Proc. Natl. Acad. Sci. USA
Vol. 76, No. 12, pp. 6552-6556, December 1979
A human thymus-leukemia antigen defined by hybridoma monoclonal antibodies (acute lymphocytic leukemia/cell surface/tumor antigens)
RONALD LEVY, JEANETTE DILLEY, ROBERT I. Fox, AND ROGER WARNKE The 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 monoclonal antibodies against human acute lymphocytic leukemia (ALL) cells was generated and screened for tumor specificity. Among 1200 primary cultures, 60 produced an anti body that could distinguish between the immunizing leukemia cells and an isologous B lymphoblastoid cell line. Of these, two produced an antibody that detects an antigen expressed preferentially on ALL cells and on a subpopulation of normal cells found in the cortex of the thymus. Other normal human lymphoid cells from lymph nodes, spleen, bone marrow, and peripheral blood express only low levels of this antigen. High levels of this "thymusleukemia" antigen were found on T-ALL cells, T-ALL-derived cell lines, and some "null" ALL cells. By contrast, B-cell leukemias, B lymphoblastoid cell lines, and normal and malignant myeloid cells contain either low or undetectable amounts of this antigen. The thymus-leukemia antigen has been isolated from the membranes of leukemia cells by detergent solubilization and subsequent immunoprecipitation with the monoclonal antibody. Preliminary biochemical characterization shows the antigen to be associated with a polypeptide of Mr -28,000. A major advance in cell surface and tumor immunology has been the introduction of techniques for the production of homogeneous monoclonal antibodies. It is now possible to sort through a complex mixture of antigens, such as cell surface molecules, with probes that see one antigen at a time. These techniques are especially applicable to the study of human cell surface antigens, particularly in the search for human tumorspecific antigens. Monoclonal antibodies can be obtained by the Klinman technique, which involves in vivo cloning of B cells by adoptive cell transfer and subsequent in vitro spleen fragment culture (1). By using this method, we demonstrated that the mouse has within its antibody repertoire the capacity to distinguish 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 technique was ultimately limited by the amount of antibody obtainable from each spleen fragment culture. With the introduction of the hybridoma technique of Kohler and Milstein (6), it became possible to obtain monoclonal antibodies in large amounts. In the present study, we generated a panel of individual hybridomas from mice immunized to human acute lymphocytic leukemia (ALL) cells. These antibodies were screened for their ability to discriminate between the leukemia cells and an isologous B lymphobastoid cell line (LCL). Among the discriminator antibodies, two define an antigen that is preferentially expressed on ALL cells and on a subpopulation of normal
MATERIALS AND METHODS Human Cells. The leukemia cells used for immunization and for screening of antibodies were derived from the peripheral blood of a child (Dom) with T-cell ALL. The patient had a mediastinal mass and a blood lymphoblast cell count of 460,000 per mm3. Greater than 95% of these blast cells formed heatstable sheep erythrocyte rosettes (7). Leukemia cells from patient Dom and a series of other patients were purified from peripheral blood or bone marrow or Ficoll-Hypaque sedimentation (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 polyclonal mixture of K and X immunoglobulin-bearing cells (9) and it contains the Epstein-Barr nuclear antigen (10) (Henry Kaplan, personal communication). The LCL-Dom cells were grown and maintained in Dulbecco's modified Eagle's medium (high glucose) (GIBCO), containing penicillin (100 units/ml), glutamine (2 mM), and 15% fetal calf serum. Other cell lines used in these studies include the T cell lines MOLT-4, 8402, and HSB2 and the B cell lines 8866, 8392, and SB. Their origins and characteristics have been described (11-13). Normal human thymus glands, lymph nodes, and spleens were obtained from fresh surgical specimens. Immunization Schemes. BALB/c mice were immunized and 1 week later were given an intraperitoneal booster with 107 glutaraldehyde-fixed (4) ALL-Dom cells. Three months later, they were given an intravenous booster with another 107 ALL-Dom cells. Three days later, 2 X 107 spleen cells from these animals were transferred intravenously to syngeneic irradiated (600 rads) mice along with another 107 ALL-Dom cells. The recipient mice had been previously hyperimmunized either with normal peripheral blood lymphocytes (PBL) (group I), LCL-Dom (group II), or nothing (group III, control). Hybridomas were prepared from the spleen cells of each recipient group 1 week after transfer. Cell Fusion and Culture. Spleen cells were fused with NSI/1-AG 4 cells (14) at a cell ratio of 2:1, respectively, by using 38% polyethylene glycol (Baker 1540) (5). After fusion, the cells were resuspended in Kennet's modification of Dulbecco's medium containing hypoxanthine, aminopterin, and thymidine (15) and dispensed into 96-well tissue culture trays (Linbro) at 1-2 X 105 cells per well along with 5 X 105 normal spleen cells. The medium was changed twice during the first 2 weeks to remove antibody released by unfused spleen cells and then was collected for testing during the third week. Culture fluids were sampled, with attention being paid to avoid contamination of each fluid by its neighbors-i.e., by washing or changing of
thymocytes. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviations: ALL, acute lymphocytic leukemia; LCL, lymphoblastoid cell line; PBL, peripheral blood lymphocytes. 6552
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Proc. Natl. Acad. Sci. USA 76 (1979)
pipettes between collections. These fluids were examined for the presence of antibody against the immunizing ALL-Doxn
cells as well as the LCL-Dom munoassay (5), using purified
cells by a cell-binding radcoim-
125I-labeled goat anti-mouse Fab
fragment as a detecting reagent. Selected cultures were subcloned by'plating limiting dilutions of the hybrid cells into the wells of 96-well plates along with normal mouse spleen cells. Immunofluorescence. Hybridoma antibodies were examined by indirect immunofluorescence on viable cells in suspension, fixed cell smears, or frozen tissue sections prepared by described methods (9). A rhodamine conjugate of the same purified goat anti-mouse Fab used in the radioimmunoassay was employed. In some cases, an F(ab')2 fragment of this purified goat antibody was employed. Labeling of Cell Membranes and Immunoprecipitation. Cell membranes were radio-iodinated by using lactoperoxidase and solubilized with Nonidet P-40 (Shell Chemical) as described (16). The solubilized 125I-labeled membrane preparation, 107 cell equivalents, was first subjected to a nonspecific immunoprecipitation by the addition of human IgG (25 Mg) and goat anti-human Fab at equivalence. To the cleared supernatant, 100-200 ,l of culture fluid containing either a specific monoclonal antibody or a myeloma protein of the same class was added. This was followed by the addition of normal mouse serum (2.5 pi) and goat anti-mouse Fab (preabsorbed on human IgG-Sepharose) at equivalence. The final precipitates were solubilized in 3% sodium dodecyl sulfate/2% mercaptoethanol/0.05 M Tris-HCI, pH 6.8/5% glycerol and boiled. Gel Electrophoresis. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (17) was performed in slab gels by using either a 5-20% gradient of acrylamide or a uniform 10% acrylamide concentration. Gels wer'e dried and autoradiographed by using Kodak XR2 film with a Cronex Lightning Plus intensifying screen (Dupont). Fluorescence-Activated Cell Sorter Analysis. Lymphocytes were incubated with monoclonal antibody (50 Ml per 5 X 106 cells) at 40C in the presence of 1 mM azide. The cells were washed in fetal calf serum and gently resuspended in 50 Ml of fluorescent goat anti-mouse Ig antibody. The stained cells were washed again in fetal calf serum, resuspended in Dulbecco's phosphate-buffered saline, and fixed in 1% formaldehyde. Analysis of staining intensity was performed by using the fluorescence-activated cell sorter as described by Loken and Herzenberg (18). RESULTS Initial Screening. A total of 1877 culture wells, of which approximately 60% contained growing hybrids at the time of fluid collection, was screened for antibody activity by radioimmunoassay. Two different target cells were used for this initial screen ALL-Dom, the immunizing leukemia cells, and LCL-Dom, an isologous B-cell LCL. A representative portion of the data from this screen is shown in Fig. 1. Antibody reactive with the immunizing ALL cells was found in 619 (33%) of the culture wells. Of these, 148 appeared to contain an antibody that distinguished between the immunizing leukemia cell and its isologous B cell line (shown as arrows in Fig. 1). With repeated testing on these and subsequently collected fluids, 60 cultures were confirmed to be continuously producing antibody with preferential reactivity for the leukemia cell. This frequency of 60/619 may be an underestimate of the true frequency of discriminators, because many of the cultures contained more than one clone. Three different spleen cell donors were used for cell fusion
in these experiments. All were radiated recipients of the same adoptively transferred immune spleen cells. However, in two
1 1 S -11S"- WNgS1E 11S
FIG. 1. Radioimmunoassay of hybridoma culture fluids. Fluids from individual culture wells were assayed for binding activity against two different target cells from patient Dom: ALL (Upper) and LCL (Lower). The data from one representative 96-well plate are displayed. groups the recipients had been hyperimmunized against normal human lymphoid antigens-either PBL (group I) or LCL-Dom (group II). This maneuver was an attempt to suppress the expansion of B cell clones reactive to normal human antigens while stimulating the expansion of clones reactive to antigens limited to the leukemia cell. A third group of recipients was not preimmunized. Similar numbers of total antibody-producing hybridomas were obtained in each group. However, a moderate advantage was noted for groups I and II in terms of the percentage of hybrids producing antibody that discriminated between the leukemia cell and the isologous B cell line-13.8 and 9.7%, respectively, vs. 5.2% in the control group. Definition of a Thymus-Leukemia Antigen. On further testing of the 60 discriminators, it became apparent that, although they failed to react with LCL-Dom, most of these antibodies were strongly reactive against human normal PBL and presumably recognize normal T cell antigens. However, two hybridomas designated 12E7 and 21D2 gave a reactivity pattern that was distinctly different, distinguishing between the leukemia 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 antibodies 12E7 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 21D2 were both derived from group II, the animals that were hyperimmunized to LCL-Dom prior to receipt of ALL-Dom immune cells. Hybridoma 12E7 that produces an IgG-1 antibody was selected for further study. Indirect immunofluorescence was performed by using antibody 12E7 on viable cell suspensions and fixed cell smears. All of the ALL-Dom cells were brightly stained on the membrane and in the cytoplasm, whereas PBL showed only faint membrane staining. Similar stained preparations were analyzed
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Proc. Natl. Acad. Sci. USA 76 (1979)
FIG. 4. Indirect immunofluorescence of antibody 12E7 on a bone marrow smear from a patient with T-ALL. An F(ab')2 fragment of a purified goat anti-mouse Fab antibody was used as a rhodamine conjugate for the second-stage reagent.
1:32 1:64 Fluid dilution
FIG. 2. Binding of antibody 12E7 to T-ALL cells (@), LCL cells (0), and PBL (A). Background of 200 cpm was subtracted from the data.
by using the fluorescence-activated cell sorter. Again, 100% of the ALL cells were very brightly stained and were easily distinguishable from the PBL of normal donors (Fig. 3 Upper). The myeloma protein produced by P3/X63 cells was used as a negative control (Fig. 3 Lower). Fluorescent staining by antibody 12E7 of a fixed bone marrow smear from a patient with T-cell leukemia is shown in Fig. 4. The leukemia cells were 100 ALL / PBL
brightly stained and were easily detectable within a background of unstained normal or weakly stained normal bone marrow cells. Various normal lymphoid tissues were examined by indirect immunofluorescence on frozen sections with antibodies 12E7 and 21D2. These antibodies gave weak staining of cells in spleen and lymph node. However, both these antibodies gave intense staining of cells in the cortex of the thymus gland (Fig. 5). By contrast, lymphoid cells in the medulla of the thymus failed to stain with antibody 21D2 or 12E7. The binding of antibody 12E7 to thymocytes in suspension is shown in Fig. 6 and compared with its binding to several types of human leukemia cells. These data show that the average density of the antigen on thymocytes is comparable to that on T-ALL cells and greater than that on the T-ALL-derived cell line, 8402. It also shows that the cells from one case of chronic lymphocytic leukemia and those from one case of "null cell" ALL contain insignificant amounts of the antigen. The reactivity of antibody 12E7, either by radioimmunoassay or by immunofluorescence, on a series of human normal and leukemia cells is summarized in Table 1. Strong reactivity of
10 20 Fluorescence intensity
FIG. 3. Fluorescence intensity profiles of ALL cells or normal PBL with monoclonal antibodies. Cells were obtained from the blood by Ficoll-Hypaque sedimentation, mixed with monoclonal antibodies, counter-stained with fluorescent rabbit anti-mouse Ig antibody, and analyzed on the fluorescence-activated cell sorter. (Upper) 12E7 antibody. (Lower) Control antibody.
FIG. 5. Indirect immunofluorescence on frozen section of normal human thymus. An F(ab')2 fluorochrome was used as in Fig. 4. Two views 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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Proc. Natl. Acad. Sci. USA 76 (1979)
1:32 1:64 Fluid 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), and a chronic lymphocytic leukemia (v).
this antibody appears to be limited to cortical thymocytes, acute lymphoid leukemias of the T-cell type, and most cases of acute lymphoid leukemias of "null cell type." Preliminary Characterization of the Thymus-Leukemia Antigen. Cell membranes were labeled with 12'I by using lactoperoxidase, were solubilized with detergent, and were immunoprecipitated with antibodies 21D2 and 12E7. Fig. 7 shows the results of gel electrophoresis of material immunoprecipitated by 12E7 from PBL, 8402 (a T-ALL derived cell line), and Dom (the original T-ALL cells used for immunization). In each case, a comparison is made with a control immunoprecipitate in which P3/X63 fluid was used instead of 12E7. Ig is included on the gel to provide molecular weight markers (L and H). Table 1. Reactivity of antibody 12E7 on normal and leukemia cells Levels of TL antigen Lymphoid cells ALL-Dom (the immunizing cell) High LCL-Dom (isologous B lymphoblastoid cell) Low Other B cell lines (SB, 8392, 8866) Low PBL Low Low Lymph node cells Spleen cells Low Bone marrow cells Low Thymocytes Cortex High Medulla Low T-All cell lines (MOLT-4, 8402, HSB2) High T cell acute leukemias and lymphoblastic lymphomas High B cell leukemias (chronic lymphocytic leukemias) Low Myeloid leukemias (acute and chronic) None "Null cell" leukemias (three out of four cases) High TL, thymus-leukemia.
IgG 12E7 P3
12E7 P3 12E7 P3
Dom 8402 FIG. 7. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of membrane molecules immunoprecipitated with antibody 12E7. 125I-Labeled cell membranes, 5 X 106 trichloroacetic acidprecipitable cpm, were immunoprecipitated either with the monoclonal 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. PBL
A specific band of -Mr 28,000 is apparent in the material immunoprecipitated from the leukemia cells. Identical results were obtained when antibody 21D2 was used in place of 12E7 (not shown). No specific bands can be seen in the material derived from PBL, nor could any be seen in material derived from chronic lymphocytic leukemia cells, LCL-Dom, or other B lymphoblastoid cell lines (not shown on this gel).
DISCUSSION In the present study, we sought to analyze possible antigenic differences between human acute leukemia cells and normal human cells. We adopted a screening strategy in which a matched pair of genetically identical cells-one leukemic and one lymphoblastoid-was used in a radioimmunoassay to search for discriminator antibodies. This approach is similar to the strategy we previously employed to screen monoclonal antibodies for recognition of human HLA polymorphic antigens (2). It requires that the antibody-producing cells be cloned prior to testing, because the presence of nondiscriminating antibodies obscure the discriminators. Furthermore, to be most effective, the pair of target cells should be as closely related as possible but different in the desired characteristic. Our initial screen eliminated most of the antibodies from consideration and allowed us to focus on a manageable number of potentially in-
Immunology: Levy et al.
teresting candidates. After a second screen with normal peripheral 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 transfer of specific B cells with antigen into irradiated recipients could greatly enhance B-cell expansion, but that the expansion could be specifically suppressed by prior immunization of the recipient (19). We attempted to stimulate the ALL-reactive B cells while suppressing the B cells reactive to normal human antigens. The maneuver resulted in a slight increase in the frequency of discriminator hybridomas. Antibodies 12E7 and 21D2 detect an antigen present on ALL cells and on a population of normal cells found in the cortex of the thymus. This tissue distribution is reminiscent of the thymus-leukemia antigen of the mouse (20). The mouse thymus-leukemia antigen is a 45,000 Mr protein that is associated on the cell surface with 32 microglobulin (21-23). By contrast, the human thymus-leukemia antigen described here appears to have a Mr of nt28,000 and it is not associated with f32 microglobulin. McMichael et al. (24) have recently described a monoclonal antibody raised against human thymocytes that reacts with the T-ALL-derived cell line MOLT-4. Their antibody immunoprecipitated a two-chain structure of Mr 45,000 and 11,000 from thymocyte cell surfaces but the 11,000 Mr polypeptide was different from 32 microglobulin; no 28,000 Mr molecule was seen (24, 25). Chechik et al. (26) have described a 43,000 Mr protein extractable from human thymocytes, T-cell lines, and T-ALL cells that copurified with a second chain of Mr 23,000. However, they found this molecule to be only intracellular. The relationship between these different human thymocyte antigens can be clarified only by a side-by-side comparison. Monoclonal antibodies, such as 12E7 and 21D2, will be useful in a number of different areas of further investigation. Because they react with cells from many, but not all, cases of ALL, they will help to categorize different subtypes of leukemia and may shed light on the tissue origin of these malignant cells. Currently, the human ALLs are subdivided into T cell, pre-B cell, and null cell types according to their expression of certain markers of normal lymphocyte differentiation (27-30). It is already clear that the antigen defined here is not limited to just the leukemias that carry other T-cell differentiation markers, because it is found on some "null" cell leukemias as well (Table 1). By using antibodies 12E7 and 21D2, leukemia cells can be detected in peripheral lymphoid tissues. These monoclonal antibodies may, therefore, provide a sensitive means of monitoring the patients with leukemia during therapy and for detecting early disease relapse by surveillance of bone marrow and blood for antigen-positive cells (31). Moreover, it should be possible to develop a radioimmunoassay for the detection of free antigen, which may be even more sensitive than the detection 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.
Proc. Natl. Acad. Sci. USA 76 (1979) Marilyn Pederson for tissue section immunofluorescence and photography and Dr. Irving Weissman for helpful discussions. This work was supported by grants from the National Institutes of Health (CA 21223-02 and AI-09072). R.L. is an investigator of the Howard Hughes Medical Institute. R.I.F. is a Fellow of the Arthritis Foundation. 1. Klinman, N. R. (1969) Immunochemistry 6,757-759. 2. Lampson, L. A., Levy, R., Grumet, F. C., Ness, D. & Pious, D. (1978) Nature (London) 271, 461-462. 3. Lampson, L. A., Royston, I. & Levy, R. (1977) J. Supramol. Struct. 6, 441-448. 4. Levy, R. & Dilley, J. (1977) J. Immunol. 119,394-400. 5. Levy, R., Dilley, J. & Lampson, L. A. (1978) in Current Topics in Microbiology and Immunology, eds. Melchers, F., Potter, M. & Warner, N. L. (Springer, Berlin), Vol. 81, pp. 164-169. 6. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495497. 7. Sen, L. & Borella, L. (1975) N. Engl. J. Med. 292,828-832. 8. Boyum, A. (1978) Scand. J. Clin. Lab. Invest. Suppl. 21 97, 91-106. 9. Levy, R., Warnke, R., Dorfman, R. F. & Haimovich, J. (1977) J. Exp. Med. 145, 1014-1028. 10. Reedman, B. & Klein, G. (1973) Int. J. Cancer 11, 499-520. 11. Royston, I., Smith, R. W., Buell, D. N., Huang, E. & Pagano, J. S. (1974) Nature -(London) 251, 745-746. 12. Huang, C. C., Hou, Y., Woods, L. K., Moore, G. E. & Minowanda, J. (1974) J. Natl. Cancer Inst. 53, 655-658. 13. Pious, D., Hawley, P. & Forrest, G. (1973) Proc. Natl. Acad. Sci. USA 70, 1397-1400. 14. Kohler, G., Howe, S. C. & Milstein, C. (1977) Eur. J. Immunol.
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