Proc. Natl. Acad. Sci. USAVol. 76, No. 12, pp. 6552-6556, December 1979Immunology

A human thymus-leukemia antigen defined by hybridomamonoclonal antibodies

(acute lymphocytic leukemia/cell surface/tumor antigens)

RONALD LEVY, JEANETTE DILLEY, ROBERT I. Fox, AND ROGER WARNKEThe Howard Hughes Medical Institute Laboratories, and the Departments of Medicine and Pathology, Stanford University Medical Center,Stanford, California 94305

Communicated by Henry Kaplan, July 9, 1979

ABSTRACT A series of mouse hybridomas producing mo-noclonal antibodies against human acute lymphocytic leukemia(ALL) cells was generated and screened for tumor specificity.Among 1200 primary cultures, 60 produced an antibody thatcould distinguish between the immunizing leukemia cells andan isologous B lymphoblastoid cell line. Of these, two producedan antibody that detects an antigen expressed preferentially onALL cells and on a subpopulation of normal cells found in thecortex of the thymus. Other normal human lymphoid cells fromlymph nodes, spleen, bone marrow, and peripheral blood expressonly low levels of this antigen. High levels of this "thymus-leukemia" antigen were found on T-ALL cells, T-ALL-derivedcell lines, and some "null" ALL cells. By contrast, B-cell leuke-mias, B lymphoblastoid cell lines, and normal and malignantmyeloid cells contain either low or undetectable amounts of thisantigen. The thymus-leukemia antigen has been isolated fromthe membranes of leukemia cells by detergent solubilizationand subsequent immunoprecipitation with the monoclonalantibody. Preliminary biochemical characterization shows theantigen to be associated with a polypeptide of Mr -28,000.

A major advance in cell surface and tumor immunology hasbeen the introduction of techniques for the production of ho-mogeneous monoclonal antibodies. It is now possible to sortthrough a complex mixture of antigens, such as cell surfacemolecules, with probes that see one antigen at a time. Thesetechniques are especially applicable to the study of human cellsurface antigens, particularly in the search for human tumor-specific antigens. Monoclonal antibodies can be obtained bythe Klinman technique, which involves in vivo cloning of B cellsby adoptive cell transfer and subsequent in vitro spleen frag-ment culture (1). By using this method, we demonstrated thatthe mouse has within its antibody repertoire the capacity todistinguish among closely related human cell surface antigens,including HLA polymorphisms (2), B- and T-cell determinants(3), and tumor-associated antigens (4, 5). However, this tech-nique was ultimately limited by the amount of antibody ob-tainable from each spleen fragment culture. With the intro-duction of the hybridoma technique of Kohler and Milstein (6),it became possible to obtain monoclonal antibodies in largeamounts.

In the present study, we generated a panel of individualhybridomas from mice immunized to human acute lympho-cytic leukemia (ALL) cells. These antibodies were screened fortheir ability to discriminate between the leukemia cells and anisologous B lymphobastoid cell line (LCL). Among the dis-criminator antibodies, two define an antigen that is preferen-tially expressed on ALL cells and on a subpopulation of normalthymocytes.

MATERIALS AND METHODSHuman Cells. The leukemia cells used for immunization and

for screening of antibodies were derived from the peripheralblood of a child (Dom) with T-cell ALL. The patient had amediastinal mass and a blood lymphoblast cell count of 460,000per mm3. Greater than 95% of these blast cells formed heat-stable sheep erythrocyte rosettes (7). Leukemia cells from patientDom and a series of other patients were purified from peri-pheral blood or bone marrow or Ficoll-Hypaque sedimenta-tion (8) and stored in 10% dimethyl sulfoxide under liquid N2.A B-cell LCL from patient Dom (LCL-Dom) was established

by Henry Kaplan. This cell line is composed of a polyclonalmixture of K and X immunoglobulin-bearing cells (9) and itcontains the Epstein-Barr nuclear antigen (10) (Henry Kaplan,personal communication). The LCL-Dom cells were grown andmaintained in Dulbecco's modified Eagle's medium (highglucose) (GIBCO), containing penicillin (100 units/ml), gluta-mine (2 mM), and 15% fetal calf serum. Other cell lines usedin these studies include the T cell lines MOLT-4, 8402, andHSB2 and the B cell lines 8866, 8392, and SB. Their origins andcharacteristics have been described (11-13). Normal humanthymus glands, lymph nodes, and spleens were obtained fromfresh surgical specimens.Immunization Schemes. BALB/c mice were immunized

and 1 week later were given an intraperitoneal booster with 107glutaraldehyde-fixed (4) ALL-Dom cells. Three months later,they were given an intravenous booster with another 107ALL-Dom cells. Three days later, 2 X 107 spleen cells fromthese animals were transferred intravenously to syngeneic ir-radiated (600 rads) mice along with another 107 ALL-Domcells. The recipient mice had been previously hyperimmunizedeither with normal peripheral blood lymphocytes (PBL) (groupI), LCL-Dom (group II), or nothing (group III, control). Hy-bridomas were prepared from the spleen cells of each recipientgroup 1 week after transfer.

Cell Fusion and Culture. Spleen cells were fused withNSI/1-AG 4 cells (14) at a cell ratio of 2:1, respectively, by using38% polyethylene glycol (Baker 1540) (5). After fusion, the cellswere resuspended in Kennet's modification of Dulbecco'smedium containing hypoxanthine, aminopterin, and thymidine(15) and dispensed into 96-well tissue culture trays (Linbro) at1-2 X 105 cells per well along with 5 X 105 normal spleen cells.The medium was changed twice during the first 2 weeks toremove antibody released by unfused spleen cells and then wascollected for testing during the third week. Culture fluids weresampled, with attention being paid to avoid contamination ofeach fluid by its neighbors-i.e., by washing or changing of

Abbreviations: ALL, acute lymphocytic leukemia; LCL, lympho-blastoid cell line; PBL, peripheral blood lymphocytes.

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pipettes between collections. These fluids were examined forthe presence of antibody against the immunizing ALL-Doxncells as well as the LCL-Dom cells by a cell-binding radcoim-munoassay (5), using purified 125I-labeled goat anti-mouse Fabfragment as a detecting reagent. Selected cultures were sub-cloned by'plating limiting dilutions of the hybrid cells into thewells of 96-well plates along with normal mouse spleen cells.

Immunofluorescence. Hybridoma antibodies were exam-ined by indirect immunofluorescence on viable cells in sus-

pension, fixed cell smears, or frozen tissue sections prepared bydescribed methods (9). A rhodamine conjugate of the same

purified goat anti-mouse Fab used in the radioimmunoassaywas employed. In some cases, an F(ab')2 fragment of this pu-rified goat antibody was employed.

Labeling of Cell Membranes and Immunoprecipitation.Cell membranes were radio-iodinated by using lactoperoxidaseand solubilized with Nonidet P-40 (Shell Chemical) as described(16). The solubilized 125I-labeled membrane preparation, 107cell equivalents, was first subjected to a nonspecific immu-noprecipitation by the addition of human IgG (25 Mg) and goatanti-human Fab at equivalence. To the cleared supernatant,100-200 ,l of culture fluid containing either a specific mono-clonal antibody or a myeloma protein of the same class was

added. This was followed by the addition of normal mouseserum (2.5 pi) and goat anti-mouse Fab (preabsorbed on humanIgG-Sepharose) at equivalence. The final precipitates were

solubilized in 3% sodium dodecyl sulfate/2% mercaptoetha-nol/0.05 M Tris-HCI, pH 6.8/5% glycerol and boiled.Gel Electrophoresis. Sodium dodecyl sulfate/polyacryl-

amide gel electrophoresis (17) was performed in slab gels byusing either a 5-20% gradient of acrylamide or a uniform 10%acrylamide concentration. Gels wer'e dried and autoradio-graphed by using Kodak XR2 film with a Cronex LightningPlus intensifying screen (Dupont).

Fluorescence-Activated Cell Sorter Analysis. Lymphocyteswere incubated with monoclonal antibody (50 Ml per 5 X 106cells) at 40C in the presence of 1 mM azide. The cells were

washed in fetal calf serum and gently resuspended in 50 Ml offluorescent goat anti-mouse Ig antibody. The stained cells werewashed again in fetal calf serum, resuspended in Dulbecco'sphosphate-buffered saline, and fixed in 1% formaldehyde.Analysis of staining intensity was performed by using the flu-orescence-activated cell sorter as described by Loken andHerzenberg (18).

RESULTSInitial Screening. A total of 1877 culture wells, of which

approximately 60% contained growing hybrids at the time offluid collection, was screened for antibody activity by radio-immunoassay. Two different target cells were used for thisinitial screen ALL-Dom, the immunizing leukemia cells, andLCL-Dom, an isologous B-cell LCL. A representative portionof the data from this screen is shown in Fig. 1. Antibody reactivewith the immunizing ALL cells was found in 619 (33%) of theculture wells. Of these, 148 appeared to contain an antibodythat distinguished between the immunizing leukemia cell andits isologous B cell line (shown as arrows in Fig. 1). With re-

peated testing on these and subsequently collected fluids, 60cultures were confirmed to be continuously producing antibodywith preferential reactivity for the leukemia cell. This fre-quency of 60/619 may be an underestimate of the true fre-quency of discriminators, because many of the cultures con-tained more than one clone.Three different spleen cell donors were used for cell fusion

in these experiments. All were radiated recipients of the sameadoptively transferred immune spleen cells. However, in two

E S S"-WNgS1E1 11S

2000 7

4000 -

6000

_ LCL

FIG. 1. Radioimmunoassay of hybridoma culture fluids. Fluidsfrom individual culture wells were assayed for binding activity againsttwo different target cells from patient Dom: ALL (Upper) and LCL(Lower). The data from one representative 96-well plate are dis-played.

groups the recipients had been hyperimmunized against normalhuman lymphoid antigens-either PBL (group I) or LCL-Dom(group II). This maneuver was an attempt to suppress the ex-pansion of B cell clones reactive to normal human antigenswhile stimulating the expansion of clones reactive to antigenslimited to the leukemia cell. A third group of recipients was notpreimmunized. Similar numbers of total antibody-producinghybridomas were obtained in each group. However, a moderateadvantage was noted for groups I and II in terms of the per-centage of hybrids producing antibody that discriminated be-tween the leukemia cell and the isologous B cell line-13.8 and9.7%, respectively, vs. 5.2% in the control group.

Definition of a Thymus-Leukemia Antigen. On furthertesting of the 60 discriminators, it became apparent that, al-though they failed to react with LCL-Dom, most of these an-

tibodies were strongly reactive against human normal PBL andpresumably recognize normal T cell antigens. However, twohybridomas designated 12E7 and 21D2 gave a reactivity pat-tern that was distinctly different, distinguishing between theleukemia cells on the one hand and the LCL-Dom and PBL on

the other (Fig. 2). These binding data, and absorption analyses(not shown), indicate that the antigen detected by antibodies12E7 and 21D2 is present on PBL, but at a level less than 5%of that on ALL cells. Of note is that hybridomas 12E7 and 21D2were both derived from group II, the animals that were

hyperimmunized to LCL-Dom prior to receipt of ALL-Domimmune cells. Hybridoma 12E7 that produces an IgG-1 anti-body was selected for further study.

Indirect immunofluorescence was performed by usingantibody 12E7 on viable cell suspensions and fixed cell smears.All of the ALL-Dom cells were brightly stained on the mem-brane and in the cytoplasm, whereas PBL showed only faintmembrane staining. Similar stained preparations were analyzed

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E

1000

1:4 1:8 1:16 1:32 1:64 1:128 1:256Fluid dilution

FIG. 2. Binding of antibody 12E7 to T-ALL cells (@), LCL cells(0), and PBL (A). Background of 200 cpm was subtracted from thedata.

by using the fluorescence-activated cell sorter. Again, 100% ofthe ALL cells were very brightly stained and were easily dis-tinguishable from the PBL of normal donors (Fig. 3 Upper).The myeloma protein produced by P3/X63 cells was used asa negative control (Fig. 3 Lower). Fluorescent staining byantibody 12E7 of a fixed bone marrow smear from a patientwith T-cell leukemia is shown in Fig. 4. The leukemia cells were

100

50

ICCD

*0lOt

St-L

ALL/ PBL

n ............

PBL

ALL

FIG. 4. Indirect immunofluorescence of antibody 12E7 on a bonemarrow smear from a patient with T-ALL. An F(ab')2 fragment of apurified goat anti-mouse Fab antibody was used as a rhodamineconjugate for the second-stage reagent.

brightly stained and were easily detectable within a backgroundof unstained normal or weakly stained normal bone marrowcells.

Various normal lymphoid tissues were examined by indirectimmunofluorescence on frozen sections with antibodies 12E7and 21D2. These antibodies gave weak staining of cells in spleenand lymph node. However, both these antibodies gave intensestaining of cells in the cortex of the thymus gland (Fig. 5). Bycontrast, lymphoid cells in the medulla of the thymus failed tostain with antibody 21D2 or 12E7. The binding of antibody12E7 to thymocytes in suspension is shown in Fig. 6 and com-pared with its binding to several types of human leukemia cells.These data show that the average density of the antigen onthymocytes is comparable to that on T-ALL cells and greaterthan that on the T-ALL-derived cell line, 8402. It also showsthat the cells from one case of chronic lymphocytic leukemiaand those from one case of "null cell" ALL contain insignificantamounts of the antigen.The reactivity of antibody 12E7, either by radioimmunoassay

or by immunofluorescence, on a series of human normal andleukemia cells is summarized in Table 1. Strong reactivity of

I I10 20

Fluorescence intensity

FIG. 3. Fluorescence intensity profiles of ALL cells or normalPBL with monoclonal antibodies. Cells were obtained from the bloodby Ficoll-Hypaque sedimentation, mixed with monoclonal antibodies,counter-stained with fluorescent rabbit anti-mouse Ig antibody, andanalyzed on the fluorescence-activated cell sorter. (Upper) 12E7antibody. (Lower) Control antibody.

FIG. 5. Indirect immunofluorescence on frozen section of normalhuman thymus. An F(ab')2 fluorochrome was used as in Fig. 4. Twoviews are shown. Staining is limited to cells in the cortex (upper area

of pictures), sparing cells in the medulla (lower area of pictures).

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Ea

1:16 1:32 1:64Fluid dilution

FIG. 6. Binding of antibody 12E7 to thymocytes (0), T-ALL cells(0), a T-ALL-derived cell line, 8402 (o), a "null cell" ALL (A), anda chronic lymphocytic leukemia (v).

this antibody appears to be limited to cortical thymocytes, acutelymphoid leukemias of the T-cell type, and most cases of acutelymphoid leukemias of "null cell type."

Preliminary Characterization of the Thymus-LeukemiaAntigen. Cell membranes were labeled with 12'I by using lac-toperoxidase, were solubilized with detergent, and were im-munoprecipitated with antibodies 21D2 and 12E7. Fig. 7 showsthe results of gel electrophoresis of material immunoprecipi-tated by 12E7 from PBL, 8402 (a T-ALL derived cell line), andDom (the original T-ALL cells used for immunization). In eachcase, a comparison is made with a control immunoprecipitatein which P3/X63 fluid was used instead of 12E7. Ig is includedon the gel to provide molecular weight markers (L and H).

Table 1. Reactivity of antibody 12E7 on normal andleukemia cells

Levels ofLymphoid cells TL antigen

ALL-Dom (the immunizing cell) HighLCL-Dom (isologous B lymphoblastoid cell) LowOther B cell lines (SB, 8392, 8866) LowPBL LowLymph node cells LowSpleen cells LowBone marrow cells LowThymocytes

Cortex HighMedulla Low

T-All cell lines (MOLT-4, 8402, HSB2) HighT cell acute leukemias and lymphoblastic lymphomas HighB cell leukemias (chronic lymphocytic leukemias) LowMyeloid leukemias (acute and chronic) None"Null cell" leukemias (three out of four cases) High

TL, thymus-leukemia.

H --

s.

28,000 Mr o

L

IgG 12E7 P3

PBL

12E7 P3

8402

12E7 P3

Dom

FIG. 7. Sodium dodecyl sulfate/polyacrylamide gel electropho-resis of membrane molecules immunoprecipitated with antibody12E7. 125I-Labeled cell membranes, 5 X 106 trichloroacetic acid-precipitable cpm, were immunoprecipitated either with the mono-clonal antibody 12E7 or with the myeloma protein secreted by P3/X63(P3), were solubilized in sodium dodecyl sulfate/2-mercaptoethanol,and were electrophoresed on a 10% slab gel. L, light chain of Ig; H,heavy chain of IgG.

A specific band of -Mr 28,000 is apparent in the materialimmunoprecipitated from the leukemia cells. Identical resultswere obtained when antibody 21D2 was used in place of 12E7(not shown). No specific bands can be seen in the material de-rived from PBL, nor could any be seen in material derived fromchronic lymphocytic leukemia cells, LCL-Dom, or other Blymphoblastoid cell lines (not shown on this gel).

DISCUSSIONIn the present study, we sought to analyze possible antigenicdifferences between human acute leukemia cells and normalhuman cells. We adopted a screening strategy in which amatched pair of genetically identical cells-one leukemic andone lymphoblastoid-was used in a radioimmunoassay to searchfor discriminator antibodies. This approach is similar to thestrategy we previously employed to screen monoclonal anti-bodies for recognition of human HLA polymorphic antigens(2). It requires that the antibody-producing cells be cloned priorto testing, because the presence of nondiscriminating antibodiesobscure the discriminators. Furthermore, to be most effective,the pair of target cells should be as closely related as possiblebut different in the desired characteristic. Our initial screeneliminated most of the antibodies from consideration and al-lowed us to focus on a manageable number of potentially in-

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teresting candidates. After a second screen with normal pe-ripheral blood lymphocytes, two antibodies, 12E7 and 21D2,emerged as especially interesting.The immunization method used in the present study was

based on the work of Moller, who showed that adoptive transferof specific B cells with antigen into irradiated recipients couldgreatly enhance B-cell expansion, but that the expansion couldbe specifically suppressed by prior immunization of the re-cipient (19). We attempted to stimulate the ALL-reactive B cellswhile suppressing the B cells reactive to normal human anti-gens. The maneuver resulted in a slight increase in the fre-quency of discriminator hybridomas.

Antibodies 12E7 and 21D2 detect an antigen present on ALLcells and on a population of normal cells found in the cortex ofthe thymus. This tissue distribution is reminiscent of the thy-mus-leukemia antigen of the mouse (20). The mouse thy-mus-leukemia antigen is a 45,000 Mr protein that is associatedon the cell surface with 32 microglobulin (21-23). By contrast,the human thymus-leukemia antigen described here appearsto have a Mr of nt28,000 and it is not associated with f32 mi-croglobulin.

McMichael et al. (24) have recently described a monoclonalantibody raised against human thymocytes that reacts with theT-ALL-derived cell line MOLT-4. Their antibody immu-noprecipitated a two-chain structure of Mr 45,000 and 11,000from thymocyte cell surfaces but the 11,000 Mr polypeptidewas different from 32 microglobulin; no 28,000 Mr moleculewas seen (24, 25). Chechik et al. (26) have described a 43,000Mr protein extractable from human thymocytes, T-cell lines,and T-ALL cells that copurified with a second chain of Mr23,000. However, they found this molecule to be only intra-cellular. The relationship between these different humanthymocyte antigens can be clarified only by a side-by-sidecomparison.

Monoclonal antibodies, such as 12E7 and 21D2, will be usefulin a number of different areas of further investigation. Becausethey react with cells from many, but not all, cases of ALL, theywill help to categorize different subtypes of leukemia and mayshed light on the tissue origin of these malignant cells. Cur-rently, the human ALLs are subdivided into T cell, pre-B cell,and null cell types according to their expression of certainmarkers of normal lymphocyte differentiation (27-30). It isalready clear that the antigen defined here is not limited to justthe leukemias that carry other T-cell differentiation markers,because it is found on some "null" cell leukemias as well (Table1).By using antibodies 12E7 and 21D2, leukemia cells can be

detected in peripheral lymphoid tissues. These monoclonalantibodies may, therefore, provide a sensitive means of moni-toring the patients with leukemia during therapy and for de-tecting early disease relapse by surveillance of bone marrowand blood for antigen-positive cells (31). Moreover, it shouldbe possible to develop a radioimmunoassay for the detectionof free antigen, which may be even more sensitive than thedetection of the leukemia cells per se.

We are grateful to Dr. Henry Kaplan for the LCL-Dom cell line,to Dr. Charles Bieber for normal human thymus tissue, and to Ms.

Marilyn Pederson for tissue section immunofluorescence and pho-tography and Dr. Irving Weissman for helpful discussions. This workwas supported by grants from the National Institutes of Health (CA21223-02 and AI-09072). R.L. is an investigator of the Howard HughesMedical Institute. R.I.F. is a Fellow of the Arthritis Foundation.

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