- Original Articles
- Open Access
Improved Anti-tumor Activity and Safety of Interleukin-13 Receptor Targeted Cytotoxin by Systemic Continuous Administration in Head and Neck Cancer Xenograft Model
© NSLIJ Research Institute 2002
- Accepted: 26 June 2002
- Published: 1 August 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.
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
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
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).
Changes in blood serum chemistry after the treatment with IL13-PE38QQR
Reference Range (Units)
150 µ g/kg
200 µ g/kg
300 µ g/kg
Histologic changes in vital organs after the treatment with IL13-PE38QQR*
150 µ g/kg
200 µ g/kg
200 µ g/kg
300 µ g/kg
Mild multi-focal necrosis, hydrophilic degeneration of pericentral vein cells
Atrophic change, Severe multi-focal necrosis, collapse of structure
Mild cell degeneration
Alveolar wall thickness
Cell hypertrophy, mild focal necrosis, basement thickness
Continuous Infusion Increased Anti-tumor Activity of IL13-PE38QQR in HN12 Tumor Xenografts
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
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.
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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