In Vivo Visualizing the IFN-ß Response Required for Tumor Growth Control in a Therapeutic Model of Polyadenylic-Polyuridylic Acid Administration.

The crucial role that endogenously produced IFN-ß plays in eliciting an immune response against cancer has recently started to be elucidated. Endogenous IFN-ß has an important role in immune surveillance and control of tumor development. Accordingly, the role of TLR agonists as cancer therapeutic agents is being revisited via the strategy of intra/peritumoral injection with the idea of stimulating the production of endogenous type I IFN inside the tumor. Polyadenylic-polyuridylic acid (poly A:U) is a dsRNA mimetic explored empirically in cancer immunotherapy a long time ago with little knowledge regarding its mechanisms of action. In this work, we have in vivo visualized the IFN-ß required for the antitumor immune response elicited in a therapeutic model of poly A:U administration. In this study, we have identified the role of host type I IFNs, cell populations that are sources of IFN-ß in the tumor microenvironment, and other host requirements for tumor control in this model. One single peritumoral dose of poly A:U was sufficient to induce IFN-ß, readily visualized in vivo. IFN-ß production relied mainly on the activation of the transcription factor IFN regulatory factor 3 and the molecule UNC93B1, indicating that TLR3 is required for recognizing poly A:U. CD11c(+) cells were an important, but not the only source of IFN-ß. Host type I IFN signaling was absolutely required for the reduced tumor growth, prolonged mice survival, and the strong antitumor-specific immune response elicited upon poly A:U administration. These findings add new perspectives to the use of IFN-ß-inducing compounds in tumor therapy.

Phase III trial of adjuvant 5-fluorouracil and adriamycin versus 5-fluorouracil, adriamycin, and polyadenylic-polyuridylic acid (poly A:U) for locally advanced gastric cancer after curative surgery: final results of 15-year follow-up.

BACKGROUND: This phase III trial was to compare 5-fluorouracil (5-FU), adriamycin, and polyadenylic-polyuridylic acid (poly A:U) against 5-fluorouracil plus adriamycin (FA) for operable gastric cancer. PATIENTS AND METHODS: From 1984 to 1989, patients who had D(2-3) curative resection were randomly assigned to receive chemotherapy or chemoimmunotherapy. Chemotherapy consisted of 12 mg/kg 5-FU every week for 18 months and 40 mg/m2 adriamycin every 3 weeks for 12 cycles. Chemoimmunotherapy consisted of FA plus 100 mg of poly A:U weekly for six cycles and was followed 6 months later by six weekly 50-mg booster injections. RESULTS: A total of 292 patients were enrolled. After excluding 12 ineligible patients, 142 and 138 patients were allocated to each treatment. Patients were balanced with prognostic variables: age, sex, tumor location, differentiation, degree of tumor invasion (T2-T4a), and lymph node status (N0-N2). During the 15-year follow-up, chemoimmunotherapy significantly prolonged overall (P = 0.013) and recurrence-free (P = 0.005) survivals compared with chemotherapy alone. The survival benefits were prominent in the subset of patients with T3/T4a, N2, or stage III. Treatments were generally well tolerated in both arms. CONCLUSIONS: These results indicate a survival advantage of chemoimmunotherapy with a regimen of FA and poly A:U in curatively resected gastric adenocarcinoma.

Effect of the double-stranded polynucleotide complex polyadenylate-polyuridylate (poly A-U) on interleukin-6 production by mouse fibroblasts.

Cultures of mouse embryonic fibroblasts (L 929) have been shown to produce a factor which promotes the growth of B cell hybridoma (hybridoma growth factor, HGF) i.e. interleukin 6 (IL-6). The aim of the present study was to investigate the effect of Poly A-U on IL-6 production by this cell type. After incubation for 48 h at 37 degrees C of confluent (1 week old) L 929 fibroblasts in the presence or in the absence of Poly A-U, IL-6-like activity in supernatants was measured by the proliferation assay of the IL-6-dependent B cell hybridoma cell line, 7TD1. Poly A-U increased IL-6 activity in supernatants in a dose-dependent manner at doses higher than 50 micrograms/ml, the maximum activity being observed at the highest concentration of Poly A-U used, i.e. 500 micrograms/ml. beta Interleukin-1 (beta IL-1) and poly-cytidylic-polyinosinic (Poly I-C) have been shown to be inducers of IL-6 in fibroblast culture and thus their effect was compared to that of Poly A-U. The IL-6 activity in supernatants induced by 500 micrograms/ml Poly I-C (58.4 +/- 16.4 U/ml; n = 4) was higher than that evoked by 100 U/ml beta IL-1 (5.7 +/- 0.4 U/ml) or 500 micrograms/ml Poly A-U (39.6 +/- 7.8 U/ml). The increased production of IL-6 by Poly A-U may explain part of its previously reported immunomodulatory effects.

Adjuvant treatment of operable stomach cancer with polyadenylic.polyuridylic acid in addition to chemotherapeutic agents: a preliminary report.

A randomized trial of polyadenylic.polyuridylic acid [poly(A).poly(U)] in addition to chemotherapy was undertaken in patients with stomach cancer following curative gastrectomy. They were randomized into a group of 108 patients receiving chemotherapy plus poly(A).poly(U) and a control group of 116 patients receiving chemotherapy alone. Chemotherapy consisted of injections of 5-fluorouracil, 12 mg/kg once weekly and adriamycin, 40 mg/m2 once every 3 weeks, continuously after operation. Poly(A).poly(U) was infused in a 100 mg dose, once a week six times from 5 days after the first injection of chemotherapeutic agents and 6 months later in a half dose similarly. At 55 months after initiation of the trial, the mean follow-up periods were 24 months for both groups. It has been revealed that patients who received the combined treatment postoperatively showed a lesser mortality and lower rate of recurrence, both reflecting significant increases in overall (P less than 0.05) and relapse-free (P less than 0.02) survivals as compared to those who received chemotherapy alone. This effect is more pronounced in patients having moderately advanced lymphnode involvement (N1) than in patients without (N0) or more advanced (N2) involvement. Thus, poly(A).poly(U) appears to be an effective agent when used postoperatively with chemotherapy in stomach cancers.

Enhanced antitumor effect of combination therapy with interleukin-2 and polyribonucleotides against adenocarcinoma 755.

Human recombinant interleukin-2 (IL-2) and interferon (IFN) inducers in combination were evaluated for their in vivo antitumor efficacy in relation to s.c.-implanted adenocarcinoma 755 in C57BL/6 mice. Two IFN inducers, polyinosinic-polycytidylic acid [poly(I)poly(C)] and polyadenylic-polyuridylic acid [poly(A)poly(U)], induced high IFN production in various tissues for a long time compared to treatment with murine interferon-beta. Especially, poly(I)poly(C) at 5 mg/kg, the maximum tolerated dose, produced the highest level of IFN in the tumor, but the tumor did not show regression. Poly(I)poly(C), however, brought about marked regression of the tumor when administered together with IL-2. This combination resulted in cure of some mice when both drugs were administered intraperitoneally. Intraperitoneal injection of IL-2 in combination with poly(I)poly(C) was more effective than intravenous injection of IL-2. The combination of IL-2 and poly(A)poly(U) also showed an enhanced antitumor effect. Thus, endogenous IFN produced by IFN inducers as well as exogenous IFN as reported previously potentiated the antitumor effect when administered together with IL-2. The degree of potentiation by combination of IL-2 and IFN inducers may depend on the level of IL-2 and IFN at the injection site.

Increase in liver-associated natural killer activity by polyribonucleotides.

Several polyribonucleotides are currently in clinical trials for the treatment of cancer or viral diseases. The present report in mice demonstrates that polyinosinic-polycytidylic acid and poly-L-lysine which has been stabilized in carboxymethylcellulose (poly (ICLC) as well as polyadenosinic-polyuridylic acid (poly AU), both potently augment natural killer (NK) activity in the liver, which is often a target organ for the formation of metastases during the progression of human cancer. Following the administration of poly ICLC (10 micrograms/mouse), greater NK activity as measured by lytic units (LU), was observed in the liver (445 LU) than in blood (63 LU) or spleen (20 LU). The high level of NK activity in the liver was in contrast to the low levels observed in untreated mice, and was maintained for at least 9 days post injection. NK activity in the blood and spleen returned to normal levels by day 6. Similar results were obtained with poly AU except that approximately 10-fold more poly AU (100 micrograms/mouse) was required to induce optimal augmentation of NK activity. Further studies demonstrated that the increase in liver-associated NK activity induced by poly ICLC was associated with a 10- to 20-fold increase in liver-associated leukocytes, termed nonparenchymal cells (NPC). Fractionation of the NPC on discontinuous density gradients of Percoll demonstrated that the NK activity mediated by NPC was associated with cells morphologically characterized as large granular lymphocytes (LGL). Further studies demonstrated that the repeated administration of poly ICLC resulted in significantly higher levels of liver-associated NK activity and total liver-associated LGL as compared to a single injection.

Muramyl dipeptide is a powerful potentiator of the antitumor action of various tumor-necrotizing agents.

The previously observed potentiation of necrosis and regression of solid immunogenic Meth A sarcoma transplants in mice after IV administration of endotoxin by addition of muramyl dipeptide (MDP) in saline was investigated further by varying time and route of administration of both agents. Equal potentiation was observed when MDP was administered 4 h before or after endotoxin, but administration 48 h or 24 h before or 24 h after endotoxin had no effect. Simultaneous administration of both agents enhanced tumor damage considerably, regardless of the route of administration of either agent. A strong potentiation of necrosis and regression was also observed upon addition of MDP to concanavalin A, poly I:C or poly A:U and, to a lesser degree, to a radio-detoxified endotoxin, purified L cell interferon, or Propionibacterium acnes. No consistent relationship could be seen between the degree of potentiation of necrosis and of regression. It was suggested that distinct mechanisms underlie the augmenting action of MDP on necrosis and regression and that enhanced production and/or action of vasoactive agents might play a role in the potentiation of necrosis. Whether the capacity of MDP to stimulate specific and nonspecific immune defense is involved in the enhancement of tumor regression remains uncertain at present.

Enhancement of natural killer cell activity and 2-5A synthetase in mice treated with polyadenylic.polyuridylic acid.

Previous studies have demonstrated that splenic natural killer (NK) cell activity of mice can be efficiently enhanced by polyadenylic.polyuridylic acid [poly(A).poly(U)]. Moreover, inoculation of mice with the duplex leads to the production of interferon (IFN). The NK boosting effect in mice was analyzed in parallel with the assay of an enzyme marker for the production and action of IFN, pppA(2'p5'A)n synthetase (2-5A synthetase). Kinetic studies revealed that splenic mononuclear cells of C3H/He mice inoculated intravenously with 10 micrograms or more of poly(A).poly(U) showed significantly enhanced cytotoxic activity in an in vitro 51Cr-release assay using NK sensitive YAC-1 target cells. Intravenous, intraperitoneal, and intramuscular administration of the duplex into mice were equally effective in obtaining such NK boosting effect. Furthermore, repeated weekly injections of poly(A).poly(U) did not induce resistance against NK boosting in the organisms. The levels of 2-5A synthetase in the organs, particularly spleens, of mice inoculated similarly with poly(A).poly(U) were greatly increased with a dose-dependent pattern. Repeated injections of mice with the duplex at 4-day intervals resulted also in significantly enhanced levels of splenic 2-5A synthetase after each injection. Thus, as a whole, enhanced NK activity accompanied increased levels of 2-5A synthetase in mice treated with poly(A).poly(U).

Enhancement of spontaneous C3H/HeJ mammary tumorigenesis by long-term polyadenylic-polyuridylic acid therapy.

Female C3H/HeJ mice received weekly s.c. injections of 2 mg polyadenylic-polyuridylic acid. Therapy was initiated at either 2 or 9 months of age. In both cases, poly-adenylic-polyuridylic acid-treated animals developed the spontaneous mammary carcinoma associated with this strain more rapidly. Because the opposite result was formerly observed for AKR spontaneous leukemia, the data indicate the polyadenylic-polyuridylic acid has no generalized antineoplastic effect upon spontaneous tumors genetically associated with specific murine strains.