Ronald M. Bukowski
Charles S. Tannenbaum
James H. Finke
The study of cytokines has evolved from the description of protein factors mediating particular cellular functions to studies at the molecular level using recombinant proteins that allow definitive identification of their structures and functions. The biologic activities of these molecules are complex, with pleiotropic and redundant actions common. Cytokines are often part of a cascade that can then lead to the synthesis and production of other mediators and result in either positive or negative regulatory effects. The antitumor activities of various cytokines have led to their use in patients with malignancy, and a large body of data now exists on their clinical effects and pharmacology. This chapter discusses six different factors, all of which have been used clinically and demonstrate the difficulties encountered in evaluating biologic agents with complex functions in vivo.
The recognition in 1965 that soluble mitogenic factors were present in conditioned supernatants from mixed lymphocyte cultures1 provided the initial observation indicating the existence of lymphokines that could stimulate cell division. In 1976, Morgan et al.2 demonstrated that normal human T lymphocytes obtained from bone marrow could be maintained in culture for periods of up to 1 year by using media from phytohemagglutinin (PHA)–stimulated mononuclear cells. Shortly thereafter, this media was also found to sustain the proliferation of antigen-specific cytolytic T cells.3 This cytokine was ultimately designated as IL-2 (Second Annual Lymphokine Workshop, 1979), and the cDNA encoding human IL-2 was isolated.4
Structure and Mechanisms of Action
IL-2 is a 15-kd glycoprotein that varies in degree of glycosylation and sialylation.5, 6 It contains a carbohydrate-binding domain that is thought to be involved in the clearance and intracellular distribution of this protein.7 In structure, IL-2 is similar to GM-CSF and IL-4.5, 6 IL-2 has four major amphipathic α helices that are arranged in an antiparallel manner (Fig. 36.1).5, 6 One disulfide bridge exists in the IL-2 protein; it provides stability of the tertiary structure and is necessary for biologic activity.8
T cells are the primary source of IL-2, and among mature T cells most of the IL-2 is produced by the CD4+ subset. In murine systems, IL-2 is produced by unprimed CD4+ cells (TH0) and by the TH1 subset of helper cells involved in delayed-type hypersensitivity responses.9
IL-2 is not constitutively produced but is induced on T-cell activation.10 Two signals are required for IL-2 gene expression. One is provided by stimulation through the T-cell receptor (TCR)/CD3 complex. The second signal appears to be provided by accessory cells that express the cell surface molecule B7, which is the ligand for CD28 or CTLA-4 molecules that are present on T cells.11 The stimulation of T cells via the TCR in the absence of costimulation (B7/CD28) can induce T-cell anergy, a state of T-cell unresponsiveness, and involves a block in IL-2 gene transcription.12 When both signals are provided, IL-2 gene expression occurs, with peak levels of IL-2 mRNA accumulation within 6 hours of stimulation.13 The induction of IL-2 gene expression is under the control of a transcriptional enhancer located approximately 300 base pairs upstream of the transcription site.14 This region contains binding sites for several DNA-binding proteins that are required for the transcription of the IL-2 gene, including nuclear factor AT (NFAT), activating protein 1 (AP-1), NF-κB, AP-3, and Oct.13
IL-2 mediates its biologic effects by binding to the IL-2 receptor (IL-2R), which is composed of three distinct chains, α (55 kd), β (75 kd), and γ (64 kd).5, 15All three chains have external domains of similar length, whereas the cytoplasmic domains vary. The β subunit (286 residues) has the largest internal domain, and the α chain has the smallest (13 residues).15, 16, 17, 18 The IL-2R that binds IL-2 with high affinity (Kd [dissociation constant] = 10 pmol/L) requires the presence of all three chains.5 Cells that express both the β and γ chains but are missing the α chain have an intermediate-affinity receptor (approximately 100-fold less than the high-affinity receptor) that is capable of signal transduction. The expression of only the α chain results in cells with a low IL-2–binding affinity and no intracellular signaling.15
Figure 36.1 Comparison of x-ray–derived and model folds of interleukin-2 (IL-2). A: Schematic drawing of the IL-2 x-ray helix bundle (3). Cylindrical helices are marked 1 to 6. Loops are drawn as loose ribbons; Pro47 (P47) is marked. The disulfide bond is noted by linked spheres. B: In the granulocyte-macrophage colony-stimulating factor–IL-4–like IL-2 model, the chain through the core helices is retraced and reconnected, the disulfide bridge is relocated, and the existence of a small β sheet is proposed; Pro65 (P65) is marked. Only helix D remains fully equivalent in sequence to x-ray helix 6. (Reproduced with permission from Bazan JF. Unraveling the structure of IL-2. Science 1992;257:410–413.)
Signaling via the IL-2R requires oligomerization of the β and γ chains.19, 20, 21 Neither of these chains has intrinsic kinase activity; however, they appear to be substrates for protein tyrosine kinases (PTKs), which associate with the IL-2R after IL-2 binding (Fig. 36.2). Multiple PTKs are involved, each associated with a specific region of the intracytoplasmic tails of the γ and β chains.21, 22 This interaction results in the activation of PTKs and the phosphorylation of multiple substrates that include the γ and β chains themselves.22 The binding of Janus kinase 3 (Jak3) to the γ chain is critical to transducing the proliferative signal via the IL-2R.23, 24, 25 The importance of Jak3 and γ chain function is illustrated by the fact that loss of either Jak3 or the γ chain results in severe combined immunodeficiency syndrome in humans.26 The proximal region of the IL-2Rβ chain, which is rich in serine residues, is also important for proliferation and cell survival through the induction of c-myc and Bcl-2/Bcl-XL, respectively.22 This region is required for activation of phosphatidylinositol 3 kinase (PI3K) and the downstream kinase Akt, which are likely involved in the expression of c-myc and Bcl-2.27, 28 Both the Zap-70 kinase, Syk, and Janus kinase 1 (Jak1) bind to the serine-rich region, and although Syk appears to be involved in c-myc induction, the role of Jak1 is not defined.29, 30 The acidic region of the IL-2Rβ chain appears to be responsible for the signaling pathway leading to c-fos and c-jun induction.31, 32 The src kinase p56lck constitutively associates with this region and is activated by IL-2 binding.31, 32 Evidence is growing of a linkage between p56lck and the Ras pathway, which is involved in the IL-2–dependent activation of c-fos and c-jun and T-cell proliferation.21, 33, 34, 35 The carboxy-terminal domain of the IL-2Rβ chain appears to be involved in signal transduction and transcription-activating factor 5 (STAT5).36, 37 STAT5 may regulate T-cell proliferation partly through the induction of the high-affinity IL-2Rα chain.38
Figure 36.2 Schematic diagram of the interleukin-2 signal transduction cascade. (MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3 kinase; PKC, protein kinase C; STAT, signal transduction and transcription–activating factor.) (Reproduced with permission from Gesbert F, Delespine-Carmagnat M, Bertoglio J. Recent advances in the understanding of interleukin-2 signal transduction. J Clin Immunol 1998;18:307–320.)
Several cell types involved in inflammation and immunity express IL-2R.5, 39, 40, 41 The IL-2Rβ chain is constitutively expressed on monocytes/macrophages and certain lymphoid cells such as natural killer (NK) cells. The majority of NK cells (90%) express the intermediate-affinity IL-2R; only a subset has the high-affinity receptor.41 Activation of T cells via the T-cell receptor complex up-regulates IL-2Rβ and induces IL-2Rα chain expression, which leads to formation of the high-affinity receptor.42 B cells can also be induced to express IL-2R and become IL-2 responsive after cross-linking of surface immunoglobulin.43 IL-2 stimulation also leads to activation of other kinases, including the serine/threonine-specific kinase Raf-1.44, 45 Evidence also exists that IL-2 binding to its receptor on T cells leads to the phosphorylation of the retinoblastoma-susceptibility gene product p110Rb, a process that is important for cell cycle progression.46
IL-2 is a major growth factor for lymphoid cells, including T cells and NK cells.2, 5, 47 IL-2 binding to IL-2R on activated T cells promotes clonal expansion of antigen-specific cells, an important component in the development of host immunity. It also plays an important role in potentiating cytotoxic activity of lymphocytes, including antigen-specific major histocompatibility complex (MHC)–restricted cytotoxic CD8+ and CD4+ T lymphocytes.48, 49 IL-2 can also enhance the cytolytic activity of NK cells, which are responsible for what has been referred to as “LAK cell activity.”50 The potentiation of cytotoxic activity is likely due to IL-2 up-regulation of the expression of proteins involved in the lytic process. IL-2 alone or in combination with other stimuli can up-regulate mRNA levels for perforin and granzyme B in both T and NK cells.5, 51 Macrophage cytotoxicity is also potentiated by IL-2.49 IL-2 is also known to stimulate cytokine secretion from mononuclear cells, including NK cells, T cells, and macrophages.49NK cells cultured with IL-2 secrete cytokines (TNF-α, IFN-γ, GM-CSF), which can facilitate inflammation and immunity by acting on monocytes and macrophages.5, 49 IL-2 can also cooperate with TCR triggering to induce IFN-γ secretion from T lymphocytes.49
IL-2 may also have a negative regulatory effect on the immune response.52, 53 IL-2 plays an important role in promoting apoptosis in T cells, a major mechanism of controlling immune responses. This concept is supported by the observation that knockout mice missing IL-2 or the α or β chains of the IL-2R display autoimmunity and lymphadenopathy.54, 55, 56 Thus, mice deficient in IL-2R signaling have abnormal accumulation of activated T cells with impaired TCR-induced apoptosis. IL-2 enhances activation-induced cell death (AICD) mediated through the Fas pathway.52, 53, 54, 55, 56 Crossing IL-2 knockout mice with TCR transgenic mice demonstrated that activated T cells from these animals were impaired in Fas-mediated AICD.57 IL-2 augments AICD by increasing the transcription of Fas ligand in antigen-stimulated T cells, partly through the induction of STAT5.53, 58 IL-2 also inhibits the transcription of the inhibitor of the Fas pathway, FLIP (FADD-like IL-1β–converting enzyme [FLICE] inhibitory protein).58 In the potentiation of AICD, IL-2 cannot be replaced by other cytokines such as interleukin-7 (IL-7) or IL-4. Thus, although IL-2 serves as a growth signal during the early phase of an immune response, it can potentiate apoptosis in T cells after repeated antigen activation, which results in termination of an immune response. IL-2 is also important for the induction of passive apoptosis in T cells. This form of apoptosis results when there is no further antigen stimulation and the induction of IL-2 and IL-2R ceases, resulting in lymphokine withdrawal.52, 53 This pathway of apoptosis is distinct from Fas-mediated AICD. Passive apoptosis results from an increase in mitochondrial permeability and cytochrome c release and can be blocked by Bcl-2.52, 53
Clinical assessment of the immunopharmacology of IL-2 has been assisted by the availability of a sensitive bioassay procedure, development of ELISAs,59 and the use of cytolytic (NK- and LAK-cell) assays to define the biologic effects of IL-2 administration. The bioassay assesses the capacity of the sera or supernatant in question to maintain the growth of an IL-2–dependent T-cell clone.
Initial studies with IL-2 used material produced by the Jurkat T-cell tumor line and purified by affinity chromatography.60 Doses of 14 to 2,000 µg were administered, and serum levels were measured using a bioassay.61 The biologic half-life varied from 6 to 10 minutes after a bolus infusion. Sustained serum levels during continuous infusion were also noted.
Several recombinant preparations of IL-2 (rIL-2) have been used clinically and are outlined in Table 36.1. They are nonglycosylated and produced in Escherichia coli. The Chiron preparation62 differs from natural human IL-2; the cysteine residue at amino acid 125 is replaced by serine, and it also lacks the N-terminal alanine. These alterations permit correct folding and maintenance of the biologic activity of this agent. Amgen rIL-263 has a similar serine substitution at position 125 and in addition has an N-terminal methionine residue. The Hoffmann-La Roche rIL-2 preparation64 lacks the amino acid substitution at position 125, has an additional N-terminal methionine residue, and has a specific activity similar to that of natural IL-2. These molecules can form only one disulfide bridge and belong to the class of proteins known as muteins, or mutationally altered and biologically active products.
Confusion has arisen because of the definitions used for units of activity and the methods used to calculate dosage (body surface area versus kilogram). At present, the accepted definition for a unit is based on an international standard described by the World Health Organization.68 A unit of IL-2 is defined as the reciprocal of the dilution that produces 50% of the maximal proliferation of murine HT2 cells in a short-term tritium-labeled thymidine incorporation assay. One milligram of Chiron rIL-2 contains 16.3 IU/mg of drug. In the past, a Cetus unit was commonly used to express doses of this cytokine, with 3 × 106 Cetus units equaling 1 mg of rIL-2. Hoffmann-La Roche rIL-2 contained 15.0 × 106 U/mg of protein. One Hoffmann-La Roche unit was reported as equivalent to one Biological Response Modifiers Program (BRMP) unit69 and approximately 3.0 IU. This situation has produced confusion in the comparing of dosages and toxicity among studies that used the various rIL-2 preparations.
TABLE 36.1 RECOMBINANT INTERLEUKIN-2 (rIL-2) PREPARATIONS
The clinical and in vitro activities of these two rIL-2 preparations have been compared by Hank et al.70 Equivalent international units of each cytokine were used, and quantitative differences were noted. A dosage of 1.5 × 106 IU/m2 per day of Roche rIL-2 was equivalent in toxicity to 4.5 × 106 IU/m2 per day of Chiron rIL-2. Equivalent amounts also differed in the induction of proliferation by various T-cell lines and binding to IL-2 receptors. These findings suggested that 3 to 6 IU of Chiron rIL-2 are required for induction of the biologic effects produced by 1 IU of Hoffmann-La Roche rIL-2.
The only preparation currently available for clinical use in the United States is Chiron rIL-2. Findings such as these suggest that one must be cautious when using doses and schedules developed with alternative preparations. The reasons for these differences in biologic activity may be related to structural and solubility differences between these proteins. Two other IL-2 preparations used clinically are natural human IL-2 (nIL-2) and Sanofi rIL-2; however, limited information is available concerning these agents.
The clinical pharmacokinetics of both the Chiron and Hoffmann-La Roche preparations have been studied, and the features of the latter more fully characterized. Like many other cytokines, rIL-2 has a short half-life. Various parameters for Jurkat cell–derived IL-2 and recombinant IL-2 preparations are outlined in Table 36.2. When rIL-2 is administered as an intravenous (i.v.) bolus, its pharmacokinetics are approximately linear, and the resulting serum levels are proportional to the dose.71 Injection of 6 × 106 IU/m2 produces serum levels of 1,950 IU/mL. The levels decrease with a t1/2α of 12.9 minutes, followed by a slower phase with a t1/2β of 85 minutes. Figure 36.3 illustrates the serum levels obtained after injection of an i.v. bolus of 6 × 106 IU/m2.72 The reported clearance rate of 117 mL/minute is consistent with renal filtration being the major route of elimination.
The variables influencing rIL-2 pharmacokinetics have been studied in animal models.33, 73, 74 The biodistribution of 125I-radiolabeled rIL-2 was studied in Sprague-Dawley rats.33 The most significant uptake was found in the kidney and liver, with the kidney cortex demonstrating the highest activity. Irradiated and splenectomized mice have been used to assess the potential role of rIL-2 binding to lymphoid cells.74 The rIL-2 was injected intravenously, and no alterations were found. In addition, the half- life of rIL-2 in nephrectomized animals rose from 2.5 to 3.5 minutes to 84 minutes, and ureteral ligation had minimal effect on the rIL-2 half-life. Finally, active IL-2 is not excreted in the urine, which implies renal tubular catabolism.
TABLE 36.2 PHARMACOKINETICS OF INTERLEUKIN-2 PREPARATIONS AFTER INTRAVENOUS BOLUS ADMINISTRATION
Figure 36.3 Interleukin-2 (IL-2) serum clearance after an i.v. bolus. A dose of 1.0 MU/m2 was given as a 5-minute i.v. bolus. Serum samples were taken, and the IL-2 bioactivity was determined. A biexponential curve has been fitted to the assay values, minimizing the sum of the squares of the percentage of deviation of the curve from the data. (Reproduced with permission from Konrad MW, Hemstreet G, Hersh EM, et al. Pharmacokinetics of recombinant interleukin 2 in humans. Cancer Res 1990;50:2009–2017.)
Other schedules of i.v. rIL-2 administration, such as continuous infusion, have also been investigated. Infusion times have varied from 2 to 24 hours, and steady-state levels are generally achieved within 2 hours (Fig. 36.4). Median steady-state levels of 123 IU/mL are produced by infusion of 6 × 106 IU/m2 over 6 hours, and the levels then fall rapidly after termination of rIL-2 infusion. The clearance rate after i.v. infusion resembles that seen with bolus administration.
The pharmacokinetics of very low doses of rIL-2 may be different than that observed with higher doses. Saturable pathways, binding to serum proteins or receptors and internalization of the receptor-ligand complex may play significant roles in altering distribution. Continued administration of rIL-2 and the accompanying lymphocytosis with increased IL-2 receptor density may potentially result in an increase of rIL-2 metabolism. In addition, alterations in renal function may change clearance of this cytokine.
Figure 36.4 Serum levels during and after a 6-hour i.v. infusion of interleukin-2 (IL-2). The patient received 1 MU/m2 over 6 hours, and the steady-state level of approximately 28 U/mL is close to the dose-normalized median level seen in all patients. Because the first blood sample was taken 60 minutes after the start of the infusion, the rising phase of the curve was not accurately determined, and the curve seen here is somewhat symbolic of the actual time course expected. (Reproduced with permission from Konrad MW, Hemstreet G, Hersh EM, et al. Pharmacokinetics of recombinant interleukin 2 in humans. Cancer Res 1990;50:2009–2017.)
Most frequently, rIL-2 is administered as a subcutaneous (s.c.) injection.72 Time to peak concentration varies from 120 to 360 minutes, and with doses of 6 × 106 IU/m2, median peak serum levels of 32.1 IU/mL and 42 IU/mL have been reported. The kinetics of lower-dose s.c. rIL-2 are different than those of high-dose i.v. administration. Studies demonstrate that IL-2 serum levels are 50- to 100-fold less than with i.v. administration.72 Some studies75 do suggest alternative clearance mechanisms at lower doses. Interleukin-2 may be bound to proteins such as soluble IL-2R (sIL-2R), α2-macroglobulin, and immunoglobulins with saturable processes in operation.75 Administration to an anephric patient on dialysis produced slightly higher IL-2 concentrations, but the pharmacokinetics of s.c. rIL-2 appeared similar to those in patients with normal renal function.75 Kirchner et al.76 examined the pharmacokinetics of subcutaneously administered rIL-2. Two schedules were investigated (20 MIU/m2 daily and 10 MIU/m2 twice daily). For the once-daily schedule, the 24-hour area under the concentration × time curve (AUC) was 627 IU/mL × 1 hour, and for the twice-daily schedule 1,130 IU/mL × 1 hour. The highest observed concentration for both schedules was similar. By 72 hours, the levels of sIL-R increased, with some reductions in AUC seen. The authors concluded that two daily doses of rIL-2 provide superior bioavailability.
TABLE 36.3 DOSES AND SCHEDULES OF RECOMBINANT INTERLEUKIN-2 (rIL-2) IN TRIALS INVOLVING PATIENTS WITH RENAL CELL CARCINOMA
Clinical Effects and Toxicity
The variables influencing serum levels and biologic activity have led to the clinical investigation of multiple schedules and doses (Table 36.3). Among the solid tumors, malignant melanoma and renal cell carcinoma (RCC) (predominantly clear cell histologic subtype)77 appear to be the most responsive to rIL-2 therapy, and in both tumor types, rIL-2 has been approved for treatment. Rosenberg et al.78 used dosages of 1.8 to 6.0 × 105 IU/kg every 8 hours for 5 days. This approach using high doses of rIL-2 administered frequently is associated with severe and life-threatening toxicity. These doses were investigated when initial studies using lower doses or less frequent administration produced limited antitumor effects. The response and survival data for this type of schedule and dose of rIL-2 in patients with renal cancer are summarized in Table 36.4. Approximately 5% of patients experienced durable complete responses.79 A comparison of results reported with various schedules and routes of rIL-2 administration is provided in Table 36.5.
The short half-life of i.v. bolus rIL-2 prompted investigation of continuous intravenous (c.i.v.) administration of this cytokine. The majority of reports have used 18.0 MIU/m2 per day. Response rates between studies vary, but in a group of 922 patients (Table 36.5), the overall response rate was 13.3%. Complete regressions occur, and in some series87 the rates are similar to those reported with high-dose bolus rIL-2.
In one trial84 patients with metastatic renal cancer were randomly assigned to receive either c.i.v. rIL-2 (18.0 MIU/m2 per day on days 1 through 5 and 12 through 16), IFN-α, or the combination. The response rate in the 138 patients receiving c.i.v. rIL-2 was 6.5%. Sixty-nine percent of individuals developed hypotension resistant to vasopressor agents. A retrospective analysis from four open-label, nonrandomized phase II trials of rIL-2 in patients with metastatic RCC showed no significant differences in overall response rate, duration, or survival between s.c. or c.i.v. routes of rIL-2 administration.88 S.c. administration was associated with a significantly lower incidence of adverse events and fewer dose reductions. Thus, the toxicity of single-agent high-dose rIL-2 given as a c.i.v. infusion is substantial. Lower doses of rIL-2 administered as a c.i.v. infusion produce less toxicity. Caligiuri et al.89 administered 0.05 to 0.6 MIU/m2 per day c.i.v. for up to 90 days; the clinical activity of this approach is unclear.
S.c. administration of rIL-2 in patients with RCC has also been examined. In the group of 290 patients (for which the data are summarized in Table 36.5), a response rate of 16.8% was noted. In the report by Buter et al.,90 two complete responses lasting 29 months and longer than 35 months were seen in patients with metastatic renal cancer, which suggests that some of these responses may be durable. Lissoni et al.91 reported a 5-year survival time in response to low-dose s.c. IL-2 to be similar to that obtained with higher doses of IL-2; as expected, responding patients live longer than patients with progressive disease. Yang et al.92 conducted a prospective randomized trial to determine the effectiveness of s.c. rIL-2 compared with bolus rIL-2. Regimens used include the following: high-dose bolus (720,000 IU/kg), low-dose bolus (72,000 IU/kg), and s.c. administration (250,000 IU/kg per day for 5 of 7 days in week 1, followed by 125,000 IU/kg per day for 5 of 7 days in weeks 2 to 6). A higher response proportion was noted with high-dose IL-2 than with low-dose i.v. and s.c. IL-2. The response rate for s.c. IL-2 was similar to that for the low-dose group. This, however, did not produce an overall survival benefit.
TABLE 36.4 CLINICAL RESULTS WITH HIGH-DOSE RECOMBINANT INTERLEUKIN-2 IN PATIENTS WITH RENAL CELL CARCINOMA
TABLE 36.5 RESULTS OF SINGLE-AGENT RECOMBINANT INTERLEUKIN-2 (rIL-2) TREATMENT OF METASTATICRENAL CELL CARCINOMA
To determine whether synergism with IFN-α would affect the dosing level of IL-2, McDermott et al.93 compared the administration of high-dose IL-2 with low-dose IL-2 plus IFN-α in patients with metastatic RCC. Significantly higher response rates were noted with high-dose IL-2 than with low-dose IL-2/IFN-α, especially in patients with bone or liver metastases or primary tumor in place. Furthermore, median and overall response durations were longer in the high-dose IL-2 group; however, this did not meet statistical significance. Similarly, Atkins et al.94 reported more responses with high-dose IL-2 than with lower-dose IL-2/IFN-α in advanced RCC, and they concluded that high-dose IL-2 is the treatment of choice in selected patients with advanced RCC. In a systematic review of 92 studies using different IL-2 routes of administration (i.v., c.i.v., or s.c.) in metastatic renal cancer, Baaten et al.95 concluded that high-dose bolus i.v. IL-2 is superior to other doses and routes of administration. Although these reports suggest that high-dose IL-2 should be the preferred IL-2 regimen for appropriately selected patients, no survival benefits have been demonstrated between the various IL-2 dosing levels in the treatment of RCC.96, 97
Because of the observed differences in response rates among the various studies of IL-2 in RCC, many investigators have documented numerous factors that correlate with disease outcome following IL-2 therapy. These include, but are not limited to, the following: prior nephrectomy and time from nephrectomy to relapse98, 99; number of organs with metastases84; presence of metastases to liver, bone, or lymph nodes94, 100; degree of treatment-related thrombocytopenia101; lymphocyte count and rebound lymphocytosis102; thyroid dysfunction103; erythropoietin production104; and absence of prior IFN therapy.101 Despite attempts to develop models to predict response to IL-2 therapy, factors that determine disease progression and resistance to IL-2 treatment remain unclear at this time.
Administration of nebulized IL-2 via inhalation has also been investigated. Aerosol therapy produces high pulmonary drug concentrations and low systemic drug levels and thereby enhances the therapeutic index.105 Huland et al.106 reported on the use of natural IL-2 administered via nebulizer. Aerosol IL-2 100,000 U was delivered five times daily and was combined with systemic IL-2 and IFN-α. The toxicity of inhaled IL-2 was reported as minimal, which allowed administration in the outpatient department, and antitumor responses were reported. These results have been updated in 116 patients with pulmonary or mediastinal metastatic disease (or both).107 Three different IL-2 formulations were used: natural nIL-2, glycosylated rIL-2 (Sanofi, Montpellier, France), and nonglycosylated rIL-2 (Chiron). Thirty-six, 12, and 68 patients received these IL-2 preparations, respectively, via inhalation. Eleven percent received only inhaled IL-2; 33% received concomitant s.c. rIL-2; and 56% were given concomitant rIL-2 and IFN-α. In 105 patients with pulmonary metastases, 16 patients responded (15.2%), of whom 3 showed complete responses. The median response duration was 15.5 months. The administration of inhaled rIL-2 has also been reported by Lorenz et al.108 and Nakamoto et al.109 In these studies, 16 patients with renal cancer were treated, and four responses (including one complete response) were noted. In another study by Huland et al.110 comparing 94 high-risk patients with RCC and pulmonary metastases treated with inhaled plus concomitant low-dose s.c. rhIL-2 to 103 comparable historical controls given IL-2 systemically, longer overall survival and progression-free survival durations were observed in the inhaled rhIL-2 group. However, the contribution of inhaled IL-2 in these studies is unclear in view of the concurrent administration systemic IL-2 as well as the administration of other cytokines. In a small study comprising 40 patients with pulmonary metastases of RCC, Merimsky et al.111 reported feasibility, tolerability, and disease-progression arrest following the administration of inhaled IL-2 alone. Nevertheless, to more accurately assess the value of this approach, randomized trials comparing results in patients receiving only inhaled cytokine with results in patients receiving systemic therapy with and without inhaled cytokine are required. The delivery of liposome-encapsulated rIL-2 was noted to be well tolerated in humans.112 This method may in the future provide an alternative delivery system. Other routes of delivery for rIL-2, such as intraarterial,108 intrapleural,113 and intraperitoneal,114 have also been examined. Results remain preliminary.
Administration of rIL-2 produces functional alterations in most organ systems. A decrease in lymphocytes occurs initially and then resolves. Subsequently, the peripheral blood lymphocyte pool (CD3+, CD56+) expands. Soluble IL-2R levels increase in the circulation, and IL-2R–positive lymphocytes also are seen.115,116 Cytolytic activity of peripheral blood lymphocytes may be enhanced during continuous infusion of high doses, which results in increased NK cell activity and the appearance of LAK cells in the circulation.117 The effects of rIL-2 on lymphocytes are mediated through specific cell surface receptors on the various subsets. The expression of high-affinity receptors and their saturation by prolonged low-dose infusion of rIL-2 has been reported.118 The possibility that these cytolytic mononuclear cells mediate the antitumor effects of systemically administered rIL-2 has been investigated. The inability to demonstrate correlations of response and development of cytolytic activity in patients treated with rIL-2, however, does not support this hypothesis.
Although most applications have been in the treatment of solid malignancies, IL-2 has also shown promise in the primary therapy of hematologic malignancies as well as in the setting of stem cell transplantation. Myeloid and lymphoid leukemic cells have been reported to be susceptible to IL-2–induced LAK activity119, 120 and have been shown to lack proliferative responses to IL-2, even when expressing the IL-2 receptor.121 Various trials demonstrated the induction of complete and partial remissions in acute myeloid leukemia following rIL-2 therapy.122, 123, 124 Furthermore, IL-2 has been successfully used in conjunction with bone marrow transplantation (BMT) for the treatment of residual malignant hematologic disease in both humans and animals. Studies with rIL-2 following BMT and donor lymphocyte infusions increased survival, induced remissions, and decreased relapse rates in various hematologic malignancies, including acute and chronic myeloid leukemias, non-Hodgkin's lymphoma, and multiple myeloma.125, 126, 127, 128 RIL-2 administration following BMT is believed to increase graft-versus-leukemia effects, possibly leading to the observed improvements in frequency and durations of remission rates.129, 130
Other effects of rIL-2 include endothelial cell activation, with increased expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and endothelial-leukocyte adhesion molecule 1 (ELAM-1).131 Secondary cytokine production (TNF-α, IFN-γ, IL-6) and increased C-reactive protein levels132,133, 134 also have been noted. Finally, rIL-2 may be immunosuppressive in certain circumstances, with decreased delayed hypersensitivity135 and neutrophil chemotaxis reported.136
The severity and nature of rIL-2 side effects are related to the dose and schedule used. The toxicities of bolus, c.i.v., and s.c. rIL-2 are outlined in Table 36.6. Uniformly, patients develop chills, fever, and malaise. A vascular leak syndrome occurs with higher dosages of rIL-2 and is characterized by weight gain, oliguria, tachycardia, and hypotension.137
When this syndrome is present, supplemental i.v. fluids, vasopressors, and diuretics may be required for management. Cardiac toxicities, including arrhythmias and myocardial infarction, and pulmonary side effects, including dyspnea and pleural effusions, may develop. Cardiovascular toxicities include not only vascular leak syndrome and hypotension but direct effects on the myocardium. Hemodynamic studies138 have demonstrated decreased mean arterial pressure and systemic vascular resistance consistent with changes noted during septic shock. Myocardial injury with creatine phosphokinase elevations139 and myocarditis secondary to lymphocyte infiltration140 have also been noted.
TABLE 36.6 TOXICITY PRODUCED BY VARIOUS SCHEDULES AND DOSES OF RECOMBINANT INTERLEUKIN-2(rIL-2)
Hematopoietic findings include anemia, thrombocytopenia, and leukopenia,142 and an increased frequency of sepsis in patients requiring central venous lines has been reported.143 This latter complication may relate to the previously noted granulocyte defect.135 Hepatic toxicities characterized by increases in serum bilirubin levels and minimal changes in transaminase levels are common. Fisher et al.80 investigated this phenomenon using technetium-labeled disofenin and noted delayed excretion and uptake consistent with cholestasis. Return to baseline levels within 4 to 6 days of rIL-2 discontinuation is usual. Gastrointestinal toxicities includes nausea, vomiting, diarrhea, and mucositis. Colon dilation,144 perforation,145 and ischemic necrosis146 have also been seen but represent uncommon manifestations of rIL-2 toxicity.
Neurologic and neuropsychiatric effects can develop acutely or chronically during rIL-2 administration. Patients receiving high-dose intensive therapy may become agitated, disoriented, and occasionally comatose.147 Increases in peritumoral edema in a series of patients with gliomas receiving rIL-2137 and increases of brain water content148 indicate that cerebral edema may be responsible. These effects are generally transient and resolve with drug discontinuation. Neuropsychiatric effects, including a decrease in cognitive function and impaired memory, have been reported in patients receiving c.i.v. rIL-2.149 These latter findings resemble the chronic central nervous system toxicity associated with IFN-α.150
Miscellaneous toxicities include dermatologic complications such as erythema, pruritus, and generalized erythroderma.151 In patients with preexisting dermatologic conditions such as psoriasis, exacerbation of the underlying condition has been described.152 Finally, hypothyroidism or hyperthyroidism has also been seen.153 The cause of this complication is uncertain, but the development of autoimmune thyroiditis secondary to induction of class II antigens in thyroid tissue has been proposed.154 The suggestion has also been made that patients developing this complication are more likely to respond.155
The etiology of rIL-2–related toxicity is uncertain but may involve interstitial lymphocyte infiltrates, vascular leaks, and secondary production of cytokines such as TNF-α.134 The side effects are generally self-limited and resolve rapidly on discontinuation of rIL-2 therapy. Rapid resolution with use of systemic glucocorticoids has also been reported.156, 157 Attempts to diminish rIL-2–related toxicity by coadministration of agents such as the phosphoesterase inhibitor pentoxifylline,158 soluble TNF-α receptor,159 and soluble IL-1 receptor160 have not been successful. In contrast, Samlowski et al.161 demonstrated a decrease in IL-2–induced dose-limiting hypotension after the administration of the superoxide dismutase mimetic (M40403) to mice. Furthermore, the coadministration of agents that either increase the sensitivity of malignant cells to IL-2 or synergize with IL-2 antitumor activities, such as thalidomide,162 IL-18,163 or IL-12,164 may allow the use of lower doses of IL-2 and thereby decrease some of the toxic effects of this cytokine (see next section).
Initial studies with rIL-2 involved single-agent therapy with or without coadministration of ex vivo–activated peripheral blood lymphocytes (LAK cells)165 or tumor-infiltrating lymphocytes.166 These studies were based on preclinical investigations demonstrating benefit of adoptive immunotherapy.167 Randomized trials comparing use of rIL-2 alone with administration of rIL-2 together with LAK or tumor-infiltrating lymphocytes have not, however, demonstrated significant increases in response rates or survival in patients with RCC or melanoma.106, 167
Cytokine combinations involving rIL-2 and a variety of other lymphokines based on studies in animal models have also been used clinically. The cytokines combined with rIL-2 have included IFN-α, interferon-β (IFN-β), IFN-γ, IL-1, IL-4, IL-12, and TNF-α.78, 164, 168, 169, 170, 171, 172 Although the combination of rIL-2 and IFN-α has been the most widely investigated and may produce higher response rates in patients with RCC173, 174 than rIL-2 alone,84 Tourani et al. concluded from a multicenter trial that the coadministration of IFN-α does not improve response or survival rates in patients with metastatic renal cancer compared with s.c. IL-2 alone.175 These observations were also reported by others.93, 94
The combination of chemotherapy and rIL-2 has also been studied. In RCC, administration of vinblastine sulfate and rIL-2 did not produce enhanced antitumor effects.176 The preclinical finding of synergistic effects for doxorubicin hydrochloride and rIL-2177 led to a series of phase I trials investigating different schedules and doses of these two agents.178, 179 Additive toxicity and no immunomodulatory interactions were seen. Finally, cyclophosphamide has been administered before rIL-2 at doses of 350 or 1,000 mg/m2.180, 181 The rationale involves the possible immunomodulatory effects of cyclophosphamide.182 No convincing evidence for enhancement of responses to rIL-2 has been seen.
Multiagent biochemotherapy combinations including rIL-2 have been investigated in patients with RCC and malignant melanoma. In patients with RCC, the combination of rIL-2, IFN-α, and fluorouracil has been used. Atzpodien et al.183 initially reported regression rates over 35%; however, response rates of 1.8%113 and 8.2%184 have been noted. Olencki et al.185 reported a 28% response rate in patients with metastatic renal cancer, but the addition of fluorouracil to rIL-2 and IFN-α significantly increased the toxicity of this therapy. Allen et al.186 also reported significant efficacy but manageable toxicity following the combination of IL-2, IFN-α, and fluorouracil in patients with metastatic renal cancer. In a similar patient population with metastatic RCC, Dutcher et al.187 noted that the addition of fluorouracil to IL-2/IFN-α failed to increase the efficacy and added new toxicity. The benefit of this regimen remains unclear.
In patients with malignant melanoma, combinations of rIL-2, IFN-α, and chemotherapy consisting of imidazole carboximide, carmustine (bischloroethylnitrosourea [BCNU]), and tamoxifen have been used.188 Response rates over 50% have been reported. Despite the high overall response rates noted using biochemotherapy, no survival benefits have been noted in patients with malignant melanoma.189
Other Recombinant Interleukin-2 Preparations
The clinical antitumor effects and toxicity of rIL-2 and its immunomodulatory activities have prompted development of different rIL-2 formulations in an attempt to diminish toxicity and enhance efficacy. Covalent binding of rIL-2 to polyethylene glycol (PEG) at amino acid sites results in an rIL-2 preparation with persistent antitumor activity in murine models.190 Phase I71 and phase II191 trials have been completed. The maximum tolerated dose (MTD) of PEG–IL-2 given as an i.v. bolus once weekly is 20 × 106 IU/m2. Pharmacokinetic studies have demonstrated a prolonged t1/2α (183 minutes) and t1/2β (740 minutes). Serum levels of 15,000 IU/mL were seen, and the clearance was 4.5 mL/minute per m2. The prolonged half-life and decreased clearance were predicted by animal models73 and may be secondary to elimination of renal clearance because of the large hydrodynamic size of PEG–IL-2. The clinical toxicity reported resembles that seen with rIL-2, and antitumor responses were noted in patients with metastatic RCC (2/31 patients). The advantages of weekly administration for an agent such as rIL-2 make the pegylated formulation attractive; however, delayed and unpredictable toxicities have been seen.
Another method of limiting the toxicity of rIL-2 is to incorporate it into liposomes. This produces altered distribution, metabolism, and elimination of the cytokine. A phase I trial of liposome-encapsulated rIL-2 has been initiated and uses escalating doses given as an i.v. bolus. Mild toxicity has been noted,192and immunologic activity, including elevated serum IL-2R levels, NK activity, and numbers of CD16+CD56+ cells, was seen. Use of this preparation may result in a decrease in overall side effects related to rIL-2 while maintaining its immunomodulatory activities. Finally, Yao et al.193 investigated the role of albumin-conjugated IL-2 in the treatment of solid tumors in an animal model and reported a significantly longer circulation time, lower kidney uptake, and increased drug localization in liver, spleen, and lymph nodes, suggesting the potential for improved efficacy and reduced toxicity using this form of IL-2.
IL-4 is a cytokine with pleiotropic actions that was first described in 1982 as a T-cell–derived factor with B-lymphocyte stimulatory activity.194, 195 Since its initial description, IL-4 has been reported to affect a wide variety of cell types196 and to have both stimulatory and suppressive effects on various responses. The genes encoding murine and human IL-4 have been cloned197, 198 and expressed in E. coli. In vivo and in vitro antitumor effects have been found, and initial trials of recombinant IL-4 are under way in patients with various malignancies.
Structure and Mechanisms of Action
IL-4 is a glycoprotein with a molecular weight between 15 and 19 kd. The human and murine forms share extensive homology;197, 199 however, unlike other cytokines, they are species specific.200 The cDNA for human IL-4 encodes a protein of 153 amino acids, which is then cleaved to yield a mature protein containing 129 amino acids.197 The IL-4 gene is on band q23-31 of chromosome 5201 and is located in the vicinity of the genes encoding interleukin 3 (IL-3) and GM-CSF.202 This gene occurs as a single copy and contains four exons and three introns.199
The human IL-4 gene has been expressed in E. coli (Schering-Plough), and milligram quantities are available. Recombinant IL-4 (Schering-Plough) has a molecular weight of 14.9 kd and contains 129 amino acids. It contains two potential glycosylation sites and six cysteine residues that form three disulfide bonds.203 The three-dimensional topology of recombinant human IL-4 (rhuIL-4) has been investigated,204 and interestingly it is similar to that described for recombinant human GM-CSF.205
The Sterling preparation of rhuIL-4 also has 129 amino acid residues and differs from the natural protein at six sites.206 It was expressed in a yeast strain (Saccharomyces cerevisiae), and amino acids 1 to 4 (Glu-Ala-Glu-Ala) are not in natural IL-4 but are a consequence of the expression system. Asp38 and Asp105 are substituted for asparagine to preclude glycosylation. These changes result in a recombinant molecule that has the same biologic activity as the fully glycosylated rhuIL-4. In vitro comparative studies of the two preparations of rhuIL-4 are not available.
IL-4 produces its effects by interaction with cell surface receptors (IL-4Rs) that are present on various hematopoietic and nonhematopoietic cells. IL-4R is up-regulated by cytokines such as IL-2, IL-4,207, 208 IFN-γ, and IL-6.209, 210 IL-4 signaling depends on binding to the IL-4R, which is composed of two chains. IL-4 actually binds the 140-kd IL-4Rα chain with high affinity (Kd 20 to 3,000 pmol/L).211, 212, 213, 214 The IL-4Rα chain is a member of the hematopoietin receptor superfamily and also serves a part of the interleukin-13 receptor.211, 215, 216, 217 IL-4 bound to the IL-4Rα then heterodimerizes with the common γ chain, which does not change the affinity of IL-4 for the receptor but is necessary for initiating signal transduction.218, 219Activation of the IL-4R leads to tyrosine phosphorylation of the α chain at multiple sites.220 Three Janus kinase members are activated by the IL-4Rα chain.221,222, 223 Studies using deletion mutants of the IL-4Rα chain indicate that different cytoplasmic regions have distinct functions, which include binding to Janus kinases, initiation of proliferation, and induction of gene expression.214, 224, 225, 226, 227 The IL-4–dependent pathway leading to proliferation is initiated by the phosphorylation of insulin receptor substrate 1 (IRS-1) by Jak1 and possibly by Janus kinase 2 (Jak2) after IRS-1 interaction with the IL-4Rα chain (residues 437–557).211, 228, 229, 230 PI3K, which is composed of an 85-kd regulatory unit and a 110-kd catalytic subunit, then binds to the phosphorylated IRS-1.229, 231,232 This lipid kinase initiates the generation of the second messenger molecules phosphatidylinositol-(3,4,5)-triphosphate and phosphatidylinositol-(3,4)-bisphosphate.233, 234 These molecules are involved in the downstream activation of protein kinase C and Akt kinase.235, 236 The IL-4 stimulation of PI3K and Akt kinase is also thought to enhance the survival of hematopoietic cells.211, 235, 236 The phosphorylation of IRS-1 by IL-4 is also known to activate the Ras/MAPK pathway, although not consistently in all cell lines tested.211 The region between residues 557 and 657 of the IL-4Rα chain is responsible for IL-4–dependent gene expression through the activation of STAT6.211, 227 In IL-4–treated cells, nuclear translocation of this transcription factor results in the expression of a number of genes, including class II MHC molecules, select immunoglobulins, and CD23.211, 214, 227, 237, 238
IL-4 is produced by activated T helper cells239 and mast cells240 and has pleiotropic effects both in vitro and in vivo (Table 36.7). Stimulation of B- and T-cell functions has been recognized, and a wide range of effects on diverse cell populations have also been reported. IL-4–deficient mice produced by genetic manipulation have provided some insights into its function.241 These animals have normal T- and B-lymphocyte development, but serum levels of immunoglobulin G1 and immunoglobulin E are decreased. Transgenic mice overexpressing IL-4,241 in contrast, have elevated levels of serum immunoglobulin G1 and immunoglobulin E.242 Thus, IL-4 may play a critical role in the development of humoral immunity, particularly immunoglobulin E. Studies with IL-4R knockout mice and STAT6 knockout mice revealed that IL-4–producing T cells play an important role in the development of an immune response to infections with helminths and other parasites. IL-4 has other functions related to the immune system, including up-regulation of the expression of MHC class II molecules on B cells. It also functions in inflammatory responses by increasing the expression of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells.
TABLE 36.7 BIOLOGIC EFFECTS OF INTERLEUKIN-4 ON HUMAN CELLS
TABLE 36.8 RECOMBINANT INTERLEUKIN-4 PREPARATIONS
In addition to these pleiotropic immunoregulatory activities, IL-4 is involved in the proliferation and maturation of dendritic cells. These cells represent antigen-processing cells that in vivo capture, process, and present foreign antigenic peptides to T lymphocytes. A variety of steps in the maturation and functioning of these cells have been identified, and in vitro a variety of cytokines are involved in their generation. The combination of GM-CSF and IL-4 generates functional dendritic cells that can endocytose antigens and stimulate T cells.243 This property of IL-4 and its control of dendritic cell proliferation are being used for the production of these cells for use in current vaccination approaches.
Antitumor activity has also been attributed to IL-4 and is suggested by a series of observations. Various murine epithelial tumor cells express IL-4R,244 and in vivo administration to mice with fibrosarcomas or spontaneous adenocarcinomas has antitumor effects. Tepper et al.245 demonstrated that IL-4 gene transfection into murine tumor cells resulted in their rejection. This appeared to correlate with the degree of eosinophil and macrophage infiltration. In another model, IL-4–producing tumor cells246 induced systemic immunity against murine spontaneous renal carcinoma cells (RENCA).
Studies with human tumors have demonstrated that rhuIL-4 inhibits the in vitro growth of various tumor cells.247, 248, 249 These include hematopoietic tumors as well as various solid tumors, such as breast cancer, ovarian cancer, and head and neck tumors.250, 251 Various reports indicate that the effects of IL-4 may be mediated by inhibition of autocrine growth factors such as IL-6252 and GM-CSF253 IL-4 may have both direct and indirect effects on hematopoietic malignancies. Solid tumors may also contain IL-4R, and in vitro inhibition by IL-4 of cell growth in a wide variety of tumors has been reported.254, 255
Two recombinant IL-4 preparations have been evaluated and are outlined in Table 36.8. They have been produced after expression of the IL-4 gene in either E. coli or yeast. The results of reported phase I trials using these preparations are summarized in Table 36.9.
Serum assays for IL-4 are performed using both biologic and ELISA methods. The traditional assay involves proliferation of human tonsillar B lymphocytes in the presence of cross-linking antibodies to immunoglobulin M. A variation264 involves induction of CD23 expression in various Burkitt's lymphoma and Epstein-Barr virus–transformed B-cell lines by IL-4. Serum may inhibit this assay and therefore must be used as a control. Finally, an ELISA using purified rabbit anti–IL-4 antibodies is available.257
TABLE 36.9 PHASE I CLINICAL TRIALS OF INTERLEUKIN-4 (IL-4)
Figure 36.5 Concentration of interleukin-4 (IL-4) over time in the serum of a patient receiving 400 µg/m2 of IL-4 as an i.v. bolus. (Reproduced with permission from Prendiville J, Thatcher N, Lind M, et al. Recombinant human interleukin-4 [rhu IL-4] administered by the i.v. and s.c. routes in patients with advanced cancer: a phase I toxicity study and pharmacokinetic analysis. Eur J Cancer 1993;29A:1700–1707.)
The pharmacokinetic behavior of IL-4 has been investigated in a variety of studies. Lotze et al.256 used a biologic assay and estimated an α distribution phase of 8 minutes and a β clearance phase of 48 minutes. Ghosh et al.257 and Prendiville et al.261 investigated the serum levels and pharmacokinetic behavior of rhuIL-4 (Sterling) after a single i.v. bolus dose, a 24-hour infusion, or s.c. administration. Serum IL-4 levels were determined using an ELISA. After i.v. bolus administration, serum levels of IL-4 increased with increasing doses, and the agent was rapidly cleared (Fig. 36.5 and Table 36.10). The half-life was between 15 and 22 minutes, and peak serum levels achieved after s.c. administration of rhuIL-4 (Fig. 36.6) were 10-fold less than after comparable i.v. administration. Serum levels produced were dose-dependent, and after s.c. administration of 400 µg/m2, rhuIL-4 bioavailability was 71% ± 14.257 A linear relationship between IL-4 dose level and AUC was found, which indicates linear pharmacokinetics for the dose ranges investigated.261 The rapid clearance and low distribution volume are consistent with binding to IL-4R on peripheral lymphocytes. The short half-life observed for rhuIL-4 is similar to that seen for other cytokines.
Clinical Effects and Toxicity
Preclinical evaluation of rhuIL-4 has demonstrated a wide range of pharmacologic and toxicologic effects in target organs, including the cardiac system, liver, spleen, and bone marrow.265 These effects were dose-related and included death, cardiac inflammation and necrosis, and hepatitis. These were seen at doses greater than 25 µg/kg per day in cynomolgus monkeys. In human trials, rhuIL-4 was safe and well tolerated at dose levels of up to 5 µg/kg per day administered subcutaneously.
The toxicity of rhuIL-4 in humans is dose-dependent. S.c. administration at low dose produces fever, headache, sinus congestion, nausea, and elevated hepatic enzyme levels.262 Anorexia, fatigue, and flu-like symptoms also are seen and generally resolve within 24 hours of rhuIL-4 discontinuation.262 Dose-limiting toxicities reported at 5 µg/kg per day include headaches and arthralgias.
At higher dose levels and with i.v. administration,256, 260 toxicity is more severe. Nasal congestion, periorbital and peripheral edema, weight gain, diarrhea, and dyspnea have been seen.256, 260 A vascular leak syndrome resembling that produced by rIL-2 has been reported,256 and gastritis with gastric ulceration was also seen.266
TABLE 36.10 PHARMACOKINETICS OF INTERLEUKIN-4 AFTER INTRAVENOUS BOLUS INJECTION
Figure 36.6 Serum levels of interleukin-4 (IL-4) following s.c. injection in patients receiving recombinant human IL-4. (Reproduced with permission from Ghosh AK, Smith NK, Prendiville J, et al. A phase I study of recombinant human interleukin-4 administered by the i.v. and s.c. route in patients with advanced cancer: immunological studies. Eur Cytokine Netw 1993;4:205–211.)
In a series of phase II trials involving patients with melanoma or RCC,260 rhuIL-4 was administered at a dose of 600 to 800 µg/m2 by i.v. bolus every 8 hours on days 1 through 5. With this high-dose intensive schedule, four patients developed cardiac toxicity characterized by electrocardiographic changes consistent with infarction and elevated creatine phosphokinase-MB fractions. One patient expired, and at autopsy myocardial infiltration by polymorphonuclear leukocytes, including eosinophils and mast cells, was observed. These findings appear to be related to frequent high-dose administration of rhuIL-4 above the recognized MTD.
The immunologic effects of rhuIL-4 are quite variable. Absolute lymphocyte counts decrease during rhuIL-4 therapy260; however, flow cytometry studies have not demonstrated consistent and reproducible changes in the distribution of lymphocyte phenotypes.257, 260, 262 Lymphocyte cytolytic activities (NK, LAK) are not augmented or induced, and occasional increases in proliferative responses produced by mitogens or rIL-2 have been reported.257
Administration of high doses of rhuIL-4 is not associated with increases in serum TNF-α or IL-1β levels but does produce significant elevations of IL-1ra.260Soluble CD23 levels also increase with rhuIL-4 therapy.260 No alterations in serum immunoglobulin levels have been noted,260 and antibody production to rhuIL-4 has not been seen.
In the phase I trials reported to date, no responses have been seen in patients with solid tumors. In patients with hematologic malignancies, tumor regression has been reported in individual patients with Hodgkin's disease and non-Hodgkin's lymphoma.263 Experience to date is limited, however. Phase II studies involving patients with malignant melanoma, non–small cell lung cancer, and acquired immunodeficiency syndrome–related Kaposi's sarcoma have been reported. The results of these studies are summarized in Table 36.11. Limited activity of rhuIL-4 has been noted, and no further investigation of its antitumor activities is planned.
TUMOR NECROSIS FACTOR
The discovery by Dr. William Coley that patients who developed streptococcal infections occasionally had clinical tumor regressions and his use of bacterial extracts to treat patients with advanced malignancies270 constituted one of the earliest applications of biologic therapy as a treatment for cancer. Shear et al.271 used an extract of Serratia marcescens and noted hemorrhagic necrosis in mice bearing transplanted tumors. The responsible ingredient was identified as an LPS from bacterial cell walls. Carswell and colleagues272 then identified in the sera of mice receiving LPS a factor, termed “tumor necrosis factor,” that produced hemorrhagic necrosis in transplanted murine Meth A sarcoma tumors. An in vitro bioassay using cytotoxicity of these sera for specific tumor cell lines was developed, followed by purification and cloning of human TNF and subsequent large-scale production of recombinant TNF-α.
Coincidentally, lymphotoxin, a cytolytic protein produced by lymphocytes, was recognized.273 It shows 30% homology with TNF-α and appears to share the same receptor.274 This cytokine has been termed “tumor necrosis factor β” (TNF-β) and, along with TNF-α has been implicated in monocyte-mediated and lymphocyte-mediated tumor cell killing.275
TABLE 36.11 PHASE II TRIALS WITH RECOMBINANT HUMAN INTERLEUKIN-4 (rhuIL-4)
Structure and Mechanisms of Action
The TNF-α gene is located on chromosome 6 in the 6p23 segment.276 It is approximately 3 kb in length and comprises four exons, the last of which encodes over 80% of the secreted protein. TNF-α production is a two-step process, with gene transcription and translation tightly controlled. The basal level of TNF gene expression in human monocytes is minimal277 and is enhanced by agents such as LPS278 or 12-O-tetradecanoylphorbol 13-acetate. PGE2 and cyclic nucleotides appear to be mediators of TNF gene regulation,279 and phosphoesterase inhibitors such as pentoxifylline block TNF production.280 Secretion of TNF-α protein is regulated by a separate process and appears to require additional signals.281 A variety of inducers have been identified and include endotoxin, calcium ionophores, and Fc-receptor cross-linking.282, 283 Inhibitors of the secretory process have also been found and include botulinum D toxin.284
Secreted TNF-α contains 154 to 157 amino acids and one disulfide bridge. It is initially synthesized as a proprotein containing 230 amino acids285 and may exist as a transmembrane surface protein that is cytotoxic to TNF-sensitive cells. Cleavage of the proprotein results in the secretion of a mature TNF protein containing 157 amino acids. Purified natural or recombinant TNF exists in solution as a trimer and under denaturing condition has a molecular weight of 17 kd.286, 287
As with other cytokines, TNF acts through specific cell surface receptors that bind both TNF-α and TNF-β.274 Most cells contain TNF receptors (TNFRs); however, the number varies from 200 to 7,000.288 Once TNF binds to its receptor, the complex is internalized and degraded.289 Studies have identified two receptor proteins (TNFR1 and TNFR2) with molecular weights of 55 kd and 75 kd290 that are also shed into the circulation. TNFR1 (p55) can trigger either an apoptotic or an antiapoptotic pathway. The binding of TNF-α to TNFR1 causes the dimerization of receptor death domains in the cytoplasmic tail.291 The adaptor molecule TNFR-associated death domain (TRADD) interacts with the activated receptor.292 This interaction then leads to the recruitment of TNF-associated factor 2 (TRAF-2) and receptor-interacting protein (RIP), which results in the activation of the transcription factors NF-κB and JNK/AP-1.292, 293, 294The signaling pathway for the activation of NF-κB via TNFR1, in which TRAF-2 and RIP can activate the NF-κB kinase NIK, which then activates the IKKα, β, γ has been relatively well characterized.292, 294, 295 This enzyme complex is responsible for the phosphorylation of the IκBα and IκBβ inhibitors, which leads to their polyubiquitinylation and degradation by the 26S proteasome.296 Activation of the death pathway results from the recruitment by TNFR1-bound TRADD of another death domain–containing protein, FADD.292, 296 FADD couples the TNFR1-TRADD complex with the activation of procaspase 8 to the active form, which in turn activates effector caspases and results in apoptosis.296 Unlike with TNFR1, the cytoplasmic domains of TNFR2 directly bind TRAF to induce NF-κB activation and promote cell survival.293, 294, 296 Because TNFR2 does not bind TRADD family members, it does not appear to play a major role in the induction of apoptosis.296 The density of TNF receptors on cells is also regulated. Type I interferons and IFN-γ increase expression,297, 298 and agents such as LPS and IL-1 down-regulate this protein.299
The probable role of TNF-α in the pathogenesis of various disease such as rheumatoid arthritis and in the toxicity produced by cytokines such as rIL-2 led to investigation of approaches to decrease toxicity. One of these uses administration of soluble TNF receptors (sTNFRs). Etanercept produced by Immunex (Seattle, WA) is a genetically engineered fusion protein consisting of two identical chains of recombinant TNFR p75 monomer fused with the Fc domain of human immunoglobulin G1.300 This preparation binds and inactivates TNF. It has been shown to be effective therapy for rheumatoid arthritis.301, 302 In addition, it has been used unsuccessfully in patients receiving high-dose rIL-2 to ameliorate toxicity.67
The in vitro activities of TNF are pleiotropic. The best recognized are antiproliferative effects on a wide range of human and murine tumor cell lines.303Cytolytic and cytostatic effects have both been described. The antiproliferative effects are measured in vitro by growth inhibition or cytotoxicity assays,304which are also used to define a unit of TNF activity. Cell surface receptors are required for these activities, but the receptor density does not correlate with the sensitivity of cells to TNF-α effects.305
In some cell types, TNF-α can induce apoptosis; however, most cells are protected from apoptosis due to the expression of NF-κB–regulated antiapoptotic genes.306, 307 The inhibition of protein synthesis of the selective inhibitors of the antiapoptotic genes makes cells susceptible to TNF-α–mediated apoptosis.296TNF-α also can affect differentiation of various cell types. One early observation indicated that TNF-α reversed adipocyte differentiation, which resulted in “dedifferentiation.”308 Proliferation of hematopoietic precursors such as colony-forming unit granulocyte erythroid monocyte-megakaryocyte (CFU-GEMM), colony-forming unit granulocyte-macrophage (CFU-GM), and erythroid burst-forming unit is inhibited,309, 310 and in HL-60 cells monocyte differentiation is promoted.311 TNF also has mitogenic properties and in normal fibroblast cultures increases DNA synthesis.312 The growth of a wide variety of other human tumor cells is stimulated by TNF.313
The immunomodulatory effects of TNF-α have also been well studied and include variable effects on T and B lymphocytes, mononuclear phagocytes, and neutrophils. Table 36.12 summarizes some of the reported effects. TNF-α appears to act as an activation signal for various classes of leukocytes during the inflammatory response.
TABLE 36.12 IMMUNOMODULATORY EFFECTS OF TUMOR NECROSIS FACTOR α (TNF-α)
Finally, TNF-α up-regulates a variety of different molecules on the surface of endothelial cells, including class I MHC antigens314 and leukocyte adhesion molecules (ELAM-1, ICAM-1).315, 316 Production of various inflammatory mediators such as IL-1317 platelet-activating factor,318 and IL-6.319 are also enhanced by TNF-α.
In view of the hemorrhagic necrosis produced by TNF preparations and the multiple in vitro effects suggesting potential antitumor properties of this agent, initial clinical trials were performed in patients with advanced malignancies. Because of endotoxin contamination of purified natural TNF-α, the various recombinant preparations listed in Table 36.13 have been used. They contain from 155 to 157 amino acid residues and have specific activities from 2.2 × 106to 4.0 × 107 U per milligram of protein. Two varieties of recombinant TNF-α (rTNF-α) are found, one containing 155 and the other 157 amino acid residues. The two types are identical except for the addition of a Val-Arg sequence at the N-terminus of the smaller molecule. The biologic activities of these preparations are similar287; however, specific activities vary.
Phase I trials of rTNF-α preparations involving over 500 patients have been conducted and have used a variety of administration routes and schedules. These are summarized in Table 36.14. The MTD identified in most trials is less than 200 µg/m2 per day and may vary with the route of administration.
The measurement of TNF-α in bodily fluids has been performed using several different methods. Bioassays for detecting cytotoxicity in various cell lines, including L-M cells,320 L-929 cells,321 and WEHI-164 cells,322 have been used and can detect TNF-α concentrations in sera as low as 50 pg/mL. Cell lysis is measured by either crystal violet dye uptake by residual viable cells320 or tritium-labeled thymidine incorporation.321 In addition, ELISA assays using polyclonal antibodies to TNF-α have been developed and can detect TNF-α at levels from 100 to 2,800 pg/mL.322 Comparison of both methods for the detection of serum levels of rTNF-α in several clinical studies322, 323 has demonstrated that they provide similar results.
TABLE 36.13 RECOMBINANT TUMOR NECROSIS FACTOR α PREPARATIONS USED IN CLINICAL TRIALS
TABLE 36.14 PHASE I CLINICAL TRIALS OF RECOMBINANT HUMAN TUMOR NECROSIS FACTOR
Pharmacokinetic studies in rats and nonhuman primates have been performed. Pang et al.324 administered 125I-labeled and unlabeled rTNF-α (Genentech) to Sprague-Dawley rats. A biexponential clearance was found, with a t1/2α of 5 minutes and a t1/2β of 30 minutes for unlabeled TNF-α. The 125I-labeled cytokine had a prolonged β phase of 280 minutes, which suggests altered receptor binding and degradation in vivo. Similar results have been reported in mice.325 In rhesus monkeys,326 short-term infusion (0.5 hour) of rTNF-α (Genentech) at various doses was administered, and two different elimination mechanisms were found. At low doses, a saturable specific process was evident. At higher dose levels, a nonspecific nonsaturable process was found. This latter process was felt probably to represent glomerular filtration of TNF-α. Similarly, in nephrectomized rats, clearance of rTNF-α was significantly reduced.327
In humans, rTNF-α has been administered by a variety of routes and schedules. Table 36.15 summarizes pharmacokinetic data for trials using i.v. administration. In the report by Blick et al.,322 rTNF-α was given as an i.v. bolus, and the half-life did not appear to change with increasing doses. The volume of distribution decreased, however, and the AUC increased when doses were escalated (Table 36.16). These data suggest a one-compartment model, as illustrated in Figure 36.7. In the reports by Moritz et al.323 and Kimura et al.,328 serum half-life and clearance increased with higher doses, which is consistent with a saturable receptor-mediated clearance mechanism. Administration of rTNF-α as a 24-hour continuous infusion329did not yield detectable serum TNF-α concentrations except at the highest dose levels (160 and 200 µg/m2 per day). Serum levels were undetectable after 60 minutes. The influence of TNFR levels in serum and tissues on rTNF-α pharmacokinetics is uncertain; however, as with other cytokines, these may play a role in determining which clearance mechanisms are operative. Induction of TNF-α clearance mechanisms is also suggested by the observations during 24-hour continuous infusion of rTNF-α.329, 330
TABLE 36.15 PHARMACOKINETICS OF HUMAN RECOMBINANT TUMOR NECROSIS FACTOR α(rHuTNF-α) AFTER INTRAVENOUS ADMINISTRATION
TABLE 36.16 PHARMACOKINETICS OF RECOMBINANT TUMOR NECROSIS FACTOR α (rTNF-α) AFTER INTRAVENOUS BOLUS ADMINISTRATION
The intramuscular (i.m.) and s.c. routes of administration for rTNF-α have also been investigated.334 Blick et al.322 administered doses from 5 to 200 µg/m2. Unlike in the case of the i.v. route, serum rTNF-α levels were not consistently detected until doses were higher than 150 µg/m2. Peak serum levels were noted at 2 hours and occasionally persisted for 24 hours. Zamkoff et al.335 administered rTNF-α (Chiron) subcutaneously at doses from 5 to 150 µg/m2 per day. No serum rTNF-α levels were detected, even at the highest doses. Thus, these routes of administration produce lower or undetectable serum levels compared with the i.v. route.
Figure 36.7 Serum disappearance of recombinant tumor necrosis factor (rTNF) after i.v. administration as measured by enzyme-linked immunosorbent assay. Symbols represent the mean blood levels for each dose for all patients at that dose. Standard error bars are shown unless insufficient data points were available. (Reproduced with permission from Blick M, Sherwin SA, Rosenblum M, et al. Phase I study of recombinant tumor necrosis factor in cancer patients. Cancer Res 1987;47:2986–2989.)
Other modes of administration such as intratumoral, intraperitoneal, and regional have also been studied. These approaches were suggested by animal studies indicating that high levels of TNF are required for antitumor effects.336 Pfreundschuh et al.337 administered escalating doses of rTNF-α as a single intralesional injection to cancer patients. The MTD was 391 µg/m2, and detectable serum levels of rTNF-α were found at higher doses, consistent with systemic absorption. Intraperitoneal instillation of rTNF-α in patients with advanced gastrointestinal tumors and ovarian carcinoma has been reported.338 Prolonged TNF-α levels in ascitic fluid without detectable serum levels were seen.
Finally, intraarterial administration of rTNF has been investigated. Hepatic artery infusion of TNF-α339 produced tumor regressions in 14% of patients with liver metastases, but the MTD of TNF-α was not altered compared with i.v. administration. Use of TNF-α in isolated limb perfusion in an attempt to increase local TNF-α concentrations without systemic exposure has also been studied.340 The rTNF-α was administered to three patients at doses of 2.0, 3.0, and 4.0 µg for 90 minutes via isolated extremity perfusion. Serum levels of TNF-α during perfusion never exceeded 62 ng/mL. Perfusate levels of TNF-α varied between 970 and 2,000 ng/mL (ELISA), with no apparent decay. Higher, more stable levels of TNF-α appear to be maintained using this route of administration than with systemic administration. When TNF-α is administered in this fashion, doses exceeding the MTD can be given; however, severe systemic toxicity occurs. The stable concentrations maintained in the perfusate are therefore of interest.
Clinical Effects and Toxicity
The role of TNF in homeostasis predicts that a sepsis-like clinical syndrome would result from its administration. In humans, the toxicity seen is dose-related except for fever and chills, which have occurred even at low doses. The fever appears rapidly after administration and generally resolves within several hours.322, 323 Additional systemic toxicities include anorexia, fatigue, malaise, and myalgia.
The initial hemodynamic effects of i.v. TNF-α include tachycardia and hypertension, followed within several hours by hypotension. This has been the dose-limiting toxicity reported in most trials, and it becomes less severe with repeated administration. The mechanisms responsible for the hypotension are unclear but may include myocardial depression,341 vascular endothelial changes,342 and secondary secretion of IL-1 and IL-6.343, 344
Hematologic toxicity includes thrombocytopenia, which has been dose-limiting in several reports.328, 335, 345 This is generally mild, and in all instances recovery occurred within 24 to 48 hours after rTNF-α discontinuation. Leukopenia has been noted at 30 to 90 minutes after injection,327, 328 and the leukocyte count quickly returns to baseline or higher (neutrophilia). These findings resemble those reported with other cytokines such as GM-CSF. The changes in leukocyte and platelet counts have generally not been associated with either bleeding or infection. Coagulation parameters such as prothrombin time and activated partial thromboplastin time remain normal, and mild elevations of fibrin degradation products have been seen.346
Hepatic toxicity is also common, with increased levels of transaminase, alkaline phosphatase, and total bilirubin reported.287 These levels have generally returned to normal or baseline during continued therapy. In several instances,328, 347 however, these changes were dose-limiting. Mild renal toxicity in the form of slight elevation of blood urea nitrogen and creatinine may occur,323, 328 but it is felt to be of little clinical significance.
Other less frequent toxicities reported include confusion, somnolence, hallucinations, and speech defects.322, 348 Pulmonary toxicity is rare, with occasional reports of dyspnea349, 350 and decreased carbon monoxide diffusing capacity.350 The metabolic changes developing during rTNF-α therapy include increases in serum triglycerides and reciprocal decreases in cholesterol.332 Although weight loss has been noted in animal studies with rTNF-α,351 this has not been a significant finding in human studies.
Administration of rTNF as a s.c. or i.m. injection has a similar toxicity spectrum.331, 335, 352, 353 In contrast to i.v. injection, these routes are also associated with pain and induration at the injection sites. S.c. administration also induces erythema and vesiculation associated with neutrophil and mononuclear cell infiltration.331, 335
Regional rTNF-α administration has similar toxicity. Hepatic artery infusion produces effects resembling those seen during i.v. administration. Isolated limb perfusion with doses of rTNF-α from 2.0 to 4.0 µg also produces systemic side effects, with hypotension, tachycardia, fever, chills, and renal toxicity noted.340Use of hydration with prophylactic dopamine in these patients has controlled the cardiovascular complications, however.
The immunologic effects of rTNF-α administration in humans have been well characterized. Chapman et al.331 reported mild elevation of the acute-phase reactant C-reactive protein during rTNF-α administration. Delayed hypersensitivity to various antigens has been examined in 26 patients, 6 of whom were anergic before rTNF-α therapy.353 Three of these latter patients then developed positive skin tests during therapy. NK and LAK activity in peripheral blood has also been studied.76, 352, 354 Significant depression at 48 hours was noted during a 120-hour continuous infusion of rTNF-α, with subsequent increases above baseline.352, 354 Decreases in IL-2–inducible LAK cells have also been reported.355 Monocyte studies356 have demonstrated increases in hydrogen peroxide production after i.v. therapy with rTNF-α. Studies of lymphocyte subsets demonstrate decreases in the percentages of CD8+ and CD56+ cells, with increases in CD4+ and CD19+ subsets.355 Secondary increases in IL-6,302 G-CSF,357 and macrophage colony-stimulating factor (M-CSF)357 serum concentrations after TNF-α administration have also been noted. The increases in these latter two hematopoietic growth factors coincided temporally with the leukocytosis seen during TNF-α infusion.
In contrast to the hemorrhagic necrosis of tumors seen in murine tumor models, the clinical antitumor effects of systemically administered rTNF have been minimal. Responses have been reported in phase I trials in patients with non-Hodgkin's lymphoma,358 gastric carcinoma,287 hepatoma,287 RCC,347 breast cancer,330 and pancreatic cancer.358 Phase II trials of rTNF treatment of most of these malignancies,359 however, have not demonstrated significant clinical activity. The apparent differences between the effects in murine and human tumors may be related to the tolerance of mice to much larger doses of rTNF-α than are tolerated by humans. Estimates are that a dose of 5 µg, which produces hemorrhagic necrosis of murine tumors, is equivalent to 1,000 µg/m2 of rTNF-α in humans.360 This represents a fivefold greater dose than the MTD of rTNF-α in humans, which is 200 µg/m2. The responses seen with intratumor injection of rTNF-α appear more frequent287 and would suggest that this differential tolerance may be an important factor in the lack of antitumor effects clinically.
The local administration of TNF-α has been investigated as a strategy to minimize systemic toxicity and serum levels, increase local concentrations, and concomitantly increase the antitumor effects of this cytokine. Intraperitoneal administration in patients with ascites and ovarian carcinoma has been evaluated in a small randomized trial.361 The recombinant human TNF-α was given at a dose of 0.06 mg/m2 intraperitoneally after paracentesis at weekly intervals (×3) and compared with paracentesis alone. Intraperitoneal instillation was well tolerated, with pain in 42.1% of patients, fever and chills in 36.9%, and hypotension in 5.3%. Responses were not seen in either group.
TNF-α has also been used to perfuse extremities and the hepatic circulation. Limb perfusion using IFN-γ, TNF-α, and melphalan in patients with nonresectable sarcomas has been reported.362 In a series of 55 patients, complete responses were seen in 18%, partial responses in 64%, and no change in 18%. Limb salvage was achieved in 84%. Systemic side effects were moderate. Another study investigating the role of TNF-α and melphalan in the therapy of soft tissue sarcoma and melanoma reported a response rate of 76% and a limb-salvage rate of 76% in the soft tissue sarcoma group and a 100% response rate and a 93% limb-salvage rate in the melanoma group, following isolated limb perfusion with TNFα and melphalan.363 In advanced limb melanoma, the presence of regional lymph node or distant metastases was associated with an increased risk of death within 1 year, even after limb perfusion.364 Hohenberger365 reported extensive tumor necrosis of recurrent synovial sarcoma tumor beds in two children following limb perfusion with high-dose recombinant TNF-α combined with melphalan. Similarly, Noorda et al.366 demonstrated a 63% overall tumor response rate and a 57% local control rate with limb preservation in patients who had unresectable soft tissue sarcomas of the limbs and underwent ILP with TNF-α and melphalan, followed by resection of the tumor remnant when possible. There has also been success in the treatment of liver metastasis with isolated hepatic perfusion (IHP) using TNF-α in combination with other agents. Numerous trials367, 368, 369, 370 have been conducted using IHP with TNF-α in combination with melphalan and other agents in the treatment of hepatic metastases. A response rate of up to 75% was demonstrated by hepatic tumor regression in patients with liver metastases following IHP with TNF-α in combination with other agents. The use of TNF-α perfusion techniques has also been explored for primary RCC,371 with uncertain clinical utility.
Preclinical studies have indicated that combining rTNF-α with other cytokines or chemotherapeutic agents may enhance the results. IFN-γ produces an up-regulation of TNFR on various cells in vitro,274 and therefore rTNF-α and IFN-γ have been combined in phase I studies. Dose-limiting toxicity has included hyperbilirubinemia,372 hypotension,373 and acute dyspnea with hypoxemia.373 The MTD for rTNF-α in these trials is less than 156 µg/m2 per day, which suggests synergistic toxicity. In murine models, the combination of rTNF-α and rIL-2 has had significant antitumor effects.374, 375 Clinical trials of this combination have been conducted to determine toxicity.78 In addition, rTNF-α has been administered with rIL-2 and IFN-α in a phase I trial.376 Patients received 40 to 120 µg/m2 of rTNF-α on days 1 through 5 and fixed doses of rIL-2 (1 to 3 × 106 IU/m2 on days 1 through 5, days 8 through 12, and days 15 through 19) and IFN-α (9 × 106 IU/m2 three times a week). Systemic toxicity was substantial, but outpatient administration was felt to be possible.
TNF-α shows synergy with a variety of chemotherapeutic compounds in vitro, including doxorubicin377 and cisplatin.378 Synergistic in vitro cytotoxicity against tumor cells has also been noted with combinations of rTNF-α and topoisomerase II inhibitors such as etoposide and dactinomycin (actinomycin D or DACT).379,380 Phase I trials of rTNF-α and etoposide have also been conducted.381 Results of these combinations suggest some clinical activity; however, more studies are needed to elucidate further the clinical utility of these combinations.
Unlike the other cytokines discussed in this chapter, which are primarily utilized for their direct antitumor actions, interleukin-11 (IL-11) has shown benefits in managing chemotherapy-induced toxicity, specifically hematopoietic toxicity. IL-11 is member of the IL-6–type cytokine family, which also includes IL-6, leukemia inhibitory factor, oncostatin M, and ciliary neurotropic factor.382, 383, 384 IL-11 was first detected in the conditioned media from the immortalized primate bone marrow stromal cell line PU-34.385 IL-11 is a megakaryocytopoietic cytokine that offers an alternative to the short-lived effects of platelet transfusion therapy in the treatment of chemotherapy-induced thrombocytopenia.
Structure and Mechanism of Action
Human IL-11 is a 199–amino acid protein, including a 21–amino acid leader sequence, with a molecular weight of approximately 19 kd.386 It is encoded by a 7-kb genomic sequence consisting of five exons and four introns.387 The IL-11 gene has been localized to the long arm of human chromosome 19 at band 19q13.3-q13.4.387 The human and murine forms of IL-11 demonstrate significant structural similarities, sharing 80% and 88% sequence homologies at the nucleotide and protein levels, respectively.388 Recombinant human IL-11 (rhIL-11) (Oprelvekin, Neumega), developed by the Genetics Institute (Cambridge, MA), has 177 amino acids, differing from the 178–amino acid native protein in that it lacks the amino-terminal proline residue. It is produced in E. coli by standard recombinant DNA methods. Similar to the native protein, the recombinant form also has a molecular mass of approximately 19 kd.
The three-dimensional structure of IL-11 has been well characterized, resembling the structures of hGH389 and human granulocyte colony-stimulating factor.390 It is composed of four α-helices connected by loops of variable lengths.383 This structure, which is also noted in IL-2,6 IL-4,391 granulocyte-macrophage colony-stimulating factor,392 macrophage colony-stimulating factor (M-CSF),393 and IL-5,394 is believed to play a role in preserving the interactions of these cytokines with specific cellular receptors.
IL-11 signaling is mediated through a hexameric receptor complex composed of two molecules of IL-11, the IL-11 receptor α-chain (IL-11Rα) and the transmembrane signal transducer glycoprotein gp130.395 Signaling in response to IL-11 is restricted to cells expressing gp130 in addition to IL-11Rα.396, 397The requirement of gp 130 in the IL-11/IL11Rα multisubunit receptor complex is not unique to IL-11 and is shared among the other members of IL-6–type cytokines, including IL-6, leukemia inhibitory factor, oncostatin M, and ciliary neurotropic factor.398, 399, 400 The use of gp130 by these different cytokines may contribute to the observed overlapping of biological activities.401, 402 Signaling through the IL-11 receptor leads to the activation of numerous proteins, including the Janus kinase signal transducer and activator of transcription (Jak-STAT) pathway,400, 403 as well as Ras and mitogen-activated protein kinase (MAPK). This activation is followed by downstream activation of various cytokine-responsive and primary response genes. Depending on the cell type, signaling through the IL-11 receptor complex can modify various cellular functions, including the production of the proinflammatory cytokines IL-1β, interferon-γ, TNF, and IL-12,404, 405 as well as cell survival through the regulation of numerous proteins involved in apoptosis and cell protection, such as NF-κB, Bcl-2, and heat shock protein.384, 406
IL-11 has pleiotropic effects in various tissues, including hematopoietic tissues, where it exerts its effects on primitive stem cells as well as mature progenitor cells.407, 408 IL-11 induces the proliferation and differentiation of primitive stem cells, including multipotential and committed hematopoietic progenitor cells from various tissues such as bone marrow and cord and peripheral blood.409, 410, 411 These effects are mediated in synergy with other cytokines and growth factors, including thrombopoietin (TPO), stem cell factor (SCF), granulocyte colony-stimulating factor (GSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3, -4, -6, -7, -12, and -13.412, 413, 414, 415, 416, 417, 418, 419
Of the various hematopoietic cell types, megakaryocytes and their precursors appear to be particularly responsive to the effects of IL-11, as demonstrated by human and animal models reporting increased proliferation and maturation of megakaryocytes after stimulation by IL-11.420, 421, 422 Synergy with TPO and SCF appear to be critical for IL-11–mediated megakaryocytopoiesis. This is highlighted by studies reporting up to 90% reduction in megakaryocytic colony formation after blocking TPO with anti-TPO antiserum or soluble TPO receptor c-Mpl423 and compete abrogation of the proliferation of hematopoietic progenitors after blocking the SCF receptor c-kit.424
Although not approved as an erythropoietic or leukopoietic agent, in addition to its role in thrombopoiesis, IL-11 has also been shown to be a stimulator of erythropoiesis and leukopoiesis. Unlike in the case of megakaryocytopoiesis, which requires IL-11 synergy with other growth factors and cytokines, IL-11 as a single agent may exert effects on erythropoiesis directly, although synergy with other cytokines has also been reported.425, 426 IL-11 exerts its effects on burst-forming unit-erythroid (BFU-E) early in erythroid development in combination with IL-3, while it can act alone later in erythroid differentiation to support the development of erythroid colony-forming units (CFU-E).425 Further supporting the role of IL-11 in erythropoiesis are observations noting a 6.4-fold increase in IL-11 levels in patients with polycythemia vera.427 Animal models have demonstrated various effects of IL-11 on the differentiation and maturation of lymphoid and myeloid progenitors. In combination with SCF, IL-11 promotes the development of B-lymphocytes and stimulates the proliferation of myeloid colonies, while the addition of IL-4 or IL-13 directs colony differentiation toward a macrophage cell lineage.416, 428 Other cell types affected by IL-11 include bone marrow stromal cells and fibroblasts,429 pulmonary and GI epithelial cells,430, 431 osteoclasts,432 osteoblasts,433 and various neuronal cells.434
The pharmacokinetics of rhIL-11 following i.v. and s.c. infusions has been characterized by Aoyama et al., who found linear pharmacokinetics for both forms of drug administration.435 Using an ELISA method developed by the Genetics Institute specifically for the detection of rhIL-11, Aoyama et al. were able to analyze blood and urine concentrations of rhIL-11 after s.c. and i.v. administrations. Following single i.v infusion at 10, 25, and 50 µg/kg, the mean plasma concentration of rhIL-11 was 53.8, 49.7, and 120 ng/mL, respectively.435 Furthermore, the bioavailability of rhIL-11 following s.c. administration at 3, 10, 25, and 50µg/kg was 67%, 65%, 62%, and 65%, respectively, when calculated on the basis of AUC after an i.v. dose of 50 µg/kg.435 The half-life following i.v. and s.c. administration was approximately 2 hours and 8 hours, respectively, irrespective of dose, indicative of absorption rate–limited pharmacokinetics after s.c. administration.435
RhIL-11 appears to be eliminated primarily by the kidney following degradation and metabolism.436, 437 Aoyama et al. failed to detect immunoreactive rhIL-11 in the urine of healthy volunteers.435 These data, in addition to results from animal models demonstrating 1% urinary excretion of intact radiolabeled rhIL-11,436 point toward degradation mechanisms accounting for the elimination of rhIL-11 before renal excretion.
Clinical Effects and Toxicity
Numerous studies to date have evaluated the toxicity and efficacy of rhIL-11 when used following chemotherapy to reduce thrombocytopenia-related complications. Toxicity related to rhIL-11 is dose-dependent and involves multiple organ systems, ranging from constitutional symptoms such as headaches, nausea, vomiting, and myalgias to more serious side effects primarily involving the cardiovascular system. In a phase I trial of rhIL-11 in patients with breast cancer, Gordon et al. determined the MTD for rhIL-11 to be 75 µg/kg per day, primarily limited by grade 2 constitutional symptoms, including arthralgias/myalgias and fatigue.438 Another phase I trial using low doses of s.c. rhIL-11 (5-15µg/kg per week) reported the most common side effects occurring in greater than 10% of patients to be (in descending order) reaction at injection site, headache, pharyngitis, nausea, asthenia, and rhinitis.439
The most serious side effect related to rhIL-11 reported by Antin et al. was severe fluid retention resistant to diuresis, which contributed to early mortality.440Although several reports noted edema and fluid retention following rhIL-11 therapy,438, 439, 441, 442 these side effects were mild and responded partially to diuretics.443 Fluid retention with rhIL-11 therapy is believed to contribute to the dilutional anemia that is noted following therapy with rhIL-11. Plasma volume expansion is believed to be secondary to rhIL-11–mediated reduced sodium excretion, a mechanism similar to that noted following interleukin-6 administration.443, 444, 445 RhIL-11 has also been noted to increase levels of the acute-phase proteins CRP, fibrinogen, von Willebrand's factor, ferritin, and haptoglobin in a dose-related manner; levels returned to normal after discontinuation of therapy.437, 438, 443 It has been suggested by Gordon et al. that such increases in acute-phase proteins can serve as a marker of rhIL-11 activity.438 Finally, rhIL-11 has been associated with cardiac side effects, including palpitations and atrial arrhythmias,441, 446, 447 although these are believed to be secondary to the volume expansion and overload induced by rhIL-11.447
Many trials have demonstrated the therapeutic efficacy of rhIL-11 in the treatment of chemotherapy-induced thrombocytopenia in cancer patients, as well as in nonmalignant diseases, including aplastic anemia, MDS, and liver cirrhosis.441, 442, 448 The efficacy of rhIL-11 is dose-dependent between 10 and 75 µg/kg per day,438 with increasing platelet counts documented at doses as low as 10 µg/kg per day.438, 442 In a randomized, placebo-controlled study in patients with advanced breast cancer receiving dose-intensive cyclophosphamide and doxorubicin, rhIL-11 decreased the requirement for platelet transfusions and also decreased time to platelet recovery when administered at 50 µg/kg per day subcutaneously for 10 to 17 days.446 In another randomized, double-blinded, placebo-controlled trial in patients with a variety of solid malignancies and lymphoma, 14- to 21-day therapy with s.c. rhIL-11 at 50 µg/kg per day was noted to decrease the need for platelet transfusions in the same patients during subsequent chemotherapeutic cycles, compared with rhIL-11 at 25 µg/kg per day of placebo.441 In both studies, the threshold for platelet transfusions was less than 20,000/µL. RhIL-11 has also shown potential therapeutic efficacy in the treatment of nonmalignant diseases, including Crohn's disease, mucositis, psoriasis, and rheumatoid arthritis. Demonstration of mucosal antiinflammatory properties in animal models449, 450, 451 led to human studies that showed efficacy of rhIL-11 in the treatment of Crohn's colitis.452, 453 Because mechanisms similar to those of intestinal mucosal damage are believed to play a role in chemotherapy-associated oral mucosal injury, rhIL-11 may also decrease the severity of oral mucositis,454, 455, 456, 457 although to date no large placebo-controlled studies have demonstrated this. Even though its antiinflammatory properties, which include reducing macrophage activity and down-regulating the production of proinflammatory mediators (e.g., TNF, IL-12, and IFN-γ), have been well documented,458 rhIL-11 has shown minimal clinical efficacy in the therapy of psoriasis and rheumatoid arthritis.439, 459 While the use of rhIL-11 in nonmalignant diseases has shown some promise, the current therapeutic indications remain for the amelioration of severe thrombocytopenia and the reduction of platelet transfusions following myelosuppressive chemotherapy. Further studies are required to clarify the utility of this cytokine in nonmalignant disease processes.
Interleukin-12 (IL-12), originally named “natural killer cell stimulatory factor” (NKSF)460 and “cytotoxic lymphocyte maturation factor” (CLMF)461 by the groups identifying its multiple activities, was first isolated as a molecule secreted from Epstein-Barr virus–transformed B-cell lines. Early characterization revealed that this protein could act synergistically with IL-2 to augment cytotoxic lymphocyte responses,462 could cause the proliferation of mitogen-activated peripheral blood lymphoblasts,463 and could induce IFN-γ secretion by resting peripheral blood lymphocytes.464
Structure and Mechanisms of Action
On purification, IL-12 was determined to be a 70-kd disulfide-linked heterodimeric protein composed of two polypeptides with approximate molecular weights of 35 kd and 40 kd.460, 465, 466 Characterization of the cDNAs encoding the subunits revealed that the p35 component was a 219–amino acid polypeptide containing seven cysteine residues and three potential N-glycosylation sites, whereas the p40 molecule was composed of 328 amino acids, ten of which are cysteines, with four possible glycosylation sites.467, 468, 469 The mature protein encoded by the p35 cDNA has an actual molecular weight of 27,500 kd but appears larger on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) due to its extensive glycosylation. Mature p40 has a calculated molecular weight of 34,700 kd and is comparatively less glycosylated than the mature p35 molecule.467, 468, 469
The heterodimeric structure of IL-12 is unique among the cytokines.470 Although transfection of cell lines with cDNAs encoding either the p35 or p40 IL-12 polypeptides results in synthesis and secretion of the individual molecules, expression of active, secreted IL-12 requires that the same cell be cotransfected with both cDNAs.471 Interestingly, expression studies suggest that the p35 gene is synthesized by numerous cell types of both hematopoietic and nonhematopoietic origin471, 472 but that active IL-12 is secreted by only those cells that also transcribe p40.471 Thus p40 likely is required for the efficient export of the p70 molecule. The p40 polypeptide is produced in large excess of the IL-12 heterodimer.472 Secreted p40 homodimers may even act as IL-12 antagonists.473 The actual physiologic significance of p40 overexpression and homodimerization, however, remains largely unknown.
The genes encoding the p40 and p35 subunits are completely unrelated and have been mapped to different chromosomes.474 The p35 gene maps to human chromosome band 3p12-3q13.2.474 It has a primary amino acid sequence that is suggestive of a richly α-helical protein and hence in this regard is similar to most other cytokines.471 Indeed, many of the amino acid positions conserved between IL-6 and GM-CSF are also shared by the IL-12 p35 subunit.475 The p40 gene has been mapped to human chromosome band 5q31-q33 and, though not homologous to any known cytokines, does strongly resemble members of the hematopoietic cytokine receptor family.474 The p40 sequence has particularly strong homologies with the extracellular regions of receptors for IL-6 and ciliary neurotropic factor468, 476 and is closely linked genetically to the M-CSF receptor.474 The p70 heterodimer thus has the characteristics of a disulfide-linked complex between a cytokine and a receptor.471
A number of different cell types are important sources of IL-12. Among normal peripheral blood mononuclear cells, monocytes and monocyte-derived macrophages are perhaps the most significant producers of the cytokine,472, 477 although the production of IL-12 by dendritic cells during antigen presentation is the crucial signal for induction of a TH1 response pattern and effective cell-mediated immunity.471, 477, 478, 479 Some studies now also suggest that IL-12 is the requisite “third signal” that participates with class I MHC/antigen complexes and B7 to induce the proliferation and activation of naïve CD8+ T cells480 and hence the cytolytic component of antitumor function. Neutrophils have also been shown to make IL-12,481 although some controversy exists as to whether nontransformed B cells are also a physiological source of the molecule.481, 482 Langerhans cells,483 murine mast cells,484 and keratinocytes485 have also been reported to produce IL-12 under some stimulatory conditions.
The most potent inducers of IL-12 are bacteria, microbial components such as LPS, and intracellular parasites.472, 478, 479 IL-12 production was found to be significantly enhanced when peripheral blood mononuclear cells were stimulated with Gram-positive and Gram-negative bacteria, endotoxin, Mycobacterium tuberculosis, Mycobacterium leprae, and Toxoplasma gondii.472 LPS has also been shown to induce IL-12 synthesis by polymorphonuclear leukocytes.479
In addition to being modulated by the components of pathogens, IL-12 synthesis is also positively and negatively regulated by various cytokines. IFN-γ and GM-CSF are both capable of stimulating IL-12 production by phagocytic cells,486 although, interestingly, the p35 and p40 polypeptide components of IL-12 seem to be differentially induced: although IFN-γ directly stimulates the accumulation of p35 mRNA by monocytes and neutrophils, it can only augment the LPS-induced synthesis of p40 mRNA by those same cell types.486, 487 Among the cytokines with inhibitory effects on IL-12 are IL-10, IL-4, and TGF-β.488, 489These products mediate their effects at the level of both RNA and protein, as they inhibit the secretion of the p70 heterodimeric protein as well as the accumulation of mRNAs encoding the p35 and p40 polypeptides.488, 489 The IL-10–mediated inhibition of IFN-γ production by T and NK cells occurs indirectly through the ability of IL-10 to prevent IL-12 synthesis by phagocytic cells.470, 488
Membrane-bound ligands present on activated T lymphocytes also stimulate phagocytes to synthesize and secrete IL-12.490, 491, 492 Although they are not yet fully defined, at least several receptor-ligand interactions have been characterized that mediate this induction. Perhaps most significant in this regard is the augmented production of IL-12 by antigen-presenting cells on engagement of their CD40 receptor by CD40 ligand (CD40L) expressed on antigen (phytohemagglutinin [PHA])–stimulated T cells.490, 492 The importance of this CD40/CD40L binding in mediation of IL-12 induction has been demonstrated for both murine and human cells. In human peripheral blood mononuclear cells, CD40/CD40L interactions stimulated IFN-γ production via an IL-12–dependent mechanism when the mononuclear cells were first optimally prestimulated with PHA.492 Direct CD28 engagement can also stimulate IFN-γ production and does so in an IL-12–dependent fashion by two different mechanisms: either by increasing CD40L expression on T cells, which then stimulate CD40 receptors and IL-12 synthesis by antigen-presenting cells, or by augmenting the levels of the IL-12 receptor β1 (IL-12Rβ1) chain on T cells and hence the capacity of IL-12 to bind to their receptors.492 CD28/B7 interactions can also enhance IFN-γ synthesis independently of IL-12. Evidence for cooperation of the CD40L/CD40 and B7/CD28 pathways for stimulating IL-12 expression comes from studies performed under conditions of low B7 expression (inadequate antigen or PHA concentrations), in which CD40L-stimulated IL-12 production was found to be enhanced by anti-CD28.492
Cells expressing IL-12 receptors (IL-12Rs) were originally identified using fluorescent anti–IL-12 antibodies to detect the cell-bound cytokine. Using this technique, IL-12Rs were observed on activated NK cells and T cells but not on B cells or resting T cells.493 When IL-12 was added to peripheral blood mononuclear cells and cross-linked after binding, anti–IL-12 antibodies immunoprecipitated a single protein of approximately 110 kd that was purported to be the IL-12 receptor.493 Subsequent expression cloning studies identified what were believed to be two distinct low-affinity IL-12 receptors, which together reportedly formed a high-affinity binding site.494 Each was characterized as having the general composition of a β-type cytokine receptor subunit and as being a gp130-like member of the cytokine receptor family.494 Thus, the functional high-affinity IL-12 receptor was originally thought to be composed of these two subunits, each of which independently exhibited a low affinity for IL-12.495
Additional studies have determined that the functional IL-12 receptor is a heterodimer composed of a β1 and β2 polypeptide and that each mediates a specific activity requisite for IL-12 responsiveness.496, 497 IL-12Rβ1 is the polypeptide that binds IL-12,498 and the β2 chain is the component that transduces the IL-12 signal into the nucleus.497, 499, 500 Normal TH1 cells express both chains and hence are fully IL-12 responsive. TH2 cells express only the IL-12Rβ1 chain, and although they thus bind IL-12 with high affinity, no reactivity to the cytokine is exhibited with the absence of signal transduction capacity.497, 501 Indeed, the selective loss of IL-12Rβ2 expression is an important correlate of TH2 cell differentiation,496 and ongoing TH1 responses can be inhibited by immunosuppressive cytokines that act by negatively regulating that molecule.502, 503, 504 IFN-γ and IFN-α have been shown to induce IL-12Rβ2 expression in the mouse and human, respectively.499 Others have reported that the expression of both IL-12Rβ1 and IL-12Rβ2 mRNA is increased in the lymph nodes of naïve mice after systemic administration of recombinant IL-12.496 The notion that the IL-12 inductive effect on IL-12Rβ2 mRNA is mediated indirectly through IFN-γ was suggested by the observation that in IFN-γ receptor–/–mice, β2 mRNA levels were significantly lower than in wild-type mice after IL-12 treatment.505 Several pathologic conditions are characterized by predominantly TH2 cell populations, and lymphocytes from such individuals display no IL-12Rβ2 chains due to their high production of IL-4, interleukin-5 (IL-5), IL-10, and TGF-β.497, 504 Antibodies to TGF-β and IL-10 restore IL-12Rβ2 chain synthesis and IFN-γ production504 by these peripheral blood lymphocytes in vitro, which supports the notion that TH1 protective responses are highly dependent on adequate expression levels of IL-12 receptor components. It is perhaps because of its potent immunostimulatory effects that IL-12 has its activity stringently regulated at both the agonist level (see earlier) and the receptor level.
The binding of IL-12 to its receptor induces dimerization of the component IL-12Rβ1 and IL-12Rβ2 chains, which leads to the interaction of their receptor-associated Jak2 tyrosine kinases.506 These kinases mediate each other's transactivation,507 which allows the now functional enzymes to phosphorylate tyrosine residue 800 in the IL-12Rβ2 chain cytoplasmic region.500 Signal transduction and transcription-activating factor 4 (STAT4) has specificity for the resulting unique phosphorylated peptide sequence on IL-12Rβ2 (GpYLPSNID, where pY represents phosphotyrosine, and the core G-pY-L is the critical motif for binding),500, 508 and it itself is phosphorylated by Jak2 on binding by its SH2 domain to this receptor site.508 Phosphorylated STAT4 molecules dimerize and migrate to the nucleus, where they bind specific DNA sequences and activate transcription of proinflammatory genes that stimulate TH1 responses.509, 510 The requirement for STAT4 in IL-12–mediated responses was demonstrated by experiments indicating that IL-12–dependent increases in IFN-γ production, cellular proliferation, and NK cell cytotoxicity were abrogated in lymphocytes from STAT4-deficient mice.511 The involvement of both Jak2 and tyrosine kinase 2 (Tyk2) in the IL-12 pathway506, 507, 512, 513 is circumstantially supported by the finding that TGF-β inhibits IL-12–induced phosphorylation of Jak2, Tyk2, and STAT4 and that TGF-β also inhibits IL-12–induced IFN-γ production.513 The possibility that Jak2 or Tyk2 molecules can independently mediate STAT4 phosphorylation is suggested by data indicating that tyrosine phosphorylation of STAT4 is not abrogated when either Tyk2 alone or Jak2 alone is inhibited.512
As discussed briefly above, IL-12 was originally isolated as a cytokine that induced the proliferation and cytolytic activity of NK cells, LAK cells, and cytolytic T lymphocytes. This stimulatory activity is now known to be specific to T cells and NK cells preactivated with either antireceptor antibody,514 mitogens, or IL-2,463 as freshly isolated peripheral blood T cells exhibit minimal responsiveness to IL-12.514 The requirement for activation is related to the absence of IL-12Rs on resting cells514 and their induction on mitogenic stimulation.514, 515, 516 T-cell activation is also necessary to induce components of the transduction pathway (STAT4) required for IL-12 signaling.514 Use of purified T-cell clones and PHA-stimulated T-cell subpopulations indicates that both CD4+ and CD8+ T-cell subsets are susceptible to IL-12 stimulation.460, 462, 466, 517, 518, 519, 520
IL-12 has a pivotal role in establishing the TH1 versus TH2 balance of a developing immune response.521, 522, 523 Dendritic cells processing foreign antigens in peripheral tissues migrate to lymph nodes and, by secreting IL-12, induce IFN-γ production by NK cells and IFN-γ and IL-2 synthesis by antigen-stimulated T cells.470, 521, 522, 523, 524, 525 Dendritic cell–derived IL-12 is also capable of acting synergistically with the induced IFN-γ to steer naïve T-cell precursors towards TH1 cellular immune responses481 and of acting synergistically with IL-2 to further augment IFN-γ production and cytotoxic lymphocyte responses.460,463, 466
In addition to stimulating TH1 activity by naïve cells,470 IL-12 also has been shown to transform preexisting TH2 responses into responses with an effective TH1 cellular component. Such activity was particularly noteworthy in a murine infectious disease model in which the characteristic detrimental TH2 response was converted into a predominantly curative cellular response after systemic administration of IL-12.526 That IL-12 can reverse TH2 responses is somewhat paradoxical, as TH2 cells secrete large quantities of IL-4, IL-5, and IL-10,470 cytokines known to strongly down-regulate the signaling IL-12Rβ2 chain of the IL-12 receptor. Whether this reversal is mediated by the purported capacity of IL-12 to induce transient, low-level production of IFN-γ by TH2 clones526, 527, 528or rather by the initiation of an overlapping TH1 immune response capable of dominating the preexisting TH2 activity is unclear. The determination that IFN-γ inhibits IL-4, IL-5, and IL-10 synthesis by TH2 cells suggests the possibility that IL-12 mediates its anti-TH2 effects indirectly via stimulation of IFN-γ production by NK cells.529
IL-12 has been shown to be an effective antitumor agent in a number of murine models, including the renal cell carcinoma RENCA,530, 531 CT-26 colon adenocarcinoma,531, 532 MCA-105 sarcoma,533 M5076 reticulum cell sarcoma, B16-F10 melanoma,530 MC38/colon carcinoma,533 KA 31 sarcoma,534 OV-HM ovarian carcinoma,535 HTH-K breast carcinoma,536 MBT-2 bladder carcinoma,537 and MB-48 transitional cell carcinoma,537 among others. Numerous studies have now demonstrated that IL-12 therapy results in inhibition of tumor growth, reduction of metastatic lesions, increased survival time, and in some models regression of and resistance to secondary challenge with the same tumor.530, 537 IL-12 is distinctive among cytokines displaying antitumor activity in that it often has proven effective even when therapy is initiated weeks after establishment of a significant tumor burden.530, 531, 537, 538 An exception to this pattern is the HTH-K breast carcinoma model, against which IL-12 mediated measurable antitumor activity only when administered 3 days but not 7 days after tumor cell inoculation.536
IL-12 has no direct cytotoxicity or antiproliferative effect on cultured tumor cells, which indicates that its antitumor effect is mediated indirectly through IL-12–inducible cellular and molecular intermediates.530 One molecule induced by and central to IL-12 activity is IFN-γ, as antibodies to that protein essentially abrogate IL-12–mediated antitumor function.533, 539, 540 Interestingly, although IFN-γ is required for IL-12 activity, administration of exogenous IFN-γ does not mediate the potent antitumor function characteristic of IL-12.541 Several factors may explain this paradox, including the differential half-lives of the two cytokines and the more limited capacity of IFN-γ to reach the tumor site: whereas IFN-γ receptors are ubiquitously expressed on numerous cell types outside the tumor environment, IL-12 receptors are limited to NK cells and activated T cells.541 Thus, compared with IFN-γ, systemically administered IL-12 is much less apt to be completely consumed by cells irrelevant to the antitumor immune response before reaching the specific effector cell types that will ultimately mediate function.541
Many investigators have shown that IL-12 enhances NK and cytolytic T-lymphocyte activity, stimulates antigen-primed T cells to proliferate and differentiate into TH1 cells, and induces NK cells and sensitized T cells to secrete IFN-γ. One study demonstrated that a preexisting CD8 and NK cell tumor infiltrate is required for maximal efficacy of IL-12–mediated antitumor therapy.541 The suggestion was made that these IL-12–responsive cells synthesize IFN-γ within the tumor bed, which induces the local molecular events required for tumor eradication.541 This hypothesis would explain why established tumors are often more susceptible to IL-12 administration than nascent tumors, because large immunogenic tumors are more apt than small ones to contain significant inflammatory infiltrates.
Data support at least three distinct mechanisms of IL-12–mediated antitumor activity, each of which requires IFN-γ as an induced molecular intermediate to execute the response. Tannenbaum et al. demonstrated that a molecular correlate of effective IL-12 antitumor activity in the murine RENCA model is the expression of two chemokines, monokine induced by IFN-γ (MIG) and IFN-γ–inducible protein 10 (IP-10), within the regressing tumor.542 These molecules have since been determined to be chemotactic for NK and activated T cells, which correlates well with immunohistologic data indicating a tremendous influx of CD8+ and CD4+ cells into the treated tumor.531, 542 The tumors undergoing therapy were also characterized by elevated levels of the cytotoxins perforin and granzyme B, which may be among the terminal effector molecules of the infiltrating CD8 T cells in this system.531, 542, 543 An integral role for the IFN-γ–inducible chemokines in IL-12–mediated antitumor activity was indicated by subsequent studies in which antibodies to MIG and IP-10 abrogated all correlates of IL-12–mediated tumor eradication: tumor shrinkage, the T-cell infiltrate, and perforin expression within the tumor bed.531 When explants of human renal tumors were treated in vitro with IL-12, a sequence of molecular events similar to those observed in the murine model was observed: explanted RCC synthesized IFN-γ and IP-10 mRNA in response to the IL-12 treatment.544 These results were also consistent with the authors' findings that biopsied renal tumors from patients enrolled in a phase I IL-12 trial variably expressed augmented levels of those molecules after therapy.544 The conclusion was thus that, as in the murine system, recombinant human IL-12 treatment of patients with RCC has the potential to induce the expression of gene products within the tumor bed that may contribute to the development of a successful immune response.
Numerous other studies support the role of enhanced cellular immunity in the IL-12 antitumor effect. Early experiments performed with this cytokine demonstrated a strict requirement for T cells, as IL-12 antitumor function was essentially abrogated in nude mice and in mice depleted of CD8 T cells.530 A negligible role for NK cells was suggested, however, by the finding that IL-12 antitumor function remained basically normal when therapy was performed in beige mice or wild-type mice depleted of NK cells by treatment with anti-asialo GM1.530 Multiple laboratories also reported a rapid and significant infiltration of IL-12–treated tumors by macrophages,542, 545 and additional studies determined that tumor-infiltrating polymorphonuclear leukocytes are also an important component of the IL-12 response.546 Perforin knockout mice have been shown to be unresponsive to IL-12 antitumor therapy,543 a result that supports the correlation originally found between IL-12 efficacy and intratumoral perforin expression.542
When the antitumor activities of intratumorally and intraperitoneally administered IL-12 were compared, both therapeutic modalities were found to lead to similar immune responses at the tumor site: both augmented IFN-γ expression, cytokine expression, chemokine expression, and inflammatory cell infiltration into the tumor bed. Systemic therapy orchestrated these responses more quickly and with greater efficacy than did local IL-12 treatment.546 Compared with systemic therapy, which immediately activated and rendered peripheral cells responsive to chemotactic signals simultaneously induced at the target site,546locally administered IL-12 required additional time to diffuse and stimulate the same sequence of events. The greater rapidity and intensity of the global immune response after systemic IL-12 treatment was thus associated with a more favorable cure rate for large subcutaneous tumors.546
A second mechanism by which IL-12–induced IFN-γ mediates antitumor activity is through its stimulation of other molecules with cytotoxic function, including nitric oxide synthase. Nitric oxide synthase is produced by endothelial cells, neurons, epithelial cells, macrophages, and tumor cells themselves, and it catalyzes the production of nitric oxide.547 Nitric oxide is known to be an important contributor to macrophage antitumor activity, and its central role in the protective process has been demonstrated by the ability of the nitric oxide inhibitor NG-monomethyl-L-arginine to abrogate IL-12 antitumor efficacy.547 IFN-γ has also been shown to induce the tryptophan degradation enzyme indolamine 2,3-dioxygenase within the tumor bed, which converts L-tryptophan to N-formyl L-kynurenine.548 RNA encoding this enzyme has been detected in IL-12–treated regressing tumor masses, and the enzyme effectively starves the tumor of that required amino acid.
A third mechanism purported to be involved in IL-12–mediated antitumor function is the IFN-γ–dependent induction of various antiangiogenic factors.549, 550,551 Growing tumors require ongoing neovascularization for nourishment, expansion, and metastatic spread.552 Several molecules, including the IFN-inducible chemokines MIG, IP-10, and platelet factor 4, have been demonstrated to inhibit IL-8–stimulated angiogenic activity in the rabbit corneal pocket assay550, 553and in several other in vivo models549, 554, 555, 556, 557 and in vitro correlates549, 558, 559 of blood vessel formation. One reported determinant of whether a specific CXC chemokine mediates angiogenic or antiangiogenic activity is the amino acid composition of its N-terminal region.560 The presence of Glu-Leu-Arg, the ELR motif, in the N-terminus of a CXC chemokine endows the molecule with chemotactic activity for neutrophils561 and angiogenic activity.556, 560 CXC chemokines lacking this motif, on the other hand, such as IP-10, MIG, and PF4, have been found not only to lack chemotactic function but also to inhibit neovascularization.560 The mechanisms by which these non-ELR CXC chemokines mediate antiangiogenic function is not certain, but in vitro studies show that nanogram concentrations of IP-10 can inhibit endothelial cell chemotaxis,558 proliferation,559 and differentiation into tubelike structures.549 The actual contribution the antiangiogenic molecules make to IL-12–mediated antitumor activity is uncertain; some workers find that IL-12 efficacy is abrogated in T-cell–depleted animals530, 546 and that IP-10 is an effective antitumor agent in euthymic but not in nude mice. Several reports have nonetheless supported a significant role for MIG and IP-10 in tumor necrosis and damage to tumor vasculature when these molecules are injected directly into the tumors of nude mice or induced in those lesions by local or systemic IL-12 therapy.562, 563
Recombinant human IL-12 manufactured by the Genetics Institute is available for clinical trials. It is a lyophilized product and is reconstituted with sterile water. Phase I trials using either i.v. or s.c. administration have been performed.
Motzer et al.564 performed a phase I trial in which IL-12 was administered subcutaneously at a fixed dose weekly for 3 weeks. An MTD of 0.5 µg/kg per week was identified, with hepatic, hematopoietic, and pulmonary toxicity being dose-limiting. The toxicities seen with IL-12 are summarized in Table 36.17. A second phase of this trial involved gradual escalation of the IL-12 dose level after an initial dose of 0.1 µg/kg. In this portion, the MTD identified was 1.25 µg/kg.
TABLE 36.17 INTERLEUKIN-12 TOXICITY
Intravenous IL-12 has also been investigated in a phase I trial. Forty patients, including 20 with renal cancer, 12 with melanoma, and 5 with colon cancer, were enrolled.565 Two weeks after a single injection of IL-12 (3 to 1,000 ng/kg), patients received an additional 6-week course of i.v. IL-12 therapy, administered 5 consecutive days every 3 weeks. The MTD was 0.5 µg/kg per day, and the toxicities included fever and chills, fatigue, nausea, and headaches. Laboratory findings included anemia, neutropenia, lymphopenia, hyperglycemia, thrombocytopenia, and hypoalbuminemia.
A phase II trial of i.v. IL-12 was then initiated using 0.5 µg/kg per day for 5 days.566 Seventeen patients were entered, and due to unexpectedly severe toxicity, the study was abandoned. The data from both the s.c. and i.v. phase I trials suggest that a single predose of IL-12 may be associated with a decrease in toxicity and permits escalation to high dosages. The antitumor effects of IL-12 in early clinical trials are summarized in Table 36.18. Responses have been seen in patients with renal cancer and melanoma but are infrequent.
Table 36.19 outlines serum IL-12 levels in patients receiving IL-12 subcutaneously.564, 567 Studies were also performed during weeks 1 and 7 of drug administration and demonstrate a decrease in IL-12 levels after prolonged administration. This has been termed an adaptive response and has been attributed to either antibody formation or an immunoregulatory feedback response. Antibody formation in response to IL-12 has not been found.564 Rakhit et al.567examined this issue in murine models and noted down-regulation of serum IL-12 levels correlated with up-regulation of IL-12R expression, which was not observed in IL-12Rβ–/–mice. These observation suggest that receptor-mediated clearance is operative and that increases in IL-12R enhance clearance of IL-12.
In preclinical studies, IL-12 induces secretion of IFN-γ by a variety of lymphoid cells. After s.c. or i.v. administration, increases in serum levels of this cytokine are also observed. Rakhit et al.567 reported that 0.5 µg/kg of IL-12 produces peak levels of 250 pg/mL, with the maximum concentration occurring approximately 24 hours after administration. IFN-γ levels then gradually decreased to baseline over the next 7 days and are minimally elevated with continuous administration of IL-12 (Table 36.20). In patients receiving escalating doses of cytokine, 1.0 µg/kg produced lower serum IFN-γ levels on day 15.564 In patients receiving i.v. IL-12, dose-dependent increases in serum IFN-γ levels have also been reported.568 Other surrogate markers such as neopterin also increase, with peak concentrations noted between 72 and 96 hours after administration of a single IL-12 dose.564 In addition to these effects, administration of IL-12 increases serum levels of IL-10,567 which results in activation of a complex immunoregulatory cytokine network (Table 36.20).
TABLE 36.18 ANTITUMOR EFFECTS OF INTERLEUKIN-12 IN PHASE I AND II TRIALS
TABLE 36.19 PHARMACOKINETICS OF INTERLEUKIN-12 AFTER SUBCUTANEOUS ADMINISTRATION: WEEK 1 VERSUS WEEK 7
Following IL-12 administration, significant lymphopenia is observed after 24 hours.568 This involves all the major lymphocyte subsets, with NK cells the most severely affected.568, 569 Augmented NK cytolytic activity and T-cell proliferative responses have also been noted.568
Bukowski et al.544 have investigated the expression of a variety of genes in peripheral blood mononuclear cells after s.c. administration of IL-12 to patients with RCC. Rapid induction of IFN-γ mRNA was found and was accompanied by subsequent induction of mRNA for IP-10 and MIG. These chemokines are IFN-γ inducible and mediate chemotaxis of T lymphocytes.542 In addition, IP-10 appears to have antiangiogenic effects and decreases proliferation of endothelial cells. Other investigators559 have also suggested that the antitumor effects of IL-12 may involve inhibition of angiogenesis.556, 559
Although no overwhelming clinical responses have been noted to date, IL-12 has shown promise in the therapy of various malignancies, including melanoma,164, 570 RCC,164, 571, 572 head and neck squamous cell carcinoma,573 cervical cancer,574 transitional cell carcinoma of the bladder,575 lymphoma,576,577 and hematologic malignancies.578 In these trials, various methods of administration have been employed, including subcutaneous,570, 571, 572intravenous,164, 570, 574 intratumoral,573 and intravesical.575
Because of its lack of efficacy when given as a single agent, combinations of cytokines have been tested. The combination of IL-12 with IL-2 or IFN-α has been shown to enhance the antitumor effects in melanoma and RCC. Alatrash et al.572 reported tolerability of subcutaneously administered IL-12 and IFN-α2b and noted the MTD for s.c. IL-12 and IFN-α2b to be 500 ng/kg and 1.0 MU/m2, respectively. The addition of IL-12 lowered the MTD for IFN-α2b by up to eightfold compared with prior studies using IFN-α2b either as a single agent or in combination with chemotherapeutic drugs.579, 580, 581, 582 The effects of this combination on cytokine expression (i.e., of IP-10, Mig, IL-5, and IFN-γ) by peripheral blood lymphocytes were similar to those noted using IL-12 alone in human571 and murine models.531 When given in conjunction with s.c. IL-2, i.v. IL-12 was well tolerated at an MTD of 500 ng/kg.164 The addition of IL-2 after approximately 3 weeks of IL-12 therapy led to the restoration and maintenance of IFN-γ and IP-10 levels in patient blood samples, which were otherwise noted to decrease to below those at the initiation of IL-12 therapy. The activation and expansion of NK cell populations was also noted with this combination. Although some disease stabilizations and partial responses were noted, phase II studies need to be conducted to further define the role of these combinations in RCC and melanoma.
TABLE 36.20 INTERFERON γ (IFN-γ) AND INTERLEUKIN-10 (IL-10) LEVELS AFTER SUBCUTANEOUS ADMINISTRATION OF INTERLEUKIN-12 (0.5 µg/kg)a
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