Induction of cellular immune responses to tumor cells and peptides in colorectal cancer patients by vaccination with SART3 peptides.

The tumor-rejection antigen SART3 possesses two antigenic epitopes (SART3(109-118) and SART3(315-323)) capable of inducing HLA-A24-restricted and tumor-specific CTLs. To determine its safety and ability to generate antitumor immune responses, 12 patients with advanced colorectal cancer were administered s.c. vaccinations of these peptides. No severe adverse events were associated with the vaccinations. Significant levels of increased cellular immune responses to both HLA-A24+ colon cancer cells and the vaccinated peptide were observed in the postvaccination peripheral blood mononuclear cells in 7 of 11 and 7 of 10 patients tested, respectively, and the higher responses were observed in those patients vaccinated with the highest dose (3 mg/injection) of the peptides. These results encourage further development of SART3 peptide vaccine for colorectal cancer patients.     

Streptococcal preparation OK-432 promotes the capacity of dendritic cells (DCs) to prime carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocyte responses induced with genetically modified DCs that express CEA

Cancer immunotherapy using dendritic cells (DCs) adenovirally transduced with the whole tumor-associated antigen (TAA) gene is an effective approach. Streptococcal preparation OK-432 is useful for stimulating DCs in terms of maturation. In this study, we established carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes (CTLs) using in vitro stimulation with adenovirally modified human DCs that express CEA. We investigated whether OK-432 stimulation could be more effective in inducing CEA-specific CTLs compared with other typical stimuli. DCs adenovirally transduced with the CEA gene were cultured under various conditions with tumor necrosis factor (TNF)-alpha, lipopolysaccharide (LPS), or OK-432. A cytotoxicity assay using peripheral blood mononuclear cell (PBMC)-derived CTLs was performed in a 4 h-51Cr release assay. OK-432 stimulated immature DCs to acquire a mature phenotype and to produce significant amounts of T-helper 1 cytokines. In all groups (immature DCs, TNF-alpha/DCs, LPS/DCs, OK-432/DCs), CEA-specific CTLs were generated. OK-432-stimulated DCs (HLA-A24) induced the most potent cytotoxic activity against CEA-expressing targets (A24) but not against controls. OK-432/DCs were able to induce markedly potent CTLs specific to target cells pulsed with CEA652 peptide (HLA-A24-restricted peptide), although others failed to induce potent CTLs. In conclusion, the CTL induction protocol using adenovirally modified DCs that express CEA after maturation with OK-432 showed a potent antitumor activity against CEA-expressing target cells, and is therefore promising for clinical applications as a cancer vaccine therapy.     

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.     

Antitumor activity of a Streptococcus pyogenes preparation (OK-432). I. Sequential effector mechanisms following a single OK-432 injection in F344 rats leading to the rejection of syngeneic MADB106 tumor cells

The effector mechanisms evoked in tumor-bearing rats following a single injection of the avirulent Su strain of type 3, group A Streptococcus pyogenes (OK-432) were sequentially examined. F344 rats challenged ip with a lethal dose of the syngeneic MADB106 mammary carcinoma could survive more than 100 days when given 50 mg OK-432/kg ip 1 day after tumor challenge. When the responsible effector mechanisms were examined in this therapeutic model, two distinct effector phases distinguished by the number of tumor cells were evident. Phase I, 1-6 days following OK-432 injection, resulted in a sharp decrease in tumor cell numbers and was related to the direct antitumor cytotoxicity of OK-432 and was coincident with an increase in the number of polymorphonuclear neutrophils. However, by day 6 a sharp increase in tumor cell numbers was again observed. Subsequently, a second phase of tumor cell destruction was observed 7-20 days following OK-432 injection and was reflected in a strong lymphocyte-mediated cytotoxicity response as well as the production of complement-dependent cytotoxic antibody against the MADB106 tumor cells. Further, the adoptive transfer of either peritoneal exudate cells or sera from the phase II animals revealed that both factors may be responsible for the antitumor activity observed in this therapeutic model. In conclusion, this study has demonstrated that the antitumor effects seen with OK-432 are due to a combination of sequential effector mechanisms leading to the eventual rejection of established tumor.     

Dendritic cells stimulated with a bacterial product,OK-432, efficiently induce cytotoxic T lymphocytes specific to tumor rejection peptide

Dendritic cells (DCs) are potent antigen-presenting cells, which have recently been applied for cancer immunotherapy using epitope peptides. Accumulating results of the clinical trials of such a strategy suggest that maturity of the applied DCs has a significant impact on the outcome of the vaccination. Here we examined the effects of penicillin-killed Streptococcus pyogenes (OK-432) on DC maturation and functions including induction of CTLs. DCs generated from peripheral blood using granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 showed immunophenotypes consistent with immature DCs (iDCs). These iDCs were further incubated with medium alone, tumor necrosis factor alpha, lipopolysaccharide, or OK-432. The immunophenotypical analysis showed DCs stimulated with OK-432 (OK-DCs) possessed significantly higher expression of CD83 compared with unstimulated DCs. Furthermore, OK-DCs showed significantly higher production of IL-12 and IFN-gamma compared with DCs with other stimulations. These results indicate that OK-432 stimulates iDCs to have a mature phenotype and to produce a significant amount of T-helper 1-type cytokines. To examine the potency of OK-DCs on the induction of specific CTLs, the tumor rejection peptide derived from carcinoembryonic antigen was used as a model antigen. The HLA-tetramer assay showed that potent CTL was induced with OK-DCs at high frequency. These results indicate that OK-432 efficiently stimulates DCs without interfering with the presentation of pulsed peptide. Furthermore, OK-432 does not activate nuclear factor kappaB through Toll-like receptor 2 or Toll-like receptor 4 in the indicator cell system; however, it induces IL-12 production through the beta(2) integrin system on DCs. These results strongly suggest that OK-432 could be applied to develop an efficient cancer vaccine using DCs pulsed with tumor rejection peptides.     

Expression of SART3 tumor-rejection antigen in gastric cancers.

We previously reported SART3 as a tumor-rejection antigen recognized by histocompatibility leukocyte antigen (HLA)-A24-restricted cytotoxic T lymphocytes (CTLs). In this study, we investigated the expression of the SART3 antigen in gastric cancers, as a candidate for use in specific immunotherapy. The SART3 antigen was detected in 9 of 10 (90%) gastric cancer cell lines, 35 of 52 (67.3%) gastric cancer tissues, and 0 of 20 non-tumorous gastric tissues. SART3-derived peptides corresponding to positions 109- 118 and 315-323 induced HLA-A24-restricted and tumor-specific CTLs from peripheral blood mononuclear cells (PBMCs) of gastric cancer patients. These peptide-induced CTLs recognized HLA-A24(+) SART3(+) gastric cancer cells, but not HLA-A24(+) SART3(-) or HLA-A24(-) SART3(+) gastric cancer cells. Therefore, the SART3 peptides could be useful in specific immunotherapy of gastric cancer patients.     

Splenectomy abolishes antitumor effect of immunotherapy with streptococcal preparation, OK-432, on mouse liver tumors

In the present study, we investigated the therapeutic effects of oral and subcutaneous administration of OK-432 prior to or following the transplantation of murine liver tumors. In addition, the effect of splenectomy on the antitumor activity of OK-432 was investigated. Mice which received OK-432 orally prior to tumor transplantation exhibited significantly lower tumor weight and significantly improved survival, when compared to the control mice. Prior subcutaneous injection of OK-432 did not show any antitumor activity. On the other hand, both oral and subcutaneous administration of OK-432, subsequent to tumor transplantation, led to a significant improvement of survival and a decrease in the number of lung metastases, although tumor weight was not affected. The anti-tumor effect of OK-432 required the presence of the spleen, since the survival of the mice with liver tumors was not improved by OK-432 if splenectomy and tumor transplantation were performed simultaneously. These results suggest that immunotherapy with OK-432 may beneficial in the treatment of liver tumors and that these effects are dependent on the presence of the spleen.     

Expression of tumor-rejection antigens in gynecologic cancers.

We recently reported the four tumor-rejection antigens (SART1(259), SART2, SART3, and ART4) that possess tumor epitopes capable of inducing HLA-A2402-restricted cytotoxic T lymphocytes (CTLs) in cancer patients. This study investigated the expression of these tumor antigens in gynecologic cancers, including 33 ovarian cancers, 38 cervical cancers, and 40 endometrial cancers. SART1(259) antigen was detected in 56%, 35%, and 30% of ovarian, cervical and endometrial cancers, while SART2 antigen was detected in 46%, 66%, and 30% of these cancers, respectively. Both SART3 and ART4 antigens were detectable in the majority of these gynecologic cancers tested. In contrast, none of these antigens was detectable in any of the normal ovarian and uterine tissues tested. Peripheral blood mononuclear cells (PBMCs) of HLA-A24(+) patients with gynecologic cancers were found to produce significant levels of interferon-gamma in response to HLA-A24(+) SART3(+) gynecologic cancer cells after having been stimulated three times in vitro with either SART3(109 - 118) or SART3(315 - 323) peptide. These PBMCs lysed HLA-A24(+) SART3(+) gynecologic cancer cells, but not HLA-A24(-) SART3(+) gynecologic cancer cells or HLA-A24(+) normal cells. Therefore, these four antigens and their peptides, including SART3 peptides, would be appropriate molecules for use in specific immunotherapy of HLA-A24(+) gynecologic cancer patients.     

Production of a cytotoxic factor in mouse peritoneal fluid by OK-432, a streptococcal preparation

A cytotoxic factor was induced by the injection of LPS into the peritoneal fluids of mice which had been previously primed with a streptococcal antitumor preparation, OK-432. No cytotoxic effect on L-929 cells was observed in the peritoneal fluids of mice singly treated with OK-432 or LPS. Various mouse and human tumor cell lines were effectively killed by this peritoneal cytotoxic factor, though normal cell lines were insensitive, which indicates that this factor is not species-specific. The highest level of cytotoxic activity was obtained when LPS was given to mice 5 days after the injection of OK-432. The optimal time for collection of peritoneal fluids for the cytotoxic factor was 2h following the LPS injection. Interferon activity was found to be negative by the plaque reduction test using L-929 cells with vesicular stomatitis virus.     

Expression of SART3 antigen and induction of CTLs by SART3-derived peptides in breast cancer patients

We recently reported the SART3 tumour-rejection antigen as possessing tumour epitopes capable of inducing HLA-class I-restricted cytotoxic T lymphocytes (CTLs). This study investigated expression of the SART3 antigen in breast cancer to explore an appropriate molecule for use in specific immunotherapy of breast cancer patients. The SART3 antigen was detected in all of the breast cancer cell lines tested, 30 of 40 (75%) breast cancer tissue samples, and 0 of 3 non-tumourous breast tissue samples. SART3 derived peptides at positions 109-118 and 315-323 induced HLA-A24 restricted CTLs that reacted to breast cancer cells from the peripheral blood mononuclear cells (PBMCs) of breast cancer patients. Therefore, the SART3 antigen and its peptides could be an appropriate molecule for use in specific immunotherapy of the majority of HLA-A24-positive breast cancer patients.     

Melphalan-induced enhancement of antitumor immune reactivity in thymocytes of adult BALB/c mice bearing a large MOPC-315 tumor

At no stage of tumor growth are thymocytes from MOPC-315 tumor bearers capable of bringing about the generation of enhanced antitumor cytotoxicity when added to immunization cultures of syngeneic normal spleen cells and "autochthonous" tumor cells. However, by Day 7 after low-dose melphalan [L-PAM (L-phenylalanine mustard)] therapy of mice bearing a large (greater than or equal to 20 mm) s.c. MOPC-315 tumor, their thymocytes exhibit such activity and it persists for at least 17 additional days. The ability of thymocytes from L-PAM-treated MOPC-315 tumor bearers to bring about the generation of enhanced antitumor cytotoxicity when added to immunization cultures of normal spleen cells and MOPC-315 tumor cells is evident over a 10-fold range of responder/stimulator cell ratios, and requires the presence of the thymocytes within the first day after initiation of the 5-day immunization cultures. In addition, immunization cultures containing normal spleen cells and thymocytes from L-PAM-treated MOPC-315 tumor bearers exhibit enhanced antitumor cytotoxicity by Day 4 after culture initiation that persists for at least 3 additional days. Thymocytes from L-PAM-treated MOPC-315 tumor bearers are able to bring about the generation of enhanced antitumor cytotoxicity only in response to stimulation with autochthonous tumor cells but not in response to stimulation with unrelated allogeneic EL4 tumor cells. The apparent specificity of the enhanced antitumor immune reactivity of thymocytes from L-PAM-treated MOPC-315 tumor bearers is not the result of extensive metastasis of tumor cells to the thymus. In fact, no tumor cells were found in the thymuses of MOPC-315 tumor bearers with methods that can detect 1 X 10(3) tumor cells, indicating that if MOPC-315 tumor cells metastasize at all into the thymus, the thymuses of mice bearing a large MOPC-315 tumor contain fewer than 1 X 10(3) tumor cells. Thus, thymocytes from mice which are engaged in the eradication of a large MOPC-315 tumor display enhanced antitumor immunity in response to stimulation with the autochthonous tumor cells. Such thymocytes may prove important to the outcome of low-dose L-PAM therapy for mice bearing a large MOPC-315 tumor, since the low-dose chemotherapy requires the contribution of T-cell-dependent antitumor immunity for its therapeutic effectiveness.     

Effect of combination therapy of radiation and local administration of OK-432 on a murine fibrosarcoma

The effects of a combination therapy of radiation and local administration of OK-432, which is one of BRMs (biological response modifiers), were studied using a radioresistant murine fibrosarcoma with weak immunogenicity. Fifty percent tumor control doses were 83.5 (79.6-87.4) Gy in animals given radiation alone and 64.3 (57.9-71.0) Gy in animals given OK-432 immediately after radiation, indicating that this biological response modifier can enhance the radiation dose effectiveness by a factor of 83.5/64.3, that is, 1.30. Histological observations of treated tumors showed that the combination therapy induced a marked infiltration with lymphocytes and prominent degeneration and necrosis of the tumor cells. Examination of subsets of the infiltrating lymphocytes using the monoclonal antibodies showed that Lyt-2 positive lymphocytes were more abundant in the group given the combination therapy than in the radiation alone group on day 5 (p greater than 0.05). Two days later, Lyt-1, Lyt-2, and L3T4 positive lymphocytes increased in the group given the combination therapy, whereas these lymphocytes disappeared in the group given radiation alone.     

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.     

Anti-tumor effect of intratumoral administration of dendritic cells in combination with TS-1 and OK-432

We investigated the in vivo anti-tumor effect of intratumoral administration of dendritic cells (DCs) after chemotherapy using TS-1, and followed by immunopotentiator OK-432. Both in Meth-A-bearing BALB/c and in SCCV II-bearing C3H/HeN mice, one week of oral administration of TS-1 affected a partial eradication of established tumors. TS-1 followed by DCs and OK-432 resulted in a marked inhibition in tumor growth, and also contributed to a greater prolongation of survival. Cytotoxic activities of tumor-infiltrating lymphocytes and draining lymph node cells against inoculated tumor cells were significantly increased by the therapy. Cytotoxic memory T cells were also induced. The same therapy was also applied to SCCV II-bearing C3H/HeJ mice in which the Toll-like receptor (TLR) 4 is mutated; no immunotherapeutic effect was observed in the mice. These findings suggest that local DC therapy in combination with TS-1 and OK-432 may well be a useful strategy for the treatment of solid tumors, and that TLR4 signaling is involved in the success of this therapy.     

Cryoimmunological therapy with local injection of OK-432 against advance or recurrent breast cancer

A total of 5 breast cancer patients, 2 with far advanced primary breast tumor and 3 with local recurrent tumors on their anterior chest wall, underwent multimodal therapy in which cryosurgery was performed in combination with local injection of the non-specific immunopotentiator OK-432. This multimodal therapy was repeated as many times as possible. In addition, all patients were treated with mild chemotherapy. In every patient who underwent cryosurgery combined with locoregional immunotherapy, eradication or reduction of tumor was observed for several months. In 3 of the patients who underwent cryosurgery, locoregional immunotherapy and systemic chemotherapy, the tumor burden decreased markedly in 2 patients even though the diameter of tumor was over 5 cm in both cases. In case 1, we examined the concentration of IFN-gamma and IL-10 before and after cryosurgery. The value of IFN-gamma/IL-10 increased from 3.0 to 6.1 after treatment. All patients experienced high fever within 2 days after surgery, but no other side effects resulted from either cryosurgery or locoregional immunotherapy. All patients maintained good QOL throughout their therapy. These results indicate that cryosurgery in combination with local injection of OK-432 should be a feasible modality against unresectable breast cancer on the chest wall, and that this therapeutic effect may be augmented by mild chemotherapy.     

OK-432 INHIBITED B16 MELANOMA GROWTH AND INDUCED A TH1 DOMINANT STATE IN TUMOR BEARING MOUSE

OK-432 was produced by culturing a low virulence Su strain of Type III, Group A streptococcus pyogens of human origin in Bernheimers basal medium supple- mented with penicillin G potassium. It has been extensively used as an adjuvant immune-potentiator for cancer therapy in Japan. Concurrent administration of OK-432 with chemotherapy has led to an improvement of the survival rate of caner patient[1]. The antitumor mechanism of OK-432 has been investigated extensively. It has been reporte…     

The combination therapy of local administration of OK-432 and radiation for 2 cases of lower bile duct carcinoma

The combination therapy of local administration of OK-432 and radiation was performed for two patients with lower bile duct carcinoma. Initially, 10KE of OK-432 was given locally, and irradiation was performed according to the schedule of 180-200 rad/5 fractions/week and given 5,000-5,220 rad totally. One case received 5KE of OK-432 locally 17 days after first injection. Both cases showed complete response and survived 4 months and 59 months without any sign of recurrence, respectively.     

Tumor Necrosis Factor Production and Colon Cancer

Tumor necrosis factor (TNF) production in B10 mice exhibiting H-2 gene heterogeneity and in C3H/ He mice differing in lipopolysaccharide (LPS) responsiveness was investigated following stimulation with OK-432. TNF-producing capacity in these mice was H-2-restricted, while their LPS responsiveness was independent of the gene. TNF production in humans was found to be HLA-B antigen-restricted. An investigation was then made of the effect of endogenous TNF induction with OK-432 on the survival rate of colorectal cancer patients. Patients in the TNF-producing group showed a trend toward having a better prognosis as compared to those in the TNF-nonproducing group. Cancer therapy formulated with consideration of host responsiveness to OK-432 may afford greater therapeutic benefit and may prolong survival.     

The management of malignant ascites with a streptococcal preparation, OK-432: relation between the clinical effect and auto-tumor cell killing activity by OK-432-induced ascites-derived lymphocytes

Twelve patients with malignant ascites caused by gastro-intestinal cancer were treated by intraperitoneal administration of OK-432. Tumor cells from these patients were separated from ascitic fluid and cultured in vitro before OK-432 therapy. OK-432 was given intraperitoneally one to two times a week at doses ranging from 5 to 20 KE suspended in saline. Mononuclear lymphocytes were collected from the fluid at various intervals throughout the therapy. The effect of ascites-derived lymphocytes on ascites-derived autologous tumor cell growth was studied in vitro using microplate assay. Nine (responders) of 12 patients showed complete disappearance or significant reduction of ascitic fluid. Ascites-derived lymphocytes slightly inhibited autologous tumor cell growth only in one case before OK-432 therapy. Lymphocytes collected from ascites after OK-432 injection significantly inhibited auto-tumor cell growth in all of 9 responders. In 3 non-responders, however, auto-tumor cell growth inhibition was found only in one case. Interestingly, lymphocytes from non-responders significantly inhibited the growth of tumor cells taken from responders. Conversely, lymphocytes from responders did not inhibit non-responder-derived tumor cell growth. These findings imply that auto-tumor killing by OK-432-induced lymphocytes may depend more on the condition of the tumor cells than on the condition of the lymphocytes, and that the measurement of auto-tumor killing activity by ascites-derived lymphocytes may be useful as an indicator in OK-432 therapy.     

Therapeutic effect of multimodal therapy, such as cryosurgery, locoregional immunotherapy and systemic chemotherapy against far advanced breast cancer]

A total of 6 breast cancer patients, 5 with local recurrent tumors on their anterior chest wall and 1 with far advanced primary breast tumor, underwent multimodal therapy in which cryosurgery was performed in combination with local injection of the non-specific immunopotentiator OK-432. This multimodal therapy was repeated as many times as possible. In addition, 3 patients whose general condition was relatively good were treated with mild chemotherapy. In every patient who underwent cryosurgery combined with locoregional immunotherapy, eradication or reduction of tumor was observed for several months. Of 3 patients who underwent cryosurgery, locoregional immunotherapy and systemic chemotherapy, the tumor burden decreased markedly in 2 patients and rapid tumor growth was suppressed in 1 patient, even though the diameter of tumor was over 5 cm in all cases. There were no side effects caused by either cryosurgery or locoregional immunotherapy. Little toxicity was observed throughout the mild chemotherapy. These results indicated that cryosurgery in combination with local injection of OK-432 should be a feasible modality against unresectable breast cancer on the chest wall, and that this therapeutic effect might be augmented by mild chemotherapy.