`
`Molecular Medicine
`
`© 2000 The Picower Institute Press
`
`Human Breast Carcinoma Cells Express Type II IL-4
`Receptors and Are Sensitive to Antitumor Activity of a
`Chimeric IL-4-Pseudomonas Exotoxin Fusion Protein in
`vitro and in vivo
`
`P. Leland,1 J. Taguchi,1 S. R. Husain,1 R. J. Kreitman,2 I. Pastan,2 and R. K. Puri1
`1Laboratory of Molecular Tumor Biology, Division of Cellular and Gene Therapies,
`Center for Biologics Evaluation and Research, Food and Drug Administration,
`Bethesda, Maryland, U.S.A.
`2Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and
`Centers, National Cancer Institute, National Institutes of Health, Bethesda,
`Maryland, U.S.A.
`Communicated by I. Pastan. Accepted December 3, 1999.
`
`Abstract
`
`Background: Human breast carcinoma cell lines ex-
`press high-affinity interleukin-4 receptors (IL-4R).
`We examined the expression and structure of these
`receptors on primary and cultured breast carcinoma
`cell lines and normal breast epithelial cells. We also
`tested the antitumor activity in vitro and in vivo of a
`fusion protein comprised of circular permuted IL-4
`and truncated Pseudomonas exotoxin, termed IL-4(38-
`37)-PE38KDEL.
`Materials and Methods: Eight different primary
`cell cultures and cell lines of human breast carcino-
`mas were examined for the expression of IL-4R by
`radiolabeled binding, reverse transcription poly-
`merase chain reaction (RT-PCR) and Northern
`analyses, and subunit structure by crosslinking
`studies. The antitumor activity of IL-4 toxin was
`tested in vitro by cytotoxicity assays and in vivo in a
`xenograft model in immunodeficient animals.
`Results: 125I-IL-4 specifically bound to primary cell
`cultures and cell lines with a Kd ranging between
`
`0.2 and 1 nM. Breast tumor cells were found to
`express IL-4R웁 and IL-13R움⬘ chains, but not IL-
`2R웂c chain. These cells were highly sensitive to
`the cytotoxic effect of IL-4(38-37)-PE38KDEL. The
`IC50 (concentration inhibiting protein synthesis by
`50%) ranged between approximately 0.005–1.5 nM.
`A normal breast epithelial cell culture was not
`sensitive to the cytotoxic activity of IL-4(38-37)-
`PE38KDEL. MDA-MB231 human breast carcinoma
`cell line formed a rapidly growing tumor in nude
`mice. Intratumor and intraperitoneal administration
`of IL-4(38-37)-PE38KDEL caused a dose dependent
`regression of established tumors. A control toxin,
`anti-Tac(Fv)-PE38KDEL, targeted to the IL-2 recep-
`tor 움 chain did not cause regression of these tumors.
`Conclusions: These results suggest that IL-4(38-
`37)-PE38KDEL may be a useful agent for targeting
`of IL-4 receptor positive human breast carcinomas
`and further studies should be performed to explore
`fully its potential.
`
`Introduction
`Breast cancer is the most common malignancy in
`women, resulting in the second most frequent
`
`Address correspondence and reprint requests to: R. K. Puri,
`Laboratory of Molecular Tumor Biology, Division of Cellu-
`lar and Gene Therapies, Center for Biologics Evaluation
`and Research, Food and Drug Administration, National In-
`stitutes of Health, Bethesda, Maryland, U.S.A. Phone: 301-
`827-0471; Fax: 301-827-0449; E-mail: PURI@CBER.FDA.GOV
`
`cause of cancer death among women in the
`United States (1). Recent studies have focused on
`the development of new potent anti-cancer
`agents for the treatment of breast cancer refrac-
`tory to contemporary chemotherapy drugs. Tar-
`geted toxins in which ligand or specific antibody
`is fused to a toxin comprise one such form of
`anticancer drug. Identification of novel tumor-
`associated antigens or receptors on human breast
`
`IMMUNOGEN 2154, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`166
`
`Molecular Medicine, Volume 6, Number 3, March 2000
`
`cancer cells may help generate targeted anti-
`breast cancer agents. Recently, various fusion
`proteins have been produced that are designed to
`target human breast cancers. For example, Hereg-
`ulin-Pseudomonas exotoxin, which the ligand
`Heregulin binds to ErbB-2, ErbB-3 and ErbB-4
`receptors, is connected to a truncated form of
`Pseudomonas exotoxin (PE). This cytotoxin is
`highly cytotoxic in vitro and in vivo to breast can-
`cer cells that overexpress ErbB-4 or ErbB-2 plus
`ErbB-3 receptors (2). A recombinant epidermal
`growth factor (EGF) Genistein conjugate, in
`which EGF was conjugated to soybean-derived
`protein tyrosine kinase inhibitor, was targeted to
`an EGF-receptor and was found to be cytotoxic to
`the EGF-receptor positive breast cancer cells (3).
`A recombinant, humanized moncloclonal anti-
`Her2 antibody (Herceptin) was able to signifi-
`cantly inhibit growth of breast cancer in an ani-
`mal model and in the clinic (4). This antibody
`synergized with paclitaxel when mediating anti-
`tumor activity against breast tumor xenograft
`models. Herceptin was recently licensed by the
`U.S. Food and Drug Administration (FDA) for
`the treatment of breast cancer. These studies
`demonstrate that these classes of biotherapeutics
`can provide an additional mode of breast cancer
`therapy, although their clinical benefits have yet
`to be completely explored. It is possible that ad-
`ditional breast tumor-associated receptors or
`antigens will be identified that may provide new
`targets for breast cancer therapy.
`We and others have identified that human
`breast cancer cell lines express elevated levels
`of the receptor for an immune regulatory cy-
`tokine, interleukin-4 (IL-4) (5–7). Although
`the functional significance of this receptor on
`breast cancer cell lines is not clear, IL-4 can in-
`hibit proliferation of these cells in vitro and in-
`duce apoptosis (5–7). It is not known whether
`these receptors are overexpressed in situ in
`breast carcinomas. We reported that IL-4 recep-
`tors were expressed in situ in renal cell carci-
`noma and AIDS-associated Kaposi’s sarcoma
`(8,9). Thus, it is likely that breast carcinoma
`may also express IL-4 receptors in vivo, be-
`cause breast cancer cell lines express receptors
`in high numbers. We also found that a variety
`of solid cancer cells overexpress high-affinity
`IL-4 receptors (IL-4R) (10–12). These receptors
`are functional because IL-4 is able to cause sig-
`nal transduction, inhibit growth, upregulate
`major histocompatibility (MHC) antigens and
`intercellular adhesion molecule-1 (ICAM-1)
`on cancer cells (10–19). IL-4R also are ex-
`
`pressed, although in low numbers, in normal
`immune cells such as T cells; B cells; mono-
`cytes; other blood cells, such as eosinophils,
`basophils, and fibroblasts; and endothelial
`cells (10,11). The significance of the overex-
`pression of IL-4R on epithelial cancer cells and
`the similarities and differences between IL-4R
`in cancer cells and immune cells is not com-
`pletely clear.
`IL-4 receptors have been shown to be com-
`prised of a 140 kDa protein originally termed
`IL-4R움 (20). Because of similarities in extracel-
`lular domains (WSXWS motif and four cysteine
`residues at a fixed location) and long intracellu-
`lar domains between the IL-4R움 and 웁 chains
`of receptors for IL-3, IL-5, and granulocyte-
`macrophage colony stimulating factor (GM-
`CSF), we have recently proposed to rename this
`chain IL-4R웁 (18,21). This recommendation
`also was based on its similarity with the IL-2R웁
`chain, which like IL-4R p140, binds IL-2, but
`does not transmit a signal on its own (22). The
`second subunit of the IL-4R system was shown
`to be a component of the IL-2 receptor system,
`the 웂 chain (22,23). Because IL-2R웂 chain also
`was shown to be a component of IL-7, IL-9, and
`IL-15 receptor systems (24–26), it was named
`웂c. Thus, the IL-4R웁 chain and 웂c form type I
`IL-4 receptors. Recently, we demonstrated by
`reconstitution experiments that a 60–70 kDa
`protein form of interleukin-13 receptor (IL-
`13R) can substitute for 웂c when mediating IL-4
`signaling and, thus, this chain forms a third
`subunit of the IL-4R system (IL-13R움⬘ also
`termed as IL-13R움1) (18,21,27,28). Conse-
`quently, IL-4R웁 and IL-13R움⬘ chains form type
`II IL-4 receptors. Whether all three chains form
`an IL-4R complex in cancer cells is not known.
`It is also not known whether breast cancer cells
`express Type I or Type II IL-4 receptors. The
`differences in subunit structure between IL-4R
`in cancer cells and normal immune cells are
`also not completely known. We demonstrated
`that the 웂c chain expressed in immune cells was
`not expressed on human solid cancer cell lines
`(11,18,29). Instead, these cells expressed the
`IL-13R움⬘ (or 움1) chain along with the IL-4R웁
`chain (29,30). Further studies on the structure
`and function of IL-4R on cancer cells are ongo-
`ing. Regardless of differences in IL-4R between
`normal and cancer cells, we have been able to
`exploit the overexpression of IL-4R on cancer
`cells by targeting them with a cytotoxic
`chimeric protein comprised of IL-4 and PE
`(31–42).
`
`IMMUNOGEN 2154, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`P. Leland et al.: IL4 Targets Toxins to Breast Carcinoma
`
`167
`
`In the present study, we employed a circu-
`larly permuted form of IL-4-toxin [IL-4(38-
`37)-PE38KDEL], which contained amino acids
`38-129 of IL-4 fused via a peptide linker to
`amino acids 1-37. These are, in turn, fused to
`amino acids 353-364 and 381-608 of PE, with
`KDEL at positions 609-612 (37). This IL4-toxin
`has potent cytotoxic activity against eight dif-
`ferent breast cancer cell lines and primary cell
`cultures. We also investigated the expression
`and structure of IL-4 receptors in breast cancer
`cell lines, primary cell cultures and a breast ep-
`ithelial cell line. We tested the antitumor activ-
`ity of IL-4(38-37)-PE38KDEL against human
`breast cancer in vivo in a xenograft model. Our
`data support further studies on the use of IL-
`4(38-37)-PE38KDEL for possible treatment of
`metastatic breast cancer.
`
`Materials and Methods
`Recombinant Cytokines and Toxins
`Recombinant circularly permuted IL-4-toxin,
`IL-4(38-37)-PE38KDEL, was produced and
`purified to ⬎95% homogeneity as described
`previously (32,33,37,38). Recombinant IL-4
`was produced as described (43).
`
`Cell Lines
`The primary cultures of human breast carci-
`noma R-BT, S-BT, and W-BT were established
`and kindly provided by Dr. Magda Sgagias,
`Surgery Branch, National Cancer Institute
`(Bethesda, MD) (44). The breast carcinoma
`cell lines (MCF-7, BT-20, SK-BR3, ZR-75-1,
`and MDA-MB231) were obtained from the
`American Type Culture Collection (ATCC),
`Rockville, MD. Dr. Sgagias also provided one
`primary epithelial cell culture from normal
`breast tissue (A-NL). The primary tumor and
`normal breast cell cultures were cultured in
`medium comprised of 움-minimum essential
`medium, HAM’s F-12, EGF, triiodothionine, N-
`2-hydroxy ethylpiperazine-N-2-ethanesulfonic
`acid (HEPES) ascorbic acid, estradiol, insulin,
`hydrocortisone,
`ethanolamine,
`transferrin,
`bovine pituitary extract, sodium selenite, glut-
`amine, gentamicin, penicillin, and strep-
`tomicin. The breast carcinoma cell lines were
`cultured in complete media, comprised of
`RPMI 1640, 10% heat inactivated fetal calf
`serum (FCS) and gentamycin. These adherent
`cell lines were routinely passaged every 4–5
`days.
`
`Animals
`Four-week-old female athymic nude mice (~20 g)
`were obtained from Frederick Cancer Center
`Animal Facilities (Frederick, MD). Animals
`were housed in filter-top cages in a laminar
`flow hood.
`
`Protein Synthesis Inhibition Assay
`The cytotoxic activity of IL-4-toxins was tested
`as previously described by determining inhibi-
`tion of protein synthesis (31). Typically, 104
`breast cancer cells were cultured in leucine-
`free medium with or without various concen-
`trations of IL-4-toxins for 20–22 hr at 37⬚C.
`Then, 1 애Ci of [3H]-leucine (NEN Research
`Products, Wilmington, DE) was added to
`each well and cells were incubated for an
`additional 4 hr. Cells were harvested and ra-
`dioactivity incorporated into cells was mea-
`sured by a Beta plate counter (Wallac, Gaithers-
`burg, MD).
`
`125I-IL-4 Binding and Displacement Assay
`IL-4 was iodinated with IODOGEN reagent
`(Pierce, Rockford, IL) according to manufac-
`turer’s instructions. The specific activity of ra-
`diolabeled IL-4 ranged between 31.5 to 212
`애Ci/애g. The IL-4 binding assay was performed
`by a previously described technique (12,17).
`Briefly, tumor cells were harvested after brief
`incubation with versene (Biowhittaker, Walk-
`ersville, MD), washed three times in Hanks
`balanced salt solution and resuspended in
`binding buffer (RPMI 1640 plus 1 mM HEPES
`and 0.2% human serum albumin). For the dis-
`placement assay, MCF-7 cells (1 ⫻ 106/100 애l)
`were incubated at 4⬚C with 125I-IL-4 (100–200
`pM) with or without increasing concentrations
`of unlabeled IL-4 or IL-4(38-37)-PE38KDEL.
`For binding assays, cells were incubated with
`various concentrations of 125I-IL-4 with or
`without 200-fold molar excess of unlabeled IL-
`4. Following a 2 hr incubation, cell-bound
`radio-ligand was separated from unbound by
`centrifugation through a phthalate oil gradient
`and radioactivity was determined with a
`gamma counter (Wallac). The number of recep-
`tors and binding affinities were determined as
`previously described (12).
`
`Affinity Cross Linking of 125I-IL-4 to Its Receptor
`MCF-7 and MDA-MB231 cells (5 ⫻ 106) were
`incubated with [125I]-labeled IL-4 in the pres-
`
`IMMUNOGEN 2154, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`168
`
`Molecular Medicine, Volume 6, Number 3, March 2000
`
`ence or absence of excess unlabeled IL-4 for
`two hr at 4⬚C. Bound [125I]-IL-4 was cross-
`linked to IL-4R with disuccinimidyl suberate
`(DSS) (Pierce Chemical company, Rockford,
`IL) at a final concentration of 2 mM for
`20 min. The cells were then lysed at 4⬚C with
`1% triton X-100 solution containing the fol-
`lowing protease
`inhibitors obtained
`from
`Sigma chemical company (St Louis, MO) and
`Boehringer-Mannheim (Indianapolis, IN): leu-
`peptin (10 애g/ml), trypsin inhibitor (100
`애g/ml), pepstatin (10 애g/ml), benzamidine
`HCl (10 mM), phenanthroline (1 mM) iodoac-
`etamide (20 mM), e-aminocaproic acid (50
`mM) and phenyl methyl sulfonic fluoride
`(PMSF) (1 mM). The resulting lysate was
`cleared by boiling in sample buffer containing
`2-mercaptoethanol and analyzed by electro-
`phoresis through a SDS-PAGE (8%) gel, as
`previously described (15). The gel was dried
`and exposed to X-ray film for 7 days to obtain
`an autoradiograph.
`For immunoprecipitation, the [125I]-IL-
`4/IL-4R cross-linked complex was immuno-
`precipitated from the lysate prepared from
`MCF-7 cells overnight at 4⬚C by incubating
`with protein A sepharose beads that had been
`preincubated with anti-웂c or anti-IL-4R웁 chain
`antibody. The resulting conjugate was washed
`twice with solubilizing buffer, diluted with re-
`ducing buffer, boiled for 5 min and analyzed
`by SDS-PAGE, as described above. The gel was
`dried and autoradiographed.
`
`Northern Analysis for IL-4R Subunits
`Total RNA was isolated using TRIZOL reagent
`(GIBCO BRL, Gaithersburg, MD). Equal
`amounts of total RNA were electrophoresed
`through a 0.8% agarose/formaldehyde dena-
`turing gel, transferred to a nylon membrane
`(S&S Nytran; Schleicher and Schuell, Keene,
`NH) by capillary action and immobilized by ul-
`traviolet crosslinking (Stratagene, Inc., La
`Jolla, CA). The membrane was then prehy-
`bridyzed for 4 hr at 42⬚C and hybridized with
`32P-labeled cDNA probes of IL-4R웁, IL-13R움⬘,
`and 웂c at 42⬚C overnight. The membranes were
`subsequently exposed to X-AR film (Eastman
`Kodak Co. Rochester, NY) to obtain an auto-
`radiogram.
`
`RT-PCR Analysis
`RT-PCR analysis was performed as previously
`described (40). Total RNA was isolated from
`
`cell lines using Tri-Reagent (Molecular Re-
`search Center, Inc. Cincinnati, OH) follow-
`ing the manufacturer’s instructions. The con-
`centration and purity of total RNA was
`determined by spectrophotometric analysis.
`One 애g of total RNA was used in the RT-PCR
`assay. RT-PCR conditions were as follows:
`95⬚C for 5 min, 1 cycle; 95⬚C for 1 min; 72⬚C for
`1 min; 72⬚C for 1 min, 30–35 cycles; and 72⬚C
`for 10 min for the final primer extension se-
`quence. RT-PCR primers for IL-4R웁: 5⬘ primer,
`5’-ATGGGGTGGCTTTGCTCTGGG-3⬘ and 3⬘
`primer, 5⬘-ACCTTCCCGAGGAAGTTCGGG-3⬘;
`for 웂c, 5⬘ primer, 5⬘-CCAGAGGTTCAGTGTTTT-
`GTGTT-3’ and 3⬘ primer, 5⬘-CAGGTTTCAGG-
`ATTTAGGGTGTA-3⬘; for IL-13R움⬘: 5⬘ primer,
`5⬘ GGAGGATACATCTTGTTTCATGG-3⬘ and 3⬘
`primer, 5⬘-GAGCTTCTTACCTATACTCATTTC-
`TTGG-3⬘. The IL-4R웁 RT-PCR cDNA prod-
`uct was 316 bp; 256 bp; for 웂c and 148 bp
`for IL-13R움⬘. A 100 bp DNA ladder (GIBCO
`BRL Life Technologies Inc., Gaithersburg,
`MD) was used as a base pair reference
`marker.
`
`Antitumor Activity of IL-4-Toxin in Nude Mice
`Implanted with Human Breast Tumor
`Human breast tumor nodules were established
`in nude mice by subcutaneous injection of
`3-4 ⫻ 106 MDA-MB231 cells in 100 애l of phos-
`phate-buffered saline (PBS) containing 0.2%
`human serum albumin (HSA) into the ab-
`domen on day 0. Palpable tumors developed
`within 3–5 days. Tumor size was calculated by
`muliplying two perpendicular diameters.
`Two routes for administration of IL-4
`(38-37)-PE38KDEL were employed: intraperi-
`toneal (i.p.) and intratumoral (i.t.). The mice
`were i.p.-injected with 100 애l excipient or
`50, 100 or 150 애g/kg twice daily for 5 consecu-
`tive days. Another group of mice was slowly
`i.t.-injected (20 애l) with excipient or an IL-
`4(38-37)-PE38KDEL dose of 250 애g/kg/dose
`on days 8, 10 and 12. Each injection was placed
`into a different area of the tumor. An addi-
`tional group of mice was i.t.-injected with
`chimeric toxin at a dose of 750 애g/kg/dose
`on days 8, 10 and 12, followed by reinjection
`with 500 애g/kg/dose on days 22, 24 and 26. A
`third group of animals were i.t.-injected with
`750 애g/kg/dose on days 22, 24 and 26, fol-
`lowed by reinjection with 500 애g/kg/dose on
`days 36, 38, and 40.
`
`IMMUNOGEN 2154, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`P. Leland et al.: IL4 Targets Toxins to Breast Carcinoma
`
`169
`
`Statististics
`The significance of differences in mean tumor
`sizes among treatment groups was analyzed by
`unpaired Student’s t-test. All p-values are pre-
`sented as two-sided analysis.
`
`Results
`Cytotoxicity of IL-4-toxins Against Breast Carcinoma
`Cell Lines and Primary Cell Cultures
`IL-4-toxins, including IL-4(38-37)-PE38KDEL,
`have been shown to have cytotoxic activity
`against cell lines that express IL-4 receptors
`(IL-4R) (32–42). However, it is not known
`whether primary cell cultures of human breast
`carcinoma express IL-4R and if they do,
`whether these cells and breast cancer cell lines
`are susceptible to the cytotoxic activity of IL-4-
`toxins. We tested four primary cell cultures of
`breast carcinoma generated from four patients
`undergoing surgical resection for their cancer,
`as previously described (44). Three of four pri-
`mary cell cultures were sensitive to the cyto-
`toxic activity of circular permuted IL-4-toxin
`and one of these three was extremely sensitive
`to IL-4(38-37)-PE38KDEL (Fig. 1A and Table
`1). The IC50 (the concentration of toxin causing
`inhibition of protein synthesis in target cells by
`50%) ranged between 0.2 to 240 ng/ml (4 pM
`to 4.8 nM). The cytotoxic activity of IL-4(38-
`37)-PE38KDEL was specific, as an excess of re-
`combinant IL-4 neutralized the cytotoxic activ-
`ity of IL-4-toxin to primary breast carcinoma
`cell culture R-BT (Fig. 1A).
`Like primary cell cultures, breast carci-
`noma cell lines were also very sensitive to the
`cytotoxic activity of IL-4-toxin. Protein synthe-
`sis was inhibited in a concentration-dependent
`manner against four of five breast cancer cell
`for IL-4(38-37)-
`lines examined. The IC50s
`PE38KDEL ranged between 0.4 ng/ml to
`75 ng/ml (8 pM to 1.5 nM) (Fig. 1B and Table 1).
`
`Inhibition of 125I-IL-4 Binding by IL-4-toxins on
`MCF-7 Breast Carcinoma Cell Line
`To determine the binding affinity of IL-4(38-37)-
`PE38KDEL to breast cancer cells, we performed
`displacement assays where [125I]-IL-4 binding
`was inhibited by either unlabeled IL-4 or IL-4
`toxin. As we reported in other cancer cell lines,
`IL-4(38-37)-PE38KDEL displaced 125I-IL-4 at a
`similar concentration as unlabeled IL-4 on MCF-
`7 cell line (Fig. 2) (39,40). The EC50 (protein
`
`concentration required for 50% inhibition of
`125I-IL-4 binding) for IL-4(38-37)-PE38KDEL
`was ~0.5 nM and for IL-4 it was ~0.4 nM. These
`data suggested
`that IL-4(38-37)-PE38KDEL
`bound to IL-4R with similar affinity to IL-4 and
`circular permutation or fusion of PE did not
`modify its binding affinity to breast cancer cells.
`
`Fig. 1. Cytotoxicity of IL-4(38-37)-PE38KDEL in
`breast tumors. Ten thousand cells from R-BT pri-
`mary breast tumor cell culture (A), MCF-7 or MDA-
`MB231 (B) breast cancer cell lines were incubated
`with various concentrations of IL-4(38-37)-
`PE38KDEL. Protein synthesis was measured after 20
`hr of culture by incorporation of [3H]-leucine
`(1 애Ci for an additional 4 hr), as described in the
`“Materials and Methods” section. For competition ex-
`periments, R-BT cells were preincubated for 45 min
`with 2 애g/ml of recombinant IL-4 before addition of
`IL-4(38-37)-PE38KDEL (A). The results are presented
`as mean ⫾ SD % control of untreated cells from qua-
`druplicate determinations. Mean total counts per
`minute (cpm) ⫾ SD incorporated in untreated R-BT
`cells was 11,765 ⫾ 1,138, in MCF-7 cells 53,924 ⫾
`8,835 and in MDA-MB231 cells 113,366 ⫾ 3,261.
`
`IMMUNOGEN 2154, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`170
`
`Molecular Medicine, Volume 6, Number 3, March 2000
`
`IL-4R expression and cytotoxicity of IL-4(38-37)-PE38KDEL on human breast carcinoma cell
`Table 1.
`lines and primary cell cultures
`
`Cells
`
`Primary breast tumor cell cultures:
`R-BT
`S-BT
`W-BT
`K-BT
`Breast cancer cell lines:
`MCF-7
`BT-20
`SK-BR3
`ZR75-1
`MDA-MB231
`
`IL-4(38-37)-PE38KDEL
`a (ng/ml)
`IC50
`
`IL-4R Expressiond
`(binding sites/cell)
`
`0.2 ⫾ 0.03b
`5 ⫾ 3
`240c
`1000
`
`0.4 ⫾ 0.13
`75 ⫾ 7
`⬍1000
`7 ⫾ 5
`1.8 ⫾ 1.6
`
`954 ⫾ 59
`NDe
`ND
`ND
`
`2263 ⫾ 157
`1703 ⫾ 144
`ND
`4687 ⫾ 118
`4598 ⫾ 167
`
`For cytotoxicity assays, 1 ⫻ 104 cells were cultured with IL-4-toxins for 20 hr at 37⬚C, pulsed with 1 애Ci of [3H]-leucine and
`further incubated for 4 hr. Cells were harvested and counted as described under Materials and Methods.
`
`aIC50, the concentration of IL-4-toxin at which 50% inhibition of protein synthesis is observed, compared with untreated
`cells.
`bThe values are presented as mean ⫾ SEM of five experiments performed in quadruplicate.
`cSingle experiment performed in quadruplicate.
`dSingle saturating concentration of 125I-IL-4 was used to calculate binding sites. Results are shown as mean
`⫾ SD.
`eND ⫽ not done
`
`IL-4R Expression on Breast Carcinoma Cell Lines and
`Primary Cultures
`We previously reported that human breast car-
`cinoma cell lines express high-affinity IL-4R
`(5). In binding studies, we found that a pri-
`mary cell culture of breast carcinoma (R-BT)
`also bound to radiolabeled IL-4 in a concentra-
`tion-dependent manner. Displacement analysis
`revealed that these receptors were of high affin-
`ity (~1 nM). MDA-MB231 and MCF-7 breast
`cancer cell lines also expressed high-affinity
`IL-4R (~0.2 nM) (Fig. 3). The number of bind-
`ing sites/cell was calculated by single point
`binding assay and found to vary in different
`cell types (Table 1).
`
`Crosslinking of 125I-IL-4 to Breast Carcinoma Cells
`The structure of IL-4R on two different breast
`carcinoma cell lines was examined next. This
`was performed by crosslinking radiolabeled
`IL-4 to surface IL-4R, followed by SDS-PAGE
`under reducing conditions (Fig. 4A). [125I]-IL-
`4 crosslinked to two prominent proteins at ap-
`proximately 155 kDa and 85 kDa on both
`breast cancer cell lines (lanes 1 and 4). In addi-
`
`tion, [125I]-IL-4 crosslinked to one protein of
`about 40 kDa. None of these bands was ob-
`served when crosslinking was performed in
`the presence of 200-fold molar excess of IL-4,
`indicating that the observed bands were in-
`volved in specific [125I]-IL-4 binding (Fig. 4A,
`lanes 2 and 5). Assuming a molecular weight
`of 15 kDa for hIL-4 and subtracting it from the
`kDa values indicated on the gel, the molecular
`weights of the [125I]-IL-4 binding proteins
`were estimated at about 140 kDa, 70 kDa, and
`25 kDa, respectively. We reported that the IL-4
`receptor shares two chains with IL-13 receptors
`in various cell lines (21,27). To investigate
`whether the IL-4 receptor may be related to IL-
`13R in breast cancer cell lines, we competed for
`binding of [125I]-IL-4 by IL-13 on the MCF-7
`cell line. As shown in Fig. 4B, like IL- 4, IL-13
`also displaced the binding of radiolabeled IL-4
`for both bands.
`To confirm the identity of [125I]-IL-4-IL-4R
`cross-linked complexes, immunoprecipitation
`with anti-IL-4R antibody was performed be-
`fore electrophoresis on SDS-gel. As shown in
`Fig. 4A (lanes 3 and 6), all three prominent
`bands were immunoprecipitated, indicating
`
`IMMUNOGEN 2154, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`P. Leland et al.: IL4 Targets Toxins to Breast Carcinoma
`
`171
`
`Fig. 2. Displacement of 125I-IL-4 binding by IL4
`and IL-4(38-37)-PE38KDEL. MCF-7 breast cancer
`cells were incubated at 4⬚C with 100 pM [125I]-IL-4
`and various concentrations of either IL-4 or IL-
`4(38-37)-PE38KDEL. After 2 hr, cells were cen-
`trifuged through a mixture of phthalate oils and
`cell pellets were counted in a gamma counter. The
`data points shown are mean ⫾ SD of duplicate de-
`terminations. A total of 1,491 ⫾ 137 cpm bound to
`1 ⫻ 106 MCF-7 cells. The SDs are shown where
`deviations are larger than the size of point symbols.
`
`that all these proteins formed an IL-4R com-
`plex on breast carcinoma cells.
`
`RT-PCR and Northern Analysis of IL-4R Subunits on
`Breast Carcinoma Cells
`We next examined the expression of IL-4R웁, IL-
`13R움⬘ and 웂c chains, all of which can form IL-4R
`complexes in different cell types. As shown in
`Figure 5, RT-PCR products of 316 bp for IL-4R웁
`and 148 bp for IL-13R움⬘ chain were expressed
`in all of the studied breast carcinoma cell lines
`and primary cell cultures. In contrast, most
`breast cancer cell lines did not express the 255
`bp 웂c product, but it was detected in R-BT pri-
`mary breast tumor cell culture (Fig. 5, lane 4).
`To confirm whether the 웂c chain was ex-
`pressed in breast cancer cell lines and primary
`cell cultures, we performed Northern analysis
`to examine mRNA for IL-4R웁, IL-13R움⬘ and 웂c
`chains. As shown in Fig. 6, IL-4R웁 and IL-
`13R움⬘ mRNA was abundantly expressed in all
`breast cancer cell lines and primary breast can-
`cer cell cultures, however, mRNA for the 웂c
`chain was not expressed in any cell lines.
`
`Fig. 3. Displacement analysis of 125I-IL-4 bind-
`ing to breast cancer cells. This was performed on
`MDA-MB231 and MCF-7 cell lines (upper panel)
`and R-BT breast cancer primary cell culture (lower
`panel). Specific binding data was utilized to gener-
`ate the Scatchard curves. Typically, for these experi-
`ments, a single cell suspension of tumor cells was
`incubated for 2 hr with increasing concentrations of
`unlabeled excess IL-4 in the presence of a fixed
`concentration (100-200 pM) of 125I-IL-4 at 4⬚C.
`Bound radioactivity was determined as described
`in the materials and methods section.
`
`These data supported our crosslinking studies
`and futher demonstrated that IL-4R웁 and IL-
`13R움⬘ chains formed an IL-4R complex on
`breast carcinoma cells and 웂c did not form a
`complex, as seen in immune cells. These data
`indicated that breast tumor cells expressed
`type II IL-4 receptors.
`
`IMMUNOGEN 2154, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`172
`
`Molecular Medicine, Volume 6, Number 3, March 2000
`
`Fig. 4. Crosslinking of 125I-IL-4 to IL-4 recep-
`tors on breast carcinoma cells. (A) MDA-MD231
`and MCF-7 cells (5 ⫻ 106) were incubated with
`125I-IL-4 in the absence (lanes 1 and 4) or presence
`of excess unlabeled IL-4 (lanes 2 and 5) for 2 hr at
`4⬚C. In both these cell lines 125I-IL-4 crosslinked
`receptors were immunoprecipitated with anti-IL-4
`receptor antibody (M-7) (lanes 3 and 6). (B) MCF-7
`cells were also crosslinked with radiolabeled IL-4
`in the absence (lane 1) and presence of excess of
`IL-13 (lane 2) or IL-4 (lane 3). Bound 125I-IL-4 was
`cross-linked to IL-4R with disuccinimidyl suberate
`(DSS). The cells were then lysed at 4⬚C with modi-
`fied RIPA buffer. The resulting lysate was analyzed
`by electrophoresis through an SDS-PAGE (7%) gel.
`The gel was dried and exposed to an X-ray film for
`7 days at ⫺80⬚C. The molecular weight markers are
`shown on the left. The positions of different recep-
`tor chains are indicated. The dark area in lane 3 (B)
`represents autoradiography exposure artifact.
`
`Antitumor activity of IL-4-toxin
`To determine the antitumor activity of IL-4-
`toxin, we established a breast tumor model in
`nude mice and then tested various routes of IL-
`4 toxin administration. First, we investigated
`whether intratumoral treatment would lead to
`regression of established breast cancer. MDA-
`MB 231 tumor cells were implanted subcuta-
`neously in immunodeficient animals. When
`
`Fig. 5. RT-PCR analysis of different receptor
`chains. The polymerase chain reaction (PCR) mix-
`ture containing specific primers was amplified as
`described in “Materials and Methods.” The num-
`bered lanes represent RNA from BT-20 (1), MCF-7
`(2), MDA-MB231 (3), R-BT (4), SK-BR3 (5) and
`ZR-75-1 (6) breast tumor cell lines. The polymerase
`chain reaction (PCR) conditions for 웁-actin were
`similar to the conditions used for IL-4R chains.
`
`the tumors reached a mean size of 18–25 mm2
`(50 mm3), the mice were injected i.t. with vary-
`ing doses of IL-4 toxin, as specified in “Mate-
`rials and Methods.” Anti-tac-immunotoxin,
`which binds to the IL-2 receptor 움 chain,
`served as a negative control since breast cancer
`cells may not express IL-2 receptors. The treat-
`ment began 8 days after tumor implantation
`and these animals received two additional in-
`jections on days 10 and 12. IL-4-toxin treat-
`ment caused a dose-dependent regression of
`breast tumors (Fig. 7A), as the antitumor ef-
`fect was more pronounced at the higher dose
`(750 애g/kg). Although the tumors began to
`grow again 10 days after the last dose of toxin,
`the growth rate of tumors in treatment groups
`was significantly slower, compared with the
`control group. For example, on day 45 after in-
`tratumor administration of IL-4-toxin, the low-
`est dose (250 애g/kg) caused a significant inhi-
`
`IMMUNOGEN 2154, pg. 8
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`P. Leland et al.: IL4 Targets Toxins to Breast Carcinoma
`
`173
`
`An additional five animals were also i.t. in-
`jected with IL-4(38-37)-PE38KDEL when tu-
`mors had almost doubled in size and reached
`36 mm2. These animals received 750 애g/kg of
`IL-4-toxin on days 22, 24 and 26, followed
`with 500 애g/kg of IL-4-toxin on days 36, 38
`and 40. The growth of these large tumors also
`was slowed, compared with control tumors
`(data not shown). These animals were sacri-
`ficed on day 52 due to large tumors.
`In another experiment, cohorts of five nude
`animals bearing subcutaneous human breast
`cancer xenografts were given intraperitoneal
`injections with escalating doses of IL-4(38-
`37)-PE38KDEL. These injections were given
`2 times a day for 5 days. As shown in Fig.
`7B, IL-4(38-37)-PE38KDEL caused regression
`of established breast cancer nodules in a
`dose-dependent manner. At the highest dose
`(150 애g/kg/dose), one of the five animals
`showed complete regression of the established
`tumor. The size of the tumor in the remain-
`ing four animals continued to be signifi-
`cantly smaller, compared with any other
`groups, including controls (p ⬍ 0.05 control
`vs. 100 애g/kg dose; p ⬍ 0.006 control vs.
`150 애g/kg dose). The tumors continued to
`grow in control animals, eventually reaching
`about 65 mm2. All animals were sacrificed due
`to large tumor burdens.
`
`Sensitivity of Breast Tumor Cultures after in vivo
`Passage and IL-4-toxin Therapy
`To determine whether breast tumor cells ac-
`quired resistance after in vivo passage or after
`IL-4-toxin therapy, tumors were excised from
`control and IL-4-toxin-treated animals. Single
`cell suspensions were prepared by enzyme di-
`gestion and the resulting cells were passaged at
`least once before cytotoxicity of IL-4(38-37)-
`PE38KDEL was determined. The sensitivity to
`IL-4(38-37)-PE38KDEL, as well as the IC50,
`was similar on cells derived from control ani-
`mals and IL-4-toxin treated animals. The IC50
`was also similar to that observed in cells not
`injected into animals (not shown).
`
`Discussion
`We have demonstrated that human breast can-
`cer cells are highly sensitive to the cytotoxic ac-
`tivity of a IL-4 receptor targeted chimeric toxin
`comprised of IL-4 and a mutated form of PE.
`Recombinant IL-4(38-37)-PE38KDEL was also
`
`Fig. 6. Northern analysis of IL-4R subunits on
`breast cancer cells. Total RNA from these cells
`was electrophoresed (10–20 애g/lane) and hy-
`bridized with a 32P-labeled cDNA probe for differ-
`ent IL-4R chains. Autoradiography was performed
`for 4 hr for IL-13R움⬘, 8 day for IL-4R 웁 chain and
`3–8 days exposure for 웂c. The numbers at the top of
`the gel represent RNA from BT20 (1), R-BT (2),
`SK-BR3 (3), ZR-75-1 (4), MCF-7 (5), and MDA-
`MB231 (6) breast cancer cell lines.
`
`bitio