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Monoclonal antibody FW6 generated against a mucin-carbohydrate of human amniotic fluid recognises a colonic tumour-associated epitope.

Mucus glycoproteins of human amniotic fluid were used to generate a monoclonal antibody (MAb) FW6, which stained the intestine of a 24 week stage fetus. In adults, 76% of colonic adenocarcinomas (13/17) showed strong expression of the FW6 epitope, but it was not detected in the histologically normal mucosae adjacent to the tumours or in normal left colon mucosa. In addition, MAb FW6 stained large cell carcinomas of the lung (2/3), gastric carcinomas (5/11), and ovary adenocarcinomas (3/4). The expression in carcinomas can also be called ectopic for testing normal tissues. MAb FW6 was also reactive with pyloric mucus glands, Brunner's glands of the duodenum, Paneth cells of the ileum, pancreatic ducts, absorptive cells of the right colon, bronchiolar glands, kidney urothelia, and with a restricted number of normal mucinous tubuli of salivary gland. It was demonstrated to be under the control of the secretion gene only in intestinal Paneth cells and absorptive cells of the right colon. Comparative histochemical analysis comprising a panel of MAbs suggests that the corresponding epitope of the MAb FW6 is a type II chain related carbohydrate structure belonging to the Lex/Ley-antigen family, but is different from short chain Lex and Ley. ImagesFigure 2

It has become increasingly evident that surface markers of tumour cells may resemble antigens of fetal cells. These observations were confirmed by the findings that tumour cells can re-express antigens which are found at an early stage of development but are suppressed during the progress of embryogenesis.
The characterisation of monoclonal antibodies (MAbs) which react with tumour-associated antigens, has brought into focus the complex carbohydrate structures of mucin glycoproteins. Such structures are expressed by both normal and malignant epithelial cells and are supposed to be highly immunogenic (Feizi, 1985). In the last decade, our knowledge of the carbohydrate structures on mucins as well as on glycolipids has been greatly enriched for this reason (Hakomori, 1985;Lloyd, 1986). Some of these carbohydrate antigens have not only been demonstrated in various carcinomas, embryonic or fetal tissues, but also in human amniotic fluid and seminal plasma (Hanisch et al., 1985;1986a, b;. Most MAbs, directed against the carbohydrate structures of tumour-associated antigens, were generated against celllines, tumour-extracts or glycolipids extracted from cell membranes. We used an alternative route by immunising with purified mucus glycoproteins from human amniotic fluid. The mucins from amniotic fluid lack any significant blood group A and B activities as previously shown (Lambotte & Uhlenbruck, 1966), but are rich sources of tumour-associated carbohydrate structures, especially sialyl-Lex and sialyl-Lea.

Monoclonal antibodies
The following MAbs were used as controls in epitope analysis, immunocytology and immunohistology: CSLEX-1 was a generous gift from Dr Terasaki (Los Angeles, USA) (Fuku-shima et al., 1984); AM-3 was kindly provided by Dr C. Hanski (Berlin, Germany) (Hanski et al., 1990). Both MAbs were directed to sialyl-Lex. MAbs anti-LeuMl and VimC6 of the cluster CD15 reacting with Lex were purchased from Becton Dickinson (Heidelberg, Germany and Boehringer Mannheim, Germany) respectively. MAb 12-4LE, directed to Ley, was prepared against the colorectal carcinoma cell line SW 1116 as described earlier (Bara et al., 1988). Anti-CEA (Dako, Hamburg, Germany) was used as control MAb for the detection of protein epitopes. Irrelevant MAbs of different isotypes were used as negative controls.
Mucin preparation Human amniotic fluid was collected during normal delivery (University Hospital of Gynecology and Obstetrics, Cologne, Germany) from six healthy women. Fluids were filtered and centrifuged and supernatants stored at -20°C until used.
For extraction of mucus glycoproteins, amniotic fluids was pooled and an equal volume of 90% aqueous phenol was added and incubated at 65°C for 15 min. The aqueous phase was extensively dialysed against water and lyophilised. The phenol-extracted material was fractionated by gel filtration on a Sephacryl S400 column (2.5 x 100 cm), equilibrated and run with 4 M guanidine hydrochloride, 1 mM EDTA and 1 mM sodium phosphate at pH 7.0 as shown previously (Hanisch et al., 1988;. The void volume representing high molecular weight glycoproteins was determined by adsorption at 280 nm. Respective fractions were pooled, dialysed against distilled water and lyophilised. Immunisation of mice and generation of hybridomas Mice (BALB/C) were first immunised by subcutaneous injection of 0.1 mg purified mucus-glycoproteins of amniotic fluid in 0.1 ml of distilled water, mixed with an equal volume of Freund's adjuvant (Boehringer Mannheim, Germany). Immunisation (0.3 mg) was repeated five times with incomplete Freund's adjuvant (Difco, Detroit, MI, USA) or with ABM-2 adjuvant system (Sebak, Aidenbach, Germany) containing 0.5 mg trehalose dimycolate, 0.5 mg monophosphoryl lipid A, 2% oil and Tween 80 in 2 ml PBS. One day after the final inoculation, spleen cells of immunised mice were fused with the HAT-sensitive X63Ag8.653 mouse myeloma cells in the presence of polyethylene glyco-1500 (Boehringer Mannheim, Germany) according to the protocols of Kohler and Milstein (1975) and Stahli et al. (1980). Clones were selected in HAT medium (1% hypoxanthine/ thymidine/aminopterine, Sigma, Deisenhofen, Germany) containing 5 x 10-5 M 2-mercaptoethanol and 10% fetal calf serum (FCS, Gibco, Karlsruhe, Germany) in the presence of mouse peritoneal macrophages. Hybridoma clones were screened for the production of monoclonal antibodies against mucins of the pooled human amniotic fluid by the ELISA technique.
Preparation of glycopeptides by protease digestion Native amniotic fluid was incubated with predigested pronase P from Streptomyces griseus (Serva, Heidelberg, Germany) at a final concentration of 0.1 mg ml-' in TBS (0.02 M Tris, 5 mM CaC12, pH 7.8) at 37°C for 48 h with a further addition of pronase P after 24 h. Then urea was added to a final concentration of 6 M, the pH was adjusted to 12 with NaOH, and phenol extraction was carried out by addition of 1 vol hot phenol (65°C, 10 min). After phase separation by further addition of 1 vol distilled water and dialysis, the freeze dried protease stable glycopeptides from amniotic fluid were fractionated by gel chromatography (2.2 x 80 cm) on Bio-Gel P30 (Bio Rad, Munchen, Germany), equilibrated with 0.01 M pyridine acetate (Hanisch et al., 1989). The eluate was collected in 3 ml fractions and registered by colorimetric analysis of hexose (phenol-sulphuric acid method), sialic acid (thiobarbituric acid method) or by ELISA using MAb anti-LeuM1. Glycopeptides found in the excluded fraction were collected and lyophilised.
Chemical and enzymatical treatment ofglycopeptides For preliminary biochemical analysis of MAb epitopes, periodate oxidation and sialic acid hydrolysis of carbohydrates were performed with coated protease-stable glycopeptides in microtitre plates. In the other experiments, soluble glycopeptides were treated in batches.
Coated glycopeptide carbohydrates were partially oxidised by incubating the wells with 1 mM or 10 mM periodate (NaIO4) in acetate buffer for 1 h at 25°C in the dark, after a short wash with 50 mM sodium acetate, pH 4.5 (Woodward et al., 1985). Following a brief rinse with sodium acetate buffer the plates were then incubated with 50 mM NaBH4 for 30 min at 22°C and washed with 0.5% BSA/PBS. Enzymatic hydrolysis of sialic acid of coated glycopeptides was performed using vibrio cholerae 0.1 U ml-' neuraminidase ( = sialidase, Behring-Werke, Marburg, Germany) in 0.05 M sodium acetate, 9 mM CaC12, 0.14 M NaCl, pH 5.5 for 1 h at 370C.
For heat treatment, mucins or glycopeptides were dissolved in 0.02 M PBS, pH 7.2, at a concentration of 1 mg ml-1, and incubated at 100°C for 1 h. Blockage of free amino groups on glycoproteins was performed in 0.1 M tetraborate buffer pH 9.3 containing 0.75 mM 2,4,6-trinitrobenzenesulphonic acid (TNBS) (Serva, Heidelberg, Germany) at 22°C for 30 min.
Glycoproteins were also treated with trifluoromethanesulphonic acid (TFMS) for 2 h at 0°C according to Sojar and Bahl (1987). The reaction mixture was neutralised with a 40% pyridine in water at -20°C, subsequently dialysed and lyophilised.
Extinction was read at 405 nm by a Flow ELISA reader. The reduction of antibody binding after chemical and enzymatic treatment of the antigen was regarded as significant, if the measured activities decreased to less than 30% of the control while more than 80% binding was assigned to a stable epitope.
Immunocytology of cell lines, blood and bone marrow smears Cell lines were cultured in 10% FCS-RPMI with penicillin and streptomycin, washed in PBS, and coated on 96-well Terasaki plates by centrifugation (200 g for 10 min). Unfixed cells, 0.025% glutaraldehyde fixed cells and 0.1% Tween 20 treated cells were incubated with hybridoma supernatant for 1 h. Binding of MAb was visualised by indirect immunoperoxidase staining.
Blood and bone marrow from patients with leukaemia or without haematological disease were taken off. The immunoreactivity of the MAbs was tested on viable cells by indirect immunofluorescence and on routinely prepared, acetone fixed smears by the APAAP method (Schwonzen et al., 1989).

Immunohistology
Tumour tissues were resected after surgery. Normal gastrointestinal tissue was obtained from seven kidney donors belonging to the following phenotypes: ALe(a), BLe(a+), BLe(a+) OLe(ab, OLe(b) Le(a + b-) individuals were considered as non-secretors and Le(ab +) as secretors (Oriol et al., 1986). Autopsies were performed within 5 min of death. Mucosa strips of pylorusduodenal junction, Ileum-right colon (or caecum) junction and sigmoid mucosae were fixed with 95% ethanol and coiled up into 'Swiss rolls' (Bara et al., 1988). Other tumour and normal tissues were first fixed in 6% neutral phosphate-buffered formalin and dehydrated in ethanol by standard histological techniques. After embedding all tissues in paraffin, serial microtome tissue sections (5,um) were used for comparative immunohistological studies. Reactivity of hybridoma tissue culture supernatants was determined using a four-step immunoperoxidase technique. After deparaffinisation in xylene, rehydration through graded ethanol, and blocking endogenous peroxidase activity with 2% H202 in absolute methanol for 30 min, the sections were incubated as follows: (1) normal swine serum (5% in PBS), (2) MAb in tissue culture supernatant or mouse ascites fluid diluted to 1/100 in PBS, (3) rabbit anti-mouse immunoglobulin (2% in PBS), (4) swine anti-rabbit immunoglobulin (2% in PBS), (5) rabbit peroxidase anti-peroxidase (PAP) complex. The slides were treated with 0.2 mg ml-' 3-amino-9ethylcarbazole (Sigma, Deisenhofen, Germany) in 0.02 M sodium acetate buffer containing 0.01% H202, then counterstained with haematoxylin, and mounted in glycerol jelly. All incubation steps were performed in a moist chamber at 21°C for 30 min, all polyclonal antibodies were purchased from Dako (Hamburg, Germany).

Results
Generation and selection of monoclonal antibodies After immunisation with the purified mucus glycoproteins (mucins), less than 10% of the antibody producing hybridomas secreted antibodies against the mucins of amniotic fluid. For detecting MAbs with carbohydrate specificity, 28 hybridomas were tested on chemically and enzymatically modified mucus glycoproteins or glycopeptides. Carbohy-drate antigens are expected to be resistant (i) protein modification by blocking of amino groups with TNBS, (ii) heat denaturation, and (iii) degradation by proteases, but to be labile to (i) oxidation by sodium periodate, (ii) deglycosylation by TFMS, or (iii) hydrolysis by glycosidases.
The hybridoma FW6 secretes antibodies of 1gM class, which bind to a TNBS-, heat-and pronase-stable epitope on mucine-derived high-molecular weight glycopeptides. The culture supernatant of the hybridoma FW6 showed no reactivity with mild (1 mM) and strong (10 mM) periodate oxidised, or with TFMS-treated glycopeptides ( Table I).
Treatment of coated mucins with vibrio cholerae neuraminidase does not reduce antibody binding of MAb FW6 in contrast to other clones, showing that terminal N-acetyl neuraminic acid is not involved in the corresponding epitope Immunohistological staining of tumour and normal tissues The results of the immunoperoxidase assay for the detection of FW6 epitope in carcinomas, their mucosae adjacent to colonic tumour, and normal tissues are summarised in Tables II-V. Testing neoplastic tissues, the FW6 epitope was strongly expressed in 13 of 17 colon carcinomas, but not in a single cell of the adjacent non-malignant tissues (Table II, Figure 2b and c). In well and moderately differentiated adenocarcinomas the FW6 epitope was typically detected on the apical cell surfaces and diffusely in the cytoplasm, as well *The test on chemical or enzymatical treated mucins of amniotic fluid was performed by ELISA technique, stable epitope = measured activity of antibody binding after chemical and enzymatical treatment of the antigen retained more than 80% binding, sensitive epitope = measured activity ofantibody binding after chemical and enzymatical treatment of the antigen decreased to less than 20% of the control; 2,4,6,- as in the secretions of carcinomatous glands. Colonic adenocarcinomas without differentiation showed predominantly intracytoplasmatic staining. MAb anti-LeuM 1 (Lex specific) stained most colon carcinomas tested, MAbs CSLEX-1 and AM-3 (both sialyl-Lex specific, manuscript in preparation for AM3) stained all colon carcinomas tested, but in contrast to MAb FW6 also reacted with the adjacent normal colonic deep crypt epithelium of few cases (Table II). MAb 12-4LE (Ley-specific) revealed different binding pattern to MAb FW6 by staining colonic adenocarcinomas, especially in the cases 4, 5 and 12. In contrast to the MAb FW6 epitope, Ley was expressed in few absorptive cells of the adjacent non-malignant mucosae in case 5. In fetal colonic mucosa in FW6 epitope was expressed on the apical cell membranes and in the cytoplasm (Figure 2a). Testing the normal colonic mucosae the FW6 epitope was detected in some absorptive cells and a few goblet cells of the right colon in secretor individuals (Le(a-b+) and Le(a-b-)) in contrast to the Lea non-secretors (Table III). FW6 antibody revealed no staining of normal left colon of secretors or non-secretors (Figure 2b).
MAb FW6 stained most cells of gastric carcinomas in five of nine cases (Table IV), but it also reacted with goblet cells and deep pyloric glands in the adjacent and normal stomach tissue ( Figure 2d). The staining of pylorus glands as well as Brunner's glands in the duodenum was independent of the ABO and the Lea/Leb blood group status ( Table V).
The FW6 epitope was not present in the villosities of normal ileum, but was detected on the Paneth cells of secretors with the blood group Leb or Le-, and not on the Paneth cells of the Lea positive individuals (Figure 2e).
MAb FW6, in addition, binds to antigens of ovary adenocarcinomas and large cell carcinomas of the lung (Table IV). Staining of bronchiolar glands and epithelia in the adjacent and normal lung tissue was noticed.
MAb FW6 did not stain mammary carcinomas, pancreatic carcinomas, prostate carcinomas and neoplasias of the testes (Table IV). In two of six normal pancreatic tissues, ductuli and not the acini showed positive cells (Figure 2f).
Normal kidney does not express the FW6 epitope, whereas normal urothelia do.

Discussion
Monoclonal antibody FW6, which identifies a periodatesensitive, neuraminidase resistant epitope on mucins from amniotic fluid, differs from our other generated antibodies in that it discriminates between adenocarcinomas of the colon and adjacent or normal colonic tissues. The FW6 epitope is   (Bara et al., 1988); L-M1 = anti-Leu MI, MAb directed to Lex; MAbs CSLEX and AM3 are directed to sialyl-Le' (Fukushima et al., 1984;Hanski et al., 1990); n = not determined; -= negative finding; positive reactions from + to + + + represent increasing percentage of immunostained tissue areas: + = 0-33%, + + = 34-66%, + + + = 67% or more. also expressed by most gastric carcinomas, ovarian carcinomas as well as some large cell carcinomas of the lung, but not by mammary carcinomas. The source of this epitope in amniotic fluid might be the secretions of the fetal gastrointestinal mucosa rather than the chorionic membrane or the fetal urine, since the FW6 epitope is strongly expressed by epithelial cells of the small intestine and right colon in the 24 week fetus. The staining pattern of MAb FW6 indicates that the expression of this epitope in adenocarcinomas could also be regarded as ectopic, for it was detected in some cells of differentiated normal glandular epithelia, such as mucus cells of pyloric glands, Brunner's glands in the duodenum, ducts of the pancreas, mucinous tubuli of salivary gland and bronchiolar glands.
Regarding the possible antigen structure of MAb FW6, recognising gastrointestinal malignancies, carbohydrate type 1 and type 2 structures related to blood group antigens have to be discussed. Immunohistological staining of normal gastric mucosa raises the possibility that the FW6 epitope is related to type 2 blood group antigens such as H type 2, Lex and Ley antigens, since it is detected in the deep area of pyloric glands regardless of the secretor status (Mollicone et al., 1985;Sakamoto et al., 1989). Thus MAb FW6 did not detect one of the type 1 antigens (Lea, sialyl-Lea, Leb), which were almost exclusively detected in the foveolar epithelia of intact gastric mucosa, corresponding to the secretor status (Mollicone et al., 1985;Sakamoto et al., 1989).
There are strong arguments against MAb FW6 defining the well known short chain Lex-antigen of the mentioned type 2 structures. First, in normal kidney epithelia MAb FW6 only stained urothelia, but no part of the nephron system. In contrast, the Lex-antigen is characteristically expressed in proximal tubules and the loop of Henle . Secondly, the monofucosylated short chain Lex-antigen and short chain sialylated Lex-antigen have been shown to be constituents of normal colonic mucosa by other investigations (Fukushima et al., 1984;Itzkowitz et al., 1986;Sakamoto et al., 1986;Yuan et al., 1987), which is confirmed by our results. Third, the FW6 epitope is not found in few Lex and sialyl-Lex positive tumours, and fourth, MAb FW6 does not stain myeloid (granulocytic) cells.
Antibody FW6 staining of colonic tissues resembles the pattern published for MAbs FH4 and FH6, which recognise extended, polyfucosylated (dimeric) Lex and sialylated polyfucosylated (dimeric) Lex respectively (Fukushi et al., 1984a, b). FH4 and FH6 antibodies gave very similar results to FW6 antibody, in that they were one of the most specific MAbs reacting with carcinomas of the colon, do not stain normal colonic mucosa , and are only bound to specific types of cells in gastric and intestinal mucosa testing normal tissues (Fukushi et al., 1984b). These Table III  Immunoperoxidase method was performed as previously described (Bara et al., 1988); positive reactions from + to + + + represent increasing intensity; CD = right colon; C = caecum; + ++ /-= simultaneous presence of strong (+ /-= weak) positive and negative zones on the same tissue sample.  MAbs had not been available for direct comparative histochemical study, but the flow cytometric pattern of MAb FW6 reaction with myeloid HL-60 cell line does not coincide with one of MAbs FH4 and FH6 tested by Symington et al. (1985). The expression of the FW6 epitope in the Paneth cells of the ileum and the absorptive cells of the right colon seems to be under the control of the secretor gene. This conflicts with the result that the FW6 epitope is found in pylorus glands, in Brunner's glands and on salivary glycoproteins from non-secretors. The expression of the FW6 epitope in the latter cases seems to be dependent on the H-gene coded fucosyltransferase. The secretions of salivary gland on the other hand do not contain H-gene coded fucosyltransferase and, accordingly, allow definitive conclusions on the secretor gene dependency of blood group related epitopes (Oriol et al., 1986). Up to now we do not have an explanation for the secretor-dependent and independent expression of the FW6 epitope found in the different organs tested.
The distribution pattern of the FW6 epitope in the normal  gastrointestinal tract is also very similar to that published for Ley. Ley-antigen expression for example is very weak or absent in normal colonic epithelium, but is strongly expressed in adenocarcinomas (Brown et al., 1984;Abe et al., 1986;Kim et al., 1986;Sakamoto et al., 1986;Bara et al., 1988). Le' and extended Ley structures have also been detected in stomach, duodenum, pancreas and lung (Mollicone et al., 1985;Sun et al., 1987;Kim et al., 1988;Pour et al., 1988;Sakamoto et al., 1989). On the other hand, there are many discrepancies in the distribution of the FW6 epitope and the Ley, extended Ley, or trifucosyl Ley determinants, which are recognised by several MAbs: 12-4LE (Bara et al., 1988), CC-1, CC-2 (Sun et al., 1987), AH6 (Abe et al., 1983), 75.12 (Blaineau et al., 1983), C14/1/46/10 (Brown et al., 1983), F-3 (Lloyd et al., 1983) and KH-1 (Kaizu et al., 1986). For example, MAbs C14/1/46/10, KH-1, CC-1, CC-2, and AH6 stained acinar cells in half the specimens of normal pancreas and pancreatic adenocarcinomas (Brown et al., 1984;Sun et al., 1987;Kim et al., 1988), whereas FW6 does not. It is questionable why MAb FW6 stains only pancreatic ducts in a few cases. This needs further investigation. Most important, the staining pattern of MAb 12-4LE does not correspond to the one of MAb FW6 in serial tissue sections of colonic adenocarcinomas. Knowledge concerning the distribution of Y-antigen and its extended derivatives outside the gastrointestinal tract and pancreas is very limited at present. However, in a limited survey by Sun et al. (1987) MAbs CC-1 and CC-2 reacted with all breast carcinomas tested, whereas MAb FW6 did not show reactivity at all.
Summarising the results of biochemical epitope analysis and histological epitope distribution of the MAb FW6, there is strong evidence for the detection of a carbohydrate antigen, which is very closely related to the Lex/LeY-antigen family, without proving identical staining pattern to a known antigen on type 2 chain precursors. The definitive structural characterisation of its carbohydrate epitope is currently under investigation. MAb FW6 might be of probable diagnostic relevance for the detection of colon carcinoma, for the FW6 epitope is demonstrated in sera from colon carcinoma patients.