EP2726872A1 - Détection d'epcam - Google Patents

Détection d'epcam

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Publication number
EP2726872A1
EP2726872A1 EP12730519.1A EP12730519A EP2726872A1 EP 2726872 A1 EP2726872 A1 EP 2726872A1 EP 12730519 A EP12730519 A EP 12730519A EP 2726872 A1 EP2726872 A1 EP 2726872A1
Authority
EP
European Patent Office
Prior art keywords
epcam
antibody
epex
epic
recognising
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.)
Withdrawn
Application number
EP12730519.1A
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German (de)
English (en)
Inventor
Gilbert Spizzo
Martin Wurm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ONCOTYROL - Center for Personalized Cancer Medicine GmbH
Original Assignee
ONCOTYROL - Center for Personalized Cancer Medicine GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by ONCOTYROL - Center for Personalized Cancer Medicine GmbH filed Critical ONCOTYROL - Center for Personalized Cancer Medicine GmbH
Priority to EP12730519.1A priority Critical patent/EP2726872A1/fr
Publication of EP2726872A1 publication Critical patent/EP2726872A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to the determination of epi ⁇ thelial cell adhesion molecule (EpCAM) in a sample and kits for use in EpCAM detection.
  • EpCAM epi ⁇ thelial cell adhesion molecule
  • EpCAM (also known as 17-1A, GA733-2, KSA, KS1/4, 323/A3; Seq ID No 1) is a calcium-independent homophilic intercellular cell adhesion molecule of 39-42 kDa which is expressed by the majori ⁇ ty of epithelial tissues.
  • the EpCAM antigen does not structural ⁇ ly resemble any of the major families of the adhesion molecules (cadherins, selectins, integrins, or cell adhesion molecules of the Ig superfamily) . It is a type transmembrane glycoprotein, consisting of an extracellular domain with two EGF-like repeats, and a short cytoplasmic domain of 26 amino acids.
  • the cDNA for EpCAM was cloned in 1989 from the lung adenocarcinoma cell line UCLA-P3 and designated KSA.
  • the gene was mapped to human chromo ⁇ some 2 by analysis of human-mouse somatic cell hybrids, by fluo ⁇ rescence in situ hybridisation and by PCR analysis (Spizzo et al . , Cancer Lett. 246 (2007), 253-261; Spizzo et al . , Br. Can. Res. Treat. 86 (2004), 207-213).
  • EpCAM shows frequent and high-level expression in numerous human malignancies including colon and breast cancers. EpCAM ex ⁇ pression can increase with disease progression in prostate cancer and cervical intraepithelial neoplasia. High EpCAM expres ⁇ sion is associated with poor disease-free and overall survival in invasive breast cancer. Silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in decreased cell proliferation of human breast cancer cell lines. Furthermore, EpCAM signalling seems to impact cell proliferation through up- regulation of the proto-oncogene c-myc. Beside its function as adhesion molecule, EpCAM can inhibit CD4 + T-cell dependent immune responses and thus enable tumour cells to evade T-cell mediated antitumour immunity (Spizzo et al . , 2007).
  • siRNA EpCAM short interfering RNA
  • EpCAM antigen has therefore attracted major interest as a target for passive and active immunotherapy.
  • Antitumour re ⁇ sponses have been observed in metastatic colorectal cancer after treatment with the EpCAM specific monoclonal antibody edre- colomab.
  • Adjuvant treatment of radically resected Dukes' C colo ⁇ rectal cancer patients using the same antibody induced a 32% relative reduction in mortality as compared to the results of surgery alone.
  • treatment with the edrecolomab antibody in a phase III study was shown to be less effective.
  • EpCAM expression can be up-regulated by chemotherapeutic agents increasing the effectiveness of EpCAM directed antibodies in terms of antibody-dependent cellular cytotoxicity.
  • the Ep ⁇ CAM antigen has become an attractive target for antitumour immune interventions (Spizzo et al . , 2007).
  • EpCAM EpCAM-binding protein
  • tu ⁇ mour patient samples Due to its marker function (and also due to its proposed function as a tumour disease target) measurement of EpCAM in tu ⁇ mour patient samples is of importance for diagnosis, prediction and monitoring of therapy in cancer patients utilising either chemotherapeutics or specific monoclonal antibodies (or other tumour treatments or combinations thereof) .
  • EpCAM in cancer patients, cell lines or other samples can be established by various methods including immunohistochemistry (Spizzo et al . , 2004; Slanchev et al . , PLoS Genetics 5 (2009), el000563) , ELISA (Abe et al . , J. Immunol. Meth. 270 (2002), 227-233; Petsch et al . , mAbs 3 (2011), 31-37); Schmetzer et al, Immunol. Lett. 143 (2012), 184-192), immunoflu ⁇ orescence, in-situ mRNA hybridisation (Gosens et al . , Modern Path.
  • EpCAM As a mi- togenic signal transducer in vitro and in vivo. This involves shedding of its ectodomain EpEx and nuclear translocation of its intracellular domain EpIC. Cleavage of EpCAM is sequentially catalysed by TACE and presenilin-2. Pharmacological inhibition or genetic silencing of either protease impairs growth-promoting signalling by EpCAM, which is compensated for by EpIC.
  • EpIC associates with FHL2, ⁇ -catenin and Lef-1 to form a nuclear complex ("EpCAM signalosome") that contacts DNA at Lef-1 consen ⁇ sus sites (thereby suggesting an association ("cross-talk") with the Wnt-pathway) , induces gene transcription and is oncogenic in immunodeficient mice.
  • EpIC was found in nuclei of colon carcinoma but not of normal tissue. Nuclear signalling of EpCAM explains how EpCAM functions in cell proliferation.
  • EpCAM is a potent signal transducer that uses com- ponents of the Wnt pathway, with a proven significance for tu ⁇ mour development and cell renewal could explain the expression of EpCAM at high levels and with high frequency in cancer- initiating cells and their progeny, and normal stem or progenitor cells.
  • EpCAM is expressed in the mammalian germline and is frequently present at the surface of human and murine stem cells and cancer stem cells. Consequently, EpCAM- specific antibodies served in many studies to isolate not only cancer stem cells but also very rare circulating tumour cells from metastatic lung, prostate, pancreatic, breast and colon cancers.
  • EpCAM signalling in normal and malignant stem-type cells is as yet unexplored (Maetzel et al . , 2009) . Accordingly, there is a significant need to analyse and elucidate signals for EpCAM cleavage and regulation of pro ⁇ teases associated with its activation in normal and malignant stem cells. Such analyses are dependent on an unambiguous iden ⁇ tification of the EpCAM species in a given sample.
  • a clear and definite distinction and distinguishing between the soluble form of total (Full-length) EpCAM i.e. the whole EpCAM molecules comprising an EpIC and an EpEx moiety, from the cleaved and shedded EpEx and EpIC domain is therefore needed. This need does not only exist in medicine but also in research.
  • the present invention should provide improved means for full-length EpCAM detection in samples, especially in samples of human tumour patients or patients being at risk of developing tumours or being suspected of having a tumour.
  • the present invention should provide means to specifi ⁇ cally determine full-length EpCAM, i.e. to discriminate between soluble full-length EpCAM and soluble EpEx.
  • the present invention provides a method for de ⁇ tecting full-length epithelial cell adhesion molecule (EpCAM) in a sample comprising the provision of a solid phase immunoassay wherein
  • either a monoclonal antibody recognising EpEx or a monoclo ⁇ nal antibody recognising EpIC is immobilised as a first an ⁇ tibody on a solid surface;
  • the first antibody is contacted with the sample to allow binding of EpCAM to the first antibody
  • the full-length EpCAM bound to the first antibody is con ⁇ tacted with a second antibody being either a monoclonal an- tibody recognising EpIC or a monoclonal antibody recognising EpEx to allow binding of full-length EpCAM bound to the first antibody to the second antibody; and
  • amino acids nos. 305 to 314 (Seq ID No 2) of the human EpCAM protein (as EpIC antibody) or amino acids nos. 75 to 88 (Seq ID No 3) of the human EpCAM protein (as EpEx anti ⁇ body) .
  • the amino acid numbering refers to the amino acid number ⁇ ing of the 314 amino acid long human EpCAM protein deposited in the UniProtKB/Swiss-Prot database under the Accession number PI 6422 (EPCAM_HUMAN; Seq ID No 1) . "Recognising" the amino acids nos. 305 to 314 or amino acids no.
  • 75 to 88 of the human EpCAM protein means that the antibody specifically binds to this re ⁇ gion.
  • Amino acids no. 305 to 314 is the most C-terminal portion of the EpCAM protein and also the most C-terminal part of the EpIC fragment of EpCAM.
  • the N-terminal Cys residue in Seq ID Nos 2 and 3 is preferably present (at the C- or N-terminus; prefera ⁇ bly at the N-terminus (especially in Seq ID No 2) in order to bind the EpCAM peptides to carriers or determine antibody bind ⁇ ing specificities) .
  • the present invention provides a highly specific determina ⁇ tion of full-length EpCAM and allows a clear and unambiguous discrimination between EpCAM and EpIC and EpEx. Since EpCAM can be cleaved in the organism to the EpIC and the EpEx fragment, binding of a molecule to an anti-EpCAM antibody which is specif ⁇ ic for an epitope in the EpEx fragment cannot unequivocally in ⁇ dicate that EpCAM is the molecule bound by the antibody; it could be EpEx as well. The same holds true for the binding to an antibody which is specific for an epitope on the EpIC fragment. Binding could indicate EpCAM as well as EpIC. Accordingly, none of the ELISA systems according to the prior art is capable of EpCAM specific determination, because none of these tests can distinguish between EpCAM and the cleavage product EpEx, because EpEx and EpCAM are two different proteins.
  • Abe et al . , 2002 disclose an ELISA where a monoclonal antibody against EpEx (EpEx was termed "sMK-1" in Abe et al . ) is immobilised in microtiter plate wells (the immobi ⁇ lised antibody was termed "M2-5") . EpEx is then bound to the im- mobilised antibody. Bound EpEx is then incubated with another monoclonal antibody against EpEx (termed “M4-10”) which recog ⁇ nises a different epitope than the immobilised antibody. It is clear that this ELISA cannot discriminate between EpEx and Ep- CAM.
  • Petsch et al . , 2011, provide an electroluminescence-based ELISA for quantification of EpCAM.
  • a biotinylated polyclonal goat-anti-human antibody which was raised against a mouse myeloma cell line NSO derived recombinant human EpEx domain (Gln24-Lys265 of EpCAM) was bound to a strep- tavidin-coated microplate. Then serum samples or serially dilut ⁇ ed recombinant EpEx spiked in human serum were bound to the im ⁇ mobilised polyclonal goat-anti-human antibody.
  • FaDu cells were stained with a mon ⁇ oclonal EpEx-antibody (HO-3; Ruf et al . , Br. J. Cancer 97 (2007), 315-321) and polyclonal EpIC-specific antibodies (affin ⁇ ity purified goat polyclonal antibody raised against a peptide mapping at the C-terminus of human EpCAM) in combination with Alexa-488 and Alexa _ 647 secondary antibodies, respectively.
  • Lo ⁇ calization of EpEx (green) and EpIC (red) was assessed with la ⁇ ser scanning microscopy.
  • a reliable, robust and very spe ⁇ cific method for determining full-length EpCAM is provided, which is specifically suited for routine testing. It allows de ⁇ termining EpCAM presence and concentrations in samples, espe ⁇ cially samples of human cancer patients or humans being suspect ⁇ ed or having a risk of having cancer, in an efficient manner.
  • any form of EpCAM can be detected and specifically identified.
  • full-length EpCAM i.e. the EpCAM molecule having an EpEx as well as an EpIC portion, can be identified and - in one and the same test - distinguished from EpCAM fragments, such as the EpEx and the EpIC being pre ⁇ sent in the same sample.
  • first antibody since it is preferred to immobilise the first anti ⁇ body to the solid surface before contact to the sample or the second antibody, it may preferably also be referred to as the "first immobilised antibody"
  • second antibody binding of the EpIC antibody
  • binding of an EpCAM species to the immobilised anti ⁇ body without binding of the second antibody proves the presence of EpEx in the sample.
  • EpIC antibody if the EpIC antibody is bound to the solid surface, binding of the EpEx antibody shows again the presence of full-length EpCAM in the sample whereas binding of an EpCAM species to the immobilised antibody without detection by the second antibody against EpEx shows presence of EpIC in the sample.
  • Determination of the binding events can be per ⁇ formed by various methods established for ELISA/immunosorbent assays.
  • binding of the EpCAM species can be determined by a reagent iden ⁇ tifying the binding event between the immobilised antibody and the EpCAM species (e.g. an antibody specifically recognising the immune complex between the immobilised immunoglobulin and the EpCAM species ("specifically" meaning that neither the (empty) immobilised antibody nor the non-bound EpCAM species is recog ⁇ nised by this antibody) .
  • the method according to the present invention is specifical ⁇ ly suited to detect soluble full-length EpCAM (sEpCAM or Ep- CAM SF ) , especially sEpCAM in the cytosol of isolated cancer cells or EpCAM in ascites, blood, plasma or serum samples isolated from humans.
  • EpCAM soluble full-length EpCAM
  • This sEpCAM species has surprisingly turned out to be a very meaningful indicator for the presence of cancer of do ⁇ nors in the course of the present invention.
  • EpCAM The role of EpCAM in cancer has recently been reclassified. Historically, EpCAM is a tumour antigen and the first one defined by antibody. In breast cancer and other solid tumours it has been shown by many groups that EpCAM is over-expressed up to lOOOfold.
  • EpCAM Overexpres- sion of EpCAM is correlated with poor prognosis in breast cancer patients.
  • EpCAM or “full-length EpCAM” always refers to an "uncleaved” or “non-cleaved” EpCAM, meaning that the EpEx part is still con ⁇ nected to the transmembranous and to the EpIC part of the EpCAM protein (Baeuerle et al . , Br. J. Cancer 96 (2007), 417-423).
  • EpCAM has oncogenic features in FaDu cells upon proteolytic cleavage into an extracellular part EpEx and a short intracellular domain EpIC. Although these results do not seem to be directly applicable to the situation in breast cancer cell lines or primary breast epithelia, it could be established in the course of the present invention that in four commercially available EpCAM over-expressing cell lines massive amounts of highly glycosylated protein is present in the cytosol. Upon cell lysis, full length glycosylated EpCAM but neither of the two EpCAM cleavage products EpEx or EpIC is found in the cytosolic lysate, proven by Western blot analysis. Also, when cells were lysed in RIPA buffer to solubilise membrane bound EpCAM; neither cleavage product could be identified.
  • the present invention identified for the first time full-length EpCAM as a monitoring target for specific tumour cell lysis in cancer patients with EpCAM over-expressing phenotype. Moreover, this invention provides for the first time a monitoring tool for EpCAM-antibody-mediated cell lysis since the EpEX antibody was designed to bind to an EpEX epitope other than that of most frequently used therapeutic antibodies (Ca- tumaxomab or Adecatumumab, these antibody bind to EGF 1 homolo ⁇ gous domain and not to the amino acids of the EpEX antibody used in this invention) . In view of these findings, the need for es ⁇ tablishing the test according to the present invention is even more evident.
  • the monoclonal antibody recognising EpIC or the monoclonal antibody recognising EpEx or both is labelled.
  • the label is preferably an enzyme (as in the "classical” ELISA) , a fluorophore, a chemiluminescent material, a radioisotope, or a coenzyme.
  • enzyme labels such as alkaline phosphatase or beta galactosidase are employed together with their appropri ⁇ ate substrates.
  • the enzyme/substrate reaction can be detected by any suitable means such as spectrophotometry.
  • the determination whether EpCAM and/or EpIC and/or EpEx have been recognised in the sample by the monoclonal antibodies is performed by using secondary antibodies binding to the monoclonal antibodies, said secondary antibodies being preferably labelled, especially with a fluorescence label, a luminescence label, a colourigenic la ⁇ bel; a radioactive label, a biotin or biotin analogue label, or combinations thereof.
  • EpCAM species bound to the immobilised mAb or the (se ⁇ cond) antibody bound to the EpCAM species can, in turn, be de ⁇ tected by having the antibody labelled directly or indirectly with a detectable marker.
  • the EpIC/EpEx-specific antibody can be contacted with a secondary antibody which is specific for the EpIC/EpEx-specific antibody. This secondary an ⁇ tibody can be labelled directly or indirectly with a detectable marker .
  • Another preferred embodiment relates to a method wherein the monoclonal antibody recognising the EpIC and the monoclonal an ⁇ tibody recognising EpEx are labelled with different labels, preferably with different fluorescence labels. Also quenching techniques can be applied. Moreover, also the EpCAM species in the sample can be labelled. It is also possible to include com ⁇ petitive techniques, e.g. by the addition of known amounts of an EpCAM species competing with the EpCAM species in the sample. For example, a labelled EpCAM can be added (e.g. a fluorescent or quencher labelled EpCAM) .
  • the solid surface contains a capturing molecule for one of the mAbs (either the EpIC or the EpEx mAb) , especially an antibody binding the EpEx or the EpIC mAb according to the present invention (e.g. a secondary antibody as disclosed above) .
  • Both mAbs according to the present in ⁇ vention can be brought into the sample to allow binding to EpCAM, EpEx or EpIC.
  • EpIC or EpEx are then bound to the surface via the capturing molecule binding the mAb recognis ⁇ ing EpEx or EpIC; if the bound species also carries the second antibody, presence of EpCAM is detected according to the present invention.
  • Presence of both mAbs according to the present invention in a form bound to the surface can be detected by various means, e.g. by different labels on the two mAbs according to the present invention or by interaction of the labels (e.g. a quenching or non-quenching effect if both mAbs are present in close distance (which is the case if both are present in a form bound to the surface captured by the capturing molecule.
  • the method according to the present invention can be performed in various practical embodiments known in the present field of surface based immunoassays.
  • the method can be provided as an enzyme linked immunosorbent assay (ELISA) .
  • ELISA enzyme linked immunosorbent assay
  • mAb monoclonal antibody
  • This term includes all forms of antibody molecules be ⁇ ing derivable (synthetically or biologically) from a monoclonal antibody, such as the whole mAb or only parts thereof (Fab, Fab' and F(ab' )2 / Fd, single-chain Fvs (scFv) , single-chain immuno ⁇ globulins (e.g., wherein a heavy chain, or portion thereof, and a light chain, or portion thereof, are fused) , disulfide-linked Fvs (sdFv) , diabodies, triabodies, tetrabodies, scFv minibodies, Fab minibodies, and dimeric scFv and any other fragments com ⁇ prising a V
  • Antigen-specific fragments may also comprise a V H H domain derived from a camelid antibody.
  • the V H H may be engi ⁇ neered to include CDRs from other species, for example, from hu ⁇ man antibodies.
  • a human-derived heavy chain V R fragment may be engineered to resemble a single-chain camelid CDR, a process referred to as "camelization" .
  • camelization a process referred to as "camelization”
  • the very nature of the antibodies used according to the present invention is mainly driven by the suitability for the detecting purposes ac- cording to the present invention.
  • the major prerequisites are the binding specificities to the EpCAM species and the detecta- bility of the binding event (e.g.
  • the term "monoclonal antibody” mainly serves to exclude EpCAM binding preparations of inhomogeneous or undetermined binding nature, such as polyclonal antibodies.
  • any sample which potentially contains soluble full-length EpCAM can be analysed and determined.
  • Preferred samples are, of course, sam ⁇ ples provided in connection with tumour diagnostics and tumour therapy. Therefore, the present method is preferably performed in samples of human tumour patients or patients being at risk of developing tumours or being suspected of having a tumour.
  • the present method is especially suitable for monitoring tumour pa ⁇ tients during therapy (especially EpCAM-specific therapies) since full-length EpCAM could be important markers for develop ⁇ ment of the tumour disease and the effectiveness of tumour ther ⁇ apy.
  • the sample is ascites, pleural effusions, blood or blood derived samples, such as serum or plasma; a tumour cell lysate, tumour tissue, or urine.
  • the major target tumour diseases are those for which EpCAM is known to be a tumour marker, i.e. expressed in the tumour cells of the patient.
  • the present invention is specifically suited for tumour diseases where presence and/or increase of Ep ⁇ CAM expression is known to correlate with poor prognosis.
  • the method according to the present invention is preferably applied in diagnosing and monitoring samples from tu ⁇ mour patients having or being suspected to have (or being at risk to have or develop) colon cancer, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer and cervical cancer, especially a cervical intraepithelial neo ⁇ plasia.
  • Solid support or “solid surface” refers to a material having a solid surface to which a mAb according to the present invention is immobilised.
  • immobilised it is meant bound covalently, or bound by noncovalent means such as hydrophobic adsorption.
  • a solid support may be the surface of a multiwell (microtiter) plate well, a bead, a membrane or a dipstick. Methods and means for covalently or noncovalently binding proteins to solid sup ⁇ ports are known in the art.
  • Suitable solid supports include la ⁇ tex, glass particles, including porous glass particles; poly- acrylamide particles; agarose; Sephadex ® (Pharmacia Fine Chemi ⁇ cals, Inc.); Sepharose ® ; bibulous materials such as glass or cel ⁇ lulose paper; plastics and polymers (e.g., in sheets, beads or microtiter wells) such as polystyrene, polyvinyl chloride, poly ⁇ styrene latex, or polyvinylidine fluoride (known as Immulon ® ) ; nylon; polymethacrylate ; etc.; silicons; metals such as gold and indium; nitrocellulose (e.g., in membrane or microtiter well form); activated beads; Protein A beads; diazotised paper; and the like.
  • plastics and polymers e.g., in sheets, beads or microtiter wells
  • polystyrene polyvinyl chloride, poly ⁇
  • the nature of the solid surface varies depending upon the specifically intended use or method.
  • the solid surface is the wall of the well or cup.
  • the solid surface is the surface of the bead.
  • the surface is the sur ⁇ face of the material from which the dipstick is made.
  • the solid surface may be the surface of latex or gelatin particles.
  • antigens When individual antigens are bound to a solid surface they may be distributed homogeneously on the surface or distributed thereon in a pattern, such as bands so that a pat ⁇ tern of antigen binding may be discerned.
  • the antibodies can al ⁇ so be bound to a surface which specifically recognises the bind ⁇ ing of a ligand (EpCAM, EpEx or EpIC) to the immobilised anti ⁇ body.
  • EpCAM EpCAM, EpEx or EpIC
  • Biochips can be designed to detect the binding of the lig ⁇ and and would therefore allow to discriminate between a bound ligand which is recognised by the second mAb according to the present invention (full-length EpCAM) and a bound ligand which is not recognised by the second mAb (EpIC or EpEx) allowing the immediate and distinguishment between full-length EpCAM and EpIC or EpEx in one and the same set-up (EpEx or EpIC would result in a binding signal only whereas full-length EpCAM results in binding and detection by the second antibody) .
  • Such techniques are e.g.
  • the present invention provides a kit for detecting full-length EpCAM in a sample comprising: a surface for binding monoclonal antibodies and performing the immunological testing according to the present invention,
  • the kit according to the present invention fur ⁇ ther comprises detection agents for detecting the monoclonal an ⁇ tibodies and/or detection means for detection of a binding event between the antibody and EpCAM in a sample.
  • the re ⁇ spective detection antibody is marked for specific detection, e.g. biotinylated .
  • the kit according to the present invention therefore already contains one of the mAbs im ⁇ mobilised to the solid surface, especially the mAb recognising EpEx immobilised to the solid surface (in this embodiment, the EpIC antibody is provided in soluble form or in a form which can be brought into such soluble form (e.g. lyophilised or frozen)) .
  • the EpIC antibody is provided in soluble form or in a form which can be brought into such soluble form (e.g. lyophilised or frozen)) .
  • the kit according to the present invention fur ⁇ ther comprises an EpCAM standard, preferably a recombinant EpCAM protein, a recombinant EpIC protein, a recombinant EpEx protein, or combinations thereof.
  • the kit according to the present invention fur ⁇ ther comprises a standard, e.g. a defined full-length EpCAM standard (preferably all three standards) , a capture antibody, a biotinylated detection antibody, e.g. a suitable biotinylated (or otherwise labelled (chromogenic, luminescent, colourigenic, fluorescent, etc.)) antibody against the monoclonal EpIC and/or EpIC antibody, containers for the sample (s) (e.g. an ELISA plate) , use instructions, written standard information, espe ⁇ cially tables of standard values of EpCAM levels in healthy hu ⁇ mans or cancer patients.
  • a standard e.g. a defined full-length EpCAM standard (preferably all three standards)
  • a capture antibody e.g. a capture antibody
  • a biotinylated detection antibody e.g. a suitable biotinylated (or otherwise labelled (chromogenic, luminescent, colourigenic, fluorescent, etc.))
  • the present invention applies more than one mAb for recognising EpIC and/or EpEx. It is specifically preferred to provide a test wherein a second monoclo ⁇ nal antibody for EpEx is provided which does not interfere with the binding of the first monoclonal antibody recognising EpEx ("does not interfere" means that these antibodies do not compete with each other (at the same binding site) ) .
  • the preferred monoclonal antibody against EpEx (binding to aa 75 to 88 of EpCAM) does not compete with catumaxomab; accordingly, ca- tumaxomab may be combined with the test kit and method of the present invention.
  • the second EpEx antibody may be used to confirm presence of EpEx in an immunosorbent assay based on an immobilised EpEx mAb.
  • Non-binding of the EpIC mAb in the test according to the present invention confirms absence of full-length EpCAM in the sample; additional testing with the second EpEx mAb can confirm presence or absence of EpEx in the same test (especially, if the second mAb for EpEx has a differ ⁇ ent label or can be differently detected than the EpIC antibody (e.g. two different fluorescence labels) .
  • EpIC antibody is immobilised, although the possibilities for providing a second EpIC antibody not com ⁇ peting with the first one are restricted and therefore less at ⁇ tractive for routine testing, specifically in view of the large number of EpEx mAbs already available.
  • a specific aspect of the present invention also deals with the monitoring of a tumour treatment which is based on an anti-EpCAM antibody, such as catumaxomab, edrecolomab, adecatumumab, oportuzumab monatox, tucotuzumab celmoleukin and citatuzumab communicatingox.
  • an anti-EpCAM antibody such as catumaxomab, edrecolomab, adecatumumab, oportuzumab monatox, tucotuzumab celmoleukin and citatuzumab communicatingox.
  • the method and kit of the present invention are specifically suitable for such monitoring.
  • Fig. 1 shows that the combination of the antibodies 9-2 and 54-7 is able to detect full-length EpCAM in lysates of cell lines (MDA-MB-231) that were previously transfected with the full-length EpCAM protein (MDA-MB EpCAM) compared to empty vector controls (MDA-MB_LV) .
  • Fig. 2 shows comparison of different antibodies: mAb 9-2 de ⁇ tects whole EpCAM in lysates of MCF-7 cells in WB; signal is similar to that of an EpIC polyclonal antibody (A-20) and of an antibody targeting the extracellular domain of EpCAM (C-10, Santa Cruz) ; and
  • mAb monoclonal mouse antibodies
  • BALB/c mice female, approx 8 weeks from Janvier (France) were used to generate hybridomas .
  • SP2/0- Agl4 cells Deutsche Sammlung von Mikroorganismen und Zellkultu- ren
  • isolated thymocytes were fused with SP2/0 cells. Positive clones were further subcloned. Immunisation, fusion and subcloning was performed by Biogenes GmbH, Berlin.
  • the first antibody 9-2 recog ⁇ nises the C-terminus of the intracellular domain of human EpCAM with the sequence C-MGEMHRELNA (Seq ID No 2) .
  • the second anti ⁇ body 54-7 is directed against the sequence C-GSKLGRRAKPEGAL (Seq ID No 3) located in the extracellular region of EpCAM.
  • Peptides for immunisation were produced by Biosyntan GmbH, Berlin.
  • Hybridoma were grown in ISF-1 medium (Biochrom AG, Berlin) and supernatants were harvested and sterile filtered. Antibodies were isolated from the supernatant by protein G chromatography and subsequently concentrated and rebuffered in phosphate buff ⁇ ered saline at pH 7, sterile filtered and aliquots thereof were stored at -35°C. Purity of mAbs was controlled by SDS-PAGE and concentration of mAbs was determined with a ND-1000 spectropho ⁇ tometer (Peqlab Biotechnologie GmbH, Er Weg) .
  • mAb 9-2 was biotinylated using sulfo-NHS-LC-Biotin from Thermo Scientific according to the manufacturer's instructions. EpCAM standard
  • Lysates of MCF-7 and Skbr-3 cells were pooled and al- iquots thereof were stored at -35°C.
  • concentration of EpCAM in aliquots was calculated by comparing with a recombinant EpCAM standard purchased from Sino Biological Inc. (Beijing).
  • the ELI SA for the detection of soluble human EpCAM (sEpCAM) in serum and ascites of cancer patients was performed using a sandwich ELISA system in standard 96well microtiter plates (BD Bioscience) .
  • 54-7 mAb directed against the extra ⁇ cellular EpCAM domain was used at a concentration of 0.5 yg/ml in PBS pH 7.0 at 4°C overnight.
  • PBS 0.05% Tween 20 wells were blocked with PBS 0.1% BSA for 20 min at RT .
  • EpCAM standard and samples were diluted in sample buffer con ⁇ sisting of PBS 0.1% BSA 0.05% Tween 20 in serial dilution and incubated for lh at RT .
  • biotinylated detec ⁇ tion antibody 9-2-bio was added at a concentration of 1 yg/ml in sample buffer for lh at RT .
  • streptavidin- peroxidase ultrasensitive Sigma was added 1:10.000 in PBS 0.5% BSA.
  • TMB Pacificn Biotech was used as substrate. Colour development was detected at 450/655 nm using a benchmark microplate reader (Biorad) .
  • MCF-7 and Skbr-3 cells were purchased from the American type cell culture collection (ATCC) .
  • BT-20 and MCF-7 cells were grown in MEM Eagle medium (PAN Biotech, Passau) supplemented with 10% FCS (PAA, Linz), 2mM glutamine, 100 U/ml of penicillin and lOOyg/ml of streptomycin (P/S, all from Invitrogen, Carlsbad, CA) .
  • Skbr-3 were grown in McCoy's 5A medium (PAN Biotech) supplemented with 10% FCS, 2mM glutamine and P/S.
  • T47-D cells were grown in RPMI 1640 medium (PAN Biotech) plus 10% FCS, 2mM glutamine and P/S.
  • lysates cells were harvest ⁇ ed in RIPA buffer ( Sigma-Aldrich, St. Louis, MO) .
  • cells were harvested in RIPA buffer, trans ⁇ ferred onto nitrocellulose membrane (Millipore, Billerica, MA) and detected using 9-2 and 54-7 antibody in combination with horseradish (HRP) -conj ugated polyclonal anti-mouse antibody and the enhanced chemiluminescence (ECL) system (Amersham Biosci- ence/GE Healthcare, Uppsala) .
  • HRP horseradish
  • ECL enhanced chemiluminescence
  • Table 1 sEpCAM (full-length) ELISA of human control sera of cancer patients and ascites of cancer patients. Values are %.
  • EpCAM in lysates of MCF-7 cells is depicted us ⁇ ing commercially available EpCAM antibodies in comparison with the 9-2 mAb.
  • the combination of the antibodies 9-2 and 54-7 is able to detect full-length EpCAM in lysates of cell lines (MDA-MB-231) that were previously transfected with the full-length EpCAM protein (MDA-MB EpCAM) compared to empty vector controls (MDA-MB_LV) .
  • Ep ⁇ CAM epithelial cell adhesion molecule
  • EpIC intracellular component of EpCAM
  • EpEx extracellular region of EpCAM
  • either a monoclonal antibody recognising EpEx or a monoclo ⁇ nal antibody recognising EpIC is immobilised as a first an ⁇ tibody on a solid surface;
  • the first antibody is contacted with the sample to allow binding of EpCAM and/or EpIC and/or EpEx to the first antibody;
  • the EpCAM and/or EpIC and/or EpEx bound to the first anti ⁇ body is contacted with a second antibody being either a mon ⁇ oclonal antibody recognising EpIC or a monoclonal antibody recognising EpEx to allow binding of EpCAM and/or EpIC and/or EpEx bound to the first antibody to the second anti ⁇ body; and
  • Ep ⁇ CAM epithelial cell adhesion molecule
  • EpIC intracellular component of EpCAM
  • EpEx extracellular region of EpCAM
  • the first antibody is contacted with the sample to allow binding of EpCAM and/or EpEx to the first antibody; and (cl) the EpCAM and/or EpEx bound to the first antibody is con- tacted with a second antibody being a monoclonal antibody recognising EpIC to allow binding of EpCAM bound to the first antibody to the second antibody;
  • the first antibody is contacted with the sample to allow binding of EpCAM and/or EpIC to the first antibody
  • ELISA enzyme linked immunosorbent assay
  • Kit for detecting EpCAM and/or EpIC in a sample comprising: a solid surface for binding monoclonal antibodies,
  • EpCAM standard preferably a recombinant EpCAM protein, a recombinant EpIC protein, a recombinant EpEx protein, or combinations thereof.

Abstract

L'invention concerne un procédé de détection de la molécule d'adhésion cellulaire épithéliale (EpCAM) de longueur totale dans un échantillon. Ce procédé consiste à effectuer un immunoessai en phase solide dans lequel - soit un anticorps monoclonal reconnaissant EpEx soit un anticorps monoclonal reconnaissant EpIC est immobilisé en tant que premier anticorps sur une surface solide ; - le premier anticorps est mis en contact avec l'échantillon pour permettre la liaison d'une EpCAM de longueur totale au premier anticorps ; et - l'EpCAM de longueur totale liée au premier anticorps est mise en contact avec un second anticorps qui est soit un anticorps monoclonal reconnaissant EpIC, soit un anticorps monoclonal reconnaissant EpEx pour permettre la liaison au second anticorps de l'EpCAM de longueur totale liée au premier anticorps ; et - à déterminer si une EpCAM de longueur totale a été reconnue dans l'échantillon par les anticorps monoclonaux.
EP12730519.1A 2011-06-28 2012-06-27 Détection d'epcam Withdrawn EP2726872A1 (fr)

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EP11171657A EP2541249A1 (fr) 2011-06-28 2011-06-28 Détection d'EpCAM
PCT/EP2012/062397 WO2013000930A1 (fr) 2011-06-28 2012-06-27 Détection d'epcam
EP12730519.1A EP2726872A1 (fr) 2011-06-28 2012-06-27 Détection d'epcam

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CN103076448B (zh) * 2012-12-31 2015-06-17 广州鸿琪光学仪器科技有限公司 一种新的子宫颈癌细胞的标记技术的制备方法及装置
EP2772758A1 (fr) 2013-02-27 2014-09-03 Oncotyrol Center for Personalized Cancer Medicine GmbH Diagnostic de tumeur EpCAM
US10787499B2 (en) 2017-02-13 2020-09-29 Regents Of The University Of Minnesota EpCAM targeted polypeptides, conjugates thereof, and methods of use thereof

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EP2041181B1 (fr) * 2006-06-08 2011-05-18 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Inhibiteurs spécifiques de protéase et leur utilisation pour le traitement du cancer
AU2010295172B2 (en) * 2009-09-21 2016-08-04 Ranju Ralhan Methods and compositions for the diagnosis and treatment of thyroid cancer

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