Immunomodulators Future prospects B. C.M.J. Takx-KShlen Introduction The biological response modifiers are sometimes called the drugs of the 90s. In this article the immunomodulators t h a t are most important today and most likely to emerge as important in the near future will be discussed. To provide background for understanding the action of immunomodulators, the basic principles of the immune system will be reviewed, with special attention given to the cytokines. The haematopoietic system is of great importance. From one pluripotent stem cell are derived the lymphoid stem cell, a progenitor of the lymphocytes, and the myeloid stem cell, a progenitor of all other blood cells. Immune response In m a i n t a i n i n g its integrity and health every living organism faces the challenge of being able to recognize and distinguish the difference between ' s e l f (its own molecules, cells and tissues) and 'nonselF (all other organisms or substances). The purpose of i m m u n e defenses is to recognize the millions of different components in the nonself world and to respond to them in a way t h a t will eliminate t h e m from the self environment and cause no damage to the self [1]. Nonself chemical and physical substances t h a t are capable of inducing specific immunologic responses are called antigens. Before a pathogenic organism can cause disease after entering the lost organism it must break t h r o u g h three levels of integrated immune defenses (Figure 1).
First level At the first level of defense, a microbe is sensed by the host as foreign and innate inflammatory defence reactions occur. These defence mech-
anisms are not specific and involve phagocytic cells (first neutrophils and later macrophages), natural killer cells, cytokines and complement. Second level At the second level highly specific immune responses occur: lymphocytes and cell p r o d u c t s (antibodies and cytokines) interact with one another and with other elements of the i m m u n e system. Macrophages from the first level process antigens and present t h e m to two groups of lymphocytes: B cells (originating from the Bursa of Fabricius; in humans, probably from bone marrow) and T cell s (derived from the thymus). These lymphocytes have antigen-specific receptors on their surfaces which can recognize millions of different antigens. Each lymphocyte can recognize only one antigen, however. Humoral i m m u n i t y is the major defence system against most bacterial and Viral infections. It is mediated by the action of B lymphocytes which, after recognizing an antigen, produce soluble components, immunoglobulins or antibodies, t h a t circulate in the serum. Complement components and cytokines are necessary for appropriate action. There are five classes of immunoglobulins, IgG, IgM, IgE, IgA and IgD. IgG and IgM are extremely important for the i m m u n e response. Cellular i m m u n i t y constitutes the major defence against parasites, fungi, cancer, viruses and a few bacteria. It is, however, also responsible for delayed allergic reactions and rejection of foreign nonself tissue transplants. The major components of cellular i m m u n i t y are T lymphocytes, phagocytes, n a t u r a l killer cells, killer cells and lymphokines produced by these cells [1].
Takx-KShlen BCMJ. Immunomodulators. Future prospects. Pharm Weekbl [Sci] 1992;14(4A): 245-52. Keywords Cytokines Haematopoietic growth factors Immunomodulators Interferons Interleukins Lymphocytes Lymphokines Monoclonal antibodies Tumor necrosis factors
B.C.M.J. Takx-K6hlen: Department of Clinical Pharmacy and Toxicology, University Hospital of Maastricht, P.O. Box 5800, 6202 AZ Maastricht, the Netherlands.
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Abstract The future role of the immunomodulators in medical practice is yet to be defined. The key question is whether these new substances will bring remarkable progress in transplantation or in the treatment of such conditions as cancer, AIDS, and autoimmune diseases, or whether they will be of only minor adjunctive importance. As background to the discussion of immunomodulating agents, the immune system is explained, with emphasis on the roles of T and B lymphocytes, macrophages, phagocytes, human leukocyte antigen and the complement system. Special attention is given to the cytokines, particularly the lymphokines. The immunomodulators can be divided into three main groups: immunosuppressive agents, such as FK 506 and rapamycin; immunostimulating agents, of which BCG vaccine is most important; and the remaining immunomodulators, which include the biological response modifiers.The last group, which encompasses the colony-stimulating factors (GM-CSF, G-CSF, and M-CSF), the interleukins, the interferons, and the tumour necrosis factors, is described in detail. Innovative research and medical applications of these cytokines, including indications, contraindications, and adverse reactions, are discussed. The role of monoclonal antibodies against endotoxins is also described.
Accepted June 1992.
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Figure
1 T cells can be divided into various subsets according to the different biological functions they perform. These cellular subsets include T4 cells,
Figure
2
immunity against extracellular pathogens (for example bacteria) plasma cell
also called CD4 (helper or inducer T cells), and T8 cells, also called CD8 cells (memory or delayed hypersensitivity T cells, cytotoxic T cells and suppressor T cells) [2]. The helper T4 lymphocytes are part of the regulatory system of the immune network. The macrophage, after processing the antigen, stimulates the antigen-specific helper T4 cell via direct cell-to-cell contact.The stimulated helper T4 cell then secretes interleukin-2 to stimulate other helper T4 cells as well as cytotoxic and delayed hypersensitivity T8 cells. Stimulated helper T4 cells can also produce lymphokines that stimulate B cells. Suppressor T8 cells play a regulatory role by inhibiting the growth and function of helper T4 cells through downregulation or negative feedback. Delayed hypersensitivity T8 cells are involved in the release of lymphokines that promote inflammation. This immune response typically occurs 24 to 48 h after the initiation of the event [2]. The cytotoxic T8 lymphocytes are responsible for antigen-specific cytotoxicity; they are capable of attaching to specific antigen-bearing cell membranes by direct cellular contact. These cytotoxic T8 cells are the lymphocytes mainly responsible for graft rejection [2]. The functions of the suppressor T8 cells include suppressed proliferation of helper T4 cells, decreased stimulation of cytotoxic T8 cells and suppressed production of antibody from plasma cells. The suppressor T8 cells also play a role in the tolerance of transplanted tissues [2]. Figure 2 illustrates the differences between humoral and cellular immune responses. The complement system initiated through a n t i g e n antibody complexes forms a cascade of events which ultimately leads to cell death. In the case of humoral immunity, the CTL (cytotoxic T8 lymphocyte) recognizes only a viral peptide, which is processed by antigen presenting cells (from the first line of defence) presented in the context of MHC molecules [3]. MHC (major histocompatibility complex), also called class I HLA
immunity against intracelluiarpathogens (for example viruses)
CTL
macrophage
~T%. OoO
"
V
=:=
9
extracellular fluid
I
cell receptor TCR viral peptide
~ J molecule ~ ) MHC class
(
virus infected cell w
246
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(human leukocyte antigen), is a class of antigens which can be found on most cell surfaces, except those of m a t u r e red blood cells. Class II HLA antigens are found on only a few cells, including B cells, activated T cells, macrophages, vascular endothelial cells, epidermal cells and sperm [2]. The MHC is very important in cases of graft rejection and graft-versus-host disease [4]. After stimulation of the T cells, lymphokines are produced. These lymphokines are polypeptide products (glycoproteins) t h a t participate in a variety of cellular responses including the regulation of the immune system. It is now clear t h a t neither the production of lymphokines nor their effects are restricted to lymphoid cells. For this reasons the t e r m lymphokine has been replaced by the more general t e r m cytokine [5]. Because lymphokines provide a molecular mechanism for communication among leukocytes they are also called interleukins (ILs). To date, 12 interleukins have been identified. N K (natural killer) cells and K (killer) cells are nonphagocytic lymphocytes not directly classifiable as T or B lymphocytes. N K and K cells are of importance in the mechanism of the immune response against tumours. Native and recombin a n t interleukin-2 can stimulate the generation of LAK (lymphokine activated killer) cells and TIL (tumour infiltrating lymphocytes). LAK cells are an ill-defined group of lymphocytes (probably K cells) t h a t display a wide range of nonspecific killing activity in vitro after stimulation of such lymphokines as IL-2. They are generated in vitro after exposure of lymphocytes to IL-2 for 3-5 days and can also be generated in vivo by administration of IL-2 [6]. TIL, a bulk of various subpopulations of lymphocytes t h a t infiltrate a tumour, have specific immunoreactivity with the tumour cells t h a t they infiltrate. They are obtained by removing cancerous tissue or a nodule from a patient and forming a cell suspension by enzymatic digestion or chopping the sample into small fragments [6]. IL-2-stimulated TIL express highly specific cytotoxic activity against autologous tumours in a subgroup of cancer patients. In mice TILs have an a n t i t u m o u r effect t h a t is 50 to 100 times as large as t h a t of LAK cells on a cell-to-cell basis [6].
Third level At the third level of defence there is concerted action of both the innate defence reactions and the products of specific acquired immune responses [1]. Immunomodulators
Immunomodulators are agents t h a t interfere with the actions of the immune system [7 8]. They can be divided into three main groups: immunosuppressive agents, immunostimulating agents and other immunomodulators (Table 1).
Irnmunosuppresants Two immunosuppressant agents, F K 506 and rapamycin, are now under investigation [9]. Other agents are beyond the scope of this paper and will not be considered. 14(4A) 1992
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Table
1
Immunomodulators Immunosuppressive agents Glucocorticoids Cytotoxics Antilymphocyte immunoglobulin - Muronomab CD3 - ATG Other - ciclosporin - F K 506 - rapamycin Immunostimulating agents BCG vaccine Levamisole Other immunomodulators Haematopoietic growth factors Interleukins Interferons Tumour necrosis factors Monoclona] antibodies
FK 506 is a macrolide immunosuppressant which has a mode of action comparable to t h a t of cyclosporin [10]. In vitro studies with helper T lymphocytes have shown that F K 506 is at least 100 times as potent as ciclosporin in selectively inhibiting the secretion of IL-2, IL-3, IL-4 and interferon-7 inresponse to different stimuli. In addition, F K 506 may also impair the expression of IL-2 receptors on nonself-antigen stimulated T cells. Although its precise molecular action is not understood, F K 506 (like ciclosporin) inhibits early, calcium-dependent events in signal transduction t h a t usually follow T cell stimulation [10]. FK 506 has greater hepatotropic properties t h a n ciclosporin, which may explain its particular success in liver transplantation. A prospective randomized study to compare F K 506 with ciclosporin immunosuppression in liver transplantation has begun in 8 centres in Europe and a multicentre study of over 650 liver graft recipients is now underway in North America. The side-effects of FK 506 and ciclosporin in h u m a n s are similar, and include nephrotoxicity. However, hirsutism, gingival hyperplasia and coarsening of facial features have not yet been reported for FK 506 [10]. Rapamycin is a powerful immunosuppressant under investigation in animals [11]. Its mechanism of action differs from that of F K 506 and is r a t h e r complex. Like FK 506, rapamycin is especially toxic in dogs. However, the vasculitic laesions t h a t develop with rapamycin are largely confined to the gastrointestinal tract, whereas vasculitis associated with FK 506 is widespread, and involves the heart. This effect is probably species-specific [11].
Immunostimulating agents BCG vaccine is currently the most important immunostimulant. Topical BCG is used as a 247
Table 2
Cytokines Group name (abbreviation)
Subtypes
Alternative names
Colony-Stimulating Factors (CSF)
G-CSF GM-CSF M-CSF multi-CSF IL-1 IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 up to IFN-a 2a IFN-~ 2b IFN-~ IFN-7 TNF-a TNF-/~
Neupogen | Leucomax LAF IL-3
Interleukins (IL)
Interferons (IFN)
T u m o u r Necrosis Factors (TNF)
therapy for recurrent superficial bladder cancer [12]. Its mechanism of action may involve an HLA-restricted T cell response. There is clear evidence that t r e a t m e n t induces HLA class II antigen expression on t u m o u r cells and there is also a direct correlation between the response to therapy and the amount of IL-2 released into the urine. Studies are being performed with topical BCG in the t r e a t m e n t of head and neck tumours and melanomas. Levamisole is a synthetic orally active agent t h a t has anthelmintic and immunomodulatory properties. Levamisole combined with fluorouracil has been associated with a one-third reduction in recurrence and risk of death in patients with surgically resected stage C colon cancer [13]. The mechanism by which levamisole affects the human immune system is complex and not yet fully understood. The majority of in vivo and in vitro data suggest t h a t levamisole acts as an immunorestorative agent. It is capable of augmenting a depressed host immune system but is incapable of hyperstimulating an intact system. The most serious adverse effect associated with levamisole is granulocytopenia [13].
Proleukin | B-cell stimulating factor 1 B-cell stimulating factor 2 Roferon-A| Intron A | cachectin lymphotoxin
probably of importance in this differentiation from the pluripotent stem cell to the more restricted colony-forming unit stem cell (CFUs). In vitro these cytokines have little activity on proliferation but together they stimulate the production of early progenitors. IL-3 and GM-CSF (granulocyte macrophage colony-stimulating factor) are multipotentional myeloid haematopoietic growth factors with overlapping and synergistic activities. IL-3 is more effective in stimulating early multipotent progenitors. GM-CSF, G-CSF (granulocyte colony-stimulating factor) and M-CSF (macrophage colony-stimulating factor) are relatively lineage-specific growth factors t h a t act on more m a t u r e progenitor cells (Figure 3). G-CSF, GMCSF, M-CSF and IL-3 have been purified, cloned and produced on a large scale with recombinant
Figure 3
Other immunomodulators To this group belong the haematopoietic growth factors, the interleukins, the interferons and the t u m o u r necrosis factors (Table 2). Because of their interactions with the immune system the monoclonal antibodies against endotoxins and against T N F a are also included in this category. Haematopoietic growth factors were originally termed colony-stimulating factors (CSFs) because they stimulate the formation of colonies of cells derived from individual bone-marrow progenitors. They show a mutual connection which is related to the hierarchy of haematopoiesis [14]. After cell division, one daughter cell from every pluripotent stem cell remains a stem cell and the other goes into a particular haematopoietic cell line, i.e., myeloid or lymphoid. IL-1 and IL-6 are 248
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DNA technology and are available for clinical use. Haematopoietic growth factors interact with blood cells at different levels in the cascade of cell differentiation from the multipotent progenitor to the circulating m a t u r e cell. Their biological effects are mediated by specific binding to high affinity receptors on the surface of target cells. These receptors are not restricted to normal progenitor cells and have also been detected on myeloid leukaemic cells as well as on nonhaematopoietic cells. II-1, IL-5 and IL-6. The functional properties of IL-1 include stimulation of thymocyte proliferation, accessory growth factor activity for certain T helper lymphocytes and B lymphocytes and stimulation of haematopoietic cell growth and differentation [16]. In addition to its important role in the immune system IL-1, together with TNF-a, is responsible for the cellular processes t h a t occur after inflammation [17]. IL-1 induces synthesis of acute phase proteins, prostaglandins and collagenase by fibroblasts and chondrocytes; it also acts as a pyrogen. Analysis of the effects of IL-1 is complicated because the molecule can induce production of other cytokines such as IL-6 [18]. At the moment little is known about IL-5. The molecule has been identified as a factor that selectively stimulates the proliferation of eosinophil precursors and the functional activity of mature eosinophils [20]. IL-6 has a broad range of actions on many cell types and has been indepently discovered and described as an interferon-like molecule (/~2) and as a B lymphocyte-stimulating factor. Several lines of evidence suggest t h a t IL-6 is involved in the pathogenesis of certain autoimmune diseases. IL-6 expression is not lymphocyte restricted and several tumours, including some cardiac myxomas, cervical cancers and bladder carcinomas, produce large amounts of this cytokine [19]. IL-1 and IL-5 are under investigation in vitro and in animals, while the first clinical studies with IL-6 are now underway. Colony-stimulating factors. T lymphocytes, monocytes or macrophages, endothelial cells and fibroblasts represent the major cellular sources of the haematopoietic growth factors. Activation by antigen, lectins, or interleukin-1 induces T lymphocytes to produce GM-CSF and IL-3. The immediate effect of G-CSF and GM-CSF is a transient fall in white blood cells occurring within minutes after a bolus injection. This is followed 6 to 12 h later by an increase in neutrophils. The early effects occurring within 24 h may be attributable to an accelerated release from a preformed pool of granulocytes in bone marrow, to demargination from endothelial adherent pools, and to a prolongation of survival in the circulation [15]. When G-CSF is administered the number of neutrophils rises. The principal cause of the increase in white blood cells after GM-CSF t r e a t m e n t is also neutrophilia, although the numbers of eosinophils and monocytes increase too. In general, the white blood cell counts r e t u r n to normal within a few days after cessation of G-CSF or GM-CSF therapy [15]. 14(4A) 1992
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Phase II and III clinical trials with IL-3 are underway in patients with Hodgkin and nonHodgkin lymphomas, after chemotherapy and autologous bone-marrow transplantation. MCSF is also being studied. GM-CSF (Leucomax | has been offered to the Dutch authorities for registration for a number of conditions accompanied by leukopenia, including myelodysplasia, aplastic anemia, AIDS, cancer chemotherapy, and bone-marrow transplantation [20 26]. Adverse reactions of GM-CSF are caused by activation of granulocytes, monocytes, macrophages and cytokines, such as IL-1 and TNF-a. At dose levels higher t h a n 10 ~ g . k g - l . d -1, GM-CSF frequently induces fever, malaise, myalgia, arthralgia, anorexia, mild elevations in transaminase levels, and a rash at the site of injection. More serious toxic effects have been observed at dose levels of 30 ~g. kg -1- d -i or higher. These include a capillary leak syndrome with pleural and pericardial effusions and ascites, and large-vessel thrombosis. G-CSF (Neupogen | is registered for use in patients with neutropenia resulting from chemotherapy [27]. Adverse reactions to G-CSF are relatively mild and include musculoskeletal pain, rash and elevated liver enzyme levels. In vitro studies have shown inconsistent stimulation of leukaemic cell clone proliferation by myeloid growth factors (including G-CSF, GMCSF and IL-3). In addition, however, these studies have shown t h a t myeloid growth factors may enhance myeloid leukaemic cell differentiation and leukaemic cell cycling [15]. The relevance of these in vitro findings has not yet been established in clinical studies. In the first study in patients with relapsed or refractory acute leukaemia, there was no evidence t h a t G-CSF accelerated regrowth of leukaemic cells [28]. Nonetheless, G-CSF, GM-CSF and IL-3 should not be given to patients with myeloid malignancies until there is more evidence of their safety. Thus, the role of haematopoietic growth factors in myeloid leukaemia and myelodysplasia remains investigational [29]. IL-4. IL-4 is a growth factor for activated B cells. It is also a growth factor for resting T lymphocytes and enhances the cytotoxic activity of cytotoxic T cells [5]. IL-2. IL-2, also called T cell growth factor, was first described in 1976 [30]. Its effects are initiated by its binding to a receptor glycoprotein or glycoprotein complex on the plasma m e m b r a n e of a responsive cell [6]. IL-2 activates and causes the multiplication of various types of white blood cells, including cytotoxic T lymphocytes, N K cells, LAK cells and TIL. These killer cells recognize, attack and destroy cancer cells. IL-2 stimulates T cells to help B cells produce antibodies. IL-2 further interacts with other cells that produce factors, such as interferon-~ and TNF, which directly attack cancer cells. IL-2 produced by recombinant DNA techniques is available as Proleukin and is registered for use in metastatic renal cell carcinoma. The overall response rate in patients with this neoplasm has been 15% to 30% [6 31]. Patients who have undergone prior nephrectomy and have a low t u m o u r 249
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Figure 4
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T-LYMPHOCYTE THE CYTOKINE NETWORK
INVOLVED IN I N F L A M M A T I O N
burden are more likely to respond t h a n those who have the cancerous kidney still in place or those who h a v e large t u m o u r masses. IL-2 with or without L A K cell therapy, in combination with chemotherapy, is being studied in patients with m e l a n o m a [32] and colorectal cancer [6]. Response r a t e s in m e l a n o m a v a r y from 15% to 25%. Research is still ongoing with combinations of IL-2 with L A K cells, TIL, interferon-a, -fl, -% T N F - a and c h e m o t h e r a p y [6 33-35]. The adverse effects of IL-2 are generally reversible b u t are frequently severe and dose-related. Constitutional s y m p t o m s such as fever, chills, headaches, muscle pain and malaise which are typical of most i m m u n o t h e r a p y products are essentially universal in patients given IL-2. Severe adverse effects include hypotension, edema, capillary leak syndrome and renal dysfunction. To avoid severe adverse effects, it is recommended t h a t IL-2 be given as a continuous infusion or subcutanously. However, there is some evidence t h a t the bolus dose schedule with its p e a k IL-2 levels m a y be superior in inducing lymphokine secretion in vivo. The additional triggering of endogenous lymphokines m i g h t be beneficial in tum o u r response. Thus, it would appear reasonable to test continous infusion of IL-2 in combination with the cells mentioned above and cytokines [33]. Interferons. Interferons were initially identified for their ability to interfere with viral replication in infected cells [5]. Interferon (IFN)-o (originally called leukocyte interferon) and interferon-fl (originally called fibroblast interferon) are structurally related polypeptides induced by viral infections. They have potent antiviral, antiproliferative and immunologic properties. Interferon-T is produced by immunologically stimulated lymphocytes. It is a m u c h more potent i m m u n o m o d u l a t o r t h a n either IFN-~ or IFN-/~, but has less antiviral activity [30]. The immunological functions affected by interferons include macrophage activation, stimulation of lymphocyte recruitment, regulation of IgE antibody secretion, antibody production, production of acute phase proteins and induction of MHC anti250
gens. A noteworthy property of I F N - a is its ability to enhance cytotoxic T lymphocyte activity and N K cell activity. IFN-T is an i m p o r t a n t element in the i n f l a m m a t o r y response to lipopolysaccharide [36]. IFN-a 2a and 2b produced by recombinant techniques are available as Roferen-A and Intron A, respectively, and h a v e been used for several y e a r s in cancer t r e a t m e n t . As single-agent therapy, I F N - a produces clinical i m p r o v e m e n t in approximately 90% of patients with hairy-cell leuk a e m i a and up to 70% of patients with earlystage chronic myeologenous leukaemia. In multiple myeloma, however, it is more effective in combination with c h e m o t h e r a p y [37]. The objective response r a t e a m o n g AIDS patients with Kaposi's sarcoma is 40%. I N F - a is also widely used in the t r e a t m e n t of renal cell carcinoma, m a l i g n a n t m e l a n o m a , certain non-Hodgkin l y m p h o m a s and hepatitis B [38 39]. However, as a single a g e n t it is not active against the most common cancer killers: breast, colon, lung and prostate cancers. A potential application of INF-~ lies in the t r e a t m e n t of p r i m a r y brain tumours. E a r l y trials h a v e shown a 10% to 20% response in the glioma subtype [37]. INF-~ has d e m o n s t r a t e d activity against several t u m o u r types in phase I and II clinical trials. However, response rates are low (up to 20% in renal cell carcinoma and 7% to 10% in melanoma). For patients with chronic granu]om a t o u s disease, INF-7 seems to be an effective and well-tolerated t r e a t m e n t t h a t reduces the frequency of serious infections [40]. The major side-effects of interferons are influenza-like s y m p t o m s and gastro-intestinal disturbances. For some patients these reactions are a reason to w i t h d r a w from therapy. TNF-a and TNF-~. T N F - a was first described as a substance t h a t caused h a e m o r r h a g i c necrosis of tumours. It was found in the s e r u m of animals primed with BCG or C. parvum and after stimulation with bacterial lipopolysaccharides [30]. Unlike IL-1, T N F - a seems to h a v e no direct effect on lymphocyte activation. T N F - a is a mediator of cachexia during chronic infections and is of importance in i n f l a m m a t o r y processes. It contributes to the pathophysiology of Gramnegative septic shock. Figure 4 illustrates the cytokine network involved in inflammation. Topical stimulation by an extrinsic stimulus (e.g., endotoxins, the lipopolysaccharide molecules in the outer layer of Gram-negative bacteria) leads to local release of various cytokines. Through their effects on the vascular endothelium, IL-1 and T N F promote inflammation, vascular damage and coagulation. IFN-~ acts synergistically with these cytokines by enhancing both their production and action [36]. TNF-~ is also a lymphokine produced by lymphocytes. It bears some structural and functional similarities to TNF-a. The clinical development of TNF-B is not as advanced as t h a t of TNF-a. Monoclonal antibodies. Anticipated applications for monoclonal antibodies in clinical t h e r a p y include cancer, infections, a u t o i m m u n e diseases, and cardiovascular diseases.
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Because of the important role of TNF and endotoxins in the pathophysiology of a n u m b e r of diseases, monoclonal antibodies against TNF-a and against endotoxins are of interest. Phase I studies are investigating the safety and pharmacokinetics of CB 00006 in patients with severe septic shock [41]. CB 00006 is a routine IgG, kappa monoclonal antibody directed against TNF-a. It neutralizes TNF-a cytotoxicity and blocks TNF-~-induced expression of MHC class I antigen. At present, it would be premature to draw any conclusions about the efficacy and safety of CB 00006. Two monoclonal antibodies against endotoxins include the recently registered h u m a n monoclohal IgM antibody against endotoxin, HA-1A (Centoxin| and the E5 monoclonal anti-endotoxin antibody, which is still under investigation. Because E5 monoclonal anti-endotoxin antibody is of murine origin and can, therefore, cause allergic reactions, it will probably be of minor importance [42]. HA-1A binds specifically to the lipid A part of the endotoxin. However, it is not clear whether this is its only action. Clinical application of HA-1A is limited to Gram-negative sepsis, where the antibody has been shown to significantly decrease mortality by 39% relative to placebo. A correct definition and diagnosis of Gram-negative sepsis and a quick and reliable endotoxin assay are therefore essential. Because HA-1A has been studied in only one multicentre trial [43], more studies are necessary to establish its clinical role [44-46]. On the other hand, the use of a promising new agent with no or few side-effects should not be limited only because of its price.HA-1A is also now under study in children with meningococcal meningitis and in patients with burns.
Conclusion Extensive research has given us detailed information about the i m m u n e system. New techniques, such as genetic engineering and monoclonal antibody production, have made available increasing numbers of agents which potentially interfere with complex immunological processes. Endogenous lymphokines are generally produced and act locally and are not demonstrable in the blood. Administration of lymphokines in dosages, by routes which drastically differ from endogenous production m a y therefore result in undesirable side-effects. Because lymphokines take part in a very complex interplay the maximal tolerated dose is not automatically the most effective dose. If side-effects are t a k e n into account, very low doses m a y have the best efficacy: side-effect ratio.In addition, therapy with one lymphokine m a y not produce an optimal response and the administration of a carefully timed combination may be preferable. Nonetheless, biological response modifiers and monoclonal antibodies m a y assume an important place in the t r e a t m e n t of such conditions as cancer, a u t o i m m u n e diseases, graft rejection, and severe infections. These agents will probably be most effective when used in combination with other drugs (e.g., cytotoxics, antibiotics and other
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cytokines). However, more research will be necessary to establish the clinical roles of these promising substances.
Acknowledgement The author t h a n k s Dr. H.C. Schouten for critically reading this manuscript.
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Colony~stimulating factors and interleukin-2. DICP Ann Pharmacother 1991;25:490-8. Crawford J, Ozer H, Stoller R, et al. Reduction by granylocyte colony-stimulating factor of fever and neutropenia induced by chemotherapy in patients with smallcell lung cancer. N Engl J Med 1991;325:164-70. Ohno R, Tomonaga M, Kobayashi T, et al. Effect of granulocyte colony-stimulating factor after intensive induction therapy in relapsed or refractory acute leukemia. N Engl J Med 1990;323:871-7. Wiltink EHH. Bloedgroeifactoren. Granulocyte colonystimulating factor en granulocyte-macrophage colonystimulating factor. Pharm Weekbl 1991;126(31):74653. Horohov DW, Siegel JP. Lymphokines. Progress and promise. Drugs 1987;33:289-95. N e ~ i e r S, Philip T, Stoter G, et al. Interleukin-2 with or without LAK cells in metastatic renal cell carcinoma: a report of a European multicentre study. Eur J Cancer Clin Oncol 1989;25 Suppl 3:$21-8. Stoter G, Shiloni E, Aamdal S, et al. Sequential administration of recombinant human interleukin-2 and dacarbazine in metastic melanoma. A multicentre phase II study. Eur J Cancer Clin Oncol 1989;25 Suppl 3:$41-3. West WH. Clinical application of continuous infusion of recombinant interleukin-2. Eur J Cancer Clin Oncol 1989;3 Suppl 3:Sll-5. Parkinson DR. Interleukin-2 in cancer therapy. Semin Oncol 1988;15 Suppl 6:10-26. Rosenberg SA. Clinical immunotherapy studies in the surgery branch of the US National Cancer Institute: brief review. Cancer Treatm Rev 1989;16 Suppl A:11521.
36 Heremans H, Bilian A. The potential role of interferons and interferon antagonists in inflammatory disease. Drugs 1989;6:957.72. 37 McManus Balmer C. Clinical use of biologic response modifiers in cancer treatment. Part I. The interferons. DICP Ann Pharmacother 1990;24:-761-7. 38 Perrillo RP, SchiffER, Davis GL, et al. A randomized, controlled trial of interferon alfa-2b alone and after prednisone withdrawal for the treatment of chronic hepatitis B. N Eng] J Med 1990;323:295-301. 39 Herzenberg H. Intel*feron alfa-2b for chronic hepatitis B. N Engl J Med 1991;324:493-4. 40 International Chronic Granulomatous Disease Cooperative Study Group. A controlled trial of interferon gamma to prevent infection in chronic gTanulomatous disease. N Engl J Med 1991;324:509-16. 41 Exley AR, Cohen J, Buurman W. Monoclonal antibody to TNF in severe septic shock. Lancet 1990;335:1275-7. 42 Speelman P, Van Deventer SJH. Behandeling van pati~nten met een door Gram-negative bacteri~n veroorzaakte sepsis met monoklonale anti-endotoxine-antistoffen. Ned Tijdschr Geneesk 1991;135:877-78. 43 Ziegler EJ, Fisher CJ, Sprung CL, et al. Treatment of Gram-negative bacteremia and septic shock with HA1A human monoclonal antibody against endotoxin. N Engl J Med 1991;324:429-36. 44 Wolff SM. Monoclonal antibodies and the treatment of Gram-negative-bacteremia and shock. N Engl J Med 1991;324:486-8. 45 Ziegler EJ, Fisher CJ, Sprung CL, Smith CR, Sadoff JC, Dellinger RP. The HA-1A monoclonal antibody for Gram-negative sepsis. N Engl Med 1991;32-5:282-3. 46 Cohen J, Glauser MP. Septic shock: treatment. Lancet 1991;338:736-9.
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First level At the first level of defense, a microbe is sensed by the host as foreign and innate inflammatory defence reactions occur. These defence mech-
anisms are not specific and involve phagocytic cells (first neutrophils and later macrophages), natural killer cells, cytokines and complement. Second level At the second level highly specific immune responses occur: lymphocytes and cell p r o d u c t s (antibodies and cytokines) interact with one another and with other elements of the i m m u n e system. Macrophages from the first level process antigens and present t h e m to two groups of lymphocytes: B cells (originating from the Bursa of Fabricius; in humans, probably from bone marrow) and T cell s (derived from the thymus). These lymphocytes have antigen-specific receptors on their surfaces which can recognize millions of different antigens. Each lymphocyte can recognize only one antigen, however. Humoral i m m u n i t y is the major defence system against most bacterial and Viral infections. It is mediated by the action of B lymphocytes which, after recognizing an antigen, produce soluble components, immunoglobulins or antibodies, t h a t circulate in the serum. Complement components and cytokines are necessary for appropriate action. There are five classes of immunoglobulins, IgG, IgM, IgE, IgA and IgD. IgG and IgM are extremely important for the i m m u n e response. Cellular i m m u n i t y constitutes the major defence against parasites, fungi, cancer, viruses and a few bacteria. It is, however, also responsible for delayed allergic reactions and rejection of foreign nonself tissue transplants. The major components of cellular i m m u n i t y are T lymphocytes, phagocytes, n a t u r a l killer cells, killer cells and lymphokines produced by these cells [1].
Takx-KShlen BCMJ. Immunomodulators. Future prospects. Pharm Weekbl [Sci] 1992;14(4A): 245-52. Keywords Cytokines Haematopoietic growth factors Immunomodulators Interferons Interleukins Lymphocytes Lymphokines Monoclonal antibodies Tumor necrosis factors
B.C.M.J. Takx-K6hlen: Department of Clinical Pharmacy and Toxicology, University Hospital of Maastricht, P.O. Box 5800, 6202 AZ Maastricht, the Netherlands.
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Abstract The future role of the immunomodulators in medical practice is yet to be defined. The key question is whether these new substances will bring remarkable progress in transplantation or in the treatment of such conditions as cancer, AIDS, and autoimmune diseases, or whether they will be of only minor adjunctive importance. As background to the discussion of immunomodulating agents, the immune system is explained, with emphasis on the roles of T and B lymphocytes, macrophages, phagocytes, human leukocyte antigen and the complement system. Special attention is given to the cytokines, particularly the lymphokines. The immunomodulators can be divided into three main groups: immunosuppressive agents, such as FK 506 and rapamycin; immunostimulating agents, of which BCG vaccine is most important; and the remaining immunomodulators, which include the biological response modifiers.The last group, which encompasses the colony-stimulating factors (GM-CSF, G-CSF, and M-CSF), the interleukins, the interferons, and the tumour necrosis factors, is described in detail. Innovative research and medical applications of these cytokines, including indications, contraindications, and adverse reactions, are discussed. The role of monoclonal antibodies against endotoxins is also described.
Accepted June 1992.
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245
Figure
1 T cells can be divided into various subsets according to the different biological functions they perform. These cellular subsets include T4 cells,
Figure
2
immunity against extracellular pathogens (for example bacteria) plasma cell
also called CD4 (helper or inducer T cells), and T8 cells, also called CD8 cells (memory or delayed hypersensitivity T cells, cytotoxic T cells and suppressor T cells) [2]. The helper T4 lymphocytes are part of the regulatory system of the immune network. The macrophage, after processing the antigen, stimulates the antigen-specific helper T4 cell via direct cell-to-cell contact.The stimulated helper T4 cell then secretes interleukin-2 to stimulate other helper T4 cells as well as cytotoxic and delayed hypersensitivity T8 cells. Stimulated helper T4 cells can also produce lymphokines that stimulate B cells. Suppressor T8 cells play a regulatory role by inhibiting the growth and function of helper T4 cells through downregulation or negative feedback. Delayed hypersensitivity T8 cells are involved in the release of lymphokines that promote inflammation. This immune response typically occurs 24 to 48 h after the initiation of the event [2]. The cytotoxic T8 lymphocytes are responsible for antigen-specific cytotoxicity; they are capable of attaching to specific antigen-bearing cell membranes by direct cellular contact. These cytotoxic T8 cells are the lymphocytes mainly responsible for graft rejection [2]. The functions of the suppressor T8 cells include suppressed proliferation of helper T4 cells, decreased stimulation of cytotoxic T8 cells and suppressed production of antibody from plasma cells. The suppressor T8 cells also play a role in the tolerance of transplanted tissues [2]. Figure 2 illustrates the differences between humoral and cellular immune responses. The complement system initiated through a n t i g e n antibody complexes forms a cascade of events which ultimately leads to cell death. In the case of humoral immunity, the CTL (cytotoxic T8 lymphocyte) recognizes only a viral peptide, which is processed by antigen presenting cells (from the first line of defence) presented in the context of MHC molecules [3]. MHC (major histocompatibility complex), also called class I HLA
immunity against intracelluiarpathogens (for example viruses)
CTL
macrophage
~T%. OoO
"
V
=:=
9
extracellular fluid
I
cell receptor TCR viral peptide
~ J molecule ~ ) MHC class
(
virus infected cell w
246
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(human leukocyte antigen), is a class of antigens which can be found on most cell surfaces, except those of m a t u r e red blood cells. Class II HLA antigens are found on only a few cells, including B cells, activated T cells, macrophages, vascular endothelial cells, epidermal cells and sperm [2]. The MHC is very important in cases of graft rejection and graft-versus-host disease [4]. After stimulation of the T cells, lymphokines are produced. These lymphokines are polypeptide products (glycoproteins) t h a t participate in a variety of cellular responses including the regulation of the immune system. It is now clear t h a t neither the production of lymphokines nor their effects are restricted to lymphoid cells. For this reasons the t e r m lymphokine has been replaced by the more general t e r m cytokine [5]. Because lymphokines provide a molecular mechanism for communication among leukocytes they are also called interleukins (ILs). To date, 12 interleukins have been identified. N K (natural killer) cells and K (killer) cells are nonphagocytic lymphocytes not directly classifiable as T or B lymphocytes. N K and K cells are of importance in the mechanism of the immune response against tumours. Native and recombin a n t interleukin-2 can stimulate the generation of LAK (lymphokine activated killer) cells and TIL (tumour infiltrating lymphocytes). LAK cells are an ill-defined group of lymphocytes (probably K cells) t h a t display a wide range of nonspecific killing activity in vitro after stimulation of such lymphokines as IL-2. They are generated in vitro after exposure of lymphocytes to IL-2 for 3-5 days and can also be generated in vivo by administration of IL-2 [6]. TIL, a bulk of various subpopulations of lymphocytes t h a t infiltrate a tumour, have specific immunoreactivity with the tumour cells t h a t they infiltrate. They are obtained by removing cancerous tissue or a nodule from a patient and forming a cell suspension by enzymatic digestion or chopping the sample into small fragments [6]. IL-2-stimulated TIL express highly specific cytotoxic activity against autologous tumours in a subgroup of cancer patients. In mice TILs have an a n t i t u m o u r effect t h a t is 50 to 100 times as large as t h a t of LAK cells on a cell-to-cell basis [6].
Third level At the third level of defence there is concerted action of both the innate defence reactions and the products of specific acquired immune responses [1]. Immunomodulators
Immunomodulators are agents t h a t interfere with the actions of the immune system [7 8]. They can be divided into three main groups: immunosuppressive agents, immunostimulating agents and other immunomodulators (Table 1).
Irnmunosuppresants Two immunosuppressant agents, F K 506 and rapamycin, are now under investigation [9]. Other agents are beyond the scope of this paper and will not be considered. 14(4A) 1992
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Table
1
Immunomodulators Immunosuppressive agents Glucocorticoids Cytotoxics Antilymphocyte immunoglobulin - Muronomab CD3 - ATG Other - ciclosporin - F K 506 - rapamycin Immunostimulating agents BCG vaccine Levamisole Other immunomodulators Haematopoietic growth factors Interleukins Interferons Tumour necrosis factors Monoclona] antibodies
FK 506 is a macrolide immunosuppressant which has a mode of action comparable to t h a t of cyclosporin [10]. In vitro studies with helper T lymphocytes have shown that F K 506 is at least 100 times as potent as ciclosporin in selectively inhibiting the secretion of IL-2, IL-3, IL-4 and interferon-7 inresponse to different stimuli. In addition, F K 506 may also impair the expression of IL-2 receptors on nonself-antigen stimulated T cells. Although its precise molecular action is not understood, F K 506 (like ciclosporin) inhibits early, calcium-dependent events in signal transduction t h a t usually follow T cell stimulation [10]. FK 506 has greater hepatotropic properties t h a n ciclosporin, which may explain its particular success in liver transplantation. A prospective randomized study to compare F K 506 with ciclosporin immunosuppression in liver transplantation has begun in 8 centres in Europe and a multicentre study of over 650 liver graft recipients is now underway in North America. The side-effects of FK 506 and ciclosporin in h u m a n s are similar, and include nephrotoxicity. However, hirsutism, gingival hyperplasia and coarsening of facial features have not yet been reported for FK 506 [10]. Rapamycin is a powerful immunosuppressant under investigation in animals [11]. Its mechanism of action differs from that of F K 506 and is r a t h e r complex. Like FK 506, rapamycin is especially toxic in dogs. However, the vasculitic laesions t h a t develop with rapamycin are largely confined to the gastrointestinal tract, whereas vasculitis associated with FK 506 is widespread, and involves the heart. This effect is probably species-specific [11].
Immunostimulating agents BCG vaccine is currently the most important immunostimulant. Topical BCG is used as a 247
Table 2
Cytokines Group name (abbreviation)
Subtypes
Alternative names
Colony-Stimulating Factors (CSF)
G-CSF GM-CSF M-CSF multi-CSF IL-1 IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 up to IFN-a 2a IFN-~ 2b IFN-~ IFN-7 TNF-a TNF-/~
Neupogen | Leucomax LAF IL-3
Interleukins (IL)
Interferons (IFN)
T u m o u r Necrosis Factors (TNF)
therapy for recurrent superficial bladder cancer [12]. Its mechanism of action may involve an HLA-restricted T cell response. There is clear evidence that t r e a t m e n t induces HLA class II antigen expression on t u m o u r cells and there is also a direct correlation between the response to therapy and the amount of IL-2 released into the urine. Studies are being performed with topical BCG in the t r e a t m e n t of head and neck tumours and melanomas. Levamisole is a synthetic orally active agent t h a t has anthelmintic and immunomodulatory properties. Levamisole combined with fluorouracil has been associated with a one-third reduction in recurrence and risk of death in patients with surgically resected stage C colon cancer [13]. The mechanism by which levamisole affects the human immune system is complex and not yet fully understood. The majority of in vivo and in vitro data suggest t h a t levamisole acts as an immunorestorative agent. It is capable of augmenting a depressed host immune system but is incapable of hyperstimulating an intact system. The most serious adverse effect associated with levamisole is granulocytopenia [13].
Proleukin | B-cell stimulating factor 1 B-cell stimulating factor 2 Roferon-A| Intron A | cachectin lymphotoxin
probably of importance in this differentiation from the pluripotent stem cell to the more restricted colony-forming unit stem cell (CFUs). In vitro these cytokines have little activity on proliferation but together they stimulate the production of early progenitors. IL-3 and GM-CSF (granulocyte macrophage colony-stimulating factor) are multipotentional myeloid haematopoietic growth factors with overlapping and synergistic activities. IL-3 is more effective in stimulating early multipotent progenitors. GM-CSF, G-CSF (granulocyte colony-stimulating factor) and M-CSF (macrophage colony-stimulating factor) are relatively lineage-specific growth factors t h a t act on more m a t u r e progenitor cells (Figure 3). G-CSF, GMCSF, M-CSF and IL-3 have been purified, cloned and produced on a large scale with recombinant
Figure 3
Other immunomodulators To this group belong the haematopoietic growth factors, the interleukins, the interferons and the t u m o u r necrosis factors (Table 2). Because of their interactions with the immune system the monoclonal antibodies against endotoxins and against T N F a are also included in this category. Haematopoietic growth factors were originally termed colony-stimulating factors (CSFs) because they stimulate the formation of colonies of cells derived from individual bone-marrow progenitors. They show a mutual connection which is related to the hierarchy of haematopoiesis [14]. After cell division, one daughter cell from every pluripotent stem cell remains a stem cell and the other goes into a particular haematopoietic cell line, i.e., myeloid or lymphoid. IL-1 and IL-6 are 248
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DNA technology and are available for clinical use. Haematopoietic growth factors interact with blood cells at different levels in the cascade of cell differentiation from the multipotent progenitor to the circulating m a t u r e cell. Their biological effects are mediated by specific binding to high affinity receptors on the surface of target cells. These receptors are not restricted to normal progenitor cells and have also been detected on myeloid leukaemic cells as well as on nonhaematopoietic cells. II-1, IL-5 and IL-6. The functional properties of IL-1 include stimulation of thymocyte proliferation, accessory growth factor activity for certain T helper lymphocytes and B lymphocytes and stimulation of haematopoietic cell growth and differentation [16]. In addition to its important role in the immune system IL-1, together with TNF-a, is responsible for the cellular processes t h a t occur after inflammation [17]. IL-1 induces synthesis of acute phase proteins, prostaglandins and collagenase by fibroblasts and chondrocytes; it also acts as a pyrogen. Analysis of the effects of IL-1 is complicated because the molecule can induce production of other cytokines such as IL-6 [18]. At the moment little is known about IL-5. The molecule has been identified as a factor that selectively stimulates the proliferation of eosinophil precursors and the functional activity of mature eosinophils [20]. IL-6 has a broad range of actions on many cell types and has been indepently discovered and described as an interferon-like molecule (/~2) and as a B lymphocyte-stimulating factor. Several lines of evidence suggest t h a t IL-6 is involved in the pathogenesis of certain autoimmune diseases. IL-6 expression is not lymphocyte restricted and several tumours, including some cardiac myxomas, cervical cancers and bladder carcinomas, produce large amounts of this cytokine [19]. IL-1 and IL-5 are under investigation in vitro and in animals, while the first clinical studies with IL-6 are now underway. Colony-stimulating factors. T lymphocytes, monocytes or macrophages, endothelial cells and fibroblasts represent the major cellular sources of the haematopoietic growth factors. Activation by antigen, lectins, or interleukin-1 induces T lymphocytes to produce GM-CSF and IL-3. The immediate effect of G-CSF and GM-CSF is a transient fall in white blood cells occurring within minutes after a bolus injection. This is followed 6 to 12 h later by an increase in neutrophils. The early effects occurring within 24 h may be attributable to an accelerated release from a preformed pool of granulocytes in bone marrow, to demargination from endothelial adherent pools, and to a prolongation of survival in the circulation [15]. When G-CSF is administered the number of neutrophils rises. The principal cause of the increase in white blood cells after GM-CSF t r e a t m e n t is also neutrophilia, although the numbers of eosinophils and monocytes increase too. In general, the white blood cell counts r e t u r n to normal within a few days after cessation of G-CSF or GM-CSF therapy [15]. 14(4A) 1992
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Phase II and III clinical trials with IL-3 are underway in patients with Hodgkin and nonHodgkin lymphomas, after chemotherapy and autologous bone-marrow transplantation. MCSF is also being studied. GM-CSF (Leucomax | has been offered to the Dutch authorities for registration for a number of conditions accompanied by leukopenia, including myelodysplasia, aplastic anemia, AIDS, cancer chemotherapy, and bone-marrow transplantation [20 26]. Adverse reactions of GM-CSF are caused by activation of granulocytes, monocytes, macrophages and cytokines, such as IL-1 and TNF-a. At dose levels higher t h a n 10 ~ g . k g - l . d -1, GM-CSF frequently induces fever, malaise, myalgia, arthralgia, anorexia, mild elevations in transaminase levels, and a rash at the site of injection. More serious toxic effects have been observed at dose levels of 30 ~g. kg -1- d -i or higher. These include a capillary leak syndrome with pleural and pericardial effusions and ascites, and large-vessel thrombosis. G-CSF (Neupogen | is registered for use in patients with neutropenia resulting from chemotherapy [27]. Adverse reactions to G-CSF are relatively mild and include musculoskeletal pain, rash and elevated liver enzyme levels. In vitro studies have shown inconsistent stimulation of leukaemic cell clone proliferation by myeloid growth factors (including G-CSF, GMCSF and IL-3). In addition, however, these studies have shown t h a t myeloid growth factors may enhance myeloid leukaemic cell differentiation and leukaemic cell cycling [15]. The relevance of these in vitro findings has not yet been established in clinical studies. In the first study in patients with relapsed or refractory acute leukaemia, there was no evidence t h a t G-CSF accelerated regrowth of leukaemic cells [28]. Nonetheless, G-CSF, GM-CSF and IL-3 should not be given to patients with myeloid malignancies until there is more evidence of their safety. Thus, the role of haematopoietic growth factors in myeloid leukaemia and myelodysplasia remains investigational [29]. IL-4. IL-4 is a growth factor for activated B cells. It is also a growth factor for resting T lymphocytes and enhances the cytotoxic activity of cytotoxic T cells [5]. IL-2. IL-2, also called T cell growth factor, was first described in 1976 [30]. Its effects are initiated by its binding to a receptor glycoprotein or glycoprotein complex on the plasma m e m b r a n e of a responsive cell [6]. IL-2 activates and causes the multiplication of various types of white blood cells, including cytotoxic T lymphocytes, N K cells, LAK cells and TIL. These killer cells recognize, attack and destroy cancer cells. IL-2 stimulates T cells to help B cells produce antibodies. IL-2 further interacts with other cells that produce factors, such as interferon-~ and TNF, which directly attack cancer cells. IL-2 produced by recombinant DNA techniques is available as Proleukin and is registered for use in metastatic renal cell carcinoma. The overall response rate in patients with this neoplasm has been 15% to 30% [6 31]. Patients who have undergone prior nephrectomy and have a low t u m o u r 249
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Figure 4
extrinsic--'~~~.~~,~ jS ~ "
stimuli
T-LYMPHOCYTE THE CYTOKINE NETWORK
INVOLVED IN I N F L A M M A T I O N
burden are more likely to respond t h a n those who have the cancerous kidney still in place or those who h a v e large t u m o u r masses. IL-2 with or without L A K cell therapy, in combination with chemotherapy, is being studied in patients with m e l a n o m a [32] and colorectal cancer [6]. Response r a t e s in m e l a n o m a v a r y from 15% to 25%. Research is still ongoing with combinations of IL-2 with L A K cells, TIL, interferon-a, -fl, -% T N F - a and c h e m o t h e r a p y [6 33-35]. The adverse effects of IL-2 are generally reversible b u t are frequently severe and dose-related. Constitutional s y m p t o m s such as fever, chills, headaches, muscle pain and malaise which are typical of most i m m u n o t h e r a p y products are essentially universal in patients given IL-2. Severe adverse effects include hypotension, edema, capillary leak syndrome and renal dysfunction. To avoid severe adverse effects, it is recommended t h a t IL-2 be given as a continuous infusion or subcutanously. However, there is some evidence t h a t the bolus dose schedule with its p e a k IL-2 levels m a y be superior in inducing lymphokine secretion in vivo. The additional triggering of endogenous lymphokines m i g h t be beneficial in tum o u r response. Thus, it would appear reasonable to test continous infusion of IL-2 in combination with the cells mentioned above and cytokines [33]. Interferons. Interferons were initially identified for their ability to interfere with viral replication in infected cells [5]. Interferon (IFN)-o (originally called leukocyte interferon) and interferon-fl (originally called fibroblast interferon) are structurally related polypeptides induced by viral infections. They have potent antiviral, antiproliferative and immunologic properties. Interferon-T is produced by immunologically stimulated lymphocytes. It is a m u c h more potent i m m u n o m o d u l a t o r t h a n either IFN-~ or IFN-/~, but has less antiviral activity [30]. The immunological functions affected by interferons include macrophage activation, stimulation of lymphocyte recruitment, regulation of IgE antibody secretion, antibody production, production of acute phase proteins and induction of MHC anti250
gens. A noteworthy property of I F N - a is its ability to enhance cytotoxic T lymphocyte activity and N K cell activity. IFN-T is an i m p o r t a n t element in the i n f l a m m a t o r y response to lipopolysaccharide [36]. IFN-a 2a and 2b produced by recombinant techniques are available as Roferen-A and Intron A, respectively, and h a v e been used for several y e a r s in cancer t r e a t m e n t . As single-agent therapy, I F N - a produces clinical i m p r o v e m e n t in approximately 90% of patients with hairy-cell leuk a e m i a and up to 70% of patients with earlystage chronic myeologenous leukaemia. In multiple myeloma, however, it is more effective in combination with c h e m o t h e r a p y [37]. The objective response r a t e a m o n g AIDS patients with Kaposi's sarcoma is 40%. I N F - a is also widely used in the t r e a t m e n t of renal cell carcinoma, m a l i g n a n t m e l a n o m a , certain non-Hodgkin l y m p h o m a s and hepatitis B [38 39]. However, as a single a g e n t it is not active against the most common cancer killers: breast, colon, lung and prostate cancers. A potential application of INF-~ lies in the t r e a t m e n t of p r i m a r y brain tumours. E a r l y trials h a v e shown a 10% to 20% response in the glioma subtype [37]. INF-~ has d e m o n s t r a t e d activity against several t u m o u r types in phase I and II clinical trials. However, response rates are low (up to 20% in renal cell carcinoma and 7% to 10% in melanoma). For patients with chronic granu]om a t o u s disease, INF-7 seems to be an effective and well-tolerated t r e a t m e n t t h a t reduces the frequency of serious infections [40]. The major side-effects of interferons are influenza-like s y m p t o m s and gastro-intestinal disturbances. For some patients these reactions are a reason to w i t h d r a w from therapy. TNF-a and TNF-~. T N F - a was first described as a substance t h a t caused h a e m o r r h a g i c necrosis of tumours. It was found in the s e r u m of animals primed with BCG or C. parvum and after stimulation with bacterial lipopolysaccharides [30]. Unlike IL-1, T N F - a seems to h a v e no direct effect on lymphocyte activation. T N F - a is a mediator of cachexia during chronic infections and is of importance in i n f l a m m a t o r y processes. It contributes to the pathophysiology of Gramnegative septic shock. Figure 4 illustrates the cytokine network involved in inflammation. Topical stimulation by an extrinsic stimulus (e.g., endotoxins, the lipopolysaccharide molecules in the outer layer of Gram-negative bacteria) leads to local release of various cytokines. Through their effects on the vascular endothelium, IL-1 and T N F promote inflammation, vascular damage and coagulation. IFN-~ acts synergistically with these cytokines by enhancing both their production and action [36]. TNF-~ is also a lymphokine produced by lymphocytes. It bears some structural and functional similarities to TNF-a. The clinical development of TNF-B is not as advanced as t h a t of TNF-a. Monoclonal antibodies. Anticipated applications for monoclonal antibodies in clinical t h e r a p y include cancer, infections, a u t o i m m u n e diseases, and cardiovascular diseases.
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Because of the important role of TNF and endotoxins in the pathophysiology of a n u m b e r of diseases, monoclonal antibodies against TNF-a and against endotoxins are of interest. Phase I studies are investigating the safety and pharmacokinetics of CB 00006 in patients with severe septic shock [41]. CB 00006 is a routine IgG, kappa monoclonal antibody directed against TNF-a. It neutralizes TNF-a cytotoxicity and blocks TNF-~-induced expression of MHC class I antigen. At present, it would be premature to draw any conclusions about the efficacy and safety of CB 00006. Two monoclonal antibodies against endotoxins include the recently registered h u m a n monoclohal IgM antibody against endotoxin, HA-1A (Centoxin| and the E5 monoclonal anti-endotoxin antibody, which is still under investigation. Because E5 monoclonal anti-endotoxin antibody is of murine origin and can, therefore, cause allergic reactions, it will probably be of minor importance [42]. HA-1A binds specifically to the lipid A part of the endotoxin. However, it is not clear whether this is its only action. Clinical application of HA-1A is limited to Gram-negative sepsis, where the antibody has been shown to significantly decrease mortality by 39% relative to placebo. A correct definition and diagnosis of Gram-negative sepsis and a quick and reliable endotoxin assay are therefore essential. Because HA-1A has been studied in only one multicentre trial [43], more studies are necessary to establish its clinical role [44-46]. On the other hand, the use of a promising new agent with no or few side-effects should not be limited only because of its price.HA-1A is also now under study in children with meningococcal meningitis and in patients with burns.
Conclusion Extensive research has given us detailed information about the i m m u n e system. New techniques, such as genetic engineering and monoclonal antibody production, have made available increasing numbers of agents which potentially interfere with complex immunological processes. Endogenous lymphokines are generally produced and act locally and are not demonstrable in the blood. Administration of lymphokines in dosages, by routes which drastically differ from endogenous production m a y therefore result in undesirable side-effects. Because lymphokines take part in a very complex interplay the maximal tolerated dose is not automatically the most effective dose. If side-effects are t a k e n into account, very low doses m a y have the best efficacy: side-effect ratio.In addition, therapy with one lymphokine m a y not produce an optimal response and the administration of a carefully timed combination may be preferable. Nonetheless, biological response modifiers and monoclonal antibodies m a y assume an important place in the t r e a t m e n t of such conditions as cancer, a u t o i m m u n e diseases, graft rejection, and severe infections. These agents will probably be most effective when used in combination with other drugs (e.g., cytotoxics, antibiotics and other
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cytokines). However, more research will be necessary to establish the clinical roles of these promising substances.
Acknowledgement The author t h a n k s Dr. H.C. Schouten for critically reading this manuscript.
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P h a r m a c e u t i s c h Weekblad Scientific edition
14(4A) 1992
