococcal strains. Of the proteins tested, plasminogen demonstrated the most substantial uptake, with uptake values ranging from 70 to 79%. A slight interaction with fibrinogen was also detected whereas no significant interaction was found with either human immunoglobulin (Ig)A, IgG, serum albumin, or mouse albumin. The results with plasminogen suggest the possibility of a new explanation of the antitumor activity described for OK-432.

Cytotoxic and cytostatic effects of the streptococcal preparation OK-432 and its subcellular fractions on human ovarian tumor cells.

Previous studies have indicated that OK-432 is a potent biologic response modifier (BRM) and that it augments immune responses to tumor cells. We studied the direct effect of OK-432 on tumor cells. Established and freshly derived human ovarian carcinoma lines were examined for their susceptibility to OK-432 or its subcellular fractions in direct cytotoxicity, cytostatic activity, and inhibition of metabolic activity. OK-432 was cytotoxic to 13 of 15 freshly derived ovarian carcinoma lines in a 24-hour chromium-51 (51Cr) release assay. The optimal effect was noticed at OK-432 concentrations between 0.1 and 1.0 Klinishe Einhert (KE) per milliliter. The cytostatic effect on two established lines and one fresh line correlated with the cytotoxic activity. In all three lines, however, the metabolic activities (DNA, RNA, and protein synthesis) were inhibited by OK-432, suggesting that cell lysis by OK-432 may not be directly correlated with the inhibition of metabolic activities. Several subcellular fractions were derived from OK-432 and only the cytoplasmic and protoplast membrane fractions showed cytotoxic activity against the OK-432-sensitive tumor cell lines, although the cytotoxicity obtained was greatly less than the whole microorganism OK-432. The direct binding of 14C-OK-432 to tumor cells was examined. Binding took place rapidly after 1 hour of incubation and reached a maximum activity at 37 degrees C. Binding in all three lines ranged between 1.7 and 2.7 pg/cell. These results demonstrate the direct cytotoxic effect of OK-432 and some subcellular fractions on human ovarian carcinoma lines. These results also show that the BRM OK-432 may exert its effect by both potentiating the antitumor response and directly inhibiting tumor cell growth.

[Study on the preoperative adjuvant therapy of cancer--relation between serum and tumor tissue levels of UFT and OK-432 after administration, and skin reactions to Su-polysaccharide (Su-Ps)].

This study was carried out with 48 patients received surgery, i.e., 23 stomach cancer, 8 colon cancer, 6 rectal cancer, 9 breast cancer etc. Patients in group A received UFT in combination with OK-432. Each of UFT or OK-432 was given to the patients in groups B or C, respectively. Changes in the skin reaction to Su-PS were measured before and after dosing, and concentrations of Tegafur and 5-FU in serum and tumor tissues were determined after administration. Analysis of the skin reaction to Su-Ps revealed that patients with positive skin reaction before surgery in group A didn't manifest depression due to sensitization by UFT therapy. Although average values of the skin reaction after dosing were slightly lower compared to those before dosing in group B, sensitization was effective. Values of the skin reaction after dosing were significantly (p less than 0.05) high compared to those before dosing in groups A and C. Concentrations of Tegafur and 5-FU in serum reached to the peak 2 hr later and were maintained high enough to expect clinical responses even at 4 hr after administration in groups A and B. Especially there was not a significant difference between groups A and B in tumor tissue levels of 5-FU, and a high effective concentration was obtained. Combination therapy of UFT with OK-432 exhibited no significant interaction between them in adjuvant immuno-chemotherapy, and satisfactory results were expected in clinical cures.

Phagocytosis of the streptococcal preparation OK-432 (picibanil) by non-phagocytic human leukemia K 562 cells and its modulation by tumor cell differentiation.

OK-432, an inactivated and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes induced a phagocytosis process in human erythroleukemic K 562 cells. This process seems to be specific to the cell line, known however as non-phagocytic, and specific to the bacterial preparation. Transmission and scanning electron microscopy confirmed phagocytosis. Increased lysosomal activity was also demonstrated by cytochemical and biochemical criteria. The induction of phagocytosis required an intact cell surface membrane and sialo-glycoproteins seemed to be implied. The phagocytosis was inversely correlated with the erythroid differentiation of the K 562 cell. Hemin-treated K 562 cells and the markedly erythroid K 562 clone showed a decreased level of phagocytosis. The phagocytosis level in a K 562 clone expressing Fc (IgG) receptors was not altered by OK-432. In addition, a weak erythroid K 562 clone expresses the same level of phagocytosis as the total population.

[Organ distribution of 99m technetium-labeled OK-432 following intravenous administration in man].

We investigated the pharmacokinetics of OK-432, au immunomodulator of streptococcus preparation which, is used in cancer patients for active nonspecific immunotherapy. First, OK-432 was labeled with 99mTechnetium in vitro. Four patients with malignancy were studied. By the method of scintigraphy using gamma camera, OK-432 administered intravenously was found to be distributed in the liver, lung and spleen, by the decreasing grade. When OK-432 was administered subcutaneously or intramuscularly in the buttocks, most of the radioactivity of 99mTechnetium remained locally at the injected site. These results suggested that OK-432 given intravenously was effectively phagocytized by cells of reticuloendothelial system (RES). Compared with other routes of administration, the intravenous route of OK-432 administration is thus considered more effective in order to stimulate RES, which is responsible for the first step of immune reaction.