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Improved Anti-tumor Activity and Safety of Interleukin-13 Receptor Targeted Cytotoxin by Systemic Continuous Administration in Head and Neck Cancer Xenograft Model
Molecular Medicine volume 8, pages487–494(2002)
IL-13 receptor (IL-13R) targeted cytotoxin, IL13-PE38QQR, has been shown to have very potent anti-tumor activity to IL-13R-expressing head and neck tumor cells in vitro and in vivo. However, its effect is limited in aggressive tumors. To further improve the anti-tumor activity and safety of IL-13 cytotoxin, we employed continuous infusion technique in animal model of head and neck cancer.
Materials and Methods
We surgically implanted continuous infusion (CI) pump intraperitoneally that released drug for 7 days, and its anti-tumor effect was evaluated. A comparison was made for antitumor activity and safety with intravenously (IV) administered IL-13 cytotoxin in a head and neck (KCCT873 and HN12) subcutaneous (SC) xenograft tumor models in nude mice. Vital organ toxicities were assessed by histologic examinations and blood serum chemistry analyses.
The 50 or 75 µg/kg/day for 7 days of IL-13 cytotoxin either by IV or CI administration did not show any difference in safety or anti-tumor activity. IV administration of 150 or 200 µg/kg/day of IL-13 cytotoxin for 7 days was lethal to nude mice, whereas 200 µg/kg/day X 7 days of CI administration was highly effective in the regression of established tumors without any toxicities. Additionally, CI administration of IL-13 cytotoxin (200 µg/kg/day) showed growth inhibition of larger HN12 tumors in nude mice.
With a CI schedule, IL-13 cytotoxin can be systemically administrated at approximately twice the dose otherwise given by daily IV bolus administration.
Desirable anti-tumor activities and unexpected toxicities of novel anti-tumor agents depend partly on drug delivery routes. Even a potentially powerful anti-tumor agent can cause unexpected serious adverse events or organ toxicities by a certain route of drug administration. To resolve these problems, therapeutic approaches such as prolonged drug release mechanisms or continuous infusion of drug have been reported to increase drug efficacy (1–4). Because of shorter half-lives of drugs, intravenous (IV) or intraperitoneal (IP) administration limits their efficacy. Moreover, multiple bolus administrations increase the susceptibility to organ toxicities. Therefore, continuous infusion (CI) of drugs can be an ideal way of systemic administration of novel cancer therapeutics.
Interleukin-13 (IL-13) is a helper T cell type 2 (Th2)-derived pleiotropic immune regulatory cytokine (5). It has predominant biological activities on B cells, monocytes, fibroblasts, and endothelial cells and plays a major role in inflammatory diseases. IL-13 may also play a prominent role in cancer because receptors for this cytokine are overexpressed. IL-13 is also an autocrine growth factor for some cancer cells (6). We first identified plasma membrane receptors for IL-13 on several human renal cell carcinoma cell lines (7,8), and since then we reported that a variety of human solid cancer cell lines including AIDS-associated Kaposi’s sarcoma (9,10), glioblastoma (11,12), prostate cancer (13), ovarian carcinoma (14), and head and neck cancer (SCCHN) (15–17) express receptor for IL-13 (IL-13R). In recent years, the receptors for IL-13R have been extensively characterized. We have demonstrated that IL-13R may exist as three different forms in different cell types (7),14,18–21). Two different chains of the IL-13R, IL-13Rα1 (also known as IL-13Rα′) and IL-13Rα2 (also known as IL-13Rα) have been cloned. The murine and human IL-13Rα1 chain was cloned first (22–24). This chain binds IL-13 with low affinity but when coupled with IL-4Rα chain (also known as IL-4Rβ) binds IL-13 with high affinity and mediates IL-13–induced signaling (14,19,20,23,25). The second chain of IL-13R, IL-13Rα2, was cloned from a human renal cell carcinoma cell line (Caki-1). This chain has 50% homology to IL-5R at the DNA level, has a short intracellular domain, and binds IL-13 with approximately 50-times higher affinity than IL-13Rα1 chain (26,27). More recently, we have reported that IL-13Rα2 chain can play an important role in receptor binding and internalization (28,29).
Based on our findings that many solid cancer cells express IL-13R, we produced IL-13 cytotoxin, termed IL13-PE38QQR, which is composed of IL-13 and a mutated form of a Pseudomonas exotoxin. IL13-PE38QQR has a potent anti-tumor activity to IL-13R expressing tumor cells in vitro (8,9,13,15,17,30,31) and in vivo (10,16,32,33). Despite the success of preclinical animal studies, the effect of systemic injection of IL-13 cytotoxin has been limited in some of the most aggressively growing head and neck tumor xenograft models (16). To achieve the optimum effect of this targeted agent, a higher dose needs to be administrated. However, systemic bolus injection of higher doses caused organ toxicities as described previously (10,33). The major organ damage caused by bacterial toxin is irreversible liver toxicity (34–36). To avoid liver toxicity, systemic drug exposure needs to be either of a lower dose or prolonged. Thus, we hypothesized that continuous release of IL-13 cytotoxin in the systemic circulation would enhance its anti-tumor effect as well as reduce organ toxicities. Therefore, in this study we employed mini-osmotic pumps that can infuse drugs continuously and compared the anti-tumor activity and safety of IL-13 cytotoxin given by IV bolus injections and by CI.
Materials and Methods
Recombinant Cytotoxin and Cell Lines
Recombinant IL13-PE38QQR was produced and purified in our laboratory (17,30). The purified protein was found to have 3200 endotoxin EU/mg protein. The final concentration of endotoxin injected to animals ranged between 3.2 and 4.8 EU/dose. The range of endotoxin is lower than the allowable limit in the clinic. Human head and neck cancer cell line WSU-HN12 (termed HN12) was a kind gift from Dr. Andrew Yeudall (National Dental and Craniofacial Research Institute, NIH, Bethesda, MD, USA) (37). KCCT873 cell line was established at Research Institute, Kanagawa Cancer Center (Yokohama, Japan) (38). Cells were cultured in Eagle’s Modified Essential Medium (HN12) or RPMI 1640 (KCCT873) containing 10% fetal bovine serum (Biowhittaker Inc., Walkersville, MD, USA), 1 mM HEPES, 1 mM L-glutamine, 100 µg/ml penicillin, and 100 µg/ml streptomycin (Biowhittaker).
Athymic nude mice 4 weeks old (about 20 g in body weight) were obtained from the Frederick Cancer Center Animal Facilities (National Cancer Institute, Frederick, MD, USA). Animal care was taken in accordance with the guidelines of the NIH Animal Research Advisory Committee. Human head and neck tumor xenografts were established in the nude mice by subcutaneous (SC) injection of cells into the flank. HN12 or KCCT873 cells (5 × 106) were injected in 150 µl of PBS. Palpable tumors developed within 3–4 days. The mice then received injections of excipient (0.2% HSA in PBS) or chimeric toxin either IV (150 µl using a 27-gauge needle through the tail vein) or CI (0.5 µl/hr for 7 days). Continuous administration was performed by loading a mini-osmotic Alzet pump (Alza, Palo Alto, CA, USA) with 100 µl IL-13 cytotoxin. The pump was surgically implanted IP on day 4 after tumor implantation. In brief, nude mice were anesthetized with ketamine and xylazine and placed in the supine position. An upper midline abdominal incision was made, and pumps were inserted from the top of the device.
Two perpendicular diameters of tumors were carefully measured by a Vernier caliper and tumor size was then calculated by multiplying the length and width of the tumor on a given day. The statistical significance of tumor regression was calculated by Student’s t test.
Organs from the experimental animals were fixed in 10% formalin and 5-µm tissue sections were prepared, and stained with hematoxylin and eosin.
A Better Anti-tumor Activity of IL13-PE38QQR by CI Compared with IV Administration in KCCT873 Tumor Xenografts
To compare the anti-tumor activity of IL-13 cytotoxin via IV administration and CI, 50 or 75 µg/kg/day of IL-13 cytotoxin was injected IV (one injection per day for 7 days) or mini osmotic pumps (350 or 525 µg/kg total infusion for 7 days) were surgically implanted IP on day 4 in KCCT873 tumor xenografts. As shown in Figure 1 (A and B), KCCT873 tumors without treatment (excipient control) grew well and mean tumor size became 162–190 mm2 by day 31. On the other hand, tumors in the treated mice began regressing during the treatment period; however, when the treatment period was over, all the tumors began growing gradually. Although in mice treated with 50 µg/kg/day of IL-13 cytotoxin by both routes showed significant regression of tumor in size (79 mm2 in IV and 66 mm2 in CI tumors) by day 31 (p < 0.008 compared to control), there was no significant difference (p = 0.14) in the anti-tumor effect between IV and CI drug administration groups.
In mice treated with 75 µg/kg/day of IL-13 cytotoxin (Fig. 1B), all tumors began to grow slowly after the treatment period. However, the antitumor activity of IL-13 cytotoxin by CI was slightly better compared with IV administration, although the difference was not statistically significant. Mice were also given 200 µg/kg/day of IL-13 toxin by CI (total 1400 µg/kg during 7 days). Two out of six tumors completely disappeared by day 12. Although one tumor recurred, by day 31 one mouse remained tumor free and the mean size of tumors was significantly smaller (50 mm2) compared with tumors in mice with excipient control-loaded pump (190 mm2) (p < 0.001) (Fig. 1C).
Evaluation of Systemic Administration of IL13-PE38QQR in HN12 Tumor Xenografts
We next examined the maximum tolerable dose of IL-13 cytotoxin by systemic administration of the drug. Four days after the implantation of HN12 tumors in nude mice, animals were treated by either IV (150 or 200 µg/kg/day for 7 days; total 1050 or 1400 µg/kg, respectively) or CI (300 µg/kg/day for 7 days; total 2100 µg/kg). As shown in Figure 2A, all the treated tumors started regressing as soon as the treatment began. However, in the 200 µg/kg/day IV treatment group (n = 4), all the mice died by day 11. In the 150 µg/kg/day IV treatment group (n = 8), seven out of eight mice died by day 12. The last mouse left was sick during the treatment period, but recovered later. The survival rate from toxicity in both IV treatment groups was poor (12.5% in 150 µg/kg and 0% in 200 µg/kg) (Fig. 2B).
In sharp contrast, all mice in the 300 µg/kg/day CI treatment group (n = 8) remained healthy throughout the experimental period. In two out of eight mice complete disappearance of tumors was observed by day 10 (Fig. 2A). Although tumors started to grow again, the mean size of the tumors on day 27 (24 mm2) was significantly smaller (p < 0.005) than the mean size of control tumors (208 mm2). The survival rate in this group was 100% compared to control group on day 30 (Fig. 2B).
To assess the organ toxicities after treatment with IL-13 cytotoxin by either IV or CI routes, blood serum chemistry and histologic analyses of vital organs were performed. Blood was drawn on day 11 (1 day after the completion of the treatment period) for serum chemistry. As shown in Table 1, after 150 µg/kg/day IV administration of IL-13 cytotoxin, blood serum potassium, CPK, LDH, and AST/ALT levels were greatly increased compared with blood samples from excipient only injected. Animals that received 200 or 300 µg/kg/day by CI also showed elevations in CPK, LDH, and AST/ALT; however, these increases were not as high as seen in IV treated mice.
Samples from vital organs such as kidney, liver, lung, and spleen were also harvested on day 10 or 11 and histology of the tissue sections was assessed. As summarized in Table 2, after 150 or 200 µg/kg/day for 7 days of IV administration of IL-13 cytotoxin, major histologic changes in vital organs were found. As expected, these histologic changes were more severe in the 200 µg/kg/day IV treated mice compared with the 150 µg/kg/day IV treated mice. Organ toxicities including notable liver necrosis, multifocal necrosis of kidney, and pulp degeneration of spleen were observed in 200 µg/kg/day for 7 days IV treated mice. Tissue sections from 100 µg/kg/day IV treated mice did not show significant histologic changes (data not shown). These histologic changes were considered to be signs of organ toxicities caused by IL-13 cytotoxin. On the other hand, the mice treated with 200 and 300 µg/kg/day of CI administration of IL-13 cytotoxin did not show severe histologic changes. Although slight abnormalities were found in liver and kidney samples from the 300 µg/kg/day CI treated mice, changes were apparently modest compared with 150 µg/kg/day IV treated mice. As shown in Figure 3, 200 µg/kg IV dose caused considerable histologic changes in vital organs. Considering 200 µg/kg/day dose delivered by either IV injections or CI were same in total dose (1400 µg/kg for 7 days), these results suggest that CI route is definitely less toxic to vital organs than IV route.
Continuous Infusion Increased Anti-tumor Activity of IL13-PE38QQR in HN12 Tumor Xenografts
Our blood serum chemistry analysis and histologic examinations of vital organs suggested that 100 µg/kg/day (IV) or 200 µg/kg/day (CI) IL-13 cytotoxin can be administrated systemically for 7 days without toxic side effects. To compare the anti-tumor effect of 100 µg/kg/day (IV) or 200 µg/kg/day (CI), HN12 cells were implanted SC into male or female nude mice. IL-13 cytotoxin was administrated for 7 days from day 4 to day 10. As shown in Figure 4A, tumors began regressing during IV treatment period in both male and female mice, and by day 10, tumors disappeared in one out of six mice in both male and female groups. Tumors started growing after the treatment period and by day 14 tumors recurred in all the mice. Although the mean size of tumors was significantly smaller (male, 71 mm2; female, 91 mm2) compared with excipient only injected control tumors (male, 184 mm2; female, 191 mm2) on the day of termination of the experiment (day 27; p < 0.0005). No complete responders to IL-13 cytotoxin were observed. No significant difference was observed in the anti-tumor activity of IL-13 cytotoxin in either male or female mice.
As shown in Figure 4B, tumors in 200 µg/kg/day CI treatment group also regressed during treatment period in both male and female mice; however, the tumors regressed more quickly than the IV treatment group (100 µg/kg/day; Fig. 4A). By day 11, tumors completely disappeared in three out of six male mice and two out of six female mice. In the rest of mice, tumors began growing gradually after the drug infusion period; recurrence was observed in one each male and female mouse. Nevertheless, two of six male mice and one of six female mice remained tumor-free until termination of the experiment (day 27). The mean size of tumors in treated mice (male, 46 mm2; female, 72 mm2) were significantly smaller compared with control mice that were implanted with excipient only loaded CI pump (male, 192 mm2; female, 216 mm2) (p < 0.0005). It appears that toxicity and anti-tumor activity of IL-13 cytotoxin was not gender specific (tumor regression, male 71% versus female 67%).
Anti-tumor Activity of IL13-PE38QQR in Large SCCHN Tumor Xenografts
Finally, to assess the anti-tumor effect of IL-13 cytotoxin in large SCCHN tumor model, HN12 tumors were implanted SC in nude mice. When tumors grew to mean size of 87 ± 12 mm2 (day 13), animals received IL-13 cytotoxin either by IV (100 µg/kg/day for 7 days) or CI (200 µg/kg/day for 7 days) routes. Excipient only injected (IV) tumors grew rapidly, reaching 199 mm2 by the end of experiment (day 29) (Fig. 5). On the other hand, tumors treated with IL-13 cytotoxin showed growth inhibition during the treatment period by both IV and CI routes. No complete responders were generated; however, large size head and neck tumors were profoundly regressed. By day 29, mean tumor size in the IV group was 113 mm2 and CI group was 60 mm2, which were significantly smaller (p < 0.0005 in both groups) when compared to control (mean tumor size 199 mm2). These data suggest that IL-13 cytotoxin mediates anti-tumor effects on large SCCHN tumors in a dose-dependent manner. The maximum tolerated dose by CI route was superior than maximum tolerated dose by IV route.
In this study, we demonstrated that systemic continuous administration decreases the toxic effects of IL-13 cytotoxin. When SC SCCHN tumor-bearing nude mice were treated with IL-13 cytotoxin by either IV or CI routes, the maximum tolerated dose by IV administration was found to be 100 µg/kg/day for 7 days, and 200 µg/kg/day for 7 days by CI route. Because CI increased the maximum tolerated dose of IL-13 cytotoxin by 2-fold, the anti-tumor therapeutic activity of IL-13 cytotoxin improved in both gender of animals.
A 50 µg/kg/day or 75 µg/kg/day dose of IL-13 cytotoxin given by IV and CI did not show any toxicities in KCCT873 tumor xenografted nude mice. Although CI treated mice showed slight superiority in anti-tumor activity, significant differences in the anti-tumor activity were not observed. HN12 xenografts treated with 150 µg/kg/day IV for 7 days had highly elevated levels of hepatic transaminases and potassium, suggesting liver toxicity. Although 300 µg/kg/day CI treated mice also showed elevated levels of these parameters, the intensity of increase was lower than in the 150 µg/kg/day IV treated mice. Histologic examinations suggested that all four vital organs—liver, kidney, lung, and spleen— were severely affected by 150 µg/kg/day IV treatment with IL-13 cytotoxin. On the other hand, organs from 300 µg/kg/day CI treatment group showed less severe toxicities compared with 150 µg/kg/day IV treated mice.
Several approaches have been tested to improve the anti-tumor activity of cytotoxins and immunotoxins and to decrease toxicity and immunogenicity of these agents. Among these approaches, site-specific PEGylation of molecule and insertion of nucleotide sequences of human immunoglobulin genes into the gene encoding mouse monoclonal antibodies have been successfully shown to prevent inadequate recruitment of host leukocytes bearing constant (Fc) region receptors (34,36,39,40). These approaches resulted into prolonged half-life of the circulating drug. In our current study, we utilized a device for CI of drug as an alternate approach to increase the availability of drug. This approach resulted in improved anti-tumor effect and decreased toxic effects. When implanted, interstitial fluid enters the CI pump via the semipermeable membrane because of the osmotic difference between the fluid and the salt solution in the pump. The fluid causes expansion of the salt layer, which compresses the flexible drug reservoir and forces solution out of the delivery portal (41). Utilizing this device, IL-13 cytotoxin can be continuously administrated systemically in body.
In summary, through CI, we successfully decreased the toxicities and increased the efficacy of IL-13 cytotoxin in tumor-bearing hosts. Because IP and IV administration of IL-13 cytotoxin has been shown to have a significant anti-tumor activity in IL-13R expressing tumors (10,16,33), its efficacy can be further enhanced by CI. We have begun Phase I/II clinical trials in patients with recurrent glioblastoma and progressive renal cell carcinoma (42). Based on our current results, we may be able to develop next generation clinical studies utilizing CI. Because IV bolus administration produces transient peak levels of drug, CI may provide constant high levels of drug exposure to tumors to enhance its systemic effectiveness.
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We thank Ms. Pamela Dover for the procurement of reagents and technical assistance, and Dr Bharat H. Joshi for providing recombinant IL13-PE38QQR. We also thank Drs Raymond P. Donnelly and David Essayan of CBER/FDA for reading this manuscript. These studies were conducted as part of a collaboration between the FDA and NeoPharm Inc. under a Cooperative Research and Development Agreement (CRADA).
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Kawakami, K., Husain, S.R., Kawakami, M. et al. Improved Anti-tumor Activity and Safety of Interleukin-13 Receptor Targeted Cytotoxin by Systemic Continuous Administration in Head and Neck Cancer Xenograft Model. Mol Med 8, 487–494 (2002) doi:10.1007/BF03402028