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     

Interferon-alpha in serum and carcinomatous pleural effusion after repeated intrapleural injections of antitumor agents

Pleural effusions and sera of two patients with lung cancer were tested after intrapleural injection of OK-432 as an anticancer drug for IFN-alpha activity by biological assay and for IFN-alpha as an antigen by radioimmunoassay. The titers by radioimmunoassay were fairly consistent with those by biological assay, but were usually higher. In Case 1, IFN-alpha was observed fairly early after administration of OK-432 and only in pleural effusions. In Case 2, induction of IFN-alpha at low level was observed late after the first administration of OK-432 both in the pleural effusion and serum and was detected only by radioimmunoassay.     

Clinical therapeutic effect of adoptive immunotherapy using IL-2-cultured autologous lymphocytes

Our method of adoptive immunotherapy (AIT) using autologous IL-2-cultured lymphocytes differs from so-called LAK therapy in several points. We (1) obtain cultured lymphocytes from effusion lymphocytes (EL) or regional lymph-node lymphocytes (RLNL), when possible, rather than peripheral blood lymphocytes (PBL), (2) use crude IL-2 to induce T cell proliferation and to maintain killer activity, (3) use sonicated autologous tumor extract as antigen (Ag) to stimulate proliferation of cytotoxic T cells, and (4) pretreat the patients with local administration of OK-432 before AIT to induce effector cells that act synergistically with transferred killer cells. Surface marker analysis showed that OKT3, IL-2 receptor, Leu 2+15- cells were elevated while Leu 11a and Leu 3+8+ cells were decreased. Culture of RLNL augmented the expression of Leu 3+8- marker. Both of PBL and RLNL responded to Ag, and their auto-tumor killing activities were augmented in about half of the patients while rarely decrease by the addition of Ag. Response rates of patients with pleural effusion due to breast cancer and those with liver metastasis of breast cancer were 94% and 60%, respectively. Moreover, the survival was prolonged in the treated patients with pleural effusion or gastric cancer patients with peritoneal dissemination.     

Intrapleural administration of OK432 in cancer patients: Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes

Summary Ten patients with carcinomatous pleural effusions were treated with single intrapleural (i.pl.) injections of the streptococcal preparation OK432 on day 0 and the effects of i.pl. OK432 on the lysis of fresh or cryopreserved autologous tumor cells isolated from the pleural effusions were observed on day 7. In eight patients tumor cells in the effusions had decreased or disappeared by day 7. The other two patients, however, had no clinical evidence of therapeutic benefit from i.pl. OK432. Effusion tumor cells were relatively resistant to lysis by autologous lymphocytes when tested in a 4-h ⁵¹Cr-release assay. Positive reactions were recorded for blood and effusion lymphocytes in two of ten untreated patients. Injection of OK432 i.pl. resulted in an induction or augmentation of cytotoxicity against autologous tumor cells and K562 in the effusions of seven of ten subjects by day 7. In contrast, autologous tumor killing activity of blood lymphocytes was not always modified by i.pl. OK432. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation enriched autologous tumor killing activity, with no reactivity in LGL-depleted, small T lymphocytes. Significant lysis of autologous tumor cells was observed with effusion LGL from seven of ten untreated patients. Seven days after i.pl. OK432 injection, effusion LGL expressed enhanced cytotoxicity against autologous effusion tumor cells, whereas T cells were still not cytotoxic to autologous tumor cells on day 7. The frequency of LGL among effusion lymphocytes was not altered by i. pl. OK432. Adherent effusion cells were not involved in lysis of autologous effusion tumor cells in either untreated or OK432-treated patients. In vitro treatment of blood and effusion lymphocytes with OK432 induced an enhancement of autologous tumor-killing activity in patients who subsequently responded to i.pl. OK432 treatment. OK432 augmented in vitro autologous tumor killing activity of LGL, whereas T cells failed to lyse autologous tumor cells even after in vitro activation with OK432. These results indicate that i.pl. administration of OK432 to cancer patients will result in an augmentation of autologous tumor killing activity of LGL in the pleural effusions, and that this could be responsible for the antitumor activity of i.pl. OK432 therapy.     

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.     

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     

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.     

Antitumor and therapeutic effects of spleen cells from tumor-bearing mice cultured with T cell growth factor and soluble tumor extract

Summary Spleen cells of BALB/c mice that had been inoculated with syngeneic plasmacytoma MOPC 104E were cultured for 11 days in T-cell growth factor (TCGF) and ultrasonicated tumor extract (USE). Cultured lymphocytes (MOPC-CL) possessed three-fold more lytic units than normal spleen cells cultured in TCGF without USE (N-CL). Moreover, the in vivo neutralization assay suggested that MOPC-CL were composed of at least two populations, one possessing tumor-specific and the other nonspecific antitumor activity. When 2×10⁷ of MOPC-CL were administered IP to mice that had been inoculated IP with 10⁵ MOPC 104E cells 5 days previously marginal prolongation of survival was observed. This effect was not augmented by the single injection of a larger number (5×10⁷) of CL, but was augmented by the repeated daily administration for 4 days (from day 5 to day 8 after the inoculation) of the same total number (5×10⁷) of CL. In addition, IP injection of the streptococcal preparation OK432 before the transfer of CL significantly enhanced the therapeutic efficacy, and resulted in a cure rate of 20%. The mechanism of this combined effect appears to involve the effect of OK432 on interleukin 2 (IL-2) regulation systems in vivo. Our culture system with TCGF and USE and our therapy system with OK432 and CL allow the clinical application of adoptive immunotherapy for the many types of solid cancers.     

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     

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.     

Streptococcal preparation OK-432 augments cytotoxic activity against an erythroleukemic cell line in humans

Summary The effects of OK-432, an inactivated and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes, were evaluated on the cytotoxicity of lymphoid cells against a natural killer (NK)-sensitive erythroleukemic cell line K562 in patients with malignant diseases. When ten patients were treated postoperatively with daily injections of OK-432, a significant degree of augmentation in the cytotoxicity of peripheral blood lymphoid cells was evoked in all the patients, and the maximum level of cytotoxicity was on the third day after the beginning of the treatment. In spite of the successive daily administration, the level of cytotoxicity declined thereafter, but stayed higher than the pretreatment level. When OK-432 was injected IP in three patients with carcinomatous peritonitis, the cytotoxic activity of ascitic lymphoid cells was significantly enhanced. Cytotoxicity of in vitro-cultured lymphoid cells taken from peripheral blood of normal donors was also augmented by the addition of OK-432.     

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.     

Search for immunobiological parameters predictive of clinical effects of OK-432 in patients with malignant ascites

Although OK-432, a potent BRM, has been known to induce the remarkable improvement of clinical conditions in cancer patients through its strong effects on their immune capabilities, no specific immune parameters have been identified to best predict the clinical outcome after the OK-432 treatment. In an attempt to identify early parameters indicative of the clinical effects, we have administered 0.1 mg of OK-432 intraperitoneally to a total of 12 patients with malignant ascites and examined peritoneal fluid and peripheral blood obtained on 4 days before, 1, 3, and 7 days after the OK-432 injection using various immunobiological assays. Four weeks later, clinical improvements were evaluated by the disappearance of malignant cells from and/or substantial decrease in ascites. Four patients (responders) showed the improvements while 8 patients (nonresponders) showed no clinical evidence for improvement. In a few parameters among the many examined, significantly different patterns of changes were noted between responders and nonresponders. Thus, in nonresponder patients MØ and T cell population returned to an initial low level after early increases (on days 1 and/or 3), while they remained increased day 1 through 7 in responders. In responder patients, the cytotoxicity of peritoneal mononuclear cells against K562 and Daudi cells were augmented on day 7, but not in nonresponder patients. Thein vitro stimulation of the mononuclear cells with OK-432 enhanced the cytotoxic activity and induced the interferon (IFN) production in the responders but not in nonresponders. These parameters will be useful for the early prediction of the expected clinical effects of OK-432.     

Intrapleural instillation of interferon γ in patients with malignant pleurisy due to lung cancer

 The effect of intrapleural instillation of recombinant human interferon γ (IFNγ) at increasing doses of (1–12) × 10⁶ U was examined in six patients with cytologically positive pleural effusion due to lung cancer. Intrapleural instillation was repeated up to three times. Clinically, no reaccumulation of pleural effusion was observed in one patient and disappearance of lung cancer cells from the pleural effusion was seen in two other patients. No severe side-effects were observed. Considerable levels of IFNγ remained in the pleural effusion as well as in patients’ serum up to 7 days after instillation of 2 × 10⁶ U and higher doses. The total cell number showed a transient decrease on day 1 of therapy. Levels of pro-inflammatory cytokines, such as tumor necrosis factor α, interleukin(IL)-1β and IL-6, in the pleural effusion remained almost stable after IFNγ instillation. On the other hand, intrapleural IL-1 receptor antagonist levels were remarkably elevated by the instillation of IFNγ. IL-2- and IL-12-inducible killer activity of pleural mononuclear cells tended to increase slightly. Despite the inability of IFNγ to control pleural effusion in this treatment schedule, IFNγ instilled by an intrapleural route had a potential local antitumor activity. Moreover, since IFNγ persists in pleural effusions for a long time after a single instillation, such a therapy in combination with other fibrogenic biological response modifiers can be promising. Received: 28 February 1997 / Accepted: 23 July 1997     

Vascular endothelial growth factor in malignant pleural effusion associated with lung cancer

The presence of vascular endothelial growth factor (VEGF) was examined by enzyme immunoassay in 60 cytology-documented malignant pleural effusions associated with primary lung cancer and 51 other benign and malignant pleural effusions. Exudative pleural effusions contained significantly higher amounts of VEGF than transudative pleural effusions. Among exudative pleural effusions, levels of VEGF in malignant pleural effusions associated with lung cancer were significantly higher than those of benign exudative pleural effusions. There was no significant difference in pleural VEGF in patients with different histological types or clinical stages of lung cancer. Serial measurement of pleural VEGF levels was performed in six lung cancer patients treated with intrapleural instillation of recombinant interferon γ, and reduction of pleural effusion was associated with decreasing pleural VEGF levels. These findings suggest that VEGF has a role in the accumulation of exudative pleural effusions, especially that of malignant pleural effusion associated with lung cancer. Received: 14 April 1999 / Accepted: 10 June 1999     

Locoregional immunotherapy of head and neck cancers utilizing allogeneic spleen cells — a report of 2 cases

Frozen-stored human spleen cells (SC) cultured with streptococcus preparation OK-432 acquired direct cytotoxicity to autologous as well as allogeneic tumor cells. The activated cells started to produce cytocidal cytokine TGIF, which is distinct from previously known cytokines.We examined the possibility of allogeneic adoptive immunotherapy (AIT) using these OK-432-stimulated spleen cells (OK-SC) in two cancer patients. Rapid necrosis of cancer tissue and remarkable decreases of tumor markers in tumor effusion were observed. There were no severe side effects.     

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.     

Human T-cell cultures with selective autotumor reactivity

Summary T-cell cultures derived from the blood of 14 patients with solid tumors were propagated with T-cell growth factor (TCGF). The cultures were initiated from lymphocytes exposed to autologous tumor-biopsy cells. TCGF was added either immediately or 3–10 days later. In the former culture type the cell yield on day 7 was considerably higher. The cytotoxic potential of the cultured cells was assayed on two occasions, between days 7 and 10 and between weeks 5 and 8. Cells of all but two cultures had the potential to lyse autologous tumor-biopsy cells.On the population level, cytotoxicity was specific for autologous tumor in those cultures that were driven to growth with TCGF after the 3rd day. These lymphocytes did not lyse allogeneic tumor-biopsy cells. In contrast, all five cultures initiated in the presence of TCGF exhibited a broader cytotoxic potential, i.e., in addition to the stimulator autologous-tumor cells, they also lysed other targets. Another difference between the two culture types was their behavior toward K562. Tested on the 7th day they all lysed K562; however, this function declined in strength or disappeared later in the cultures exposed to TCGF after the 3rd day.Reexposure of the lymphocytes to autologous tumor-biopsy cells after 2 weeks of culture period, but not on the 7th day, induced DNA synthesis. This secondary response was specific inasmuch as allogeneic tumor cells had little or no effect.One of the autotumor restimulated cultures was tested for cytotoxic potential. It increased against the autologous but not against other tumors or K562 cells.     

Bone metastasis as a prognostic factor in breast cancer patients with liver metastasis given OK-432-combined adoptive immunotherapy via the hepatic artery

The outcome in 31 patients with liver metastases from breast cancer given OK-432-combined adoptive immunotherapy via the hepatic artery was analyzed. Patients received intraarterial OK-432, a streptococcal preparation, followed by the transfer of autologous lymphocytes cultured with autologous tumor extract and interleukin-2 for 9–13 days. Liver lesions were evaluable in 11 of the 12 patients with bone metastasis (group A) and in 16 of the 19 patients without bone metastasis (group B). Complete response (CR) in the liver was attained in 8 patients in group A, but in only 1 in group B (p < 0.01). In group A, radiological features of all metastatic foci of bone improved after CR in the liver. Moreover, the median survival time (MST) of group A (20 months) was longer (p=0.06) than that of group B patients with extra-hepatic metastasis (n=12; MST=6 months), while group B patients with liver metastasis alone (n=7) showed a MST similar to that of group A. Thus, loco-regional immunotherapy via the hepatic artery was found to be useful in controlling both liver and bone metastasis from breast cancer. Moreover, in breast cancer patients with liver metastasis, bone metastasis appears to be a prognostic factor associated with good response to this immunotherapy.Abbreviations MST median survival time - CR complete response - PR partial response - MDP metyl-diphosphonate     

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.