The structures of tumor necrosis factor (TNF) and of interleukin-1 (IL-1), as indicated by their amino acid sequences (1-6) bare no similarity to each other. However, recent information on the function of these cytokines suggests that their mechanisms of action are closely related. TNF-alpha and the structurally homologous lymphokine, lymphotoxin, also named TNF-beta, were initially identified by their ability to mediate in vitro cytotoxic effects on some cultured tumor cells and to induce in mice hemorrhagic necrosis in certain transplantable tumors (7-9). Yet, it has later been found that TNF exerts, besides cytotoxic activity, many other effects of the quite heterogeneous nature on cell functions (10). Several of these effects appear indistinguishable from those of IL-1. Both TNF and IL-1 have been found, for example, to stimulate the growth of fibroblasts and to induce in these cells the synthesis of collagenase, prostaglandin E.sub.2 and interferon beta-2 (11-16), to decrease in adipocytes the activity of lipoprotein lipase (17, 18), to activate osteoclasts (19, 20), and to increase in endothelial cells adhesivity for blood leukocytes and synthesis of a cell surface protein which is probably involved in that adherence (21-23). Like TNF, IL-1 is cytotoxic to some tumor cells (24).
Among the many possible effects of the cytotoxins (CTXs), no doubt the one with the most far-reaching bearing on functioning of the cell is cell death, resulting from the cytotoxic activity of the CTXs. Cells treated by interferons (IFNs) are found to exhibit a significant increase in vulnerability to killing by the CTXs (10, 30) due to an increase in TNF receptor expression induced in those cells by IFNs (28, 30, 33, 34). There are several indications that vulnerability to the cytotoxic effect is subjected to regulation by mechanisms which, at least in part, are independent to those controlling the response to other effects of CTXs. Thus, comparison of effects of the CTXs on cells of different cultured lines has revealed marked differences in vulnerability from one cell line to another; tumor cells being in general more vulnerable than normal ones. These differences did not correlate to the level of receptors to the CTXs, nor to the effectiveness at which non cytotoxic effects could be induced in the cells. Cell killing by the CTXs is enhanced not only by IFNs, but also by metabolic blockers, such as inhibitors of RNA and protein synthesis (10 ). Prior art relating to the fact that TNF and IL-1 function through binding to high affinity cell surface receptors is disclosed in references (26) to (32).
Inhibitors of RNA and protein synthesis, sensitize certain cells to the cytotoxic effect of tumor necrosis factor (TNF). Treating cells with TNF, for a few hours, in the absence of such inhibition decreases the sensitivity to killing by subsequent application of TNF together with the inhibitors. Such decrease in vulnerability to killing by TNF could be observed also when treating cells with preparations of leukocyte-produced cytokines which were effectively depleted of TNF and lymphotoxin activity (10).
It is thus known that TNF has a cytocidal activity against tumor cells in culture. It is further known that the killing by TNF is markedly potentiated by sensitizing agents, particularly by agents inhibiting the synthesis of RNA and of proteins, i.e. metabolic blockers. Thus, such sensitizing agents increase the vulnerability of tumor cells to the cytocidal activity of TNF. It is also known that IFNs have a potentiation effect on the cytocidal activity of TNF, similar to the effect of a sensitizing agent.
It was also recently revealed that TNF, which was believed to have a selective antitumor function, may also mediate destructive effects on normal tissues. Thus TNF, with or without a sensitizing agent or another potentiating material, while being potentially effective as a therapeutic material against tumor cells, may also be potentially deleterious to normal cells.
It has previously been shown (50) that treating cells with crude leukocyte-produced cytokine preparations can result in decreased vulnerability to the cytotoxic effect which the cytotoxins (TNF and the related protein-lymphotoxin) in such preparations can exert. That protective effect was observed when applying preparations of the cytotoxins for a few hours and then applying them again in the presence of cycloheximide (CHI). The extent of cell death in case of treatment with the leukocyte-produced cytokine preparations prior to the treatment with the same preparations in the presence of CHI, was much lower than that observed in applying these preparations right away in the presence of CHI. The active ingredient in the leukocyte-produced cytokine preparations was not known.
The identification, in accordance with the present invention, of IL-1 as the leukocyte-produced cytokine desensitizing cells to the cytotoxic effect of TNF (i.e. decreasing the responsiveness to the destructive effect of TNF) and the studies which followed on both in vitro and in vivo systems have enabled to attain the present invention.
USPTO Backgrounds
USPTO Backgrounds