©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Chimeric Protein Composed of Interleukin 13 and Pseudomonas Exotoxin Is Highly Cytotoxic to Human Carcinoma Cells Expressing Receptors for Interleukin 13 and Interleukin 4 (*)

Waldemar Debinski (1)(§), Nicholas I. Obiri (2), Ira Pastan (3), Raj K. Puri (2)

From the (1)The Milton S. Hershey Medical Center, Division of Neurosurgery, Department of Surgery, and Department of Microbiology and Immunology, Pennsylvania State University, Hershey, Pennsylvania 17033, the (2)Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892-4555, and the (3)Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Chimeric proteins provide a unique opportunity to target therapeutic bacterial toxins to a subset of specific cells. We have generated a new recombinant chimeric toxin composed of human interleukin 13 (hIL13) and a Pseudomonas exotoxin A (PE) mutant, PE38QQR. The hIL13-PE38QQR chimera is highly cytotoxic to cell lines derived from several human epithelial carcinomas such as adenocarcinoma of stomach, colon, and skin. The cytotoxic action of hIL13-PE38QQR, which can only occur upon internalization of ligand-receptor complex, is blocked by an excess of hIL13 but not of hIL2. This action is not solely hIL13-specific because an excess of hIL4 also blocks the cytotoxicity of hIL13-toxin. Conversely, hIL13 blocks the cytotoxicity of a hIL4-PE38QQR chimera. Binding studies showed that hIL13 displaces competitively I-labeled hIL4-PE38QQR on carcinoma cells. These results indicate that IL4 and IL13 compete for a common binding site on the studied human cell lines. Despite this competition, hIL4 but not hIL13 decreased protein synthesis in malignant cells susceptible to the cytotoxicity of both hIL13- and hIL4-PE38QQR. Our results suggest that a spectrum of human carcinomas express binding sites for IL13. Furthermore, hIL13 and hIL4 compete reciprocally for a form of the receptor that is internalized upon binding a ligand. Thus, cancer cells represent an interesting model for studying receptors for these two growth factors. Finally, hIL13-PE38QQR may be a useful agent in the treatment of several malignancies.


INTRODUCTION

Recent reports have described the isolation and cloning of lymphocyte growth factor interleukin 13 (IL13)()(1, 2) . IL13 is a potentially glycosylated peptide of M 12,000, and it bears a limited but significant homology to IL4 at the N and C termini of the protein chain. Both ends of IL4 are important for binding to receptor (3, 4). Functional responses of normal blood cells to IL13 are pleiotropic and often, but not always, overlapping with those observed using IL4(5, 6) . Contrary to IL4, IL13 is not species-specific and does not regulate T lymphocytes(1, 2) . It has been suggested that receptors for hIL4 and hIL13 share a subunit that is important for ligand-induced intracellular signaling(7) . This is based on observation that an antagonist of IL4, a mutated form of hIL4, blocked the mitogenic effect of both hIL4 and hIL13 on pre-leukemic TF 1 cells. As hIL13 weakly displaced labeled hIL4 from binding sites, nonmutated hIL4 competition for hIL13 sites was not reported(7) . In fact, our experiments showed lack of displacement of labeled hIL13 by a wild-type hIL4 on TF 1 cells and a very weak or nil displacement of hIL13 by hIL4 on two renal cell carcinoma cell lines(8) . Also, a possibility has been raised that hIL13R may use the subunit of the hIL2R, as has been observed with hIL4R and hIL7R(9, 10, 11) .

We have recently reported that a wide range of human cancer cell lines express receptors for hIL4; these include hematopoietic and epithelial malignancies(12, 13) . When the hIL4R was targeted with PE-based chimeric toxins, we observed a specific killing of cancer cells expressing hIL4R(12, 14) . We have also shown that the PE-containing chimeric toxins have prominent antitumor activities in a human solid tumor xenograft model(15) .

PE is a single-chain bacterial protein made up of three major domains (for review, see Ref. 16). A characteristic feature of PE is that the N-terminally located domain Ia binds to the -macroglobulin receptor, and the ligand-receptor complex undergoes receptor-mediated internalization (endocytosis) to allow intracellular routing and processing of the toxin(16) . Domain II is a site of proteolytic cleavage that activates PE and is necessary to catalyze the translocation of the toxin into the cytosol. The C-terminal domain, domain III, contains the REDLK sequence that directs the processed fragment of the toxin to the endoplasmic recticulum and possesses an ADP ribosylation activity that inactivates elongation factor 2 and leads to cell death(16) . Since domain Ia of PE binds to many types of eukaryotic cells, several mutated forms of the exotoxin have been constructed that do not bind to its ubiquitous receptor and can be attached at a gene level to various internalized ligands. One of the derivatives of PE used in the present study is PE38QQR in which domain Ia (amino acids 1-252) and amino acids 365-380 of PE are deleted, and lysine residues at positions 590 and 606 are replaced by glutamine and at 613 replaced by arginine(17) .

Because hIL4R are richly represented among malignancies and because of the similarities between hIL4 and hIL13, we used human cancer cell lines to study the interaction of hIL13 and hIL4 with their receptors. We have used a novel recombinant chimeric toxin in which hIL13 is fused to PE38QQR, since fusion proteins containing PE are a powerful mean of plasma membrane receptors analysis(18) .


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and DNA ligase were obtained from New England Biolabs (Beverly, MA), Life Technologies, Inc., and Boehringer Mannheim. [H]Leucine and I were purchased from Amersham Corp. Fast protein liquid chromatography columns and media were purchased from Pharmacia Biotech Inc. Oligonucleotide primers were synthesized at Pharmacia's Gene Assembler at the Research Center, Hôtel-Dieu Hospital, Montreal. PCR kit was from Perkin-Elmer.

Plasmids, Bacterial Strains, and Cell Lines

Plasmids carry a T7 bacteriophage late promoter, a T7 transcription terminator at the end of the open reading frame of the protein, a f1 origin of replication and gene for ampicillin resistance(19) . The gene encoding hIL13 (a generous gift of Dr. René de Waal Malefyt of the DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA to W.D.; plasmid JFE14-SR) was PCR-cloned into plasmid pWDMH-QQR to produce hIL13-PE38QQR or into plasmid pSGC242FdN1(20) , to make hIL13 alone. hIL4 was cloned into an expression vector in a similar way to hIL13 using plasmid pWDMH4 (12) as a template for PCR amplification. Recombinant proteins were expressed in Escherichia coli BL21 (DE3) under control of the T7 late promoter(12) . Plasmids were amplified in E. coli (HB101 or DH5 high efficiency transformation) (Life Technologies, Inc.) and DNA was extracted using Qiagen kits (Chatsworth, CA).

Plamids Construction

The chimeric gene encoding hIL13-PE38QQR (phuIL13-Tx) was constructed as outlined (see Fig. 1). hIL13 was PCR-amplified from plasmid JFE14-SR in its mature form. New sites were introduced for the restriction endonucleases NdeI and HindIII at the 5` and 3` ends of the hIL13 gene, respectively. The 336-base pair DNA fragment was obtained during 25 cycles of PCR and digested with appropriate restriction enzymes. The digested fragment was subcloned into the vector obtained by digestion of plasmid pWDMH4-QQR (15) with NdeI and HindIII to produce plasmid phuIL13-Tx. The 5` end of the gene fusion was sequenced and showed the correct DNA of hIL13.


Figure 1: Scheme for cloning plasmid phuIL13-Tx, which encodes hIL13-PE38QQR. prohIL13, propeptide of hIL13; mhIL13, mature hIL13; N, NdeI; H, HindIII; p1, primer 1; p2, primer 2.



Expression and Purification of Recombinant Proteins

E. coli BL21 (DE3) cells were transformed with plasmids of interest and cultured in 1.0 liter of Terrific Broth (Life Technologies, Inc.). hIL13 and hIL13-PE38QQR were localized to the inclusion bodies. The procedure for the recombinant proteins isolation from the inclusion bodies was described previously(12) . After dialysis, the renatured protein of hIL13-PE38QQR was purified on Q-Sepharose Fast Flow and by size exclusion chromatography on Sephacryl S-200 HR (Pharmacia). The initial step of hIL13 or hIL4 purification was conducted on SP-Sepharose Fast Flow (Pharmacia).

Protein concentration was determined by the Bradford assay (Pierce ``Plus,'' Rockford, IL) using bovine serum albumin as a standard.

Protein Synthesis Inhibition Assay

The cytotoxic activity of chimeric toxins, such as hIL13-PE38QQR, were tested as follows. Usually 1 10 cells/well were plated in a 24-well tissue culture plate in 1 ml of medium, and various concentrations of the chimeric toxins were added 20-28 h following cell plating. After 20 h of incubation with chimeric toxins, [H]leucine was added to cells for 4 h, and the cell-associated radioactivity was measured. For blocking studies, rhIL2, -4, or -13 was added to cells for 30 min before the chimeric toxin addition. Data were obtained from the average of duplicates, and the assays were repeated several times.

To evaluate the effects of cytokines on the protein synthesis, the assays were performed as follows. 2 10 cells/well were plated in a 24-well tissue culture plate in 1 ml of medium, and the cytokines were added 20 h following cell plating. After 72 h of incubation with the cytokines, [H]leucine was added to the cells for 4 h, and the cell-associated radioactivity was measured.

Competitive Binding Assay

We determined the binding ability of hIL13 as compared with hIL4-PE38QQR. The recombinant hIL4-PE38QQR was labeled with I using the lactoperoxidase method exactly as described previously(19) . The specific activity of I-hIL4-PE38QQR was 6.2 µCi/µg of protein. Binding assays were performed by a standard saturation and displacement curves analysis. A431 epidermoid carcinoma cells were seeded at 10 cells/well in a 24-well tissue culture plates at 24 h before the experiment. The plates were placed on ice, and cells were washed with ice-cold PBS without Ca or Mg in 0.2% bovine serum albumin, as described previously(19) . Then, increasing concentrations of hIL13 or hIL4-PE38QQR were added to cells and incubated 30 min prior to the addition of fixed amount of I-hIL4-PE38QQR for 2-3 h. After incubation, the cells were washed twice and lysed with 0.1 NAOH, and the radioactivity was counted in a counter.


RESULTS

To construct the chimeric toxin, the coding region of the hIL13 gene was fused to a gene encoding a mutated form of PE, PE38QQR (Figs. 1 and 2A). The chimeric gene is in the bacterial vector under the control of a bacteriophage T7 late promoter; the protein was expressed in E. coli BL21 (DE3) as described previously (12) (Fig. 1). hIL13 alone was subcloned into the same expression plasmid. hIL13 and hIL13-PE38QQR were expressed at high levels in bacteria as seen in SDS-polyacrylamide gel electrophoresis analysis of the total cell extract (Fig. 2B). After initial purification on SP-Sepharose (hIL13) or Q-Sepharose (hIL13-PE38QQR), the renatured recombinant proteins were applied onto a Sephacryl S-200 HR Pharmacia column (Fig. 2B). hIL13 and hIL13-PE38QQR appeared as single entities demonstrating the very high purity of the final products. The chimeric toxin migrated within somewhat lower than expected for 50-kDa protein M range, which may be related to the hydrophobicity of the molecule. The biologic activity of the rhIL13 was exactly the same as one obtained from Sanofi Elf Bio Recherche(8) .


Figure 2: A, Schematic drawing of multi-domain proteins: (i) PE, (ii) its derivative, PE38QQR, and (iii) hIL13-PE38QQR. Circles, structural domains of PE; domain Ia (amino acids 1-252), binding domain; domain II (amino acids 253-364), place of the proteolytic cleavage shown by an interrupted line; the dispensable domain Ib (amino acids 365-404); and domain III (amino acids 405-613), the ADP-ribosylating domain. PE38QQR, domain Ia and amino acids 365-380 in Ib are deleted, plus the three lysine residues in domain III at positions 590, 606, and 613 are changed to two glutamines and arginine (QQR) (17). The square symbolizes hIL13. B, expression in E. coli and purification of hIL13 and hIL13-PE38QQR. 15% nonreduced SDS-polyacrylamide gel electrophoresis stained with Coomassie Blue. Total cell extract of transformed E. coli BL21 (DE3) was loaded at 2 µl/lane (sucrose suspension of the pellet obtained from 1.0 liter of culture; total volume was 75 ml). Sephacryl S-200 HR-purified proteins were loaded at 5.5 µg/lane of hIL13 and 11 µg/lane hIL13-PE38QQR, respectively.



hIL13-PE38QQR Is Cytotoxic to Many Cancer Cell Lines

There have been no reports about the presence of IL13R on human solid cancers besides on renal cell carcinomas(8) . Therefore, we tested several established cancer cell lines to determine if hIL13-PE38QQR is cytotoxic to them. We examined cancers derived from colon, skin, and stomach; the cancer cells were sensitive to hIL13-PE38QQR with IC values ranging from less than 1 ng/ml to 300 ng/ml (20 pM to 6.0 nM). A colon adenocarcinoma cell line, Colo201, was very responsive with an IC of 1 ng/ml (Fig. 3). A431 epidermoid carcinoma cells were also very sensitive to the action of hIL13-toxin; the IC for hIL13-PE38QQR ranged from 6 to 10 ng/ml. A gastric carcinoma CRL1739 cell line responded moderately to the hIL13-toxin with an IC of 50 ng/ml. Another colon carcinoma cell line, Colo205, had a poorer response with an IC of 300 ng/ml. The cytotoxic action of hIL13-PE38QQR was specific as it was blocked by a 10-fold excess of hIL13 on all cells (Fig. 3). These data suggest that a specturm of human cancer cells possess hIL13 binding sites and such cells are sensitive to hIL13-PE38QQR chimeric toxin.


Figure 3: Cytotoxic activity of hIL13-PE38QQR on several cancer cell lines and an inhibition of this cytotoxicity by hIL13. hIL13 was added at a concentration of 1.0 µg/ml. The dashed line shows 50% of [H]leucine incorporation. hIL13-PE38QQR; , with hIL13.



Because the hIL13R has been suggested to share subunit of the IL2R(11) , we explored further the specificity of hIL13-PE38QQR action on A431 and CRL1739 cells, the two cell lines with different sensitivities to the chimeric toxin (Fig. 4). The cells were treated with hIL13-PE38QQR with or without rhIL2 (a gift from Cetus) at a concentration of 1.0 or 10 µg/ml. The rhIL2 did not have any blocking action on hIL13-PE38QQR on the two cell lines, even at 10,000-fold molar excess over the chimeric toxin (Fig. 4). These results indicate that the cell killing by the hIL13-toxin is independent of the presence of hIL2.


Figure 4: Failure to block the cytotoxicity of hIL13-PE38QQR on cancer cells by 1.0 µg/ml or 10 µg/ml of hIL2. The dashed line shows 50% of [H]leucine incorporation. hIL13-PE38QQR; , with hIL2 (1 µg/ml); , with hIL2 (10 µg/ml).



hIL4, Unlike hIL2, Blocks the Action of hIL13-PE38QQR

We also added native hIL4 to cells and then treated with hIL13-PE38QQR, as another measure of specificity (Fig. 5). Unexpectedly, we found that hIL4 inhibited very potently the cytotoxic activity of the hIL13-toxin. This phenomenon was seen on all of the tested cell lines, which includes Colo201, A431 (Fig. 5), and CRL1739.()To investigate the possibility that hIL13 and hIL4 may compete for the same binding site, we also treated the cells with hIL4-based recombinant toxin, hIL4-PE38QQR (15) (Fig. 5). The cytotoxic action of hIL4-PE38QQR had already been shown to be blocked by an excess of hIL4 but not of hIL2(15) . In the present experiment, we examined hIL13 and found that it blocked potently the cytotoxic activity of hIL4-PE38QQR (Fig. 5). Also, the action of another hIL4-based chimeric toxin, hIL4-PE4E(12) , was blocked by an excess of hIL13 on Colo201 and A431 cells. Thus, the cytotoxicity of hIL13-PE38QQR is blocked by an excess of hIL13 or hIL4, and the cytotoxic action of hIL4-PE38QQR is also blocked by the same two growth factors. However, IL2 does not block the action of either chimeric toxin. These results strongly suggest that hIL4 and hIL13 have affinities for the same binding site.


Figure 5: Blocking the cytotoxicity of hIL13-PE38QQR by hIL4 and blocking the cytotoxicity of hIL4-PE38QQR by hIL13 on cancer cell lines. hIL4 was added at a concentration of 0.25 µg/ml and hIL13 at a concentration of 1.0 µg/ml. The dashed line shows 50% of [H]leucine incorporation. hIL13-PE38QQR; , with hIL4; , hIL4-PE38QQR; , with hIL13.



This conclusion was supported by the observation of one cytokine blocking the effect of a mixture of the two chimeric toxins. Fig. 6illustrates experiments in which we used both hIL13- and hIL4-PE38QQR at equimolar concentrations on A431 cells. When the cells were incubated with both chimeric toxins concomitantly, the cytotoxic action was preserved, and additive effect was observed as expected. An excess of hIL13 efficiently blocked the action of a mixture of the two chimeric toxins (Fig. 6). Moreover, neither hIL13 (Fig. 6) nor hIL4 blocked the cell killing by another mixture composed of hIL13-PE38QQR and TGF-PE40, a chimeric toxin that targets the epidermal growth factor receptor (TGF-based chimeric toxin, TGF-PE40)(21) . The same was observed on Colo201 cells.


Figure 6: hIL13 blocks the cytotoxic action of a mixture of hIL13-PE38QQR and hIL4-PE38QQR, but it does not block the cytotoxicity of a mixture of hIL13-PE38QQR and TGF-PE40. The dashed line shows 50% of [H]-leucine incorporation. , with hIL13.



Blocking the Action of Chimeric Toxins by the Cytokines Is Due to the Competition for Binding Sites

It was important to determine whether the binding of hIL4 chimeric toxin is affected by hIL13, since receptor binding is the first step in the cytotoxic action of chimeric protein. To investigate this, we performed competitive binding assays. The iodination of hIL4-PE38QQR and the binding experiments were carried out as described under ``Experimental Procedures.'' hIL4-PE38QQR competed for the binding of I-hIL4-PE38QQR to A431 cells with an apparent IC of 4 10M (Fig. 7). In addition, hIL13 also competed for the I-hIL4-PE38QQR binding site with a comparable potency to that exhibited by the chimeric protein. More extensive binding studies have shown that hIL13 competes for hIL4 binding sites on human renal carcinoma cell lines also(8) .


Figure 7: Competitive binding assay on A431 cells. Data are expressed as a percentage of total I-hIL4-PE38QQR binding to cells. The points are the average of experiments performed in duplicate.



We excluded the possibility of an influence of hIL13 or hIL4 on the process of receptor-mediated endocytosis and post-binding PE cellular toxicity steps by adding the following to cells: (i) native PE (PE binds to the -macroglobulin receptor), (ii) TGF-PE40, and (iii) a recombinant immunotoxin C242rF(ab`)-PE38QQR(20) . C242rF(ab`)-PE38QQR binds a tumor-associated antigen that is a sialylated glycoprotein(19) . We observed the expected potent cytotoxic actions of these recombinant toxins and neither hIL13 nor hIL4 blocked these actions on A431 and Colo205 cells.

hIL4 and hIL13 Compete for a Common Binding Site on Carcinoma Cells but May Evoke Different Biological Effects

Despite being competitors for the same binding site, some differences were observed in hIL13- and hIL4-induced cellular effects. Protein synthesis was inhibited in A431 epidermoid carcinoma cells in a dose-dependent manner by hIL4 alone (Fig. 8) or by a ADP ribosylation-deficient chimeric toxin containing hIL4(15) . This effect of hIL4 or enzymatically deficient chimeric toxin can be best seen with a prolonged time of incubation (24 h) and requires concentrations of hIL4 many fold higher than that of the active chimeric toxin in order to cause a substantial decrease in tritium incorporation. When, however, A431 cells were treated with various concentrations of hIL13, no inhibition (or stimulation) of protein synthesis was observed, even at concentrations as high as 10 µg/ml of hIL13 for a 72-h incubation (Fig. 8). The same lack of response to hIL13 was found on renal cell carcinoma cells PM-RCC.()Thus, hIL13 and hIL4 possess a common site but may transduce differently in carcinoma cells expressing this common site, such as A431 and PM-RCC cells.


Figure 8: hIL4, but not hIL13, inhibits protein synthesis in A431 epidermoid carcinoma cells. Data represent the average of quadruplicates. Standard deviations are marked as vertical bars.




DISCUSSION

We have made a novel recombinant chimeric toxin, hIL13-PE38QQR, composed of hIL13 and a mutated PE, PE38QQR. This chimeric toxin exerts a potent cytotoxic activity on a spectrum of human carcinomas. The cytotoxic activity of hIL13-PE38QQR, which can only occur subsequent to the internalization of a ligand-receptor complex(16) , is competed for by hIL13 and also by hIL4. Thus, there is a common internalized binding site for the two cytokines on many human carcinoma cells.

Our present results add new information on the interrelatedness between receptors for IL13 and IL4, since we demonstrate unequivocally that the wild-type hIL4 is capable of inhibiting the action of hIL13 chimeric toxin on human carcinoma cells. The inhibition is most likely conditioned by the binding of hIL4 to hIL13R, and not by other conceivable mechanisms, because hIL2 does not block the cytotoxic effect of hIL13 toxin. However, hIL4 has been shown to be a very weak competitor for hIL13 binding sites on renal cell carcinomas(8) . This contrasts the potent blocking activity of hIL4 on the action of hIL13-toxin on the currently studied cancer cell lines. As to this point, Puri and co-workers have recently found that the action of hIL13-PE38QQR is poorly blocked by the wild-type hIL4 on the very same renal cell carcinomas on which hIL4 also poorly or not at all displaced labeled hIL13(8) . Thus, the binding data may actually predict the competition for the activity of chimeric toxin. These data together suggest the presence of different cytokine receptors variants among various human malignant cells. The nature of the receptor forms on cancer cells is under further investigation.

On the other hand, two independent observations demonstrate that hIL13 competes well for hIL4 binding sites: (i) hIL13 antagonizes the cytotoxic action of hIL4-PE38QQR and (ii) hIL13 displaces labeled hIL4 chimeric toxin on tumor cells. Thus, hIL13 blocks internalization of the hIL4-toxin by a direct binding to the hIL4R; this finding supports original observation that hIL13 competes for hIL4 binding sites(7) .

The relationship between hIL13R and hIL4R is a matter of extensive investigations(7, 8, 11, 24) . Our current data and previous studies(7, 8, 12) suggest a possible form of the common for hIL13 and hIL4 internalized receptor on cancer cells. There are several lines of evidence suggesting the presence of (i) more than one binding protein and (ii) a common receptor by virtue of having a common receptor component(s). First, our results showed a potent killing of phytohaemagglutinin-activated peripheral blood lymphocytes by a hIL4-based chimeric toxin (12) but not by hIL13-toxin. This finding has been substantiated by the demonstration of a negligible amount of hIL13 binding sites on phytohaemagglutinin-activated peripheral blood lymphocytes, which in turn bind hIL4 avidly(8) . Second, there is no parallelism between the cytotoxic potencies of hIL13- and hIL4-PE38QQR on most of the studied cancer cell lines of various origins. Third, a truncated chain of the major hIL4R 140-kDa protein(22) , which contains the extracellular and transmembrane portions of the receptor(23) , mediates the binding and internalization of hIL4 that is not blocked by hIL13 (7). Fourth, the cross-linking experiments revealed interesting patterns of binding proteins for hIL13 and hIL4(8) . For example, the is not a component of hIL3R and, unexpectedly, the chain is not associated with hIL4R on renal cell carcinomas(8) . Furthermore, hIL4 cross-linked to two proteins: the already identified 140-kDa protein and a novel 70-kDa plasma membrane protein(8) . hIL13 cross-linked to only one protein of 70 kDa, which is similar in size to a lower molecular weight hIL4-cross-linked protein (8). It is thus plausible that a common internalized species of the hIL13R and hIL4R is the hIL4R 140-kDa protein complexed with a 70 kDa hIL13-responsive subunit different from . Since there are so many carcinomas expressing receptors for hIL13 and hIL4 (12-15), it will be important to establish the exact nature of interrelatedness between the two types of receptors and their possible molecular arrangements. This should help in designing strategies targeting these internalized receptors for anticancer therapy using, for example, chimeric toxins.

We have demonstrated the lack of hIL13 activity while hIL4, at high concentrations, inhibited the protein synthesis in two carcinoma cell lines. Differential effects of hIL13 and hIL4 have recently been reported(24) . The reason for this phenomenon is not clear at present. It is possible that hIL13 and hIL4 and their binding sites(s) may resemble other growth factors in that the formation of binding sites is determined by the expression of different protein variants and modulated by ligands(25) . In consequence, various cellular responses can be seen with the same ligands. Alternatively, the number of receptors for the cytokines responsible for intracellular signaling is significantly different on the studied cancer cell lines. This may be the case for A431 epidermoid carcinoma cells, which has 1000 binding sites/cell for hIL4 (15) and only 200 binding sites/cell for hIL13(8) . Further studies are needed to identify the reason for dissimilar effects of hIL13 and hIL4 on cancer cells.

In conclusion, we have demonstrated that a variety of human carcinoma cell lines express hIL13R because hIL13-toxin is highly cytotoxic to these cells. This cytotoxicity is hIL13- and hIL4-specific, indicating that the two cytokines bind to a common form of the receptor that is present on the studied cell lines. Finally, our data suggest that hIL13-PE38QQR merits evaluation as a therapeutic agent for the treatment of human carcinomas, since binding sites for hIL13 on normal cells appear to be limited even if the cells are responsive to hIL13(8) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed. Tel.: 717-531-4541; Fax: 717-531-3858; E-mail: debinski@debin.nsr.hmc.psu.edu.

The abbreviations used are: IL, interleukin; h, human; R, receptor; , -subunit of the interleukin 2 receptor; PE, Pseudomonas exotoxin A; r, recombinant; PCR, polymerase chain reaction; IC, 50% inhibitory concentration; TGF, transforming growth factor.

W. Debinski, unpublished data.

R. K. Puri, unpublished data.


ACKNOWLEDGEMENTS

We thank Sonia Girouard for excellent technical assistance and Pamela Leland for performing some of the binding and cytotoxicity experiments. The readiness of the DNAX Research Institute to share recombinant material is greatly appreciated.


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