Human Trophoblast Cell-Surface Antigens Defined by Monoclonal Antibodies (Choriocarcinoma/Fetal Cells/Fluorescence-Activated Cell-Sorter) MARC LIPINSKI*, DAVID R

Proc. Nati Acad. Sci. USA Vol. 78, No. 8, pp. 5147-5150, August 1981 Medical Sciences

Human trophoblast cell-surface antigens defined by monoclonal antibodies (choriocarcinoma/fetal cells/fluorescence-activated cell-sorter) MARC LIPINSKI*, DAVID R. PARKS, ROBERT V. ROUSE, AND LEONARD A. HERZENBERG Departments of Genetics and Pathology, Stanford University School of Medicine, Stanford, California 94305 Communicated by L. L. Cavalli-Sforza, April 17, 1981

ABSTRACT A series ofmonoclonal antibodies has been raised ford University and from Janice Chou ofthe National Institutes against the human choriocarcinoma cell-line, BeWo. Four anti- ofHealth. They were grown in monolayers in 50% Waymouth's gens, Trop-1, -2, -3, and -4, are defined on normal and malignant medium (Irvine Scientific, Santa Ana, CA)/40% balanced salt trophoblast cells. Trop-1 and Trop-2 appear to be specifically ex- solution/10% newborn calf serum (Irvine Scientific) supple- pressed on syncytio- and cytotrophoblasts, whereas Trop-3 and mented with NaHCO3 to 7.5% final concentration and gluta- Trop4 are also detected on various tumor cell lines, normal lym- mine. The HT1080C and HeLaS3 cell lines were obtained from phocytes, and monocytes. Anti-Trop-I and anti-Trop-2 antibodies Douglas Wallace, Stanford University and grown as monolayers might prove useful for detection and isolation of fetal trophoblast in glutamine-supplemented RPMI 1640 (Irvine Scientific)/ cells circulating in pregnant women's blood and for diagnosis and 15% newborn calf serum. The adherent cells were suspended therapy in patients having choriocarcinomas and other germ-cell by using 0.25% trypsin/EDTA (GIBCO). All the other cell lines neoplasms. (including the hybridomas) were grown in suspension in RPMI The trophoblast is a fetal-derived tissue located at the interface 1640/15% newborn calf serum. between the fetus and the maternal circulation. It develops from Production of Monoclonal Antibodies. Two BALB/cN fe- the inner layer of young proliferative cells, called cytotro- male mice were immunized and boosted 3 weeks later with 106 phoblasts, from which originates the outer layer ofsyncytiotropho- cells of the BeWo choriocarcinoma cell line injected intraper- blasts that are in direct contact with the maternal circulation. itoneally. Three days after the boost, spleen cells were har- Trophoblast cells sometimes give rise to tumors called vested and fused to NS-1 myeloma cells as described (7) at a 4:1 choriocarcinomas. ratio in the presence of 50% polyethylene glycol 1500 (BDH The passage offetal nucleated cells into the mother has been Chemicals, Toole, England). After the fusion, 4 X .105 cells per documented, such cells having been detected in maternal blood well were incubated in 0.2 ml of hypoxanthine/aminopterin/ samples either directly (1) or after enrichment with a fluores- thymidine. cence-activated cell sorter (FACS) (2, 3). The histological type Initial Screening of Hybrid Production and Cloning. On the ofthese cells, however, has not been determined, and it is not 11th day after the fusion, all 150 cultures showed growth. Cul- known whether they form a homogenous population. The prox- ture supernates were harvested and screened for antibodies to imity of trophoblast cells to mothers' blood makes them good the immunizing BeWo cell line by using a radioimmunoassay; candidates for at least some of the fetal cells found in maternal 2 x 104 BeWo cells were plated in wells of microtiter plates circulation and, indeed, syncytiotrophoblast cells have been (Costar, Division of Data Packaging, Cambridge, MA), and al- observed in maternal lungs (4). Much smaller cytotrophoblast lowed to adhere to the plastic overnight, and then washed with cells might not be trapped in the lung capillary network and radioimmunoassay buffer. The cells were incubated for 1 hr would continue to circulate. Specific antibodies to trophoblasts with 20 ,ul of supernate and then washed three times. Anti- could be used to identify FACS-sorted cells or as fluorescent BeWo antibodies were detected by adding, for 1 hr, 30,000 cpm reagents to label them so they can be separated with the FACS. of an "2I-labeled goat anti-mouse immunoglobulin antiserum Xenogeneic antisera have been raised to human trophoblast absorbed on a human immunoglobulin immunosorbent. Incu- cells (5), but using these for isolating rare trophoblasts from bations were at room temperature. After three more washes, maternal blood cells is greatly hampered by their evident com- the cells were lysed in the presence of 1% Nonidet D. The ly- plexity, which requires that they be extensively absorbed. sates were harvested with a cotton tip swab and assayed in a The emergence of hybridoma technology, which overcomes gamma scintillation counter (Micromedic Systems, Division of the technical limitations of conventional serology (6), has per- Rohm and Haas, Philadelphia, PA). Individual viable cells from mitted us to raise monoclonal antibodies to human trophoblast positive cultures of interest were directly deposited into mi- cells. This communication deals with the generation and char- croculture wells with the FACS. In a previous report (8), the acterization ofa series ofsuch monoclonal antibodies to various FACS was used to sort individual hybrids on the basis of their antigens expressed on normal and malignant trophoblast cells. binding of fluorescent microspheres coupled with antigen. In Four new antigens, named Trop-1, Trop-2, Trop-3, and Trop- the present case, however, the cells were selected on the basis 4, are defined with these antibodies. of viability only. Growing clones were tested 1 week later for production of the desired monoclonal antibody. Monoclonal MATERIALS AND METHODS antibody isotypes were determined by reaction with mouse allotype-specific I-labeled monoclonal antibodies (9). Clones 162- Cell Culture. The BeWo, JEG, Reid, and JAR choriocarci- 21.2, 162-25.3, and 162-28.2 are IgG2a (Igh-la allotype), and noma cell lines were obtained from Howard Sussman of Stan- clones 16243.4 and 162-46.2 are IgGl (Igh4a allotype) (10).

The publication costs ofthis article were defrayed in part by page charge Abbreviation: FACS, fluorescence-activated cell sorter. payment. This article must therefore be hereby marked "advertise- * Present address: Laboratoire d'Immunologie Clinique, Institut Gus- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. tave-Roussy, 94800 Villejuif, France. 5147 Downloaded by guest on September 27, 2021 5148 Medical Sciences: Lipinski et al. Proc. Natl. Acad. Sci. USA 78 (1981) Table 1. Distribution of Trop antigens on normal and tumor cells Trop-2 Trop-4 162-25.3 162-28.2 Trop-1 and Trop-3 and Target cell 162-21.2 162-46.2 162-10.2 162-43.4 Choriocarcinoma BeWo + + + + > JEG + + + + Reid + + ND ND JAR + - ND ND .:t \ Fibrosarcoma T (HT1080C) + - + + Cervix carcinoma (HeLa S3) - - + + Colon carcinoma 1 2 3 4 HT18 - - ND ND HT29 - - ND ND Fluorescence intensity Melanoma (Shroet) - - ND ND FIG;, 1. Immunofluorescence staining-of peripheral blood lympho- Neuroblastoma cytes. Target cells were incubated for, 30 min with 50 ,ul of medium (----), hybridoma supernate. containing anti-Trop-3 162-10.2 (...), or (TG8) - - ND ND anti-Trop-4 162-28.2 (-) antibodies. The reaction was revealed by Erythroleukemia a fluoresceinated, goat anti-mouse immunoglobulin antiserum and (K562) + - + + analyzed on a FACS. Fluorescence units are arbitrary and given on a Lymphoid logarithmic.(base 10) scale. Ramos (B lymphoma) - - + + Molt-4 (T) - - + + staining. Enzy~mes used were Pronase and neuraminidase (Cal- Normal blood and - - + biochem/Behring), trypsin a-chymotrypsin (Worthing- Lymphocytes +* and a mixture of 17 Monocytes - - + + ton), glycosidases (from Turbo cornutus, Platelets - - + Miles). FACS Cells were as described with Erythrocytes - - Analysis. analyzed (11) a modified FACS-II (Becton Dickinson FACS Systems, Sun- Thereactivity of anti-Trop monoclonal antibodies was tested against nyvale, CA) fitted with a logarithmic amplifier (12). various target cells by indirect immunofluorescence and FACS anal- Tissue Section Immunoperoxidase Staining. Portions ofpla- ysis (or by radioimmuno binding assay). Target cells were incubated centas were obtained from abortions carried out for rea- with 50 p.l of antibody containing hybridoma supernate, and the re early action was revealed by incubation with a fluoresceinated (or 125I-labeled) anti-mouse immunoglobulin. Intensity of fluorescence (or ra- Table 2. Blocking studies with anti-Trop monoclonal antibodies dioactivity) bound per target cell was compared with background flu- Mean orescence (or radioactivity) obtained from target cells incubated with anti-mouse immunoglobulin only. Results were scored as positive Blocking Staining fluorescence (+) or negative (-). ND, not done. antibody antibody intensity Blocking * Two subsets with different antigen densities. Experiment 1 - - 1.7* _ - 1.7t Indirect.Immunofluorescent Staining. Target cells (2 .x 105) - 162-46.2 2.1* obtained from cultured cell lines or mononuclear cells from 162-25.3 162-46.2 1.8* Yes Ficoll-Paque (Pharmacia) preparations of human peripheral 162-21.2 162-46.2 2.1* No blood. samples were incubated for 45 min on ice in 100 ,ul of - 162-25.3 2.7t RPMI 1640 containing 50 ,u1 ofhybridoma supernate. After two 162-46.2 162-25.3 2.Ot Yes washes, the cells were incubated in the presence ofa saturating Experiment 2 - amount of a fluorescein-coupled goat anti-mouse immunoglob- - 162-21.2 33t ulin antiserum absorbed on human immunoglobulins. The cells 162-46.2 162-21.2 3.2t No were washed twice, resuspended, and immediately analyzed - - 1.5* on a FACS. - - 1.3t Blockdng Studies. For investigating the proximity or identity - 162-28.2 3.4t of two- antigenic determinants recognized by two monoclonal 162-43.4 162-28.2 2.2t Yes antibodies of distinct isotypes, the target cells were incubated - 162-43.4 2.8* first with 50 ,ul ofone monoclonal antibody for 30 min and then 162-28.2 162-43.4 1.4* Yes with 50 A.1 of the second monoclonal antibody for another 30 Target cells were incubated for 30 min with 50 ,ul of hybridoma su- min. After washing, the reaction of the latter with the target pernatant containing blocking antibody. Supernate (50 Dl) containing antigen was detected with a fluorescent goat anti-mouse IgG2a the staining antibody was then added to the incubation medium. After or IgGl specific for the second monoclonal antibody used. 30 min more, the reaction was revealed with a fluoresceinated goat Enzymic Treatments. For studies ofthe enzyme sensitivity anti-mouse IgG1 or IgG2a. The cells were analyzed on the FACS. Mean the the monoclonal fluorescence intensities are given on a logarithmic (base 10) scale. of antigenic determinants recognized by Antibodies are of the IgGl (162-46.2 and 162-43.4) or IgG2a (162-25.3, antibodies, 2 x 106 target cells were incubated in RPMI 1640 162-21.2, and 162-28.2) isotype. at 37°C in the presence of various enzymes (see Results) and * Stained with fluoresceinated anti-yl. then washed and processed for indirect immunofluorescent t Stained with fluoresceinated anti-y2a. Downloaded by guest on September 27, 2021 Medical Sciences: Lipinski iet aL Proc. NatL Acad. Sci. USA 78 (1981) 5149

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I 0 r I SF C FIG. 2. Placenta section immunoperoxidase staining. Formalin-fixed paraffin-embedded sections of a 10-week placenta were stained with anti- Trop-1 (a), anti-Trop-2 (b) and 11-4.1 (control) (c) antibodies and then incubated with a peroxidase-coupled anti-mouse immunoglobulin. Both cytotrophoblast (CT) and syncytiotrophoblast (ST) stain positively with anti-Trop-1 and anti-Trop-2 antibodies.

sons independent of this study. Normal liver, kidney, spleen, Sections ofplacenta were stained with all six monoclonal an- thymus, and lymph nodes were obtained from portions of sur- tibodies and with 11-4.1, a mouse immunoglobulin (of 2a sub- gical specimens not required for pathologic examination. Tissue class) that does not react with human tissues as a control. All was frozen, cut, and stained as described (13) except that the four Trop antigens were detectable on both frozen and fixed last stage applied was peroxidase-coupled rabbit anti-mouse im- paraffin-embedded sections. Fig. 2 illustrates staining with munoglobulin (Dako, distributed by Accurate Chemical and anti-Trop-1 and anti-Trop-2 antibodies. Study offrozen sections Scientific, Hicksville, NY), which was followed by diamino- ofnormal adult liver, kidney, spleen, thymus, and lymph nodes benzidine (Sigma) at 1 mg/ml in phosphate-buffered saline with with anti-Trop-1 and anti-Trop-2 antibodies did notproduce any 10 A1 of30% H202 per ml. After a 2- min incubation, the slides detectable staining. were washed, incubated with 0.5% CuS04 in isotonic saline for We were unable to immunoprecipitate any I-labeled protein 5 min, washed, dehydrated, and mounted in Permabond. For- from lactoperoxidase-labeled choriocarcinoma cells or cell malin-fixed paraffin-embedded placenta was stained in the membranes. However, the Trop-3 antigen could be precipi- same way. Undiluted hybridoma culture supernates were used tated with the 162-10.2 antibody from I-labeled peripheral for this staining. blood lymphocytes. NaDodSOJpolyacrylamide gel electro- phoresis analysis indicates that Trop-3 consists ofa single chain RESULTS protein of Mr 200,000 (data not shown). An indication that We examined the cellular reactivity of six distinct anti-BeWo the other three Trop antigens are also associated with proteins monoclonal antibodies by indirect immunofluorescence stain- was obtained by protease and glycosidase treatment of cells ing and FACS analysis using a series ofhuman tumor and lym- bearing these antigens. Results shown in Table 3 indicate that phoid cell lines and normal peripheral blood mononuclear cells the staining for Trop-1 and Trop-3 antigens was virtually ab- (Table 1). Three antibodies, 162-10.2, 162-28.2, and 162-43.4, rogated, while the staining for Trop-2 and Trop-4 was greatly stained all cell lines tested, as well as blood lymphocytes and decreased, after incubating 2 x 106 cells for 2 hr with 500 ,ug monocytes. However, these antibodies detect two different an- of Pronase. Treatment ofthe cells with 500 ,ug oftrypsin drast- tigens; distinct peripheral blood lymphocyte FACS staining ically decreased the staining for Trop-1, Trop-2, and Trop-3, profiles were obtained with 162-10.2 on the one hand and with whereas Trop-4 was hardly affected. The staining for all four 162-28.2 and 162-43.4 on the other hand (Fig. 1). The FACS antigens was partially decreased after treatment with 500 ,ug of staining profiles on all cells tested with 162-28.2 and 162-43.4 a-chymotrypsin. No decrease in staining was obtained by pre- were similar. As each antibody also blocked the staining of the treating the cells with neuraminidase or a mixture of 17 gly- target with the other (Table 2), both antibodies probably react cosidases (data not shown). with the same or proximal determinants of the same antigenic molecule. This antigen, called Trop4, does not distinguish lymphocyte subpopulations. In contrast, Trop-3, detected by 162- Table 3. Percentage decrease of staining after treatment of 10.2, is present on about half of the lymphocytes with a con- target cells with proteases siderably higher density than on the other half (Fig. 1). Treatment The other three monoclonal antibodies appear to be troph- Antigen a-Chymotrypsin Trypsin Pronase oblast-specific (Table 1). 162-21.2 reacts with an antigen des- Trop-1 38 81 99 ignated Trop-1. 162-25.3 and 162-46.2 both react with a second Trop-2 42 86 61 antigen referred to as Trop-2. They crossblock each other but Trop-3 41 91 100 do not block the staining of Trop-1 with 162-21.2 (Table 2). Trop-4 48 7 90 Trop-1 is present on all four choriocarcinoma cell lines and also, at a much lower density, on the fibrosarcoma and the K562 cell Target cells were incubated for 2 hr with 500 jg of enzyme and, the lines. It is absent from normal blood cells and various other tu- reaction was stopped by washing the cells with serum-containing medium. Cells were then divided into aliquots and stained with anti-Trop mor cell lines (Table 1). The expression of Trop-2 is restricted antibodies, and the reaction was revealed by incubation with a flu- to three ofthe four choriocarcinoma cell lines. (Table 1). It could oresceinated goat anti-mouse immunoglobulin antiserum and sub- not be detected on any ofthe other normal or tumorcells tested. jected to FACS analysis. Downloaded by guest on September 27, 2021 5150 Medical Sciences: Lipinski et al. Proc. Nad Acad. Sci. USA 78,(1981.) DISCUSSION We foresee several possible clinical applications for tropho- We have reported the production of monoclonal antibodies blast-specific reagents such as the anti-Trop-1 and anti-Trop-2 raised against.the human neoplastic trophoblast cell line BeWo. monoclonal antibodies described here. They could be used to Four newly recognized.antigens, called Trop-l, Trop-2, Trop- investigate the passage of fetal trophoblast cells into maternal 3, and Trop4, have been defined with six of these antibodies circulation during pregnancy and, ifpresent, to sort them with on the cell surfaces of normal and malignant trophoblast cells. a FACS for possible prenatal diagnostic use. They could also be Two main cell distribution patterns have been observed by test- coupled to radionuclide probes to detect and then deliver local ing the reactivity of the monoclonal antibodies against a panel irradiation to primary tumors and metastases in patients with. of human cell lines and peripheral blood cells. The expression choriocarcinomas and'other germ-cell neoplasms.. They might of the Trop-1 and Trop-2 antigens appeared to be virtually re- also be able to immunologically target cytotoxic agents coupled stricted to cells of trophoblast origin as they reacted only with to the antibodies or antibody fragments as has been described normal trophoblast cells and choriocarcinomas, with the excep- (14, 15). Finally, studies performed in the rat have indicated that tions of the fibrosarcoma cell line HT1080C and the erythro- xenogeneic antitrophoblast antisera, when injected into mated leukemia cell line K562, which expressed a low density ofTrop- female rats after being absorbed with lymphoid cells, showed 1. Conversely, monoclonal antibodies to Trop-3 and Trop4 an- an abortifacient activity (16). Thus, the anti-Trop-1 and anti- tigens displayed a wide range of reactivity and were positive Trop-2 monoclonal antibodies described here could have useful with various solid tumor and lymphoid cell lines and with nor- investigative, diagnostic, and therapeutic applications. mal nucleated peripheral blood cells. Interestingly, FACS anal- We thank Marc Fellous and Philip Avner for their participation in ysis ofimmunofluorescent staining showed that, whereas Trop- the testing ofthe distribution ofthe Trop-1 and Trop-2 antigens, Jeffrey 4 was homogenously distributed, Trop-3 on the contrary was Ledbetter for his help in the immunoprecipitation of the Trop-3 anti- not evenly expressed. on peripheral blood lymphocytes and gen, Jennifer Royce for her excellent technical assistance, and Jean monocytes; about one-half of the total lymphocyte and mono- Anderson for preparation ofthe manuscript. This work was supported, in part, by National Institutes ofHealth Grants HD-13025, GM-28428, cyte populations expressed a higher antigen density (as much and GM-17367. D.R.P. is a recipient of a Senior Fellowship from the as a 4-fold effect in some individuals) than the other half. American Cancer Society, California Division. In previously reported studies with xenogeneic antitropho 1. Schroder, J. (1975) J. Med. Genet. 12, 230-242. blast antisera, trophoblast surface antigens were classified into 2. Herzenberg, L. A., Bianchi, D. W., Schroder, J., Cann, H. M. two major groups (5). The first group (TA1) included antigens & Iverson, G. M. (1979) Proc. NatL Acad. Sci. USA 76, 1453-1455. present on trophoblast and some tumor cell lines; the second 3. Iverson, G. M., Bianchi, D. W., Cann, H. M. & Herzenberg, L. group (TA2) included antigens widely expressed on normal and A. (1981) J. Prenatal Diag. 1, 61-73. tumor tissues. It is difficult to compare monoclonal antibodies 4. Park, W. W. (1958) J. Pathol Bacteriol. 75, 257-265. with conventional antisera because of the complexity ofthe lat- 5. Faulk, W. P., Temple, A., Loving, R. E. & Smith, N. (1978) Proc. Nati Acad. Sci. USA 75, 1947-1951. ter. It appears, however, that the anti-Trop-1 and anti-Trop-2 6. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495-497. monoclonal antibodies, on one hand, and the anti-Trop-3 and 7. Oi, V. T. & Herzenberg, L. A. (1980) in Selected Methods in Cel- anti-Trop4 monoclonal antibodies, on the other hand, define lular Immunology, eds. Mishell, B. B. & Shiigi, S. M. (Freeman, antigens related to the TAl and TA2 groups, respectively. San Francisco), pp. 351 372. Enzymatic studies indicate that all four antigens are associ- 8. Parks, D. R., Bryan, V. M., Oi, V. T. & Herzenberg, L..A. (1979) ated with polypeptides. The staining for all four was greatly Proc. Nati Acad. Sci. USA 76, 1962-1966. 9. Oi, V. T. & Herzenberg, L. A. (1979) Mol Immunol 16, decreased after a 2-hr incubation of the target cells with large 1005-1017. amounts (500 ,Ag) of proteases. By titrating the Pronase sensi- 10. Herzenberg, L. A. & Herzenberg, L. A. (1978) in Handbook of tivity of the four antigens, we found that Trop-3 was the most Experimental Immunology, ed. Weir, D. M. (Blackwell, Oxford, Pronase-sensitive antigen; its staining was abolished by treating England), 3rd Ed., pp. 12.1-12.23. the target cells for, 60 min at 370C with as little as 30 ,ug of Pro- 11. Herzenberg, L. A. & Herzenberg, L. A. (1978) in Handbook of nase, whereas Trop,2 and Trop-4 were comparatively more Pro- Experimental Immunology, ed. Weir, D. M. (Blackwell, Oxford, England), 3rd Ed., pp. 22.1-22.21. nase resistant and Trop-1. showed an intermediate sensitivity. 12. Micklem, H. S., Ledbetter, J. A., Eckhardt, L. A. & Herzen- A protein consisting of a single chain of Mr 200,000 was pre- berg, L. A. (1980) in Regulatory T lymphocytes, eds. Pernis, B. cipitated from peripheral blood lymphocytes with anti-Trop-3 & Vogel, H. J. (Academic, New York), pp. 119-132. antibodies. 13. Rouse, R. V., van Ewijk, W., Jones, P. P. & Weissman, I. L. The function these antigens fulfill at the surface oftrophoblast (1979) J. Immunol. 122, 2508-2515. and other cells is unknown. In this it is note- 14. Krolick, K. A., Villemez, C., Isakson, P., Uhr, J. W. & Vitetta; currently regard, E. S. (1980) Proc. Natl Acad. Sci. USA 77, 5419-5423. worthy that among the six monoclonal antibodies, only the two 15. Youle, R. J. & Neville, D. M. Jr., (1980) Proc. NatL. Acad. Sci. anti-Trop4 antibodies added to the culture medium ofa mixed USA 77, 5483-5486. lymphocyte, reaction inhibit the allogeneically stimulated 16. Beer, A. E., Billingham, R. E. & Yang, S. L. (1972)J. Exp. Med. proliferation. 135, 1177-1184. Downloaded by guest on September 27, 2021