ICANCER RESEARCH 45. 783-790. February 1985| Isolation and Characterization of a Monoclonal Antibody Specific for Epithelial Cells Anne W. Hamburger,1 Yvonne A. Reid, Barbara Pelle, George E. Milo,2 Inge Noyes, Henry Krakauer, and J. Peter Fuhrer Cell Culture Department, American Type Culture Co/lection, Rockville, Maryland [A. W. H., Y. A. Ft., B. P./,' Comprehensive Cancer Center. Ohio State University, Columbus, Ohio [G. E. M., I. N.¡;and the National Institute of Allergy and Infectious Diseases [H. K.¡and the National Cancer Institute, Bethesda. Maryland [J. P. F.] ABSTRACT monoclonal antibodies have been isolated (9, 15, 19, 29), rela tively few monoclonal antibodies specific for epidermal cell sur Monoclonal antibodies were generated to antigens on human face antigens have been described (18). foreskin keratinocytes to identify epithelial-specific molecules. Cell cultures of human epidermis retain epidermal-specific Spleen cells from BALB/c mice, immunized with membrane functions and antigens (10, 11, 20). Thus, these keratinocyte preparations from primary expiants of foreskin epithelial cells, cultures could serve as immunogens in attempts to produce were fused with the NS-1 mouse myeloma line. Hybridoma monoclonal antibodies specific for keratinocyte cell surface an supernatants were screened for the desired immunological reac tigens. We now describe the isolation of hybridomas secreting tivity using enzyme-linked immunosorbant binding assays. Hy- monoclonal antibodies which bind selectively to cultured normal bridomas secreting antibodies reacting with epithelial cells, but keratinocytes and show a high specificity for both cultured and not fibroblasts or lymphocytes, were cloned by limiting dilution, primary epithelial tissues. and two stable clones producing immunoglobulin M K antibodies were selected for study. Evaluation of fixed paraffin-embedded human tissue by an indirect immunoperoxidase technique re MATERIALS AND METHODS vealed that the antibodies bound most strongly to normal strati fied squamous and transitional epithelium, and squamous and Cells and Media. The following human cell lines were obtained from transitional cell carcinomas. Antibodies from the cloned hybri- the American Type Culture Collection, Rockville, MD: B-lymphoblastoid domas also reacted with primary cell cultures of foreskin keratin lines: CCL 213 (Daudi), CCL 114 (RPMI 766); T-lymphoblastoid lines: CCL 119 (CCRF-CEM), CRL 1552 (Molt-3); melanoma: CRL 1424 (G- ocytes, pulmonary epithelium, fetal liver, and amnion cells, but 361), HTB 64 (Malme-64); neuroblastoma: CCL 127 (IMR 32), HTB 11 not with primary cultures of nonepithelial cells. Further testing (SK-N-SH); adrenal adenocarcinoma: CCL 105 (SW 13); carcinoma of by enzyme-linked immunosorbent assays revealed that the an tibodies reacted with some long-term cell lines derived from the pharynx: CCL 138 (Detroit 562); adenocarcinoma of the colon: CCL 220 (Colo 320 DM), CCL 218 (WiDr); adenocarcinoma of the prostate: epithelial tumors. Nonepithelial cell lines were not stained by the HTB 81 (DU 145), CRL 1425 (PC-3); carcinoma of the lung: CCL 185 antibodies. Indirect immunofluorescent studies indicated that (A549, epidermoid carcinoma), HTB 54 (CALU-1, adenocarcinoma); tran staining was confined to the cell surface. These antibodies may sitional cell carcinoma of the bladder: CRL 1473 (HT-1197), HTB-2 (RT- prove useful in studies of differentiation markers of human epi 4); squamous cell carcinoma: CRL 1623 (SCC-15); carcinoma of the thelial cells. pancreas: CRL 1420 (MIA-PaCa-2), CRL 1469 (PANC-1, ductal carci noma); carcinoma of the cervix: CCL 2 (HeLa); renal leiomyoblastoma: CRL 1440 (G-402); Wilms1 tumor: CRL 1441 (G-401); choriocarcinoma: INTRODUCTION CCL 98 (BeWO); adenocarcinoma of the breast: CRL 1500 (ZR 75-1), CRL 1504 (ZR-75-30); foreskin fibroblasts: CRL 1521 (CCD-76), CCL Specific molecular markers which identify cell types in epithelial 109 (Detroit 550); fibrosarcoma: CCL 121 (HT-1080); and Ewing's sar tissue are important for the characterization of cells grown in coma HTB 87 (SK-ES-1 ). A primate cell line from African green monkey culture and for studies of phenotypic changes in carcinogenesis, kidney cells, CCL 70 (CV 1), was also used. The human melanoma cell and they may have applications in clinical diagnosis and organ line WM-9 and a line of normal human lung fibroblasts were obtained transplantation. from the Wistar Institute. A line of epithelial cells derived from normal BALB/c mouse skin (BK-1 ) was obtained from Dr. Stuart Yuspa, National Several proteins have been identified as markers of epithelial Cancer Institute. Primary cultures of human foreskin epithelial cells, lung tissue. Autoantibodies in the serum of patients with bullous fibroblasts, pulmonary epithelia, and epithelial cells from fetal liver were pemphigus and pemphigus vulgaris have been used to detect prepared in the laboratory of Dr. George Milo. Undifferentiated prolifer epidermal-associated antigens and to study epidermal differen ating keratinocytes were prepared as follows (22). Foreskin tissue was tiation (25). Epidermal-specific histocompatibility antigens, be cut into 4-mm pieces in 4 ml of HBSS3 minus calcium and magnesium lieved responsible for immunogenicity of skin allografts, include at pH 7.2 at 21°. Tissue pieces were rinsed 3 times, recovered, and the SKA antigens in mice (3) and a non-H-2 alloantigen (epa) placed in 3 ml of prechilled HBSS solution containing 0.125% trypsin identified on murine epidermal cells (26). Keratin, a major differ (Worthington Enzymes, Freehold, NY) and 0.02% EGTA. After 24 hr, the entiation product of epidermal cells, is also considered to be an tissue was removed, and the upper epidermal layers were mechanically epithelial-specific marker (7, 27). While several keratin-specific separated from the dermal tissue and the attached stratum basalis. The lower layer containing the dermis and stratum basalis was placed in 3 1Recipient of support in part from Contract NO102655 from the National Institute ml of HBSS minus calcium and magnesium containing 0.2% trypsin and of Allergy and Infectious Diseases. To whom requests for reprints should be 3 The abbreviations used are: HBSS. Hanks' balanced salt solution; FBS, fetal addressed 2 Recipient of support from Grant NCI RO1CA 25907-04. bovine serum; EGTA, etnyleneglycol bis(/i-aminoethylether)-N,/V,N',W '-tetraacetic Received May 5. 1983: accepted November 5. 1984. acid; ELISA, enzyme-linked immunosorbent assay. CANCER RESEARCH VOL. 45 FEBRUARY 1985 783 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. EPITHELIAL-SPECIFIC MONOCLONAL ANTIBODIES 0.02% EGTA, pH 3.9 to 4.2.' The tissue suspension was reincubated at diaminobenzidine and 0.03% hydrogen peroxide. Sections were coun- 8°for an additional 24 hr. The dermal pieces free of the stratum basalis terstained in hematoxylin:eosin. Cytocentrifuge preparations of single- were removed. Freely floating cells from the stratum basalis were re cell suspensions of human tissue or cell lines were fixed in absolute covered by centrifugation at 650 x g. These cells were seeded at 5000 methanol and stained in the same manner. cells/25-sq cm flask in Eagle's minimal essential medium supplemented Immunofluorescence Studies. Indirect immunofluorescence studies with 10% FBS and 0.75 rriM calcium (total free calcium). using monoclonal antibodies were performed on cells growing on glass Keratinocytes were serially passaged by treatment of confluent mon- coverslips or on 5-/¿mcryostat sections of human tissue. Cells were olayers with 0.25% trypsin in HBSS:0.02% EDTA at pH 7.2. The cells initially fixed in -20° methanol and acetone as described by Sun ef al. were seeded in Eagle's minimal essential medium supplemented with (27) and stained with the appropriate monoclonal antibody and then 10% FBS and 0.75 ÕTIMcalciumat 106 cells/25 sq cm (1:2). The cells at fluorescein-conjugated goat anti-mouse IgM (Cappel Laboratories, Coch- primary passage exhibited a 75 to 90% attachment efficiency. ranville, PA). Cells were also stained with a monoclonal antibody to All tumor cell lines were grown in monolayer culture as described (2). intermediate filaments (TIB 131) obtained from the American Type Cul The lymphoblastoid lines were grown in RPMI 1640 with 20% FBS. ture Collection or a rabbit anti-human keratin heteroantibody that was Purified human peripheral blood mononuclear cells and bone marrow the generous gift of Dr. T. T. Sun, New York University. In later experi cells were separated by buoyant density centrifugation on Ficoll:Hypaque ments, cells were stained with both antibodies and then fixed in 95% by standard techniques. ethanol:5% glacial acetic acid and mounted in Gelvatol as described (24). The NS-1 cell line (obtained from Mark Lostrum, Fred Hutchinson The cells were viewed using a Zeiss epiilluminated fluorescent micro Cancer Center, Seattle, WA) was grown as a stationary suspension scope. culture in RPMI 1640 with 20% FBS and treated with 10~8 M 8-azagua- Analysis of Monoclonal Antibodies. Antibodies were tested for class nine 3 weeks prior to its use in fusions. Hybridoma cell lines were and subclass by Ouchterlony immunodiffusion using antisera specific for maintained as described for NS-1 cells. the major mouse immunoglobulin classes and for the subclasses of IgG Generation of Hybridoma Cell Lines. BALB/c mice were immunized and light chains. Immunoglobulin class was confirmed by sodium dodecyl by 2 i.p. injections (administered 15 weeks apart) of 100 ^g of membrane- sulfate gel electrophoresis of metabolically labeled antibodies as de enriched preparations (4) of primary cultures of human keratinocytes in scribed (13,16, 23). complete Freund's adjuvant. The third, and final, injection of 100 ßQof membrane protein in phosphate-buffered saline was administered i.v. 3 weeks after the second injection. Three days after the final injection, the RESULTS spleens were excised, and spleen cells were fused with NS-1 cells in the presence of 40% Polyethylene Glycol 4000 as described previously (5). Generation of Monoclonal Antibodies.