.
Cancer
Br. J. Cancer
(1991),
(1991), 63, Suppl. XIV, 29-32 63,
Suppl.
XIV,
29-32
f,-"%
Macmillan Press Ltd., 1991
©
Macmillan
Press
Ltd.,
1991~~~~
Monoclonal antibodies defining the cluster-5A small cell lung carcinoma antigen R. Waibell, C.J. O'Hara2 & R.A. Stahel' 'Division of Oncology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland; and 2Department of Pathology, New England Deaconess Hospital, Boston, Massachusetts 02115, USA.
Summary The cluster-5A antigen on small cell lung cancer has been previously identified by the murine IgG2. monoclonal antibody SWA20. We now describe four new murine monoclonal antibodies, SEN3 (IgG,), SENl (IgG3), SEN12 (IgG2b) and SEN31 (IgG,) which recognise the same antigen. The antibodies have similar binding characteristics as antibody SWA20 on cell lines and on a limited number of frozen sections of human tissues when examined by immunofluorescence and immunohistochemistry. Radioimmunoassays on fixed cells showed competitive binding of all antibodies with radiolabelled antibody SWA20. Solid phase radioimmunoassays with the new antibodies on biochemically treated target cells showed similar characteristics to antibody SWA20, including sensitivity to treatment with periodate and neuraminidase. Immunoblotting experiments with all antibodies revealed similar bands at molecular weights of 180, 100 and 50 kD. Binding studies after incubation of cells with inhibitors of glycoprotein synthesis suggest the cluster-5A antigen to be a N-linked sialoglycoprotein.
We have previously reported on the murine monoclonal antibody SWA20 which recognises a tumour-associated antigen on small cell lung cancer (SCLC) (Waibel et al., 1988). The antigen is a sialoglycoprotein strongly expressed on the surface membrane of one third to one half of small cell lung carcinomas (SCLC). The antigen has little or no expression on normal epithelial tissues and no expression on normal neural and hemopoietic tissues. At the First International Workshop on Small Cell Lung Carcinoma antigens, the antigen defined by antibody SWA20 was classified as cluster-5A antigen (Souhami et al., 1988). Since that time, the selectivity of expression of this antigen in SCLC, but not in other lung tumours has been confirmed by extensive immunohistochemical studies on bronchial biopsies and resected tumour tissues (Maier et al., 1989). In an effort to define new small cell lung cancer antigens and to expand the number of reagents which might be useful for therapy of SCLC lung cancer, new monoclonal antibodies have been generated in our laboratory. The analysis of cell line reactivity of four of the new antibodies suggested that they might recognise the same or a similar antigen as antibody SWA20. Investigations presented here demonstrate that the four antibodies SEN3, SENI 1, SEN12 and SEN31 bind to the cluster-5A antigen. Further studies on the nature of cluster-5A antigen suggest the antigen to be an N-linked
sialoglycoprotein. Materials and methods Cell lines All cell lines were grown in RPMI supplemented with glutamine and 10% FCS. The SCLC cell lines NCI-H249, NCIH417, NCI-H446, NCI-H524 and NCI-H526 were provided by Dr D.N. Carney, Mater Private Hospital, Dublin. Other cell lines employed for screening of antibody reactivity were generated in our own laboratory or obtained from ATCC or from sources that have been described previously (Waibel et al., 1988).
Antibody generation and characterisation Our procedure for antibody generation and selection of hybrids has been reported (Stahel et al., 1985). Hybridomas of Correspondence: R.A. Stahel, Division of Oncology, University Hospital, CH-8091 Zurich, Switzerland.
the SEN clones SEN3 IgGl, SENl IgG3, SEN12 IgG2b and SEN31 IgGl were obtained by injection of viable SW2 cells into Balb/c mice and fusion of the spleen cells with mouse X63Ag8.653 myeloma cells. Antibodies which by indirect immunofluorescence displayed a ring pattern of reactivity with SW2, but were negative with leukaemia, adenocarcinoma or squamous cell carcinoma cell lines were chosen initially. Further selection was for lack of reactivity with normal blood and bone marrow and for IgG isotype which was determined by Ouchterlony double diffusion assays and with a dipstick assay (Amersham).
Immunofluorescence staining Cell lines were examined for antibody binding by indirect immunofluorescence and by solid phase radioimmunoassay. The procedure for immunofluorescence has been reported. Briefly, cells were aliquoted at 2 x 105 tube, incubated with 25 gsl of supernatant for 60 min at 37'C, washed, and incubated with 50 p1l of fluorescein-conjugated goat antimouse antibody.
Immunoperoxidase staining of tissues Frozen tissues were cut into 5 1lm sections, mounted on glued slides and treated with methanol peroxide and 2% swine serum as blocking reagent. The sections were covered with antibody and incubated for I h at room temperature. After washing peroxidase-conjugated rabbit anti-mouse diluted 1:30 and peroxidase swine anti-rabbit 1:60 were added sequentially. The reaction was localised with 3,3'-diaminobenzidine tetramonohydrate. Slides were counterstained with hematoxylin. Control studies included substitution with an irrelevant antibody (anti-leucocyte common antigen; Dako corp.). Antibody purification and analysis Antibodies SWA20, SEN3, SENl 1, SEN 12 and SEN31 were purified as follows: A 50% ammonium sulphate fraction was taken from culture supernatant and absorbed onto a protein A column in PBS (for IgG2a, IgG2b, IgG3) or in 3.3 M NaCl, 50 mM Tris pH 8.9 for IgGi. The absorbed IgG was eluted with 50 mM acetate buffer containing 150 mM NaCl at pH 6 for IgG, or pH 4.5 for IgG2a and IgG2b and glycine/HCI buffer containing 150 mM NaCl at pH 3 for IgG3. The antibodies were dialysed against 25 mM acetate pH 6 for SENl SEN12, SEN31 and against 25 mM acetate pH 5.5 for SEN3 and SWA20. The antibodies were then applied to a 1,
30
R. WAIBEL et al.
Mono-S cation-exchange column (Pharmacia) and were eluted with a NaCl-gradient. Their purity was checked by SDS gel analysis and isoelectric focusing.
Solid-phase radioimmunoassays Target cells were fixed to 96-well plates. The plates were coated with poly-L-lysine and 5 x 104 cells fixed to each well using glutaraldehyde. Plates were stored at 4°C in PBS containing 1% BSA and 0.2% sodium azide. Binding was determined by incubating the washed cells (Tris-buffered saline (TBS), 5% non-fat milk and 1% gelatine) for 1 h with saturating amounts of monoclonal antibody. After three washings the bound antibody was detected with 4 yCi ml' 1251I goat anti-mouse IgG (Amersham). After a final washing individual wells were cut out and counted in a gamma counter. The binding ratio was defined as: (Counts bound with antibody-background)/(counts bound with PBS-background). For competitive radioimmunoassays, unlabelled antibodies were added at saturating concentrations, incubated for 1 h and washed as before. Radiolabelled SWA20 (iodinated by the lodogen method) was then added at a concentration giving one-half maximal binding, incubated for 1 h, washed and processed as before. Antibody SWAl to cluster-w4 antigen served as a control (Smith et al., 1989). Enzymatic and periodate treatment of antigens For periodate treatment of antigens cells were incubated with 5 mM periodate at pH 7 at room temperature for 15 min. The reaction was stopped with ethylene glycol. For enzyme treatment the cells were pre-incubated for 1 h at 37°C with one unit of neuraminidase (Clostridium perfringens) or one unit of trypsin or 10 mg ml-' of mixed glycosidases. Mixed glycosidases is a mixture of exoglycosidases from Charonia lampas containing 0.3 U 10 mg-' mannosidase, galactosidase, fucosidase, 1.7 U 10 mg-' acetylglucosaminidase and 0.5 U 10 mg-' acetyl-galactosaminidase. The cells were then incubated at saturating concentrations with the antibodies washed and processed as before.
FACScan analysis of antigen expression of SW2-cells treated with modulators ofglycoprotein biosynthesis SW2 cells (5 x 104) were treated with 1 U ml-' neuraminidase overnight, the cells washed and incubated with normal medium or medium supplemented with inhibitors of glycoprotein synthesis. Tunicamycin and swainsonine were used at 1 fig ml P-hydroxy-norvaline was used at 10 mM and monensin at 2 tg ml-'. The cells were cultured for 3 days, washed, exposed to test antibodies (SWA20, SEN3, SEN31) or a control antibody (SWA1 1) for 1 h. Cells were washed and incubated with fluorescein-labelled goat anti-mouse antibody for 30 min and cellular binding analysed on a FACScan (Becton-Dickinson). 1,
Immunological detection of antigens transferredfrom SDS gels onto nitrocellulose Electrophoresis was accomplished on 7.5% SDS-polyacrylamide gels under reducing conditions using the buffer system of O'Farrel. Aliquots of 5 x 106 cells were used. The cells were lysed in 20 mM N-dodecyl-N,N-dimethylammonio-3propanesulfonate (Serva) in ice-cold PBS for 30 min and centrifuged for 1 h at 100,000 g, containing as enzyme inhibitors 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin and 50 1tg ml-' pepstatin A (Fluka). After electrophoresis, gels were washed for 30 min in 4 M urea (Waibel et al., 1987) and the proteins were transferred electrophoretically onto 0.2pm nitrocellulose in a two-step procedure, beginning with elution of low molecular weight molecules at low current density, followed by prolonged electrotransfer at high current density in transfer buffer. After electrotransfer, the sheets were quenched in TBS con-
taining 5% nonfat dry milk and incubated overnight at 4'C with monoclonal antibody supernatants. After washing, the sheets were incubated with affinity-purified goat anti-mouse IgG, alkaline phosphatase conjugate (Bio-Rad) diluted 1:3000 and the colour was developed with 5-bromo-4-chloro3-indoyl phosphate p-toluidine salt and nitrotetrazolium blue chloride (Fluka) in 100 mM ethanolamin, 1 mM MgCl at pH9.8. Immunoblot of tumour cells digested with neuraminidase SW2 cells were incubated with 1 unit neuraminidase (Clostridium perfringens) for 4 h, washed and cultivated in RPMI and 10% FCS. The antigen reappearance was chased for 48 h. Aliquots of cells were removed at 0 h, 4 h, 8 h, 24 h and 48 h post-treatment, lysed, electrophoresed, blotted and developed with SWA20 and alkaline phosphatase conjugated goat anti-mouse as before. Results
Characterisation of antibodies The antibodies SEN3, SENI 1, SEN12 and SEN31 are all IgG antibodies with isoelectric points close to neutral pH. Binding ratios to the SCLC cell line SW2 were determined in a solid phase radioimmunoassay and ranged between four for SENIl and 13 for SEN31. A solid phase radioimmunoassay was used to examine competitive binding between the new antibodies and radiolabelled antibody SWA20 on the SCLC cell line SW2. The antibody SWAl 1, a murine IgG2, directed against cluster-w4 antigen was used as a control. The antibodies SEN3, SENl 1, SEN12 and SEN31, but not antibody SWAl 1 competed with antibody SWA20 for cellular binding (Table I). These results suggest that the new antibodies recognise a related epitope on the cluster-5A antigen. Binding of antibodies to cell lines and tissues Indirect immunofluorescence staining of viable SCLC cells with the antibodies SEN3, SENl1, SEN12 and SEN31 demonstrated a ring type staining. All antibodies had the same pattern of reactivity in cell lines when examined by indirect immunofluorescence on live cells. Strong staining was seen with the four SCLC cell lines SW2, OHI, NCI-H69, and NCI-H249, whereas the three SCLC cell lines NCI-N417, NCI-H446 and NCI-H526, the lung adenocarcinoma cell line SCL52, the mesothelioma cell line SPC1 11, the lung large cell carcinoma cell line SLC6, the breast carcinoma cell line MCF-7, the renal cell carcinoma cell line FOHN and the leukaemia cell line K562 were negative. None of the antibodies reacted with buffy coat of peripheral blood cells or Ficoll preparations of bone marrow cell aspirates. A comparative analysis of antibody binding to a limited number of frozen sections of human tissues was also performed. No significant differences in tissue binding were found as demonstrated in Table II. Significant staining was seen in three of seven small cell lung carcinomas, but not on normal tissues. Table I Binding competition between radiolabelled antibody SWA20 and supernatant of unlabelled SCLC antibodies using a solid phase radioimmunoassay with SW2 cells Percent bindinga Unlabelled antibody 100 None .9 SWA20 22 SEN3 4 SENI1 SEN12 25 SEN31 4 SWAI 1 107 aExpressed as percentage binding of SWA20 in the absence of competing antibody.
ANTIBODIES DEFINING CLUSTER-5A ANTIGEN Table II Comparative examination of antibody reactivity in frozen sections by immunoperoxidase
staining
Tissues SCLC
Antibodies SEN12
SEN3D SWA20 a@@OOOOO @000000 @000000 @0@0000 *@@000 SEN3
SENJJ
Kidney 0 0 0 Liver 0 0 0 Bronchus 0 0 2' Colon 0 0 0 Skin 0 0 0 Brain 0 0 0 a0, strong positive staining; 0, occasional positive cells; 0, no staining.
Characterisation of antigens SDS-PAGE analysis was performed on a detergent extract of SW2 cells followed by Western blotting of proteins from SDS gels to nitrocellulose membranes. Antibody SWA20, SEN3, SEN 11, SEN12 and SEN31 had the same pattern of reactivity revealing a small band around 180 kD, a prominent band around 100 kD and a third smaller band around 50 kD. The control antibody SWAl 1 recognised a different band with strong reactivity around 40 kD (Figure 1). Solid phase radioimmunoassays were used for analysis of antibody binding to SW2 cells digested enzymatically or oxidised with periodate. The sensitivity of the antigen binding was similar for antibody SWA20 and all four new antibodies. The antigen was resistant to trypsin under the conditions tested. High glycosylation seems to protect the antigen from protease digestion. Mixed glycosidases, a mixture of exoglycosidases without neuraminidase activity, were unable to influence the antigen recognition. The epitope was susceptible to neuraminidase digestion, which decreased binding of all antibodies between 54 and 77%. Periodate treatment decreased the reactivity of the antigen markedly for SEN3, SENl 1 and SEN31 and to a lesser degree for SEN12 and SWA20 (Table III). To analyse the re-synthesis of the antigen after neuraminidase digestion, SW2 cells were incubated for 4 h with 1 U ml-' neuraminidase, washed and cultivated in RPMI and 10% FCS. The re-synthesis of the antigen was followed for 48 h by Western blotting. Aliquots of cells were removed at 0 h, 4 h, 8 h, 24 h and 48 h, extracted with detergent, run on a SDS gel and blotted onto nitrocellulose. The reappearance of the antigen was probed with antibody SWA20. The results are depicted in Figure 2. Initial reactivity appeared after 4 h at the 100 kD band. The high molecular band at 180 kD reappeared 8 h post-treatment. To examine whether the carbohydrate epitope of the clus-
84a4 58
0
0 0 0
0 0
0
0
Sensitivity of antigen recognition to enzyme and periodate
treatment using a solid phase radioimmunoassay on the SCLC cell line
SW2
Monoclonal antibody SEN3 SENJJ SEN12 SEN31 SWA20 Enzyme 121 124 87 93 100 Trypsin (1 U) Neuraminidase (0.1 U) 56 54 69 77 64 Mixed glycosidase (lOmg) 113 117 127 115 113 Periodate (5 mM) 8 20 52 18 80 The results are expressed as percent binding on untreated target cells.
180 116
84 58
49
19~~~~~~~1 180
116
Table III
0
0 e 0
37
M
2
3
4
5
C
Figure 2 Immunoblot of SW2 cell extracts developed with antibody SWA20. Cells were incubated with neuraminidase for 4 h, washed and cultivated in RPMI and 10% FCS. Molecular weight markers (M) in thousands are: A2-macroglobulin (180,000), pgalactosidase (116,000); fructose 6-phosphate kinase (84,000); pyruvate kinase (58,000); fumarase (48,500); lactic dehydrogenase (36,500); and triosephosphate isomerase (26,000). Lanes contain detergent extract of digested cells lysed 0 h post-treatment (lane 1), lysed 8 h post-treatment (lane 2), lysed 24 h post-treatment
(lane 3) and lysed 48 h post-treatment (lane 4). 4 antigen of MW 180,000 and 100,000.
49
27-
M 1 2 3 4 5 Figure 1 Immunoblots of SW2 cell extracts developed with cluster-5A antibodies and cluster-w4 antibody as a control. Lanes are developed with SEN3 (lane 1), SENl 1 (lane 2), SEN12 (lane 3), SEN31 (lane 4) and SWA20 (lane 5). Antibody SWA Il was used as a control (lane C). M. molecular weight markers (see legend to Figure 2).
ter-SA antigen might be a N-linked oligosaccharide the influence of different inhibitors of glycoprotein biosynthesis on antigen re-synthesis after neuraminidase digestion was studied. Neuraminidase treated cells were cultured in the presence of inhibitors for 3 days and the antigen expression determined by FACS analysis using the antibodies SWA20, SEN3 and SEN3 1. Antibody SWA 11 directed against a protein epitope was used as a control. The results are summarised in Table IV. Tunicamycin which prevents N-glycosylation inhibited antigen re-synthesis, thus demonstrating the carbohydrate epitope to be N-linked to asparagine of the protein backbone. The threonine analogue P-hydroxynorvaline was used
32
R. WAIBEL et al.
Table IV Influence of inhibitors of glycoprotein synthesis on antigen
expression Percent cells positive with SWA20 SEN3 SEN31 SWAJJ 78 65 57 98
Inhibitor None Tunicamycin (1 tLgml-') 6 14 50 20 Swainsonine (I lg ml-') 8 7 20 95 2 2 6 81 P-Hydroxynorvalin (10 mM) Monensin (2 Lg ml-') 8 5 n.d. 84 SW2 cells were incubated with inhibitors of glycoprotein synthesis following pre-treatment with neuraminidase. After 3 days the percent of cells expressing the cluster-5A antigen was determined by FACS using the antibody SWA20. Antibody SWAI I to cluster-w4 antigen serviced as control.
to prevent the transfer of N-linked oligosaccharide-units to Asn-X-Thr sequences. In the presence of this inhibitor, only Asn-X-Ser can become glycosylated (Polonoff et al., 1982). No antigen reactivity was seen with this inhibitor, demonstrating that the carbohydrate epitope is linked to Asp-X-Thr and not to Asp-X-Ser. A decrease of antigen recognition was also seen in the presence of swainsonine, an inhibitor of Golgi mannosidase-II which prevents the formation of complex glycoproteins. This suggests that complex oligosaccharide structures are needed for antigen recognition. Monensin, a sialyltransferase inhibitor prevented resialylation of the epitope and thus antigen recognition.
Discussion
The results presented in this paper confirm that the new murine IgG antibodies SEN3, SENIl, SEN12 and SEN31 recognise the SCLC antigen designated cluster-5A. This antigen which was initially defined by the antibody SWA20 is a tumour-associated sialoglycoprotein expressed on a proportion of SCLC, but not on non-small cell lung carcinoma cell lines and tissues (Waibel et al., 1988; Maier et al., 1989). Indirect evidence for the recognition of a cluster-5A antigen by the new antibodies was first obtained by the comparative examination of antibody binding to cell lines and human tissues. Solid phase radioimmunoassays demonstrated direct binding competition between the new antibodies and anti-
body SWA20 suggesting binding to a closely related epitope. Additional evidence for the identity of the antigens with cluster-SA antigen was obtained by Western blotting. All new antibodies and SWA20 reacted with bands of 180, 100 and 50 kD on the SCLC cell line SW2. Similarities were also seen in the sensitivity of antigen recognition to treatment of fixed SCLC cells with neuraminidase and periodate. Recently we were able to further confirm the close identity of the epitopes recognised by antibodies SWA20 and antibodies SEN3, SENI1, SEN12, and SEN31 by immunological methods (Zwicky et al., 1990). Blocking experiments with a purified polyclonal goat-anti-SWA20-idiotype serum have shown inhibition of binding of all cluster-5A antibodies to antigen positive SCLC cells. Synthesis and expression of large complex type asparaginelinked oligosaccharides are consistently observed following neoplastic transformation (Dennis et al., 1988). These large oligosaccharides typically exhibit increased sialylation and increased branching. To determine whether analogous alterations of the oligosaccharide structure are part of the epitope recognised by the antibodies to cluster-SA antigen, we performed a series of analyses on antigen re-synthesis after enzymatic destruction of the sialylated epitope. Antigen resynthesis was blocked by tunicamycin, an inhibitor of normal processing of asparagine-linked glycoproteins and ,B-hydroxynorvalin which prevents the transfer of the oligosaccharide part of lipid intermediates to asparagin-x-threonine. Swainsonine which promotes synthesis of hybrid-type oligosaccharides instead of complex-type oligosaccharides blocked the re-synthesis as well. These results suggest that sialylated complex-type oligosaccharides, N-linked to asparagine-xthreonine are an important feature of the antigen. We are currently exploring the use of cluster-SA antigen as a target for the therapy of SCLC. The availability of a series of antibodies of different isotypes directed to this antigen should allow us to easily select individual reagents based on important properties, such as stability in vitro, retention of immunogenicity after radiolabelling and antibody affinity to use for delivery of toxins (Warwrzynczak et al., 1990). We appreciate the technical assistance of Gudrun Christiansen. This work was supported by the Swiss Cancer League grants FOR.802. 87.1 and FOR.302.89.2.
References
DENNIS, J.W. & LAFERTE, S. (1988). Asn-linked oligosaccharides and the metastatic phenotype. In Altered Glycosilation in Tumor Cells. Reading, C.L., Hakomori, S. & Marcus, D. (ed.) p. 257. A.R. Liss: New York. MAIER, A., SCHMIDT, U., WAIBEL, R., HARTUNG, W. & STAHEL,
R.A. (1989). Expression of small cell carcinoma antigens of cluster-S and cluster-5A in primary lung tumors. Br. J. Cancer, 59, 692. POLONOFF, E., MACHIDA, C.A. & KABAT, D. (1982). Glycosilation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acids analogues and by tunicamycin. J. Biol. Chem., 257, 14023.
SMITH, A., WAIBEL, R., WESTERA, G., MARTIN, A., ZIMMERMANN,
A.T. & STAHEL, R.A. (1989). Immunolocalisation and imaging of small cell cancer xenografts by the IgGu monoclonal antibody SWAI I. Br. J. Cancer, 59, 174. SOUHAMI, R.L., BEVERLEY, P.C.L. & BOBROW, L. (1988). (eds) The first international workshop on small cell lung cancer antigens. Lung Cancer, 4, 1. STAHEL, R.A., SPEAK, J.A. & BERNAL, S.D. (1985). Murine monoclonal antibody LAM2 defines cell membrane determinant with preferential expression on human small cell carcinoma and squamous cell carcinoma. Int. J. Cancer, 35, 11.
WAIBEL, R., O'HARA, C.J., SMITH, A. & STAHEL, R.A. (1988). A tumour-associated membrane sialoglycoprotein on human small
cell carcinoma identified by the IgG2, monoclonal antibody SWA20. Cancer Res., 48, 4318. WAIBEL, R., O'HARA, C.J. & STAHEL, R.A. (1987). Characterization of an epithelial and a tumor-associated human small cell lung carcinoma antigen. Cancer Res., 47, 3766. WAWRZYNCZAK, E.J., DERBYSHIRE, E.J., HENRY, V.R. & 4 others (1990). Selective cytotoxic effects of a ricin a chain immunotoxin made with the monoclonal antibody SWA1I recognising a human small cell lung cancer antigen. Br. J. Cancer, 62, 410. ZWICKY, C., WAIBEL, R., JAKSCHE, H. & STAHEL, R.A. (1990). Polyclonal anti-idiotypic antibody mimicking the small cell lung carcinoma (SCLC) cluster-5A antigen interacts with a panel of antibodies to the same antigen and induces specific immune response in animals. Proc. Amer. Assoc. Cancer Res., 31, 281.
Cancer
Br. J. Cancer
(1991),
(1991), 63, Suppl. XIV, 29-32 63,
Suppl.
XIV,
29-32
f,-"%
Macmillan Press Ltd., 1991
©
Macmillan
Press
Ltd.,
1991~~~~
Monoclonal antibodies defining the cluster-5A small cell lung carcinoma antigen R. Waibell, C.J. O'Hara2 & R.A. Stahel' 'Division of Oncology, Department of Medicine, University Hospital, CH-8091 Zurich, Switzerland; and 2Department of Pathology, New England Deaconess Hospital, Boston, Massachusetts 02115, USA.
Summary The cluster-5A antigen on small cell lung cancer has been previously identified by the murine IgG2. monoclonal antibody SWA20. We now describe four new murine monoclonal antibodies, SEN3 (IgG,), SENl (IgG3), SEN12 (IgG2b) and SEN31 (IgG,) which recognise the same antigen. The antibodies have similar binding characteristics as antibody SWA20 on cell lines and on a limited number of frozen sections of human tissues when examined by immunofluorescence and immunohistochemistry. Radioimmunoassays on fixed cells showed competitive binding of all antibodies with radiolabelled antibody SWA20. Solid phase radioimmunoassays with the new antibodies on biochemically treated target cells showed similar characteristics to antibody SWA20, including sensitivity to treatment with periodate and neuraminidase. Immunoblotting experiments with all antibodies revealed similar bands at molecular weights of 180, 100 and 50 kD. Binding studies after incubation of cells with inhibitors of glycoprotein synthesis suggest the cluster-5A antigen to be a N-linked sialoglycoprotein.
We have previously reported on the murine monoclonal antibody SWA20 which recognises a tumour-associated antigen on small cell lung cancer (SCLC) (Waibel et al., 1988). The antigen is a sialoglycoprotein strongly expressed on the surface membrane of one third to one half of small cell lung carcinomas (SCLC). The antigen has little or no expression on normal epithelial tissues and no expression on normal neural and hemopoietic tissues. At the First International Workshop on Small Cell Lung Carcinoma antigens, the antigen defined by antibody SWA20 was classified as cluster-5A antigen (Souhami et al., 1988). Since that time, the selectivity of expression of this antigen in SCLC, but not in other lung tumours has been confirmed by extensive immunohistochemical studies on bronchial biopsies and resected tumour tissues (Maier et al., 1989). In an effort to define new small cell lung cancer antigens and to expand the number of reagents which might be useful for therapy of SCLC lung cancer, new monoclonal antibodies have been generated in our laboratory. The analysis of cell line reactivity of four of the new antibodies suggested that they might recognise the same or a similar antigen as antibody SWA20. Investigations presented here demonstrate that the four antibodies SEN3, SENI 1, SEN12 and SEN31 bind to the cluster-5A antigen. Further studies on the nature of cluster-5A antigen suggest the antigen to be an N-linked
sialoglycoprotein. Materials and methods Cell lines All cell lines were grown in RPMI supplemented with glutamine and 10% FCS. The SCLC cell lines NCI-H249, NCIH417, NCI-H446, NCI-H524 and NCI-H526 were provided by Dr D.N. Carney, Mater Private Hospital, Dublin. Other cell lines employed for screening of antibody reactivity were generated in our own laboratory or obtained from ATCC or from sources that have been described previously (Waibel et al., 1988).
Antibody generation and characterisation Our procedure for antibody generation and selection of hybrids has been reported (Stahel et al., 1985). Hybridomas of Correspondence: R.A. Stahel, Division of Oncology, University Hospital, CH-8091 Zurich, Switzerland.
the SEN clones SEN3 IgGl, SENl IgG3, SEN12 IgG2b and SEN31 IgGl were obtained by injection of viable SW2 cells into Balb/c mice and fusion of the spleen cells with mouse X63Ag8.653 myeloma cells. Antibodies which by indirect immunofluorescence displayed a ring pattern of reactivity with SW2, but were negative with leukaemia, adenocarcinoma or squamous cell carcinoma cell lines were chosen initially. Further selection was for lack of reactivity with normal blood and bone marrow and for IgG isotype which was determined by Ouchterlony double diffusion assays and with a dipstick assay (Amersham).
Immunofluorescence staining Cell lines were examined for antibody binding by indirect immunofluorescence and by solid phase radioimmunoassay. The procedure for immunofluorescence has been reported. Briefly, cells were aliquoted at 2 x 105 tube, incubated with 25 gsl of supernatant for 60 min at 37'C, washed, and incubated with 50 p1l of fluorescein-conjugated goat antimouse antibody.
Immunoperoxidase staining of tissues Frozen tissues were cut into 5 1lm sections, mounted on glued slides and treated with methanol peroxide and 2% swine serum as blocking reagent. The sections were covered with antibody and incubated for I h at room temperature. After washing peroxidase-conjugated rabbit anti-mouse diluted 1:30 and peroxidase swine anti-rabbit 1:60 were added sequentially. The reaction was localised with 3,3'-diaminobenzidine tetramonohydrate. Slides were counterstained with hematoxylin. Control studies included substitution with an irrelevant antibody (anti-leucocyte common antigen; Dako corp.). Antibody purification and analysis Antibodies SWA20, SEN3, SENl 1, SEN 12 and SEN31 were purified as follows: A 50% ammonium sulphate fraction was taken from culture supernatant and absorbed onto a protein A column in PBS (for IgG2a, IgG2b, IgG3) or in 3.3 M NaCl, 50 mM Tris pH 8.9 for IgGi. The absorbed IgG was eluted with 50 mM acetate buffer containing 150 mM NaCl at pH 6 for IgG, or pH 4.5 for IgG2a and IgG2b and glycine/HCI buffer containing 150 mM NaCl at pH 3 for IgG3. The antibodies were dialysed against 25 mM acetate pH 6 for SENl SEN12, SEN31 and against 25 mM acetate pH 5.5 for SEN3 and SWA20. The antibodies were then applied to a 1,
30
R. WAIBEL et al.
Mono-S cation-exchange column (Pharmacia) and were eluted with a NaCl-gradient. Their purity was checked by SDS gel analysis and isoelectric focusing.
Solid-phase radioimmunoassays Target cells were fixed to 96-well plates. The plates were coated with poly-L-lysine and 5 x 104 cells fixed to each well using glutaraldehyde. Plates were stored at 4°C in PBS containing 1% BSA and 0.2% sodium azide. Binding was determined by incubating the washed cells (Tris-buffered saline (TBS), 5% non-fat milk and 1% gelatine) for 1 h with saturating amounts of monoclonal antibody. After three washings the bound antibody was detected with 4 yCi ml' 1251I goat anti-mouse IgG (Amersham). After a final washing individual wells were cut out and counted in a gamma counter. The binding ratio was defined as: (Counts bound with antibody-background)/(counts bound with PBS-background). For competitive radioimmunoassays, unlabelled antibodies were added at saturating concentrations, incubated for 1 h and washed as before. Radiolabelled SWA20 (iodinated by the lodogen method) was then added at a concentration giving one-half maximal binding, incubated for 1 h, washed and processed as before. Antibody SWAl to cluster-w4 antigen served as a control (Smith et al., 1989). Enzymatic and periodate treatment of antigens For periodate treatment of antigens cells were incubated with 5 mM periodate at pH 7 at room temperature for 15 min. The reaction was stopped with ethylene glycol. For enzyme treatment the cells were pre-incubated for 1 h at 37°C with one unit of neuraminidase (Clostridium perfringens) or one unit of trypsin or 10 mg ml-' of mixed glycosidases. Mixed glycosidases is a mixture of exoglycosidases from Charonia lampas containing 0.3 U 10 mg-' mannosidase, galactosidase, fucosidase, 1.7 U 10 mg-' acetylglucosaminidase and 0.5 U 10 mg-' acetyl-galactosaminidase. The cells were then incubated at saturating concentrations with the antibodies washed and processed as before.
FACScan analysis of antigen expression of SW2-cells treated with modulators ofglycoprotein biosynthesis SW2 cells (5 x 104) were treated with 1 U ml-' neuraminidase overnight, the cells washed and incubated with normal medium or medium supplemented with inhibitors of glycoprotein synthesis. Tunicamycin and swainsonine were used at 1 fig ml P-hydroxy-norvaline was used at 10 mM and monensin at 2 tg ml-'. The cells were cultured for 3 days, washed, exposed to test antibodies (SWA20, SEN3, SEN31) or a control antibody (SWA1 1) for 1 h. Cells were washed and incubated with fluorescein-labelled goat anti-mouse antibody for 30 min and cellular binding analysed on a FACScan (Becton-Dickinson). 1,
Immunological detection of antigens transferredfrom SDS gels onto nitrocellulose Electrophoresis was accomplished on 7.5% SDS-polyacrylamide gels under reducing conditions using the buffer system of O'Farrel. Aliquots of 5 x 106 cells were used. The cells were lysed in 20 mM N-dodecyl-N,N-dimethylammonio-3propanesulfonate (Serva) in ice-cold PBS for 30 min and centrifuged for 1 h at 100,000 g, containing as enzyme inhibitors 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin and 50 1tg ml-' pepstatin A (Fluka). After electrophoresis, gels were washed for 30 min in 4 M urea (Waibel et al., 1987) and the proteins were transferred electrophoretically onto 0.2pm nitrocellulose in a two-step procedure, beginning with elution of low molecular weight molecules at low current density, followed by prolonged electrotransfer at high current density in transfer buffer. After electrotransfer, the sheets were quenched in TBS con-
taining 5% nonfat dry milk and incubated overnight at 4'C with monoclonal antibody supernatants. After washing, the sheets were incubated with affinity-purified goat anti-mouse IgG, alkaline phosphatase conjugate (Bio-Rad) diluted 1:3000 and the colour was developed with 5-bromo-4-chloro3-indoyl phosphate p-toluidine salt and nitrotetrazolium blue chloride (Fluka) in 100 mM ethanolamin, 1 mM MgCl at pH9.8. Immunoblot of tumour cells digested with neuraminidase SW2 cells were incubated with 1 unit neuraminidase (Clostridium perfringens) for 4 h, washed and cultivated in RPMI and 10% FCS. The antigen reappearance was chased for 48 h. Aliquots of cells were removed at 0 h, 4 h, 8 h, 24 h and 48 h post-treatment, lysed, electrophoresed, blotted and developed with SWA20 and alkaline phosphatase conjugated goat anti-mouse as before. Results
Characterisation of antibodies The antibodies SEN3, SENI 1, SEN12 and SEN31 are all IgG antibodies with isoelectric points close to neutral pH. Binding ratios to the SCLC cell line SW2 were determined in a solid phase radioimmunoassay and ranged between four for SENIl and 13 for SEN31. A solid phase radioimmunoassay was used to examine competitive binding between the new antibodies and radiolabelled antibody SWA20 on the SCLC cell line SW2. The antibody SWAl 1, a murine IgG2, directed against cluster-w4 antigen was used as a control. The antibodies SEN3, SENl 1, SEN12 and SEN31, but not antibody SWAl 1 competed with antibody SWA20 for cellular binding (Table I). These results suggest that the new antibodies recognise a related epitope on the cluster-5A antigen. Binding of antibodies to cell lines and tissues Indirect immunofluorescence staining of viable SCLC cells with the antibodies SEN3, SENl1, SEN12 and SEN31 demonstrated a ring type staining. All antibodies had the same pattern of reactivity in cell lines when examined by indirect immunofluorescence on live cells. Strong staining was seen with the four SCLC cell lines SW2, OHI, NCI-H69, and NCI-H249, whereas the three SCLC cell lines NCI-N417, NCI-H446 and NCI-H526, the lung adenocarcinoma cell line SCL52, the mesothelioma cell line SPC1 11, the lung large cell carcinoma cell line SLC6, the breast carcinoma cell line MCF-7, the renal cell carcinoma cell line FOHN and the leukaemia cell line K562 were negative. None of the antibodies reacted with buffy coat of peripheral blood cells or Ficoll preparations of bone marrow cell aspirates. A comparative analysis of antibody binding to a limited number of frozen sections of human tissues was also performed. No significant differences in tissue binding were found as demonstrated in Table II. Significant staining was seen in three of seven small cell lung carcinomas, but not on normal tissues. Table I Binding competition between radiolabelled antibody SWA20 and supernatant of unlabelled SCLC antibodies using a solid phase radioimmunoassay with SW2 cells Percent bindinga Unlabelled antibody 100 None .9 SWA20 22 SEN3 4 SENI1 SEN12 25 SEN31 4 SWAI 1 107 aExpressed as percentage binding of SWA20 in the absence of competing antibody.
ANTIBODIES DEFINING CLUSTER-5A ANTIGEN Table II Comparative examination of antibody reactivity in frozen sections by immunoperoxidase
staining
Tissues SCLC
Antibodies SEN12
SEN3D SWA20 a@@OOOOO @000000 @000000 @0@0000 *@@000 SEN3
SENJJ
Kidney 0 0 0 Liver 0 0 0 Bronchus 0 0 2' Colon 0 0 0 Skin 0 0 0 Brain 0 0 0 a0, strong positive staining; 0, occasional positive cells; 0, no staining.
Characterisation of antigens SDS-PAGE analysis was performed on a detergent extract of SW2 cells followed by Western blotting of proteins from SDS gels to nitrocellulose membranes. Antibody SWA20, SEN3, SEN 11, SEN12 and SEN31 had the same pattern of reactivity revealing a small band around 180 kD, a prominent band around 100 kD and a third smaller band around 50 kD. The control antibody SWAl 1 recognised a different band with strong reactivity around 40 kD (Figure 1). Solid phase radioimmunoassays were used for analysis of antibody binding to SW2 cells digested enzymatically or oxidised with periodate. The sensitivity of the antigen binding was similar for antibody SWA20 and all four new antibodies. The antigen was resistant to trypsin under the conditions tested. High glycosylation seems to protect the antigen from protease digestion. Mixed glycosidases, a mixture of exoglycosidases without neuraminidase activity, were unable to influence the antigen recognition. The epitope was susceptible to neuraminidase digestion, which decreased binding of all antibodies between 54 and 77%. Periodate treatment decreased the reactivity of the antigen markedly for SEN3, SENl 1 and SEN31 and to a lesser degree for SEN12 and SWA20 (Table III). To analyse the re-synthesis of the antigen after neuraminidase digestion, SW2 cells were incubated for 4 h with 1 U ml-' neuraminidase, washed and cultivated in RPMI and 10% FCS. The re-synthesis of the antigen was followed for 48 h by Western blotting. Aliquots of cells were removed at 0 h, 4 h, 8 h, 24 h and 48 h, extracted with detergent, run on a SDS gel and blotted onto nitrocellulose. The reappearance of the antigen was probed with antibody SWA20. The results are depicted in Figure 2. Initial reactivity appeared after 4 h at the 100 kD band. The high molecular band at 180 kD reappeared 8 h post-treatment. To examine whether the carbohydrate epitope of the clus-
84a4 58
0
0 0 0
0 0
0
0
Sensitivity of antigen recognition to enzyme and periodate
treatment using a solid phase radioimmunoassay on the SCLC cell line
SW2
Monoclonal antibody SEN3 SENJJ SEN12 SEN31 SWA20 Enzyme 121 124 87 93 100 Trypsin (1 U) Neuraminidase (0.1 U) 56 54 69 77 64 Mixed glycosidase (lOmg) 113 117 127 115 113 Periodate (5 mM) 8 20 52 18 80 The results are expressed as percent binding on untreated target cells.
180 116
84 58
49
19~~~~~~~1 180
116
Table III
0
0 e 0
37
M
2
3
4
5
C
Figure 2 Immunoblot of SW2 cell extracts developed with antibody SWA20. Cells were incubated with neuraminidase for 4 h, washed and cultivated in RPMI and 10% FCS. Molecular weight markers (M) in thousands are: A2-macroglobulin (180,000), pgalactosidase (116,000); fructose 6-phosphate kinase (84,000); pyruvate kinase (58,000); fumarase (48,500); lactic dehydrogenase (36,500); and triosephosphate isomerase (26,000). Lanes contain detergent extract of digested cells lysed 0 h post-treatment (lane 1), lysed 8 h post-treatment (lane 2), lysed 24 h post-treatment
(lane 3) and lysed 48 h post-treatment (lane 4). 4 antigen of MW 180,000 and 100,000.
49
27-
M 1 2 3 4 5 Figure 1 Immunoblots of SW2 cell extracts developed with cluster-5A antibodies and cluster-w4 antibody as a control. Lanes are developed with SEN3 (lane 1), SENl 1 (lane 2), SEN12 (lane 3), SEN31 (lane 4) and SWA20 (lane 5). Antibody SWA Il was used as a control (lane C). M. molecular weight markers (see legend to Figure 2).
ter-SA antigen might be a N-linked oligosaccharide the influence of different inhibitors of glycoprotein biosynthesis on antigen re-synthesis after neuraminidase digestion was studied. Neuraminidase treated cells were cultured in the presence of inhibitors for 3 days and the antigen expression determined by FACS analysis using the antibodies SWA20, SEN3 and SEN3 1. Antibody SWA 11 directed against a protein epitope was used as a control. The results are summarised in Table IV. Tunicamycin which prevents N-glycosylation inhibited antigen re-synthesis, thus demonstrating the carbohydrate epitope to be N-linked to asparagine of the protein backbone. The threonine analogue P-hydroxynorvaline was used
32
R. WAIBEL et al.
Table IV Influence of inhibitors of glycoprotein synthesis on antigen
expression Percent cells positive with SWA20 SEN3 SEN31 SWAJJ 78 65 57 98
Inhibitor None Tunicamycin (1 tLgml-') 6 14 50 20 Swainsonine (I lg ml-') 8 7 20 95 2 2 6 81 P-Hydroxynorvalin (10 mM) Monensin (2 Lg ml-') 8 5 n.d. 84 SW2 cells were incubated with inhibitors of glycoprotein synthesis following pre-treatment with neuraminidase. After 3 days the percent of cells expressing the cluster-5A antigen was determined by FACS using the antibody SWA20. Antibody SWAI I to cluster-w4 antigen serviced as control.
to prevent the transfer of N-linked oligosaccharide-units to Asn-X-Thr sequences. In the presence of this inhibitor, only Asn-X-Ser can become glycosylated (Polonoff et al., 1982). No antigen reactivity was seen with this inhibitor, demonstrating that the carbohydrate epitope is linked to Asp-X-Thr and not to Asp-X-Ser. A decrease of antigen recognition was also seen in the presence of swainsonine, an inhibitor of Golgi mannosidase-II which prevents the formation of complex glycoproteins. This suggests that complex oligosaccharide structures are needed for antigen recognition. Monensin, a sialyltransferase inhibitor prevented resialylation of the epitope and thus antigen recognition.
Discussion
The results presented in this paper confirm that the new murine IgG antibodies SEN3, SENIl, SEN12 and SEN31 recognise the SCLC antigen designated cluster-5A. This antigen which was initially defined by the antibody SWA20 is a tumour-associated sialoglycoprotein expressed on a proportion of SCLC, but not on non-small cell lung carcinoma cell lines and tissues (Waibel et al., 1988; Maier et al., 1989). Indirect evidence for the recognition of a cluster-5A antigen by the new antibodies was first obtained by the comparative examination of antibody binding to cell lines and human tissues. Solid phase radioimmunoassays demonstrated direct binding competition between the new antibodies and anti-
body SWA20 suggesting binding to a closely related epitope. Additional evidence for the identity of the antigens with cluster-SA antigen was obtained by Western blotting. All new antibodies and SWA20 reacted with bands of 180, 100 and 50 kD on the SCLC cell line SW2. Similarities were also seen in the sensitivity of antigen recognition to treatment of fixed SCLC cells with neuraminidase and periodate. Recently we were able to further confirm the close identity of the epitopes recognised by antibodies SWA20 and antibodies SEN3, SENI1, SEN12, and SEN31 by immunological methods (Zwicky et al., 1990). Blocking experiments with a purified polyclonal goat-anti-SWA20-idiotype serum have shown inhibition of binding of all cluster-5A antibodies to antigen positive SCLC cells. Synthesis and expression of large complex type asparaginelinked oligosaccharides are consistently observed following neoplastic transformation (Dennis et al., 1988). These large oligosaccharides typically exhibit increased sialylation and increased branching. To determine whether analogous alterations of the oligosaccharide structure are part of the epitope recognised by the antibodies to cluster-SA antigen, we performed a series of analyses on antigen re-synthesis after enzymatic destruction of the sialylated epitope. Antigen resynthesis was blocked by tunicamycin, an inhibitor of normal processing of asparagine-linked glycoproteins and ,B-hydroxynorvalin which prevents the transfer of the oligosaccharide part of lipid intermediates to asparagin-x-threonine. Swainsonine which promotes synthesis of hybrid-type oligosaccharides instead of complex-type oligosaccharides blocked the re-synthesis as well. These results suggest that sialylated complex-type oligosaccharides, N-linked to asparagine-xthreonine are an important feature of the antigen. We are currently exploring the use of cluster-SA antigen as a target for the therapy of SCLC. The availability of a series of antibodies of different isotypes directed to this antigen should allow us to easily select individual reagents based on important properties, such as stability in vitro, retention of immunogenicity after radiolabelling and antibody affinity to use for delivery of toxins (Warwrzynczak et al., 1990). We appreciate the technical assistance of Gudrun Christiansen. This work was supported by the Swiss Cancer League grants FOR.802. 87.1 and FOR.302.89.2.
References
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R.A. (1989). Expression of small cell carcinoma antigens of cluster-S and cluster-5A in primary lung tumors. Br. J. Cancer, 59, 692. POLONOFF, E., MACHIDA, C.A. & KABAT, D. (1982). Glycosilation and intracellular transport of membrane glycoproteins encoded by murine leukemia viruses. Inhibition by amino acids analogues and by tunicamycin. J. Biol. Chem., 257, 14023.
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A.T. & STAHEL, R.A. (1989). Immunolocalisation and imaging of small cell cancer xenografts by the IgGu monoclonal antibody SWAI I. Br. J. Cancer, 59, 174. SOUHAMI, R.L., BEVERLEY, P.C.L. & BOBROW, L. (1988). (eds) The first international workshop on small cell lung cancer antigens. Lung Cancer, 4, 1. STAHEL, R.A., SPEAK, J.A. & BERNAL, S.D. (1985). Murine monoclonal antibody LAM2 defines cell membrane determinant with preferential expression on human small cell carcinoma and squamous cell carcinoma. Int. J. Cancer, 35, 11.
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cell carcinoma identified by the IgG2, monoclonal antibody SWA20. Cancer Res., 48, 4318. WAIBEL, R., O'HARA, C.J. & STAHEL, R.A. (1987). Characterization of an epithelial and a tumor-associated human small cell lung carcinoma antigen. Cancer Res., 47, 3766. WAWRZYNCZAK, E.J., DERBYSHIRE, E.J., HENRY, V.R. & 4 others (1990). Selective cytotoxic effects of a ricin a chain immunotoxin made with the monoclonal antibody SWA1I recognising a human small cell lung cancer antigen. Br. J. Cancer, 62, 410. ZWICKY, C., WAIBEL, R., JAKSCHE, H. & STAHEL, R.A. (1990). Polyclonal anti-idiotypic antibody mimicking the small cell lung carcinoma (SCLC) cluster-5A antigen interacts with a panel of antibodies to the same antigen and induces specific immune response in animals. Proc. Amer. Assoc. Cancer Res., 31, 281.
