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Production and characterisation of a monoclonal antibody (Cx-99) against cervical carcinoma.

An IgG1 monoclonal antibody (MAb Cx-99) has been established which recognises a surface antigen on epithelial cells, but not on fibroblastic or hematopoietic cells. Immunohistochemical studies showed that this antigen was present in all 37 squamous cell carcinomas (SCC) including 33 cervical SCC, and 30 of the 32 adenocarcinomas examined; most of the 33 cervical SCC were stained extensively. It was also detected in the culture medium of cervical cancer cell lines. In the normal cervix, this antigen was restricted to the undifferentiated basal cells. This observation suggests that the widespread expression of the antigen was triggered by oncogenesis. The MAb Cx-99 recognised an epitope on an asialyted glycoprotein which has an apparent molecular weight of 37 kilodaltons (kD) (and 2 minor proteins at 18 and 27 kD) and an isoelectric point (pI) of 5.3. It may have potential for studies on differentiation and oncogenesis and for diagnostic applications. ImagesFigure 1Figure 2

Squamous cell carcinoma (SCC) of the uterine cervix is a malignancy that is highly prevalent worldwide. In Taiwan, where this study was conducted, cervical SCC has a very high prevalence and is the leading cause of death of all female cancers (Chen, 1986). Conventional examinations still have limitations with regards to sensitivity and specificity in diagnosis and to monitoring of the disease. Thus, an additional tumour marker would be helpful.
Tumour markers used for studying cervical carcinoma include carcinoembryonic antigen (CEA), TA-4, epithelial membrane antigen (EMA), antigens recognised by MAb 17.13 or MAbs CE400-413, and cytokeratins (Rutanen et al., 1978;Kato et al., 1979;Bamford et al., 1983;Randen et al., 1987;Koprowska et al., 1986;Bobrow et al., 1986). There has been limited success in the early diagnosis and monitoring of the disease. Most of these markers have considerable crossreactivity with normal human tissues, usually in a sporadic way, therefore limiting their functional roles. We present here the derivation and characterisation of a monoclonal antibody which recognises a surface antigen of human cervical carcinoma. It seems to have potential for diagnostic application. This antibody was produced through immunisation with tissue fragments of cervical carcinomas and through systemic screenings using a variety of human cell lines and tissues.
Normal and malignant human tissues were collected postoperatively. One aliquot was stored frozen, and another aliquot was fixed in 10% buffered-formalin and then embedded in paraffin. Some specimens were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde before embedding.
Preparation of membrane-enrichedfractions of human cervical carcinoma Carcinoma tissues were collected from four patients with cervical SCC after radical hysterectomy. After excision of the necrotic areas, 12 gm of fresh tissues were washed and minced finely, placed into 1:3 (w/v) deionised distilled water, and homogenised three times with a blender at 4°C for 10 s each time. The homogenate was then sonicated three times, each for 20 s (Ultrasonic 2000 Artek System Co., NY). The tissue sediment was discarded, and the unsedimented material was centrifuged at 100,000 g for I h. The precipitate (0.5 ml) was diluted with phosphate-buffered saline (PBS) and stored in 10 aliquots (I ml each) for immunisation.
The homogenate was sonicated then stirred at 4°C for 2 h. The sonicated solution was centrifuged at 100,000 g for I h, and the supernatant was stored in 1 ml aliquots at -20°C. The protein content was determined spectrophotometrically (Lowry et al., 1951), and each aliquot was adjusted to contain 2.5 mg of protein per ml.
Production of monoclonal antibody Six-week-old Balb/c mice were immunised subcutaneously with 1 ml of an emulsion containing equal amounts of the membrane-enriched fraction and complete Freund's adjuvant, and then they were boosted intraperitoneally with a mixture of membrane-enriched fraction and incomplete Freund's adjuvant given every 2 weeks for five injections; the final booster was given intravenously.
Three days after the last injection, lymphocytes were taken to make the monoclonal antibody by the cell fusion technique as previously reported (Tsai et al., 1985). In brief, spleen cells (1.6 x 108) were fused with 2 x 107 mouse myeloma cells (NS-1) in 1 ml of 50% polyethylene glycol 4000 (Merk Darmstadt, Germany), and then cultured in HATcontaining RPMI-1 640 medium in 96-well microplates (Nunc, Denmark). Fourteen days after the fusion, supernatants were tested by ELISA against two cervical cancer cell lines, CC7T and SIHA, and the fibroblast cell line FS-4. Positive cultures were further selected by the immunohistochemical staining on tissue sections of cervical SCC and of normal cervix. Hybridomas whose antibodies reacted much stronger against cancerous tissues than normal cervical epithelium were selected. Subcloning was performed by the limiting dilution method. In addition, ascitic fluid containing the antibodies was prepared by intraperitoneal injection of 5 x 106 hybridoma cells into Balb/c mice which had been previously primed with 0.5 ml pristane (Sigma, MO, USA) 2 weeks ago. The antibody was purified by precipitation twice with 45% saturated ammonium sulfate followed by a DEAEion exchange column chromatography, and it was stored at -70°C until used.
The reactivities toward leukaemia cell lines and isolated blood cells were determined by the IF method after fixing the cells onto microscopic slides, as described by others (Johnstone & Thorpe, 1987). Mononuclear leukocytes were prepared by density gradient centrifugation using Ficoll-Hypaque (English et al., 1974).

Immunohistochemistry
The histochemical staining was performed according to Hsu's method (Hsu & Raine, 1981). Briefly, sections of tissues were reacted with culture supernatants for 30 min at room temperature after removal of endogenous peroxidase by preincubation for 30 min at room temperature with methanol containing 0.3% H202, followed by diluted biotin-labelled horse anti-mouse IgG (1:400 Vector Lab, Burlingame, CA). The avidin-biotin-peroxidase complex (ABC, Vector Lab) was then applied, followed by development in 3-amino-9ethyl carbazol (AEC)-3% H202 solution. The NS-1 culture supernatant was used as the negative control.

Immunoglobulin isotype
Aliquots of 0.1 ml of goat anti-mouse IgG (50 sg ml-' in PBS) were coated onto polyvinyl 96-well microtiter plates and incubated sequentially with each of the following for 1 h: 0.1 ml of culture supernatant; 50 p11 of rabbit antiserum specific for mouse immunoglobulins (Ig) A, M, GI, G2a, G2b or G3; 50 p1 of diluted peroxidase-labelled goat antirabbit antibody; and 100 tl of substrate ABTS (monoAb-ID EIA kit, Zymed, USA).

SDS-PAGE, isoelectrofocusing (IEF) and immunoblotting
Tissue extracts were subjected to 13.5% SDS-polyacrylamide slab gel electrophoresis under non-reducing or reducing (2mercaptoethanol) conditions as described by Laemmli (1970), using 30-40 jig protein in each lane. Then the protein was transferred electrophorectically onto nitrocellulose paper according to the method of Towbin et al. (1979) (Hoefer Scientific Instruments, USA). The membrane strips were blocked overnight with 1% BSA and incubated with undiluted hybridoma supernatants for 1 h. The strips were incubated for 30 min with diluted alkaline-phosphatase-conjugated rabbit anti-mouse IgG antibody (Sigma, MO), followed by 60 min incubation with substrates containing 25 mg of fast red (Sigma, MO) and 10 mg of alpha-nathyl-phosphate (Sigma, MO) in 50 ml of 50 mM Tris-HCl, pH 8.2. All incubations were performed at room temperature.
The IEF was performed by polyacrylamide IEF gel electrophoresis at 100 V for 15 min, 200 V for 15 min, and 450 V for 1 h (Mini IEF Cell, Bio-Rad Mode 111). The separated proteins were electrotransferred onto the nitrocellulose paper in 0.7% acetate in distilled H20, pH 2.8, at 1 A for 40 min (Hoefer Instrument), then used in the reactions described above.
Other biochemical characterisations The CC7T cells, 5 x 105, were treated with the following reagents in different concentrations: trypsin (0.01 -1 %), periodic acid (0.05-5%) (Sigma, MO) and neuraminidase (0.01-1.0 U ml 1) (Behringwerke AG, Marburg). After incubation at 37°C for 30 min, these cells were washed three times, put in poly-L-lysine coated 96-well plate, centrifuged at 2,000 r.p.m. for 10 min, and fixed with 0.5% glutaraldehyde in PBS at room temperature for 15 min. Then the ELISA assay was performed as described above, and immunohistochemical studies were performed on tissue sections pretreated with these reagents.

Specificity
Purified antigens of CEA (DAKO, Glostrup, Denmark) were used to coat a 96-well plate at 0.05 jig per well respectively.
MAb Cx-99 were added and the reactivities were determined by the ELISA assay described above. In excluding crossreactivity with the TA-4 (SCC) Antigen, 50 ng of the standard TA-4 (SCC) Antigen from the diagnostic kit (Dainabot, Tokyo) was mixed with MAb Cx-99 in four diluted concentrations, then quantitations of the standard TA-4 (SCC) Antigen from these mixtures was performed by radioimmunoassay according to the diagnostic kit.
Detection of the MAb-eliciting antigen in culture medium from cervical cancer cell lines First, a competitive inhibition ELISA test was performed. The wells coated with SIHA cells were reacted with culture supernatant containing MAb Cx-99 as the control. To test for antigen shedding by the various cancer cell lines, 50 pl of spent medium from these cell lines in conventional culture containing 10% FCS were reacted with 50 p1 of the culture supernatant containing MAb Cx-99 at 37°C for 2 h, and the mixtures were subjected to reaction with coated SIHA cells by ELISA assay to determine whether the MAb was still Uterine myoma 5 5 Reactivity was expressed on an arbitrary degree of staining: 0, < 5% staining of the cancerous area; +, 5-30%; + +, 30-50%; + + +, 50-70%; + + + +, 70-100%.
alive. Second, a confirmation experiment by ELISA assay on serum-free medium was performed. The SIHA cells were cultured in serum-free medium containing growth factors for 4-6 days. Then 50 LI of this medium containing 75 ,ug ml-' of protein were coated in each well, incubated at 4°C overnight, reacted with the purified ascitic MAb Cx-99 in various dilutions from 10-2 (38.4 ,tg ml-' of protein) to 10-6, and assayed by ELISA.

Results
Production of MAb Cx-99 After fusing, 78 of 556 wells containing hybridoma cells showed a 4-fold or higher absorbance above background when reacted with the cervical carcinoma cell lines CC7T and SIHA. The hybridomas whose supernatants also showed a positive reaction to the foreskin fibroblast cell line FS-4 were excluded. After secondary screening by immunoperoxidase staining on tissues of cervical carcinomas, nine wells that showed stronger staining with the cervical carcinomas than with normal tissues were isolated for subcloning. The MAb Cx-99, which showed the strongest activity and seemed stable, was finally selected for further characterisation.
Determination of antibody specificity by immunohistochemistry Using the immunoperoxidase method, all 22 cases of invasive squamous cell carcinoma and all 11 cases of cervical intraepithelial neoplasia III (CIN III) of the uterine cervix were stained with MAb Cx-99 (Table I). Nineteen of the 22 invasive cervical SCC had more than half of their cancerous areas stained, and no differences in staining were found between keratinised and nonkeratinised types. Islands of SCC were clearly stained in primary cerivcal lesions as well as in metastatic lymph nodes (Figure la-c). Of the 11 cases of CIN III, nine had staining on the entire epithelium of anaplastic cells ( Figure Id). The reactivity to MAb Cx-99 in all four cases of CIN I and two of the four cases of CIN II was confined to the dysplastic areas, which were located at the lower to middle layers of epithelia, but in two cases of CIN II, the staining pattern was similar to that of CIN III (Data not shown).
All stromal tissues including fibroblasts, blood cells, blood vessels were not stained. In contrast to the widespread re-activity in the cervical carcinoma, normal cervix was stained in only some discrete areas, including the basal cells of the ectocervical epithelium and the subcolumnar cells of the endocervical glands at the transformation zone (Figure le and f).
All SCC other than cervical carcinoma and most of the adenocarcinomas, including ovarian carcinoma, stained immunohistochemically with MAb Cx-99, whereas most of the other types of tumours did not (Table I). In one case of synovial sarcoma, reactivity was present only in the epithelial components. Some normal epithelial tissues including endometrial glands, fallopian tube, colon, the ductal epithelia of the liver and kidney, sweat glands, trophoblasts of normal placentas and some foetal tissues were also stained (Table II). In contrast to the extensive reactivity of ovarian carcinomas, normal ovarian tissues were not reactive.
The patterns and intensities of staining in frozen sections or in glutaraldehyde-paraformaldehyde-fixed tissues were similar to those of formalin-fixed sections.

Reactivity of MAb Cx-99 with cell lines and isolated leukocytes
The MAb Cx-99 reacted with all five cervical cancer cell lines as determined by ELISA on coated cells in 96-well plates (Table III). Similar results could also be observed in one of three hepatoma cell lines, two of six colon cancer cell lines, and the trophoblast-like TL cell line but not in any of the stromal cell lines or three different leukaemia cell lines (Table  III).
Characteristics of the antigen recognised by MAb  The isotype of MAb Cx-99 is IgGI. Analysis of the antigen that reacts with MAb Cx-99 by SDS-PAGE under nonreducing or reducing (2-ME) conditions revealed a similar pattern, i.e., a band corresponding to a relative molecular mass of 37 kD, and fainter bands of 27 kD and 18 kD (Figure 2a). By IEF and blotting with MAb Cx-99, a single band at pI 5.3 was obtained (Figure 2b). Treatment of cancer tissues or cells with periodic acid or trypsin destroyed their reactivity with MAb Cx-99, but treatment with neuraminidase did not (Figure 3). Identical results were found in tissues stained after treating with these reagents. Therefore, the epitope of MAb Cx-99 seems to be a glycoprotein without sialic acid residue. Further immunoassay tests showed that a c d e f Figure 1 Immunoperoxidase staining of invasive cervical SCC with MAb Cx-99. a, large-cell non-keratinised type; b, keratinised type; c, pelvic lymph node showing a metastasis; d, CIN III; e, nornal ectocervical epithelium; f, normal endocervix. Bars: a to e=8.6 p; f=5.61 .
Detection of the antigen in culture medium of cell lines Competitive inhibition tests showed that culture medium from certain cell lines, including CC7T, ME180 and SIHA, decreased the reactivity between MAb Cx-99 and the SIHA cells, whereas others, such as HEK, Caski, HeLa and Huh-7, did not (Figure 4a). Further studies utilising SIHA supernatant generated in serum-free medium as the coated antigen in the ELISA assay also demonstrated the presence of the shedding antigen in the culture supernatant (Figure 4b).

Discussion
We report the establishment of a MAb to cervical carcinoma, designated MAb Cx-99. The eliciting antigen is present only in epithelial cells, but not in fibroblastic or hematopoietic cells, and it is found immunohistologically in 100% of the 37 SCCs and 94% of the 32 adenocarcinomas examined; most b ..7.
s.:. .... aOnly the basal cells were positive. of these tissues were stained extensively. In contrast to the diffused expression of this antigen in all of the 22 invasive cervical SCC, it was present only in the undifferentiated basal cells of the cervix and reserve cells of the transformation.
These cell types are rapidly replicating in normal differentiation and are the source of the cells that undergo malignant transformation (Dallenbach-Hellweg & Poulsen, 1990). In CIN III, MAb Cx-99 stained the entire epithelial layer, which was composed of anaplastic cells. These findings identify a phenotypic similarity between cervical cancer cells and immature cells. We suggest that cervical carcinoma cells retain the capability of expressing the antigen reactive with MAb Cx-99, which is normally found in the basal cells, during oncogenesis. This antigen may be valuable in identifying undifferentiated epithelial cells and for studies on differentiation and oncogenesis. immunostaining in cancer tissues provides a favourable basis for its application in vivo, in tumour localisation (Tsai et al., 1988). The reactivity pattern of Cx-99 may also open the possibility of using this antibody on smears for the early detection of cervical carcinoma, because reactivity was found from CIN lesions to invasive carcinomas. Finally, since the antigen recognised by MAb Cx-99 can be detected in the culture medium, it may be present in the serum of patients with cervical cancer or with adenocarcinomas. The epitope that reacts with MAb Cx-99 is carried on an asialyted glycoprotein with a relative molecular mass of 18-37 kD and a pI of 5.3. It apparently does not crossreact with CEA and TA-4 (SCC) Antigen. Also, its physiochemical and antigenic properties are distinct from those of other reported epithelial cell-associated antigens. Tissue polypeptide antigen (TPA) is a 45 kD complex related to cytokeratins number 8, 18 and 19 that is also located in the basal cells of the normal squamous epithelium; however it has rarely been found in keratinised cervical SCC and is absence in the normal endocervical columnar cells (Stegner et al., 1986). MAb 17.13 reacts with a basal cell antigen, but is not present in adenocarcinomas (Randen et al., 1987). MAb 17-lA is a 37 kD glycoprotein, but unlike MAb Cx-99, the epitope that it recognises contains sialic acid residues. Also, the SDS-PAGE banding pattern of the molecule reactive with MAb 17-lA was changed under reducing condition (Gottlinger et al., 1986); therefore, its epitope is structurally different from that recognised by MAb Cx-99 which was not changed under reducing conditions in SDS-PAGE. MAb MOv18 and 19 have a relative molecular weight similar to the antigen that reacts with MAb Cx-99, but they are different in immunohistochemistry (Miotti et al., 1987). The other antigens of SCC include human milk fat globulin (HMFG 1 and 2), epithelial membrane antigen (EMA), VM-2, MAbs CE 400-413, cytokeratins of low molecular weight such as CAM 5.2, and antigens reactive with antibodies SQM1 and Ca 1. However, all of these antigens are different from the antigen recognised by MAb Cx-99 in molecular weight, biochemical characteristics and tissue distribution (Fray et al., 1984;Bamford et al., 1983;Mortenn et al., 1985;Koprowska et al., 1986;Bobrow et al., 1986;Boeheim et al., 1985;Ashall et al., 1982). In summary, we have established a MAb that recognises an antigen present only in epithelial cells. Based on its pattern of reactivity, we suggest that MAb Cx-99 may be tion ELISA test, 1 X and 10 x dilutions of the culture medium were used. A: SIHA culture medium was replaced by PBS (positive control). B: MAb Cx-99 was replaced by PBS (negative control). b, ELISA assay of SIHA supernatant generated in serum-free medium. This antigen preparation was used to coat the wells, and is reacted with MAb Cx-99 (0). Replacing the antibody with PBS eliminated the reactivity with SIHA supernatant-coated wells (absorbance <0.09). The titration curve of MAb Cx-99 with SIHA cells is also shown (*).
useful for the rapid diagnosis of cervical carcinomas and the detection of micrometastases or microinvasion. Since it is shed by cell lines in culture, it may be present in the sera of patients with SCC or with adenocarcinomas.