Induction of gene expression for nitric oxide synthase by immunomodulating drugs in the RAW264.7 murine macrophage cell line

 We have elucidated the direct effects of PSK (a protein-bound polysaccharide) and OK-432 (a streptococcal preparation), both immunomodulating drugs, on the gene expression for an inducible nitric oxide synthase and on the production of nitric oxide (NO) in the RAW264.7 murine macrophage cell line. As determined by northern blot analysis, both immunomodulating drugs were potent inducers of gene expression for inducible NO synthase when cells were costimulated with interferon-γ (IFNγ). Expression of mRNA for the enzyme occurred in a dose-dependent manner after 3 h, when 10 – 50 μg/ml PSK or 0.001 – 1 KE/ml OK-432 was used. Furthermore, NO was also produced in response to these drugs, as detected by the Griess reagent reaction. The enhancement of NO synthesis was thought to be mediated, in part, through tumor necrosis factor α (TNFα) induction by these agents, since a neutralizing antibody to TNFα significantly suppressed NO production in RAW264.7 cells stimulated with PSK or OK432 in combination with IFNγ. We speculate that NO production may play a role in tumoricidal and microbicidal activities of PSK or OK-432 in vivo. Received: 9 August 1995 / Accepted: 1 April 1996     

The neutrophil as an information transmitter in tumor inhibition by a streptococcal biological response modifier,OK-432

 Effective treatment of a rat transplanted ascites tumor by i. p. injection of a streptococcal biological response modifier, OK-432, was abrogated by selective in vivo depletion of neutrophils by a monoclonal antibody, RP-3. The mechanisms by which neutrophils participate in the therapeutic action of OK-432 were studied with Winn’s assay using peritoneal exudate cells periodically obtained from rats i. p. injected with this biological response modifier. Intraperitoneal resident macrophages were first activated with OK-432, and within 3 h, tumor-inhibitory activity had moved to the early exuded neutrophils. However, 6 h after injection, exuded macrophages were the only cells involved in tumor inhibition. Considered together with other findings, it is likely that, in this system, neutrophils may transmit information from resident macrophages to exuded inflammatory macrophages in a series of responses induced by i. p. injection of OK-432. Received: 29 April 1996 / Accepted: 27 July 1996     

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     

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.     

Changes in immunological parameters in lung cancer patients undergoing immunotherapy with streptococcal preparation OK-432

Summary We have studied the immunological status of patients treated with streptococcal preparation OK-432. Two KE of OK-432 was injected intramuscularly once every week for more than three years unless the patients died. The natural killer (NK) activity in those patients who underwent curative surgery for lung cancer and had no sign of recurrence was significantly increased (P < 0.01) during the OK-432 treatment. However, the NK activity in the patients who had persistent disease (non-resected cases, incompletely resected cases or recurrent cases) was not significantly increased in comparison with that before the immunotherapy. Also, in the cases with no clinical symptoms of recurrence, both the lymphoblastogenetic reactions to the mitogens and the IL-2 production were significantly enhanced(P < 0.01) during the administration of OK-432. Reactions in the SU-PS (polysaccharide taken from the cell-wall fraction of theStreptococcus pyogenes SU strain and containing 7.2% of protein) skin-test appeared to significantly correlate with the immunological status of the patients under OK-432 therapy, but the PHA and PPD skin reactions showed no definitive enhancement. The survival rate of the patients whose SU-PS skin tests were positive during the OK-432 immunotherapy was significantly higher (P < 0.01) than that of the patients with negative reactions.     

The concurrent administration of OK432 augments the antitumor vaccination effect with tumor cells by sustaining locally infiltrating natural killer cells

 The effect of a local injection with a streptococcal preparation OK432 on the antitumor vaccination with tumor cells was investigated. Natural killer (NK) cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells after an intraperitoneal (i.p.) injection with syngeneic B16 melanoma cells. Furthermore, a concurrent i.p. injection with OK432 efficiently sustained the locally infiltrating NK cells. The OK432 treatment also sustained the augmented NK and lymphokine-activated killer activities in the peritoneal exudate cells. This treatment also increased the ability of the locally infiltrating NK cells to produce interferon γ in response to the tumor cells. In addition, the concurrent i.p. injection with OK432 in combination with the tumor cells enhanced the capacity of the spleen cells to turn into anti-(B16 melanoma) cytotoxic T lymphocytes after in vitro restimulation. This augmenting effect of OK432 was dependent on NK cells. Moreover, the concurrent injection with OK432 at the time of antitumor vaccination significantly enhanced the protective immunity against B16 melanoma at the rechallenge. Taken together, these findings indicate that a concurrent local injection with OK432 in combination with tumor cells efficiently augments the antitumor vaccination effect, in part, by sustaining the locally infiltrating activated NK cells. Received: 6 March 1996 / Accepted: 30 May 1996     

Adjuvant immunotherapy with intrapleuralStreptococcus pyogenes (OK-432) in lung cancer patients after resection

A prospective randomized study to evaluate the effect of adjuvant intrapleural OK-432 immunotherapy after resection of lung tumor was conducted in 93 patients with primary lung cancer. Among them, 46 patients had had intrapleural OK-432 injection, 47 had not. In the meantime, serial measurements of serum immunosuppressive acidic protein, of serum interleukin-2 receptor and of the sub-population of the peripheral blood cells and lymphocytes were performed in all these patients. Patient characteristics in these two groups (sex, age, histological type, pathological stage, type of operation, and performance status) were compatible. The results showed that adjuvant intrapleural OK-432 injection after resection had no beneficial effect on a patient's survival time. Patients who received intrapleural OK-432, had an increase in blood leukocytes, granulocytes and monocytes and serum immunosuppressive acidic protein level. But the cell numbers of total T cells, suppressor/cytoxic cells, helper/inducer cells and natural killer cells of peripheral blood were decreased in the OK-432 positive group. Over half of the patients had transient 1- or 2-day febrile reactions after intrapleural OK-432 injection. It was concluded that neither clinical observation nor immunological monitoring of peripheral blood could demonstrate a beneficial effect from intrapleural OK-432 immunotherapy after complete resection of the tumor.     

A case report of immunotherapy on a patient with advanced gastric cancer by adoptive transfer of OK-432-reactive HLA-matched allogeneic lymphocytes

 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. Received: 19 May 1997 / Accepted 18 November 1997     

Peripheral natural killer cell activity and intraperitoneal soluble p55 tumor necrosis factor receptor in patients with malignant ascites: two possible indicators for response to intraperitoneal combined tumor necrosis factor α and interferon γ treatment

 Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i. p.) TNFα and IFNγ once weekly over 3 – 8 weeks in order to evaluate the effect of locoregionally administered TNFα/IFNγ on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E₂ values in ascites were measured immediately before and 24 h after each TNFα/IFNγ infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i. p. TNFα/IFNγ application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFα/IFNγ treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i. p. TNFα/IFNγ therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. Received: 28 September 1995 / Accepted: 16 November 1995     

The presence of a common antigen between human gastric cancer cells and OK-432

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.Abbreviations PAP peroxidase anti-peroxidase - anti-Su anti-Su Streptococcus, Su-strain - TIL tumor infiltration of lymphocytes     

Induction of endogenous tumor necrosis factor by OK-432 in ovarian cancer patients with ascites

To four ovarian cancer patients with malignant ascites, 10 KE of OK-432 was intraperitoneally administered four times at 2 day intervals for priming, and 40 KE of OK-432 was given on the 13th day after the first injection for triggering. The changes in blood monocyte and peritoneal macrophage levels and the production of tumor necrosis factor (TNF) by blood mononuclear cells (BMCs) and ascitic lymphoid cells (ALCs) were examined. In the two patients in whom TNF was induced in the ascites, TNF production by BMCs and ALCs was noted during priming. After triggering, increases in both the number of peritoneal macrophages and TNF production by ALCs were noted. In the other two patients, in whom TNF was not detected in the ascites, the ratio of peritoneal macrophages to ALCs did not change throughout the study period, and TNF production by the ALCs was not augmented. These findings suggest that OK-432 can exert a primary effect on both peritoneal macrophages and blood monocytes, and that OK-432 triggering can promote an increase in primed peritoneal macrophages and the release of TNF from these cells.     

Bacillus Calmette-Guérin plus interleukin-2 and/or granulocyte/macrophage-colony-stimulating factor enhances immunocompetent cell production of interferon-γ, which inhibits B16F10 melanoma cell growth in vitro

 Although immunotherapy with bacillus Calmette Guérin (BCG) is an established adjuvant treatment for malignant melanoma, the mechanism of its role in this process is unclear. To investigate the possible contribution of tumor-inhibitory cytokines induced by BCG, B16F10 melanoma cell growth in culture was assessed in response to purified cytokines and conditioned media of BCG-stimulated splenocytes. Interferon-γ (IFNγ) was the most potent single agent (IC₅₀≈50 pg/ml). Tumor necrosis factor α was substantially weaker (IC50>10 ng/ml) but provided synergy with IFNγ. None of the other cytokines such as interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-12, or granulocyte/macrophage-colony-stimulating factor had direct antitumor activity against B16F10 melanoma cells. However, when IL-2 and/or GM-CSF were combined with BCG either by exogenous addition or through endogenous production by novel cytokine-secreting recombinant BCG (rBCG), a substantial increase in INFγ production by splenocytes was observed. Antitumor activity of this conditioned medium directly correlated with IFNγ concentration and was completely blocked by neutralizing antibody to IFNγ. These results suggest that BCG may exert part of its antitumor action on melanoma through the induction of IFNγ, which can be greatly enhanced through the concomitant addition of IL-2 and/or GM-CSF. Furthermore, by utilizing rBCG that secrete these cytokines, it may be possible to potentiate the antitumor effect of BCG directly at the site of BCG inoculation. Received: 29 January 1996 / Accepted: 9 April 1996     

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide

Summary The present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is,l-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C.N ^G-Monomethyl-l-arginine, a specific competitive inhibitor ofl-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations ofl-arginine (0.125–1 mM) in the culture medium.d-Arginine, however, did not substitute forl-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited byN ^G-Monomethyl-l-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased byN ^G-Monomethyl-l-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited byN ^G-Monomethyl-l-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent onl-arginine-dependent nitric oxide, and that macrophageassociated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.     

Differential modulation by interferon γ of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens

 Human melanoma is a highly immunogenic tumor capable of inducing a specific immune response. A number of melanoma-associated antigens have been characterized during the past several years and can be classified into two groups: differentiation antigens  –  present also in normal melanocytes  –  and tumor-specific antigens, which, with the exception of testis, are present only in tumor cells. In a previous publication [Kirkin A. F., Petersen T. R., Olsen A. C., Li L., thor Straten P., Zeuthen J. (1995) Cancer Immunol Immunother 41:71] we have described the production of clones of cytotoxic T lymphocytes (CTL) against the highly immunogenic human melanoma cell line FM3. Using these clones we have defined four previously unknown melanoma-associated antigens, which could be subdivided into differentiation and progression antigens. In the experiments reported in this paper, we have further compared CTL clones from different groups and shown that the sensitivity of melanoma cells to CTL that recognize differentiation or progression antigens is differentially modulated during tumor progression as well as by the lymphokines interferon γ (IFNγ) and interleukin-10 (IL-10). The interaction of CTL clones recognizing progression antigens was strongly increased after treatment of melanoma cells with IFNγ, while the recognition by CTL clones specific for differentiation antigens either was unchanged or significantly decreased. IL-10 treatment of melanoma cells induced up-regulation with respect to recognition by CTL clones specific for differentiation antigens without affecting the recognition of melanoma cells by CTL clones specific for progression antigens. Using cellular systems at different stages of tumor progression, we demonstrated that the progressed state of melanoma cells is associated with increased sensitivity to recognition by CTL clones detecting progression antigens, and with decreased sensitivity to CTL clones recognizing differentiation antigens. Mimicking tumor progression, treatment with IFN-γ induced apparent down-regulation of differentiation antigens. A hypothesis is suggested in which IFN-γ plays different roles in the immune response against poorly immunogenic and highly immunogenic melanoma cells, increasing the progression of poorly immunogenic tumor cells or promoting a strong immune response and regression of highly immunogenic melanoma cells. Received: 23 November 1995 / Accepted: 7 March 1996     

Enhancement of the photodynamic antitumor effect by streptococcal preparation OK-432 in the mouse carcinoma

 Biological response modifier antitumor effects are enhanced by the activation of the host defense mechanisms. We have investigated the antitumor effect of photodynamic therapy (PDT) and/or local administration of a biological response modifier, the streptococcal preparation OK-432, on transplanted NR-S1 mouse squamous cell carcinoma. Hematoporphyrin oligomers (20 mg/kg body weight) were used to photosensitize PDT. A pulsed Nd:YAG dye laser, tuned at 630 nm, was used as the light source. The laser power was 15 mJ cm⁻² pulse⁻¹, and the irradiation time was 40 min. The photosensitizer was injected intraperitoneally 48 h before laser irradiation. Where used, OK-432 was injected into the tumor either 3 h prior to PDT or immediately afterwards. The antitumor effects were evaluated 48 h after each protocol by (a) estimating the area of tumor necrosis (%) in hematoxylin/eosin-stained specimens, and (b) bromodeoxyuridine immunohistochemistry. Furthermore, the tumor sizes were evaluated 3, 7 and 10 days after each protocol, and the survival time after each protocol was evaluated as well. The anti-tumor effect of PDT was enhanced by administration of OK-432 3 h before PDT, whereas the administration of OK-432 immediately after PDT did not potentiate a PDT antitumor effect. Treatment with OK-432 alone had little effect on tumors. Photodynamic therapy in combination with local administration of OK-432 3 h before PDT is considered to be a useful treatment modality. Received: 23 July 1999 / Accepted: 31 May 2000     

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     

Restoration of macrophage tumoricidal activity by bleomycin correlates with the decreased production of transforming growth factor β in rats bearing KDH-8 hepatoma cells

 To explore the mechanisms of immuno-modulatory activities of bleomycin, we investigated interferon γ (IFNγ) mRNA expression, tumor necrosis factor α (TNFα) production, nitric oxide (NO) production and macrophage tumoricidal activities in rats bearing KDH-8 hepatoma cells, which secreted a large amount of transforming growth factor β (TGFβ), and these processes in KDH-8 tumor-bearing rats treated with bleomycin. We found that IFNγ mRNA expression, TNFα production, NO production and macrophage cytotoxic activities were lower in the KDH-8-bearing rats than in normal rats. On the other hand, low-dose bleomycin restored the macrophage cytotoxic activities, NO production, IFNγ mRNA expression and TNFα production in the KDH-8-bearing rats. In vitro experiments showed that KDH-8-derived TGFβ decreased the IFNγ mRNA expression and TNFα production in splenocytes, and NO production in peritoneal macrophages. These results suggest that low-dose bleomycin restored the cytokine production and macrophage tumoricidal activities in the KDH-8-bearing rats by decreasing KDH-8-derived TGFβ. Received: 14 October 1996 / Accepted: 22 July 1997     

Diverse manifestations of tumorigenicity and immunogenicity displayed by the poorly immunogenic B16-BL6 melanoma transduced with cytokine genes

 We evaluated the in vivo response to the poorly immunogenic B16-BL6 (BL6) murine melanoma genetically altered to secrete interleukin-2 (IL-2), IL-4, interferon γ (IFNγ) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). Three parameters were evaluated: (1) tumorigenicity, (2) vaccination of naive animals, and (3) assessment of antitumor reactivity of T cells derived from tumor-draining lymph nodes (TDLN). Secretion of IL-2 abrogated the tumorigenicity of BL6, while IFNγ and IL-4 partially reduced tumorigenicity, and GM-CSF had no effect. Protective immunity to wild-type tumor challenge could not be achieved by vaccination with irradiated cytokine-secreting tumors, although IL-2 and IL-4 secretion appeared to retard the growth of the challenge inoculum significantly. An alternative method to evaluate the immunogenicity of the cytokine-secreting tumors was to measure the ability of T cells obtained from TDLN to mediate regression of wild-type tumor in adoptive immunotherapy. Neither IL-2 nor IFNγ secretion resulted in the induction of immune T cells. By contrast, GM-CSF and IL-4 secretion were found to induce immune T cells in the TDLN with GM-CSF being superior to IL-4. The combined secretion of GM-CSF and IL-4 did not lead to enhanced induction of immune T cells. GM-CSF secretion was found to up-regulate B7-1 expression in TDLN, consistent with an increase in the population of antigen-presenting cells. These studies demonstrated that reduced tumorigenicity by cytokine secretion did not correlate with increased immunogenicity. With the cytokines examined, there was limited capability of developing protective immunity against the BL6 tumor. Nevertheless, GM-CSF and IL-4 secretion significantly enhanced T cell immune reactivity to the poorly immunogenic BL6 tumor. Received: 30 January 1996 / Accepted: 22 March 1996     

Peri-operative immunotherapy with OK-432

Pre- and postoperative intradermal administration of OK-432 enhanced the SU-PS skin reaction in patients with gastric cancer, but failed to prevent a fall in the NK activity induced by the operation.The change in NK activity was not associated with a change in the proportion of Leu 7-positive cells, but was related to Leu 11a-positive cells. Intradermal injection of OK-432 increased the proportion of Leu 7-positive cells in the patients in whom they accounted for less than 20% of lymphocyte population. The case was the same with Leu 11a-positive cells.Intravenous injection of OK-432 tended to increase suppressor-inducer T cells (CD4⁺2HA⁺ cells), B cells and Leu 7-positive cells. Particularly, the proportions of OK-M₁-positive cells and MHC class II antigen-positive cells increased in all patients. Immunotherapy with OK-432 given intravenously at a dose of 0.1 KE appeared to be safe because no side effects were essentially observed.     

The effect of local immunotherapy for breast cancer using a mixture of OK-432 and fibrinogen supplemented with activated macrophages

OK-432 is an immunomodulatory agent prepared from a strain ofStreptococcus pyogenes. We have previously reported that intratumoral injection of a mixture of OK-432 and fibrinogen (hereinafter referred to as OK/fbg) is very effective in the local immunotherapy for colorectal cancer. However, we found that the intratumoral injection of OK/fbg into tumor tissues of breast cancers did not always induce a strong antitumor effect. With conventional OK/fbg treatment, tumor necrosis observed in breast cancer tumors was significantly less than that in colorectal cancer tumors; the formation of fibrin meshwork and macrophage infiltration, in particular, were poor.In this study, the OK/fbg mixture was supplemented with activated macrophages for local immunotherapy of breast cancers. Macrophages were prepared from peripheral blood of breast cancer patients and activated with 0.05 mg/ml of OK-432. Between 2–7 days before operation, a single intratumoral injection of the above mixtures was done.The addition of activated macrophages to the OK/fbg mixture resulted in marked degrees of fibrin meshwork formation, macrophage infiltration and cancer cell necrosis.These findings suggest that the recruitment of macrophages in tumor stroma and their activation are necessary for sufficient induction of antitumor immunity, and supplementation of activated macrophages at the site of immune reaction may be an alternative method for reinforcement of the antitumor effect of local immunotherapy.Abbreviations mØ macrophages - OK/fbg mixture of OK-432 and fibrinogen - OK/fbg/mØ mixture of OK-432, fibrinogen and macrophages - OK/mØ mixture of OK-432 and macrophages - fbg/mØ mixture of fibrinogen and macrophages