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  • A61K38/19—Cytokines; Lymphokines; Interferons
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• • A—HUMAN NECESSITIES
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Definitions

• SUMMARY Lipopolysaccharide or variants thereof can be used together with such anti-tumor agents as exogenous TNF as effective anti-tumor agents at non-toxic levels.
• Inhibitors such as indomethacin which block agents, such as prostaglandin which act against immune system activation or agents which act as against suppressor cell activation such as cyclophosphamide can be used as well.
• Fig. 1 shows synergism in causing necrosis and regression of tumors with LPS and TNF, also with TNF and LPS with INDO (Indomethacin).
• Fig. 2 shows correlation between tumor necrosis and subsequent regression of tumors.
• Fig. 1 Synergism between TNF and LPS is causing necrosis and regression of tumor transplants in mice. Animals were treated in addition to TNF and LPS with INDO.
• Fig. 2 Correlation between tumor necrosis and subsequent regression of tumors, the data obtained with LPS (0) relates the degrees of necrosis in individual mice to the regression of the same tumors. In the experiments with TNF we had not assessed individual mice for necrosis and regression but only groups of identically treated animals. The percent of tumor regression in these animals was related to the group's average degree of necrosis.
• LPS Lipopolysaccharide
• LPS has long been known to elicit anti-tumor reaction in experimental animals and man. LPS causes acute partial necrosis of certain murine and human tumor transplants in mice and allows one third of the treated animals to completely reject the tumor. The response can be divided into two phases, an early non-immunologic necrosis phase and a subsequent immunologic rejection phase.
• TNS tumor necrosis serum
• TNF is a very efficient inducer of tumor necrosis but is also very toxic to animals. LPS-induced shock seems to be mediated by TNF (B. Beutler et al, Nature 316:552-554, 1985). For the cure of a tumor a mouse seems to have to pay a smaller penalty on toxicity when TNF is administered within TNS than when administered in purified form. TNF kills tumor cells in tissue culture whereas LPS has no effect on cultured tumor cells directly. Our findings suggest LPS acts on tumors through different pathways only one of which is mediated by TNF. In combination the different defense systems are more effective than each by itself. TNS contains other factors which affect the fate of tumors. The increased presence of IL-1 or IL-2 like factors and; of interferon has been identified and indirect evidence suggests the presence of other, as yet unidentified factors.
• TNS has been found to stimulate the activity of NK cells, a cell that kills tumor cells on contact. It induces also the production of substances such as prostaglandins which paralyze the defense capacity of the tumor host.
• Interleukin 1 IL-1
• IL-1 Interleukin 1
• LPS-reactive macrophages facilitates the activation of lymphocytes which are the carriers of the immunological response against tumors.
• IL-1 facilitates the recruitment of lymphocytes, causes them to produce lymphokines which participate in the process of the immune response (such as IL-2 and TRF) and is therefore essential for the generation of effector cells which produce either antibodies to tumor associated surface membrane structures or which seek out tumor cells via these membrane antigens and kill them.
• lymphokines also initiate downregulatory mechanisms which block the immune response. This downregulatory pathway is mediated by cells called suppressor cells, of which suppressor T-cells are the most prominent.
• LPS can be viewed as inducing reactions that inhibit malignant growth (TNF production, enhancement of immune function) and reactions which favor malignant growth (immune suppression). It would appear, that effective immunotherapy must be designed to support the former reaction and to curb the latter.
• Our laboratory has identified negative feedback responses which favor tumor growth and we have demonstrated that these feedback mechanisms can be controlled leading to substantial improvement of tumor rejection.
• Another relevant objective of immunotherapy is the least possible damage caused to the treated tumor host, that is to say low toxicity to the host.
• Tumor necrosis factor is a highly toxic molecule and its therapeutic dose in mice and humans is limited by its lethal effects. This laboratory has established a treatment regimen in which TNF can be applied in low (not toxic) doses in conjunction with other reagents in non toxic doses.
• LPS itself is a good candidate to complement TNF function since LPS induces in the host the release of several lymphokines such as IL-1, IFN, IL-2 and those not identified as yet which may replace LPS.
• LPS presumably through mediators such as IFN causes increased production of prostaglandins (PG) in the tumor host.
• PG prostaglandins
• PG inhibits the function of the immune system (including NK cell activity).
• Inhibition of PG synthesis with Indomethacin increases the tumor rejection induced by TNF and LPS from 30 to 60 percent.
• LPS causes the generation of suppressor cells.
• Suppressor cell activity can be inhibited by administration of cyclophosphamide and this drug given together with TNF, LPS and INDO increases tumor rejection in mice up to 100%.
• LPS Bacterial lipopolysaccharide
• TNF tumor necrosis factor
• TNF tumor necrosis factor
• Effective therapeutic use of TNF is limited, however, by its toxicity.
• We show here, that the efficacy of TNF can be substantially increased by combining its application with low doses of LPS.
• Our data suggests that LPS exerts its anti-tumor effects by engaging more than one defense mechanism.
• Characteristic for the activation of a biological system is a concommitant induction of negative feedback mechanisms which antagonize the initial stimulus. Interference with the negative feedback response may substantially increase biological reactions.
• LPS prostaglandin E
• PGE prostaglandin E
• analogues of LPS in low doses to stimulate the systems to produce natural anti-tumor factors.
• Meth A tumors were maintained in BALB/c mice by weekly passages. For transplants, 10 cells/0.2 ml, were injected intradermally into two sites of the shaved abdominal skin of (BALB/c x C57BL/6) F1 mice with a 30 gauge needle. The grade of tumor necrosis was scored 48 hours after LPS treatment in a grading system consisting of grades 0,1,2 and 3 according to Carswell et al. Proc.Natl. Acad. Sci., Supra. Tumor rejections were scored four weeks later. Grade 3 reflects total or near total necrosis of the tumor, grade 2 covers necrosis areas down to approximately one-half of the tumor, and grade 1, covers all necrosis areas smaller than one-half of the tumor.
• BALB/c mice and (BALB/c x C57BL/6) F1 mice were purchased from Jackson Laboratories, Bar Harbor, Maine.
• TNF or LPS was injected intraveneously. Indomethacin (100 ug/animal) was injected intraveneously together with LPS and/or TNF and subsequently added to the drinking water (20 ug/ml). Cyclophosphamide (30 mg/kg) was injected interperitoneally. Freshly prepared BALB/c mouse serum was injected intraveneously twice, two and 5 days after LPS injection.
• TNF is an effective inducer of tumor necrosis but its therapeutic dose range is small. Substantial degrees of necrosis can be achieved with the injection of 10 ug TNF. There is a rapid decline of necrosis at lower doses. Higher doses were toxic and not administered here. LPS, in a non-toxic dose range, is a less potent inducer of tumor necrosis than purified TNF. Added together, however, the two drugs synergize. They cause a more effective tumor necrotization together than either one of the two substances alone.
• LPS lymphocytes
• IFN interferon
• NK cell activity G. Trinchieri, et al, J. Exp. Med., 147: 1314-1332, 1978; M. Chun et al, J. Exp. Med., 150: 426-431, 1979; M. Chun et al, J. Immunol., 12:331-334, 1981.
• IFN interferon
• Another factor is interleukin-1, a mediator that stimulates the antigen dependent response of lymphocytes (M.K.
• IFN activates NK cells.
• NK cell activation contributes to the response of tumor-bearing mice to LPS, and studied the activation of NK cells in vitro.
• PGE prostaglandin E
• the production of PGE may be blocked with indomethacin (INDO) (H.R. Strausser et al, Int. J. Cancer, 15:724-730, 1975) and suppression of PGE production by INDO increases NK cell activation by IFN 5-50 times (M. Chun et al, J. Scan. Immunol., Supra).
• INDO indomethacin
• LPS alone.
• the degree of necrosis induced with LPS is also increased by treatment of tumor bearing mice with INDO.
• LPS may also stimulate antigen-specific lymphocyte reactions through the release of IL-1 (M.K. Hoffman et al. Nature, 263:416-417, 1976; S.K. Durmm et al. Am. Rev. Immunol., 3 : 262-368 , 1985; M.K. Hoffman et al, J. Immunol., 122:1371-1375, 1979).
• An immune response of T lymphocytes against tumor-associated antigens has been described by Berendt et al. as a consequence of LPS treatment (M.J. Berendt et al, J. Exp. Med., 148:1550-1559, 1978; M.J. Berendt et al, J. Exp.
• Cyclophosphamide (Cy) injected two days after LPS and INDO allows almost all experimental animals to reject their tumors.
• Cy was chosen to delay injection of Cy as compared to the injection of LPS and INDO because preliminary experiments had shown Cy to interfere with the formation of tumor necrosis induced by LPS.
• TNF tumor necrosis factor
• LPS-induced tumor regression are two distinct phases of LPS mediated anti-tumor activities (M.J. Berendt et al, J. Exp. Med., 148:1550-1559, 1978; L.J. Old et al, Current enigmas in cancer research. Lectures, Series 67:273-315, Academic Press, New York and London, 1973), it would appear that TNF mediates only the early phase of tumor necrosis induction.
• TNF may not be the only factor in LPS-induced TNS to account for effective necrotization of tumors because TNF synergizes with LPS also in causing tumor necrosis. It is unlikely that this can be attributed to the release of additional TNF. LPS causes necrosis only in rather high concentrations but synergizes with TNF in low concentrations. This would suggest that in order to produce TNF, LPS is required in high doses, while in low doses it causes the release of factors that synergize with TNF (LPS itself does not act directly on tumor cells) (E.A. Carswell et al, Proc. Nat. Acad. Sci. USA., 72:3666-3670, 1975).
• LPS is thus an extremely effective anti-tumor therapeutic substance in mammals as shown against Meth-A tumor transplants in mice. Its main disadvantage, its toxicity, seems to be related to the production of TNF (B. Beutler et al, Nature, 316:552-554, 1985) whose sufficient production (judged by the degree of necrosis achieved) requires high doses of LPS. When TNF is used from an external source much lower doses of LPS are needed to complement TNF action.
• LPS provides an example as to how these defense systems may interact to achieve an optimized attack against malignancies and the study of LPS action may help in designing new strategies in the treatment of malignancies by activating the body's means of defense in a concerted fashion.
• this method may prove effective when used together with other chemotherapeutic agents or chemical equivalents or variants of the natural agents induced by LPS.
• purified natural TNF, IL-I, IL-2 or IFN may be used or recombinant material.
• combination therapy between LPS and factors whose: release it induces such as TNF, IL-1, IL-2 or IFN is indicated as is combination therapy between factors whose release LPS causes: TNF, IL-1, IL-2, IFN.
• Combination therapy involves direct (TNF) effects on tumor cells and indirect effects (mediated by the immune system). It does not relate to a combined effect of different factors directly on tumor cells such as seen in the synergism between TNF and IFN on L cells in vitro.
• TNF effects may also be shown by other factors with the ability to cause necrosis of tumor cells and which may be released by different cell types.
• LPS effects may also be shown by other microbial or non microbial products which cause the release of immunoregulatory factors in mammals.
• chemotherapeutic agents or natural factors which block the negative feedback inhibition against immune system activation.
• Indomethacin is only one example from a group of prostaglandin inhibitors such as aspirin or Voltaren (Ciba-Geigy Co., Basel, Switzerland).

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