The potential role of cytokines in cancer therapy.

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The potential role of cytokines in cancer therapy By Richard M. Schultz Lilly Research Laboratories; Indianapolis, Indiana 46285, USA

Introduction Historical perspectives IL-2 2. 2.1 Molecules. 2.2 Mechanism of antitumor action 2.3 Clinical aspects Interferons 3 3.1 Molecules. 3.2 Mechanism of antitumor action 3.3 Clinical aspects Myeloid colony stimulating factors 4 4.1 Molecules. 4.2 Biological activities 4.3 Clinical aspects Concluding remarks . 5 Acknowledgments References I

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U. Bachrach et al., Progress in Drug Research / Fortschritte der Arzneimittelforschung / Progrès des recherches pharmaceutiques © Birkhäuser Verlag Basel 1992

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Introduction

Drugs can only repress symptoms. They cannot eradicate the disease. The true remedy for all diseases is nature's remedy ... Nature has provided, in the white corpuscles as we call them - a natural means of devouring and destroying all disease germs ... Stimulate the phagocytes., they devour the disease, and the patient recovers - unless, of course, he's too far gone. Sir Ralph Bloomfield Bonnington in the "Doctor's Dilemma" by George Bernard Shaw, 1902

For most non-lymphoid tumors, the standard therapeutic triad of surgery, chemotherapy, and radiotherapy appears to be approaching the limits of its potential for the successful eradication of advanced stages of neoplastic disease. Therefore, new approaches to therapy are continuously being sought, including investigations on the use of cytokines, which can be used to expand subpopulations of tumoricidal cells or to exert direct antitumor effects on the cell. Recently, genetic engineering techniques have allowed for the large-scale production of these molecules generated during the immune response. The broad spectrum of biologic activities displayed by cytokines has stimulated considerable interest in the oncology community because of their potential application as a new "fourth modality" of cancer treatment. Cytokines may (a) enhance the antitumor response(s) of the host through augmentation and restoration of effector mechanisms; (b) increase the host defenses against intercurrent infections by reversing myelosuppression or enhancing the function of antimicrobial effector cells; (c) improve tolerance of the host to damage by cytotoxic modalities of cancer treatment; (d) induce alterations in tumor cell membranes that render the cells more susceptible to treatment; or (e) arrest tumor cell growth by inducing maturation/differentiation. Cytokines that modify the host resistance to malignant disease also have potential to augment effectiveness of other cancer treatment techniques. This article provides an overview of the current status of three types of cytokines (interleukin-2, interferons, and myeloid colony stimultating factors) and provides some basic information about these important molecules. The interaction of cytokines in regulating immune function has previously been the topic of several ex-

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cellent reviews [1-5]. Although the ultimate role of cytokines in cancer treatment is still unkown, it is certain that the role will be significant. 1.1

Historical perspectives

During the last century, "nonspecific" immunotherapy with a variety of bacterial extracts, viruses, and chemicals has been utilized with the hope of stimulating the host's antitumor immune response. In fact, the earliest attempts at immunotherapy occurred in the late 1800s when Dr. Coley used a toxin after noting that some patients remained tumor-free when experiencing severe infections [6, 7]. These nonspecific approaches have occasionally proven useful under defined conditions in experimental tumor models [8-10]. Despite early promising results in trials of nonspecific immunostimulants, such as Bacillus Calmette-Guerin (BCG) [11-13], none of these approaches has been shown to be effective in controlled trials [14-15]. The lack of predictive ability of the animal models possibly involves their frequent reliance on the use of tumors transplanted into normal syngeneic recipients. Clearly, normal animals are not comparable to animals bearing autochthonous neoplasms [16,17]. Defects in normal surveillance mechanisms may lead to the development of autochthonous tumors, and corrections of such defects may require a totally different form of biological modification than that required to assist the normal host in controlling an implanted neoplastic growth and its subsequent metastasis [18]. In addition, many of the studies involving animal tumor systems were actually investigations of prophylaxis because they involved stimulation of the host before or simultaneously with tumor implantation. Often the transplant consisted of a very small number of cells, since immunotherapy was not strikingly effective in animals bearing palpable tumors. These experimental results developed into the dogma that the "old" immunotherapy could only work for minimal residual disease after the tumor was debulked by other treatment modalities. Evaluating new approaches utilizing cytokine therapy will be greatly augmented by the development of appropriate and relevant animal models in tumor systems as closely as possible related to those of man. Human cancer patients are often immunosuppressed not only by progressive tumor growth [19], but also by various modalities of cancer

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treatment, including surgery [20], radiotherapy [21], and chemotherapy [22]. Since there is a relationship between immunocompetence and prognosis in cancer [23], it has been hypothesized that reversing the immunocompetence associated with a poor prognosis would thus improve the prognosis [24]. Dr. Ehrlich, in a 1900 address [25], noted: "We are brought more and more to the conviction that the blood serum is the carrier of substances innumerable as yet little known or conceived of." In 1968 Marcfarlane Burnet proposed that inherent host defense against neoplasia was effected by means of autopharmacological mediators of immunological surveillance [26]. Dumonde later (1969) introduced the term lymphokine to bring together these non-antibody lymphocyte-derived factors, that act as messengers between cells [27]. Lymphokines have pleiotropic biochemical and cellular effects, such as induction of antiviral activity, stimulation of lymphocyte and myeloid progenitor cell growth, activation of macrophage functions in nonspecific host defense and direct cytocidal activity. The lymphokine is produced by one cell and can affect the cell itself in an autocrine fashion, or it can travel to nearby or distant cells in a paracrine or endocrine manner. The clinical significance of lymphokines/ cytokines arises from their role in mediating both intrinsic and extrinsic pathways of immunoregulation, inflammation, as well as inherent and adaptive mechanisms of host defense against microbial pathogens and neoplastic diseases. Lymphokines are, by definition, naturally-occurring substances (proteins) possessing a biological activity that is assessed by an in vitro procedure. Lymphokines may alter the relationship between the tumor and host by modifying the host's biological responses to tumor cells, with resultant therapeutic effects. A common source of lymphokines is the supernatant fluid from peripheral blood lymphocytes or lymph node cells that have been stimulated with antigens or mitogens [4, 28, 29]. Lymphokine activity is also demonstrated in supernatants from lymphoblastoid lines in permanent culture [30, 31]. The first attempts were made in the middle 1970s to utilize relatively crude lymphokine preparations, derived from established B lymphoblastoid cell lines, in the treatment of cancer [32-35]. Activities detected in these preparations included leukocyte migration inhibition factor (MIF), macrophage activating factor (MAF), skin inflammatory factor, lymphotoxin activity and lymphocyte mitogenic activity. Prelimi-


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nary studies by Papermaster and coworkers [32, 33, 36] demonstrated that intratumoral injection of lymphokine preparations derived from the cultured human lymphoblastoid cell line, RPMI 1788, produced local tumor regression in humans and in mice. Historically, cytokine activities have been detected in many biological preparations, but because of extremely minute concentrations, chemical instability and other factors, the isolation and purification of individual cytokines has been very difficult. The tremendous advances in molecular biology over the past 12 years have allowed for the large-scale production of cytokines, the isolation and purification of these proteins and eventually their production by genetic engineering. Scientists now have the capability to clone individual cytokine genes and produce, through prokaryotic hosts, huge quantities of highly purified products for analysis. These cloned products can be purified to homogeneity and the reactions that they engender can be understood in the absence of other cytokine substances that could complicate the interpretation of experimental results. This revolution in molecular biology enables scientists to produce the highly purified reagents necessary to begin to better understand the immunological mechanisms involved in cancer growth and metastasis. It is critical to understand the therapeutic mechanism of cytokines that are currently available and how best to translate their therapeutic potential into clinical efficacy. 2

IL-2

Interleukin-2 (IL-2) was first described more than a decade ago as a growth factor for activated and certain malignant T-Iymphocytes [37]. Over the next several years, the biochemical and molecular analyses of IL-2 enabled identification and cloning of the IL-2 gene. Production of large quantities of IL-2 by recombinant DNA techniques, using the human IL-2 gene functioning within Escherichia coli, allowed for molecular, preclinical and clinical research to proceed. Although natural IL-2 is glycosylated, the biological, pharmacological and clinical behavior of E. coli-produced IL-2, which is nonglycosylated, seems for practical purposes to be indistinguishable from that of the natural molecule.

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2.1

Molecules

Natural IL-2 is a glycoprotein, with a molecular weight of 15,400, containing 133 amino acids. It is produced by T cells, activated by exposure to plant lectins or specific antigens [38~0]. The interaction of IL-2 with target cells occurs via a specific cell surface receptor, comprised of a bimolecular complex [41]. The 55 kD component (also known as TAC antigen) was identified first and was recognized by a monoclonal antibody [42]. The TAC molecule is not present on resting lymphocytes, but appears after cell activation. A 75 kD molecule that also functions as an IL-2 receptor was discovered on cells that do not express the TAC molecule (mainly non-T-lymphocytes with natural killer [NK] markers). These cells can respond to IL-2 and express TAC on their membranes [43]. The 75 kD molecule binds IL-2 at approximately 100-fold lower affinity (~= 10-9 M) than the high-affinity receptor. TAC, the other component of the high-affinity receptor, will also bind IL-2, although at low affinity (~ of 10-8 M). The association of both 55 (the f3 chain) and 75 kD (the a chain) receptor molecules on the surface of cells allows for rapid IL-2 binding with high affinity (~ of approximately 10- 11 M) and slow dissociation. Thus, higher concentrations of IL-2 are needed to activate cells expressing only the a chain receptors. It appears that the role of the f3 chain, which is induced during cell activation, is to interact with the a chain to produce a receptor exquisitely sensitive to even low concentrations of IL-2. The immunologic consequences of IL-2 therapy, therefore, include the amplification of whatever activated T cells are present in the patient. Cells that constitutively express the low affinity 75 kD molecule, including natural killer (NK) cells and lymphokineactivated killer (LAK) cells, require high IL-2 concentrations for activation of antitumor effector function. 2.2

Mechanism of antitumor action

IL-2 treatment induces several effector cell populations that may participate in defense against cancer. These include natural killer (NK) cells [44, 45], monocytes [46, 47], lymphokine-activated killer (LAK) cells [48, 48 a] and cytotoxic T cells, including tumor-infiltrating lymphocytes (TIL) [49-51]. IL-2 stimulates proliferation of activated T- lymphocytes [52], activated B-Iymphocytes [53] and NK cells [54].


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IL-2 treatment also induces a spectrum of "secondary" cytokines, including interferon-y and tumor necrosis factor (TNF) [55-58], which likely mediate additional effects. The antitumor activity of IL-2 in animal models has been reviewed in detail elsewhere [59-62]. IL-2 is active during the early stages of tumor growth (day 3) in both immunogenic and nonimmunogenic murine tumors. However, IL-2 is only active in the advanced stages of tumor growth (day 10) in animals with weakly immunogenic tumors. The antitumor effect in nonimmunogenic tumors appears to be mediated primarily by LAK cells and in weakly immunogenic tumor by LAK cells as well as T-cells [59]. A number of principles have emerged from the animal tumor models that have served as a basis for the design of early clinical investigations in humans. In general, the greatest antitumor effects (measured as increased cure rates or prolonged survival) were obtained when a) IL-2 treatment was given at a time of low tumor burden [63] and b) treatment was prolonged over several days or weeks, yielding sustained, lower levels rather than brief, high-peak levels [64]. It was also clear that IL-2 treatment alone did not exhibit antitumor activity in many animals models. The activity of IL-2 could be enhanced, however, in these models by the addition of ex vivo - activated LAK cells [65] or in vitro cultivated TIL [66]. In animal modeis, the adoptive transfer of TIL have antitumor reactivity 50-tOO times more potent than do LAK cells [59]. 2.3

Clinical aspects

Knowledge of the central role of IL-2 in the immune response and theories of the importance of immune surveillance in the control of neoplastic cells has led to the hope that IL-2 may be useful in cancer therapy. The infusion of IL-2 in humans was associated with increases in mature T-cells [58] and induced expression of IL-2 receptors in both T-cells and monocytes [67]. IL-2 treatment also induced the expansion of cells with NK markers and in vivo activation of LAK activity. In addition, increased expression of HLA-DR antigen occurred in tumor, endothelial cells and in perivascular T-cells following IL-2 therapy [68]. When IL-2 is administered by intravenous bolus in humans, a high peak plasma level was noted with a rapid initial plasma clearance (six

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to seven minutes) [69, 70]. However, a more prolonged later clearance was observed, suggesting that the initial rapid clearance of IL-2 was due to movement into an extravascular compartment, from which it returns more slowly to the plasma. West provided data that antitumor responses could be maintained with less toxicity by administering IL-2 by continuous infusion [71]. Initial clinical testing of recombinant IL-2 demonstrated two critically important phenomena [72]: 1) A minority of patients showed measurable antitumor effects (complete and partial responses). 2) Patients treated with these high-dose IL-2 regimens had severe, at times life-threatening, systemic toxicities, which were probably immunemediated. The toxicity of systemically administered IL-2 included fever, chills, nausea, vomiting, diarrhea, hypotension, cutaneous erythema, fluid retention, life-threatening pulmonary edema, eosinophilia, anemia and moderate to severe hepatic and renal dysfunction [73]. Most IL-2 toxicities resolved within hours to days following discontinuation of IL-2 therapy. Approximately 20 % of patients with metastatic melanoma and renal cell carcinoma showed measurable antitumor responses [73-75]. These were usually partial responses that lasted for a few months, but occasionally, prolonged, complete remissions occurred. It is not presently clear whether the addition of in vitrQ activated LAK cells or TIL to this regimen provides a sufficiently large boost to the antitumor effect of IL-2 treatment alone to warrant theincreased cost, complexity and patient care [76, 77]. There is general consensus now that the initial high dose IL-2 + LAK protocols had unacceptable toxicity for the therapeutic results achieved [77 a]. Despite the clinical antitumor effects for IL-2 in some patients, Borden and Sondel stressed that improvements are needed to obtain more meaningful clinical benefit and improved therapeutic indices [75]. For example, testing so far has focused on Phase II trials in patients with measurable disease, despite murine studies showing greatest therapeutic efficacy of IL-2 for nonimmunogenic tumors when there is only a microscopic tumor burden [78]. Similarly, studies in animal models have suggested additive or synergistic effects by combining IL-2 with other agents [reviewed in 62]. Aside from clinical studies of recombinant IL-2 with lymphoid cells, combination studies using other lymphokines, cytokines and cytotoxic chemicals have occurred during the past few years. These strategies to improve IL-2 ac-


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tivity include combinations with interferons, tumor necrosis factor, or monoclonal antibodies. A recent study reported 6 of 21 (29%) patients with advanced renal cell carcinoma responded to the combination of IL-2 and ,8-inteneron [79]. In addition, attempts have been made to decrease T-suppressor cells by injecting low-dose cyclophosphamide prior to IL-2 administration [80]. The growing body of knowledge emerging from in vitro and animal models may provide clinicians with critical clues to guide the use of combinations among the various lymphokines/ cytokines and cytotoxic modalities and thereby, improve therapeutic activity. 3

Interferons

The interferons (IFN) are a family of secreted proteins that were originally characterized by their ability to interfere with virus infection [81]. They do not directly inactivate viruses or prevent virus absorption to cells, but they do prevent viral replication by a host cell mechanism requiring RNA and protein synthesis. Aside from host defense against virus infections [82-85], interferons also have been shown to inhibit cell proliferation, modulate cell differentiation and activate various cell types of the immune system [86-92]. Potentially useful effects of interferons in antitumor therapy include antiproliferative activity on tumor cells, enhancement of macrophage/monocyte and NK cytotoxicity and induction or augmentation of expression of membrane antigens on tumor cells. The interferons are synthesized and secreted in a variety of cell types not only in response to viruses, but also to bacteria and bacterial products [93-95], protozoa [96], chlamydia [97], fungal extracts [98, 99], dsRNA [100] and synthetic compounds including anionic polymers [101]. The initial studies with interferons were plagued with inability to produce sufficient material for in vivo tests. Taniguchi and coworkers [102] successfully cloned ,8-interferon in 1979. Although crude preparations of interferons were administered to cancer patients as early as 1966 [103]. It was not until 1981 that supplies of human interferons produced in bacteria using recombinant DNA techniques and purified to homogeneity became available, permitting evaluation of the effects of single interferon species in the absence of contaminating proteins.

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3.1

Molecules

Interferons are a group of proteins and glycoproteins, subdivided into three major classes ( a, p and y) according to differences in biologic, antigenic and physicochemical properties. These have now been confirmed to result from significant differences in primary amino acid sequence. Interferons a and p are stable at low pH, whereas treatment of interferon-y at pH 2 neutralizes its activity. Interferons-a and -p can be induced in cells of diverse origin by viruses or synthetic polyribonucleotides. In contrast, interferon-y (immune interferon) is produced by T cells induced by stimulation with mitogens or specific antigen. Only single genes have been found coding for human interferon-p or -y, whereas two families of more than 20 chemically-related interferon-a proteins have now been defined [104-107]. Both interferon-a and -pare 166 amino acids in length with an additional 20 amino acid secretory peptide present on the aminoterminal end [l08]. Interferon-yis 143 amino acids in length and also contains a 20 amino acid secretory peptide [109]. Although interferon -p and interferon-yare glycosylated when produced by eukaryotic cells, biologic differences from the nonglycosylated proteins produced in E. coli have not yet been identified [110]. All three classes of interferons possess antiviral, anti proliferative and immunomodulatory activities that may playa part in antitumor activity. Interferon receptors have been found on the surface of most cells, although cell lines lacking receptors have been identified [111]. Interferons-a and -p have been shown to compete for the same high-affinity receptor, while interferon-y binds to a separate receptor, suggesting similarities in the tertiary structures of interferons-a and -P[112]. The receptor for interferon-yhas recently been cloned and sequenced [113]. The presence of the appropriate receptor partially governs sensitivity of a particular cell to interferon a, p, or y. 3.2

Mechanism of antitumor action

Interferon preparations have been shown to inhibit the growth of a number of tumor cells and established cell lines in vitro (114-116]. Interferon also inhibits the growth of normal cells in culture and there is no convincing evidence that tumor cells are more susceptible than normal cells to the cytostatic effect of interferons [115]. Although all


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interferons have antiproliferative activity, interferon-y is considered more potent than interferon-a/p per antiviral unit [117-120]. Furthermore, there is evidence for a synergistic potentiation of the antiproliferative activity between Interferon-yand interferon-a or -P[120], suggesting differences in their mechanisms of action. Other direct antitumor effects include the ability otinterferons to induce cell differentiation, enhanced expression of cell surface. antigens and oncogene expression in tumors [reviewed in ref. 89]. Some tumors with resistance to the cytostatic effect of interferons in vitro have exhibited sensitivity in vivo, implicating a contribution of host response to the antitumor effect [121-124]. For example, interferon-sensitive and interferon-resistant Friend leukemia cells showed equal response rates to partially-purified murine interferon-a/ P preparations in vivo [122, 123]. Similarly, L 1210 leukemia cells that were resistant to interferon in vitro demonstrated sensitivity in vivo to interferon treatment [124]. In addition to direct anticellular mechanisms, interferons may affect tumors through a variety of other actions. They activate and enhance the activity of several antitumor effector cells, including NK cells [125-128] macrophages/monocytes [129-131] and sensitized T cells [132]. Antibody-dependent, cell-mediated cytotoxicity, mediated by subpopulations of these effector cells, can also be boosted by interferons [133, 134]. Interferon-y is significantly more potent than other interferons at enhancing NK activity [135] and at activating macrophages [136]. In fact, macrophage-activating factor (MAF) and interferon-y have been shown to be the same substance [129]. It is important to note that doses of interferons lower than that maximally tolerated, indeed as much as 100-fold lower, have demonstrated optimal enhancement of NK cell and monocyte cytotoxic activity [137-139]. The antitumor activity of interferons in animal models has been reviewed in detail elsewhere [115,140-143]. In vivo administration of interferon inhibited the growth of a wide variety of transplantable murine ascitic or solid tumors of different origins (spontaneous, viral or chemically-induced) in various strains of syngeneic and allogeneic mice. Interferon treatment was only effective when continued after tumor inoculation. Optimal antitumor effects occurred when contact between interferon and tumor cells was maximal, and therapy was most effective when the tumor burden was low. In addition, prophylactic interferon treatment markedly delayed the appearance and re-

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duced the frequency of mice bearing spontaneous lymphomas [144] and mammary carcinomas [145]. The studies to date with animal tumor models suggest that the major clinical utility of interferon would be in the adjuvant setting to eliminate residual disease after the bulk of the tumor had been eliminated by conventional therapeutic means and that interferon would not be useful in the treatment of bulky, disseminated tumors. Interferon was not able to reproducibly induce a significant regression in a clinically detectable animal tumor [146]. 3.3

Clinical aspects

Interferon-a has been extensively studied over the past decade in clinical research and the most impressive results have occurred in the hematologic malignancies [reviewed in ref. 147]. The most impressive therapeutic results were achieved and later broadly confirmed, with using interferon-a for therapy of hairy cell leukemia, a rare B-cell disorder for which no effective therapy existed previously [148]. Even though interferon-a is effective in reversing the clinical manifestations of hairy cell leukemia, it does not appear to be curative since a significant percentage of patients relapse within 6 months after cessation of therapy. Although therapeutic activity has been reported, other hematologic malignancies are not as sensitive as hairy cell leukemia to relatively low dosages of interferon-a [149]. However, objective responses have been observed in 40-50% of patients with lowgrade non-Hodgkin's lymphoma [150, 151] and multiple myeloma [152, 153] and in > 50% of patients with chronic myeloid leukemia [154-156].

Antitumor activity for interferon-a has been quite limited for solid tumors [90, 149, 157-160]. Small, but definite responses to interferon therapy have been observed in renal cell carcinoma, malignant melanoma, and Kaposi's sarcoma. Trials with interferon-p and -yare lagging behind interferon-a. Given the in vitro and animal model data that interferon -p and interferon-y may be more active than interferon-a in solid tumors and given recent observations of synergism between various interferons for antiproliferative activity [120, 161-163], it will be of interest to determine the clinical value of other interferons in solid tumors and of combination studies with other cytokines and cytotoxic agents. In addition, further studies need to address the effects of interferons in patients with smaller tumor burden


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and in the adjuvant setting in patients with micrometastatic disease [164]. Aside from biological response modification, interferons have been shown to potentiate the cytotoxic effects of 5-fluorouracil (5FU) in a concentration-dependent fashion in vitro [165-169]. A mechanism for synergy between these agents may entail enhanced thymidylate synthase inhibition by 5FU when cells have had prior exposure to interferon [168]. Gewert and coworkers showed that interferon treatment can decrease thymidine kinase activity, inhibiting thymidine incorporation into DNA and decreasing the rate of phosphorylation of thymidine in Daudi cells [170]. Elias and Sandoval demonstrated that enhanced 5-fluoro-2-deoxyuridine 5-monophosphate (FdUMP) accumulation occurs in interferon-treated HL-60 cells without an increase in 2-deoxyuridine-2'monophosphate (dUMP) [169]. In addition, in a murine model, interferon was shown to protect host tissues against 5FU-induced toxicity, allowing delivery of higher doses of the antimetabolite [171]. Several clinical trials have been initiated to exploit this potential synergy between interferons and 5FU [172-174]. Although the preliminary results are encouraging, additional randomized trials are necessary to clarify the effectiveness of combinations of 5FU and interferons in cancer treatment. The major side effects with interferon-a have been those of a flu-like illness (fever, chills, muscle aches, headache, gastrointestinal upset and fatigue). These effects have been reviewed by Quesada and coworkers [175]. Several cardiovascular effects have been reported, including hypotension, cardiac dysrhythmia, tachycardia and premature ventricular extrasystole. Transient leukopenia and neurotoxic changes, including somnolence, confusion and overall mental and motor slowing have been reported at higher dose levels. These toxicities are reversible within days after cessation of interferon-a. 4

Myeloid colony stimulating factors

Blood cell development is regulated by a group of glycoprotein hormonal growth factors, collectively known as colony-stimulating factors (CSFs), that control the differentiation and proliferation of progenitor cells in the bone marrow. The term CSF derives from the in vitro observation that these factors stimulate progenitor cells of different hematopoietic cell lineages to form discrete colonies of recog-

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nizable maturing cells. Four hemopoietic CSFs have been described that are capable of stimulating the production of granulocytes and macrophages. These have been identified and classified according to the major cell lineage on which they exhibit their most obvious effects. Thus, IL-3 (multi CSF) targets the early cell lineage, granulocyte-macrophage CSF (GM-CSF) targets both the granulocyte and macrophage lineages, macrophage CSF (M-CSF) targets only macrophages and granulocyte CSF (G-CSF) targets only granulocytes. It has become abundantly clear that these definitions are too simplistic, since these molecules can act synergistically with each other and also induce other growth factors or modulators of various cell classes. For example, GM-CSF and IL-3 induce production of G-CSF by human monocytes [176]. Another example is that M-CSF induces interleukin-l from macrophages, which in tum stimulates fibroblasts to synthesize GM-CSF and G-CSF [177] and keratinocytes to produce CMCSF [178]. These extensive "networking" interactions extend tremendously the actions of growth factors. These cross-induCtions of CSFs make it difficult to attribute an activity to a particular CSF in a mixed culture system in vitro or when administered in vivo. 4.1

Molecules

GM-CSF. Metcalf and his associates first defined the hematopoietic properties of GM-CSF, which included stimulated growth in vitro of bone marrow granulocyte and/or macrophage progenitor cells to form colonies [179, 180]. Growth of erythroid and megakaryocyte progenitors is also supported by GM-CSF under the appropriate conditions [181, 182]. Human GM-CSF is a glycoprotein containing 127 amino acids. The molecular weight of GM-CSF varies according to the degree of glycosylation, ranging from 14 to 35 kD. The molecule is produced by T-Iymphocytes, natural killer cells, marrow stroma, activated macrophages, fibroblasts, endothelial cells, keratinocytes and astrocytes [183-191]. T-Iymphocytes are probably the major source of GM-CSF. The coding sequence for GM-CSF has been isolated and cloned [192], and large amounts of recombinant GM-CSF have been prepared for use in preclinical and clinical studies. The factor produced by expression of cDNA in bacteria is not glycosylated [193], but glycosylation of human GM-CSF is not required for bioactivity in vivo.


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Two different GM-CSF receptors have been identified. Normal hematopoietic cells exhibit a singular class of high affinity (kD in pmol range) receptors for GM-CSF [194-196]. Mature neutrophil and eosinophil granulocytes express between 293 and 1000 receptors per cell. G-CSF. Human G-CSF was first purified to homogeneity from the human bladder carcinoma cell line, 5637 [197]. The polypeptide contains four cysteine residues and has no N-glycosylation sites. Neuraminidase treatment and O-glycanase treatment of natural G-CSF reduced its molecular mass from 19,600 to 18,000 Da, indicating O-glycosylation of the native molecule. The gene was cloned from this cell line by Souza and colleagues [198]. The recombinant protein has been successfully expressed in E. coli and has a molecular weight of 18,000. Glycosylation is not required for in vitro or in vivo biologic activity. The cellular sources for G-CSF include monocytes, fibroblasts and endothelial cells [176, 199,200]. G-CSF receptors range in number from 100-200 to a maximum of 1000-2000 on cells of the neutrophil granulocyte lineage. Only one class of high affinity receptors (apparent kD 60-100 pM) has been reported with receptor numbers increasing with differentiation [201, 202]. Receptors are present in low· numbers on monocytes, macrophages and promonocytes, but absent on eosinophils, lymphocytes and erythroid cells. The apparent molecular weight of the G-CSF receptor was 150,000 as determined by chemical cross-linking [201]. IL-3. Interleukin-3 was initially purified from conditioned media from the WEHI-3B murine myelomonocytic leukemia cell line [203]. Both the murine [204, 205] and human [206, 207] genes for IL-3 have now been cloned. Interleukin-3 is a complex glycoprotein, ranging in size from 14 to 28 kD. It is a product of activated T-helper cells that have been stimulated by antigens or lectins [208-210]. IL-3 is probably the least restricted of the CSFs with regard to cell lineage, supporting progenitors at early stages of hematopoietic development, but needing other factors for the process of terminal differentiation [211]. The progenitor cell targets include myelomonocytic, erythroid, platelet and even lymphoid precursors [212]. M-CSF. Human M-CSF was purified from urine [213] and was the first of the CSFs to be biochemically defined. The molecule was comprised of a dimer of two identical polypeptide subunits with variable degrees of complex N-linked glycosylation to yield a native glycopro-

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tein of 47-76 kD. The genes from murine [214] and human [215] M-CSF have now been cloned and expressed. M-CSF is produced by monocytes and endothelial cells. The receptor for M-CSF has been well characterized and is a product of the fms protooncogene located on chromosome 5 [216]. 4.2

Biological activities

Chemotherapy-induced neutropenia poses a basic problem in cancer therapy. The mature neutrophil provides the primary cellular defense against bacteria and is an important component of the acute inflammatory response. Since myelosuppression limits the maximum dose of some cytotoxic drugs that.can be administered, it seems likely that better antitumor effects could be achieved if marrow damage by these drugs could be ameliorated [217]. Indeed, there is evidence that the dose intensity of chemotherapy can determine the clinical outcome. GM-CSF. Preclinical studies in mice [218] and non-human primates [219,220] showed that circulating numbers ofneutrophils and macrophages were augmented by the administration of recombinant human GM-CSF. The most notable change was an increase in neutrophil counts. In normal animals, continuous or daily infusion of GM-CSF led to three distinct biological responses; as detected by changes in the peripheral blood neutrophil counts. The first change was a transitory neutropenia (>90% decrease in neutrophil count) which occurred over the first 5 to 120 min of intravenous GM-CSF treatment. This was followed by a neutrophilia which lasted for about 72 hr and is presumably the result of a mobilization of neutrophils from storage pools. A third phase consisted of a further increase in neutrophil counts and occurred after 72-96 hr of GM-CSF treatment. In this phase, newly formed neutrophils were released from the stimulated bone marrow. Moreover, GM-CSF treatment has been shown to stimulate recovery of neutrophils one week earlier than controls in monkeys receiving ablative irradiation and autologous marrow grafting [221,222]. GM-CSF appears to control the proliferation of CSF-dependent cells through an action during the G, phase of the cell cycle [223]. Aside from the ability to induce proliferation of myeloid progenitor cells, GM-CSF also enhances the function of terminally differentiated cells such as neutrophils, monocytes, macrophages and eosino-


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phils. The direct effects of GM-CSF on human neutrophils include enhanced phagocytosis [224, 225], superoxide production [226, 227], increased expression of cell surface adhesion proteins [228, 229], inhibition of neutrophil motility [230, 231], and changes in the number or affinity of surface receptors [232? 233]. GM-CSF also stimulates neutrophil antibody-dependent cytotoxicity for human target cells [234] and has priming effects on neutrophils which are not observed unless the cells are subsequently stimulated with a chemotactic agent [232, 233, 235-237]. GM-CSF also exerts its influence on the function of mature macrophages. It enhances the killing of Leishmania tropica by murine peritoneal macrophages [238] and cytotoxicity of the malignant melanoma cell line A375 by human monocytes [239]. GM-CSF, in a monocyte model of human immunodeficiency virus (HTLV-III), substantially restricted virus expression [240]. G-CSF. G-CSF stimulates granulocyte-macrophage progenitors, acts synergistically with IL-3 to stimulate megakaryocyte and blast colonies and interacts with G-CSF, GM-CSF and IL-3 to stimulate high proliferative potential colony forming cells. On mature neutrophils, G-CSF has been shown to enhance the specific binding of the chemotactic bacterial peptide fMet-Leu-Phe and promote chemotaxis [241], to augment neutrophil-mediated antibody-dependent cellular cytotoxicity [242, 243] and to prime for enhanced oxidative metabolism [244, 245] and release of arachidonic acid metabolites [246]. Animal studies have demonstrated that G-CSF has potent effects in producing neutrophilia and decreasing the severity of chemotherapyinduced granulocytopenia [247]. IL-3. IL-3 induces proliferative responses in precursors of all myeloid lineages, including eosinophils, basophils, megakaryocytes, neutrophils and monocytes. Like the other myeloid growth factors, IL-3 induces differentiation, as well as proliferation, with relatively selective effects on mature mast cell and eosinophil function [207]. It acts synergistically with a number of other hematopoietins, including G-CSF, GM-CSF, M-CSF, erythropoietin and interleukin-l [212, 248-250]. IL-3 induces basophil histamine release [251], stimulates monocyte cytotoxicity through a tumor necrosis factor- (TNF) dependent mechanism [252] and augments calcium ionophore-induced generation of leukotriene C4 by human eosinophils [253].

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Interleukin-3 is the only CSF that is able to stimulate mast cell proliferation in vivo [254]. A dramatic dose-related effect on splenic levels of mast cells was observed (up to 100-fold increase in numbers). IL-3 was also tested on Chemotherapy-induced myelosuppression in monkeys [255]. There was no significant impact on neutrophil recovery, although eosinophils and basophils increased. Concomitant treatment with IL-3 and GM-CSF was not better than GM-CSF alone. Krumwieh and Seiler administered IL-3 daily by intravenous bolus injection to cynomolgous monkeys [256]. Pharmacokinetic studies in these animals indicated a half-life of 5-10 min. IL-3 alone did not increase neutrophil counts. However, when IL-3 was given for 8 days followed by 5 days of GM-CSF, synergistic myelomonocytic responses were observed. M-CSF. M-CSF acts primarily on stem cells of the macrophage lineage. Initial observations suggested that human M-CSF had only marginal activity on human macrophage progenitors. However, when human marrow progenitors are exposed to picogram concentrations of GM-CSF, their responsiveness to M-CSF is markedly enhanced [257]. Similar synergy has been noted with other growth factors [258, 259]. The production of M-CSF by stomal cells in the bone marrow microenvironment may playa critical role in the self-renewal of early hematopoietic progenitors [260]. M-CSF also enhances the functional activity of mature macrophages. It has been shown to increase the capacity of murine macrophages to secrete oxygen reduction products [261], induce production of a variety of monokines, including interleukin-l, interferons and TNF [246, 262] and enhance monocyte differentiation in serum-free cultures [263]. 4.3

Clinical aspects

The capacity of the myeloid colony-stimulating factors to stimulate blood cell production and maturation has enormous potential in management of both spontaneous and iatrogenic hematologic disorders that are characterized by too few or poorly functioning white blood cells [reviewed in refs 264-272]. One of the most promising applications for the myeloid CSFs is their potential to decrease or eliminate the neutropenia associated with cancer chemotherapy. Suppression of granulocyte production by cytotoxic drugs leaves the patients


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highly susceptible to serious bacterial infection, making neutropenia the dose-limiting toxicity of most cytotoxic drugs [273]. The ability to stimulate granulocyte production by myeloid growth factor administration in conjunction with antineoplastics could decrease the morbidity and mortality of chemotherapy, or permit the administration of higher or more frequent (and potentially more curative) doses of cytotoxic drugs. GM-CSF. The immediate effect of GM-CSF administration in humans is an acute transient fall in numbers of circulating neutrophils, monocytes and eosinophils [274]. The white blood cell counts return to normal after 4-6 hr. The transient leukopenia is due to margination of neutrophils and monocytes predominantly in the pulmonary vasculature as a result of enhanced expression of cellular adhesion molecules [228, 229]. Thereafter, GM-CSF induces a dose-dependent granulocytosis and monocytosis in humans [275-278]. This rise in granulocyte counts occurs within 20 to 48 hr, reaches a maximum within 10 days and is maintained at a constant level during the duration of GM-CSF treatment. In 17 patients with advanced cancer, both intravenous bolus or continuous infusion of GM-CSF over a 5- to 9-day period resulted in a twofold to fourfold increase in absolute neutrophil counts and increases in eosinophils and monocytes [279]. GM-CSF administration after the first cycle of chemotherapy significantly shortened the periods of neutropenia with a trend toward higher neutrophil nadirs in patients with sarcomas [280]. Brandt and coworkers reported a study of 19 patients with breast cancer and melanoma given high-dose chemotherapy followed by autologous bone marrow transplantation [281]. Following chemotherapy and reinfusion of bone marrow, patients received a 14-day continuous infusion ofGM-CSF at escalating doses to sequential patient groups. The period of neutropenia was reduced at doses of GM-CSF above 8 pg/kg/day. In addition, there were fewer episodes of septicemia, hepatotoxicity and nephrotoxicity in patients receiving GM-CSF compared to historical controls. Recently, Herrmann and associates have administered subcutaneous recombinant human GM-CSF to patients with solid tumors with and without autologous bone marrow support after their second cycle of chemotherapy [282]. These patients were given a single daily s.c. dose of GM-CSF (250 pg/m2/day) beginning 48 hr after high-dose chemotherapy and continuing for 10 days. There was a significant reduc-

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tion in the duration of neutropenia and hospitalization and decreased antibiotic requirements. Although the initial clinical trials are encouraging, the potential value of human GM-CSF in reversing the leukopenia associated with cancer treatment and in augmenting host defense against infection remains to be determined. In general, the results of phase 1 trials have suggested that continuous infusion of recombinant human GM-CSF produces optimalleucocyte increments with least toxicity [283]. In studies to date, the toxicities were similar and included fever, myalgia, bone discomfort, dyspnoea, pruritis and headache. At higher doses, substantial toxicity might appear, including thrombosis of major vessels with pulmonary embolism, pericardial or pleural effusion and other signs of capillary leak syndrome. Some ofthe toxic manifestations may be secondary to GM-CSF's ability to induce several key inflammatory cytokines, including interleukin-l [284, 285], tumor necrosis factor [286], granulocyte colony-stimulating factor [287] and macrophage colony-stimulating factor [288]. It remains to be clearly demonstrated whether treatment with GMCSF can accelerate cancer chemotherapy treatment by shortening the intervals between the single cycles and whether in these regimens, increased doses of chemotherapy can be used~ If this is indeed the case, additional long-term studies will be required to find out whether shortened intervals of chemotherapy and the application of higher doses of cytotoxic drugs will lead to an improvement in response rates and long-term results. Future clinical trials may also be anticipated based upon combinations of hematopoietic growth factors designed to ensure optimum regeneration and functional activation ofthe diversity of hematologic and immunologic differentiation pathways. G-CSF. G-CSF has also been tested for amelioration of the myelosuppressive side effects of intensive chemotherapy. Morstyn and colleagues showed that G-CSF treatment can shorten the duration of the neutrophil nadir after melphalan chemotherapy for a variety of malignancies [289]. Patients did not respond as well if they had been extensively pretreated with chemotherapy or radiotherapy. In addition, G-CSF has been shown to reduce the neutropenia caused by combination chemotherapy in small cell lung cancer [290, 291] and transitional cell carcinoma of the bladder [292]. Gabrilove and coworkers noted that the addition of G-CSF increased the percentage of patients


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able to receive their subsequent planned course of chemotherapy without delay from 29 % to 100 % [292]. The concept that dose intensity is the most important determinant in the effectiveness of chemotherapy in a responsive neoplasm [217, 293] has prompted Bronchud and colleagues to study the effect of intensive doxorubicin therapy for patients with metastatic ovarian and breast cancer [294]. The conventional upper dose of doxorubicin as a single agent is 75 mg/m2 every three weeks. In this study [294], doses of up to 150 mg/m2 could be given every two weeks. The nadir in granulocyte count occurred earlier and was shorter than in control patients, returning to safe levels well before 14 days. Although the number of patients studied was small, it was encouraging that all patients receiving 125 mg/m2 or greater responded to single agent therapy. Additional studies are required with G-CSF administration for longer periods of time in randomized studies to determine whether G-CSF not only causes a reduction in the period of neutropenia, but also protection against bacterial and fungal infections. IL-3 and M-CSF. Investigations of recombinant IL-3 in humans are currently in early phase I trials. Kurzrock and colleagues treated patients with IL-3 daily for 28 days at doses ranging from 30 to 1,000 pg/m2 [250]. A total of 24 patients with bone marrow failure caused by myelodysplastic syndrome, aplastic anemia, or prolonged high-dose chemotherapy were entered on this study. Responses were observed in all lineages, although the type and degree of response varied tremendously between individuals. Similarly, Ganzer and associates observed dose-related increases in white blood cell, platelet, neutrophil, eosinophil and lymphocyte counts in all patients receiving daily subcutaneous doses of IL-3 (30-500 pg/m2) [295]. Increases in reticulocyte and basophil counts were also noted. Side effects in these studies were minimal and consisted mainly of low-grade fever, mild headaches and local erythema at the injection site. In vitro and animals studies have suggested that the most important clinical applications of IL-3 may be in synergistic combinations with other growth factors [212, 248-250] or to expand early progenitor cells in vitro [296]. Human M-CSF has been purified from human urine and served as the source of M-CSF for the first clinical trials of any purified hematopoietic growth factor. Motoyoshi and coworkers treated 24 cancer patients with two consecutive courses of the same chemotherapeutic regimen [297]. Each cycle was followed by either a 2-hr intravenous

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infusion of human serum albumin or partially purified urinary M-CSF for five days. M-CSF treatment was demonstrated to hasten the recovery of adequate neutrophil counts, although this effect is minimal and not yet reported to be associated with a decreased risk of infection. The ultimate potential of M-CSF awaits the results of higher dose trials with the· recombinant factor, which are ongoing. The enhancement by M-CSF of the functional properties of macrophages may provide a clinical role for M-CSF in the augmentation of nonspecific host defenses against infectious diseases and cancer. 5

Concluding remarks

The use of cytokines represents the emerging fourth modality of cancer treatment. The cytokines discussed in this brief review represent only a minute fraction of the many agents that can be expected to become available in the near future for development toward clinical trial. For example, sixteen hematopoietic growth factors have now been produced in recombinant form and are potentially available for clinical use either alone or in various combinations [298]. Adequate testing of these agents will be a formidable logistical problem for clinicians. Recombinant cytokines are toxic when administered at high doses in vivo [299-302]. The toxic systemic effects suggest that orchestration of local inflammation is their true biologic role and exaggerated production or exogenous addition may be highly injurious. Currently, efforts are underway to minimize cytokine toxicity while maintaining therapeutic efficacy. In conclusion, a measure of cautious optimism can be expressed in considering the future of cytokines in cancer therapeutics. Additional studies need to clarify key mechanism(s) of antitumor action for each factor in order to develop optimal therapeutic protocols. A different set of guidelines are needed than those used for the evaluation of chemotherapeutic agents, since it is becoming apparent that the optimal therapeutic dose is not necessarily the same as the maximal tolerated dose. To date, most clinical trials with cytokines have been predicated on a "more is better" philosophy, despite bell-shaped immunomodulatory and therapeutic response curves from preclinical models [303]. In addition, preclinical models suggest that these factors will act optimally in a minimal residual disease setting, especially when combined with the existing treatment modalities.


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The past decade of research and major developments in recombinant DNA technology have demonstrated that there is indeed a role for biologic products of the human genome in cancer therapy. The promise of cytokines is great, but the problems of selection and evaluation of agents, determination of optimal protocols and selection of appropriate combination modalities are exquisitely complex. Dr. Ehrlich concluded his address in 1900 with the vision that: "We no longer find ourselves lost on a boundless sea, but that we have already caught a distinct glimpse of the land which we hope, nay which we expect, will yield rich treasures for biology and therapeutics" [25]. Hopefully, we will realize these rich treasures in the near future. Acknowledgments

The author wishes to thank Dr. David E. Seitz for advice and critical review of the manuscript.

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