DK162844B - Antibody against a human carcinoma-associated antigen, such a human carcinoma-associated antigen, an anti idiotypic antibody which is directed against the antibody, an anti-anti-idiotypic antibody which is directed against the anti-idiotypic antibody, a diagnostic agent which comprises the antibody or the antigen, a process for preparing a monoclonal antibody against the carcinoma-associated antigen, a process for, etc. - Google Patents

Description

DK 162844 B

The present invention relates to an antibody to a human carcinoma-associated antigen, such a human carcinoma-associated antigen, an anti-idiotypic antibody directed to the antibody, an anti-anti-idiotypic antibody directed to the anti-idiotypic antibody, a diagnostic agent. comprising the antibody or antigen, method for producing a monoclonal antibody against a carcinoma-associated antigen, method for in vitro diaanostication of human carcinoma using the diagnostic agent, use of the antibody to prepare an agent. for in vivo diagnosis of human carcinoma, a pharmaceutical composition containing the antibody or antigen and use of the antibody, antigen, an anti-idiotypic antibody, or the anti-idiotypic antibody for the manufacture of a drug for the treatment of a human carcinoma. .

The ability to develop more precise methods for the detection and diagnosis of cancer by identifying and characterizing tumor-associated antigens (i.e., antigens expressed by tumors) is of great medical interest.

Monoclonal antibodies against tumor-associated antigens may play a significant role in the detection of cancer because of their specificity. Until now, most of the monoclonal antibodies produced against cancer-associated antigens have been of mouse origin and are expressed by hybridomas resulting from a fusion of spleen cells from a mouse that has been immunized with a human cancer cell line or cells from a cancer patient, with a mouse-much-30 pocket cell line. Immunogenicity in the mouse is a requirement for antigens to be recognized by mouse monoclonal antibodies and they do not necessarily correspond to antigens recognized by human antibodies. In addition, the therapeutic value of these mouse monoclonal antibodies may be limited, as patients recognize these antibodies as foreign proteins and may therefore develop an opposite immune response to the mouse antibody. The result may be a neutralization of the therapeutic effect and the triggering of potentially dangerous allergic reactions.

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Human hybridoma antibodies may be more promising as diagnostic and therapeutic agents for administration to cancer patients under the assumption that human monoclonal antibodies are less immunogenic in humans than heterologous antibodies and are capable of recognizing the relevant antigens. .

Problems related to the specificity of mouse monoclonal anti-tumor antibodies are highlighted by the colonic adenocarcinoma antibody 17-A1, which has been used in diagnosis and therapy, but has now been shown to respond with both normal and Tumor Tissue of Hybridoma 5 Suppl. 1, 1986, 15 Special Edition on Ca-17-Al, ed. Z. Steplewski).

The immune response of patients to administered mouse monoclonal antibody has been described by several researchers (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 20).

Colo-rectal cancer is one of the most common cancers and one of the main causes of cancer deaths. The prognosis for this type of cancer has not improved for a long time, and therefore new methods are needed for detection of colo-rectal cancer and adjuvant therapy at the same time as surgery thereof.

Therefore, there is a need for a human carcinoma tumor-associated antigen, in particular an antigen which is not substantially expressed by normal tissue, and antibodies to such an antigen for diagnostic and therapeutic purposes.

The present invention relates to an antibody to a human carcinoma-associated antigen which by immunohistochemical

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3 analysis on frozen, acetone-fixed tissue sections binds to colon adenocarcinoma, ovarian adenocarcinoma, renal adenocarcinoma, breast carcinoma, pulmonary adenocarcinoma, non-seminomal testis carcinoma, breast tubules, breast ductuli and prostate and does not bind to pulmonary epithelial carcinoma, sarcoma, malignant melanoma, B lymphoma, thymoma, ovarian stroma, ovarian epithelium, renal glomeruli, renal tubules, pulmonary alveoli, bronchial epithelium, testis, epidermis, tonsillar lymphatic tissue, tonsillar mouse epithelium, blood vessel.

10 Monoclonal antibodies produced by hybridoma cells or transformed B cell lines derived from cancer patients actively immunized with autologous tumor antigens are described in EP 151,030. However, compared immunohistochemical assays with the antibody of EP 151,030 and the antibody of the invention have shown a significant and significant difference between these two antibodies. Thus, the antibody described in EP 151,030 reacts with smooth muscle, whereas the antibody of the invention does not react with smooth muscle. Therefore, the antibody of the invention does not recognize the same epitopes as the antibody described in EP 151,030. Since smooth muscle cannot be described as human carcinoma-associated, the antibody of the invention has a higher cancer specificity than the antibody mentioned above.

The antibody may be a polyclonal antibody, but is most advantageously a monoclonal antibody as these tend to have a higher specificity than polyclonal antibodies, making them suitable for accurate diagnostic assays. Due to the above considerations, the antibody, when intended for injection into 30 patients, should preferably be of human origin, ie. be a monoclonal antibody produced by a fusion product of a human lymphocyte and a human cell line or a fusion product of a human lymphocyte and, for example, a mouse myeloma cell line, and not a fusion product of a mouse lymphocyte and a mouse myeloma cell line, have been usual in the preparation of monoclonal antibodies to human

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4 tumor-associated antigens. Such monoclonal antibodies may target different epitopes on the antigen. One example of a suitable monoclonal antibody is the antibody designated C-OU1 and produced by human hybridoma cell line B9165, which was deposited in the European Collection of Animal Cell Cultures (ECACC), Center for Applied Microbiology and Research,

Porton Down, Salisbury, Wiltshire, United Kingdom, with the deposit number ECACC 87040201.

The monoclonal antibody of the invention may be prepared by a method comprising the steps of: (a) antibody producing cells isolated from a cancer patient; (b) cells producing the antibody are fused with cells from an appropriate human fusion cell line, and the resulting hybridoma cells producing the antibody are selected and cloned; and (c) the cells of step (b) are grown in an appropriate medium for producing the antibody and the antibody 20 is harvested from the culture medium.

The antibody producing cells used for fusion to the human fusion cells are preferably spleen or lymph node cells. The fusion of antibody producing cells and human fusion cells can be performed essentially as described by Kohier and Milstein, Nature 256. 1975, p. 495, or Kohler, Immunological Methods Vol. II, Academic Press, 1981, pp. 285-298, viz. preferably in the presence of a fusion promoter such as polyethylene glycol. Hybridomas produced by this fusion technique are described in EP 178 30 891, which describes hybridomas produced by fusing lymphocytes isolated from a regionally draining lymph node from a patient having a vulvar carcinoma to a drug-resistant human lymphoblastoid B cell line. In the present invention, the lymphocytes are isolated from 35 mesenteric lymph nodes which drained the tumor area into the

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5 patients with colo-rectal cancer, and merged with the human fusion cell line W1-L2-729-HF2. The human fusion cell line used is of a type which is unable to survive in selective medium; one type of cell line that is often used for cell fusions is a line that lacks the enzyme hypoxanthine-guanine phosphoribosyl transferase and is therefore unable to grow in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium). .

Selection of hybridoma cells producing an antibody to the carcinoma antigen can then be performed by culturing non-fused antibody-producing cells, non-fused human fusion cells, and putative fused cells in a selective medium (such as HAT), in which the unfused human fusion cells unable to grow and eventually become extinct. The 15 non-fused antibody producing cells can only survive for a limited time, after which they also become extinct. On the other hand, well-fused cells continue to grow as they have inherited permanent growth properties from the original human fusion cells and ability to survive in the selective medium from the original antibody producing cells.

The resulting antibody producing cells can be grown in vitro after cloning in a suitable medium such as RPMI 1640. This results in the production of monoclonal antibodies 25 with a very high purity as they are secreted into the culture supernatant by the cells. The antibodies can then be isolated by conventional methods such as centrifugation, filtration, precipitation, chromatography or a combination thereof.

For purposes that do not require a very specific antibody, the antibody may be a polyclonal antibody. This can be made by injecting a suitable animal (e.g., a rabbit, mouse or goat) with a substantially pure preparation of the antigen, followed by one or more booster injections at appropriate intervals (e.g., from two weeks 35 to one month). to six months before the first blood-

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6 ning. As this established immunization regimen is continued, blood from the animals is then taken for approx. one week after each booster immunization, and antibody is isolated from the serum in conventional manner, e.g., as described in Harboe and Ingild, 5 Scand. J. Immun. 2 (Suppl. 1), 1973, pp. 161-164.

For some purposes, it may be advantageous that the antibody be a hybrid antibody containing one combination site specifically directed to one epitope of the antigen of the present invention, and another specifically targeted to another epitope of the same antigen, an epitope of another antigen or a pharmaceutical. The term "combination site" is intended to denote the antigen recognition structure in the variable region of the antibody molecule. Hybrid antibodies enable specific procedures to detect the antigen in a sample and to target a pharmaceutical or other biologically active molecule or other antigen to the site of the tumor where the reagent is most effective. In an advantageous embodiment, the second antigen with which 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 which the hybrid antibody can be reactive is preferably selected from cytotoxic or antineoplastic agents (cf. Collier, R. J. and Kaplan, D. A., Scientific American 25 251, 1984, p. 44).

The hybrid antibody can be produced by hybrids between two monoclonal cell lines producing the two relevant antibodies, or it can be produced by chemically binding fragments from the two antibodies.

30 The antibody can for various purposes, cf. below, be an anti-idiotypic antibody, i.e. an antibody directed to the site of an antibody reactive with the epitope on the antigen. The anti-idiotypic antibody is directed to an antibody reactive with the antigen of the invention. The anti-idiotypic antibody can

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7 is prepared by a procedure similar to that described above for the monoclonal or polyclonal antibody. The antibody may also be an anti-anti-idiotypic antibody directed against the above-defined anti-idiotypic antibody.

In another aspect, the invention relates to an isolated human carcinoma-associated antigen which has an estimated molecular weight of approx. 43 kD and an isoelectric point in the range of approx. pI 5.4-6.2, and having at least one epitope reactive with a monoclonal antibody of the invention, namely the antibody produced by the human hybridoma cell line B9165 (ECACC 87040201), or an analog thereto.

In the present context, the term "analog" refers to a protein or polypeptide having a similar amino acid composition or sequence to the present antigen, taking into account minor variations which do not adversely affect the antigenicity of the analog. The analog polypeptide or protein may originate from a source other than carcinoma tissue such as from a recombinant organism, or it may be wholly or partially of synthetic origin. The term further denotes any derivative of the antigen such as an immunogenic sub-sequence thereof.

Immunocytochemical analysis by standard procedures using a monoclonal antibody with specific reactivity to the antigen shows the presence of the novel antigen in human colon carcinoma and breast carcinoma tissue, but not in duodenal adenocarcinoma, melanoma or Burkite lymphoma tissue -cytes (see Table I below).

30 Immunohistochemical analysis by standard procedures using a monoclonal antibody with specific reactivity to the antigen shows the presence of the novel antigen in colon adenocarcinoma, ovarian adenocarcinoma, renal adenocarcinoma, lung adenocarcinoma, lung.

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8 adenocarcinoma and non-seminomal testis carcinoma tissues, but not in lung epithelial carcinoma, sarcoma, malignant melanoma, B lymphoma or thymoma tissues or in normal tissues other than breast tubules, breast ductuli or prostate epithelium (see.

5 Tables HA and IIB below).

Therefore, it can be concluded that the novel antigen is an antigen which is essentially absent in normal human tissue and has been found to be expressed by carcinoma tissue, especially adenocarcinoma tissue, but not by other malignant tissues, which have been determined by standard immunocytochemical and immunohistochemical assays. In particular, the novel antigen appears to be an antigen associated with colon adenocarcinoma.

A tumor-associated antigen expressed by colon carcinoma is described in EP 199 586. However, this antigen is different from the antigen of the present invention in characteristics such as molecular weight and isoelectric point, and it is described to be present in both normal and malignant colon tissue (i.e., it does not appear to be tumor specific), whereas the antigen of the present invention has been found to be present in several different malignant tissues as mentioned above.

The antigen of the invention may be obtained, for example, by isolation from extracts of carcinoma cells or extracts of carcinoma tumors by affinity chromatography on a non-solubilized antibody directed against the antigen by methods known to those skilled in the art (cf. Johnstone, A.

Thorpe, R., Immunochemistrv in Practice. Blackwell, Oxford 1987). Further purification by one or more additional 30 protein purification procedures (e.g., size exclusion chromatography, ion exchange chromatography, ligand affinity chromatography, hydrophobic interaction chromatography, electrophoresis on gels with or without denaturation, and various precipitation procedures) may be necessary to obtain sufficient anti-35 pure form for detailed molecular

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9 for use in diagnostic or therapeutic applications.

However, to ensure a sufficient yield of the antigen, it may be advantageous to prepare the antigen by 5 recombinant DNA techniques. These may include: (a) isolating a nucleotide sequence encoding the antigen, for example, by establishing a cDNA or gene library of human carcinoma cells and screening for positive clones in accordance with conventional methods; (b) inserting this sequence into a suitable replicable expression vector; (c) transforming a suitable host microorganism with the vector prepared under step (b); (d) the microorganism prepared under step (c) is grown under appropriate conditions for expression of the antigen; and (e) the antigen is harvested from the culture.

The steps (a) - (e) of the process can be performed by standard methods, for example, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, 1982.

Once the gene encoding the antigen has been isolated, it may be possible to determine the DNA sequence by standard procedures, e.g., as described by Sanger et al.

Science 214. 1981, p. 1205, or Gilbert, Science 214.

1981, p. 1305, and the amino acid sequence can be deduced on the basis of the DNA sequence. The antigen or sub-sequence thereof can then be prepared by conventional peptide synthesis, for example liquid or solid phase peptide synthesis, with solid phase peptide synthesis being the preferred procedure (cf. RB Merryfield, J. Am. Chem. Soc. 85. 1963, p. 2149, or Stewart and Young, Solid Phase Peptide Synthesis. Freeman & Co., San Francisco, USA, 1969). In solid phase synthesis, the amino acid sequence is constructed by coupling an initial amino acid to a solid support and then successively adding the other amino acids in the sequence by peptide bond until the desired length is achieved.

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In a further important aspect, the present invention relates to a diagnostic agent comprising an antibody of the invention as described above, an anti-idiotypic antibody as described above, or an anti-anti-idiotypic antibody as described above.

Although in some cases, such as when the diagnostic agent is to be used in an agglutination assay, in which solid particles to which the antibody is coupled, agglutinate in the presence of a carcinoma antigen in the sample undergoing testing is not necessary. to label the antibody, it is preferred for most purposes to provide the antibody with a marker to detect bound antibody.

In a double antibody ("sandwich") assay, at least one of the antibodies may be provided with a marker. Substances which can be used as markers in this context can be selected from enzymes, fluorescent substances, radioactive isotopes and ligands such as biotin.

Examples of enzymes that can be used as marker s tofers are peroxidases, e.g., horseradish peroxidase, or phosphatases, e.g., alkaline phosphatases. Since enzymes are not directly detectable, they must be combined with a substrate to form a detectable reaction product, which may be, for example, fluorescent or colored. Examples of useful substrates are H2O2 / o-phenylenediamine, H2O2 / azinodiethylbenz-thiazoline sulfonic acid, H2O2 / diaminobenzidine and p-nitrophenylphosphate. Such reaction products can be detected with the naked eye as a color appearance or change or by a spectrophotometer.

Examples of fluorescent substances that can be used as marker markers are H 2 O 2 / p-hydroxyphenylacetic acid and methyl lumelliferyl phosphate. Such substances can be detected by a fluorescence spectrophotometer in a manner known per se.

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Examples of radioactive isotopes that can be used as marker substances are 1-125, S-35 and P-35. The radioactivity emitted by these isotopes can be measured in a gamma counter or a scintillation counter in a manner known per se.

In a preferred embodiment, the diagnostic means comprises at least one antibody coupled to a solid support. This can be used in a double antibody assay, in which case the antibody bound to the solid support is not labeled, while the unbound antibody is labeled. The solid support may consist of a polymer or may comprise a matrix on which the polymer is placed. The solid support may be selected from a plastic, e.g., latex, polystyrene, polyvinyl chloride, 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 be of any convenient form. It may thus be in the form of a plate, for example a thin layer plate or preferably a microtiter plate, a strip, film, paper or microparticles such as latex balls or the like.

X instead of being directly coupled to the solid support, the monoclonal antibody may be coupled to a spacer immobilized on a solid support. Examples of spacers include Protein A.

It should be noted that practically all methods or uses based on intact antibodies can instead be performed using fragments of the antibodies, e.g., F (ab ') 2 "or Fab fragments (cf. Delaloye, B. et al., J. Clin. Invest. 87, 1986, p. 301).

In addition, for use in a sandwich assay, the diagnostic agent may comprise another antibody. This second antibody may be labeled and / or coupled to a solid support as described above. In this embodiment, it can

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12, one or both of the antibodies may be a monoclonal antibody as described above.

Alternatively, the diagnostic agent of the invention may comprise a carcinoma antigen as defined above. This agent can be used to detect the presence of antibodies against the carcinoma antigen in a sample. The diagnostic agent may also exhibit any of the features described above for diagnostic agents comprising an antibody of the invention, although this diagnostic agent detects bound antibody instead of binding an antibody to the antigen.

In a further aspect, the invention relates to a method for in vitro diagnosis of human carcinoma expressing the antigen of the invention, by which method a sample 15 of a body fluid from a putative cancer patient is contacted with a diagnostic agent of the present invention, which agent comprises an antibody according to the invention, after which the presence of bound antigen is determined. The antibody may be labeled and / or bound to a solid support as exemplified above. The body fluid can be selected from blood, plasma, serum, lymph, lung examinate, urine and gastrointestinal fluids.

In a preferred embodiment of the method, the sample is incubated with a first monoclonal antibody coupled to a solid support, and then with a second monoclonal or polyclonal antibody provided with a marker. An example of this embodiment is the sandwich ELISA (enzyme-linked immunosorbent assay) described in Voller, A. et al., Bull. World Health Organ. 53. 1976, p.55.

In an alternative embodiment (a so-called competitive binding assay), the sample can be incubated with a monoclonal antibody coupled to a solid support and then with a labeled carcinoma antigen, the latter competing for binding sites on the antibody with the carcinoma antigen which possibly included in the sample.

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An alternative embodiment relates to a method for in vitro diagnosis of human carcinoma expressing the antigen 5 of the invention, by which method a tissue sample from a putative cancer patient is contacted with a diagnostic agent of the present invention, comprising an antibody of the invention , after which the locations of the sample to which antibody is bound are determined. One such immunohistochemical method can be performed according to well-established procedures, for example, as described below in Example 2.

A further embodiment of a diagnostic method of the invention relates to a method of locating 15 tumors (especially carcinomas) in vivo by the antibody of the invention. This method comprises administering a diagnostically effective amount of an antibody of the invention, which antibody is labeled to enable detection thereof, and then determining the sites for localization of bound antibody. The antibody can be labeled with a radioactive isotope and then injected and located by known methods, for example, a gamma ray detector with an appropriate configuration (cf. Mach, J.-P. et al., Nature 248.

1974, p. 704).

A further embodiment relates to a method for in vitro diagnosis of human carcinoma expressing the antigen of the invention, in which method a sample of a body fluid from a putative cancer patient is contacted with a diagnostic agent of the present invention, which agent comprises the antigen or anti-idiotypic antibody of the invention, after which the presence of bound antibody to the antigen of the invention present in the body fluid is determined.

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In a further aspect, the invention relates to a pharmaceutical composition for the treatment of human carcinoma, comprising a antigen of the invention or an antibody of the invention as well as a pharmaceutically acceptable excipient.

The excipient used in the composition of the invention can be any pharmaceutically acceptable carrier. This carrier may be any carrier commonly used in the preparation of injectable compositions, e.g., a diluent, suspending agent, etc., such as isotonic or buffered saline. The composition may be prepared by mixing a therapeutically effective amount of the antigen or antibody with the carrier in an amount which provides the desired concentration of antigen or antibody in the composition.

In some cases it may be advantageous to link the antigen or antibody to a carrier, especially a macromolecular carrier. The carrier is usually a polymer to which the antigen or antibody binds by hydrophobic non-covalent interaction, such as a plastic, e.g., polystyrene, or a polymer to which the antigen or antibody binds covalently, such as a polysaccharide, or a polypeptide, e.g. bovine serum albumin, ovalbumin or "keyhole limpet hemocyanin". In addition, the carrier may advantageously be selected from a pharmaceutical, e.g., a cytotoxic or antineoplastic agent, to which the antigen or antibody is coupled. The carrier may also be another antibody directed against a cytotoxic effector mechanism, e.g., cytotoxic cells. The carrier should preferably be non-toxic and non-allergenic. The antigen 30 or antibody may be coupled multivalent to the macromolecular carrier, as this may give the preparation an increased immunogenicity.

For oral administration, the composition may be in the form of tablets, capsules, granules, pastes, gels, a mixture or suspension optionally provided with a coating.

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15 for prolonged delivery or a coating which protects the antibody or antigen from passage through the stomach.

Solid formulations, e.g., granules, tablets and capsules, may contain fillers, e.g., sugars, sorbitol, mannitol and silicic acid; binders, for example cellulose derivatives such as carboxymethyl cellulose and polyvinylpyrrolidone; disintegrants, for example starch, sodium bicarbonate and calcium carbonate; lubricants, eg magnesium stearate, talc and calcium stearate. Semi-solid formulations, e.g., pastes or gels, may include a gelling agent such as an alginate, gelatin, carrageenan, tragacanth gum and pectin, a mineral oil such as liquid paraffin, a vegetable oil such as corn oil, sunflower oil, rapeseed oil and grape seed oil, as well as a thickening agent. , rubber, gelatin, etc. Liquid formulations, ie. mixtures and suspensions may comprise an aqueous or oily carrier, e.g., water, or a mineral oil such as liquid paraffin, a vegetable oil such as corn oil, sunflower oil, rapeseed oil, grape seed oil, etc. The antibody or antigen of the invention may be suspended in it. liquid carrier in accordance with usual practice.

The prolonged release coating may be, for example, an enteric coating which may be selected from shellac, cellulose acetate esters such as cellulose acetate phthalate, hydroxypropyl methyl cellulose esters such as hydroxypropyl methyl cellulose phthalate, polyvinyl acetate acetate acetate, and polyvinyl acetate acetate esters such as polyvinyl acetate and

The composition may also be intended for rectal administration, for example 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 of the invention or an anti-idiotypic antibody of the invention to prepare a medicament for treatment.

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16 of human adenocarcinoma or use of an antibody or anti-idiotypic antibody of the invention for the manufacture of a medicament for the treatment of human carcinoma.

Therapy of cancers, especially carcinomas expressing the carcinoma antigen of the invention, can be performed by a variety of methods known to those skilled in the art. An antibody to the antigen (especially a human monoclonal antibody for the reasons set forth above) can be injected into cancer patients 10 to fight the tumor directly or via various effector mechanisms, e.g., complement-mediated cytotoxicity or antibody-dependent cell-mediated cytotoxicity. The antibody may be modified prior to injection into the patient as indicated above, for example by coupling to pharmaceuticals (thereby transporting them 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 cytotoxic cells. It is believed that hybrid antibody containing one combination site for antigen and another against another antigen or pharmaceutical as described above can also advantageously be used to provide a two-way attack against the tumor in question.

The antibody of the invention can be used in extracorporeal devices to remove circulating tumor antigen or immune complexes containing tumor antigen, thereby restoring the anticancer immune response, allowing the immune system to recover following the paralysis induced by the tumor antigen or immune complexes.

The carcinoma antigen of the invention can be used for immunization to provoke an anticancer immune response in the body. The antigen can also be used in vitro to produce effector cells against cancer, the antigen being cultured with leukocytes from a cancer patient.

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The carcinoma antigen can be used in extracorporeal devices to remove antitumor antibody or immune complexes in a similar manner as using the antibody.

Furthermore, the antibody to the carcinoma antigen can be administered to elicit an anti-idiotypic or anti-idiotypic immune response. An anti-idiotypic antibody (whether monoclonal or polyclonal or a fragment thereof) produced against an antibody that reacts with the carcinoma antigen of the invention may express epitopes similar to those of the antigen, and may therefore be similarly used for immunization to establish an anticarcinoma immune response. Such antibodies may also be used in extracorporeal devices 15 as described above for the antigen and the primary antibody.

Anti-anti-idiotypic antibodies can be used in a manner similar to that described for primary anti-tumor antibodies.

The invention will be explained in more detail below with reference to the drawing, in which Figure 1 shows immunocytochemical staining with C-OU1 of COLO 201 cells (colon adenocarcinoma cells, positive) and staining of the same cells with a non-responsive human hybridoma IgM (negative); FIG. 2A and B show immunohistochemical staining with C-OU1 of a frozen tissue sample from colon adenocarcinoma tissue (2A) and normal tonsillar tissue (2B); FIG. Figures 3A and B show an electron microscopic analysis of the 3Q reaction of C-OU1 with Colon 137 cells (colon adenocarcinoma cells, positive) (3A) and HUTU 80 (duodenal adenocarcinoma cells, negative) (3B); FIG. 4 shows an immunoblot analysis of tissue extracts separated by isoelectric focusing; and

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18 FIG. Figure 5 shows an analysis of carcinoma antigen by SDS-PAGE blotting by labeling with C-OU1. Extract of Colon 137 (colon adenocarcinoma cells, positive) (5A) and extract of HUTU 80 (duodenal adeno-5 carcinoma cells, negative) (5B).

The invention is further illustrated by the following examples.

EXAMPLE 1

Preparation of Human Monoclonal Antibody (c-oui)

Mesenteric lymph nodes that drained the tumor area in patients with colo-rectal cancer were finely chopped under sterile conditions. Residues were removed by filtration through cotton and the lymphocytes were purified by centrifugation on Ficoll-Isopaque (Boehringer-Mannheim, Mannheim, Federal Republic of Germany).

The lymphocytes were fused to the human fusion cell line W1-L2-729-HF2 (hereinafter referred to as HF2) (from Tecniclone Int., Santa Ana, California, USA) as described by Kohler, Immunological Methods Vol. II, Academic Press, 1981, pp. 285-298. The ratio of HF2 to lymphocytes (107) 20 was 1: 2.

After washing the HF2 and the lymphocytes together in RPMI-1640 medium, followed by centrifugation, the cell pellet was resuspended in 0.5 ml of 50% PEG (polyethylene glycol) 6000 over 1 minute with constant shaking. Prior to dilution of PEG with RPMI-1640, cells were incubated for an additional 2 minutes. Then, the fusion product was washed and resuspended in solution medium [RPMI-1640, 10% FCS (fetal calf serum) added to HAT (2x10-4 M hypoxanthine, 4x10 ~ 7 M aminopterin, 3.2x10 ~ 6 M thymidine)]. The cells were plated 30 with 2x105 cells at 200 µl per ml. well in 96 well microtiter plates. The cells were kept in selective medium for two

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19 * weeks. Further cultivation was performed in RPMI-1640 + 10% FCS added with hypoxanthine and thymidine. Growing hybrids appeared 10 days to 4 weeks after fusion. Cloning was performed by limiting dilution without feeder cells.

Five supernatants from growing clones were analyzed for immunoglobulin production by ELISA (enzyme-linked immunosorbent 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 hydrogen carbonate, pH 10 9.6. Coated wells were washed with PBS-Tween (phosphate buffered saline - 0.05% Tween 20) and incubated for 2 hours at room temperature with supernatants diluted 1:10 in PBS-Tween. Development was made with alkaline phosphatase (AP) -coupled antibody specific for 15 IgM, IgA or IgG (Dakopatts, Copenhagen), diluted 1: 3000 in PBS-Tween. After incubation for 1 hour at room temperature, the substrate p-nitrophenyl phosphate (PNPP), 1 mg / ml 10% diethanolamine, 1 mM MgCl 2, pH 9.6 was added. Optical density was measured at 405 nm after 1 hour of incubation at 37 ° C. Standard 20 curves for quantification were constructed with dilution of IgM (Cappel) or IgG (Kabi AB, Stockholm,

Sweden). Hybrids producing immunoglobulin (Ig) as assayed by ELISA were propagated by transfer to 24-well macroplates (Nunc A / S, Denmark) and the supernatants were further assayed by immunocytochemical analysis for reaction with tumor cells or by immunohistochemistry. chemical analysis for tumor tissue reactions as described below.

The hybridoma cell line B9165 (ECACC 87040201), selected by the methods described below, was shown by ELISA to produce between 1 and 5 µg IgM per ml. ml when grown for 2 weeks without changing medium.

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EXAMPLE 2

Antigen Characterization by Immunocytochemical Analysis

The anti-tumor reactivity assay was performed by immunocytochemical analysis and by immunohistochemical analysis. Immunocytochemical analysis was performed on cell smear preparations prepared from various human tumor cell lines and peripheral human blood leukocytes. Cells were fixed to slides by treatment with formol acetone (9.5% formaldehyde, 43% acetone in 86 mM phosphate buffer, pH 7.2).

Ca. 50 µl of C-OU1 supernatant (from hybridoma B9165; ECACC 87040201) was applied to the fixed cell smear preparation and incubated overnight at 4 ° C in a humid room before rinsing and incubating for 1 hour at room temperature 15 with horseradish peroxidase (HRP) label. rabbit anti-human IgM (Dakopatts) diluted to 1:80 in PBS-Tween. Finally, peroxidase substrate (0.01% H 2 O 2 and diaminobenzidine at 0.6 μg / ml in PBS) was added. The smears were counterstained slightly with hematoxylin and mounted. Table I 20 shows the results obtained by analysis of c-OU1 on smear preparations of different cells.

TABLE I

Reactivity of C-OUl Assayed by Immunocytochemistry 25 Type of Cell Name Reaction 1. Colon Adenocarcinoma Colon 137 Positive 2. Colon Adenocarcinoma COLO 201 Positive 3. Melanoma HU 373 Negative 30 4. Breast Carcinoma MCF-7 Positive 5. Duodenal Adenocarcinoma HUTU 80 Negative 6. Burkitt's lymphoma EB-2 Negative 7. Human peripheral blood PBL Negative leukocytes

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A selective reactivity is evident. Alternatively, the living cells were incubated with the hybridoma antibody at 4 ° C, followed by the enzyme-labeled anti-Ig antibody. The cells were then plated on slides, fixed with glutaraldehyde (0.17% in PBS) and incubated with substrate. FIG. Figure 1A shows staining of living COLO 201 (colon adenocarcinoma cells) cells with C-OU1, whereas Figs. IB shows a lack of staining when a non-reactive human hybridoma IgM is used in the first layer. The method used is the accepted standard procedure for labeling molecules exposed on the cell surface, and the one shown in FIG. 1 seat marking matches this.

b) Immunohistochemical analysis

Immunohistochemical analysis was performed on frozen tissue sections fixed in acetone. Endogenous IgM was blocked by incubation with Fab 'fragments of anti-chain antibody (obtained from Dakopatts, Copenhagen, Denmark) before incubation with the hybridoma antibody C-OU1 (0.5 µg / ml) (the Fab' fragments were prepared as described by B. Nielsen et al., Hybridoma 6 (1), 1987, pp. 103-109) (the hybridoma antibody was concentrated by precipitation with 2M ammonium sulfate). The bound hybridoma antibody was then visualized as described above during the immunocytochemical analysis.

In this case, the same antibody preparation was labeled with peroxidase as that used to prepare Fab 'fragments. FIG. 2A shows that after application of C-0U1, only the tumor cells are stained in a section of a colon adenocarcinoma. FIG. 2B shows a lack of staining of tonsil larva. Tables IIA and B summarize the reactivity analyzed on a variety of tissues, Table IIA showing the results from malignant tissues and Table IIB showing the results from non-malignant tissues.

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TABLE IIA

Reactivity of C-0U1 (0.5 µg / ml) on frozen, acetone-fixed tissue sections of human malignant tissues analyzed by immunohistochemical techniques 5 Tissue Result

Colon adenocarcinoma 19/21 a)

Ovarian adenocarcinoma 2/2

Renal adenocarcinoma 1/2 10 Breast carcinoma 7/9

Lung adenocarcinoma 7/7

Lung epithelial carcinoma 0/6

Non-Seminomal Testis Carcinoma 1/1

Sarcoma 0/3 15 Malignant melanoma 0/7 B lymphoma 0/1

Thymoma 0/1 a) Number of positives / number tested

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TABLE IIB

Reactivity of C-0U1 (0.5 µg / ml) on frozen, acetone-fixed tissue sections of human normal tissues analyzed by immunohistochemical techniques 5 Tissue Result

Ovarian stroma Negative

Ovarian Epithelium Negative

Renal Glomeruli Negative 10 Renal Tubules Negative

Breast tubules Positive

Chest ductuli Positive

Lung alveoli Negative

Bronchial epithelium Negative 15 Testis Negative

Epidermis Negative

Tonsillar lymphatic tissue Negative

Tonsillar epithelium Negative

Smooth muscles Negative 20 Blood vessels Negative

Prostate Epithelium Positive

Normal colon epithelium showed binding of all analyzed human IgMs, monoclonal antibodies, myeloma IgMs, and 25 normal polyclonal human IgMs. Thus, this general binding of IgM to normal colonic epithelium was considered to be nonspecific, an interpretation further supported by analysis by electron microscopy and isoelectric focusing (see below). This non-specific binding of antibody is also consistent with the knowledge of those skilled in the art.

c) Electron microscopy

For electron microscopy, 105 / ml adenocarcinoma cells were grown in RPMI-1640 medium, 10% FCS, in plastic glass tube tubes

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24

for 3 days until a monolayer was obtained. The cells were fixed in 0.1% (v / v) glutaraldehyde in 0.1 M PBS, pH

7.2, for 30-60 minutes at 4 ° C and then washed in PBS with added bovine serum albumin (BSA) and lysine-HCl overnight 5 at 4 ° C. The cells were dehydrated in from 30% to 90% ethanol at progressively lower temperatures down to -20 ° C, then infiltrated into Lowicryl K4M (Chemische Werke Lowi, Federal Republic of Germany) at -35 ° C and polymerized overnight at -35 ° C. C under ultraviolet light 10 and then for an additional 2 days at ambient temperature.

Ultrathin (50-60 nm) sections of the cellular Lowicryl mounted on coated nickel grids were used. The immuno-labeling procedure, which consisted of allowing the grids to flow with the sections down on top of different solutions, included the following steps: 1) the grids were applied to drops of 1% (w / v) NaBH4 in PBS for 10 minutes; 2) after washing in 0.1% (w / v) BSA-Tris for 2x5 minutes, the grids were transferred to drops of 3% BSA-Tris (15 minutes); 3) the grids were placed on drops of C-OU1 antibody for 20 hours at room temperature; 4) the grids were washed in 0.1% BSA-Tris for 2x5 minutes; 5) the grids were transferred to rabbit anti-human IgM droplets dissolved in 3% BSA-Tris for 1 hour at room temperature; 6) after washing in 0.1% BSA-Tris for 2x5 minutes, the grids were applied to drops of goat anti-rabbit IgG labeled with gold probes (Janssen Pharmaceuticals) at 15 nm, dissolved in 3% BSA-Tris and incubated for 30 min. minutes at room temperature; 7) the grates were washed in 0.1% BSA-Tris for 2x5 minutes and in redistilled water for 2x5 minutes and dried; and 8) the ultra-thin sections were stained with 1% 30 uranyl acetate for 10 minutes and 0.4% lead citrate for 2 minutes at room temperature.

Tween 20 and 0.5 M NaCl were added to all antibody and wash solutions.

The ultrathin sections were examined in a JEOL 100-CX-35 electron microscope operating at 80 kV.

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Experiments to assess the specificity of the immunocytochemical reactions included omission of the primary antibody and replacement of the primary antibody with a human IgM (Cappel).

FIG. Figure 3A shows the particular pattern of labeling of colon adenocarcinoma cells (Colon 137) in regions around the ends of intermediate filaments; 3B the failure to label (by a similar procedure) duodenal adenocarcinoma cells (HUTU 80). Again, sections of colon adenocarcinoma cancer tissue showed only labeling of the tumor cells associated with intermediate filaments (not shown). Normal colon epithelium showed no labeling. Thus, electron microscopy demonstrates the presence of the target antigen in association with cytoplasmic structures.

D) Isoelectric focusing

Molecular characterization of the target antigen was performed by isoelectric focusing. Tumor cells or cancerous tissue and normal tissue were solubilized by ultrasonication (4x30 seconds on ice) in extraction buffer (75 mM NaCl, 75 mM 20 KCl, 10 mM Hepes, 5 mM EDTA, 5% 2-mercaptoethanol, 5 mg / l Trasylol, 01 mM Lenpeptin, 0.01 mM Pepstatin). After ultrasonication, urea was added up to 6 M together with 80 mg of sucrose and the homogenate incubated for 30 min at 37 ° C. Insoluble material was removed by centrifugation (5 minutes at 15,000 x g).

Isoelectric focusing was performed with the extracts applied to 1% agarose thin layer gel (Agarose IEF, Pharmacia) containing 6 M urea, 3% (vol / vol) servalites 3-10 and 1% (w / v) servalites 4-6 (Serva). As a backing, 30 gel-based films (LKB) were used. Focusing was performed at 1500 V / hr before electrophoretic transfer of the proteins to nitrocellulose. Residual binding sites were blocked by incubating the nitrocellulose in 0.1 M Tris-HCl, pH 7.5,

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26 containing 0.1 M NaCl, 2 mM MgCl 2 and 0.05% Tween 20. The nitrocellulose was cut into strips, incubated for 2 hours at room temperature with the hybridoma antibody C-OUl diluted in PBS-Tween to ca. 200 ng / ml. After washing with PBS-Tween 5, the nitrocellulose strips were incubated with alkaline phosp-hatase-conjugated Ftab ') 2 anti-IgM antibody (Jackson Immunoresearch) followed by substrate (a solution of 5-bromo-4-chloro-indoxyl phosphate and nitro blue tetrazolium).

FIG. 4, left, shows staining of extracts of a 10 colon adenocarcinoma tumor; 4, to the right, an extract of normal colonic epithelium. I. staining of total protein with India ink; II. staining with C-OUl; and III. staining with a different hybridoma IgM. It is evident that C-OUl exhibits distinct staining of acidic proteins (pI 5.4-6.2) in the tumor extract but not in the normal colon extract.

e ^ SDS-PAGE or Western blottina

Extracts of Colon 137 (colon adenocarcinoma cells) and HUTU 80 (duodenal adenocarcinoma cells) were prepared by solubilization with detergents (2% SDS, 4M urea, 10 mM 20 iodoacetamide) and incubated for 15 minutes before PAGE on 5 to 20% gradient gels. The proteins were then electrophoretically transferred to nitrocellulose sheets. The nitrocellulose sheets were cut into 3 mm strips and incubated overnight at 4 ° C with C-OUl followed by incubation for ‘2 hours with AP-25 rabbit anti-human IgM (Sigma). The blots were washed in PBS and fixed by incubation for 15 minutes 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. Molecular weight was calculated from the mobility of the following, previously stained molecular weight markers: β-galactosidase (116 K), fructose-6-phosphatase kinase (84 K), pyruvate kinase (58 K), fumarase (48.5 K), lactic acid dehydrogenase (36.5 K), triosephosphatase isonerase (26.6 K).

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The results of FIG. Figure 5 shows that C-0U1 reacts with a protein with a molecular weight of 43,000 found in the extracts of colon adenocarcinoma and not in extracts from duodenal adenocarcinoma.

Claims (25)

An antibody to a human carcinoma-associated antigen, characterized in that by immunohistochemical analysis on frozen, acetone-fixed tissue sections, it binds to 5 colon adenocarcinoma, ovarian adenocarcinoma, renal adenocarcinoma, breast carcinoma, lung adenocarcinoma. - Seminomal testis carcinoma, breast tubules, breast ductuli and prostate epithelium and does not bind to pulmonary epithelial carcinoma, sarcoma, malignant melanoma, B lymphoma, thymoma, ovarian stroma, ovarian epithelium, renal glomeruli, renal tubules , pulmonary alveoli, bronchial epithelium, testis, epidermis, tonsillar lymphatic tissue, tonsillar epithelium, smooth muscle or blood vessels. An antibody according to claim 1, characterized in that it is a monoclonal antibody. An antibody according to claim 1, characterized in that it is made from a human lymphocyte. An antibody according to claim 1, 20, characterized in that it is a polyclonal antibody. An antibody according to claim 3, characterized in that it is made from a human-human hybrid cell injection. The antibody of claim 5, characterized in that the human-human hybridoma cell line is B9165 (ECACC 87040201). An antibody, characterized in that it is cross-reactive with an antibody according to claim 6. DK 162844 B 29 Isolated human carcinoma-associated antigen, characterized in that it has an estimated molecular weight of approx. 43 kD and an isoelectric point in the range of approx. and having at least one epitope reactive with a monoclonal antibody produced by the human hybridoma cell line B9165 (ECACC 87040201), or an analogue thereof. Antigen according to claim 8, characterized in that it is substantially pure form. 10 B lymphoma, thymoma, ovarian stroma, ovarian epithelium, renal glomeruli, renal tubules, pulmonary alveoli, bronchial epithelium, testis, epidermis, tonsillar lymphatic tissue, tonsillar epithelium, smooth muscle or blood vessels. An antibody according to claim 1, characterized in that it is a hybrid antibody containing a combination site specifically directed to an epitope on an antigen of claim 8 or 9, and a second targeted specifically to another epitope. on the same antigen, an epitope on another antigen, for example, a differentiation antigen of cytotoxic T cells, or a pharmaceutical selected, for example, from cytotoxic and antineoplastic agents. Anti-idiotypic antibody, characterized in that it is directed to an antibody according to any one of claims 1-7 and 10. Anti-anti-idiotypic antibody, characterized in that it is directed to an anti-idiotypic antibody according to claim 11. Diagnostic agent, characterized in that it comprises an antibody according to any one of claims 1-7 or 10, an anti-idiotypic antibody according to claim 11 or an anti-anti-idiotypic antibody according to claim 12. Diagnostic agent according to claim 13, characterized in that the antibody is provided with a label, eg an enzyme, fluorescent substance, radioactive isotope or ligand such as biotin. Diagnostic agent according to claim 13, 5, characterized in that the antibody is coupled to a solid support either directly or via a spacer, eg Protein A, the solid support eg comprising a polymer or a matrix coated with a polymer, wherein the polymer e.g. is selected from plastics, cellulose (e.g. nitrocellulose paper), silicone, silica, and a polysaccharide such as agarose or dextran, and the solid support, for example, is in the form of a plate, strip, film, microparticles or paper. Diagnostic agent, characterized in that it comprises an antigen 15 according to claim 8 or 9. Diagnostic agent according to claim 16, characterized in that the antigen is provided with a label, eg an enzyme, fluorescent substance, radioactive isotope or ligand such as biotin. Diagnostic agent according to claim 16, characterized in that the antigen is coupled to a solid substrate either directly or via a spacer, eg Protein A, wherein the solid substrate comprises, for example, a polymer or a matrix coated with a polymer, wherein the polymer is e.g. 25, selected from plastics, cellulose, silicone, silica and a polysaccharide such as agarose or dextran, and the solid support, for example, in the form of a plate, strip, film, microparticles or paper. A method of producing a monoclonal antibody against a colon carcinoma-associated antigen, characterized in that lymphocytes from mesenteric lymph nodes drain the end of the tumor region of a patient having colon cancer, these lymphocytes are fused with a human cell line suitable for use in a fusion for the purpose of creating human-human hybridomas, the hybridoma cells produced are screened for hybridomas secreting a monoclonal antibody that specifically binds to colon adenocarcinoma, ovarian adenocarcinoma, adenocarcinoma, breast carcinoma, pulmonary adenocarcinoma, non-seminomal testis carcinoma, breast tubules, breast ductuli and prostate epithelium and which do not bind to frozen, acetone-fixed tissue sections of lung epithelial carcinoma, sarcoma, malignant melanoma, A method for in vitro diaanosticerina of human carcinoma expressing an antigen according to claim 8 or 9, characterized in that a sample of a body fluid from a suspected cancer patient is contacted with a diagnostic agent according to any of the claims 13-15, comprising an antibody according to any one of claims 1-7 or 10, after which the presence of bound antibody is determined. Method for in vitro diagnosis of human carcinoma expressing an antigen according to claim 8 or 9, characterized in that a tissue sample from a putative 25 cancer patient is contacted with a diagnostic agent according to any one of claims 13-15. , which agent comprises an antibody according to any one of claims 1-7 or 10, after which the sites on the sample are determined to which antibody is bound. Use of an antibody according to any one of claims 1-7 or 10 for the preparation of an agent for in vivo diagnosis of human carcinoma expressing an antigen according to claim 8 or 9. DK 162844 B 32 A method for in vitro diaanosticerina of human carcinoma expressing an antigen according to claim 8 or 9, characterized in that a sample of a body fluid from a putative cancer patient is contacted with a diagnostic agent according to any of the claims 15-17, comprising an antigen according to claim 8 or 9, or with a diagnostic agent according to any one of claims 13-15, comprising an anti-idiotypic antibody according to claim 11, after which the presence of the bound is determined. antibody to an antigen according to claim 8 or 9 contained in the body fluid. Pharmaceutical composition for the treatment of human carcinoma, characterized in that it comprises an antigen 15 according to claim 8 or 9 or an antibody according to any one of claims 1-7 or 10 and a pharmaceutically acceptable excipient. Use of an antigen according to claim 8 or 9, an anti-idiotypic antibody according to claim 11, an antibody according to any of claims 1-7 or 10 or an anti-idiotypic antibody according to claim 12 for the preparation of a drug for the treatment of human carcinoma.