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     

Sensitivity of ovarian tumor cells to effector cells generated by various biological response modifiers

The sensitivity of freshly derived human ovarian tumors (FOT) to various allogeneic cytotoxic effector cells stimulated by recombinant interleukin 2 (rIL-2), recombinant interferon alpha 2 (rIFN-alpha 2), OK-432, and concanavalin A was examined using the 51Cr release assay. Peripheral blood lymphocytes (PBL) of normal female donors were used as source of effector cells. Incubation of PBL with these biological response modifiers for 24 h generated effector cells with high natural killer activity, and only 20% (1/5) of the FOT examined were susceptible to lysis. By contrast, 83% (5/6) of the FOT were sensitive to lymphokine-activated killer (LAK) cells generated by rIL-2. OK-432 and concanavalin A activation of PBL also generated cytotoxic cells, though the cytotoxic activity against FOT was much less than that obtained by LAK cells. The addition of OK-432 to LAK culture medium containing rIL-2 generated effector cells with higher cytotoxicity against FOT than cultures with IL-2 alone. However, the addition of rIFN-alpha 2 in LAK culture medium resulted in the generation of effector cells with lower cytotoxicity. The addition of rIL-2, rIFN-alpha 2, or OK-432 to LAK cells during the in vitro cytotoxicity assay had no significant effect. When FOT target cells were pretreated with OK-432 they became more sensitive to LAK than nontreated tumor cells. However, pretreatment with rIL-2 or rIFN-alpha 2 did not influence cytolysis. These results suggest that the generation of LAK cells in vitro using rIL-2 plus OK-432 may be a more effective way to prepare these cells for adoptive immunotherapy in the treatment of ovarian cancer.     

Therapeutic efficacy of sequential therapy with OK-432, cyclophosphamide,IL2-cultured lymphocytes andin vivo IL2 against advanced murine plasmacytoma

BALB/c mice inoculated IP with a syngeneic plasmacytoma MOPC104E were treated with a combination of a streptococcal preparation, OK-432 (1 KE, 0.1 mg/mouse), low-dose of cyclophosphamide (CPA, 1 mg/kg) and adoptive transfer of tumor-bearer-spleen cells (2 x 10(7) cells) cultured with IL2 and sonicated tumor extract (adoptive immunotherapy; AIT). The consecutive protocol of OK-432 (day 8, 9 post inoculation) - CPA (day 10) - AIT (day 11) was the most effective. Rate of complete remission was highest when recombinant (r-) IL2 was injected to the mice after AIT. Moreover, another bacterial preparation, Nocardia rubra cell wall skeleton and another low-dose chemotherapy, Mitomycin C could be used successfully instead of OK-432 or CPA. Transfer test of intraperitoneal cells (tumor cells plus host cells) of mice on day 11 post inoculation (on the day of AIT) revealed that OK-432 augmented the susceptibility of peritoneal cells to cultured lymphocytes in inhibition of transplantability, and that CPA after OK-432 augmented the anti-tumor effect of tumor-bearer-spleen cells which act synergistically with cultured lymphocytes. This therapy schedule seems to be the best model to augment the effect of AIT with minimal side effect.     

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     

Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

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 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     

Effective Induction of Human NK Cells with OK-432 and Further Augmentation of Their Cytolytic Function by rIL-2

Low concentrations of exogenously added recombinant interleukin 2 (rIL-2) were able to augment OK-432-induced natural killer (NK) cell activity. This kind of augmenting effect depended on the dose of rIL-2 and manifested itself only in PBMC stimulated with OK-432 (OK-MC) followed by rIL-2; augmentation did not happen in the reverse order. The existence of CD16⁺/CD25⁺ (IL-2 receptor positive; IL-2R⁺) and CD57⁺/CD25⁺ double positive cells which possess NK cell surface markers in OK-MC markedly increased in a long-term culture (12 days). A strong positive correlation was observed between the IL-2-dependent augmentation of NK activity and the quantitative changes in cell populations that possessed NK cell phenotypes. Treatment of the day-12-OK-MC with monoclonal anti-CD56 antibody plus complement could almost completely abrogate the augmented NK cytotoxicity. Furthermore, this augmenting effect was detectable within 4 hr after addition of rIL-2 at single cell level, suggesting that the effect did not require NK cell's DNA synthesis. Thus it was suggested that OK-432 could promote and upregulate the expression of IL-2 receptor (CD25) on CD56⁺ NK cell populations. Moreover, it was considered that the interaction of low concentration rIL-2 with IL-2 receptors on OK-432-activated NK cells could augment their lytic function.     

Augumentation of splenic antitumor immunity by local immunotherapy in gastric cancer patients

We previously reported that the antitumor effect of OK-432, a streptococcal preparation, was markedly augmented when this agent was injected into tumors together with fibrinogen. In order to elucidate the effect of this treatment on the spleen, we assessed splenic function in gastric cancer patients receiving preoperative local immunotherapy with OK-432 and fibrinogen. Immunohistochemical studies of the spleen at 7 days after intratumoral injection therapy revealed numerous macrophages phagocytizing OK-432 in the splenic sinuses. Phenotypic analysis of splenocytes by flow cytometry revealed an increase in the CD4/CD8 ratio and in the expression of HLA-DR, CD25, and Leu M3 by splenic T cells of the patients treated with OK-432 plus fibrinogen when compared to patients treated with OK-432 alone or untreated patients. Splenic T cells from patients treated with OK-432 plus fibrinogen showed significantly higher cytotoxicity against Daudi and K562 cells than T cells from control patients (p<0.05), and culture of these splenic T cells with recombinant IL-2 induced the expansion of lymphokine-activated killer cells. These results demonstrate that local immunotherapy with a mixture of OK-432 and fibrinogen effectively augumented splenic antitumor immunity in gastric cancer patients.     

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     

Phase IB trial of picibanil (OK-432) as an immunomodulator in patients with resected high-risk melanoma

 The microbial immunostimulant OK-432 has been studied intensively in preclinical systems and has shown promise as an anticancer agent in trials that have been conducted over the past 20 years in Japan. To date, no systematic dose response evaluation of this agent has defined its dose-limiting toxicity or immunobiological activity. A phase IA study has been conducted in 25 patients with metastatic cancer at the University of Pittsburgh Cancer Institute Melanoma Center, establishing 30 KE as the maximal tolerable dosage, on the basis of cutaneous reactions. Subsequently, 48 patients with resected high-risk melanoma participated in a phase IB study of OK-432. This study has evaluated the immunomodulatory activity of OK-432 at five dosages ranging from 1 KE to 20 KE, administered ID twice weekly for 3 months. A formal analysis of the treated population in comparison to the randomized control group has been conducted, and profound immunological effects have been defined in the group of patients treated with OK-432. Patients who participated in this trial had a significant depression of OK-432- inducible cytokine production (interleukin-1β, interferon γ, and tumor necrosis factor α) at baseline. Treatment with OK-432 reversed this deficit for interferon γ (IFNγ) production in a dose-dependent manner, and mitigated the inhibition for interleukin-1 (IL-1) across all dosage groups. The impact of OK-432 upon other immunological functions of the treated cohorts is more variable, with durable suppression of mononuclear cell superoxide production, and in vitro cytotoxicity to tumor. Immunological characteristics of the entire cohort demonstrate a strong and significant correlation of elevated blood CD16⁺ cell counts and natural killer activity with early tumor progression and death due to melanoma. Favorable prognosis is associated with monocyte capacity to produce tumor necrosis factor (TNF), and polymorphonuclear leukocyte formylmethionyl-leucylphenylalanine-inducible superoxide release. This study reveals several new immunological correlates of tumor progression and lethal outcome in resected high-risk melanoma. It demonstrates that the depressed IL-1, TNF, and IFNγ release associated with melanoma may be mitigated by treatment with OK-432. This study has defined treatment and dose response patterns of immunomodulation associated with one of the most complex immunological agents yet evaluated in phase IB trials, in a well-defined population of high-risk patients with resected melanoma. Received: 2 October 1996 / Accepted: 28 January 1997     

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.     

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.     

Recruitment of T lymphocytes and induction of tumor necrosis factor in thyroid cancer by a local immunotherapy

Summary To elucidate the mechanism of action for intratumoral injection of immunopotentiators, infiltrating mononuclear cells and tumor necrosis factor (TNF) were assayed by immunostaining tissue samples of differentiated thyroid cancer resected with or without presurgical local application of OK-432, a streptococcal preparation. Frozen sections of resected specimens were stained with monoclonal antibodies using either a conventional or a modified immunoperoxidase method. The tumors injected with OK-432 showed increased T lymphocyte infiltration and HLA-DR expression on cancer cells as compared to the non-injected controls. Among these T cells, the CD4⁺ subset was more numerous than the CD8⁺ population. In four out of the seven cases constituting the injected group, numerous TNF-positive cells were seen in clusters or lines as well as scattered, while none of the seven cases in the control group was associated with a considerable amount of these cells. In their morphology and distribution pattern, these TNF-positive cells appeared to be of macrophage lineage. Thus local injection of OK-432 in thyroid cancer was shown to recruit T lymphocytes of predominantly the CD4⁺ subset and to induce in situ production of TNF, a known potent tumoricidal cytokine. The present data warrant further studies in this direction besides wider clinical intratumoral application of the reagent.     

In vivo effects of human recombinant tumor necrosis factor alone and in combination with other biological response modifiers on human digestive organ cancer xenografts transplanted in nude mice

The present study was designed to evaluate the effect of rTNF alone or in combination with other BRMs on human digestive organ cancers. Six kinds of human digestive organ cancer xenografts (esophageal, stomach, colonic, pancreatic, bile duct, and liver cancers: EC-YO, GC-YN, CC-KK, PC-HN, BDC-SN and Li-7, respectively) were transplanted in nude mice, and rTNF was administered at 10³, 5 × 10³, or 10⁴U/head directly into the tumor 3 times a week for 2 weeks. EC-YO was the most sensitive to rTNF, and intratumoral administration of rTNF at 10³ U/head caused tumor regression. PC-HN, CC-KK and GC-YN were relatively sensitive to rTNF, and their growth was significantly inhibited by rTNF at 5 × 10³ U/head, however, the tumors regrew after treatment. Li-7 and BDC-SN were resistant to rTNF. The effects of rTNF in combination with recombinant interferon- (rIFN-), recombinant interleukin-2 (rIL-2), or streptococcal preparation OK-432 were assessed in mice transplanted with GC-YN. All combinations of rTNF at 5 × 10³ U/head and other BRMs were more effective than rTNF alone, and GC-YN tumors were completely regressed after treatment with a combination of rTNF and rIFN- or rTNF and OK-432. However in all cases, the combination of rTNF at 10³ U/head and any other BRM did not improve the effect. Furthermore, the adverse effects of the combinations were more serious than those of rTNF alone.TNF may still be a useful cytokine, because it can induce the regression of tumors. However, for its clinical application, a method should be developed to reduce its side effects.     

Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

In vitro augmentation of cytotoxicity by N-CWS in peritoneal polymorphonuclear leukocytes (PMNs) induced by PSK,OK-432 or N-CWS

Peritoneal polymorponuclear leukocytes (PMNs) were collected from the peritoneal cavity of C3H/He mice 6 hrs after intraperitoneal (i.p.) injection of 2.5 mg/head of PSK, 1 KE (100 µg)/head of OK-432 or 200 µg/head ofNocardia rubra cell wall skeleton (N-CWS). Withoutin vitro stimulation, these PMNs did not show cytotoxicity to syngeneic MM46 mammary carcinoma cells in⁵¹Cr release assay. Cytotoxicity of these PMNs was augmented by the addition of 25 µg/ml of N-CWS but not of PSK or OK-432 to cultures for the assay at the beginning of the culture. H₂O₂ production of PSK-induced PMNs was increased by thein vitro addition of 25 µg/ml of N-CWS but not of PSK. These results suggest that PSK as well as OK-432 and N-CWS can induce PMNs capable of responding further to N-CWS as the second stimulant.     

Intratumoral interleukin-2 immunotherapy: Activation of tumor-infiltrating and splenic lymphocytes in vivo

Direct intratumoral injection of interleukin-2 (IL-2) was evaluated in a murine model. Balb/c mice received 5 × 10⁴ Line 1 alveolar carcinoma cells (L1C2) by subcutaneous injection. On the third day following tumor implantation, mice received injections of IL-2 (5 × 10³–5 × 10⁴ units) or diluent twice daily, either by i. p. or intratumoral injection, 5 days/week for 3 weeks. Intratumoral injection of 5 × 10⁴ units IL-2 significantly reduced tumor volume (P <0.05 versus control), increased median survival time (P = 0.0001), and resulted in a 23.5% cure rate (P = 0.008). There were no long-term survivors in the other treatment groups. Both tumor-infiltrating lymphocytes (TIL) and splenic lymphocytes isolated directly from IL-2-treated mice demonstrated enhanced cytolytic activity compared to diluent-treated controls. To determine whether non-T-cell-mediated antitumor responses were active in our model, intratumoral immunotherapy was evaluated in athymic Balb/cnu/nu mice. In order to decrease the recruitment of lymphocyte precursors, nude mice were splenectomized and received cyclophosphamide prior to tumor injection and IL-2 therapy. Intratumoral IL-2 immunotherapy also significantly decreased tumor volume in these immunodeficient mice (P <0.02), but did not lead to long-term survival. We conclude that both TIL and splenic lymphocytes are activated in vivo in response to intratumoral IL-2 immunotherapy, suggesting that intratumoral therapy with IL-2 activates both local and systemic antitumor responses.Supported by the Tobacco-Related Disease Research Program of the University of California, the Cancer Research Coordinating Committee, the Jonsson Cancer Center Foundation, and Veterans Administration Medical Research Funds     

Therapeutic and life-prolonging effect of intrapleural injection with a streptococcal preparation,OK-432, and IL2-cultured effusion lymphocytes to breast cancer patients with malignant pleural effusion

We developed a local AIT using PEL cultured with TCGF combined with preadministration of OK-432. Twenty-six patients of breast cancer with pleural effusion have been treated with this therapy since 1983. PEL expanded and tumor cells collapsed by day 9 in culture with TCGF. Cultured PEL possessed significantly higher cytotoxic activity against autologous tumor cells than PBL cultured in the same condition (p < 0.05), but there was no difference between their cytotoxic activities against K562. The proliferation rate of PEL obtained after intrapleural administration of OK-432 was higher than that obtained before OK-432 (p < 0.01). Moreover, the cytotoxic activities against both autologous tumor and K562 of cultured PEL obtained after OK-432 administration was significantly (p < 0.05) higher than those cultured PEL obtained before.Cultured PEL (1 x 10⁸ - 6 x 10⁹) were transferred into the pleural cavity after the intrapleural administration of OK-432 (1–5 KE). The volume of pleural effusion increased temporarily after the administration of OK-432 but significantly (p < 0.01) decreased after AIT. Tumor cells disappeared cytologically in 22 patients at the last puncture of pleural effusion. Pleural effusion disappeared completely in 19 of 26 patients and decreased by more than 50% in volume in 6 patients. Performance status improved in 22 patients. The response rate for OK-432-combined AIT in the present study was 96%. The survival period of the patients treated by OK-432-combined AIT in this trial was significantly (p < 0.002) prolonged compared to that of the patients receiving chemotherapy alone. The side effects were fever and general malaise after OK-432 administration but no critical toxicity was observed.     

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.