Effects of interferons and other cytokines on tumors in animals: A review

Pharmac. Ther. Vol. 52, pp. 307-330, 1991 0163-7258/91 $0.00 + 0.50 Printed in Great Britain. All rights reserved © 1992 Pergamon Press Ltd

Associate Editor: S. PESTKA

EFFECTS OF INTERFERONS AND OTHER CYTOKINES ON TUMORS IN ANIMALS: A REVIEW

HILARY THOMAS a n d FRANCES R. BALKWILL*

Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U.K.

Abstract--The term cytokine describes a group of protein cell regulators involved in the control of cell growth and differentiation in embryogenesis, immunity and inflammation. They are of low molecular weight, are produced locally, and 'act in an autocrine or paracrine manner. In the past decade their use as cancer therapy has become a reality. Thirty years ago mice were treated with the antiviral protein interferon (IFN) which not only produced a reduction in the incidence of virus-induced tumors but also slowed the development of transplantable tumors. This was one of the first indications that cytokines can be negative regulators of cell growth. Here we outline current knowledge of the actions of IFNs and other cytokines in animal models, and draw parallels with clinical trials to illustrate the invaluable nature of this preclinical and mechanistic work.

C O N T E N T S

1. Introduction 1.1. Why are animal models necessary? 1.2. Cytokines 1.3. Problems with using cytokines in animal models

1.3.1. Species specificity of some cytokines 1.3.2. Comparison with human clinical studies 1.3.3. Wide variation in scheduling and differing pharmacokinetics between

humans and animals 1.3.4. Few tumor lines have been studied 1.3.5. Purity of cytokine preparations 1.3.6. Immunogenicity of some syngeneic tumors

2. Animal Models 2.1. Nude mouse and human tumor xenografts 2.2. Murine transplantable tumors 2.3. Carcinogen-induced tumors 2.4. Models of metastases

2.4.1. Spontaneous 2.4.2. Experimental 2.4.3. Nude mice

2.5. Transgenic mice 3. Cytokines and Animal Models

3.1. Interferons 3.1.1. Interferon-u 3.1.2. Interferon-~ 3.1.3. Interferon-), 3.1.4. Interferons and chemotherapy 3.1.5. Interferon therapy and other modalities

3.2. Interleukin- 1 3.2.1. Nude mouse xenografts 3.2.2. Transplantable tumors

3.3. Interleukin-2 3.3.1. Toxicity 3.3.2. Nude mouse xenografts 3.3.3. Transplantable tumors 3.3.4. Transplantable tumor studies and combinations with IL-2 3.3.5. Clinical studies with IL-2

308 308 308 308 308 309

309 309 3O9 309 309 309 311 311 311 311 311 312 312 312 312 312 315 315 316 317 317 317 317 317 318 318 319 319 32O

*Corresponding author.

JFT $2/3--D 307

308 H. THOMAS and F. R. BALKWlLL

3.4. Interleukin-4 3.4.1. Transplantable tumors 3.4.2. Chemically-induced and spontaneous tumors

3.5. Interleukin-6 3.5.1. Clinical correlates

3.6. Tumor necrosis factor 3.6.1. TNF and cachexia 3.6.2. Nude mice and murine tumors 3.6.3. Nude mouse and human tumor xenografts 3.6.4. Transplantable tumors 3.6.5. Combination therapy 3.6.6. Clinical correlates

4. Summary References

320 320 320 320 321 321 322 322 322 323 323 324 325 325

1. INTRODUCTION

1.1. WHY ARE ANIMAL MODELS NECESSARY?

The availability of an increasing number of cytokines for therapeutic use has meant that a new mode of cancer therapy has become feasible within the past decade. Clinical trial results have been varied and the number of patients for whom cytokine therapy is appropriate remains proportionately very small. Consequently the need for data from animal models, to predict efficacy, toxicity and mechanisms, is greater than ever.

1.2. CYTOKINES

Cytokines are a group of proteins of low molecular weight and short half-life that are involved in regulation of cell growth, differentiation and proliferation. They have properties which are likely to be important in both cancer therapy and endogenous immunity to malignant cells, and other characteristics which suggest they may have a role in cancer cau- sation (Table 1). There are certain limitations both in understanding the mechanisms of cytokine action, and extrapolating from different animal models to human malignancy. Consequently interpretation of results is only useful when considered in the context of a broad base of in vitro and in rive work in animal models (Table 2).

An outline of the characteristics of the different cytokines identified so far is given in Tables 3 and 4.

Those cytokines in clinical and in vivo use will be discussed individually and in combination, as used in

TABLE 1. Cytokines in Cancer

Advantages As inhibitors of tumor cell growth Toxic for tumor cells Stimulate host rejection of tumor Alter host/tumor relationship e.g. vasculature/nutrient supply Stimulate haematopoiesis after myelosuppressive therapy

Disadvantages As autocrine growth factors Deletions or abnormalities of cytokine genes Contributing to neoplastic progression

each animal model. Particular emphasis has been placed on the interferons (IFNs), of which there is greatest experience.

1.3. PROBLEMS WITH USING CYTOKINES IN ANIMAL MODELS

1.3.1. Species Specificity o f some Cytokines

Some cytokines, such as interferons (IFNs), are strongly species specific whereas others (including tumor necrosis factor (TNF), interleukin- 1 (IL- 1) and interleukin-2 (IL-2)) cross species barriers. Human IFNs induce 2,5A synthetase activity in human tumor xenografts but do not enhance murine NK activity or alter the level of the enzyme in murine tissue. Simi- larly murine IFNs do not induce 2,5A synthetase in human tissues (Balkwill et al., 1982). The availability of purified murine cytokines has facilitated their study and demonstrated their marked potency, though they are still less readily available than human cytokines.

There are limitations when extrapolating from animal model work using human cytokines to therapeutic application, related to the species specificity of the different cytokines. Murine and human TNF have very similar activity profiles in the mouse but the former is more toxic to mice and stimulates thymo- cytes, unlike recombinant human TNF (rhTNF). Complementary DNA clones encoding two distinct receptors have now been isolated from a mouse macrophage cDNA library mTNF-RI and mTNF- R2. mTNF-R1 has similar affinity for h and mTNF but mTNF-R2 shows strong specificity for mTNF-~ suggesting that the activities seen with hTNF in mice or murine cell lines are only mediated by mTNF-R1 (Lewis et al., 1991).

TABLE 2. Current Problems with Cytokine Therapy

Identifying markers predictive of response Understanding mechanisms of action Role of endogenous tumor cytokine production Systemic use of locally acting agents Understanding network and development of rational cytokine combinations Dose-response relationships

Effects of IFNs on tumors in animals

TABLE 3.

309

Molecular Chromosomal weight location of Characteristics

Cytokine (kDa) gene of receptor

IL-P, 17.5 2q(q12-q21) IL-lfl 17.3 2q(q13-q21) IL-2 15-20 4q(q26-q27) IL-3 14-30 5q(q23-3 l) IL-4 15-19 5q(q23-31) IL-5 45 (homodimer) 5q(q23-q31) IL-6 26 7p(p21-24) IL-7 25 8q(q 12-q 13) IL-8 8.5 IL-9 32-39 5 IL-10 c 18.7 IL-l 1 c 23 TNF 17 ( x 3) 6p21.3

active form trimer Lymphotoxin 18 ( x 3) 6q21.3 IFN-~t 16-27 9q(p22-13) IFN-fl 20 9q22 IFN-~ 20-24 12q24. l

280 and 60kDa shared with IL-I/] 280 and 60 kDa shared with IL-~, 55 kDa and 70 kDa MW--120 kDa 50-60 kDa 46.5 kDa 114 kDa 80 kDa 70 kDa

85 kDa 55 kDa shared with LT

85 kDa 55 kDa shared with TNF 95-110 kDa same as IFN-~ at least 2 classes of receptor 90-95 kDa

Table 5 lists those cytokines which are cross species reactive and those which are species specific.

1.3.2. Comparison with Human Clinical Studies

Many studies have demonstrated that cytokine therapy prolongs survival of tumor bearing mice, but partial and complete remission as defined in clinical studies (greater than 50% regression and complete disappearance of all assessable tumor, respectively) may not be seen, or can only be assessed at POSt mortem. In animal models the end points of tumor stasis, reduction in growth rate, or a reduction in number of metastases, may be useful in terms of understanding mechanisms, but would not necess- arily translate into a useful clinical outcome.

1.3.3. Wide Variation in Scheduling and Differing Pharmacokinetics between Humans and Animals

Due to the incompletely defined mode of action of most cytokines and the apparent lack of a dose-response relationship in many studies, both clinical and animal studies have attempted to define the optimal mode of administration and regimen. A comparison of the toxicity of anticancer agents in mouse, rat, hamster, dog, monkey and man was devised based on a formula in which surface area to volume ratios between species were taken into ac- count (Freireich et al., 1966). This formula was applied to calculate appropriate doses when using recombinant human IFN-~ to treat a human ovarian tumor xenograft in nude mice. Using this method of calculation comparable peak plasma and intraperitoneal levels of rhlFN-~, were obtained in mouse and human (Malik et al., 1991).

1.3.4. Few Tumor Lines have been Studied

Only a small number of tumor lines have been studied and many behave as highly malignant

tumors. This makes them poor models for those tumors which are responsive to cytokine therapy in clinical practice.

1.3.5. Purity o f Cytokine Preparations

Early work with cytokines, IFNs in particular, necessitated the use of crude preparations which were almost certainly contaminated with other cytokines. However purified preparations are now widely available and this is no longer an important consideration. Endotoxin, a lipopolysaccharide constituent of the cell walls of gram- negative micro-organisms, exerts many of its effects via the expression of cytokines, and is extremely potent. It may be present in recombinant cytokine preparations and thereby influence the response to treatment.

1.3.6. lmmunogenicity o f some Syngeneic Tumors

Some syngeneic tumors are highly immunogenic. When treated with cytokines a response to the allo- graft, and not the tumor itself, may be seen.

2. A N I M A L MODELS

2. I. NUDE MOUSE AND HUMAN TUMOR XENOGRAFTS

An advantage of the human tumor xenograft system over murine transplantable tumors is that the histology and ultrastructure of their human counter- part is maintained in the mouse, as well as chromosome number, DNA content, tumor markers and hormone secretion (Feibig, 1988; Mattern et al., 1988).

310 H. THOMAS and F. R. BALKWILL

Cytokine

TABLE 4. Cytokine in vitro and Cell Regulatory Properties

Properties

IFN=,~

IFN-/} IFN-y

IL-I

IL-2

IL-3 (Multi-CSF)

IL-4

IL-5

Defense against viruses and other intracellular parasites Cytostasic for various cells including tumor cells Stimulates B cell proliferation and differentiation Inhibits T cell proliferation Enhances cytotoxicity of: Macrophages, Neutrophils, NK cells, T lymphocytes Induces fever Enhance Class I and possibly Class II MHC expression ~ As for IFN alpha except does not stimulate B cell or inhibit T cell proliferation Properties of IFNs alpha and beta in addition to: Mitogenic for various cells 2 Inhibits B cell proliferation Stimulates lymphokine-activated-killer cells Activation of macrophages Increasing cell surface receptors for other cytokines and the Fc portion of IgG Acts synergistically with IL-2 to enhance B-cell proliferation and IgG synthesis Induces Indoleamine-dioxygenase (IDO) (this in turn depletes tryptophan) defense mechanism against parasite and possibly tumor growth 3 Regulator of immunity, wound healing and inflammation Induces cellular antiviral state Cytotoxic or cytostatic for various cells including tumor cells Stimulates B cell proliferation and differentation Activates T cells--chemotaxis, augments eytotoxicity, increases stability of erythrocyte binding, changes membrane viscosity, involved in T-cell activation and IL-2 production Induces ICAM-1 Alters properties of endothelial surfaces mitogenic for fibroblasts and glial cells Promotes breakdown of bone and cartilage Mediates fever in vivo Induces acute phase proteins and other cytokines (IFN beta, IL-6, CSFs, TNF and further IL-I) and regulatory proteins Promotes haematopoiesis Produced by T cells Mitogenic for various cells Activates macrophages Stimulates B cell proliferation and differentation Activates T-cells--stimulates their proliferation and differentiation Stimulates NK cell activity Stimulates LAK activity--highly cytotoxic to tumor cells and tumor cell lines Activates macrophages Stimulates production of other cytokines--including IFN ~,, TNF and lymphotoxin A potent stimulator of esinophil functions Stimulates multi-potent stem cell growth Mitogenic for various cells Stimulates esinophil activity Inhibits lymphokine-activated killer activity Stimulates B cell differentiation Stimulates in vivo hematopoiesis Enhances antibody-dependent cytotoxicity and phagocytic ability Increases production of peroxide anions Mitogenic for various cells Activates and stimulates proliferation of B cells Stimulates isotype selection--IgGl and IgE Induces IgE receptors on B cells Increases Class II MHC expression of B cells Activates T cells Enhances in vitro survival of T cells and cytotoxic activity of CTL Inhibits IL-2 stimulated CTL and LAK activity Activates macrophages Primarily concerned with eosinophil regulation, and may contribute to the eosinophilia seen with cytokine therapy Mitogenic for various cells Stimulates eosinophil activity Activates macrophages Murine IL-5 activates and stimulates proliferation and differentiation of B cells Stimulates isotype selection--IgA

Continued opposite

Effects of IFNs on tumors in animals 311

T ~ L E 4, Continued . . . . .

Cytokine Properties

IL-6

IL-7

IL-8

TNF-~

Expressed by a variety of lymphoid and non-lymphoid ceils Mitogenic for various cells Cytostatic for various cells Stimulates B and T cell proliferation 5 Induced by IL-I and TNF 6 and expression enhanced by IFN beta, PDGF, cyclohexamide and viruses Stimulates liver cells to produce acute-phase proteins Stimulates immunoglobulin production by B cells Autocrine factor for myelomas Induces antibody secretion by pre.activated normal and Epstein-Barr virus transformed human B cells without first inducing cellular proliferation 7 Mitogenic for various cells Stimulates B-cell proliferation s Stimulates T-cell proliferation Stimulates granulocyte activity Induces chemotactic migration of cells Similar properties to lymphotoxin, (otherwise known as TNF r) which is produced by mitogen- stimulated lymphocytes and interleukin-I Induces cellular anti-viral state Mitogenic for various cells Cytotoxic or cytostatic for various cells including tumor cells Activates macrophages Stimulates granulocyte activity Stimulates eosinophil activity Enhances Class I MHC expression Role in recruitment of the inflammatory process, Gram negative sepsis, the cachexia of chronic disease and of malignancy 9 Direct action on growth and differentiation of T and B cells

References: IFeilous et al., 1982; 2Balkwill, 1985b; 3Takikawa et al., 1990; 4Le Beau et al., 1989; 5Ulich et al., 1989; 6De Filippi et al., 1987; 7Hirano et al., 1986; SYoung et al., 1991; 9Balkwill et al., 1987a.

hormone secretion (Feibig, 1988; Mattern et al., 1988).

Not all human tumor xenografts thrive in the nude mouse system and the ' take rate' varies with factors such as age, species of mouse and health of the recipient (Fidler, 1986). One limitation of this system for the in vivo testing of cytokines is that nude mice do not have a competent immune system. In addition, with some cytokines, such as IL-2, a xenogeneic response may be induced.

2.2. MURINE TRANSPLANTABLE TUMORS

Transplantable tumors also have certain limitations. They are often derived from cell lines and

T^Bt~ 5. Species Reactivity o f Cytokines

Cross species reactive Interleukin-2 Tumor necrosis factor alpha Lymphotoxin Granulocyte-eolony stimulating factor Macrophage-colony stimulating factor Interleukin-6*

Species specific Interleukin-3 lnterleukin-4 Interleukin-5 IFN-,y t IFN-fl? IFN-~, Granulocyte-macrophage colony stimulating factor

*Human IL-6 works in murine models but the converse is not true.

tlFN-~ and p will act on subhuman primates, bovine and ovine tissues.

produce rapidly growing tumors which serve as a model for poorly differentiated or anaplastic tumors. This behaviour is not analogous to those tumors in which responses to cytokines have been described. Many transplantable tumors are highly immunogenic.

2.3. CARCINOGEN-INDUCED TUMORS

Some murine and rat models have been developed by using mutagens to induce tumors. However many of these may also be highly immunogenie.

2.4. MODELS OF METASTASES

2.4.1. Spontaneous

Here transplantable tumor cells are injected locally and the animal is then observed for progression of metastases. The primary tumor may be excised to prolong survival of the mouse and observe development of spontaneous metastases. The advantages of this model are that a wide range of histological types is available and it resembles metastatic tumors in the clinical situation. The disadvantages are that the tumors are derived from small numbers of cells, are highly anaplastic and often immunogenie in nature.

2.4.2. Exper imen ta l

Here a single cell suspension of tumor cells is injected into the mouse (usually via the tail vein). Tbe number of metastatic deposits is counted and used as

312 H. THOMXS and F. R. BALKWILL

an indication of therapeutic efficacy. This model assesses the role of NK cells in relation to various cytokines. The deposits themselves usually develop in a specific site and are useful in the study of the metastatic process without the delay necessitated in the spontaneous model. However the metastases arise after intravenous injection and then entrapment-- they are consequently not an optimum model for the mechanism in humans where cells are shed into the lymphatics or bloodstream from the primary site.

2.4.3. Nude Mice

Xenografts grown within nude mice at the site of tumor origin in the human will metastasize although this rarely occurs when they are grown at inappropriate sites. This fact limits the usefulness of this model.

2.5. TRANSGENIC MICE

A wide range of transgenic mouse models of malignancy express an oncogene and develop spontaneous tumors of various histological types (Jenkins and Copeland, 1989). These mice are suitable for therapeutic studies and have fewer limitations than most models when assessing the immune system.

In relation to cytokines, this system has also been used to assess the influence of human cytokine genes inserted into transgenic mice, some of which subsequently overexpress the cytokine of interest. One such system is a mouse which constitutively expresses the interleukin-5 (IL-5) gene. These mice appear macroscopically normal apart from splenomegaly and their eosinophil counts which were significantly higher in transgenics than non-transgenic littermates. This model has helped understanding of the role of IL-5 in the induction of eosinophilia differentiation (Dent et al., 1990).

As yet the transgenic mouse system has not been widely used to assess the therapeutic potential of cytokines but it has the advantage of models in which tumors arise spontaneously in immunocompetent animals which therefore more closely resemble the development of malignancy in humans. The disadvantages are that most transgenic mice are heterozygous and in many models not all those expressing the transgene develop tumors, with the potential 'wastage' of up to three-quarters of the mice. Furthermore the time to development of a tumor may be as long as 18-24 months leaving a long period between establishing the colony and carrying out therapeutic experiments.

3. CYTOKINES AND ANIMAL MODELS

3.1. INTERFERONS

Interferons (IFNs) comprise a multigene family whose protein products are defined as ~t, fl and y on the basis of their antigenicity, physicochemical

properties and amino acid sequence (Dijkmans and Billiau, 1985; Henco et al., 1985; Pestka, 1986; Pestka et aL, 1987). Most cells in the body have receptors for all three IFN types but express more of those for IFN-7 (Aguet and Mogensen, 1983; Langer and Pestka, 1988).

3.1.1. Interferon-or

Interferon-~t (IFN-~t) consists of about 16 subtypes of molecular weights 16-27 kDa (Pestka et aL, 1987). The genes are located on the long arm of chromosome nine. The receptors are 95-I 10 kDa.

3.1.1.1. Human tumor xenografts in nude mice. Human tumor xenografts have been used to look at the direct antitumor activity of a wide range of IFN types and subtypes and to determine optimal doses and regimens (Crane et aL, 1978). The more frequently used xenografts are those from human breast, colonic, bowel, melanoma and ovarian tumors and osteosarcomas. The antitumor effect is more noticeable when therapy is commenced shortly after tumor injection (Hoffman et al., 1985) which supports the contention that IFNs are best used in the presence of minimal residual disease or in an adjuvant setting. Regression of established tumors is rare in these xenograft models. Repeated doses, comparable with maintenance regimens in humans, are more effective than single doses and the total dose given over a longer period of time is more effective.

One of the first studies to look at the effects of different forms of IFN-~t showed that there was a marked difference in the antitumor activity of IFN-~ 2 and a hybrid IFN, IFN-~tA/D, in breast cancer models (Balkwill et al., 1985). In a comparison between the in vivo and in vitro activity of IFN-7 and IFN-a on twelve different human tumor xenografts (breast, bowel and ovary) in nude mice significant tumor stasis was seen in nine out of twelve different adenocarcinomas treated with IFN-~t, while none of the twelve responded significantly to recombinant human IFN°~/. However, in vitro, when the cells from these xenografts were grown as soft colonies in agar, both IFNs inhibited colony development in all three lines tested (Balkwill et al., 1989). Work using two cecal adenocarcinomas and a breast xenograft demonstrated that murine IFN also inhibited their growth, but did not induce 2,5A synthetase or enhance HLA expression in human tumor cells in vitro (Balkwill and Proietti, 1986).

Growth inhibition of human osteosarcoma xenografts in nude mice has been established in a number of studies (Brosjo et aL, 1985, 1987, 1989; Hoffman et aL, 1985). Histologically there is mineralization and partial replacement of the tumor by normal bone tissue and this was initially thought to be due to induction of differentiation by IFN-~. More recently antibodies to murine and human type I collagen have demonstrated that normal bone is host derived. This


suggests that IFN-~t induces the osteosarcoma cells to interact with the host cells and induce heterotopie bone formation (Forster et al., 1988).

In xenografts, Sidky and Borden (1987) showed that IFN had a direct effect on tumors by inhibiting tumor-induced angiogenesis. The effect on angiogenesis was species specific, suggesting that the signal for angiogenesis is provided by the tumor cells themselves.

A summary of studies using different forms of IFN-ct in nude mouse human tumor xenografts is given in Table 6.

3.1.1.2. Transplantable tumors. In transplantable tumor models, the indirect antitumor effects of IFN-~s are well documented (Gresser, 1989). IFNs are more effective against low tumor load and not against established tumors, the anti-tumor effects are dose-related and the effects are apparent as increased survival but rarely tumor regression or cure. In the wide number of experiments using tumor cell lines, the treatments were most effective if given regularly each day and when the tumor load was low. In many solid tumors no effect was seen and where an effect was seen it was before or shortly after implantation. Tumor stasis or growth inhibition were the usual outcome, in very few models did IFN therapy effect a cure. IFNs are ineffective as tumor prophylaxis if mice are pretreated but IFN pretreatment of tumor cells can diminish their tumorigenic capacity when subsequently inoculated in vivo (Paraf et al., 1983).

A B-cell lymphoma stable subline--38C13 (SIR-l) resistant to the antiproliferative effects of IFN-~ was isolated. The subline is completely resistant to IFN-~t in vitro but is more sensitive to IFN-ct than the parental cell line in vivo. When injected into mice the mean and long-term survival of IFN-~ treated mice bearing the SIR-I mutant was significantly greater than for animals with the IFN-~-sensitive 38C13 cell line. These results suggest that the antitumor effects of IFN-~ for this cell line are mediated by activation of host defences or other effects of IFN-~ on the host, such as induction of other cytokines. The in vitro resistance to IFN-a results in a tumor phenotype that appears to be more readily recognized by host defences and eliminated (Reid et al., 1989).

Dvorak and Gresser (1989), showed that subcutaneous tumors of a Friend leukemia cell subline, which were resistant to mlFN-~t/fl in vitro, underwent necrosis when treated with peritumoral and intratumoral injections of mlFN~t/fl. Histologically there was evidence of pronounced vascular endothelial cell damage prior to this necrosis.

IFNs-~t and ~ are known to have synergistic antiproliferative effects in vitro and have been studied

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314 H. TrlOMAS and F. R. BALKWILL

both in vitro and in vivo using the Renca model (an established spontaneously arising murine renal cancer). HuIFN-~A/D (a hybrid human IFN-~ that has activity on murine cells), MuIFN-~ and / / all produced dose-dependent inhibition of Renca tumor in vitro, whereas IFN-7 produced very little effect. When IFN-g was combined with very low doses of IFN-7 (1-10 units/ml) this yielded significantly more growth inhibition than either cytokine alone. To determine whether the synergy was due to direct antiproliferative effects the combination was used to treat i.p. Renca nude and euthymic mice. Potent antitumor effects were seen in euthymic mice and three quarters were immune to rechallenge. The combination of IFN-~ and IFN- 7 only slightly increased mean survival times in athymic mice and no long term survivors were obtained. It appears that this combination directly inhibits the growth of this tumor but a major effect of IFNs in vivo is to induce an anti-Renca immune response (Sayers et al., 1990).

3.1,1.3. Metastatic models. Friend erythroleukemia cells (FLC) strain 3C18, which metastasize to liver and spleen, were given intravenously to investi- gate the role of cytokines in metastasis in DBA/2 mice. Administration of murine IFN-ct/fl has been shown to have a marked antitumor effect in DBA/2 mice. When 3C18 FLC resistant to IFN-~/fl in vitro, were cocultured with murine hepatocytes, a soluble factor was released from IFN-treated hepatocytes which appeared to inhibit FLC multiplication. This factor was isolated and found to be distinct from IFN-~:, IL-l-a and fl and transforming growth factor-ill and /t2 (TGF-fll and f12). This suggests that this inhibitory effect is mediated by an IFN-induced inhibitor of FLC multiplication (Yasui et al., 1990).

The FLC multiply extensively in the livers of syngeneic DBA/2 mice and not at all in the livers of allogeneic C57B1/6 mice. This resistance appears to depend on IFN-~t, which is undetectable in the serum of syngeneic DBA/2 mice or newborn C57B1/6 mice unlike older C57B1/6 mice. Intravenous inoculation of FLC into 3-week old mice was followed by detection of IFN-~ in the serum and the cells failed to multiply in the liver. Gresser et al. (1990a) concluded that different host mechanisms are of varying importance depending on the site of tumor implantation and that IFN<t is important when FLC are injected intravenously.

To compare the efficacy of IFN-~/fl in immune- competent and immunodeficient mice in a metastatic model, this same group used newborn DBA/2 mice, adult nude mice, beige DBA/2 mice (the murine equivalent of Chediak-Higashi syndrome) and Balb/c scid/scid mice (which have severe combined immune deficiency without T or B cells). The antitumor effect of IFN was markedly reduced or abolished in all these groups of immunodeficient mice. Immune-

competent DBA/2 mice had a prolonged survival time with IFN-~/fl and in those mice who responded there was resistance to a second challenge with FLC. To determine the phenotype of the effector cells, mice were treated with antibodies to asialo-GM 1, CD4 or CD8 antigens, and cyclosporin A or silica. IFN u/fl treatment was much less effective in all these mice suggesting a variety of effector cell types participated in the IFN-induced suppression of visceral metastases. An intact immune system appears to be essential to obtain optimal therapeutic effects of IFN-u/fl in this experimental model (Gresser et al., 1990b).

However, hybrid rHuIFN-~A/D with equal activity on murine and human cells, was studied in another metastatic model (Colon 26). IFN-uA/D inhibited experimental pulmonary metastases and prolonged the survival of BALB/c, nude mice and beige mice (Ramani and Balkwill, 1989a).

3.1.1.4. Gene therapy: Future possibilities. Human IFN-~t 5 complementary DNA was inserted into a bovine papilloma virus plasmid vector (BMGNeo) with a neomycin resistance gene. The recombinant plasmid was transfected into NIH3T3 fibroblasts. A fibroblast clone secreting a large amount of hlFN into the supernatant was selected by a monoclonal antibody. Presence of the gene and its expression were confirmed by Southern and Northern blot analyses respectively. The clone was found to suppress proliferation of a hlFN-~-sensitive chronic myelocytic leukemia cell line (KU812) during cocuiti- vation in vitro. When nude mice bearing KU812 tumors were implanted with hIFN-at-producing fibroblasts, tumor growth was significantly suppressed (Ogura et al., 1990).

3.1.1.5. Clinical studies with IFN or. The first large scale clinical trial involved relatively impure leucocyte IFN. The IFN was given in an adjuvant setting to patients with operable osteogenic sarcoma and the treated group showed a survival benefit (Strander, 1986). Subsequent prospective trials failed to show any improvement in survival for the IFN-treated group (Ito et aL, 1990) and indeed IFNs are not generally effective in advanced solid tumors. Of the three IFNs, IFN-ct is the only one with an established role in the therapy of human cancer, particularly in hairy cell leukemia, chronic myeloid leukemia and carcinoid tumors, and more recently in maintenance therapy after conventional treatment in follicular lymphoma and multiple myeloma (Price et aL, 1991; Baccarini et aL, 1989).

Most cytokines do not appear to follow the principles of cytotoxic chemotherapy where dose- intensity appears to relate to response rate. In general, protocols involving IFNs, and to a lesser extent IL-2, are likely to be used to prolong remission and prevent relapse in patients with minimal tumor load. Enhancing the therapeutic ratio is the aim of many current clinical trials, and is already being


investigated in much of the animal model work (Malkovska and Sondel, 1990).

Parallels can be drawn between the effects of IFNs on transplantable tumors and nude mouse human tumor xenografts where low volume disease is most sensitive and low dose, frequent administration is most effective. IFNs are effective in solid tumor xenografts in nude mice, but comparatively higher doses of IFNs are used. Further, the hlFNs are essentially targetted to the tumors as they are not able to bind murine IFN receptors. The apparent effects of growth inhibition or tumor stasis may be clinically useful if, as it appears, this translates into survival in those malignancies (mentioned above) where IFNs are effective.

3.1.2. Interferon-f l

Interferon-fl (IFN-fl) has a molecular weight of 20 kDa, is found on the long arm of chromosome nine and shares a receptor with IFN-~c Used as a single agent in murine tumors it has had disappointing results and only mild local antitumor effects have been described. However, in combination, its synergistic effect may be biologically useful.

3.1.2.1. Nude mice xenografts . Nude mice bearing bilateral xenografts of human breast carcinoma cells (MCF-7 and BT20) were treated with 2 or 45-day cycles of intralesional injections of human natural IFN-/3 (nlFN-fl) alone or in combination with human natural IFN-v (nIFN-v). Injections were given to one of the two tumors in each animal to assess both local (in the injected tumor) and systemic (in the contralateral tumor) therapeutic effects. When nIFN- fl was used as a single agent there were mild local antitumor effects and virtually no systemic effects. The combination produced marked antiproliferative effects, which were assumed to be the result of synergy. These ranged from complete regression, shown histologically, to degrees of growth inhibition. Local effects were more obvious than systemic, and nIFN-fl proved more effective than the recombinant form. MCF-7 tumors were more sensitive to nIFN<t, whereas nIFN-fl was more effective on BT20 tumors (Ozzello et al., 1990) confirming the wide variation in sensitivity between individual tumors.

3.1.2.2. Transplantable tumors. IFN-fl administered intratumorally into a glioblastoma multiforme (GM) xenograft, led to growth inhibition, however this effect on a subcutaneous transplant of a tumor not normally found at such a site is unlikely to be clinically useful in a disease where access to the tumor is difficult (Tanaka et al., 1983).

As yet there is no indication that IFN-fl has better activity than IFN-ct and for clinical purposes it appears that the former will remain the most widely used.

3.1.3. Interferon-y

Interferon-~ (IFN-~) has a molecular weight of 20-24 kDa, is found on the long arm of chromosome twelve and has 90 and 95 kDa cell surface receptors. It is not cross-species reactive. IFN-~, is produced during an immune response by antigen specific T cells, and by natural killer cells recruited by IL-2. It has immunoregulatory effects which are outlined in Table 3 (Trinchieri and Perussia, 1985).

3.1.3.1. Nude mouse xenografts . Administration of rhlFN-~ strongly induced expression of HLA-DR on the tumor cells in two of three adenocarcinoma xenografts tested (Balkwill et al., 1987b).

Relatively high doses of IFN-y caused growth inhibition of melanoma cell lines injected into nude mice (Trotta and Harrison, 1987).

In human ovarian cancer xenografts growing as ascites or bulky solid tumors IFN-~ was given intraperitoneally. Both forms responded with significant increases in survival time and ascites resolved in two of the three ascitic models. The activity of r-hlFN-), was dose and schedule dependent and histologically the treated tumors showed increased necrosis and loss of cellular organization with large areas of hypo- cellular epithelial mucin. These changes were pre- ceded by a fall in tumor tryptophan and a rise in tumor kynurenine, consistent with increased metabolism of tryptophan (Malik et al., 1991). Tryptophan depletion is implicated in the in vitro antiproliferative action of IFN-y against certain human tumor cell lines (Ozaki et al., 1988; De la Maza and Peterson, 1988).

3.1.3.2. Transplantable tumors. Much of the animal work involving IFNs has been determined by clinical trials and consequently the bias has favoured IFN-cc In recent years there has been little use of IFN-), as a single agent in animal models and much of the data is outlined under relevant combinations below. One early study involved the treatment of C57B1/6 mice inoculated with a continuous line of murine osteogenic sarcoma cells. A seven day course of 30,000--60,000 U/day of Type I(a) IFN completely inhibited or delayed the appearance of tumors in experimental animals, whereas only 600 U/day (100- fold less) of Type II(~) IFN was required to inhibit tumor development (Crane et al., 1978). However with this and other experiments where IFN-~ showed increased efficacy it is important to remember that the IFN-~, preparations used were very impure and probably contained the contaminating cytokines which could have contributed to the anticancer effect. Recombinant IFN-~ was also found to be very potent in inhibiting tumor growth of a chemically induced fibrosarcoma (CE-2) transplanted into BALB/c mice (Giovarelli et al., 1986).


3.1.3.3. Carcinogen-induced tumors. Early work with IFN- 7 involved impure preparations. These were found to be ineffective against transplanted myeloma tumors that had been growing for 17 days and were palpable before the start of therapy (Heremans et aL, 1983). However when administered three days before the carcinogen 3-methylcholanthrene and continued for four months, partially pure murine IFN-y inhibited the induction of subcutaneous fibrosarcomas and lung adenomas in CF1 mice (Salerno et al., 1972).

3.1.3.4. Metastat ic models. In experimental lung metastasis models of colorectal cancer and melanoma cell lines (Kondo et al., 1987), IFN-~ had undoubted antitumor effects. This suggests that the antitumor activity of IFN-~ may be site dependent. This is borne out by the finding that using IFNs to treat the murine reticulum cell sarcoma M5076 IFN-ct was more effective against hepatic metastases whereas IFN-7 was more effective against tumors growing subcutaneously (Brunda et al., 1987b). Therapy with human lymphoblastoid IFN (hlFN-ct) or rhlFN-y inhibited experimental pulmonary metastases of the human melanoma cell line, DX3-azac, in BALB/c nude mice and significantly prolonged survival. The human IFN did not affect the state of differentiation of the melanoma cells in rive, as measured by melanin content, but both IFNs inhibited the development of colonies of DX3-azac cells in vitro (Ramani and Balkwill, 1989b).

Contrary to the findings of an antitumor effect with treatment in rive, pretreatment of Colon 26 cells with recombinant mlFN-7 in vitro significantly increased the number of lung tumor nodules when cells were injected intravenously into immunocompetent BALB/c mice and BALB/c nude mice. This did not happen in NK depleted or NK-deficient mice and the effect did not appear to be related to altered MHC expression. It appears that pretreatment with rmlFN- 7 renders colon 26 cells resistant to in vivo NK-cell lysis by a mechanism independent of MHC expression (Ramani and Balkwill, 1987). IFN- 7 exerted its effects by increasing the pulmonary retention of cells during the first 6 hr after tumor cell injection. During this time all cells visualized in the lung were trapped in pulmonary capillaries. This suggests that low doses of IFN-y generated at the site of the tumor by host-infiltrating cells or during cytokine therapy could enhance the survival of tumor cells in the circulation and enhance their metastatic potential (Kelly et al., 1991).

3.1.3.5. Genes transfected into cells. The IFN- 7 gene has been inserted into CMS-5 tumor cells (a weakly immunogenic cell line of BALB/c origin) and different colonies isolated, cloned and expanded into cell lines which produced varying levels of IFN- 7. The cells were injected intradermally. IFN- 7 secretion abrogated their tumorigenicity and produced a persistent and specific antitumor immunity. In contrast

to unmodified CMS-5 tumor-bearing mice, IFN-~,- producing tumors induced a long-lasting state of T-cell immunity as shown by rejection of a tumor challenge. It appears that localized secretion of IFN-~, induces potent antitumor immune responses (Gansbacher et al., 1990).

A cell line constitutively producing IFN-~ was established from a malignant murine neuroblastoma, C1300, by retroviral transfection of mouse IFN-~ cDNA. The cells showed enhanced high-level expression of MHC Class I antigens at the cell surface and high mRNA levels. In vitro growth of these cells was not affected by their IFN-~ production but subcutaneous growth in syngeneic A/J mice was related to levels of IFN-~ production. Tumors induced by a low-producer line grew at a rate similar to those produced by the parental line, but high producers were strongly suppressed. The authors concluded that the reduced tumorigenicity of the IFN-7 high producer line was due to augmented specific antitumor immunity, in which cytotoxic T lymphocytes appeared to be important (Watanabe et al., 1989).

3.1.3.6. Clinical work with I F N 7. With the possible exception of chronic myelogenous leukemia and ovarian cancer (Kurzrock et al., 1987; Allavena et al., 1990) IFN-v has demonstrated limited antitumor activity. However it does appear to enhance immune responses at intermediate and low doses (Maluish et al., 1988).

In one recent clinical study there were fifteen complete and two partial responses in 58 evaluable ovarian cancer patients after intraperitoneal therapy with 20 x 106 U/m 2 IFN-~, twice weekly for 2--4 months (Pujade-Lauraine et al., 1991).

3.1.4. lnterferons and Chemotherapy

Early work in the clinical field demonstrated a synergistic effect when using IFNs in combination with chemotherapeutic agents in hematological malignancies (Chirigos and Pearson, 1973; Gresser et al., 1987). Later studies extended these findings to human tumor xenografts of solid tumors (Balkwill and Moodie, 1984; Carmichael et al., 1986). There are an increasing number of trials involving a combination of IFN-ct and 5-fluouracil and some of these suggest that adjuvant chemotherapy in Duke's Stage C colon cancer prolongs survival (Beart et al., 1990). In one trial using IFN-~ in combination with 5-FU, a response rate of 76% was achieved in previously untreated patients with metastatic disease (Wadler et al., 1989). IFN-~ alone is ineffective in colorectal cancer and 5-FU alone shows a response rate of 5-30% varying with the regimen used.

The synergy between 5FU and IFN-~ has been examined in a number of transplantable tumor cell lines. It was demonstrated in the Cole 205 cell line by Birsic et al. and in HL60 and adenocarcinoma 38 that


the antitumor effect was reversed by addition of exogenous thymidine, suggesting that IFN-~ inhibits thymidilate synthetase activity in tumor cells (Birsic et aL, 1988; Elias and Crissman, 1988), Cells resistant to 5FU often express high thymidilate synthetase levels and IFN may reverse this phenotype by block- ing the compensatory increase in thymidylate synthetase activity (Gewert et al., 1981).

IFN-~ has been administered alongside 5-fluouracil (5FU) to human colon carcinoma cell lines established in nude mice. HulFN-y had cytostatic and cytolytic effects against human colon carcinoma cells without any effect on mouse macrophages whereas murine IFN-~ had no direct cytotoxic effects against any of the cell lines but did activate tumoricidal properties in mouse macrophages. Human colon carcinoma cells were transplanted into the spleens of nude mice which were treated three days later with 5-FU and human or mouse rlFN-~. The best therapeutic effects were seen with the combination of 5-FU and mlFN-7. In this model the antitumor effects seen suggest a direct effect of 5-FU and activation of host defense mechanisms by IFN-~ (Morikawa et al., 1989).

3.1.5. Interferon Therapy and other Modalities

Several investigators have shown that the antitumor effects of IFNs can be enhanced by other treatment modalities, such as hyperthermia and radi- ation (Namba et aL, 1984; Onishi et al., 1989).

Thus there is evidence that IFNs can regulate tumor growth and alter the relationship between host and tumor. They appear to act through direct regulation of tumor cells, stimulation of a host response to the tumor and by inhibiting angiogenesis, the supply of nutritional factors (e.g. tryptophan) and production of paracrine growth factors. Their role in cancer therapy is evolving and appears likely to be important in maintenance therapy of minimal residual disease or as an adjunct to other modalities.

3.2. INTERLEUKIN- 1

3.2.1. Nude Mouse Xenografts

IL- 1 • and/~ are cytostatic for some tumor cell lines in vitro (Onozaki et al., 1985). In a study of the effects of IL-I~ against two murine tumors, in euthymic and nude mice, IL-I~ therapy led to a hemorrhagic response, suggesting the possible role of a non-T cell dependent mechanism in this model (Braunschweiger et al., 1988). Intraperitoneal human recombinant IL-1/~ has been used to treat three intraperitoneal human ovarian cancer xenografts in nude mice, and its actions compared with recombinant human TNF (S. T. A. Malik and F. R. Balkwill, submitted for publication). The results show that IL-I~ markedly enhanced the peritoneal adhesion and implantation of two of these ascitic tumors with no effect on

survival, whilst in a TNF resistant tumor, IL-I/~ had a marked antitumor effect. These results may indicate that, like TNF, IL-I~ can influence the metastatic behaviour of human cancers.

3.2.2. Transplantable Tumors

IL-1 has antitumor activity in some syngeneic tumor models (eg Nakamura et al., 1986). Peri- tumoral injection of recombinant IL-1/~ in mice transplanted subcutaneously with Friend erythroleukemia cells (FLC) resulted in marked inhibition of tumor growth and increased survival, although in vitro treatment of FLC barely inhibited cell multiplication. IL-I/~ induced marked morphological changes in established tumors which were very similar to those seen with TNF-~ and ~. When studied by Nuclear Magnetic Resonance spectroscopy it appears that alterations in tumor vasculature may be the primary event in solid tumors treated with IL-1/~ and TNF (Belardelli et al., 1990).

Subcutaneous injection of ILI-/~ in mice transplanted with highly metastatic FLC resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. Com- bined treatment with IL-1]~ and IL-2 produced a synergistic antitumor effect: Sixty percent of mice injected with highly metastatic FLC survived as compared with no control mice. Combined treatment exerted no antitumor activity in DBA/2 mice injected with antibody to Thyl.2 antigen or in nude mice, suggesting an important role for T-cells during combined IL-I/IL-2 therapy. In vitro treatment of FLC cells with IL-I/~ resulted in slight inhibition of proliferation, whereas IL-2 did not influence it. The results suggest that local combined treatments can induce potent, host-dependent antitumor effect against highly malignant tumors (Belardelli et al., 1990).

Many of the systemic manifestations of infection can be attributed to IL-1. Its local production may contribute to vascular damage in inflammatory, autoimmune and cardiac disease. In malignant disease it may act as an autocrine growth factor for leukemia cells, particularly acute myelogenous leukemia (AML). It also shares many of the biological activities of TNF and LT. However TNF/IL-I combinations have synergistic effects (see TNF section).

3.3. INTERLEUKIN-2

The characteristics of IL-2 (IL-2) are described in Tables 3 and 4. It is encoded by a single gene with 60% amino acid sequence homology between murine and human IL-2.

The receptor consists of a 55 kDa ~ chain and a 70 kDA ~ chain. Low affinity binding to the two chains results in high affinity binding and a biological effect.


Lymphoid cells can be activated by IL-2 to lyse a wider range of tumor cells than NK cells and this is termed lymphokine activated killer (LAK) cell activity. It is probably mediated by diverse cell populations including T cells, NK cells and macrophages. Antitumor responses in animals may be improved by concomitant use of LAK-cells, tumor-infiltrating lymphocytes (TILs), chemotherapy or other cyto- reductive measures and regional or persistent administration (Stern and Smith, 1986; Hadden, 1988; Kornfeld et al., 1985).

3.3.1. Toxici ty

There is little data on the toxicity of IL-2 in animals although it has been established that mice can toler- ate up to 2 x 105 units daily (Ettinghausen and Rosenberg, 1986). However, Papa found that mice receiving half this dose thrice daily (a total of 1.5 times that used by Ettinghausen) developed ascites, splenomegaly and lymphadenopathy which resolved on stopping IL-2 (Papa et al., 1986). rhlL-2 and rhTNF were administered in a sequential schedule to 30 dogs with a variety of spontaneous neoplasms. Dose escalation of both drugs was performed and dose-limiting toxicities were mainly gastrointestinal, although weakness and malaise were seen above this maximally tolerated dose (Moore et al., 1991). No significant hematological toxicity was seen and objec- tive tumor responses were seen in oral mucosal melanoma and cutaneous mastocytoma. In animal models IL-2 has also been coupled to polystyrene beads in an attempt to deliver increased concen- trations of the cytokine to tumor sites while maintain- ing low systemic levels and thereby increasing the therapeutic index (Crum and Kaplan, 1991).

3.3.2. Nude Mouse Xenografts

Studies have been reported in nude mice where LAK cell activity can be induced by infusion of human recombinant IL-2 (which is cross species reactive), confirming that LAK activity can be mediated by non-T-cell populations (Ortaldo et al., 1986). It is also possible adoptively to transfer xenogeneic LAK cells into nude mice, and compare their relative efficacy to in vivo induced mouse LAK cells following injection of IL-2 alone. The limitation of this ap- proach is that the LAK cells may exhibit a xenogeneic response.

Adoptive cellular immunotherapy of the human ovarian cancer cell line NIH OVCAR-3, implanted intraperitoneally into nude mice, involved human lymphoid and monocytoid effector cells which had been activated in vitro with rIL-2 and IFN-? (Ortaldo et al., 1986). Mice were injected intraperitoneally with tumor cells on day 0 and therapy began the next day with either recombinant human IL-2 alone, in vitro IL-2 activated human peripheral blood mononuclear cells (PBMC's) alone, or activated PBMC's and IL-2

simultaneously. The results showed that IL-2 or LAK cells alone had no antitumor activity, but that weekly injections of IL-2 plus LAK cells for 4 weeks prolonged the median survival of mice from 35 days (control group), to 60 days. Further experiments to characterize the subpopulations of human PBLs mediating LAK activity, showed that IL-2-activated large granular lymphocytes and T cells were equally effective in mediating antitumor activity. Similar results were reported by Marincola et al. in nude mice with human pancreatic cancer transplanted into the pancreas (Marincola et al., 1989). In the group of mice treated with IL-2 alone, deaths, apparently due to toxicity, were reported in 37% of mice. The side effects were similar to those reported in IL-2/LAK trials in man (Rosenberg et al., 1987). Interestingly, this form of toxicity was not noted in the IL-2/LAK group, the major cause of early mortality in this group being pulmonary embolism at the time of injection of LAK cells. IL-2 alone or LAK cells alone again had no therapeutic effect, but the combination prolonged the survival of mice bearing pancreatic tumor implants. A significant antitumor effect on pancreatic tumor transplanted subcutaneously was also noted with the IL-2/LAK combination. Shimizu et aL investigated IL-2/LAK therapy of subcutaneous human endometrial cancer xenografts. In contrast to the previous two reports, no antitumor effect was noted with IL-2/LAK combination therapy. The addition of lentinan (a polysaccharide with T-cell activating properties isolated from an edible Japanese mushroom) to the IL-2/LAK regime, led to a significant antitumor effect (Shimizu et al., 1989). These three investigations suggest, as did Rosenberg's original work, that for adoptive cellular immunotherapy to work, the concomitant injection of IL-2 and adoptively transferred LAK cells is required for antitumor activity, and can be enhanced by other immunomodulating agents. However, recent data from human trials has shown that prolonged continuous infusion of IL-2 alone may lead to in vivo activation of host antitumor mechanisms in those who respond (West, 1988).

This is also suggested by the work of Bubenik et al. (1988) who transfected the human IL-2 gene into a fibroblast cell line. IL-2 secreting clones were isolated and IL-2 transfectants, or control fibroblasts, were injected peritumorally into nude mice bearing subcutaneous HeLa cell xenografts. The growth ofxeno- grafts was significantly inhibited by the IL-2 transfectants but not the control fibroblasts. Histo- logical examination of the regressing tumors showed intensive infiltration by mononuclear cells of the peritumoral stroma and necrosis of tumor. The nature of the cell populations in the peritumoral areas was not fully defined, but this experiment suggests that LAK activity can be generated in nude mice in vivo.

The role of endogenous IL-2 production in restrict- ing tumor development in vivo was demonstrated by


the study of Saito et al. (1989). Pretreatment of nude mice with IL-2 inhibited the rate of tumor take with the human hepatoma cell line HCC-H, and this effect could be abolished by treatment of mice with anti- asialo GM1 antiserum, suggesting that augmented NK cell activity mediated this effect (Saito et al., 1989). In a rare report of a direct antiproliferative effect of IL-2 on tumor cells, the hormone dependent MCF-7 breast cancer cell line was shown to be growth inhibited in vitro and in vivo by low concen- trations of IL-2 (Paciotti and Tamarkin, 1988).


The foundations for much of the clinical work using IL-2 were laid by the work of Rosenbcrg and colleagues at the National Cancer Institute. They studied the effect of IL-2 alone, or in combination with LAK cells on the development of metastases from both immunogenic and non-immunogenic tumors. They outlined the pharmacokinetics (Dono- hue et al., 1984), defined the toxicity in mice, and determined whether amelioration with agents such as corticosteroids would affect efficacy (Ettinghausen et al., 1985).

In syngeneic murine tumor models concomitant infusion of in vitro IL-2-activated lymphoid cells with IL-2 led to regression of tumors, whilst IL-2 or LAK cells alone did not (Rosenberg, 1986). The majority of experiments using LAK cells and IL-2 involved artificially produced pulmonary and hepatic metastases of a number of different tumor cell lines syngeneic to C57B1/6 mice. Tumor cell colonies in the lungs or liver were visible microscopically 3 days after intravenous tumor cell injection and macroscopically by 10 days. This form of therapy inhibited development of, and destroyed, established micrometastases and led to increased survival time in some experiments. LAK cells needed to proliferate in vivo as well as in vitro, and the efficacy of LAK was directly related to the number of LAK cells transferred, the number of LAK cell infusions, and the dose of IL-2. LAK cells plus IL-2 were more effective than either alone.

Many of the studies involving transplantable tumors used other agents, particularly other cytokines, in combination with IL-2, and theses are discussed below.

3.3.4. Transplantable Tumor Studies and Combi- nations with 1L-2

Many in vivo and clinical studies over recent years have used combinations of cytokines and demonstrated synergistic activity (Truitt et al., 1989).

3.3.4.1. With flavone acetic acid. Renca (a murine renal cell cancer) has been successfully treated with the combination of flavone acetic acid (FAA) and IL-2. Subsequent in vivo experiments demonstrated

that FAA induces the expression of the genes for IFNs-~ and/~ as well as TNF (Sayers et al., 1990).

3.3.4.2. With IFN-ot. The combination of IL-2 and IFN-~ has synergistic effects both in vitro and in vivo, producing tumor responses at non-toxic doses. In an attempt to enhance the antitumor effect of IL-2, the hybrid recombinant protein, human IFN-~A/D, known to be active on murine cells in vitro, was used (Rehberg et al., 1982; Brunda et al., 1986). The combination invariably showed an antitumor effect and the effects were synergistic in the Lewis lung tumor model and M5076 and B16F10 tumors. In the latter two models IL-2 as a single agent was ineffective (Brunda et al., 1987a). The best responses were seen when the two agents were given simultaneously to animals bearing colon 38 tumors (Truitt et al., 1987).

C57/BL6 mice were injected via the tail vein with the weakly immunogenic methylcholanthrene- induced fibrosarcoma MCA-106 and treated with intraperitoneal IL-2 combined with rlFN-gA/D. En- hanced tumor reduction was seen in therapy of both early and advanced pulmonary metastases. This was compared with IL-2 and LAK cells (again both given intraperitoneally) and found to be superior to any of the agents given alone. Treatment of advanced metastases with IFN and IL-2 resulted in early deaths without visible tumor, suggesting toxicity. Efficacy was comparable to the more complex and expensive IL-2/LAK, although there was increased toxicity. Thus IL-2 and IFN-ct appear to act synergistically, producing tumor responses at non-toxic doses. This has important clinical implications (Kim and Warnaka, 1991).

3.3.4.3. With IFN-fl. Combination therapy with recombinant murine IFN-fl and rhlL-2 had a significant antitumor effect on subcutaneous adenocarcinoma-755 and colon-38, although this particular combination was no more effective than rhlL-2 alone against Lewis lung carcinoma in C57BL/6 mice. In the first two tumors, which are sensitive, a marked increase in L3T4+,Lyt-2+ and asialo GMI + cells was noted in the peritoneal cavity, although this was not seen in Lewis lung carcinoma (Iigo et al., 1989).

3.3.4.4. With IL-l- f l . Mice transplanted subcutaneously with Friend erythroleukemia cells (FLC) and treated with peritumoral injection of recombinant IL-l-fl were found to have prolonged survival and inhibition of metastases in liver and spleen (Ciolli et al., 1991). Treatment with IL-2 alone had no effect although the two cytokines in combination were highly effective in both increasing survival time and producing tumor regression. The effect persisted when the primary tumors were removed suggesting independent mechanisms for the combination acting on metastasis and primary tumor progression. The therapy was more effective on established tumors


than early-tumor transplanted mice (in contrast with IFN therapy) and the effect was abolished or markedly reduced by treatment with antibodies to CD4 or CD8, suggesting that these mediate the antitumor effects and not NK cells and the mechanism is distinct from that of IFN-c¢ (Ciolli et aL, 1991). This particular combination has yet to be used in clinical trials.

3.3.5. Clinical Studies with IL -2

injection of a neutralizing monoclonal antibody to IL-4 reversed the non-tumorigenic phenotype of the transfectants. When transfectants and various non- transfectant cell lines were mixed and injected/n vivo, suppression of tumor growth was again seen showing that expression of IL-4 by the tumor cell itself was not necessary. Histologic examination of tumor sites showed that the transfectant induced an inflammatory infiltrate composed of eosinophils and macrophages (Tepper et al., 1989).

Clinical trials using IL-2 alone, or with LAK cells generated by in vitro incubation with IL-2, at first showed encouraging results, particularly in renal cell carcinoma and melanoma (Lotze et al., 1984; Rosenberg et al., 1987).

In humans the toxicity of IL-2 both as a single agent and in combination with LAK or TIL cells or other cytokines is marked, with numerous side-effects which are not easily defined in rodents. Side-effects are abrogated by steroids but these also diminish the antimetastatic activity of IL-2. Attempts have been made to limit this by using continuous infusion therapy in place of bolus treatment but where toxicity is reduced it appears efficacy is also compromised. The principles which apply to IFN therapy, of increased efficacy in minimal residual disease, are also true when considering IL-2 therapy, although here responses have occasionally been documented in bulky, metastatic disease. Many of the trials currently being reported have dismal response rates and the pooled data from one group showed an overall response rate in over 600 patients of only 8% (Dillman et al., 1991), whilst in melanoma another group reported one partial response in 33 patients (Dutcher et al., 1991). Marked toxicity, disappointing clinical trial results, critical editorials in the medical press, inadequate data supporting the use of IL-2 alone and too skeletal an understanding of its relevant mechanisms in malignant disease have meant difficulty in obtaining FDA approval for IL-2 (Dutcher et al., 1991; Quirt and Tannock, 1990).

3.4. INTERLEUKIN-4

Interleukin-4 (IL-4) is a multi-functional cytokine produced by the T m subset of helper T iymphocytes. Its characteristics are described in Tables 3 and 4. It is encoded by a single gene located on the long arm of chromosome 5. There is no cross-species reactivity. The receptor is 50--60 kDa in molecular weight.

3.4. I. Transplantable Tumors

In an assessment of the antitumor potential of IL-4, tumor cells were transfected with the gene for this cytokine (Tepper et al., 1989). The IL-4 transfectants showed greatly decreased tumorigenicity. Re- duced tumor growth correlated with the level of IL-4 production in the various transfected cell lines and

3.4.2. Chemically-induced and Spontaneous Tumors

rlL-4 has been found to trigger host reactivity against a chemically induced fibrosarcoma and against a cell line established from a mammary adenocarcinoma which arose spontaneously in a 20- month-old BALB/cAncr mouse. The cytokine was administered daily subcutaneously around tumor- bearing lymph nodes and as the dose was increased from 0.001 fg to 1 ng/day appreciable inhibition of the growth of both tumors was seen. rlL-4 had no direct antitumor activity but it appeared that host immune reactivity was of fundamental importance in this lymphokine-activated tumor inhibition. The effect was abolished by sublethal irradiation, treatment with cyclosporin A, and suppression of CD4 + lymphocytes. This rlL-4 induced tumor inhibition appeared to depend on the recruitment of several intercellular mechanisms, rlL-4 tumor inhibition re- suited in a state of long-lasting and specific immune memory with the growth of a second contralateral tumor being significantly impaired, rlL-4 had greater ability to trigger lymphokine-activated tumor inhibition than most effective doses of rlL-2, r lL-1/ /and IFN-~ in both the poorly immunogenic CE-2 fibrosarcoma and the TS/A adenocarcinoma (Bosco et aL, 1990).

3.5. INTERLEUKIN-6

Interleukin-6 (IL-6) has a molecular weight of 26 kDa. The gene is on the short arm of chromosome seven and there is an 80 kDa receptor. Human IL-6 is effective in mice but the converse is not true. Its properties are described in Table 3.

The capacity of several recombinant cytokines to induce IL-6 in vivo in normal and tumor-bearing (TB) mice was investigated by McIntosh et al. (1989a) using a number of different carcinogen induced tumors in syngeneic B6 mice. IL-6 was induced by a number of recombinant human cytokines, including TNF, IL-2 and IFN-~A/D as well as rmIFN-~. rhTNF was the most potent inducer and TB animals consistently produced more IL-6 than non-TB mice. rhIL-2, rh IFN-aA/D and rmIFN-7 induced comparable levels in non-TB and TB mice. In untreated TB mice levels of IL-6 were proportional to the level of tumor burden in all four tumor types. IL-6 was at no time measurable in the sera of untreated normal mice.


The role of de novo IL-6 in tumor-bearing animals is uncertain but it is known to have immune and metabolic effects which may be important in host response to malignancy.

Purified human rlL-6 given alone at relatively high doses, comparable to therapeutic levels of IL-2, was found to mediate substantial reductions in the number of pulmonary and hepatic micrometastases from four distinct syngeneic tumors (Mule et al., 1990). The IL-6 therapy resulted in no observable toxicity, (including no altered wet lung weights) nor death of the treated mice at the dose regimens used. Sublethal total body irradiation before treatment prevented the IL-6 antitumor effect suggesting that it acts via a radiosensitive host component and not directly on the tumor itself. In addition, the combination of IL-6 with TNF administered to mice with an established weakly immunogenic, syngeneic tumor at a subcutaneous site, resulted in marked tumor regression and cure rates. This study was the first demonstration of tumor regression mediated by recombinant IL-6 in vivo (Mule et al., 1990).

Castleman's disease is a syndrome of lymph node hyperplasia with plasma cell infiltration, hyper- gammaglobulinemia and an increase in serum level of acute phase proteins. B cells in the germinal centers of hyperplastic lymph nodes found in the disorder generate IL-6 (Yoshikazi et al., 1989). A high titer recombinant retroviral vector was used to introduce the coding sequences of murine IL-6 into mouse hematopoietic cells. Congenitally anemic W/Wv mice were reconstituted with bone marrow cells trans- duced with the retroviral vector. They developed a syndrome characterised by anemia, granulocytosis, hypoalbuminemia and polyclonal hypergamma- globulinemia, with marked splenomegaly and peripheral lymphadenopathy. These findings are very similar to those described in multicentric Castleman's disease and suggest that inappropriate synthesis of IL-6 has an important role in the pathogenesis of this disorder (Brandt et al., 1989).

A transgenic mouse which overexpresses IL-6 was found to have an IgG1 plasmacytosis, again suggesting the role of this cytokine in B cell development (Suematsu et al., 1989).

3.5.1. Clinical Correlates

The in vivo work undertaken by Rosenberg's group (Mclntosh et al., 1989a) is paralleled by the finding that cancer patients treated with high dose recombinant IL-2 had significantly raised levels of mRNA for IL-6 in their circulating PBMCs (Kasid et al., 1989).

The potential role of IL-6 as an autocrine growth factor for human multiple myeloma (Kawano et al., 1988) raises the possibility that agonists or antibodies to IL-6 may be useful to treat the condition. Of those cytokines released during an immune response, IL-I, TNF~ and IL-6 seem to be the major mediators of intermediary metabolism. They act together to de-

crease food intake, increase resting energy expendi- ture and alter metabolism (Klasing, 1988). IL-6 is another cytokine which allows communication between stromal cells and the immune system. It is a potent growth factor for certain cells and may play an autocrine role in the evolution of multiple myeloma. As yet there is little clinical evidence that it has growth inhibitory or antitumor activity in other cancers, but as its production is regulated by IL-1 and TNF such properties would be expected.

3.6. TUMOR NECROSIS FACTOR

Tumor Necrosis Factor (TNF) and lymphotoxin (LT) are cytokines with a central role in immunity and inflammation. TNF exists biologically as a trimer consisting of 17kDa non-glycosylated polypeptide subunits (Wingfield et al., 1987). The term TNF was first coined to describe a macrophage derived protein induced by endotoxin administration in BALB/c mice primed with BCG. In 1975 Carswell demonstrated induction of hemorrhagic necrosis in the Meth A sarcoma model (Carswell et al., 1975). By 1984 human TNF had been cloned and sequenced (Pennica et al., 1984) since which time production of the recombinant form has been possible. A macrophage derived protein was characterized by Cerami and his coworkers on the basis of its ability to suppress lipoprotein lipase activity in mice and in 3T3-L1 adipocytes in vitro (Kawakami and Cerami, 1981). The protein responsible for lipoprotein lipase suppression was purified from a murine macrophage cell line (RAW264.7), and shown to be identical to TNF (Beutler et al., 1985). Lymphotoxin, otherwise known as TNF-fl, is released by T lymphocytes in delayed type hypersensitivity reactions and was shown to have antitumor activity in vitro by Granger and Williams (1968). In humans the TNF and LT genes are closely linked and located on the short arm of chromosome 6. A single class of high affinity receptor is shared by LT and TNF and has been found on a variety of normal and malignant cells (Kull et al., 1985; Aggarwal et al., 1985, 1987). The physiological role of TNF is still uncertain, although inappropriate production has been implicated in the pathophysiology of infectious and autoimmune dis- eases, inflammation and cancer (Fiers, 1991).

The antitumor activity of TNF has been studied in experimental murine tumor models and in nude mice bearing human tumor xenografts. It has direct antiproliferative effects on tumor cells and can also be an essential mediator and activator of other cellular antitumor mechanisms. The activation of tumoricidal macrophages (Hori et al., 1987), enhancement of NK and natural cytotoxic cell activity (Ortaldo et al., 1986), cytotoxic T cell activity (Nakano et aL, 1989) and activation of neutrophils (Shau, 1988a,b) can all be mediated by TNF. The experimental data suggests that TNF has dose-dependent growth inhibitory effects if given intratumorally. The cytotoxic action of


TNF or LT is independent of protein synthesis and there are a number of pathways whereby cells can be killed: Activation of phospholipases, induction of proteases, and DNA damage.

3.6.1. TNF and Cachexia

The first experiments to suggest that TNF had properties other than antitumor activity were studies on cachexia in rabbits with chronic parasitic infections (Trypanosoma bruceii). The mechanisms responsible for decreased serum albumin levels in patients with cachexia-associated infection, inflammation and cancer are unknown. However the associ- ation of an elevated TNF in the presence of cachexia led Brenner et al. to assess the regulation of albumin gene expression by TNF in vivo. They transfected chinese hamster ovary (CHO) cells with the functional gene for TNF and inoculated these into nude mice as a model of cachexia. The animals subsequently manifested decreased serum albumin levels, albumin synthesis and albumin mRNA levels. Indeed albumin mRNA levels had decreased by about 90% before weight loss was evident, which was not the case in controls. The mRNA levels of several other genes were not altered and it appears the TNF-c~ selectively inhibits the expression of albumin in this model before weight loss (Brenner et al., 1990).

The influence of TNF and IL-1 on experimental cancer cachexia was investigated with a transplantable methylcholanthrene induced sarcoma in C57BL/6J mice. The mouse was passively immunized on alternate days with antibodies to TNF or the IL-1 receptor. Both antibodies inhibited tumor growth significantly as assessed by tumor weight and food intake was improved in tumor-bearing immunized animals. The combination of the two antigens had no additive effects suggesting that they act through a common mechanism. It appears that both these cytokines are involved in cancer cachexia and that in this model they also promote tumor growth (Gelin et al., 1991).

The development of cachexia was investigated in rats given intermittent intravenous boluses of rhTNF or slow continuous infusion. This reduced food intake of treated rats when compared with saline treated rats. The group given intermittent rhTNF developed tolerance to these effects at four days unlike those given a continuous infusion. Of the continuously infused mice fifty-six percent were dead within eight days. It appears that slow continuous secretion of sublethal amounts of TNF may mediate the cachexia of cancer (Darling et al., 1990).

Treatment of tumor-bearing rats (which are more sensitive to the effects of TNF than non-tumor- bearing rats) with repeated exposure to rhTNF resulted in tolerance and ameliorated cachexia. Non- tumor-bearing (NTB) rats were treated with TNF intraperitoneally and developed tolerance to the cachectic effects of TNF. TB rats given the same

repeated exposure showed less weight loss and a relative increase in food intake compared with saline treated controls. This same amelioration was seen in the antineoplastic effects of TNF, due to the development of tolerance, as evidenced by increased survival (Sheppard et al., 1990).

In a similar study in mice NTB mice were treated with escalating doses of intraperitoneal TNF and then challenged with a lethal dose of TNF two days later. All the pre-treated mice survived whereas all of the control mice, who had not been pretreated, died. This method was then used to treat TB mice but the ability to administer higher doses of TNF intravenously to TNF pretreated tumor bearing mice did not improve therapeutic efficacy. The results suggest that TNF tolerance occurs in TB mice and reduces both toxicity and therapeutic efficacy (Fraker et al., 1990).

3.6.2. Nude Mice and Murine Tumors

Constitutive production of small amounts of TNF by neoplastic cells affects their in vivo tumorigenicity. TNF-resistant derivatives were isolated from the TNF-sensitive murine fibrosarcoma cell lines L929s and WEHII64c113s. Some TNF-resistant subclones were found to produce TNF constitutively in vitro whereas other TNF-resistant subclones did not. TNF-sensitive and non-producing resistant cell lines produced fast-growing tumors on subcutaneous inoculation into nude mice whereas TNF-producing cells had reduced tumorigenicity. Prior to whole body y irradiation of the recipient facilitated increased tumor take but not in vivo growth rate of the cell lines. This suggests involvement of host mechanisms in the tumor implantation. The mechanisms appeared to act only locally and histological examination re- vealed that tumors induced by TNF-resistant, TNF- producing subclones did not show invasiveness in host tissues and were frequently encapsulated. This was in contrast with the cell lines which did not produce TNF, which were invasive (Vanhaesebroeck et al., 1991).

In contrast to the studies described above Chinese hamster ovary (CHO) cells transfected with the gene for human TNF showed a greatly enhanced ability to invade peritoneal surfaces and metastasize in nude mice compared with control cells containing the vector alone. In situ hybridization confirmed that the CHO/TNF cells were transcribing TNF after in vivo injection and their enhanced metastatic activity was abrogated by neutralizing antibodies to human TNF. This study suggests that a cytokine gene can confer a metastatic phenotype on the recipient cell (Malik et al., 1990).

3.6.3. Nude Mouse and Human Tumor Xenografts

One disadvantage in assessing the role of TNF in human tumor xenografts growing on nude mice is


that the contribution of the T-cell response cannot be assessed. In the syngeneic meth-A sarcoma model TNF causes hemorrhagic necrosis of the tumor, but this effect is greatly decreased in nude mice, suggesting a requirement for T-cells in the predomi- nantly vascular damage seen (Haranaka et al., 1984). This has been confirmed by adoptive transfer experiments (Palladino et al., 1987). Similarly the TNF induced red-cell influx in the murine SAI fibrosarcoma is less pronounced in T-cell deficient mice (Havell et al., 1988).

The antitumor efficacy of local/Iocoregional administration of TNF against subcutaneous human tumor xenografts (Balkwill et al., 1986), led to the study of the antitumor effects of intracavitary TNF administration, particularly in intraperitoneal human ovarian cancer xenograft models (Balkwill et al., 1987c; Creasey et al., 1986; Rubin et al., 1989). Intraperitoneal therapy of three intraperitoneal human ovarian cancer xenografts with rhTNF significantly prolonged the survival of nude mice bearing two of the three tumors (Balkwill et al., 1987c). In other studies TNF therapy cured nude mice of intraperitoneal cancer when administered intraperitoneally but not intravenously (Creasey et al., 1986).

In spite of the positive survival effect rhTNF was found to have against human ovarian cancer xenografts growing intraperitoneally in nude mice, it promoted adhesion of tumor cells to the peritoneum and the establishment of tumor nodules beneath the mesothelial surface (Malik et al., 1989).

Although the effect of TNF on tumor vasculature in the nude-mouse human tumor xenograft models has not been so extensively studied as in murine models the phenomenon of hemorrhagic necrosis appears to be less pronounced.


The most widely used tumor for the study of the antitumor effects of TNF is the murine Meth A sarcoma model (Carswell et al., 1975; Haranaka et al., 1984; Palladino et aL, 1987). Hemorrhagic necrosis is induced within 24 hr of administration of TNF serum or rTNF in a dose-dependent fashion (Carswell et al., 1975). Early and intraperitoneal tumors are resistant to the effects of TNF suggesting that the development of tumor vasculature, or a host immune response to the tumor, are essential to hemorrhagic necrosis (Manda et al., 1987; Palladino et al., 1987).

The evidence that TNF is therapeutic in vivo is based not on the ability of TNF to cause tumor regression in vivo but on its ability to cause hemorrhagic necrosis of the centres of established tumors. TNF-induced intratumor hemorrhaging was quantified by measuring the intratumor extravasation of 51Cr-labelled syngeneic red cells against time. The mice were injected intravenously with TNF and at particular time intervals afterwards killed and the

tumors excised. It was demonstrated that the therapeutic action of TNF against established immunogenic sarcoma does not depend on the ability of TNF to destroy tumor cells /n vivo but on its ability to destroy, directly or indirectly, the tumor's vasculature. Thereby destruction of most of the tumor center results from ischemia (Havell et al., 1988).

North and Havell have shown that the endotoxin- induced reaction seen in the SAI sarcoma model is associated with intratumor production of TNF. This endotoxin-induced hemorrhagic reaction and subsequent complete tumor regression can be inhibited by infusing the host with an adequate quantity of an antibody capable of neutralizing the antitumor activity of TNF. It appears that endotoxin-induced regression of the ring of tumor surrounding the hemorrhagic reaction, unlike the hemorrhagic necrosis itself, is dependent on host immunocompetence (North and Havell, 1989).

Intravenous TNF led to a rapid decrease in tumor blood flow in murine colon 26 tumors (Bibby et al., 1989). Following administration of flavone acetic acid (FAA) endogenous TNF was produced and a similar effect was seen (Mahadevan et al., 1990). With a radioactive tracer and a clearance technique, FAA was given intraperitoneally and led to progressive and sustained decrease in blood flow in colon 26 grown subcutaneously in syngeneic mice. One hour after administration an increase in the half-life clearance value was seen for intratumoral ~33Xe which peaked at 3 hr. This finding is abolished in tumor-bearing mice pretreated with antiTNF antiserum compared with controls. In vitro FAA induces TNF secretion from murine peritoneal cells and splenocytes, which suggests that in this model tumor vasculature shutdown induced by FAA is mediated by TNF (Mahadevan et al., 1990). Data from many of these murine tumor models suggests that selective damage to tumor vasculature is the mechanism whereby the antitumor effect of TNF is mediated (Gerlach et al., 1989).

Mclntosh et al. (1990) studied the effects of TNF on primary autochthonous sarcomas induced in C57B1/6 mice by 3-methylcholanthrene; on spontaneous mammary tumors in C3H/HEN mammary tumor virus positive mice; and on the rejection of normal tissue transplants at different stages of matu- rity, in C57B1/6 mice. The tumors underwent necrosis with a neutrophil infiltrate, and reduced in size, but no effect was observed on vascularized normal tissue transplants.

3.6.5. Combination Therapy

3.6.5.1. With IFNs. The nitrosomethylurea-induced rat mammary tumor model was used to study the effects of parenteral rhTNF and rat IFN-~. Com- bined treatment was found to induce significant tumor regression over 4 weeks whereas individual

JP'T 52/3--E


treatment did not affect the overall rate of tumor growth, although there was an initial reduction in size. It was concluded that this combination may therefore represent a useful form of treatment for human breast cancer (Shah et al., 1989).

The in rive antitumor effects of natural hTNF and natural hlFN-~ were studied in nude mice with established intraabdominal tumor from a human tumor cell line RPMI 4788. Treatment with the two cytokines in combination significantly prolonged survival of the nude mice and complete regression was seen in five different human tumor xenografts given intratumoral hTNF and hlFN-~. No significant signs of toxicity were seen and the results suggest that the synergism may allow treatment at a relatively low dose range (Naomoto et al., 1989).

The combination of mlFN<t/fl and natural hTNF was used to prevent the development of hepatic metastases in a colon 26 murine hepatic metastasis model. The inhibitory effects of the two cytokines were found to be synergistic with significant augmentation of NK activity (Sanada et al., 1990).

3.6.5.2. With IL-2. Combination therapy utilizing rhTNF-~, rhlL-2 and rh lFN-~A/D was assessed against weakly (MCA-106) and non-immunogenic (MCA-102) sarcomas at subcutaneous and visceral sites. In the MCA-106 sarcoma the combination of all three cytokines together gave substantial improve- ments in cure rates, tumor regression, survival pro- longation compared with any other cytokine or combination, in both visceral and subcutaneous sites. No antitumor effect was seen with the nonimmunogenic MCA-102 tumor. Various synergistic mechanisms have been hypothesized, and this work, although using a tumor markedly different from a human tumor, nevertheless suggests possible combinations for clinical trials (Mclntosh et al., 1989b). In combination with IL-2, TNF completely inhibited subcutaneous tumors although either cytokine alone had little activity. Maximal antitumor effects were seen at the maximal tolerated dose (MTD) of TNF, although the IL-2 dose could be reduced by as much as 90% of the MTD (Winkelhake et al., 1989). Most in vivo work showing synergy between IL-2 and TNF has demonstrated an immunological component, in agreement with the absence of antitumor activity of IL-2 in nude mice bearing murine tumors.

Recombinant IL-6 and TNF act synergistically against several murine tumors, which is of particular interest since this cytokine is induced by TNF in rive (Fiers, 1991).

TNF kills cells when combined with IFN-? even when they are not susceptible to either agent in isolation. The mechanism has yet to be established but is probably not related to the upregulation of TNF receptor expression by IFN-?. In a phase I clinical trial 36 patients with advanced cancer were treated with overlapping continuous intravenous in-

fusions. They received a fixed dose of IFN- 7 with TNF escalation between patients. The dose-limiting toxicity at the maximal tolerated dose of TNF (205/zg/m z) with IFN-~ (at doses from 100-250/~g/ m2/day) was hypotension. The addition of IFN-~, to TNF produced a greater than 3-fold increase in toxicity, suggesting again that these cytokines act synergistically in vivo (Demetri et al., 1989).

3.6.6. Clinical Correlates

, One century ago William Coley, a New York surgeon, on looking back through case notes found documentation of complete regression of an inoper- able sarcoma in a patient after two episodes of erysipelas (Coley, 1893). He subsequently attempted to reproduce the infection in patients, with some succumbing to the infection. He had greater success with a mixed filtrate of erysipelas cultures and Bacil- lus prodigiosus. This preparation became known as Coley's mixed toxins and over the subsequent 40 years was used on over 1200 patients. The work was reviewed by his daughter who concluded that more than 270 patients had achieved complete regression of their tumors (Coley Nauts et al., 1953). The principle active agent was thought to be endotoxin (bacterial lipopolysaccharide) which induces endogenous TNF (North and Havell, 1989) and LT as well as IL-I and other cytokines. Other inducers of cytokines are also likely to have been present. In experimental animal models, tumor necrosis induced by bacterial endotoxin was thought to be related to the effects seen in humans. Extrapolating from murine tumors to the clinical use of TNF has been questioned because of the immunogenicity of chemically induced tumors like the Meth A sarcoma. The antitumor effects of TNF were significantly greater in mice with immunogenic tumors than those of low immunogenicity (Asher et al., 1987).

In clinical trials the toxicity of rhTNF has been severe and as a single agent it has had disappointing results. A study was carried out to determine its maximum tolerated dose, pharmacokinetics and anticancer effect on 18 patients with advanced cancer (Selby et al., 1987). Dose levels from 9 × 103 units m 2 with incremental dose escalations to 1.2 × 106 were used. Patients were very carefully monitored and TNF levels in serum measured by an ELISA method. One third of patients experienced febrile symptoms, which were not related to dose in incidence or severity. Hypotension, abnormal liver enzymes and a neutrophil leucocytosis were all dose-related. Objec- tive disease response was seen in only 3 patients--all three were partial responses in patients with lymphomas. No evidence of elevated endogenous TNF was found in patients with malignant disease (Balkwill et al., 1987a). A further Phase II study in 18 patients showed no responses and it was later established that inadequate doses had been administered (Frei and Spriggs, 1989). The dose-response depen-


dence is currently limited by the severe toxicity in man and the fact that any patients selected for trials are heavily pretreated and are likely to have been exposed to endogenous TNF in the course of infectious complications of therapy. Intratumoral injection of rhTNF has shown response rates of 25-40% which is closer to the more promising findings in human tumor xenografts where intraperitoneal and intratumoral injection is more straightforward (Balkwill et al., 1986). A recent study demonstrated disappearance of malignant ascites in 20 out of 26 patients with advanced cancer, although there was no decrease in tumor volume (Raeth et al., 1991).

Sixteen patients with colorectal carcinoma were treated with i.v. bolus infusions of TNF. Compli- cations were severe, with more hepatotoxicity than had been previously documented and retinal vein thrombosis as a late event. No responses were seen in 14 evaluable patients (Kemeny et al., 1990).

In a Phase I trial of 53 patients with advanced malignancies dexamethasone was given to ameliorate toxicity without significant differences in symptoma- tology. One partial response lasting nine months was seen in a patient with colorectal cancer (Schiller et al.,

1991). As yet the place of TNF as a therapeutic agent in the treatment of malignancy is limited, however it may have a role in a few specific indications which appear to include locoregional therapy.

In vivo production of TNF in tumors has been studied using in situ hybridization. Local expression of the TNF gene has been demonstrated in 35 of 68 ovarian tumors studied. A minority of cells in the epithelial areas of the tumor contained TNF mRNA (M. S. Naylor, personal communication). From im- munohistochemistry and morphological studies it appeared that the tumor cells were transcribing the TNF gene and TNF protein was detected in a tumor lysate. The production of TNF may influence the biology of the tumor, contribute to neoplastic progression and have important implications for therapy (Naylor et al., 1990).

4. SUMMARY

The now vast body of knowledge about cytokines has largely been obtained from a combination of in vitro and in vivo work. These proteins are an integral part of the immune system and as such appear to have a central role in the prevention and development of malignancy. They are found in a wide variety of hematological and solid tumors and by defining their mechanisms of action more precisely, through the use of a range of animal tumor models, we are more likely to understand the processes involved in cancer and thereby both prevent and treat it.

REFERENCES

AC~ARWAL, B. B., EESSALO, T. E. and HAgs, P. E. (1985) Characterisation of receptors for human tumour necrosis

factor and their regulation by interferon-y. Nature 318: 665-667.

AGGARWAL, B. B., AYER, R. A., PENNlCA, D., GRAY, P. W. and GOF.DDEL, D. V. (1987) Human tumour necrosis factors: Structure and receptor interactions. In: Tumour Necrosis Factor and Related Cytotoxins, CIBA Foun- dation Symposium, No. 131, pp. 39-51. John Wiley and Sons, Chichester.

AGUET, M. and MOGENSEN, K. E. (1983) Interferon receptors. In Interferon, pp. 1-22, Gresser, I. (ed.) Academic Press, London.

ALLAVENA, P., PECCATORI, F., MAGGIONI, D., ERROI, A., SIRONI, M., COLOMBO, N., LISSONl, A., GALAZKA, A., METERS, W. and MANOIONI, C. (1990) Intraperitoneal recombinant gamma interferon in patients with recurrent ascitic ovarian carcinoma: Modulation of cytotoxicity and cytokine production in tumour-associated effectors of major histocompatibility antigen expression on tumor ceils. Cancer Res..SO: 7318-7323.

ASHER, A., MOLE, J. J., REICrIEgT, M. M., SmLONI, E. and ROSENBERG, S. A. (1987) Studies on the antitumour efficacy of systemically administered recombinant tumour necrosis factor against several murine tumours in vivo. J. Immunol. 138: 963-974.

BACCARINI, M., FANIN, R., FASOLA, G. and GALLIZiA, C. (1989) Maintenance treatment of multiple myeloma. Eur. J. Haematol. Suppl. 51: 145-151.

BALKWILL, F. R. (1985) The regulatory role of IFNs in the human immune response. In: IFNs and Impact in Biology and Medicine, Taylor-Papadimitriou, J. (ed.) Oxford, Oxford Medical Publications.

BALKWILL, F. R. and MOODIE, E. M. (1984) Positive interactions between human IFN and cyclophosphamide or adriamycin on a human model system. Cancer Res. 44: 904-908.

BALKWlLL, F. R. and PRO1ETTI, E. (1986) Effects of mouse IFN on human tumour xenografts in the nude mouse host. Int..L Cancer 38: 375-380.

BALKWILL, F. R., TAYLOR-PAPADIMITRIOU, J., FANTES, K. H. and SEBESTENY, A. (1980) Human lymphoblastoid interferon can inhibit the growth of human breast cancer xenografts in athymic (nude) mice. Fur. J. Cancer 16: 569-573.

BALKWILL, F. R., MOOOIE, E. M., FREEDMAN, V. and FANTES, K. H. (1982) Human IFN inhibits the growth of established human breast tumours in the nude mouse. Int..L Cancer 30: 231-235.

BALKWILL, F. R., GOLDSTEIN, L. and STEBBING, N. (1985) Differential action of six human IFNs against two human carcinomas growing in nude mice. Int..I. Cancer 35: 613-617.

BALKWILL, F. R., LEE, A., ALDAM, G., MOOoIE, E., THOMAS, J. A., TAVERNmR, J. and FmRS, W. (1986) Human tumour xenografts treated wtih recombinant human tumour necrosis factor alone or in combination with interferons. Cancer Res. 46: 3990-3993.

BALKWILL, F., OSBORNE, R., BURKE, F., NAYLOR, S., TALBOT, D., DURmN, H., TAVERNtER, J. and FtERs, W. (1987a) Evidence for tumour necrosis factor/cachectin production in cancer. Lancet ii: 1229--1232.

BALKWILL, F. R., STEVENS, M. H., GRIFFIN, D. B., THOMAS, J. A. and BODMER, J. G. (1987b) IFN gamma regulates HLA-DR expression on solid tumours /n vivo. Eur. ,L Cancer clin. Oncol. 23: 101-106.

BALKWILL, F. R., WARD, B. G., MOODIE, E. and FmRs, W. (19870 Therapeutic potential of turnout necrosis factor alpha and gamma IFN in experimental human ovarian cancer. Cancer Res. 4"/: 4755-4758.

BALKWILL, F. R., GRIFFIN, D. B. and LEE, A. E. (1989) IFNs alpha and gamma differ in their ability to cause tumour stasis and regression /n vivo. Fur..L Cancer 25: 1481-1486.


BEART, R. W. JR, MOERTEL, ~ .G . , WIEAND, H. S., LEIGH, J. E., WINDSCmTL, H. E., ~¢AN-HEEgDEN, J. A., FITZ- GIBBONS, R. J. JR and WOLFF, B. G. (1990) Adjuvant therapy for resectable colorectal carcinoma with fluorouracil administered by portal vein infusion. A study of the Mayo Clinic and the North Central Cancer Treatment Group. Arch. Surg. 125: 897-901.

BELARDELLI, F., PROETTI, E., CIOLLI, V., SESTILI, P., CAgPINELLI, G., DI VITO, M., FEgRETO, A., WOODROW, D., BOgASCHI, D. and PODO, F. (1990) Interleukin- 1 fl induces tumour necrosis and early morphologic and metabolic changes in transplantable mouse turnouts. Similarities with the anti-tumour effects of tumour necrosis factor or ft. Int. J. Cancer 44: 116-123.

BEUTLER, B., MILSARK, I. W. and CERAMI, A. C. (1985) Identify of tumour necrosis factor and the macrophage secreted factor cachectin. Nature 319: 516-518.

BmBY, M. C., DOUBLE, J. A., LOADMAN, P. M. and DUKE, C. V. (1989) Reduction of tumour blood flow by flavone acetic acid: A possible component of therapy. J N C I 81: 2 ! 6-220.

BIRSlC, W., D'ORo, L., CHAROENSIRI, S. and KATOH, A. (1988) The combined effect of IFN and 5 F-U on tumour cell metastasis in the nude mouse. Dis. Col. Rectum 32: 340-343.

BOSCO, M., GIOVARELLI, M., FORNI, M., MODESTI, A., SCARPA, S., MASU~LLI, L. and FORNI, G. (1990) Low doses of IL-4 injected prelymphatically in tumour-bearing mice inhibit the growth of poorly and apparently non-immune- genie tumors and induce a tumor-specific immune memory. J. Immunol. 145: 3136-3143.

BRADLEY, N. J., DARLING, J. L., OKTAR, N., BLOOM, H. J., THOMAS, D. G. and DAVIES, A. J. (1983) The failure of human leucocyte interferon to influence the growth of human glioma cell populations: In vitro and in vide studies. Br. J. Cancer 48: 819-825.

BRANDT, S. J., BODINE, D. M., DUNBAR, C. E. and NIENHUIS, A. W. (1989) Dysregulated interleukin-6 expression pro- duces a syndrome resembling Castleman's disease in mice. J. Clin. Invest. 86: 592-599.

BRAUNSCHWEIGER, P. G., JOHNSON, C. S., KUMAR, N., ORD, V. and FURMANSKI, P. (1988) Antitumor effects of recombinant human interleukin I alpha in RIF-1 and Pane02 solid tumors. Cancer Res. 48:6011-6016.

BRENNER, D. A., BUCK, M., FEITELBERG, S. P. and CHOJKIER, D. J. (1990) Tumour necrosis alpha inhibits albumin gene expression in a routine model of cachexia. J. clin. Invest. 85: 248-255.

BROglO, O., BAUER, H. C. F., BROSTROM, L. A., NILLSONNE, U., NILSON, O. S., REINHOLT, F. P., STRANDER, H. and TRIBUKAIT, B. (1985) Influence of human alpha interferon on four human osteosarcoma xenografts in nude mice. Cancer Res. 45: 5598-5562.

BROglO, O., BAUER, H. C. F., BROSTROM, L. A., NILLSON, O. S., REINHOLT, F. P. and TRIBUKAIT, B. (1987) Growth inhibition of human osteosarcomas in nude mice by human IFN alpha: Significance of dose and tumour differentiation. Cancer Res. 47: 258-262.

BROgIO, O., BAUER, H. C., NILLSON, O. S., HALVORSEN, D., REINHOLT, F. P. and TRIBUKAIT, B. (1989) Effect of human IFN alpha and IFN gamma on growth, histology and DNA content of human osteosarcomas in nude mice. J. IFN Res. 9: 475-489.

BRUNDA, M. J., TARNOWSKI, D. and DAVATELIS, V. (1986) Interaction of recombinant IFNs with recombinant interleukin-2: Differential effects on natural killer cell activity and interleukin-2 activated killer cells. Int. J. Cancer 37: 787-793.

BRUNDA, M. J., BELLANTONI, D. and SULICH, V. (1987a) In vide antitumour activity of combinations of IFN alpha and interleukin-2 in a murine model. Correlation of efficacy with the induction of cytotoxic

cells resembling natural killer cells. Int. J. Cancer 40: 365-371.

BRUNDA, M. J., SULICH, V. and BELLANTONI, D. (1987b) The anti-tumour effect of recombinant IFN alpha or gamma is influenced by tumour location. Int. J. Cancer 40: 807-810.

BUnENIK, J., VOI~NOK, N. N., KmLER, J., PRASSOLOV, V. S., CHUMAKOV, P. M., BUBENIKOVA, D., SIMOVA, J. and JANDLOVA, T. (1988) Local administration of cells containing an inserted IL-2 gene and producing IL-2 inhibits growth of human tumours in nu/nu mice. Immunol. Lett. 19: 279-282.

CARMICHAEL, J., FERGUSSON, R. J., WOLF, C. R., BALKWILL, F. R. and SMYTH, J. F. (1986) Augmentation of cytotoxicity of chemotherapy by human alpha IFNs in human non-small cell lung cancer xenografts. Cancer Res. 46: 4916-4920.

CAR.SWELL, E. A., OLD, L. J., KASSEL, R. J., GREEN, S., FIORE, N. and WILLIAMSON, B. (1975) An endotoxin-induced serum factor that causes necrosis of tumours. P N A S U.S.A. 72: 3666-3670.

CHIRIGOS, M. A. and PEARSON, J. W. (1973) Cure of murine leukaemia with drug and IFN treatment. J. natn. Cancer Inst. 51: 1367-1368.

CIOLLI, V., GABRIELE, L., SESTILI, P., VARANO, F., PROIETI'I, E., GRESSER, 1., TESTA, U., MONTESORO, E., BULGARIN1, D., MARIANI, G., PESCHLE, C. and BELAgOELLI, P. (1991) Combined interleukin 1/interleukin 2 therapy of mice injected with highly metastatic Friend leukaemia cells: Host antitumour mechanisms and marked effects on established metastases. J. exp. Med. 173: 313-322.

COLEY, W. B. (1893) The treatment of malignant tumours by repeated inoculations of erysipelas: With a report of ten original cases. Am. J. med. Sci. 105:48%511.

COLEY NAUTS, H., FOWLER, G. A. and BOGATKO, F. H. (1953) A review of the influence of bacterial infections and of bacterial products (ColBy's Toxins) on malignant tumors in man. Acta Med. Scand. (suppl) 274-7: 29-97.

CRANE, J. L., GLASGOW, L. A., KERN, E. R. and YOUNGNER, J. S. (1978) Inhibition of murine osteogenic sarcomas by treatment with Type I or Type II interferon. J N C I 61: 871-874.

CREASEY, A., REYNOLDS, M. T. and LAIRD, W. (1986) Cures and partial regression of murine and human tumours by recombinant human tumour necrosis factor. Cancer Res. 46: 5687-5690.

CRUM, E. D. and KAPLAN, D. R. (1991) In vide activity of solid phase interleukin-2. Cancer Res. 51: 875-879.

DARLING, G., FRAKER, D. L., JENSEN, J. C., GORSCI-IBOTH, C. M. and NORTON, J. A. (1990) Cachetic effects of recombinant human tumour necrosis factor in rats. Cancer Res. 50: 4008-4013.

DE CLERCQ, E., GEORGIADF.S, J., EDYS, V. J. and Sores, H. (1982) Effect of human and mouse interferon and of polyriboinosinic acid and polyribocytidylic acid on the growth of human fibrosarcoma and melanoma tumours in nude mice. Fur. J. Cancer din. Oncol. 11: 1273-1276.

DE FILLn'I, P., POUVART, P., TAWRNmR, J., FmRS, W. and CON~N% J. (1987) Induction and regulation of mRNA encoding 26 kDa protein in human cell lines treated with recombinant human tumour necrosis factor. P N A S U.S.A. 84: 4557-4561.

DE I~ MAZA, L. M. and I~gSON, E. M. (1988) Dependence of the in vitro antiproliferative activity of recombinant human gamma IFN on the concentration of tryptophan in culture media. Cancer Res. 48: 346-350.

DEMETRI, G. D., SPmGGS, D. R., SHERMAN, M. L., AR~UR, K. A., IMAMVgA, K. and Kta~, D. W. (1989) A phase I trial of recombinant human tumour necrosis factor and IFN-g: Effects of combination cytokine administration in vide. J. din. Oncol. 7: 1545--1553.


DENT, L. A., STRATH, M., MELLOR, A. L. and SANDERSON, C. J. (1990) Eosinophilia in transgenic mice expressing interlenkin-5. J. exp. Med. 172: 1425-1431.

DDKMANS, R. and BILL1AU, A. (1985) An introduction to the genes of the IFN system. In: IFNs: Their Impact in Biology and Medicine, pp. 1-18, TAYLOR-PAPADIMITRIOU (ed.) Oxford Medical Publications, Oxford.

DILLMAN, R. O., CHURCH, C., BARTH, N. M., OLDHAM, R. K., SCHWATZBERG, L., ARNOLD, J., BIRCH, R. and W~ST, W. H. (1991) High-dose continuous infusion IL-2 in 693

cancer patients---experience of the national biotherapy study group (NBSG). In: Proceedings of the American Society of Clinical Oncology, Houston, TX., 19-21 May, 1991. p. 212, PERRY, M. C. (ed.), ASCO, Philadelphia.

DoNomm, J. H., LOTZE, M. M. T., ROBB, R. J., ROSENSTEIN, M., BRAZEL, R. M., JAFFE, C. S. and ROSENBERG, S. A. (1984) In vivo administration of purified Jurkat-derived interleukin-2 in mice. Cancer Res. 44: 1380-1386.

DUTCHER, J. P., (JAYNOR, E. R., BOLDT, D. H., DOROSHOW, J. H., BAR, M. H., SZNOL, M., MIER, J., SPARANO, J., FISHER, R. I., WEns, G., MARGOLIN, K., ARONSON, F. R., HAWKINS, M. and ATKINS, M. (1991) A phase II study of high-dose continuous infusion interleukin-2 with lymphokine-activated killer cells in patients with metastatic melanoma. J. clin. Oncol. 9: 641--648.

DVORAK, H. F. and GRESSER, I. (1989) Microvascular injury in pathogenesis of IFN-induced necrosis of subcutaneous tumours in mice. JNC1 81: 497-502.

ELIAS, L. and CRISSMAN, H. A. (1988) IFN effects upon the adenocarcinoma 38 and HL-60 cell lines: Antiprolifera- tive responses and synergistic interactions with halo- genated pyrimidine antimetabolites. Cancer Res. 48: 4868-4873.

ETTINGHAUSEN, S. E. and ROSENBERG, S. A. (1986) The adoptive immunotherapy of cancer using lymphokine activated killer cells and recombinant interleukin-2. Springer Semin. in Immunopathol. 9, 51-71.

ETTINGHAUSEN, S. E., LIPFORD, E. H., MULE, J. J. and ROSENEERG, S. A. (1985) Recombinant interleukin-2 stimulates in vivo proliferation of adoptively transferred lymphokine activated killer cells. J. Immunol. 135: 3623-3635.

FEImG, H. H. (1988) Comparison of tumour responses in nude mice and in patients. In: Human Tumour Xenografts in Anticancer Drug Development, pp. 25-30, WlNOGRAO, B., PECKHAM, M. J. and PINEDO, H. M. (eds) Springer- Verlag, Berlin.

FELLOUS, M., Nm, U., WALLACH, D., MERLIN, G., RUBINSaXlN, M. and REVEL, M. (1982) IFN-dependent induction of mRNA for the major histocompatibility antigens in human fibroblasts and lymphoblastoid cells. Proc. HatH. Acad. Sci. U.S.A. 79: 3082-3086.

FIDLER, I. J. (1986) Rationale and methods for the use of nude mice to study the biology and therapy of human cancer metastases. Cancer Metastasis Rev. 5: 29-49.

FIEm, W. (1991) Turnout necrosis factor, characterisation at the molecular cellular and in vivo level. FEBS Lett. 285: 199-212.

FORSTER, S., TRIFFIT, J. T., BAUER, H. C, F., BROSJO, O., NILLSON, O. S., SMITH, R. and SYKES, B. (1988) IFN-inhibited human osteosarcoma xenografts induce host bone in nude mice. J. Bone Mineral Res. 3: 199-202.

FRAKER, D. L., SHEPPARD, B. S. and NORTON, J. A. (1990) Impact of tolerance on antitumour efficacy of tumour necrosis factor in mice. Cancer Res. 50: 2261-2267.

Fm~I, E. and SPRIGGS, D. (1989) Tumour necrosis factor: Still a promising agent, d. clin. Oncol. 7: 291-294.

FREIREICH, E. J., GEHAN, E. A., RALL, D. P., SCHMIDT, L. H. and SKIPPER, H. E. (1966) Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey and man. Cancer Chemoth. Rep. 50: 219-244.

GANSBACHER, B., BANNEILII, R., DANIELS, B., ZIE.R, K., CRONIN, K. and GILaOA, E. (1990) Retroviral vector-

mediated gamma-IFN gene transfer into tumour cells generates potent and long-lasting antitumour immunity. Cancer Res. 50: 7820-7825.

GELIN, J., MOLDAWER, L. L., LONROTH, C., SHERRY, B., CHIZZONI~, R. and LUNDHOLM, K. (1991) Role of endogenous Tumour Necrosis Factor and Interleukin-I for experimental turnout growth and the development of cancer cachexia. Cancer Res. 51: 415-421.

GERLACH, H., LIEBERMAN, H., BACH, R., CrODMAN, O., BRETT, J. and STERN, D. (1989) Enhanced responsiveness of endothelium in the growing/motile state to tumour necrosis factor/cachectin. J. exp. Med. 170: 913-931.

GEWERT, D. R., SHAH, S. and CLEIvmNS, M. J. (1981) Inhi- bition of cell division by IFNs: Changes in the transport and intracellular metabolism of thymidine in himen lymphoblastoid (Daudi) cells. Eur. J. Biochem. 116: 487-492.

GIOVARELLI, M., COFANO, F., VECCHI, A., FORNI, M., LANDOLVO, S. and FORNI, G. (1986) Interferon-activated tumor inhibition in vivo, Small amounts of interferon- gamma inhibit tumour growth by eliciting host systemic immunoreactivity. Int. J. Cancer 37: 141-148.

GRANGER, G. A. and WILLIAMS, T. W. (1968) Lymphocyte cytotoxicity in vitro: Activation and release of a cytotoxic factor. Nature 218: 1253-1254.

GRESSER, I. (1989) Antitumour effects of IFN. Acta Oncol. 28: 347-353.

GRESSER, I., MAURY, C. and TOVEy, M. (1987) Efficacy of combined IFN cyclophosphamide therapy after diagnosis of lymphoma in AKR mice. Fur. J. Cancer 14." 97-99.

Gm~SSER, I., MAURY, C., BANDU, M.-T. and BELARDELLI, F. (1990a) Importance of IFN alpha in the resistance of allogeneic C57B1/6 mice to the multiplication of Friend Erythroleukaemia cells in the liver. Cancer Res. 45: 364-37 I.

GRESSER, I., MAURY, C., CARNAUD, C., DE MAEYER, E., MAUNORY, M.-T. and BELARDELLi, F. (1990b) Anti- tumour effects of IFN in mice injected with IFN sensitive and IFN-resistant Friend erythroleukaemia cells. VIII. Role of the immune system in the inhibition of visceral metastases. Int. J. Cancer 46: 468-474.

HADDEN, J. W. (1988) Recent advances in the preclinical and clinical immunopharmacology of interleukin-2: Emphasis on IL-2 as an immunorestorative agent. Cancer Detect. Prey. 12(1-6): 537-552.

HARANAKA, K., SATOMI, N. and SAKURAI, A. (1984) Anti- tumor activity of routine tumour necrosis factor (TNF) against transplanted murine tumours and heterotrans- planted human tumours in nude mice. Int. J. Cancer 34: 263-267.

HAVELL, E. A., FIERS, W. and NORTH, R. J. (1988) The antitumour function of tumour necrosis factor (TNF). 1. Therapeutic action of TNF against an established murine sarcoma is indirect, immunologically dependent, and limited by severe toxicity. J. exp. Med. 167: 1067-1085.

HENCO, K., BROSIUS, J., FUJISAWA, A., FUJ1SAWA, J. I., HAYNES, J. g., HOCHSTADT, J., KOVACIC, T., PASEK, M., SCHAMBOCK, A., SCHMID, J., TODOKORO, K., WALCHLI, M., NAGAT, S. and WEiSSMAN, C. (1985) Structural relationship of IFN alpha genes and pseudogenes. J. tool. Biol. 185: 227-260.

HEREMANS, H., BILLIAU, A. and DE SO,mR, P. (1983) Effect of IFN on the growth of tumours in experimental routine models. In: The Biology of the 1FN System. pp. 425-430, DE MAEYER, E. and SCHELLEKENS, H. (eds)s Elsevier Science Publishers B. V., Amsterdam.

HIRANO, T., YASUKAWA, K., HARADA, H., TAGA, T., WATANABE, Y., MATSUDA, T., KASHIWAMURA, S., NAK~IMA, K., KOVAMA, K. and IWA~tA~SU, A. (1986) Complementary DNA for a novel human interleukin- (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 324: 73-76.


HOFFMAN, V., GROSCtmTH, P., MORAN'r, R., CSERHATI, M., HONEGGER, H. P. H.-P. and VON HOCHSTETTER, A. (1985) Effects of leukocyte IFN on human osteosarcoma growth in vitro and in the nude mice. Eur. d. Cancer clin. Oncol. 21: 859-863.

HoRn, K., ErmKE, M. J., MACE, K. and MImCH, E. (1987) Effect of recombinant human turnout necrosis factor on the tumouricidal activation of murine macrophages: Synergism between tumour necrosis factor and g-IFN. Cancer Re,. 47: 5868-5874.

hGO, M., NISmKATA, K., NAr, mIMA, Y. and MOIYAMA, M. (1989) Effects of interleukin-2 and IFN-beta treatment on lymphocyte, in various tumour bearing mice. J. BioL Regul. Homeost. Agents 3:108-111.

ITO, H., MURArO, MI, K. and YANACAWA, T. (1980) Effect of human leukocyte IFN on the metastatic lung tumour of osteosarcoma. Cancer 46: 1562-1565.

JENKINS, N. A. and COI'ELAND, N. G. (1989) Transgenic mice in cancer research. Important Adv. OncoL 61-77.

KASID, A., DI~CTOR, E. P. and ROSENBER~3, S. A. (1989) Induction of endogenous cytokine-mRNA in circulating peripheral blood mononuclear cells by IL-2 administration to cancer patients. J. lmmunol. 143: 736-739.

KAWAKAMI, M. and CERAMI, A. (1989) Studies ofendotoxin- induced decrease in lipoprotein lipase activity. J. Exp. Med. 154: 631-639.

KAWANO, M., HIRANO, T., MATSUDA, T., TAGA, T., HORII, Y., IWATO, K., ASAOKU, H., TNG, B., TANABE, O. and TANAKA, H. (1988) Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 332: 83-85.

KELLY, S. A., GSCHMEISSNER, S., EAST, N. and BALKWILL, F. R. (1991) Enhancement of metastatic potential by ~,-interferon. Cancer Res. 51: 4020-4027.

KEMENY, N., CHILDS, B., LARCHIAN, W., ROSADO, K. and KELSEN, D. (1990) A phase II trial of recombinant tumor necrosis factor in patients with advanced colorectal carcinoma. Cancer 66: 659-663.

KIM, B. and WARNAKA, P. (1991) Enhanced survival of I EN alpha augmented IL-2 therapy of pulmonary metastases: efficacy comparable to interleukin-2 and lymphokine activated killer cells. J. Surg. Res. 50: 40~6.

KLASING, K. C. (1988) Nutritional aspects of leukocytic cytokines. J. Nutr. 118: 1436-1446.

KONDO, H., TANAKA, N., NAOMOTO, Y. and ORITA, K. (1987) Antitumour effect of combined intraperitoneal administration of human recombinant interferon-/~ and interferon-~, against intraabdominal carcinomatosis in nude mice. Acta Med, Okayama 42: 69-75.

KORNFELD, H., BERMAN, J. S., BEER, D. J. and CENTER, D. M. (1985) Induction of human T-lymphocyte motility by IL-2. J. Immunol. 134: 3887-3890.

KULL, F. C. JR, JACOBS, S. and CUATRECASAS, P. (1985) Cellular receptor for 1251-labeled tumor necrosis factor: specific binding, affÉnity labeling, and relationship to sensitivity. Proc. natn. Acad. Sci. U.S.A. 82: 5756--5760.

KURZROCK, R., TALPAZ, M., KANTARJIAN, H., WALTERSs R., SAKS, S., TRUJILLO, J. M. and GUTTERMAN, J. U. (1987) Therapy of chronic myelogenous leukaemia with recombinant IFN gamma. Blood 70: 943-947.

LANGER, J. and PESTKA, S. (1988) Interferon receptors. Immunol. Today 9: 393-400.

LE BEAU, M. M., LEMONS, R. S., ESPINOSA, R., LARSON, R. A., ARAI, N. and ROWLEY, J. D. (1989) Interleukin-4 and interleukin-5 map to human chromosome 5 in a region encoding growth factors and receptors and are deleted in myeloid leukaemias with a del (5q). Blood 3: 647-650.

LEWIS, M., TARTAGLIA, L. A., LEE, A., BENNETT, G. L., RICE, G. C., WONG, G. H. W., Cr~N, E. Y. and GOEDDEL, D. V. (1991) Cloning and expression of eDNA, for two distinct routine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. natn. Acad. Sci. U.S.A. 88: 2830--2834.

LOTZE, M. T., ROBB, R. J., Sro~m~ow, S. O., FRANA, L. W. and ROSENBERG, S. A. (1984) Systemic administration of interleukin-2 in humans. J. Biol. Resp. Modifiers 3: 475--482.

MAHADEVAN, V., MALIK, S. T. A., M~oFa~, T., Fmas, W. and HART, I. (1990) Role of tumour necrosis factor in flavone acetic acid-induced tumour vasculature shutdown. Cancer Re,. 50: 5537-5542.

MALIK, S. T. A., GRIFFIN, D. B., FmRS, W. and BALKWILL, F. R. (1989) Paradoxical effects of tumour necrosis factor in experimental ovarian cancer. Int. J. Cancer 44: 918-925.

MALIK, S. T. A., NAYLOR, M. S., EAST, N., OLIFF, A. and BALKWiLL, F. R. (1990) Cells secreting tumour necrosis

fac tor show enhanced metastasis in nude mice. Eur. J. Cancer 26: 1031-1034.

MALIK, S. T. A., KNOWLES, R. G., EAST, N., LANDO, D., STAMP, G. and BALKWILL, F. R. (199 0 The antitumour activity of interferon-~, in ascitic and solid tumor models of human ovarian cancer. Cancer Re,., in press.

MALKOVSKA, V. and SONDEL, P. M. (1990) Prospects for interleukin-2 therapy in haematologic malignant neoplasms. Monogr. natn. Cancer Inst. 10: 69-72.

MALUISH, A. E., URBA, W. J., LONGO, D. L., OVERTON, W. R., COGGIN, D., CRISP, E. R., WILLIAMS, R., SHERWIN, S. A., GORDON, K. and STEiS, R. G. (1988) The determi- nation of an immunologically active dose of interferon gamma in patients with melanoma. J. clin. Oncol. 6: 434-445.

MANDA, T., SHIMOMURA, K., MUKUMOTO, S,, KOBAYA$HI, K., MIZOTA, T., HIRAI, O., MATSUMOTO, S., OKU, T., NISHI- GAKI, F., MORI, J. and KIKUCm, H. (1987) Recombinant tumour necrosis factor-a: Evidence of an indirect mode of activity. Cancer Re,. 47:3707-3711.

MARINCOLA, F. M., DAPoZzo, L. F., HOWlCK, J. C., DRUCKER, B. J., HOUGH, K. L. and HOLDER, W. D. (1989) Prolonged survival of nude mice hearing human pancreatic cancer and treated with lymphokine- activated killer cells and interleukin-2. Curr. Surgery 46: 304-307.

MASUDA, S., FUKUMA, H. and BEPPU, Y. (1983) Antitumour effect of human leucocyte interferon on human osteosarcoma transplanted into nude mice. Eur. J. Cancer din. Oncol. 19: 1521-1528.

MATTERN, J., BAK, M., HALN, E. W. and MANERED, V. (1988) Human tumour xenografts as model for drug testing. Cancer Metastasis ReD. 7: 263-284.

MCINTOSH, J. K., JAaLONS, D. M., MULE, J. J., NORDAN, R. P., RUDIKOFF, S., LOTZE, M. T. and RO~ENBERG, S. A. (1989a) In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumour-bearing mice. J. lmmunoL 143, 162-167.

MCINTOSH, J. K., MULE, J. J., KROSNICK, J. A. and ROSENaERG, S. A. (1989b) Combination cytokine immunotherapy with tumour necrosis factor alpha, interleukin-2, and recombinant hybrid alpha IFN and its synergistic antitumour effects in mice. Cancer Res. 49: 1408-1414.

MCINTOSH, J. K., MULE, J. J., TRAVIS, W. D. and ROSENBERG, S. A. (1990) Studies of effects of recombinant human tumor necrosis factor on autochthonous tumor and transplanted normal tissue in mice. Cancer Res. 50: 2463-2469.

MOORE, A. S., TI-IEILEN, G. H., NEWELL, A. D., MADEWELL, B. R. and RUDOLF, A. R. (1991) Preclinical study of sequential tumour necrosis factor and interleukin-2 in the treatment of spontaneous canine neoplasms. Cancer Res. 51: 233-238.

MORIKAWA, K., FAN, D., DENKINS, Y. M., LEViN, B., GUTTERMAN, J. U., WALKER, S. M. and FIDLER, I. J. (1989) Mechanisms of combined effects of gamma IFN and 5-fluouracil on human colon cancers implanted into nude mice. Cancer Res. 49: 799-805.


MULE, J. J., MCINTOSH, J. K., JABLONS, D. M. and ROSEN- BERG, S. k . (1990) Antitumour effect of recombinant interleukin 6 in mice. J. exp. Med. 171: 629-636.

NAKAMURA, S., NAKATA, K., KASHMOTO, S., YOSHIDA, H. and YAMADA, M. (1986) Antitumour effect of recombinant human interleukin 1 alpha against routine syngeneic tumors. J. Cancer Res. 77: 767-773.

NAKANO, K., OKUGAWA, K., FURUICm, H., MATSUL Y. and SOHMURA, Y. (1989) Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor. Cell Immunol. 120:154-164.

NAMBA, M., YAMAMOTO, S., TANAKA, B., KANAMORI, T., NOBUHARA, M. and KIMOTO, T. (1984) In vitro and in rive studies on potentiation of cytotoxic effects of anticancer drugs or cobalt 60 gamma ray by IFN on human neoplastic cells. Cancer 54(10): 2262-2267.

NAOMOTO, Y., TANAKA, N. and ORITA, K. (1989) Anti- tumour effect of natural human tumour necrosis alpha and natural human IFN alpha in combination against human cancer transplanted into nude mice. Acta &led. Okayama 43:211-221.

NAYLOR, M. S., MALIK, S. T. A., STAMP, G. W. H., JOBLIN, T. and BALKWILL, F. R. (1990) In situ detection of tumour necrosis factor in human ovarian cancer specimens. Eur. J. Cancer 26: 1027-1030.

NORTH, R. J. and HAVELL, E. A. (1989) Glucocorticoid- mediated inhibition of endotoxin-induced intratumour tumor necrosis factor production and tumor haemor- rhagic necrosis and regression. J. exp. Med. 170: 703-710.

OGURA, H., TANI, K., OZAWA, K., NAGATA, S., ASANO, S. and TAKAKU, F. (1990) Implantation of genetically manipulated fibroblasts into mice as antitumour alpha- IFN therapy. Cancer Res. 50: 5102-5106.

ONISHI, T., MACHIDA, T., MORI, Y., IIZUKA, N., MASUDA, F., MOCHIZUKI, S., TSUKAMOTO, H. and HARADA, N. (1989) Hyperthermia with simultaneous administration of IFN using established human renal carcinoma heterotrans- planted in nude mice. Br. J. Urol. 63: 227-232.

ONOZAKI, K., MATSUSHIMA, K., AGGARWAL, B. B. and OPPENHEIM, J. J. (1985) Human interleukin- 1 is a cytocidal factor for several tumor cell lines. J. lmmunol. 135: 3962-3968.

ORTALDO, J. R., PORTER, H. R., MILLER, P., STEVENSON, H. C., OZOLS, R. F. and HAMILTON, T. e . (1986) Adoptive cellular immunotherapy of human ovarian carcinoma xenografts in nude mice. Cancer Res. 46: 441~4419.

OZAKI, Y., EDELSTEIN, M. P. and DUCH, D. S. (1988) Induc- tion of indoleamine 2,3 dioxygenase: a mechanism of the antitumour activity of IFN gamma. PNAS U.S.A. 85: 1242.

OZELLO, L., HABtV, D, V. and DE ROSA, C. M. (1990) Antiproliferative effects of natural IFN beta alone and in combination with natural IFN gamma on human breast carcinomas in nude mice. Breast Cancer Res. Treat. 16: 89-96.

PACIOTT1, G. F. and TAMARKIN, L. (1988) Interleukin-2 differentially affects the proliferation of a hormone- dependent and a hormone-independent human breast cancer cell line in vitro and in rive. Anticancer Res. 8: 1233-1239.

PALLADINO, M. A. JR, REFAAT SHALABY, M., KRAMER, S. M., FERRAIOLO, B. L., BAUGHMAN, R. k., DELEO, A. B., eRASE, n., MARAFINO, B., AGGARWAL, B. B. and EIGARI, I. S. (1987) Characterization of the antitumor activities of human tumour necrosis factor alpha and the comparison with other cytokines: induction of tumour-specific immunity. J. Immunol. 138: 4023-4032.

PAPA, M., MULE, J. J. and ROSENBERG, S. A. (1986) Anti- tumour efficacy of lymphokine-activated killer cells and recombinant interleukin-2 in rive. Successful immune- therapy of established pulmonary metastases from weakly immunogenic and nonimmunogenic murine tumours of

three distinct histological types. Cancer Res. 46: 4693-4978.

P ~ , A., PmLUPS, N., SIMOmN, G., DE MAYER- GtaGNAP, O, J. and DE MAEVER, E. (1983) Differential cytostatic effects of IFN on routine BALB. c B and T-cell turnouts. J. IFN Res. 3: 253-260.

PENNICA, D., NEDWlN, G. E., HAYFLiCK, J. S., SEEBURG, P. H., DERYNCK, R., PALLADINO, M. A., KOHN, W. J., AC, GARWAL, B. B. and GOEODEL, D. V. (1984) Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin. Nature 312: 724-729.

PESTKA, S. (1986) Interferon from 1981 to 1986. In: Methods in Enzymology, pp. 3-14, PESTKA, S. (ed.), Academic Press, New York.

PESTKA, S., LANGER, J. A., ZOON, K. C. and SAMUEL, C. E. (1987) Interferons and their actions. A. Rev. Biochem. 56: 727-777.

PRICE, C. G., ROHATINER, A. Z., STEWARD, W., DEAKIN, D., BAILEY, N., NORTON, A., BLACKLEDGE, G., CROWTHER, D. and LISTER, T. A. (1991) IFN alpha 2b in the treatment of follicular lymphoma: preliminary results of a trial in progress. Ann. Oncol. 2 (Suppl. 2): 141-145.

PUJADE-LAURAINE, E,, GUASTELLA, J. P., COLOMBO, N., NAMER, N., FUMOLEAU, P., MONNIER, A., NOOY, M. A., FALKSON, G., MIGNOT, L., BUGAT, R., OLIVIERA, C. M. D., MOUSSEAU, M., NETTER, G., OBERLING, F., COIFFIER, B. and BRANDEL, Y. M. (1991) Intraperitoneal human rIFN- gamma as treatment of residual carcinoma (OC) at second look laparotomy (SLL). In: Proceedings of the American Society of Clinical Oncology, Houston, TX, 19-21 May, 1991, p. 195, PERRY, M. C. (ed.), ASCO, Philadelphia.

QUIRT, I. C. and TANNOCK, I. F. (1990) Interleukin-2 for metastatic melanoma: treating polyuria with insulin? J. clin. Oncol. 8: 1125-1127.

RAETH, U., SCHMID, H., HOFMANN, J., W1EOENMANN, B., KEMPENI, L., SCHLICK, E. and KAUFMANN, M. (1991) Intraperitoneal (i.p.) application of recombinant human tumour necrosis factor (rHuTNF) as an effective pallia- tive treatment of malignant ascites from ovarian and gastroenteropancreatic carcinomas. In: Proceedings of the American Society of Clinical Oncology, Houston, TX, 19-21 May, 1991, p. 181, PERRY, M. C. (ed.), ASCO, Philadelphia.

RAMANI, P. and BALKWILL, F. R. (1987) Enhanced metastases of a mouse carcinoma after in vitro treatment with murine IFN gamma. Int. J. Cancer 40: 830-834.

RAMANI, P. and BALKWILL, F. R. (1989a) Action of recombinant alpha IFN against experimental and spontaneous metastases in a murine model. Int. J. Cancer 43: 140-146.

RAMANI, P. and BALKWILL, F. R. (1989b) Human interferons inhibit experimental metastases of a human melanoma cell line in nude mice. Br. J. Cancer 58: 350-354.

REHBERG, E., KELDER, B., HOAL, E. G. and PESTKA, S. (1982) Specific molecular activities of recombinant and hybrid leukocyte interferons. J. biol. Chem. 257:11497-11502.

REID, T. R., RACE, E. R., WOLFF, B. H., FROEDMAN, R. M., MERIGAN, T. C. and BASHAM, T. Y. (1989) Enhanced in rive therapeutic response to IFN in mice with an in vitro IFN-resistant B Cell lymphoma. Cancer Res. 49: 4163-4169.

ROSENBERG, S. A. (1986) Adoptive immunotherapy of cancer using lymphokine activated killer cells and recombinant interleukin-2. In: Important advances in Oncology, pp. 55-91, DEV1TA, V. T., BELLMAN, S. and ROSENBERG, S. A. (eds). Philadelphia: J. B. Lippincott.

ROSENBERG, S. A., LOTZE, M. T., MUUL, L. M., CHANG, A. E., AVLS, F. P., LEITMAN, S., LINEHAN, M., ROBERTSON, C. N., I .~, R. E., Rums, J. T., SvapP, C. A., SIMPSON, C. G. and WroTE, D. E. (1987) A progress report on the treatment of 157 patients with advanced cancer using lymphokine activated killer cells and interleukin-2 or


high-dose interleukin-2 alone. N. Engl. J. Med. 137: 1735-1742.

RUBIN, B. Y., ANDERSON, S. L., LUNN, R. M., HELLERMANN, G. R. and SM1TH, L. J. (1989) Fragmentation of cellular DNA is a nonspecific indicator of responsiveness to tumor necrosis factor. J. Biol. Resp. Mod. 8: 553-559.

SAITO, H., MOKIZANE, T., INAGAKI, Y., KUMAGAI, N., WATANABE, T., NAKAMURA, T., SATOH, I., SAWAGUCHI, K., IWABUCm, N., TSUCmMOTO, K. and TSUCmYA, T. (1989) Decrease of transplantability by the immunopotentiators, OK-432 and interleukin-2: Experiments on a human hepatoma cell line in nude mice. Fur. J. Cancer 25: 79-89.

SALERNO, R. A., WHITMORE, C. A., GARCIA, I. M. and HuEnHER, R. J. (1972) Chemical carcinogenesis in mice inhibited by IFNs. Nature New Biol. 239: 31-32.

SANADA, E., FUCHIMOTO, S. and ORITA, K. (1990) Synergistic antiproliferative effect of the combination of natural human tumour necrosis factor-alpha and natural murine IFN alpha/beta against colon-26 adenocarcinoma hepatic metastases in a murine model. Acta Med. Okayama 44: 217-222.

SAYERS, T. J., WILTROUT, T. A., McCORMICK, K., HUSTED, C. and WILTROUT, R. H. (1990) Antitumour effects of IFN-a and IFN-g on a routine renal cancer (Renca) in vitro and in vivo. Cancer Res. 50: 5414-5420.

SCHILLER, J. H., STOKER, B. E., WITT, P. L., ALBERTI, D., TOMBES, M. B., ARZOOMANIAN, R., PROCTOR, R. A., MCCARTHY, D., BROWN, R. R., VOSS, S. D., REMICK, S. C., GKEM, J. L., BORDEN, E. C. and TRUMP, D. L. (1991) Biological and clinical effects of intravenous TNF-a administered three times weekly. Cancer Res. 51: 1651-1658.

SELBY, P., HOBBS, S., VINER, C., JACKSON, E., JONES, A., NEWELL, D., CALVERT, A. H., McELWAIN, T., FEARON, K., HUMPHREYS, J. and SmGA, T. (1987) Tumour necrosis factor in man: Clinical and biological observations. Br. J. Cancer 56: 803-808.

SHAH, P., VAN DER MEIDE, P. H., BORMAN, T., SCHROEDER, N., BLkSS, J. M. and COOMBES, R. C. (1989) Effect of human recombinant tumour necrosis factor and rat gamma IFN on nitrosomethylurea-induced mammary tumours. Br. J. Cancer 59: 206-209.

SHAu, H. (1988a) Cytostatic and tumoricidal activities of tumor necrosis factor-treated neutrophils. Immanol. Left. 17: 47-51.

S ~ u , H. (1988b) Characterisation and mechanism of neutrophil-mediated cytostasis induced by tumour necrosis factor. J. lmmanol. 141: 234--238.

SHEPPARD, B. C., VENZON, D., FRAKER, D. L., LANGSTEIN, H. N., JENSEN, J. C. and NORTON, J. A. (1990) Prolonged survival of tumor-bearing rats with repetitive low-dose recombinant tumour necrosis factor. Cancer Res. 50: 3928-3933.

SmMIZU, H., INOUE, M. and TANIZAWA, O. (1989) Adoptive cellular immunotherapy ofendometrial cell line xenografts in nude mice. Gynecol. Oncol. 34: 195-197.

SIDKY, Y. A. and BORDEN, E. C. (1987) Inhibition of anglo- genesis by IFNs: effects on tumour and lymphocyte induced vascular response. Cancer Res. 47: 5155-5161.

STERN, J. B. and SMITH, K. A. (1986) IL-2 induction of G1 progression and c-myb expression. Science 233: 203-206.

STRANDER, H. (1986) Interferon treatment of human neo- plasma. Adv. Cancer Res. 46: 1-203.

SUEMATSU, S., MATSUDA, T., AOZASA, K., AKIRA, S., NAKANO, N., OHNO, S., MIYAZAKI, J., YAMAMORA, K., HIRANO, T. and KISHIMOTO, T. (1989) IgGl plasmacytosis in interleukin-6 transgenic mice. Proc. ham. Acad. Sci. U.S.A. 86: 7547-7551.

TAKIKAWA, O., HABARA-OHKUBO, A. and YOSHIDA, R. (1990) IFN-gamma is the inducer of indoleamine 2,3 dioxygenase in allografted tumour cells undergoing rejection. J. Im- munol. 145" 1246-1250.

TANAKA, N., NAGAO, S., TOGHO, A., SEKIGUCHI, F., KOHNO, M., OGAWA, H., MATSUI, T. and MATSUTANI, M. (1983)

Effects of human flbroblast IFN on human gliomas transplanted into nude mice. Gann 74: 308-316.

~PPER, R. I., PATTENGALE, P. K. and LEDER, P. (1989) Murine Interleukin-4 displays potent anti-tumour activity in vivo. Cell 57: 503-512.

TlUNCH~I, G. and PF~USSIA, B. (1985) Immune IFN: a pleiotropic lymphokine with multiple effects. Immunol. Today 6: 131-136.

TROTTA, P. P. and HAgmSON, S. D. (1987) Evaluation of the antitumor activity of recombinant human gamma-IFN employing human melanoma xenografts in athymic nude mice. Cancer Res. 47: 5347-5353.

TRUITT, G. A., S ~ N , L. L. and Bo~rr~MPO, J. (1987) Recombinant interleukin-2(IL-2) alone and in combination with IFN alpha, is a potent antitumour agent against colon 38 and Lewis lung carcinomas in mice. In: Proceedings of the American Society of Clinical Oncology, Houston, TX. 19-21 May, 1991, p 369, PERRY, M. C. (ed.), ASCO, Philadelphia.

TRUITT, G. A., BRUNDA, M. J., LEVITT, D., ANDERSON, T. D. and SHERMAN, M. I. (1989) The therapeutic activity in cancer of IL-2 in combination with other cytokines. Cancer Sure. 8: 875-889.

ULICH, T. R., DEL CASTILLO, J. and Guo, K. Z. (1989) In vivo haematologic effects of recombinant interleukin-6 on haematopoiesis and circulating numbers of RBCs and WBCs. Blood 73:108-110.

VANHAESEBROECK, B., MAP, EEL, M., VAN ROY, F., GROOTEN, J. and FmRS, W. (1991) Expression oft.he tumour necrosis factor gene in tumour cells correlates with reduced tumorigenicity and reduced invasiveness in vivo. Cancer Res. 51: 2229-2238.

WADLER, S., SCHWARTZ, E. L., GOLDMAN, M., LYVER, A., RADER, M., ZIMMERMAN, M., ITRI, L., WEINBERG, V. and WIERNIK, P. H. (1989) Fluorouracil and recombinant alpha 2a IFN: An active regimen against advanced colorectal carcinoma. J. clin. Oncol. 7: 1769-1775.

WATANABE, Y., KURIBAYASHI, K., MAYATAKE, S., NISHIHARA, K., NAKAYAMA, E., TANIYAMA, T. and SAKATA, T. (1989) Exogenous expression of mouse IFN gamma eDNA in mouse neuroblastoma CI300 cells results in reduced tumorigenicity by augmented anti-tumour immunity. Proc. natn. Acad. Sci. 86: 9456-9460.

WEST, W. H. (1988) Continuous infusion recombinant interleukin-2 (rIL-2) and adoptive cellular therapy of renal cell carcinoma and other malignancies. In: First lnterleukin.2 Internatwnal Symposium, p. 21. Eurocetus, Amsterdam.

WINGFIELD, P., PAIN, R. H. and CR^IG, S. (1987) Turnout necrosis factor is a compact trimer. FEBS Lett. 211: 179-184.

WINKELHAKE, J. L., STAMPFL, S. and ZIMMERMAN, R. J. (1989) Synergistic effects of combination therapy with human recombinant interleukin-2 and tumour necrosis factor in murine tumour models. Cancer Res. 47: 3948-3953.

YAMAOKA, T., TAKADA, H., YANAGI, Y., KATAOKI, T. and SAKURAI, Y. (1985) The antitumour effects of human lymphoblastoid IFN on human renal cell carcinoma in athymic nude mice. Cancer Chemother. Pharmac. 14: 184-187.

YASUI, H., PROmTTI, E., VIGNAUX, F., EID, P., GRESSER, I. (1990) Inhibition by mouse alpha/beta-IFN of the multiplication of alpha/beta-IFN resistant Friend Erythro- leukaemia cells cocultured with mouse hepatocytes. Cancer Res. 50: 3533-3539.

YOSHIKAZl, K., MATSUDA, T., NtSaIMOTO, N., KURITAN, T., TAEHO, L., AOZASA, K., NAKAHATA, T., KAWAI, H., TAGOH, H., KOMORI, T., KISHIMOTO, S., HIRANO, T. and KISHIMOTO, T. (1989) Pathogenic significance of interleukin-6 in Castleman's disease. Blood 74: 1360-1367.

YOUNG, J. C., GISHIZKY, M. L. and WITTE, O. N. (1991) Hyperexpression of interleukin-7 is not necessary or sufficient for transformation ofa pre-B lymphoid cell line. Mol. cell. Biol. l h 854-863.

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Effects of interferons and other cytokines on tumors in animals: A review