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     

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.     

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     

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     

Monocyte activation by an oral immunomodulator (bestatin) in lymphoma patients following autologous bone marrow transplantation

 Bestatin (ubenimex), an inhibitor of aminopeptidase, is an oral immunomodulator that binds to CD13 (aminopeptidase N) on macrophages/monocytes. To examine its immunomodulatory effect after high-dose therapy and autologous bone marrow transplantation (BMT), a dose-finding phase Ib trial was conducted with 30 Hodgkin’s disease and non-Hodgkin’s lymphoma patients who received no drug (control), 10 and 30 mg (low dose), or 90 and 180 mg (high dose) of bestatin daily for 60 days following autologous BMT. Bestatin administration was initiated when the absolute neutrophil count was greater than 250/mm3 on 2 consecutive days. The serum neopterin levels, an indicator of monocyte/macrophage activation, increased in the high-dose group compared to the control group (not significantly) and the low-dose group (significantly). Similarly, the colony-stimulating activity in the sera was significantly increased in the high-dose group compared to the control and low-dose groups. We also examined the expression of cell-surface markers on monocytes in these patients by fluorescent cytometry analysis. There was no significant difference either in the frequency or absolute number of monocytes (CD14⁺) among the three groups at any time. However, a significant increase in the frequency of CD16(FcgRIII)-positive monocytes (a marker of activation) was observed in the high-dose group compared to controls from day 14 to day 60 after the start of bestatin administration. Further, the frequency of HLA-DR⁺ monocytes (another marker of activation) was significantly increased in the high-dose group. These results indicate that bestatin at higher doses (90 and 180 mg daily), but not lower doses, activates macrophages/monocytes, as demonstrated by phenotypic marker (HLA-DR and CD16) up-regulation, and this provides augmentation of neopterin and colony-stimulating activity in the serum of patients following autologous BMT. Received: 24 June 1996 / Accepted 13 September 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 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.     

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     

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.     

Predictive indicators for the therapeutic effect of OK-432 in patients with chronic active type B hepatitis

Twenty-two patients with chronic type B hepatitis were treated with OK-432. Immunological parameters were serially measured to find predictive indicators for the seroconversion from hepatitis B envelope antigen(HBe Ag) to anti-HBe. In patients who achieved the disappearance of HBe Ag associated with or without the appearance of anti-HBe, the numbers of CD8⁺DR⁺ and CD4⁺DR⁺T cells in peripheral blood increased gradually during OK-432 therapy and then reduced subsequently to the seroconversion from HBe Ag positive to anti-HBe positive. Increases of DR-positive T cells in numbers were significantly correlated with increased amounts of IFN- produced in response toin vitro OK-432 stimulation.In vitro OK-432-stimulated IFN- production and the increase of CD8⁺DR⁺T cells in number in peripheral blood could be proposed as predictive indicators for the disappearance of HBe Ag.     

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.     

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.     

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     

Active immunization of metastatic melanoma patients with interleukin-2-transduced allogeneic melanoma cells: evaluation of efficacy and tolerability

 From January 1994 to July 1996 we immunized metastatic melanoma patients with HLA-A2-compatible, interleukin-2 (IL-2)-secreting, immunogenic melanoma lines in an attempt to induce a systemic reaction that might also affect distant melanoma lesions. Twelve patients (6 male and 6 female) aged from 28 to 72 years, affected with visceral and/or subcutaneous (s.c.) melanoma metastases, were treated. Two different HLA-A2⁺ melanoma lines were transduced with the human IL-2 gene (14932/IL-2 and 1B6/IL-2) and used as vaccine. Two groups of 4 patients each were injected s.c. with 5×10⁷ and 15×10⁷ irradiated 14932/IL-2 melanoma cells respectively, whereas a third group received 5×10⁷ cells of the second line (1B6/IL-2). All patients received the vaccine on days 1, 13, 26; if no progression was evident, further immunizations were administered at monthly intervals. All patients were assessable for clinical response after at least three injections of the vaccine. In 4 cases a stabilization of disease lasting from 2 to 6 months was observed; in 2 of them a mixed type of response to treatment was noted with simultaneous evidence of regressing and non-responding lesions in the same patients. No signs of clinical response were found in the remaining patients. Nine patients died of disease between 3 and 24 months after the onset of therapy, whereas 3 were alive 3 months after the end of therapy. The local and systemic side-effects of treatment were mild. These results indicate that vaccination with cells bearing the appropriate antigens and releasing IL-2 locally can produce weak clinical responses, but also indicate that better results may be achieved through modifications of the vaccine, the schedule of immunization and/or a more appropriate selection of patients. Received: 20 December 1996 / Accepted: 27 February 1997     

Co-expression of immunogenic determinants by the same cellular immunogen is required for the optimum immunotherapeutic benefit in mice with melanoma

 Tumor-associated T cell epitopes are recognized by T cells in the context of determinants specified by class I loci. Since the rejection of foreign histocompatibility antigens is known to enhance tumor immunity, immunization with a cellular vaccine that combined the expression of both syngeneic and allogeneic class I determinants could have important immunological advantages over a vaccine that expressed either syngeneic or allogeneic determinants alone. To investigate this question in a mouse melanoma model system, we tested the immunotherapeutic properties of B16 melanoma × LM fibroblast hybrid cells in C57BL/6J mice with melanoma. Like C57BL/6J mice, B16 cells expressed H-2K^b class I determinants and (antibody-defined) melanoma-associated antigens. LM cells, of C3H mouse origin, formed H-2K^k determinants along with B7.1, a co-stimulatory molecule that can activate T cells. The B16 × LM hybrid cells co-expressed H-2K^b and H-2K^k class I determinants, B7.1 and the melanoma-associated antigens. C57BL/6J mice with melanoma, immunized with the semi-allogeneic hybrid cells, developed CD8-mediated melanoma immunity and survived significantly (P<0.005) longer than mice with melanoma immunized with a mixture of the parental cell types. The failure of melanoma immunity to develop in mice injected with the mixture of parental cells indicated that co-expression of the immunogenic determinants by the same cellular immunogen was necessary for an optimum immunotherapeutic effect. Augmented immunity to melanoma in mice immunized with the semi-allogeneic hybrid cells points toward an analogous form of therapy for patients with melanoma. Received: 19 May 1997 / Accepted: 23 July 1997     

Characterisation of the complement-regulatory proteins decay-accelerating factor (DAF,CD55) and membrane cofactor protein (MCP,CD46) on a human colonic adenocarcinoma cell line

 To avoid destruction by complement, normal and malignant cells express membrane glycoproteins that restrict complement activity. These include decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and protectin (CD59), which are all expressed on colonic adenocarcinoma cells in situ. In this study we have characterised the C3/C5 convertase regulators DAF and MCP on the human colonic adenocarcinoma cell line HT29. DAF was found to be a glycosyl-phosphatidylinositol-anchored 70-kDa glycoprotein. Blocking experiments with F(ab′)₂ fragments of the anti-DAF monoclonal antibody BRIC 216 showed that DAF modulates the degree of C3 deposition and mediates resistance to complement-mediated killing of the cells. The expression and function of DAF were enhanced by tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β). Cells incubated with interferon γ (IFNγ) did not alter their DAF expression. Two MCP forms were expressed, with molecular masses of approximately 58 kDa and 68 kDa, the lower form predominating. MCP expression was up-regulated by IL-1β, but not by TNFα or IFNγ. Expression of DAF and MCP promotes resistance of colonic adenocarcinoma cells to complement-mediated damage, and represents a possible mechanism of tumour escape. Received: 18 July 1995 / Accepted: 4 January 1996     

The prognostic significance of immune changes in patients with renal cancer,melanoma and colorectal cancer,treated with interferon α2b

 In the present study we evaluated the response rate and the immunorestorative properties of interferon α2b (IFnα2b) administered to patients with advanced renal cell carcinoma (RCC), melanoma (MEL) or colorectal cancer (CC). We studied the immune status and correlated it with clinical responses. Thirty-five patients with advanced RCC, and 14 with MEL were treated with recombinant INFα2b. The dose was increased progressively from 5×10⁶ IU/day in the first week (three times every week) to 10×10⁶ IU/day in the second week and thereafter to 15×10⁶ IU/day subcutaneously. In patients with CC INFα2b was given at 5×10⁶ IU/day every other day (three times every week); these patients also received (together with INF) leucovorin 200 mg m^–2 day^–1 in a 1-h i. v. infusion every week, and mid-infusion 400 mg/m² 5-FU was administered as an intravenous bolus every week. The response rate was as follows: for RCC, 6 patients achieved partial response (PR), 10 stable disease (SD), and 21 progressed (PD); for MEL, 5 patients achieved PR and 9 PD; for CC, 6 achieved PR, 5 SD, and 9 PD. In all patients blood was withdrawn prior to INFα2b treatment and then monthly. T lymphocytes, after isolation from peripheral blood, were tested for proliferation in the autologous mixed-lymphocyte reaction and allogeneic mixed-lymphocyte reaction, interleukin-2 (IL-2) production, expression of IL-2 receptors during the allogeneic-mixed-lymphocyte reaction, and the production of IL-1 by peripheral blood monocytes. Striking increases were demonstrated in all parameters 2 months after treatment with INFα2b. In comparison to normal controls, all patients with the malignant neoplasms presented decreased (>45%) mean values of the immunological parameters under investigation (P 0.0001). Responders (patients with RCC, MEL, and PR) presented lower mean values of all the parameters studied than did non-responders (P 0.0001). Patients with CC presented the lowest mean values of the parameters than did the other patients (RCC, MEL) (P 0.0001). After therapy with INFα2b, patients with RCC experiencing PR showed a mean increase of more than 30% (P 0.0001). Patients with SD showed a mean increase of about 20% (P 0.0001), and those with PD showed a 6% increase in the immunological parameters under investigation. Patients with MEL experiencing PR showed a mean increase of more than 30% and patients with PD a decrease of more than 10% (P 0.0001). All patients, regardless of the clinical response, achieved an increase of more than 60% (P 0.0001). Administration of IFNα2b resulted in a marked potentiation of a deficient cellular immune response in vitro in those patients with RCC and MEL who responded to the treatment. On the other hand, non-responders demonstrated a decrease in the examined parameters and, in some, deterioration of the already depressed immunological functions was observed. This observation can have prognostic significance regarding clinical response of INF. In contrast, our findings show that the immune stimulation associated with INFα treatment in all our CC patients did not predict an improved clinical outcome. There are several theoretical explanations for this discrepancy. Received: 30 November 1996 / Accepted: 25 June 1996     

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.