[CANCER RESEARCH56, 2590-2595, June 1, 19961 Interleukin 2 (IL.-2) Receptor Expression and Sensitivity to Diphtheria Fusion Toxin

DAB38@IL-2 in Cultured Hematopoietic Cells1

Gian G. Re,2 Cory Waters, Louis Poisson, Mark C. Willingham, Kazuo Sugamura, and Arthur E. Frankel

Departments of Pathology and Laboratory Medicine (G. G. R.. M. C. W.J and Medicine (A. E. Fl, Medical University of South Carolina, Charleston, South Carolina 29425; Seragen. Inc., Hopkinton, Massachusetts 0J748 (C. W.. L P.]; and Department ofMicmbiology, Tohoka University School ofMedicine, Sendai 980, Japan (K. 5.1

ABSTRACT genotypic characteristics of the patients' cancers and offer a pure cell population for physiological studies. To date, a number of these cell The high-affinity interleukin 2 (IL-2) receptor is a heterotrimer con lines have been evaluated for IL-2R status (Table 1). Also, in those slating of a, @,and @‘subunits.We examined the concentration of subunit studies, different methods have been used, making direct comparisons mRNAforeachofthe threeproteinsubunitsonhumanhematopoleticcell lines, human peripheral blood mononuclear cells, and murine fibroblasts difficult. transfected with CDNASencoding the human IL-2 receptor subunits. In As a result of these shortcomings, despite the wealth of data, the most cultured hematopoietic cells, there was abundant ‘ysubunitmessage. relationships between IL-2R subunit expression, affinity, inhibition of In contrast, there was variable expression of both a and @Jsubunit protein synthesis, and subsequent cell killing by IL-2 toxins are still message. Sensitivity ofcells to the diphtheria fusion toxin DAB@,IL-2 was poorly understood. In principle, the response of malignant cells to not related to expressionof any single IL-2 receptorsubunit mRNA. receptor-targeted IL-2 toxin therapy reflects the ability of the IL-2R Rather,the greatestsensitivitywasobservedfor cellspossessingallthree on the cell surface to bind the toxin with adequate affinity and to subunit mRNAs. Cells displaying 0 and ‘ysubunit mRNA showed a transport it to an intracellular compartment where it can exert its lethal reduced but significant sensitivity to the fusion toxin. In contrast, cells action. In turn, these properties depend on the relative composition of with a and ‘ysubunit mRNA, but missing the @3subunit mRNA, were the IL-2R in a, j3, and ‘ysubunits.To begin to understand the basis for Insensitive to DAB@IL-2. The data correlate with the requirement for an Intermediate or a high-affinity receptor for cell Intoxication. A critical the IL-2 toxin therapeutic efficacy, we have developed a Northern blot concentration of the @3subunitmay be required for toxin Internalization assay for the concomitant measurement of the a, @,and y subunit and hillIng, mRNA in IL-2R-bearing cells and have applied it to study the rela tionships between IL-2R subunit expression and inhibition of protein synthesis of tumor cell lines by DAB38@IL-2. A positive correlation INTRODUCTION could be used as a predictor of response in patients undergoing IL-2 Many patients with hematopoietic neoplasms fail conventional and toxin therapy. We report that inhibition of cell protein synthesis directly correlated with the expression of the @3subunit mRNA, and high-dose chemoradiotherapy, and new agents with distinct mecha nisms of cytotoxicity have been sought. One class of such agents is that it was greatly enhanced by the additional presence of the a represented by toxins consisting of peptide ligands chemically or subunit mRNA, provided that the y subunit mRNA was expressed. genetically fused to peptide toxins targeted against specific receptors. IL-23 is a polypeptide hormone which binds its specific receptor MATERIALS AND METHODS (IL-2R) on normal activated T cells, B cells, monocytes, and macro phages and controls their proliferation and differentiation (1). The Materials. Restriction enzymes were obtained from Promega (Madison, IL-2R is a heterocomplex between three membrane-bound proteins, WI). [32P]dCTPand [3Hjleucine were obtained from Amersham (Arlington including a Mr 55,000 a subunit, a Mr 75,000 13 subunit, and a Mr Heights, IL). RPMI 1640 media, leucine-free RPM! 1640, penicillin, strepto 64,000 y subunit, and exists in forms exhibiting differing affinities for mycin, fetal bovine serum, and dialyzed fetal bovine serum were obtained from IL-2 (2). The high-affinity IL-2R is composed of all three subunits. In Life Technologies, Inc. (Grand Island, NY). Cell lines were obtained from the American Type Culture Collection (Rockville, MD) or Seragen. DAB38@IL-2 addition, cells can display an a-y heterodimer with low binding obtained from Seragen has been described previously (29). L929 murine affinity for IL-2 and a @3-yheterodimer with intermediate binding fibroblasts stably transfected with expression vectors for human a and (3chains @ affinity (3). Signal transduction and receptor internalization requires (La-@-2), for a, @,andy chains (La-@--y-4), and for and ‘ychains(Lfry-l) the 13and @ysubunits (4). A number of IL-2R-targeted toxins have have been described (2). been produced and tested (5—8).The outcomes of such testing have Northern Blots. Whole cell RNA was isolated using the acid guanidinium been varied, mainly because the criteria for the successful application thiocyanatephenol-chloroformextractionmethod(30).In the standardassay, of IL-2-toxins have yet to be developed. aliquots of 6 @&gRNA/lanewere electrophoresed on formaldehyde-agarose The IL-2R has been identified on fresh malignant cells in a number gels. The RNA was visualized by UV transillumination and photographed with of studies. However, a consistent set of data regarding the relative a Polaroid camera to document RNA integrity and equal loading. Then it was frequency and density of the LL-2R subunits could not be derived transferred to mtrocellulose by capillary blotting with lOX SSC [lX SSC = 0.15 MNaCl, 0.015 MNa citrate (pH 7.0)]. On transfer, the membrane because of the lack of suitable reagents and the differing methods used was visualized in UV light and photographed to document complete and (9—19).Cell lines derived from clonogenic tumor cells of patients uniform transfer. The prehybridization was omitted. Hybridization was carried with leukemias and lymphomas retain many of the phenotypic and out in 40% formamide 2X SSC, 40 nmi sodium phosphate (pH 8.0), and 250 ILg/mlsonicated, heat-denatured salmon sperm DNA overnight at 50°C.Blots Received 12/21/95; accepted 3/27/96. were washed two times for 5 mm at room temperature with 2X SSC/0.l% The costs of publication of this article were defrayed in part by the payment of page SDS; then they were washed twice with 0.1X SSCIO.l% SDS at 50°C,dried, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and exposed to X-ray film with two intensifying screens for various times I This work was supported by a grant from the Medical University of South Carolina (5—48h).Plasmids pSRA4, pSRB5, and pSRG1 containing cDNA for the a, (G. G. R.). p,and-ysubunitsoftheIL-2R,respectively,havebeendescribedpreviously 2 To whom requests for reprints should be addressed, at RS714, Medical University of (2). These were propagated in XL1-blue Escherichia coli and purified by South Carolina, 171 Ashley Avenue, Charleston, SC 29425. Phone: (803) 792-3423. cesium chloride density gradient sedimentation. Inserts were released by XbaI 3 The abbreviations used are: IL-2, interleukin 2; IL-2R, interleukin 2 receptor; ICso, 50% inhibitory concentration; PBMC. peripheral blood mononuclear cell; RT, reverse digestion, resolved by agarose gel electrophoresis, and recovered by electro transcription. elution. 32P probes were generated by a random-primed labeling method 2590

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linesCell Table 1 IL-2R expression on hematopoietic cell lineTypeaa13yAssayRef.HUT1O2AU+‘)+i―itCross-link20IARC3OILymphoma+++Ab21,22YrALL++ND23CR11.2ATh++ND24KG1AML+++Ab,

RT-PCR18Sez627Sezary+@4T,@CAb23MT-iATh+—NDCross-link20L54OCyHodgkin's+NDNDAb24HEL92.l.7AML±++Ab,

RT-PCR18HH@@TCL-+NDCross-link; Ab25YT2C2ALL-+NDCross-link20MLA144Gibbon

T-lymphoma—+NDCross-link20SKW6.4ALL-+NDCross-link, Ab26Reh 6ALL—+NDCross-link17KM3ALL—+NDCross-link17MOLT4T-ALL-±NDCross-link,

Ab17,27K562CML 17HPB-ALLALL--NDAb27JurkatALL——NDAb15,27HL-60AML-M3--NDAb15PeerTALL-—NDAb15CEMTALL--NDAb15HUT78TALL——NDAb15U937AML-M4-NDNDAb19C46Burkitt's--ND28blast—±+Cross-link, Ab, RT-PCR15,

a ATh, adult T-cell leukemia; ALL, acute lymphoblastic leukemia; Hodgkin's, Hodgkin's disease; TALL, T-cell acute lymphoblastic leukemia; CTCL, cutaneous T-cell lymphoma; AML, acute myeloblastic leukemia; CML, chronic myelogenous leukemia; Sezary, Sezary syndrome; Burkitt's, Burkitt's lymphoma; Ab, antibody. b [email protected];±weaklypresent;—,absent. C ND, not determined

(Boeringer-Manheim) and were purified by G-75 Sephadex chromatography; 0.8 X 106 Cerencov cpm 32P-labeledprobe were used per ml hybridization solution (31). Densitometry was performed using a Kodak DCS digital camera A to quantitate mRNA levels for the a, j3, and y subunits. Cytotoxicity Assays. Cells were seeded in 96-well plates from Costar (Cambridge, MA) at a concentration of l0@'/wellin100 ,d complete medium. Prior to assay, MLA-l44 cells, which produce IL-2, were first washed in cold, acidified (pH 5) RPMI medium to remove bound IL-2. DAB38@,IL-2was added at 12 different concentrations (l0 12_l0_6 M) in complete medium. Cells cultured with medium alone were included as the control. Following an 18-h incubation at 37°Cina 5% CO2 atmosphere, the plates were centrifuged for 5 mm at 230 x g, and the medium removed and replaced with 100 @dleucine free medium containing 0.25 @Ci[‘4C]leucine(New England Nuclear, Boston, MA). Cells were then incubated at 37°Cfor 90 mm and collected on glass fiber filters using a cell harvester (Skatron, Sterling, VA). Filters were washed, dried, and counted. All determinations were performed in triplicate or quin tuplicate. The IC@ DAB38@,lL-2, reducing protein synthesis by 50%, was —1.6 determined by extrapolation. .@

@ HUTIO2 120 @ RESULTS ug3TFI .TT'l@1 rrl-1 @@@ Distribution of IL-2R Subunits on Cell Lines. First, we identi 3.5— .@ 8.0 fled and characterized cell lines that could be used as positive and 1.7— -‘@ [email protected] @I4.0 negative controls. The CTCL cell line HUT1O2/6TG is known to be @ sensitive to DAB38@IL-2 and to express at the plasma membrane the a V a, f3, and @‘proteinsubunits (29), whereas the fibrosarcoma cell line @ SW684 is known to be insensitive and to be devoid of any IL-2R —1.6 ugRNA2.0 4.0 6.0 subunits (32). Triplicate samples of 3, 6, and 9 p.g HUT1O2 whole cell Fig. 1. Northern blot of the IL2R a, (3, and y subunit mRNA in HUT1O2 and SW684 RNA were electrophoresed next to 3, 6, and 9 @tgSW684 RNA and cells. A, triplicate sets of 3, 6, and 9 @gHUT1O2(Hf/i) and SW684 (SW) total cell RNA separately hybridized to the a, (3, and ‘yprobes(Fig. lA). With a 19-h were electrophoresed next to each other and transferred to nitrocellulose membranes. Each exposure at all three concentrations of HUT1O2 RNA, the a probe membrane was separately hybridized to the probes for the IL-2R a, (3,and ‘jsubunitsand subsequently rehybridized to the GAPD gene probe, as indicated. Top panel, UV fluo detected a major band doublet at 1.7 kb, a minor band at 3.6 kb, and rescent image of the RNA stained with ethidium bromide after transfer onto the mem a faint band at approximately 7.5 kb. The @3probedetected a major braise.The positions of the 285, 185, and 55 rRNAs, 45 RNA, and molecular size markers are indicated. B, triplicate sets of 0.2, 0.6, 2.0, and 6.0 @zgHUT1O2RNA were separately band at 4.0 kb and a minor band at 11 kb at all three concentrations hybridized to the a, (3,and @‘probesand subsequently rehybridized to the GAPD gene of HUT1O2 RNA. The ‘yprobedetected a major band at 1.8 kb, a probe, as in A. Numbers, sizes in kb of the detected mRNA species. Fortuitously, the 285 minor band at 4.0 kb, and a faint band at 8.0 kb at all three concen rRNA migrating upstream of the (3subunit mRNA displaces the (3mRNA band downward as the amount of RNA loaded in the lane is increased, resulting m a skewed mobility trations of HUT1O2 RNA. As judged from the intensity of the bands, pattern. Inset at right, densitometric analysis of the blot, showing a linear relationship the mRNA expression level was -y > @3> a. SW684 RNA did not between band densities and amount of RNA for all four probes. 2591

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IL-2 RECEFFOREXPRESSION

@ m c@a — @ 1'- I—';,, — ‘° 1-@

Fig. 2. Northern blot analysis of tumor cell lines for the expression of the a, @,and y subunit mRNA of the IL-2R. A and B, two representative assays using 6.0 @gwholecell RNA/lane. The method is the same as in Fig. 1. Numbers, sizes in kb of the detected mRNA species. Top of each blot, names of the corresponding cell lines. Ioonztl) xQO@Zc') zo@2Zu, C91-a and C91-b, two independent RNA isolates from the same cell line (see text). •: ..@ : •@

—1.6

yield any signal at any concentration for any probe. Background was cells for IL-2R subunit expression (Fig. 2 and Table 2). Two repro imperceptible. sentative Northern assays are presented in Fig. 2 (A and B) to illustrate Second, to define better the sensitivity of the Northern assay, we our analytical procedures and to document the reliability and repro probed increasing amounts of HUT1O2 total cell RNA (0.2, 0.6, 2.0, ducibility of the measurements. Each assay always comprised a re and 6.0 @.ag;Fig.1B). On an overnight exposure, the mRNA for the ‘y hybridization with the constitutively expressed GAPD gene to correct and j3 subunits could be detected in as little as 0.2 @gtotalRNA, 0.03 for loading differences and RNA degradation. Also, it included trip of the RNA normally loaded in a lane. The a subunit was detected licate nitrocellulose filters for contemporaneous, but separate hybrid only in 0.6 i.@gRNA. Densitometry of the autoradiograph showed that ization with the a, @3,and ‘ysubunit probes. Each filter always there was linearity between the signal and amount of RNA (Fig. 1B, contained the IL-2R-positive (HUT1O2) and -negative (SW684) con inset). trols. In Fig. 14, the HUT1O2, MJ, H9, HUT78, YT2C2, U937, HL6O, Next, we examined RNA from a number of malignant and normal Daudi, THP-l, U1l8, and SW684 cell lines were examined. In Fig.

Table 2 Correlation between IL-2R subunit status and sensitivity to DAB389IL-2 Mean values ±SD are followed by the number of measurements in parentheses.

a mRNA corrected 13mRNAcorrected ‘,9mRNA corrected Cells― IC50 (M) (%HUT1O2) (%HUTIO2) (%HUTIO2) PBMC, OKT3 activated I X 10—12(3) 43 ±20(3) 108 ±44(3) 111 ±22(3) HUTIO2/6TG 2 X 10—12(3) 100 (17) 100 (17) 100 (17) MJ 4 X 10—12(4) 279 ±25 (3) 90±27(3) 98± 17(3) MT-2 1 X 10― (3) 192 ±54(3) 127±7 (3) 50±7 (3) C9 l/PL I X l0@―(4) 83±9 (5) 34± 11(5) 77±8 (5) C1OIMJ I X l0@―(3) 197 146 102 HH 4 x 10—10(4) 0 (4) 129 ±31(4) 57±8 (4) YT2C2 2 X l0@ (3) 0 137 66 C82l5 2 X i0@ (3) 0 17 63 MLA-l44 (monkey) 6 X l0@ (3) 0 (3) 109± 18(3) 99± 11(3) PBMC, resting 8 X lO_8 (2) 0 (2) 35±7 (2) 83±5 (2) SKW6.4 9 x l0@@(3) 0 12 54 MT-I >lO@ (3) 116±19(2) 0 (2) 48±1 (2) H9 >l0@ (4) 0 (3) 16±7 (3) 162 ±50(3) HUT78 >l0@ (3) 0 (4) 8±2 (4) 96±5 (4) U937 >l0@ (3) 0 (4) 0 (4) 128± 15(4) HL-60 >l0@ (3) 0 (3) 0 (3) 46±4 (3) Daudi >l0@ (3) 0 (3) 0 (3) 40±2 (3) SW684 >iø—@(3) 0 (17) 0 (17) 0 (17) HTIO8O >l0@ (4) 0 (2) 0 (2) 0 (2) MCF7 >iø—@(3) 0 (2) 0 (2) 0 (2) Ul 18MG >i@—@(2) 0 (3) 0 (3) 0 (3) NCI-H929 >l0@ (3) 0 (2) 0 (2) 0 (2) ThP- I >l0@ (3) 0 (4) 0 (4) 0 (4) Figures without SD indicate single measurements. a CTCL cell lines are HUT1O2/6TG, Mi, HH, H9, and HUT78. All cell lines are MT-2, C91/PL, and MT-l. ALL cell lines are YT2C2, C8215, and SKW6.4. Gibbon non-Hodgicin's lymphoma cell line is MLA-l44. Burkitt's lymphoma cell line is Daudi. U937 is a histiocytic lymphoma cell line. HL-60 is an acute promyelocytic leukemia cell line. 1'HP-l is an acute myelogenous leukemia cell line. Ul 18MG is a glioblastoma cell line and SW684 and HT1O8Oare fibroblast cell lines. NCI-H929 is a myeloma cell line. 2592

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0 0 The IC50 for HUT1O2 cells was 2 X 10 12M, and repeated testing on y = -0.0154x - 7.931 y = -0.0256x -73122 separate days gave values within 30%. Other cell lines were less R2 0.4528 R2 0.5459 sensitive, as shown in Table 2. Cells missing a but containing (3 and P=O.0016 e-4 P = 0.0003 I0 ,l subunit mRNA had reduced but measurable sensitivity to 0 C-) DAB389IL-2. C, C, 0 0 IL-2R Subunit Content and DAB@IL-2 Sensitivity of Trans. ..1@8 —J-8 fected Fibroblasts. The L929 murine fibroblasts stably expressing the human a and (3 chains (La-(3-2), a, (3, and y chains (La-j3-y-4), and (3 and ‘ychains(L(3--y-l) of IL-2R were assayed for sensitivity to -12 DAB38@IL-2(Fig. 4). The IC50s for DAB389IL-2 were 5 X iO@ Mfor @@ 0 100 200 300 0 50 100 150 La-(3-2 cells, 9 X i0 M for L(3-y-l cells, and 9 X 10 12 M for ALPHA mRNA (% HUTIO2) BETA mRNA (% HUTIO2) La-(3-y-4 cells. Northern analysis demonstrated that La-j3-2 transfec 0 y = -0.Olllx - 7.8861 tants had a and (3 subunit mRNAs but no y mRNA. L(3-y-1 cells had R2= 0.0439 13and-ymRNAsbutnoa mRNA.La-(3-'y-4cellshadallthreesubunit mRNAs. e-4 P=0.3893 I0 C.) C, DISCUSSION 0 -J-8 Suitable antibodies that would enable simultaneous detection of each of the three IL-2R subunits have been generally unavailable. This -12 has led to a number of studies that have attempted to evaluate IL-2R expression on various cell types with only a subset of reagents. In 0 100 200 GAMMA mRNA (% HUTIO2) addition, these reports vary in the method of detection (Table 1). Some of the methods use RT-PCR, affinity cross-linking, and anti Fig. 3. Linear regression analysis of the relationship between logIC@and a, @,and‘,‘ mRNA levels. body binding. The most sensitive assay, RT-PCR, amplifies rare mRNA species among total cell RNA. The procedure can detect one molecule of mRNA/l06 cells (33), but it is troublesome to use 2B, the HUT1O2, C91, MT-i, NCI-H929, and SW684 cell lines were quantitatively. Cross-linking depends on the affinity of the ligand for examined. C91-a and C91-b represent two independent RNA isolates. the receptor and the specific activity of the reagent and is less C91-a and C91-b were virtually identical for the hybridization with sensitive, because it requires several hundred to 1000 receptors/cell both the a, @,)@and GAPD probes. MT-l mRNA was analyzed in the for detection (34). Antibody binding to cell surface IL-2R using a cell Fig. 2, both A and B, blots. One can appreciate that, although the sorter or fluorescence microscopy is even less sensitive and re hybridization signals for the a, y, and GAPD probes varied from Fig. quires l0@molecules/cell (35). 2A to Fig. 2B, their intensities relative to the HUT1O2 controls were To overcome some of these shortcomings, using a highly son virtually identical. Following densitometry, mRNA levels of each cell sitive Northern assay with cDNA probes for the full subunit line were corrected for GAPD and expressed as the percentages of complement of the receptor (a, (3, and y) we have conducted a HUT1O2 a, @3,and y subunit mRNA, respectively. We analyzed a comprehensive study on a large number of malignant and nonma total of 24 different specimens, including four nonlymphoid cell lines lignant hematological specimens to correlate the relative IL-2R (SW684, Ui 18, HT1O8O, and MCF-7), 12 T-cell-derived cell lines (HUT1O2, MJ, MT-2, C91, C1OIMJ, H9, HUT78, HH, YT2C2, C8215, MLA-144, and MT-i), three B-cell-derived cell lines c,@ (SKW6.4, Daudi, and NCI-H929), three myeloid-derived cell lines (U937, HL6O, and THP-l), and quiescent and OKT3-activated PB MCs. The corresponding mRNA levels for the a, @3,and@ysubunitsof @8O the IL-2R are presented in Table 2, along with the IC50 for DAB38@,IL-2.The a subunit was detected in 7 of the 24 cell prepa rations, and there was a 6-fold range in mRNA levels. Similarly, (3 subunit expression was found in 14 of 24 cell lines, with a 17-fold 60 range in amounts. Eighteen preparations possessed -y chain mRNA, and there was only a 4-fold range in amounts on positive cell lines. 40 The relationship between the logIC50 and the mRNA data correspond ing to the first 19 cell lines of Table 2 (from the most sensitive to the negative control SW684) was examined by regression analysis (Fig. 20 3). A moderately significant (P = 0.0016) inverse correlation was found between logIC50 (DAB389IL2 concentration for half-maximal cell killing) and the expression level of the a subunit mRNA. A strongly significant (P = 0.0003) inverse correlation was found be -8 -9 -10 -11 -12 -13 tween logIC50 and the level ofexpression of the (3subunit mRNA. No correlation (P = 0.3893) was found between the logIC50 and the LOG DAB,JL2 CONCENTRATION (M) expression of the @‘subunitmRNA. Fig. 4. Cell cytotoxicity assay with DAB38,,IL-2. •,L@y2 cells; U, La-@-@y-4cells; Sensitivity of Cell Lines to DAB@,IL-2. The inhibition of protein A, La-y-I cells. Each assay point was repeated in triplicate, and the value shown is the mean. SD was less than 30% value. ID@(@wascalculated as the concentration of toxin synthesis by DAB38@IL-2 yielded a sigmoid curve when plotting the reducing protein synthesis by 50% and was determined from the mean of two or three toxin concentration versus the percentage of control protein synthesis. separate experiments. 2593

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subunit mRNA expression with sensitivity to intoxication by an (YT2C2, C82i5, and SKW6.4). A cell line of acute myeloblastic IL-2 fusion toxin, DAB389IL-2 (Table 2). Northern hybridization leukemia (HL-60), a single histiocytic lymphoma cell line (U937), has been reported to be able to detect as few as one mRNA and a B-cell, non-Hodgkin's lymphoma cell line (Daudi) had only molecule/cell (36), however, our assay demonstrated a 30-fold -Vsubunit mRNA. These results are comparable to observations on increased sensitivity, because we were able to detect (3 and y fresh lymphoid and myeloid tumor cells (9—19). The finding of mRNA in 0.03 of the amount of RNA that is normally loaded in a variable a subunit mRNA expression in CTCL cell lines parallels lane (Fig. 1B). Several observations can be made from Table 2. the immunocytochemical observations on both fresh Sezary cells First, the cells more sensitive to DAB389IL-2 contained all three and leukemic infiltrates of skin biopsies (9, 14, 38). receptor subunit mRNAs, and their sensitivity progressively de Similar to PBMCs and leukemic cell lines, transfectants possessing creased with the loss of the a, (3, and y subunit mRNA. Second, the the heterotrimeric a-(3-'y IL-2R were most sensitive to DAB38@IL-2. presence of the -y subunit mRNA alone was insufficient to confer The IC50 similar to that of HUT1O2/6TG cells suggests that no sensitivity to DAB389IL-2, as demonstrated by U937, HL6O, and additional components are required for efficient IL-2R-targeted tox Daudi cells. To impart any degree of sensitivity, this receptor icily. The f3and y subunit-transfected cells behaved indistinguishably subunit mRNA had to be associated with at least the (3 subunit from leukemic cells possessing (3and -ysubunits and were moderately mRNA, as exemplified by the first 12 cell lines of Table 2. MT-i sensitive to DAB389IL-2, but less so than high-affinity, IL-2R-bearing cells, which expressed ‘yanda subunit mRNA but no (3 subunit cells. Finally, cells transfected with a and (3but no y subunit (La-(3-2) mRNA, were insensitive to DAB389IL-2. Third, the presence of the were relatively insensitive to DAB38@IL-2,consistent with the require a subunit mRNA greatly enhanced the mediation of cytotoxicity, ment of the ‘ychainfor efficient ligand internalization (2). Diphtheria as demonstrated by OKT3-activated PBMCs and HUT1O2, MJ, toxin must reach the endosomal compartment for translocation to the MT-2, C9iIPl, and C1OIMJ cells. However, this subunit mRNA cytosol and inhibition of protein synthesis (39). appeared not to be obligatory for sensitivity, because the HH, Enrollment in current CTCL clinical trials with DAB389IL-2 re YT2C2, C82l5, and MLA-144 cell lines and resting PBMCs quires expression of an IL-2R that is detected with commercially displayed varying degrees of sensitivity without its presence. All available monoclonal reagents that measure IL-2Ra alone (38). Our of the above are consistent with the notions that: (a) the -ysubunit results suggest that (3 subunit mRNA content may be a more critical alone cannot bind DAB389IL-2; (b) the ‘ysubunit mediates inter factor in determining the sensitivity to fusion protein. The CTCL cell nalization of the receptor/DAB389IL-2 complex and is necessary line MT-i, for example, which did not have (3 subunit mRNA, was for cell intoxication; (c) the (3 subunit is necessary to impart any insensitive to fusion toxin, despite the presence of detectable a and ‘y degree of sensitivity, provided that it is expressed with the y subunit mRNA. Northern blots of fresh CTCL tumor samples with a, subunit; (d) the /3 subunit confers an intermediate affinity for f3,andy subunitprobesmaymoreaccuratelypredictclinicaloutcome DAB389IL-2 to the (3-y heterodimer and along with y is required than current IL-2R a antibody staining alone. for its internalization to produce cell killing; (e) the a--y het We have used established tumor cell lines as a model system to erodimer either cannot be internalized or has an affinity too low to study the IL-2R subunit mRNA requirements for cell intoxication by mediate cell intoxication; and, finally, (f) the a-f3-'y heterotrimer DAB38@[email protected] of mRNA by Northern analysis represent has the highest affinity and is associated with the most efficient average levels of message within a cell population and do not describe cell killing. This was supported by regression analysis, because a mRNA expression in individual cells. These measurements only re significant inverse correlation between logIC50 and mRNA was motely reflect the actual protein level in the plasma membrane, found only for the (3 (P = 0.0003) and a (P = 0.0016) subunits, because translatability of the message, processing, and turnover of the underscoring the necessity of the (3 subunit for ligand binding and protein are not known. In addition, they do not provide clues as to internalization and the auxiliary nature of the a subunit. Paradox receptor assembly at the cell surface. Nevertheless, mRNA measure ically, no correlation was found for the y subunit (P = 0.3893), ments have been informative. Regression analysis has shown that celi despite the necessity of this subunit for the internalization of the killing correlates with subunit mRNA expression levels. We have IL-2R. This result can be interpreted in light of the current under determined that, provided the y subunit mRNA is ptesent, cell killing standing of the function and distribution of the y subunit. The ‘y directly depends on the (3 subunit mRNA level. Cell killing is greatly subunit is shared among a number of lymphokine receptors, in enhanced by the additional, but not obligatory, presence of the a cluding IL-2R, IL-4R, IL-7R, IL-i lR, and IL-l5R (37) and is subunit mRNA. These observations, if applied to clinical settings, abundantly expressed in certain IL-2R-negative cells, such as may provide principles on which to evaluate patients with IL-2R- U937, HL6O, and Daudi cells (Table 2). Therefore, ‘ysubunit bearing cancers for their responsiveness to an IL-2 fusion toxin mRNA expression may not necessarily correlate with cell killing therapy. Furthermore, they may assist in determining whether it is by DAB389IL-2 in a regression analysis of all cells that express the possible to improve patient response to DAB38@IL-2 by up-regulating 7 mRNA. the IL-2R subunit expression with one of a number of substances (19, Our results on the IL-2R mRNA subunit expression of cell lines are 40—44).This would expand the application of IL-2R-directed fusion in general agreement with published literature (Table 1). In HU'F78 toxins. cells, we found low levels of (3 subunit mRNA (8%), whereas the antibody-binding assay did not detect the (3subunit on the cell surface REFERENCES (15). This difference may be accounted for by the greater sensitivity of the Northern analysis. 1. Waldmann, T. The multi-subunit interleukin-2 receptor. Annu. Rev. Biochem., 58: 875—911,1989. Some pattern of IL-2R subunit mRNA expression could be 2. 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Gian G. Re, Cory Waters, Louis Poisson, et al.

Cancer Res 1996;56:2590-2595.

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