EP0403559A1 - Ameliorations dans le domaine des antigenes - Google Patents

Ameliorations dans le domaine des antigenes

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Publication number
EP0403559A1
EP0403559A1 EP89904148A EP89904148A EP0403559A1 EP 0403559 A1 EP0403559 A1 EP 0403559A1 EP 89904148 A EP89904148 A EP 89904148A EP 89904148 A EP89904148 A EP 89904148A EP 0403559 A1 EP0403559 A1 EP 0403559A1
Authority
EP
European Patent Office
Prior art keywords
antigen
antibody
kda
fragments
core
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP89904148A
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German (de)
English (en)
Inventor
Peter Stern
Nicholas Hole
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Cancer Research Campaign Technology Ltd
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Cancer Research Campaign Technology Ltd
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Priority claimed from GB888805240A external-priority patent/GB8805240D0/en
Priority claimed from GB888821078A external-priority patent/GB8821078D0/en
Application filed by Cancer Research Campaign Technology Ltd filed Critical Cancer Research Campaign Technology Ltd
Publication of EP0403559A1 publication Critical patent/EP0403559A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • THIS INVENTION relates to a new antigenic peptide, isolatable from human trophoblast cell membranes, antibodies to the antigenic peptide, methods of producing the antigen and antibody and the use of the antigen and antibody in diagnosis and in the production of vaccines.
  • Trophoblast demonstrates some functional properties of neoplastic tissue, namely invasiveness of host tissue and escape from immunological surveillance.
  • Several monoclonal antibodies to trophoblast membrane proteins have been described. In terms of cancer research, the rationale behind this approach has been to identify 'oncofetal' antigens present on both trophoblast and neoplastic cells (Johnson, 1984). If such antigens were restricted to neoplastic tissues, then these reagents would be potentially useful in diagnosis, tumour localisation and drug targeting. Of those monoclonal antibodies that do identify trophoblast oncofetal antigens, relatively few have been fully characterised.
  • PLAP placental alkaline phosphatase
  • Native glycoprotein is N-terminus blocked and resistant to digestion with V8 protease, pepsin, chymotrypsin or chemical cleavage with 75% formic acid, hydroxylamine or N-chloro-succinimide.
  • High sensitivity amino acid analysis reveals most abundant residues as approximately 10% glutamic acid, 12% serine, 16% glycine, 9% threonine and 15% alanine.
  • the N-linked carbohydrate structures are not susceptible to endo beta-galactosidase digestion. g.
  • the present invention also provides proteolytic fragments of 5T4 antigen as well as the 42 KDa core and fragments thereof.
  • the 5T4 glycoprotein of the invention can be isolated and purified from human trophoblast cells by recovering the syncytiotrophoblast glycoproteins from human placenta, subjecting these glycoproteins to purification by either antibody affinity chromatography or a combination of other chromatographic methods and isolating 5T4 antigen as described in more detail below.
  • the glycoprotein can be prepared by synthetic or semi-synthetic techniques, synthetic techniques involving building up the polypeptide core structure by building up the polypeptide chain by conventional peptide synthesis followed by introducing any appropriate glycosylation by chemical or biological methods.
  • the 42 KDa polypeptide core can be produced by recombinant DNA techniques, utilising a synthetic or naturally-occurring DNA encoding the 42 KDa polypeptide core.
  • DNA will comprise a first DNA sequence encoding the 42 KDa polypeptide core of 5T4 antigen and a second DNA sequence, not normally found in association with the first sequence, but under whose influence, the first sequence can express the 42KDa core in a suitable host cell.
  • Suitable techniques include incorporating the selected DNA in a plasmid, transforming a suitable host cell with this plasmid and expressing the DNA in the host cell.
  • the glycosylation of the genetically engineered 42 KD apolypeptide core can then follow by appropriate chemical or biological methods.
  • the present invention includes DNA encoding and capable of expressing the 42 KDa polypeptide core as well as plasmids including it and host cells transformed with such plasmids.
  • a further aspect of the present invention provides antibodies that recognise the 5T4 glycoprotein, or fragments thereof or the 42 KDa core or fragments thereof.
  • Such antibodies may be monoclonal or polyclonal antibodies.
  • the antibodies may be prepared by conventional techniques. Polyclonal antibodies may be obtained by using the 5T4 glycoprotein of the invention or fragments thereof or the 42 KDa core or fragments thereof as immunogen injected into small or large animals from whose blood the polyclonal antibodies are recovered by conventional methods.
  • Monoclonal antibodies can be prepared utilising the 5T4 glycoprotein of the invention or fragments thereof or the 42 KDa core or fragments thereof as immunogen in a host animal, immortalising antibody producing cells of the host animal and recovering monoclonal antibody from the immortalised cells.
  • the 5T4 glycoprotein of the invention As an alternative to the use of the 5T4 glycoprotein of the invention, or its 42 KDa core or fragments thereof as immunogen in the raising of antibodies, one can also use a natural product including the 5T4 glycoprotein of the invention, isolatable from trophoblast cells.
  • This material is known as syncytiotrophoblast glycoproteins, (StMPM), which can be isolated from human placenta by known methods.
  • StMPM syncytiotrophoblast glycoproteins
  • the 5T4 glycoprotein of the invention can be isolated from the StMPM by either antibody affinity chromatography or a combination of other chromatographic methods.
  • One particular monoclonal antibody that we have isolated and tested is one prepared by hybridoma techniques using StMPM wheat germ agglutin (WGA) glycoprotein as immunogen and which has become known as 5T4.
  • the antigens (5T4 glycoprotein, fragments thereof, the 42 KDa core and fragments thereof) of this invention and antibodies (that recognise antigens of this invention) are useful as diagnostic tools and in the production of vaccines.
  • the purified 5T4 antigen for example allows the production of a family of related antibodies which recognise different epitopes of 5T4 antigen.
  • these antibodies are of interest: i) in the development of contragestional vaccines since the antigen is expressed very early on in pregnancy; ii) in foetal typing by the detection of foetal cells in the mother's bloodstream; iii) as an early warning signal in situations of danger or damage to the foetus e.g.
  • pre-eclampsia in tumour screening and diagnosis in vitro and/or in vivo - in this respect it may offer significant advantage over antibodies to PLAP since the antigen is not found in pregnancy serum; v) in routine monitoring of the female population with respect to premalignant conditions known as cervical intraepithelial neoplasia CIN 1, 2 and 3 detected in cervical biopsies.
  • premalignant conditions known as cervical intraepithelial neoplasia CIN 1, 2 and 3 detected in cervical biopsies.
  • the labelling intensity corresponds to the severity of the dysplasia with invasive carcinomas of the cervix strongly labelled.
  • the present invention includes compositions comprising the antigen or antibody of the invention together with a carrier or diluent.
  • a carrier or diluent The exact nature of the carrier or diluent will depend upon the ultimate application of the antigen or antibody and, in the case where the antigen is to be used as a vaccine (or antibody as a passive vaccine) the carrier will be a parenterally acceptable liquid carrier.
  • the carrier may be liquid or solid and solid carriers for the antibody also represent a particularly important aspect of the present invention where the antibody is to be used as a means of purifying the naturally-occurring antigen by techniques of affinity chromatography.
  • the antigens and antibodies, immobilised or not, may be linked with radioisotopes or other revealing labels for localisation and/or therapy or conjugated with anti-tumour reagents for therapy.
  • the antigen and antibody can be derivatised for use in different forms of assay for antigen concentration.
  • the present invention includes a diagnostic test kit containing, as a solid component, an immobilised antigen or antibody of the invention and more specifically can contain, depending upon the specific type of assay to be used, an antigen and an antibody of the invention, one of which bears a revealing label.
  • the antigen of the invention can be used in methods of in vitro or in vivo diagnosis targeting antibody while the antibody of the invention may be similarly used to target antigen. Such methods are of particular use in the diagnosis of various types of cancer, particularly for mass screening of cervical smears.
  • 5T4 antigen has a relatively limited tissue distribution. It appears to be a pan-trophoblast marker which is expressed by all types of trophoblast examined as early as 9 weeks of development. It is specific for this tissue type within the placenta except for the amniotic epithelium which is also antigen positive. On the basis of immunoperoxidase staining of frozen sections from normal tissue, 5T4 antigen is also expressed by certain epithelial cell types. It should be noted that several 'trophoblast-characteristic ' antigens, such as PLAP, are in fact found in normal tissues at trace concentrations (Mclaughlin, 1986).
  • 5T4 antigen was found in placental plasma membrane in at least a 1000-fold higher concentration than that found in other normal tissues tested. However, this level of sensitivity would not necessarily detect expression in minor subpopulations of cells within a given tissue.
  • HMFG1 and 2 Tumadimitriou et al., 1981; Wilkinson et al., 1984
  • CA 1 , 2 and 3 Bowell et al., 1985
  • 5T4 is reactive with tumour cell lines of a diverse, but select origin, including those of a developmental nature, such as choriocarcinoma and embryonal carcinoma.
  • tumour cell lines of such apparent diversity of tissue origin are not clear; the normal cell line types tested are all of embryonic origin.
  • the lack of reactivity with tumour cell lines derived from lung, bronchus and lymphoid tissue is consistent with the immunohistology of the normal tissue types.
  • Other antigen positive tumour cell lines may have been derived from an epithelial component of normal tissue or represent reexpression of embryonic antigen on tumour cells.
  • trophoblast antigens have been reported to exhibit a pattern of expression by tumour cell types apparently not detected in the normal cell counterpart (McLaughlin et al., 1982).
  • Rettig et al. (1985) a series of six monoclonal antibodies were generated against choriocarcinoma cells, one of which was reactive with neoplastic, but not normal, kidney cells; the other mAbs did not demonstrate such a selective expression.
  • 5T4 antigen does however appear to be novel. On the basis of reactivity in dot-blots and other criteria, we have specifically excluded PLAP and transferrin as the 5T4 antigen.
  • the 5T4 antigen is carried by glycoprotein molecules of 72kD on syn ⁇ ytiotrophoblast microvillous plasma membranes but appears on molecules of similar molecular weight from several different cell lines including some choriocarcinomas.
  • the molecules are sialylated and have approximately 30kD of the apparent molecular weight due to N-linked carbohydrate structures as judged from removal of the latter by N-glycanase endoglycosidase.
  • 5T4 appears to exist on the cell surface as a monomeric protein. Firstly, 5T4 antigen elutes with an apparent molecular weight in gel filtration of 120kD, an increase consistent with the addition of a detergent shell, and inferring that 5T4 is not associated non-covalently with any other large molecules. Additionally, reduction with 2-mercaptoethanol does not substantially alter the apparent molecular weight of the 5T4 radio immunopreoipitate, as would be the case if it were disulphide bonded to another protein.
  • the pattern of expression of 5T4 is similar to that of the family of mucin type glycoproteins (Swallow et al., 1987), but with clear differences from those defined by the CA or HMFG series of antigens (Wiseman et al., 1984; Burchell et al., 1983). These latter glycoproteins are defined by several monoclonal antibodies which have been shown to be reactive with a wide range of malignant tumour cells but also reactive with certain specialized normal epithelia.
  • StMPM was purified from full term human placentae, obtained within one hour post partum, by the method of Smith et al. (1974).
  • the StMPM pellet was solubilised in 0.5% DOC in tris-buffered saline (TBS, 0.15M NaCl, 25mM tris, pH 8.0) containing 0.1mM phenylsulphonylmethyl fluoride (PMSF) and centrifuged at 14,000g for 10 minutes.
  • the WGA-reactive glycoproteins were then purified by incubation of the supernatant with WGA-Sepharose (5mg ligand/ml Sepharose) for one hour at room temperature.
  • the beads were washed extensively in TBS/0.5%DOC, and the specifically bound glycoproteins eluted in 5ml of 0.2M N-acetyl glucosamine (Sigma) in TBS.
  • the eluted fraction was extensively dialysed against 30mM ammonium bicarbonate (pH 7.9), and lyophilised.
  • a male BALB/c mouse was immunised by six intra-peritoneal injections of WGA-purified StMPM glycoproteins (100-200 ⁇ g/injection).
  • Spleen cells were fused with NS1 murine myeloma cells (Kohler and Milstein, 1975), and the cells plated out in 24-well Linbro plates at 7 ⁇ 10 5 cells/well. After two weeks, wells were assayed for StMPM reactive antibody by immunodotting. Positive clones were picked directly and further subcloned by limiting dilution. The antibody subclass was determined by double radial diffusion using a monoclonal isotype typing kit (Serotec, Bicester, U.K.). Antibody 5T4 was obtained by this technique
  • AV-3 cells Near confluent cell cultures of AV-3 cells were radiolabeled for 15-18 hours with %-glucosamine (20 nCi/ml) (Amersham International) in RPMI containing 10% dialysed FCS. Metabolically labeled cells were collected and immunoprecipitated as follows : cells were removed from tissue culture flasks by incubation in 0.1M EGTA-PBS, washed in PBS (Dulbeccos-A) and then solubilized for 30 minutes at 4°C in 0.5% (v/v) NP40 in tris-buffered saline (TBS, 0.15M NaCl, 25mM Tris, pH 8.0) containing 0.1mM PMSF.
  • TBS tris-buffered saline
  • Non-solubilized cellular components were removed by centrifugation at 14,000g and the amount of radioactivity incorporated into protein was determined following precipitation with 10% trichloroacetic acid.
  • Cell surface labelling by the lactoperoxidase- 125 I method together with the techniques of immunoprecipitation and SDS-PAGE were carried out as previously described; high molecular weight standards
  • red blood cell membrane proteins or 14 C-methylated protein mixtures were used as marker proteins (Thompson et al., 1984; Stern et al., 1984; 1986). Tritiated sodium borohydride labelling of cell surface glycoproteins was carried out as described by Axelsson et al. (1978).Autoradiography and fluorography were as described in Thompson et al. (1984.) using pre-flashed Fuji X-ray film.
  • Immunoperoxidase staining of frozen tissue sections was carried out by the method of Bulmer and Sunderland (1983) . Tissues were obtained as soon as possible post mortem, always within 12 hours , and processed immediately. Indirect immunofluorescence with cell suspensions was as described previously (Thompson et al. , 1984) . A monoclonal antibody we have isolated , directed against a widely expressed human antigen ( mAB 1 D2 ) , was used as positi ve control .
  • EBSS Earle' s buffered saline solution
  • bovine serum albumin 0.1% sodium azide
  • the suspensions were plated out at 50 ⁇ l (10 5 cells) /well in microtitre plates. 50 ⁇ l mAb/well were added and incubated at room temperature for one hour. The cells were washed and 5 ⁇ 10 5 CPM of 125 I-labelled (Fab' ) 2 fragments of sheep anti-murine immunoglobulin (Amersham International) added.
  • Fab' 125 I-labelled
  • Tissues were obtained at post mortem held within 12 hours of death , andproc ⁇ ssed immediately .
  • 1 0-20g o f tissue was finel chopped , rinsed , and homogenised in 1 0-20ml o f ice-cold phosphate buffered saline containing 5mM MgCl 2 and 0.1mM PMSF with 20 strokes of a Dounce homogeniser.
  • the homogenate was centrifuged at 10,000g for 20 minutes, the pellet discarded and the supernatant centrifuged at 100,000g for 1 hour.
  • This pellet was solubilised in 0.5% (w/v) DOC/TBS containing 0.1mM PMSF and unsolubilised material pelleted by centrifugation at 14,000g.
  • the protein concentration of the supernatant was determined by the method of Lowry et al. (1951). Membranes from 12-hour old placentae were prepared identically and acted as positive controls.
  • StMPM protein was solubilised in 6.5ml of 1.0% (w/v) DOC/TBS containing 0.1mM PMSF, centrifuged at 100,000g for 30 minutes, and the supernantant fractionated over S200 Sephacryl (Pharmacia).
  • Column size was 90x2cm, running buffer was 0.1% (w/v) NaDOC/TBS containing 0.1mM PMSF.
  • Flow rate was 17ml/hour.
  • Fraction size was 3.3ml.
  • the column was calibrated with the following proteins; Equine ferritin (Sigma), IgG (Kabi), transferrin (Sigma), Bovine serum albumin (Sigma) and ovalbumin (Sigma). Fractions were assayed for 5T4 antigen in ELISA and immunodot.
  • Elisa plates were activated by one hour incubation with 100ul/well of PBS containing 0.25% gluteraldehyde (BDH), the plates washed with PBS, and 100ul/well of undiluted or 10-fold diluted fractions from gel filtration bound to the plates by overnight incubation at 4°c. Following washing, the plates were incubated with 1% BSA/TBS as blocking agent. ELISA was then carried out as described (Johnson et al., 1981). Immunodotting on nitrocellulose was carried out using the Bio-Rad Dot-Blot apparatus. Fractions from gel filtration were loaded at 10ul and 100ul/dot.
  • NaDOC solubilised plasma membrane protein was loaded in the range of 50 ⁇ g-12.5ng protein/dot.
  • the following antigens were loaded at 1 ⁇ g protein/dot; transferrin (Sigma), PLAP (Gift of Dr.P.J.McLaughlin), human placental la ⁇ togen (HPL) (Sigma), calmodulin (Sigma), IgG (Miles Ltd.), albumin (Miles Ltd.) and normal human sera.
  • transferrin Sigma
  • PLAP Gaift of Dr.P.J.McLaughlin
  • HPL human placental la ⁇ togen
  • HPL human placental la ⁇ togen
  • calmodulin Sigma
  • IgG Meats Ltd.
  • albumin Methylactin
  • StMPM membranes (approx. 1mg protein) were treated overnight at 37°C with either 2mg trypsin (Boehringer), 2mg pronase (Boehringer), 0.1U neuraminidase (Behringwerke) in 300ul of PBS or 10 units/ml ⁇ T-glycanase (Genzyme) in buffer containing final concentrations as follows: 0.17% SDS; 0.2M tris-HCl, pH 8.7; 10mM 1,10-phenanthroline hydrate (in methanol); 1.25% NP-40 (Plummer et al., 1984).
  • All procedures were carried out at room temperature. All buffers contained 0.1 mM phenyl methyl sulphonyl fluoride (PMSF).
  • PMSF phenyl methyl sulphonyl fluoride
  • the microvillous membranes from one placenta (approx. 1 g weight) were solubilised in 100 ml of 1% nonidet P40 (NP-40) in TBS (20 mM Tris, 150 mM sodium chloride. pH 8.0) for 30 minutes on a multimixer. ⁇ nsolubilised material was pelleted at 100,000 g for 30 minutes.
  • the IgGl 5T4 mAb was purified by high salt protein A affinity chromatography (loading buffer 1.5 M glycine, 3 M NaCl, pH 8.9. Elution buffer 100 mM citrate, pH 6.0) and bound to CNBr-activiated Sepharose (Pharmacia).
  • the mAb 5T4 affinity column was washed with 5 column volumes of 1% NP40/TBS and 5 column volumes of TBS.
  • the bound 5T4 glycoprotein was eluted with 8 M urea. Fractions were assessed by immunodot (Stern et al, 1986), protein assay (Lowry, Rosebrough, Farr and Randall, 1951) and SDS-PAGE.
  • the monoclonal antibody 5T4 is a murine IgG1. All work detailed in this study was carried out using subclone 5T4.B8. The preliminary screen by immunodot showed that the antigen recognised was none of the following major proteins associated with the trophoblast; IgG, transferrin, PLAP, HPL, albumin, calmodulin nor was it detectable in serum.
  • Figure 1 illustrates antigen expression in term villous placenta as assessed by immunohistology of frozen sections. Villous trophoblast is strongly labelled by mAb 5T4, whereas the stroma is negative. There is specific labelling of the amniotic epithelium and extravillous cytotrophoblast of the chorion laeve but not of the amniotic mesenchyme or maternal decidua ( Figure 1 c,d).
  • mAb 1D2 labels all parts of villi (fig 1a)
  • mAb H316 labels trophoblast but is not specific for this tissue type (fig 1b; Stern et al., 1986); negative controls are unlabelled (Figs 1e, f).
  • Extravillous cytotrophoblast in the placental bed is also labelled by mAb 5T4; no other element of the term placenta is 5T4 antigen-positive. Similar analysis of first trimester villous tissue has shown antigen expression by both syncytiotrophoblast and cytotrophoblast (data not shown).
  • 5T4 was unreactive with the following non-pregnant tissues examined in immunohistology; spleen, heart, brain, liver, lung, bronchus, skeletal muscle, testis or ovary. Glomeruli in the kidney, villi of the small intestine, bladder epithelium, basal layer of the epidermis, endometrial glands of non-pregnant uterus and endocervical glands showed some specific labelling with mAb 5T4. Some small vessels in various tissues appeared to be weakly stained. Table I summarises 5T4 reactivity assayed by immunohistology of frozen tissue sections.
  • 5T4 was not specifically reactive with any other tissue tested (ovary, testis, kidney, brain, liver and muscle) at all antigen concentrations used (up to 50ug/dot). From this it was concluded that these normal non-gestational tissues express 5T4 antigen at approximately 1000-fold lower concentration than full-term placenta on a weight of crude membrane protein basis. This relative level of expression is comparable with PLAP as measured using mAb H317 (Table II).
  • 5T4 antigen expression by cell lines of normal and neoplastic derivation was assessed by indirect immunofluorescence and a more quantitative radiobinding assay (Table III). By comparison of reactivity with negative control xenogeneic cell lines, radiobinding indices of greater than 1.5 were considered to indicate postive expression of antigen. Trypsinisation was necessary to remove some attached cell lines from the substratum and it was noted where compared that this procedure tended to reduce the binding index compared with EGTA removal (data not shown) .
  • Normal leukocytes were 5T4 antigen negative and "normal" types represented by cell lines of amnion, embryonic lung fibroblasts and embryonic intestine origin were labelled by 5T4 antibodies.
  • Tumour cell lines of myeloid origin were all 5T4 antigen negative; 6/6 tumour cell lines of gestations! or developmental origin were positive. 11/15 carcinomas of other histological types and origins were positive, as was one glioma and 1/3 Wilms tumour lines tested.
  • 5T4 was unreactive with reduced and unreduced western blots of StMPM.
  • the molecular species bearing the 5T4 antigen was identified as a
  • glycoprotein can be labelled by reduction with tritiated sodium borohydride either after periodate oxidation of sugar residues or galactose oxidase/neuraminidase treatment. These latter treatments change the relative mobility in SDS-PAGE as compared with 125 I labelled 5T4.
  • antigen Figure 3
  • AV-3, Tera-2, MRC-5, Hep-2, HN5, HT29 cell lines all express a molecule of similar molecular weight to that on StMPM as judged by SDS-PAGE of immunoprecipitates of surface iodinated cells; the antigen has been immunoprecipitated from AV-3 cells metabolically labelled with tritiated glucosamine (data not shown).
  • Isolated StMPM membranes were digested with trypsin, pronase, neuraminidase or N-glycanase, the components solubilised and subjected to immuno-dot assay. Both proteases and N-glycanase destroyed 5T4 antigenicity, whilst neuraminidase did not (Table IV).
  • the effects of various fixatives on 5T4 antigenicity as expressed by Tera-2 cells was assessed by solid-phase radiobinding assay. Neither Bouins' fixative, buffered formalin, gluteraldehyde or absolute ethanol were found to significantly affect 5T4. binding index relative to PBS control (data not shown).
  • 5T4 shows specific labelling of villous trophoblast and extravillous cytotrophoblast of the chorion laeve as well as amniotic epithelium.
  • Positive control mAb 1D2 labels all cell types;
  • mAb H316 labels trophoblast of the chorion laeve and amniotic epithelium.
  • Normal mouse serum shows no labelling.
  • a panel of normal, non-neoplastic and neoplastic tissues were used. Fresh tissue samples were quenched in iso-pentane, c ⁇ led in liquid nitrogen for a few minutes until viscous. 6 micron thick cryostat sections were cut, air dried for 10 minutes and then fixed in acetone. An avidin-biotin immunoperoxidase technique was employed for the screening of the hybridoma culture supernatant 5T4.
  • TBS peroxidase was visualised using a freshly prepared and filtered solution of diaminobenzidine tetrahydrochloride (DAB-Sigma) in TBS containing 0.03% hydrogen peroxidase. (6 minutes). Sections were washed in tap water and counter stained in Coles haematoxylin, dehydrated, cleared and mounted (Ralmount-R.A. Lamb). The immunohistochemical results were interpreted with reference to a set of controls run in parallel with each test. These included sections treated with DAB only to show endogenous peroxidase, omission of the primary antibody and replacement of the primary antibody with one of the same class but of unrelated specificity.
  • DAB-Sigma diaminobenzidine tetrahydrochloride
  • the distribution of positive reactions with MAb 5T4 in normal and non neoplastic tissues is summarised in Table I.
  • the villous syncytiotrophoblast from first and third trimester placentae and an ectopic (tubal) pregnancy showed strong membrane positivity.
  • Placental site trophoblast displayed both membrane and cytoplasmic reactions.
  • the stroma of chorionic villi and foetal blood vessels were negative.
  • Table II summarises the distribution of MAb 5T4 in neoplastic tissues. Many of the malignant epithelial tumours displayed positive reactions in the neoplastic cells. Of note, were carcinomas of breast (5/5), lung (5/5), stomach (6/7) and pancreas including one of the ampulla of Vater (4/4). Also positive, albeit in only a limited number of cases available, were carcinomas of endometrium and cervix.
  • Cystadenocarcinomas of the ovary produced variable reactions. In three of the four positive cases the majority of tumour cells were positive, and in the other the majority were negative.
  • Undifferentiated mesenchyme may also be positive.
  • the cystic epithelium of mature teratomas often displayed a focal weak to moderate reaction.
  • Syncytiotrophoblast of choriocarcinomas and a complete hydatidiform mole was strongly positive. Much of the trophoblast of placental site tumours showed moderate or strong labelling on both cell membranes and within the cytoplasm. Single examples of fibrosarcoma and leiomyosarcoma showed that whilst most tumour cells were negative, there were focal and weak reactions in a few cells. Malignant melanomas (2) and malignant lymphomas (3) were negative.
  • the stroma of some tumours showed weak and focal reactions. This was also noted in the endothelium lining mainly small blood vessels in many tissues and tumours.
  • the cellular location of binding with mAb5T4 in tumours may be either membranous or cytoplasmic or a combination of both.
  • MAb 5T4 gives reactions in trophoblast which are similar to other antitrophoblast antibodies.
  • our detailed immunohistochemical analysis tends to suggest that the antigen recognised is distinct from HCG,HPL,PLAP and those which react with mAb 18A/C4 and 18B/A5 (Loke, University of Cambridge).
  • 5T4 will react with some non-HCG producing tumours and gives intense reactions with syncytiotrophoblast of term placenta.
  • Antibodies to both HCG and HPL are unreactive with the basal layer of stratified squamous epithelium and normal or non-neoplastic endocervical glands.
  • mAb 5T4 The failure of mAb 5T4 to react in fixed and routinely processed paraffin sections is a characteristic of some other reported antibodies directed against membrane-associated antigens, notably anti-PLAP, 18A/C4 and 17.1A antibodies.
  • Cervical carcinoma arises from dysplastic precursor lesions in the reserve cells in the basal layer of the stratified metaplastic epithelium. These areas develop from proliferating basal cells which have undergone some form of transformation, and gradually spread throughout the whole epithelium. Cervical carcinomas thus develop from a series of atypical changes which progress in continuum to a stage of carcinoma in situ. This is probably the final premalignant state before the lesion invades the underlying stroma becoming microinvasive.
  • the dysplastic variations have been categorised as a series of changes in cervical intraepithelia neoplasia (CIN). They have been graded from 1-3, with CIN 1 representing less than a third of the dysplastic involvement, located in the basal layer. CIN 2 with a third to two thirds involvement, and CIN 3 two thirds to full thickness involvement, equivalent to carcinoma in situ. At any stage, the lesion may regress back to normality. 90% of cervical neoplasms are squamous cell carcinomas and have a contrasting aetiology and of epidemiology to adenocarcinomas (present in nulliparous women) which comprise the remaining 8-10%.
  • Placental tissue was washed in PBS (phosphate buffered saline). 1 cm 3 of tissue was then embedded in PBS (phosphate buffered saline).
  • Frozen cervical specimens were selected from a store at the Royal Liverpool Women's Hospital (RLWH). These samples, from cone or punch biopsies routinely submitted for histology, were embedded in polycel, snap-frozen and stored at -70oC. The specimens were cut at 7um thick using a cryostat and placed on slides (cleaned with ethanol and coated with poly-L-lysine). The pathological assessment was obtained from the records of examination of the specimens subjected to fixation in formal buffered saline, dehydration wax embedding and haemotoxin/eosin staining. Immunohistology
  • the first layer test mAb was applied (ascites fluid diluted 1/100 in 1% BSA/PBS) and incubated for 1 hour at room temperature in a moist box. Subsequently, each slide was washed individually x3 in 1% BSA/PBS before having the second layer peroxidase conjugated rabbit anti-mouse (R anti-MIg) (Dako) diluted 1:50 in 1% BSA/PBS +10% normal human serum (NHS). After 1 hour incubation (same conditions as before), the slides were washed 2 ⁇ 1% NHS, 1% BSA/PBS and developed using 3'3' diaminobenzidine, 5 ug/10 mis PBS + 0.02% H 2 O 2 ).
  • R anti-MIg second layer peroxidase conjugated rabbit anti-mouse
  • NHS normal human serum
  • the reaction was stopped after 10 minutes by rinsing with tap water.
  • the slides were counterstained using Meyer's Haemalin dehydrated by passing up graded alcohols, fixed in xylene and mounted. All experiments were performed using positive W6/32) and negative (10.2.16) control ascites fluid.
  • Tissue sections were selected on the basis of routine diagnosis, being placed into the appropriate groups according to the pathology of the epithelium.
  • the groups were categorised as follows: normal, metaplastic, HPV infected, CIN 1, CIN 2 or CIN 3, with or without HPV infection, invasive carcinoma and common non-malignant cervical inflammatory disorders.
  • Over a 100 biopsy specimens were investigated and each experiment was performed with a positive and negative control (W6/32 and 10.2.16 respectively), read independently by two observers - some material was poorly preserved and not included in the summary. Placental villous sections were included in each experiment to ensure that the procedure was working optimally.
  • the degree of labelling was assessed as anything above that shown in the negative control, eliminating the possibility of false positives becoming included into the study. A subjective estimation of the intensity of the labelling was also made. Experiments were repeated at least once on greater than 50% of the specimens.
  • Table VII summarises the extent of 5T4 labelling from the basal epithelium to the surface in 66 cone or punch biopsies from the ectocervix.
  • the data can be grouped in several categories.
  • the sections of "normal" ectocervix, squamous metaplasia and HPV infection without evident dysplasia exhibited an overlapping phenotype in intensity and range of distribution of 5T4 antigen.
  • 9/17 showed labelling confined to the basal cells of the epithelium; 6 showed faint labelling throughout the epithelial layers and only 2 demonstrated significant labelling to level C3.
  • 5/5 examples of squamous cell carcinoma showed positive intense labelling of the malignant cells and surrounding stroma.
  • the final group of miscellaneous conditions includes hyperplasia, chronic inflammation, cervicitus, acanthotic epithelium and radiation induced atypia. These specimens were selected on the basis of their conventional pathology and exhibited a range of labelling.
  • the inflammatory infiltration response did not increase 5T4 expression per se; the single example of acanthotic epithelium was clearly labelled as were 2/3 of the hyperplastic epithelia. This arbitrary grouping shows some tendency to higher levels of 5T4 expression in the centre layers but appears different from the CIN 2 and CIN 3 groupings.
  • tumour marker specific for cervical cancer may revolutionise current methods for screening, by offering the potential for the tumour specific Ag to be detected in serum and mucous samples and solubilised biopsies.
  • the quantitative assessment of 5T4 antigenicity in cervical smear material using radio or other immunoassay with 5T4 monoclonal antibodies may be used as a means of assessing the degree of dysplastic cells in a specimen. This procedure can be highly efficient in mass screening and assigning further investigative procedures.
  • Patillo R.A., Ruckert, A., Hussa, R. , Bernstein, R. and Delfs, E. (1971) The JAr cell line - continuous human multihormone production and controls. In Vitro, 6, 398.
  • tumour-associated epithelial mucins are coded by and expressed hypervariable gene locus PUM. Nature, 328, 82.

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Abstract

On a identifié dans les trophoblastes humains une nouvelle glycoprotéine 5T4. L'antigène et les fragments d'antigène et, plus particulièrement, les anticorps qui reconnaissent l'antigène et ses fragments sont utiles pour diagnostiquer et soigner le cancer, notamment pour les contrôles de routine des frottis cervicaux.
EP89904148A 1988-03-04 1989-03-03 Ameliorations dans le domaine des antigenes Pending EP0403559A1 (fr)

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GB888821078A GB8821078D0 (en) 1988-09-08 1988-09-08 Improvements relating to antigens

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ATE123652T1 (de) 1995-06-15
EP0336562B1 (fr) 1995-06-14
IE890720L (en) 1989-09-04

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