Two proteins share immunological epitopes on the tumor-associated antigen 17-1A

Cancer Letters 144 (1999) 101±105

Two proteins share immunological epitopes on the tumorassociated antigen 17-1A Ying-Hua Chen*, Tianwei Yu, Yun Bai, Nanming Zhao School of Life Science and Engineering, Tsinghua University, Beijing 100084, PR China Received 12 April 1999; received in revised form 17 May 1999; accepted 18 May 1999

Abstract The mouse monoclonal antibody (mAb) 17-1A which recognizes the tumor-associated antigen 17-1A (also called EGP-40 or EpCAM) was successfully used in adjuvant therapy for colorectal carcinoma. In the 17-1A antigen analysis, we isolated not only a protein of 33 kDa (P33) which was reported as the tumor associated antigen 17-1A, but also a protein of 65 kDa (P65) using af®nity chromatography from cell lysates of HCT, and another protein of 50 kDa (P50) from lysates of human colorectal tumor tissues. The mAbs 17-1A and M79 (mAb M79 recognizes a different epitope on the 17-1A antigen) both could bind P33 and P50, but only M79 bound to P65 in an enzyme-linked immunosorbant assay (ELISA). These results indicate that P33 and P50 share at least two epitopes, and a common immunological epitope exists among P33, P50 and P65, suggesting that the two new proteins (P50 and P65) are related to the tumor-associated antigen 17-1A. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tumor-associated antigen; EpCAM; 17-1A; Common immunological epitope

1. Introduction The murine monoclonal antibody (mAb) 17-1A against human tumor-associated antigen 17-1A (EpCAM) can mediate antibody-dependent cellmediated cytotoxicity (ADCC) against human colorectal tumor cells [1] and has protective effect on nude mice xenografted with human colorectal carcinoma cells [2]. It has been shown that injection of mAb 17-1A can decrease recurrence rate and death rate of patient with colorectal cancer after surgery [3,4]. A 17-1A cancer vaccine is also in clinical development [5]. Monoclonal antibodies against 17-1A antigen recognize various normal epithelial tissues and carcinomas of different tissue origin by immunohistochem* Corresponding author. Fax: 186-10-627-855-05. E-mail address: [email protected] (Y.H. Chen)

ical staining [6]. The 17-1A antigen, a 33-kDa glycoprotein (P33), was isolated and identi®ed from several human colorectal tumor cell lines [6,7]. In this study, we used M79-sepharose and Con-A-sepharose columns in af®nity chromatography to purify 17-1Arelated proteins, and found that two proteins share immunological epitopes present on the tumor-associated antigen 17-1A. 2. Materials and methods 2.1. Human colorectal carcinoma cell lines and tissues Human colorectal carcinoma cell lines HCT and HT-29 were grown in DMEM medium (Gibco) supplemented with 10% fetal calf serum (Gibco),

0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00205-0

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2 mmol/l glutamine, 100 units/ml penicillin (Sigma) and 50 mg/l streptomycin (Sigma). Cells were harvested by digestion using 0.05% EDTA and 0.05% trypsin (Sigma) in DMEM medium (Gibco). Four, fresh human colorectal tumor tissue samples were obtained from the Sino-Japan Friendship Hospital in Beijing. 2.2. Antibodies Mouse mAbs 17-1A and M79 recognizing two different epitopes on 17-1A antigen [described in Ref. 1] were kindly provided by Prof. G. Riethmuller (Munich, Germany). Peroxidase-conjugated rabbit anti-mouse immunoglobulins were obtained from Dako (Denmark). FITC-conjugated goat anti-mouse IgG was obtained from Sigma, USA. 2.3. Isolation of proteins by af®nity chromatography and silver-staining Using standard procedure from Pharmacia, mAb M79 (4 mg) was coupled to 2 ml CNBr-activated sepharose 4B. The concanavalin A (Con A)-sepharose column (5 ml) was obtained from Sigma. The tumor tissues were homogenized in lysis buffer (5 % vol/vol) for 3 min and lysed for 1 h at 48C. The lysis buffer comprise of 50 mmol/l Tris±HCl, 0.5% NP-40, 0.18 mg/l PMSF, 1.57 mg/ml benzamidin±HCl, 0.1 mg/ml trypsin inhibitor and 0.1% leupeptin (all reagents purchased from Sigma). Cell lysates were prepared by centrifugation at 800 £ g for 30 min and at 10 000 £ g for 45 min, then ®ltrated by 0.22 mm membrane (Gelman, USA). With a low ¯ow-rate, tissue lysates were run through M79-sepharose af®nity chromatography column. The column was washed with 20 ml phosphate-buffered saline (PBS), eluted with 0.5 mol/l acetic acid at 48C. Eluates were neutralized immediately with 1 mol/l Tris (pH 9.6). Cell lysates were also run through con A-sepharose column. The column was washed with 40 ml PBS, eluated with 100 mmol/l a-methyl-d-mannose, 20 mmol/l Tris±HCl, 100 mmol/l NaCl, 0.1% Triton X100, 1 mmol/l CaCl2 (all reagents purchased from Sigma). Eluates from con A-sepharose column and unbound materials of cell lysates were run through M79-sepharose column separately, and eluted as described above. Eluates were subjected to electrophoresis in a 12.5% sodium dodecyl sulphate (SDS)-

polyacrylamide gel under reducing conditions and then examined by silver-staining. 2.4. Enzyme-linked immunosorbent assay (ELISA) The isolated proteins (P33, P50 and P65) were coated overnight on a microtiter plate at 48C, separately. Non-speci®c binding was blocked by incubation with 0.3% gelatine (Sigma) in PBS for 1 h at room temperature. After washing twice with PBS containing 0.1% Tween 20, mouse mAb M79 or 171A were added and incubated for 1 h at room temperature. The plate was washed again with PBS-Tween 20. Peroxidase-conjugated rabbit anti-mouse antibody was added. After further washing, freshly prepared O-phenylenediamine-peroxide solution was added and the optical density was measured (l ˆ 490 nm). 2.5. Flow cytometry analysis HT-29 cells (1 £ 106 ) were incubated with mAb 171A or M79 (10 mg/ml) for 45 min in a 50 ml ®nal volume. Cells were washed with PBS-supplemented 2% FCS and incubated with 50 ml FITC-conjugated goat anti-mouse IgG (Sigma, 1:50 dilution in PBS). After further washing with PBS plus 2% FCS, cells were ®xed with 1% paraformaldehyde in PBS and analyzed on a FACScan (Becton-Dickinson, USA). In the blockade test with isolated proteins, mAbs 17-1A and M79 were incubated with the proteins

Fig. 1. Isolation of proteins from cell and tissue lysates by af®nity chromatography using M79- and Con A-sepharose columns. The proteins were subjected to SDS-PAGE under reducing conditions and silver staining. Lane A, protein weight-marker; Lane B, P33 isolated from HCT cell lysates by M79-column; Lane C, P50 isolated from lysates of human colorectal tumor tissue by M79column; Lane D, P65 isolated from HCT cell lysates by con-Aand M79-columns.

Y.H. Chen et al. / Cancer Letters 144 (1999) 101±105

(5 mg/ml) separately for 30 min at 48C before incubation with cells. 3. Results By af®nity chromatography using M79-sepharose column, a 33-kDa protein (P33) was isolated from HCT cell lysates (Fig. 1B). Moreover, a 50-kDa protein (P50) was isolated from lysates of human colorectal tumor tissues (Fig. 1C), and a very weak band of 33 kDa (data not shown) was also detected by silver-staining. A 65-kDa protein (P65) was also found by af®nity chromatography using Con A-

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sepharose column and then M79-sepharose column (Fig. 1D). In a previous study, we found a 33-kDa protein (P33) from HT-29 cell lysates by af®nity chromatography using M79-sepharose column [8]. To test the interaction of both mAb 17-1A and M79 with these isolated proteins, an ELISA was performed. mAbs 17-1A and M79 recognizing two different epitopes on the 17-1A antigen could bind P33 and P50 (Fig. 2a,b), but only M79 bound to P65 (Fig. 2c). These results indicate that P33 and P50 share at least two epitopes, and a common immunological epitope exists among P33, P50 and P65, suggesting that the two new proteins (P50 and P65) are related to the tumor-associated antigen 17-1A.

Fig. 2. Identi®cation of interaction of mAbs 17-1A and M79 with the isolated proteins by ELISA. a: A, mAb 17-1A binding to P33; B, mAb M79 binding to P33. b: A, mAb 17-1A binding to P50; B, mAb M79 binding to P50. c: mAb 17-1A binding to P65; B, mAb M79 binding to P65. Control: gelatine instead of these antigens by coating the microtiter plate.

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Fig. 3. Inhibition of mAb 17-1A (a) and M79 (b) binding to HT-29 cells by P50, in ¯ow cytometry analysis. FACS-histogram overlays: A, PBS (control); B, mAb (10 mg/ml); C, mAb (10 mg/ml) plus P65 (5 mg/ml).

To know whether P50 or P65 could inhibit the binding of mAb 17-1A and M79 to HT-29 cells, the binding of both mAbs to HT-29 cells were examined by ¯ow cytometry analysis. P50 showed an enhancement effect on the binding of both mAbs (Fig. 3). P65 did not inhibit the binding of mAb 17-1A to HT-29 cells (Fig. 4a), and on the contrary, could weakly upmodulate the binding of M79 (Fig. 4b). 4. Discussion Mouse monoclonal antibodies against the tumor-

associated antigen 17-1A have been used in adjuvant therapy for human colorectal tumors and have produced good curative effects [3,4]. In this study, we isolated three different proteins, two of which are not reported before. The isolation of P33 is consistent with the report that a 33-kDa glycoprotein (or 37 kDa, under non-reducing conditions) had been isolated from cell lines HT-29, WiDr, DLD-1 [4,5]. In our study, P33 was not isolated by Con A-sepharose column, which suggests that P33 does not contain ad-mannosyl or a-d-glucosyl groups. The interaction between P50 and both mAbs 17-1A and M79 (recog-

Fig. 4. Inhibition of mAb 17-1A (a) and M79 (b) binding to HT-29 cells by P65, in ¯ow cytometry analysis. FACS-histogram overlays: A, PBS (control); B, mAb (10 mg/ml); C, mAb (10 mg/ml) plus P65 (5 mg/ml).

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nizing two different epitopes on 17-1A antigen) suggests P33 and P50 share at least two epitopes. P65 only bound mAb M79, indicating a common immunological epitope exists among P33, P50 and P65. These results demonstrate that the two new proteins (P50 and P65) are related to the tumor-associated antigen 17-1A, at least on a serological level. Up to now, we have no experimental data to explain the enhancement effect of P50 and P65 on the binding of the mAbs to HT-29. This will the subject of future investigation in. One possibility is that P50 binds directly to a certain cell surface antigen on the HT29 cells and thus making more binding sites on the cells available for the antibodies. HT-29 cells in part should be mutated during cultivation so that the cells showed different behavior in the blockade test. The slight enhancement of M79 binding to HT-29 cells by P65 could be explained by a similar proposal. Another explanation may be that P65 contains two or more M79 epitopes, thus by the linkage of P65, more antibodies could bind to a single cell. 17-1A antigens extracted in different laboratories are not congruent. Gottlinger et al. reported that 171A antigen was a 33-kDa glycoprotein [6,7]. Ross et al. reported that the 17-1A antigen contained a 30- and a 40-kDa subunit [9]. There is still another report that 17-1A antigen is a 41-kDa protein [9]. Tumor associated antigen GA733 is also recognized by mAb 171A [10]. Now we report a 50-kDa protein which is a putative 17-1A antigen and a 65-kDa protein which is related to the 17-1A antigen. The difference among the reports on 17-1A antigen indicates that there may exist a group of cell surface antigens that are recognized by mAb 17-1A and related antibodies. Gottlinger et al. reported a wide distribution of 171A antigen (EpCAM) among normal and malignant tissues. Based on the ®ndings by us and others, we can propose a hypothesis that a 17-1A tumor-associated antigen family may exist, and the success of the adjuvant therapy is based on the interaction of the anti-171A mAbs with a set of proteins, for example, P33, P50 and P65. More studies should be carried on about the biochemical properties of P50 and P65, and their relationship with P33.

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Acknowledgements The authors wish to thank Prof. Dr. G. Riethmuller (Munich, Germany) for his kindly providing the antibodies. This work was supported by Tsinghua University and Tsinghua Novel Hi-Tech Investment Holdings Limited.

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