Internalization Property of Interleukin-4 Receptor Chain Increases Cytotoxic Effect of Interleukin-4 Receptor- targeted Cytotoxin in Cancer Cells

Although the receptor for interleukin-4 (IL-4R) is highly expressed on solid human cancer cells, its significance and internalization function is still unclear. To address these issues, we reconstituted Chinese hamster ovarian (CHO-K1) cells with various components of the IL-4R by transient transfection and performed internalization assays using radiolabeled IL-4. Radiolabeled IL-4 internalized through the IL-4R chain in a time-dependent manner. When the IL4R chain was cotransfected with the IL-13R 1 or c chain, the IL-4 internalization level was identical to IL-4R transfectants, suggesting that the IL-4R chain plays a major role in IL-4 internalization. These results were confirmed by determining the cytotoxicity of a chimeric protein composed of IL-4 and a mutated form of Pseudomonas exotoxin [IL4(38–37)-PE38KDEL] in CHO-K1 cells transfected with increasing concentrations of IL-4R cDNA. To use the internalization property of the IL-4R chain in the context of IL-4R-targeted cytotoxin therapy, we transiently transfected pancreatic and brain tumor cells with IL-4R chain. Surprisingly, these tumor cells acquired 4–75-fold higher binding activity to IL-4 compared with control cells. Consequently, the cytotoxic activity of IL-4 toxin to these cancer cells was enhanced 5–13-fold compared with control cells as assessed by protein synthesis inhibition and clonogenic assays. Taken together, a combination approach that involves transfer of the IL-4R gene and IL-4R-targeted cytotoxin therapy may serve as a novel approach for cancer therapy. INTRODUCTION Targeting of cell surface antigens or receptors expressed on tumor cells is a modern and effective approach for cancer therapy (1–5). In the last decade, many investigators have focused on the identification of tumor-specific targets and approaches that use these targets for cancer therapy. In this context, we have identified the IL-4R as a specific tumor cell surface target for novel cytotoxic agents (5). IL-4R has been shown to be overexpressed in a wide variety of murine and human tumor cells in vitro and in vivo (6–9). It was found that the IL-4R system could exist in three different types. Type I IL-4Rs consist of a major Mr 140,000 protein (IL-4R , also known as IL-4R ) and the IL-2R chain ( c). Type II receptors are composed of IL-4R and IL-13R 1 (also known as IL-13R ) chains. In type III IL-4R, all three chains may form a functional IL-4R complex (10–14). The significance of expression of IL-4Rs on solid tumor cells is unrecognized; however, we and others have observed that solid human tumors, including malignant melanoma, breast carcinoma, ovarian carcinoma, mesothelioma, glioblastoma, renal cell carcinoma, head and neck carcinoma, and AIDS-associated Kaposi’s sarcoma, express IL-4R (6–9, 15–24). We and others have investigated the mechanism of IL-4R internalization after binding to ligand (25–28). Among the IL-4R components, IL-4R chain seems to play a premier role in ligand binding (14), and a recent study showed that IL-4R and c chains are responsible for ligand-induced internalization in human T cells (28). However, we have reported that the IL-4R system in solid tumor cells is composed of IL-4R and IL-13R 1 chains (type II IL-4R; Refs. 11–14, 22, 24). This observation suggests that in solid tumor cells, internalization of IL-4-IL-4R could occur through IL-4R and IL-13R 1 chains or IL-4R chain alone. To study this phenomenon, we reconstituted Chinese hamster ovary (CHO-K1) cells by transient transfection with various chains of IL-4R and examined the internalization characteristics of these transfectants. We found that the IL-4R chain by itself can internalize after binding to I-IL-4. To target IL-4Rs on human solid cancer cells, we have produced a recombinant agent that binds to IL-4R on tumor cells (5, 8). This molecule is a chimeric protein composed of circular permuted IL-4 and a truncated form of a powerful bacterial toxin called Pseudomonas exotoxin [IL4(38–37)-PE38KDEL], and we have shown that this toxin is highly cytotoxic to IL-4RReceived 6/6/01; revised 10/3/01; accepted 10/3/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 These studies were conducted as part of a collaboration between the Food and Drug Administration and Neurocrine Biosciences Inc. under a Cooperative Research and Development Agreement (CRADA). 2 To whom requests for reprints should be addressed, at Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, NIH Building 29B, Room 2NN10, 29 Lincoln Drive MSC 4555, Bethesda, MD 20892. Phone: (301) 827-0471; Fax: (301) 8270449; E-mail: [email protected]. 3 The abbreviations used are: IL, interleukin; IL-4R and IL-13R, interleukin-4 and interleukin-13 receptor, respectively; c, common chain; PE, Pseudomonas exotoxin A; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR. 258 Vol. 8, 258–266, January 2002 Clinical Cancer Research Research. on October 14, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from positive tumor cells in vitro (8, 16, 20–24) and in vivo (23, 24, 29, 30). On the basis of these preclinical developments, IL-4 toxin is being tested in the clinic. In our initial study, IL4(38– 37)-PE38KDEL was infused over a 4–8-day period into recurrent malignant high-grade glioma by one to three stereotactically placed catheters; in six of nine patients, IL-4 toxin mediated extensive necrosis of tumor without systemic toxicity (31). Because in this Phase I clinical trial the numbers of patients were small, no correlation between dose and tumor response could be determined. However, ongoing Phase I/II clinical trial supported these observations and demonstrated an advantage of this form of therapy. Again, no dose-dependent tumor response was clearly observed. The long-term effect of this tumor necrosis is being evaluated and may not be apparent until a Phase III controlled clinical trial is undertaken. However, results to date indicate that IL-4 toxin can be safely given to patients with glioblastoma multiforme and that not all patients seem to respond to IL-4 toxin therapy. Although in tissue culture 100% of glioma samples express IL-4R mRNA, it is possible that the level of surface expression of IL-4R protein is different in different samples. In vitro data suggest that the cytotoxicity of IL-4 toxin depends on the number of IL-4 binding sites/cell: the higher the receptor numbers, the higher the sensitivity to IL-4 toxin. Therefore, it would be of interest to determine whether the IL-4R chain can increase sensitivity to IL-4 toxin. An increase in sensitivity may help patients with cancer by gene transfer of this chain followed by IL4(38-37)PE38KDEL therapy. A new Phase I clinical trial for the therapy of renal cell and breast cancer therapy began recently. In this trial, IL-4 toxin will be administered i.v. every alternate days for 3 injections. Although we did not test the IL-4R expression level in fresh or fixed tumors, theoretically, a higher expression level of IL-4R chain may be desirable to achieve maximum effects of systemic treatment. In addition, although IL-4 toxin is highly cytotoxic to cancer cells that express IL-4R, its cytotoxic efficacy is limited in cell types that express no or low levels of this receptor. On the basis of previous studies that have shown the importance of correlation between receptor number on the cell surface and efficacy of receptor-targeted toxin therapies (32–38), we hypothesized that transfer of the IL-4R gene into these cells may increase the sensitivity to IL-4 toxin. Because in clinical trials not all patients’ tumors seem to respond, it is possible that not all cells in a tumor sample express IL-4R chain or that a majority of tumor cells express lower levels of IL-4R chain. Therefore, patients may benefit further by transfer of gene encoding this chain, which may sensitize all cancer cells to IL-4 toxin therapy. To demonstrate this, human pancreatic (SU.86.86 and COLO587) and primary brain tumor (BT10 and BT12) cell lines were transfected with IL-4R chain, and cytotoxicity assays were performed. IL-4R transfectants dramatically enhanced the sensitivity to the cytotoxic effect of IL-4R-targeted