LV10202B - Human carcinoma-associated antigen and antibody binding to the antigen - Google Patents
Human carcinoma-associated antigen and antibody binding to the antigen Download PDFInfo
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- LV10202B LV10202B LV940022A LV940022A LV10202B LV 10202 B LV10202 B LV 10202B LV 940022 A LV940022 A LV 940022A LV 940022 A LV940022 A LV 940022A LV 10202 B LV10202 B LV 10202B
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Abstract
The innovation relates to a tumor associated antigen, the antibody to this antigen, a diagnostical remedy containing the antigen or antibody, the remedial composition containing the antigen or antibody and the application of the antigen or antibody for various diagnostical or therapeutic purposes.
Description
LV 10202
HUMAN CARCINOMA-ASSOCIATED ANTIGEN AND ANTIBODY BINDING TO THE ANTIGEN
The present invention relates to a tumour-associated antigen, an antibody directed against the antigen, a diagnostic aģent comprising the antigen or the antibody, a pharmaceutical composition comprising the antigen or the antibody, and the use of the antigen or the.antibody for a variety of diagnostic or therapeutic purposes.
The possibility of developing more precise methods of detecting and diagnosing cancer by identifying and characterizing tumour-associated antigens (i.e. antigens expressed by tumours) is of great medical interesi.
Monoclonal antibodies against tumour-associated antigens may play an important role for the detectioņ of cancer because of their greater specificity. To datē most of the monoclonal antibodies raised against cancer-associated antigens have been of mouse origin, being expressed by 2. hybridomas resulting from a fusion of spleen celis from a mouse immunized vvith a human cancer celi line or celis from a cancer patient with a mouse myeloma celi line. lmmunogenicity in the mouse is a requirement for antigens recognized by murine monoclonal antibodies and they do not necessarily correspond to antigens recognized by human antibodies. In addition, the therapeutic value of these murine monoclonal antibodies may be limited since patients recognize these antibodies as foreign proteīns and may therefore develop an adverse immune response against the murine antibody. The result may be a neutralization of the therapeutic effect and triggering of potentially dangerous allergic reactions.
Human hybridoma antibodies may be more promising as diagnostic and therapeutic aģents for administration to patients with cancer under the assumption that human monoclonal antibodies are less immunogenic in humāns than heterologous antibodies and are capable of recognizing the relevant antigens.
Problems related to the specificity of murine monoclonal anti-tumour antibodies are illustrated by the colin adenocarcinoma antibody 17-A1, vvhich has been used in diagnosis and therapy, but has now been found to react with normai as well as tumour tissue (Hybridoma 5 Suppl. 1, 1986, special issue on Ca-17-A1, ed. Z.Steplevvski).
The immune response in patients against administered murine monoclonal antibody has been described by numerous investigators (e.g. H.F.Sears et al., Lancet 1985, i:762-765; and M.S.Mitchell et al., Prog.Cancer Res. Ther. 21,1982, Raven Press, New York).
Colo-rectal canser is one of the most frequently occurring cancers and one of the major causes of death from cancer. The prognosis of this cancer type has not improved for a long period of time, and novel methods for the detection of colo-rectal cancer and adjuvant therapy concomitantly with surgery thereof are therefore needed.
Therefore, .a need exists for a human carcinoma tumour-associated antigen, in particular one vvhich is sabstantially not expressed by normai tissue, and antibodies against such an antigen for diagnostic and therapeutic purposes.
Accordingly, the present invention relates to a human carcinoma-associated cytoplasmatic antigen vvhich has at least one epitope vvhich in LV 10202 frozen tissue sections fixed in acetone of colin adenocarcinoma, ovarian adenocarcinoma, rēnai adenocarcinoma, mammary carcinoma, lung adenocarcinoma, non-semonomal testis carcinoma, mammary tubules, mammary ductuli and epithelium is exposed so that it binds to the monoclonal antibody produced by the human-human hybridoma celi line B9165 (ECACC 87040201) and vvhich is not so exposed in frozen tissue sections fixed in acetone of lung epithelial carcinoma, sarcoma, malignant melanoma, B-lymphoma, thymoma, ovarian stroma, ovarian epithelia, rēnai glomeruli, rēnai tubules, lung alveoles, bronchial epithelium, testis, epidermis, tonsillary lymphatic tissue, tinsillary epithelium, smooth muscles or blood vessels, vvhich antigen has an apparent molecular vveight of about 43,000 and an isoelectric point in the range of about 5.4-6.2, or an analogue thereof.
The term "analogue" is used in the present context to indicate a protein or polypeptide of a similar amino acid composition or sequence as the present antigen, allovving for minor variations vvhich do not have an adverse effect on the immunogenicity of the analogue. The analogous polypeptide or protein may be derived from another source than carcinoma tissue such as from a recombinant organism or may be partially or completely of synthetic origin. The term is further intended to mean any derivative of the antigen such as an immunogenic subsequence thereof. lmmunocytochemical analysis by Standard procedures using a monoclonal antibody with specific reactivity to the antigen indicates the presence of the novel antigen in human colon carcinoma and mammary carcinoma tissue, but not in duodenal adenocarcinoma tissue, melanoma or Burkitt's lymphoma tissue or human peripheral blood leukocytes (cf. Table I belovv).
Immunohistochemical analysis by Standard procedures using a monoclonal antibody vvith specific reactivity to the antigen indicates the presence of the novel antigen in colon adenocarcinoma, ovarian adenocarcinoma,. rēnai adenocarcinoma, mammary adenocarcinoma, lung adenocarcinoma and non-seminomal testis carcinoma tissues, but not in lung epithelial carcinoma, sarcoma, malignant melanoma, B-lymphoma or thymoma tissue or in normai tissues except for mammary tubules, mammary ductuli or prostate epithelium (cf. Tables IIA and IIB belovv).
It is therefore concluded that the novel antigen is one vvhich is substantia!ly not found in normai human tissue and vvhich has been found to be expressed by carcinoma tissue, in particular adenocarcinoma tissue, but not by other malignant tissues as determined by Standard immunocytochemical and immunohistochemical analyses. In particular, the novel antigen seems to be one associated with colon adenocarcinoma. A tumour-associated antigen expressed by colon carcinoma is disclosed in EP 199 586. This antigen, hovvever, differs from the antigen of the present invention in characteristics such as molecular vveight and isoelectric point, and it is indicated to be present in both normai and malignant colon tissue (i.e. it does not appear to be tumour-specific) vvhereas the antigen of the present invention has been shovvn to be present in several different malignant tissues, as indicated above.
The antigen of the invention may for instance be obtained by isolation from extracts of carcinoma celis or extracts of carcinoma tumours by affinity chromatography on an insolubilized antibody raised against the antigen according to procedures known to those skilled in the art (cf. Johnstone, A. & Thorpe, R., lmmunochemistry in Practice, Blackvvell, Oxford 1987). Further purification by one or more additional protein purification procedures (e.g. size exclusion chromatography, ion exchange chromatography, ligand affinity chromatography, hydrophobic interaction chromatography, electrophoresis in gels with or vvithout denaturaration, and various precipitation procedures) may be necessary to obtain the antigen in sufficiently pure form for detailed molecular characterization and for use for diagnostic or therapeutic purposes.
In order to ensure an adequate supply of the antigen, it may, however, be advantageous to producē the antigen by recombinant DNA techniques. These may comprise (a) isolating a nucleotide sequence coding for the t antigen, e.g. by estačlisling a cDNA or gene library of human carcinoma celis and screening for positive clones in accordance with conventional methods; (b) inserting said sequence in a suitable, replicatable expression vector; (c) transforming a suitable host microorganism with the vector produced in step (b); (d) cultivating the microorganism produced in step (c) under suitable conditions for expressing the antigen; and (e) harvesting the antigen from the culture.
Steps (a)-(e) of the process may be carried out by Standard methods, e.g. as described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Apring Harbor, 1982.
Once the gene coding for the antigen has been isolated, it may be possible to establish the DNA sequence by standart procedures, e.g. as described by Sanger et al., Science 214, 1981, p.1205, or Gilbert, Science 214, 1981, p, p.1305, and the amino acid sequence may be deduced on the basis of the DNA sequence. The antigen or a subsequence thereof may then LV 10202 be produced by conventional peptide synthesis, e.g. Iiquid or solid phase peptide synthesis, soiid phase peptide synthesis being the preferred procedure (cf. R.B. Merryfield, J.Am. Chem.Soc. 85,1963, p.2149, or Stewart and Young, Solid Phase Peptide Synthesis, Freeman & Co., San Francisco Co., USA, 1969). In solid phase synthesis, the amino acid sequence is constructed by coupling an initial amino acid to a solid support and then sequentially adding the other amino acids in the sequence by peptide bonding until the desired length has been obtained.
In another aspect, the present invention relates to an antibody vvhich in immunohistochemical analysis on frozen tissue sections fixed in acetone binds to colon adenocarcinoma, ovarian adenocarcinoma, rēnai adenocarcinoma, mammary carcinoma, lung adenocarcinoma, non-seminomal testis carcinoma, mammary tubules, mammary ductuli and prostate epithelium and does not bind to lung epithelial carcinoma, sarcoma, malignant melanoma, B-lymphoma, thymoma, ovarian epithelia, rēnai glomeruli, rēnai tubules, lung alveoles, bronchial epithelium, testis, epidermis, tonsillary lymphatic tissue, tonsillary epithelium, smooth muscles or blood vessels and vvhich binds to a human carcinoma-associated cytoplasmatic antigen vvhich has an apparent molecular vveight of about 43,000 and an isoelectric point in the range of about 5.4-6.2 and vvhich has at least one epitope vvhich is reactive with a monoclonal antibody produced by the human hybridoma celi line B9165 (ECACC 87040201).
The antibody is advantageously a monoclonal antobody since these tend to be of a higher specificity than polyclonal antibodies, making them useful for accurate diagnostic determinations. Due to the considerations outlined above, the antibody when intended for injection into patients should preferably be of human prigin, that is, be one produced by a fusion product of a human lymphocyte and a human celi line or a fusion product of a human lymphocyte and, for instance, a murine myeloma celi· line, rather than a fusion product of a murine lymphocyte and a murine myeloma celi line, vvhich has hitherto been usual for the production of monoclonal antibodies against human tumour-associated antigens. Such monoclonal antibodies may be raised against different epitopes on the antigen. One example of a useful monoclonal antibody is the one denoted C-OU1 produced by the human hybridoma celi line B9165 vvhich was deposited on 2 April, 1987, in the European Collection of Animal Celi Cultures (ECACC), Centre for Applied Microbiology and Research, Porton Down, Salisbury, VViltshire, Great Britain, with the accession number ECACC 87040201. 6.
The monoclonal antibody of the invention may be prepared by a method comprising a) isolating antibody-producing celis from a cancer patient, b) fusing celis producing the antibody with celis of a suitable human fusion celi line, and selecting and cloning the resulting hybridoma celis producing said antibody, and c) grovving the celis of step b) in a suitable medium to producē said antibody, and harvesting the antobody from the growth medium.
The antibody-producing celis used for fusion to the human fusion celis are preferably spleen or lymph node celis. The fusion of antobody-producing celis and human celis may be performed substantially as described by Kohler and Milstein, Nature 256, 1975, p. 495, or Kohler, Immunological Methods Vol. II, Academic Press, 1981, pp. 285-298, that is, preferably in the presence of a fusion promoter such as polyethylene glycol. The human fusion celi line employed is preferably of a type unable to survive in selective medium; one type of celi line frequently used for celi fusions is one vvhich lacks the enzyme hypoxanthine-guanine phosphoribosyltransferase and which is consequently unable to grow in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium).
The selection of hybridoma celis vvhich producē an antibody against the carcinoma antigen may then be carried out by culturing unfused antibody-producing cellsm, unfused human fusion celis and supposedly fused celis in a selective medium (such as HAT) in vvhich the unfused human fusion celis cannot grovv and eventually die out. The unfused antibody-producing celis can only survive for a limited period of time after vvhich they also die out. On the other hand, successfully fused celis continue to grovv as they have inherited permanent grovvth properties from the parent human fusion celis and the ability to survive in the selective medium from the parent antobody-producing celis.
The resulting antibody-producing celis may be grovvn in vitro after cloning in a suitable medium, such as RPMI 1640. This results in the production of monoclonal antibodies of a very hign purity as these are secreted into the culture supernatant by the celis. The antibodies may then be isolated by conventional methods such as centrifugation, filtration, precipitation, chromatography, or a combination thereof.
In an specific embodiment, the present invention thus relates to a method of producing a monoclonal antibody for a colonic carcinoma-associated cytoplasmatic antigen vvhich has an apparent molecular vveight of LV 10202 about 43,000 and an isoelectric point in the range of about 5.4-6.2 and vvhich has at least one epitope vvhich is reactive with a monoclonal antibody produced by the human hybridoma celi line B9165 (ECACC 87040201), said method comprising providing lymphocytes derived from mesenteric lymph nodēs draining the tumor region in a patient having a colonic cancer, fusing these lymphocytes with a human celi line suitable for use in a fusion for the purpose of creation of human-human hybridomas, screening the hybridoma celis obtained for hybridomas that secrete a monoclonal antibody vvhich specifically binds to colon adenocarcinoma, ovarian adenocarcinoma, rēnai adenocarcinoma, mammary carcinoma, lung adenocarcinoma, non-seminomal testis carcinoma, mammary tubules, mammary ductuli and prostate epithelium and does not bind to lung epithelial carcinoma, sarcoma, malignant melanoma, B-lymphoma, thymoma, ovarian stroma, ovarian epithelia, rēnai glomeruli, rēnai tubules, lung alveoles, bronchial epithelium, testis, epidermis, tonsillary lymphatic tissue, tonsillary epithelium, smooth mascles or blood vessels.
For purposes not requiring monoclonality, the antibody may be a polyclonal antibody. This may be prepared by injecting a suitable animal (e.g. a rabbit, mouse or goat) with a substantially pure preparation of the antigen follovved by one or more booster injections at suitable intervāls (e.g. two vveeks to a month) up to six months before the first bleeding. Then, vvhile continuing this established immunization regimen, the animals are bled about one week after each booster immunization, and antibody is isolated from the serum in a conventional manner, e.g. as described in Harboe and Ingild, Scand. J.lmmun. 2 (Suppl. 1), 1973, pp.161-164.
For some purposes, it may be an advantage that the antibody is a hybrid vvhich contains a combining site directed specifically against an epitope of the antigen of the invention and another directed specifically against another epitope of the same antigen, an epitope of another antigen or a pharmaceutical. The term "combining site" is understood to mean the antigen recognition structure in the variable region of the antibody molecule. Hybrid antibodies make special procedures possible for detecting the antigen in a sample and for targeting a pharmaceutical or other biologically active molecule or another antigen to the site of the tumour vvhere the reaģent has the greatest effect. In an advantageous embodiment, the other antigen with vvhich the hybrid antibody is reactive is a differentiation antigen of cytotoxic T-cells (cf.Staerz et al., Nature 314, 1985, p.628). The pharmaceutical with vvhich the hybrid antobody may be reactive is preferably selected from cytotoxic or antineoplastic aģents (cf. Collier, R.J. and Kaplan, D.A., Scientific American 251,1984, p.44).
The hybrid antibody may be produced by hybrids between two monoclonal celi lines producing the two relevant antibodies or may be produced by chemically linking fragments of the two antibodies.
The antibody may, for different purposes, vide below, be an anti-idiotypic antibody, i.e. an antibody directed against the site of an antibody which is reactive with the epitope on the antigen. The anti-idiotypic antibody is directed against an antibody which is reactive with the antigen of the invention. The anti-idiotypic antibody may be prepared by a similar method to that outlined above for the monoclonal or polyclonal antibody. The antibody may also be an anti-anti-idiotypic antibody directed against the anti-idiotypic antibody defined above.
In a further important aspect, the present invention relates to a diagnostic aģent vvhich comprises an antibody of the invention as described above, a fragment of an antibody as described in the follovving, an anti-idiotypic antibody as described above, or anti-anti-idiotypic antibody as described above.
Although in some cases, such as when the diagnostic aģent is to be employed in an agglutination assay in vvhich solid pārticies to vvhich the antibody is coupled agglutinate in the presence of a carcinoma antigen in the sample subjected to testing, no labelling of the antibody is necessary, it is preferred for most purposes to provide the antibody or the fragment of the antibody vvith a label in order to detect bound antibody or fragment of antibody. In a double antibody ("sandvvich") assay, at least one of the antibodies may be provided vvith a label. Substances useful as labels in the present context may be selected from enzymes, fluorescent substances, radioactive isotopes and ligands such as biotin.
Examples of enzymes vvhich may be used as label substances are peroxidases, e.g.. horseradish peroxidase, or phosphatases, e.g. alkaline phosphatases. As enzymes are not directly detectable, they must be combined vvith a substrate to form a detectable reaction product vvhich may, for instance, be fluorescent or coloured. Examples of useful substrates are Hi Oz /o-phenylene diamine, Hz 0* /azinodiethylbenzthiazoline sulphonic acid, ΗΑ Oi /diaminobenzidine and p-nitrophenylphosphate. Such reaction Products may be detected by the naked eye as a colour emergence or change, or by means of a spectrophotometer. LV 10202
Examples of fluorescent substances useful as label substances are Hi Oz /p-hydroxyphenylacetic acid and methylumbelliferyl phosphate. Such substances may be detected by means of a fluorescence spectrophotometer in a manner knovvn per se.
Examples of radioactive isotopes useful as label substances are 1-125, S-35 and p-35. The radioactivity emitted by these isotopes may be measured in a gamma-counter or a scintillation counter in a manner knovvn per se.
In a favoured embodiment, the diagnostic aģent comprises at least one antibody or fragment of antibody coupled to a solid support. This may be used in a double antibody assay in vvhich case the antobody or fragment of antibody bound to the solid support is not labelled vvhile the unbound antibody or fragment of antibody is labelled. The solid support may be composed of a. polymer or may comprise a matrix on vvhich the polymer is applied. The solid support may be selected from a plastic, e.g. Iatex, polystyrene, polyvinylchloride, nylon, polyvinylidene difluoride, or cellulose, e.g. nitrocellulose, silicone, silica and a polysaccharide such as agarose or dextran.
For use in a diagnostic assay, the solid support may have any convenient shape. Thus, it may be in the form of a plate, e.g. a thin layer or, preferably, microtiter plate, a strip, film, paper or microparticles such as latex beads or the like.
Rather than being coupled directly to the solid support, the monoclonal antibody or fragment of antibody may be coupled to a spacer immobilized on a solid support. Examples of spacers include Protein A.
It should be noted that practically ali methods or applications based on intact antibodies could instead be performed using fragments of the antibodies, e.g. F(ab')A or Fab fragments (cf. Delaloye, B. et al., J.Clin. Invest. 87, 1986, p.301). Therefore, in a number of embodiments a fragment of the antibody of the invention can be used as an alternative to the vvhole antibody.
For use in a sandvvich assay, the diagnostic aģent may additionally comprise another antibody. This other antibody may be labelled and/or coupled to a solid support as described above. In this embodiment, either or bOth of the antibodies may be a monoclonal antibody as described above.
Alternatively, the diagnostic aģent of the invention may comprise a carcinoma antigen as defined above. This aģent may be used to detect the presence of antibodies against the carcinoma antigen in a sample. The diagnostic aģent may othervvise exhibit any of the features described above 10. for diagnostic aģents comprising an antibody of the invention or a fragment thereof, although they will detect bound antibody rather than binding of an antibody to the antigen.
In a stili further aspect, the invention relates to a method of in vitro diagnosing human carcinoma, comprising contacting a sample of a body fluid from a suspected cancer patient with a diagnostic aģent according to the invention comprising an antibody of the invention or a fragment of said antibody, and determining the presence of antigen bound to the antobody or fragment of antibody. The antibody may be labelled and/or bound to a solid support as exemplified above. The body fluid may be selected from blood, plasma, serum, lymph, lung expectorate, urine and gastrointestinal fluīds.
In a favoured embodiment of the method, the sample is incubated with a first monoclonal antibody coupled to a solid support and subsequently with a second monoclonal or polyclonal antibody provided with a label. An example of this embodiment is the sandvvich ELISA (enzyme linked immuno sorbent assay) described in Voller, A. et ai., Buli. World Health Organ, 53, 1976, p.55.
In an alternative embodiment (a so-called competitive binding assay), the sample may be incubated with a monoclonal antibody coupled to a solid support and subsequently with a labelled carcinoma antigen, the latter competing for binding sites on the antibody with any carcinoma antigen present in the sample.
An alternative embodiment relates to a method of in vitro diagnosihg human carcinoma, comprising contacting a tissue sample from a suspected canser patient with a diagnostic aģent of the invention comprising an antibody of the invention or a fragment of said antibody, and determining the sites on the sample to which antibody or fragment of antibody is bound. Such an immunohistochemical method may be carried out according to well-established procedures, e.g. as described belovv in Example 2.
The diagnostic aģent of the invention may be used in a method of localizing tumours (in particular carcinomas) in vivo by means of the antibody of the invention. This method comprises administering a diagnostically effective amount of an antibody of the invention which is labelled so as to permit detection thereof, and determining the sites of localization of bound antibody. The antibody may be labelled by means of a radioactive isotope and subsequently injected and localized by knovvn methods, e.g. a gamma ray detector of a suitable configuration (cf.Mach, J.-P. et al., Nature 248,1974, p.704). LV 10202 A stili further embodiment relates to a method of in vitro diagnosing human carcinoma, comprising contacting a sample of a body fluid from a suspected cancer patient with a diagnostic aģent of the invention comprising the antigen or the anti-idiotypic antibody of the invention, and determining the presence of bound antibody against the antigen of the invention present in said body fluid.
In yet aspect, the invention relates to a pharmaceutical composition for the treatment of human carcinoma, which comprises an antigen according to the invention or an antobody, a fragment of an antibody, an anti-idiotypic antibody or an anti-anti-idiotypic antibody according to the invention and a pharmaceutically acceptable excipient.
The excipient employed in the composition of the invention may be any pharmaceutically acceptable vehicle. This vehicle may be any vehicle usually employed in the preparation of injectable compositions, e.g. a diluent, suspending aģent etc. such as isotonic or buffered saline. The composition may be prepared by mixing a therapeutically effective amount of the antigen, antibody or fragment of antibody with the vehicle in an amount resulting in the desired concentration of the antigen, antibody or fragment of antibody in the composition.
In some cases it may be advantageous to couple the antigen, antibody or fragment of antibody to a carrier, in particular a macromolecular carrier. The carrier is usually a polymer to vvhich the toxin is bound by hydrophobic non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the antigen, antibody or fragment of antibody is covalently bound, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or keyhole limpet hermocyanin. Furthermore, the carrier may advantageously be selected from a pharmaceutical, e.g. a cytotoxic or antineoplastic aģent, to vvhich the antigen, antibody or fragment of antibody is coupled. The carrier may also be another antibody directed against a cytotoxic effector mechanism, e.g. cytotoxic celis. The carrier should preferably be non-toxic and non-allergenic. The antigen, antibody or fragment of antibody may be multivalently coupled to the macromolecular carrier as this may provide an increased immunogenicity of the composition.
For oral administration, the composition may be in the form of a tablet, capsule, granulate, paste, gel, mixture or suspension optionally provided with a sustained-release coating or a coating vvhich protects the antigen from passage through the stomach. 12.
Solid formulations, i.e. granulates, tablets and capsules, may contain fillers, e.g. sugars, sorbitol, mannitol and silicic acid; binders, e.g. cellulose derivatives such as carboxymethyl cellulose and polyvinylpyrrolidone; disintegrants, e.g. starch, sodium bicarbonate and calcium carbonate; lubricants, e.g. magnesium stearate, talc and calcium stearate. Semisolid formulations, i.e. pastes or gels, may comprise a gelling aģent such as an alginate, gelatin, carrageenan, tragacanth gum and pectin, a mineral oil such as liquid paraffin, a vegetable oil such as corn oil, sunflovver oil, rape oil and grape kernel oil, as well as a thickener such as a starch, gum, gelatin, etc. Liquid formulations, i.e. mixtures and suspensions, may comprise an aqueous or oily vehicle, e.g. vvater, or a mineral oil such as liquid paraffin, a vegetable oil such as corn oil, sunflovver oil, rape oil, grape kernel oil, etc. The antigen of the invention may be suspended in the liquid vehicle in accordance with usual practice.
The sustained-release coating may, e.g., be an enteric coating vvhich may be selected from shellac, cellulose acetate esters such as cellulose acetate phthalate, hydroxypropylmethyl cellulose esters such as hydroxypropylmethyl cellulose phthalate, polyvinyl acetate esters such as polyvinyl acetate phthalate, and polymers of methacrylic acid and (meth)acrylic acid estera.
The composition may also be adapted for rectal administration, e.g. as a suppository. Such a suppository may contain conventional excipients such as cocoa butter or other’glycerides.
Finally, the invention relates to the use of an antigen according to the invention or an anti-idiotypic antibody of the invention for preparing a medicament for the treatment of human adenocarcinoma, or to the use of an antibody, a fragment of an antobody or an anti-anti-idiotypie antibody according to the invention for preparing a medicament for the treatment of human carcinoma.
Therapy of cancers, in particular carcinomas, expressing the carcinoma antigen of the invention may be carried out by a variety of procedures knovvn to those skilled in the art. An antibody or a fragment of an antibody against the antigen (in particular a human monoclonal antibody or a fragment thereof for the reasons stated above) may be injected into cancer patients to combat the tumour directly or via various effector mechanisms, e.g. complement-mediated cytotoxicity or antibody-dependent cell-mediated cytotoxicity. The antibody or the antibody fragment may be modified prior to injection into the patient as indicated above, e.g. by coupling to LV 10202 pharmaceuticals (thus transporting those to the site of their activity), or to another antibody directed against a cytotoxic effector mechanism such as antigens on cytotoxic T-cells or on other cetotoxic celis. It is contemplated that hybrid antibody containing one combining site for the antigen and another against another antigen or a pharmaceutical as described above may also advantageously be used to provide a two-way attack on the tumour in question.
The antibody of the inventiOn or a fragment thereof may be used in extra-corporal devices for removing circulating tumour antigen or immune complexes containing tumour antigen, thereby reconstituting the anticancer immune response by allovving the immune system to recover from paralysis induced by said tumour antigen or immune complexes.
The carcinoma antigen of the invention may be used for immunization in order to provoke an anticancer immune response in the body. The antigen may further be used in vitro for raising effector celis against cancer by culturing the antigen with leukocytes from a cancer patient.
The carcinoma antigen may be used in extra-corporal devices for removing anti-tumour antobody or immune complexes in a similar way ti that employing the antibody.
The antibody against the carcinoma antigen or a fragment thereof may turthermore be administered to provoke an anti-idiotypic or anti-anti-idiotypic immune response. An anti-idiotypic antibody (whether monoclonal or polyclonal or a fragment of these) raised against an antibody reacting with the carcinoma antigen of the invention may express epitopes similar to those of the antigen and may therefore be used in a similar fashion for immunization to raise an anti-carcinoma immune response. Such antibodies may further be used in extra-corporal devices as described above for the antigen and primary antibody.
Anti-anti-idiotypic antibodies may be used in ways similar to those described for primary anti-tumour antibodies.
BRIEF DESCRIPTION OF THE DRAVVINGS
The invention will be described in further detail in the follovving Examples and with reference ti the appended dravvings, vvhere
Fig. 1 shows immunocytochemical staining with C-OU1 of COLO 201 celis (colonic adenocarcinoma celis, positive) and staining of the same celis with a non-reacting human hybridoma IgM (negative); 14
Fig. 2A and B show immunohistochemical staining with C-OU1 of a frozen tissue specimen from colonic adenokarcinoma (2A) and normai tonsillar tissue (2B);
Fig. 3A and B show an electron mocroscopic analysis of the reaction of C-OU1 with Colon 137 celis (colonic adenocarcinoma celis, positive) (3A) and HUTU 80 (duodenai adenokarcinoma celis, negative) (3B);
Fig. 4 shovvs an immuno-blot analysis of tissue extracts separated by isoelectric focusing; and
Fig. 5 shovvs an analysis of carcinoma antigen by SDS-PAGE blotting by labelling with C-OU1. Extract of Colon 137 (colon adenocarcinoma celis, positive) (5A) and extract of HUTU 80 (duodenai adenocarcinoma celis, negative (5B). EXAMPLE 1
Production of human monoclonal antibody (C-OU1)
Mensenteric lymph nodēs draining the tumour region in patients with colo-rectal cancer vvere minced under sterile conditions. Debris were removed by filtration through cotton wool and the lymphocytes were purified by centrifugation on Ficoll-isopaque (Boehringer-Mannheim, Mannheim, Federal Republic of Germany).
The lymphocytes were fused with the human fusion celi line VV1-L2-729-HF2 (in the follovving referred to as HF2) (from Tecniclone Int., Santa Ana, California, USA) according to Kohler, Immunological Methods Vol. II, Academic Press, 1981, pp.285-298. The ratio betvveen the HF2 and lymphocytes (10 )was1:2.
After vvashing the HF2 and the lymphocytes together in RPMI-1640 medium follovved by centrifugation, the celi peliet was resuspended in 0.5 ml of 50% PEG (polyethylene glycol) 6000 over a period of 1 minūte vvith constant shaking. Before dilution of the PEG vvith RPMI-1640, the celis were incubated for another 2 minūtes. Then the fusion product was vvashed and resuspended in solution medium [RPMI-1640, 10% FCS (fetal calf serum) supplemented vvith HAT (2x10"^ M hypoxanthine, 4x10'^ M aminopterin, 3.2x10*c M thymidine)]. The celis vvere plated at 2x10* celis in 200 μ\ per vvell of 96-well microtiter plates. The celis vvere maintained in selective medium for two vveeks. Further culturing vvas carried in RPMI-1640 + 10% FCS supplemented vvith hypoxanthine and thymidine. Grovving hybrids appeared LV 10202 10 days to 4 weeks after tusion. Cloning was performed by limiting dilution vvithout feeder celis,
Supernatants from vvells with growing clones were analyzed for immunoglobulin production by ELISA (enzyme linked immuno sorbent assay) on microtiter plates coated with rabbit anti-human Ig (H and L chain) (Dakopatts, Copenhagen, Denmark) diluted 1:10,000 in 0.1 M bicarbonate, pH 9.6. Coated vvells were vvashed with PBS-Tvveen (phosphate buffered saline - 0.05% Tvveen 20) and incubated for 2 hours at room temperature with supernatants diluted 1:10 in PBS-Tween. Development was carried out with alkaline phosphatase (AP)-coupled antibody specific for IgM, IgA or IgG (Dakopatts, Copenhagen) diluted 1:3000 in PBS-Tween. After incubation for 1 hour at room temperature, the substrate p-nitrophenylphosphate (PNPP), 1 mg/ml 10% diethanolamine, 1 mM MgCli , pH 9.6, was added. Optical density was measured at 405 nm after 1 hour of incubation at 37 ° C. Standard curves for quantification were constructed with dilution of IgM (Cappel) or IgG (Kabi AB, Stackholm, Svveden). Hybrids producing immunoglobulin (Ig) assayed by ELISA were propagated by transfer to 24-well macroplates (Nunc A/S, Denmark), and the supernatants were further analyzed in immunocytochemical analysis for reaction with tumour celis or in immunohistochemical analysis for reactions with tumour tissues as described belovv.
The hybridoma celi line B9165 (ECACC 87040201) selected by the methods described belovv vvas shovvn by ELISA to producē betvveen 1 and 5 j*g of IgM per ml vvhen allovved to grovv for tvvo vveeks vvithout any change of medium. EXAMPLE 1
Antigen characterization a) lmmunocytochemical analysis
The analysis for anti-tumour reactivity vvas carried out by immunocytochemical analysis and by immunohistochemical analysis. lmmunocytochemical analysis vvas performed on celi smears prepared from different human tumour celi lines and peripheral human blood leukocytes. Celis vvere fixed on slīdes by treatment vvith formol-acetone (9.5% formaldehyde, 43% acetone in 86 mM phosphate buffer, pH 7.2). 16.
Approximately 50 μλ of C-OU1 supernatant (from the hybridoma B9165; ECACC 87040201) was placed on the smear of fixed celis and incubated overnight at 4°C in a humidified chamber before rinsing and incubation for 1 hour at room temperatur wlth horseradish peroxidase (HRP)-labelled rabbit anti-human IgM (Dakopatts) diluted to 1:80 in PBS-Tween. Finaly, peroxidase substrate (0.01% Hz 0i and diaminobenzidine at 0.6 juxj/ml in PBS) was added. The smears were lightly counterstained with hematoxylin and mounted. Table 1 shovvs the results obtained by analysis of C-OU1 on smears of various celis. TABLE 1
Reactivity of C-OU1 assayed by immunocytochemistry
Type of call Name Reaction 1 .Colon adenocarcinoma Colon 137 Positive 2.Colon adenocarcinoma COLO 201 Positive 3.Melanoma HU 373 Negative 4.Mammary carcinoma MCF-7 Positive 5.Duodenal adenocarcinoma HUTU 80 Negative 6.Burkitt's lymphoma RB-2 Negative 7.Human peripheral blood leukocytas PBL Negative A selective reactivity is apparent. Altematively, the live celis were incubated with the hybridoma antibody at 4 ° C, followed by the enzyme-labelled anti-lg antibody. The celis were then smeared on slides, fixed with glutaraldehyde (0.17% in PBS) and incubated with substrate. Fig. 1A shovvs the staining of live COLO 201 celis (colonic adenocarcinoma celis) with C-OU1 vvhile Fig. 1B shovvs the lack of staining vvhen using a non-reactive human hybridoma IgM in the first layer. The method employed is the accepted Standard procedure for labelling molecules exposed on the celi surface, and the labelling seen in Fig. 1 is in accordance vvith this. b) Immunohistochemical analysis LV 10202
Immunohistochemical analysis was performed on frozen tissue sections fixed in acetone. Endogenous IgM was blocked by incubation with Fab1 fragments of anti- μ* -chain antibody (purchased from Dakopatts, Copenhagen, Denmark) before the incubation with the hybridoma antibody C-OU1 (0.5μ£)/ητιΙ) (the Fab' fragments were prepared according to B. Nieisen et ai., Hybridoma 6 (1), 1987, pp. 103-109) (the hybridoma antibody was concentrated by precipitation with 2M ammonium suiphate). The bound hybridoma antibody was then visualized as described above for the immunocytochemicai analysis. In this case the same antibody preparation was labeiled with peroxidase as that used for preparing Fab' fragments. Fig.2A shovvs that after application of C-OU1, only the tumour celis are stained in a section of a colon adenocarcinoma. Fig. 2B shovvs the lack of staining of tonsillar tissue. Table IIA and B summarize the reactivity as analyzed on a variety of tissues, Tabie lla shovving the results from malignant tissues and Table IIB shovving the results from non-malignant tissue.
TABLE IIA
Reactivity of C-OU1 (0.5Mg/ml) on frozen, acetone fixed tissue sections of human malignant tissues analyzed by immunohistochemical technigues
Tissue
Result
Colon adenocarcinoma Ovarian adenocarcinoma Rēnai adenocarcinoma Mammary carcinoma Lung adenocarcinoma Lung epithelial carcinoma Non-seminomal testis carcinoma Sarcoma Malignant melanoma B-lymphoma Thymoma 19/21 a) 2/2 1/2 7/9 7/7 0/6 1/1 0/3 0/7 0/1 0/1 18 . a) No. positive/ No. tested
TABLE IIB
Reactivity of C-OU1 (0.5>cg/ml) on frozen, acetone fixed tissue sections of human normai tissues analyzed by immunohistochemical techniques
Tissue
Result
Ovarian stroma Negative Ovarian epithelia Negative Rēnai glomeruli Negative Rēnai tubules Negative Mammary tubules Positive Mammary ductuli Positive Lung alvaoles Negative Bronchial epithelium Negative Testis Negative Epidermis Negative Tonsillary lymphatic tissue Negative Tonsillary epithelium Negative Smooth muscles Negative Blood vessels Negative Prostate epithelium Positive
Normai colon epithelium showed binding of ali analyzed human IgM, monoclonal antibodies, myeloma IgM as well as normai polyclonal human IgM. This general binding of IgM to normai colon epithelium was thus judged to be non-specific, an interpretation which was further supported by analysis by electron microscopy and isoelectric focusing (see belovv). This non-specific binding of antibody is also in accordance with the knowledge of persons skilled in the art. LV 10202 c) Electron microscopy
For electron microscopy, 10b/ml adenocarcinoma celis were cultivated in RPMI-1640 medium, 10% FCS, in tubes with plastic cover slīps for 3 days until a monolayer had been obtained. The celis were fixed in 0.1% (v/v) glutaraldehyde in 0.1 M PBS, pH 7.2, for 30-60 minūtes at 4° C and then vvashed in PBS supplemented with bovine serum albumin (BSA) and lysine-HCL overnight at 4 0 C. The celis were dehydrated in from 30% to 90% ethanol at progressively lower temperatures to -20 0 C, then infiltrated in Lowicryl K4M (Chemische Werke Lowi, Federal Republic of Germeny) at -35° C and polymerized overnight at -35° C under ultraviolet light and then for additionally 2 days at ambient temperature.
Ultrathin (50-60 nm) sections of the cell-containing Lowicryl mounted on coated nickel grids were used. The immunomarking procedure consisting in floating the grids with the sections downwards on top of different Solutions comprised the follovving steps: 1) the grids were placed on drops of 1% (w/v) NaBH<j in PBS for 10 minūtes; 2) after vvashing in 0.1% .(w/v) BSA-Tris for 2x5 minūtes, the grids were transferred to drops of 3% BSA-Tris (15 minūtes); 3) the grids were placed on drops of C-OU1 antibody for 1 hour at room temperature; 4) the grids were vvashed in 0.1% BSA-Tris for 2x5 minūtes; 5) the grids were transferred to drops of rabbit anti-human IgM dissolved in 3% BSA-Tris for 1 hour at room temperature; 6) after vvashing in 0.1% BSA-Tris for 2x5 minūtes, the grids were placed on drops of goat anti-rabbit IgG labelled with gold probes (Janssen Pharmaceuticals) of 15 nm, dissolved in 3% BSA-Tris, incubated for 30 minūtes at room temperature; 7) the grids were vvashed in 0.1% BSA-Tris for 2x5 minūtes and in redistilled vvater for 2x5 minūtes and dried; and 80 the ultrathin sections vvere stained vvith 1% uranyl acetate for 10 minūtes and 0.4% lead citrate for 2 minūtes at room temperature.
Tvveen 20 and 0.5 M NaCI vvere added to ali antibody and vvashing Solutions.
The ultrathin sections vvere examined in a JEOL 100-CX electron microscope operating at 8- kV.
Experiments to assess the specificity of the immunocytochemical reactions included omission of the primary antibody and substitution of human IgM (Cappel) for the primary antibody. 20.
Fig. 3A shows the distinct pattern of the labelling of colon adenocarcinoma celis (Colon 137) in reģions around the ends of intermediate filaments and Fig. 3B the lack of labelling (by a similar procedure) of duodenal adenocarcinoma celis (HUTU 80). Sections of colon adenocarcinoma cancer tissue shovved labelling only of the tumour celis again associated with intermediate filaments (not shown). Normai colon epithelium shovved no labelling. Electron microscopy thus reveals the presence of the target antigen in association with cytoplasmic structures. d) Isoelectric focusing
Molekular characterization of the target antigen was carried out by isoelectric focusing. Tumour celis or cancer tissue and normai tissue were solubilized by ultrasonication (4x30 seconds on ice) in extraction buffer ( 75 mM NaCI, 75 mM KCI, 10 mM Hepes, 5 mM EDTA, 5% 2-mercaptoethanol, 5 mg/l Trasylol, 0.01 mM Lenpeptin, 0.01 mM Pepstatin). After ultrasonication, urea was added to 6 M together with 80 mg of sucrose and the homogenate was incubated fpr 30 minūtes at 37 ° C. Insoluble material was removed by centrifugation (5 minūtes at 15.000 x g).
Isoelectric focusing was performed with the extracts applied onto 1% agarose thin-layer gel (Agarose IEF, Pharmacia) containing 6 M urea, 3% (w/v) servalytes 4-6 (Serva). As support gel-based film (LKB) was used. Focusing was performed for 1500 V/h before electrophoretic transfer of the proteīns onto nitrocellulose. Remaining binding sites were blocked by incubation of the nitrocellulose in 0.1 M Tris-HCI, pH 7.5, containing 0.1 NaCI, 2 mM MgCli and 0.05% Tvveen 20. The nitrocellulose was cut into strips, incubated for 2 hours at room temperature with the hybridoma antibody C-OU1 diluted in PBS-Tween to about 200 ng/ml. After vvashing with PBS-Tween, the nitrocellulose strips were incubated with alkaline phosphatase conjugated Fiab'^-anti IgM antibody (Jackson Immuno-research) follovved by substrate (a solution of 5-bromo-4chloroindoxyl phosphate and nitro blue tetrazolium).
Fig. 4, left panei, shovvs the staining of extracts of a colon adenocarcinoma tumour and Fig.4, right panei, an extract of normai colon epithelium. I. staining of total protein with India ink; ii. staining with C-OU1; and III. staining with a different hybridoma IgM. Evidently, C-OU1 shovvs distinct staining of acidic proteīns (pl 5.4-6.2) in the tumour extract, but not in the extract of normai colon. LV 10202 e) SDS-PAGE and VVestern blotting
Extracts of Colon 137 (colon adenocarcinoma celis) and HUTU 80 (duodenal adenocarcinoma celis) were prepared by solubilization with detergents (2% SDS, 4M urea, 10 mM iodoacetamide) and incubated for 15 minūtes before PAGE on 5 to 20% gradient gels. The proteīns were then electrophoretically transferred to nitrocellulose sheets. The nitrocellulose sheets were cut into 3 mm stripe and incubated overnight at A°C with C-OU1 followed by incubation for 2 hours with AP-rabbit anti-human IgM (Sigma). The blots were vvashed in PBS and fixed by incubation for 15 minūtes with 0.2% glutaraldehyde in PBS. Alkaline phosphatase was visualized by incubation for 1 hour at 37° C with substrate, nitro blue tetrazolium and 5-bromo-4-chloroindoxyl phosphatase. The molecular vveight was calculated from the mobility of the follovving prestained molecular weight markers: β-galactosidase (116K), fructose-6-phosphatase kinase (84 K), pyruvate kinase (58 K), fumarase (48.5 K), lactic dehydrogenase (36.5 K), triosephosphatase isonerase (26.6 K).
The results in Fig. 5 show that C-0U1 reacts with a protein with a molecular vveight of 43,000 found in the extracts of colon adenocarcinoma and not in extracts of duodenal adenocarcinoma.
Claims (39)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK169987A DK169987D0 (en) | 1987-04-03 | 1987-04-03 | HUMAN TUMOR-ASSOCIATED ANTIGEN, CA-OU1 |
| EP88105299A EP0285173B1 (en) | 1987-04-03 | 1988-03-31 | Human carcinoma-associated antigen and antibody binding to the antigen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| LV10202A LV10202A (en) | 1994-10-20 |
| LV10202B true LV10202B (en) | 1995-08-20 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LV940022A LV10202B (en) | 1987-04-03 | 1994-01-27 | Human carcinoma-associated antigen and antibody binding to the antigen |
Country Status (2)
| Country | Link |
|---|---|
| AT (1) | ATE95426T1 (en) |
| LV (1) | LV10202B (en) |
-
1988
- 1988-03-31 AT AT88105299T patent/ATE95426T1/en not_active IP Right Cessation
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1994
- 1994-01-27 LV LV940022A patent/LV10202B/en unknown
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| LV10202A (en) | 1994-10-20 |
| ATE95426T1 (en) | 1993-10-15 |
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