Norimichi Kan

Cyclophosphamide given after active specific immunization augments antitumor immunity by modulation of Th1 commitment of CD4+ T cells.

Background and Objectives: In order to evaluate the regulatory effect of cyclophosphamide (CPA) on active specific immunization (ASI)‐induced antitumor immunity, we examined the timing of CPA (100 mg/kg) with ASI, and focused on whether CPA given after ASI augments antitumor immunity by modulation of Th1 commitment of CD4+ T cells.

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

SummarySpleen 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.

The therapeutic effect of OK-432-combined adoptive immunotherapy against liver metastases from breast cancer

SummaryWe studied the therapeutic effect of OK-432 combined with adoptive immunotherapy in 19 cases of liver metastases from breast cancer. Of the 14 patients who received intraarterial OK-432 injection and transfer of cultured lymphocytes, 9 responded to this therapy, whereas no patients responded to intravenous administration. The minimum cell number for a therapeutic response was 8×108 cells. Metastatic lesions other than those in the liver regressed after therapy in 4 patients. The serum carcinoembryonic antigen level paralleled the therapeutic effect. There were no severe side-effects accompanying this therapy. These results indicate that intraarterial adoptive immunotherapy combined with OK-432 is effective as a new therapeutic approach against liver metastases from breast cancer.

Relationship between immunological parameters and survival of patients with liver metastases from breast cancer given immuno-chemotherapy

SummaryWe treated 33 patients with liver metastases from breast cancer by immuno-chemotherapy including adoptive cell transfer between 1987 and 1992. In this study, we examined the change of immunological parameters in the peripheral blood lymphocytes and interleukin-2 (IL-2)-cultured lymphocytes, in primary vs. metastatic breast cancer patients and before vs. after treatment. Moreover, we examined their correlation with therapeutic response and survival after treatment. The immunological parameters used werein vitro natural killer cell activity (% lysis of K562),in vitro autologous tumor-killing activity (% lysis against autologous freshly isolated tumor cells), and proliferation of lymphocytes stimulated with IL-2 and autologous sonicated tumor extract antigen in mixed culture (IL-2-enhanced MLTR). When compared with primary breast cancer patients, patients with liver metastases showed a significant decrease in % lysis of K562 and autologous tumor cells. After treatment, the stimulation index in IL-2-enhanced MLTR increased significantly from the pretreatment level and correlated with survival after treatment. Moreover, non-specific immunological parameters (performance status, lymphocyte count, and transferred cell count and proliferation rate of cultured lymphocytes) were significantly associated with response and prognosis.

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.

Interferon‐γ‐producing Tumor Induces Host Tumor‐specific T Cell Responses

We investigated the mechanism of host immune responses against two interferon‐γ (IFN‐γ) gene‐transduced tumors, plasmacytoma MOPC104E(Muγ) and mammary cancer SC115(Kγ), which originally had weak immunogenicity. Both IFN‐γ‐producing tumor cells had reduced tumorigenicity and were rejected by syngeneic mice. The rejection was completely blocked by in vivo treatment with anti‐CD8 or anti‐IFN‐γ monoclonal antibodies. While anti‐CD4 monoclonal antibody also blocked the rejection of SC115(Kγ), it enhanced the initial tumor growth of MOPC104E(Muγ). Specific protection against subsequent challenge with the respective parental tumor cells was demonstrated in mice which rejected the IFN‐γ‐producing tumor cells. Cultured lymphocytes derived from immunized mouse spleens had cytotoxic T cell activity against parental tumor cells, as well as against cells that produced IFN‐γ, These findings indicate that the antitumor effects are mediated by cytotoxic T cells and, partly, by helper T cells, and that locally secreted IFN‐γ plays an important role in generating these effector cells.

Comparison between crude Interleukin-2 and recombinant Interleukin-2 in maintaining killing activity of cultured lymphocytes

Peripheral blood lymphocytes, regional lymph node lymphocytes or malignant effusion lymphocytes from cancer patients were incubated with crude IL-2 (cIL-2) for 13 days. These effectors, which frequently expressed IL-2 receptor (IL-2R), proliferated well and possessed augmented killing activity against fresh autologous tumor cells and K562. However, when recombinant IL-2 (rIL-2) was added for the last 4 days of culture instead of cIL-2, IL-2R expression and killing activity against fresh autologous tumor cells decreased significantly (P<0.05). Phenotypic analysis indicated that cIL-2 significantly promoted the expansion of the cytotoxic population (CD8+ .11b−)(P<0.05). The decreases in killing activity and IL-2R expression were restored by 0.004% PHA plus rIL-2, but not in the presence of rIFN-γ, rIL-1α, rIL-lβ, rIL-4 or rIL-6. PHA-free cIL-2 maintained killing activity, but not IL-2R expression.We conclude that some factors in cIL-2 and a low dose of PHA-P are necessary for the maintenance of killing activity and IL-2R expression of cultured lymphocytes in the late phase of culture.

The Induction of Specific Antitumor Immunity by In Vivo Treatment with Interleukin-1 and Sonicated Tumor Extract in a Murine Model

BALB/c mice were pretreated intraperitoneally with interleukin-1 (IL-1) and sonicated tumor extract (SE) from plasmacytoma MOPC104E, 10, 7, and 4 days prior to the intraperitoneal or subcutaneous inoculation of MOPC104E cells, following which significant suppression was observed. The mean survival time and tumor diameter on day 21 were 46.7 days and 0 mm, respectively, in contrast to the 20.9 days and 20.4 mm of control mice. Mice pretreated with IL-1 and SE from MOPC104E (MOPC-SE) were not suppressed following fibrosarcoma MethA inoculation, which indicates the tumor specificity of immunity in this model. This systemically operating antitumor immunity was also achieved by the intramuscular administration of IL-1, or when tumor challenge was performed on day 7 or 14. Moreover, MOPC104E-specific delayed-type hypersensitivity was detected in these mice. The results of this study suggest the possibilities of a new type of active specific immunotherapy, which could prove useful as postsurgical adjuvant therapy for cancer patients.

Factors influencing the response and survival of patients with liver metastases from breast cancer receiving OK-432-combined adoptive immunotherapy

SummaryThe response and survival of 26 patients with liver metastases from breast cancer, who received OK-432-combined adoptive immunotherapy from 1984 to 1990, were evaluated. OK-432-combined adoptive immunotherapy was comprised sequential treatment via the hepatic artery with a streptococcal preparation, OK-432 (1–5 KE), and adoptive transfer of lymphocytes expanded in T-cell growth factor and sonicated tumor extract antigen. Seventeen (65%) patients responded to the therapy. The median survival time of all patients after treatment was 13 months (range, 2–63 months). Of the 20 prognostic factors analyzed, performance status (PS) alone was related to response (P<0.01). The response rate of the patients with a PS of 0–2 was 83% but only 25% in those with a PS of 3 or 4. In univariate analysis, 11 factors significantly influenced the survival: tumor response; size of primary tumor; menopausal status; PS; serum bilirubin, albumin, lactate dehydrogenase and glutamate-oxalate transaminase (aspartate aminotransferase); the extent of liver involvement; and the number and the proliferation rate of transferred lymphocytes. The MST was 22.8 months for the responders versus 2.8 months for the nonresponders (P<0.01). In multivariate analysis, the most important factor associated with survival was the tumor response, as well as PS, liver involvement, lactate dehydrogenase and albumin. These results suggest that OK-432-combined adoptive immunotherapy can be considered a candidate for a randomised control study and these factors should be used for stratification.

A new model of active specific immunotherapy using interleukin-1 and sonicated tumor supernatant in murine tumor system

The possibility of active specific immunotherapy using interleukin‐1 (IL‐1) plus sonicated tumor supernatant (SS) was examined in a murine tumor model. The growth of intraperitoneally or subcutaneously inoculated plasmacytoma MOPC104E, which is syngeneic to BALB/c mice, was significantly suppressed by intraperitoneal pretreatment with IL‐1 and SS from MOPC104E cells (MOPC‐SS), on days 10, 7, and 4 before tumor inoculation. Pretreatment with IL‐1 plus MOPC‐SS or MethA‐SS (SS from MethA cells) suppressed the growth of subcutaneous tumor of only the corresponding tumor cells, indicating the development of tumor‐specific immunity in vivo. The splenic cells of immunized mice with IL‐1 and MOPC‐SS showed tumor neutralizing activity. However, their tumor neutralizing activity was abrogated when they were treated in vitro with anti‐Thy1.2 or anti‐L3T4 plus complement. Moreover, when combined with indomethacin per oral, IL‐1 plus MOPC‐SS significantly suppressed the growth of established subcutaneous tumor and prolonged survival of postoperative mice. These results suggest that this new type of active specific immunotherapy could be a useful method for cancer immunotherapy, especially when combined with oral indomethacin.