NO176903B - Antibody or binding fragment thereof for the diagnosis of human carcinoma, or as a carrier of pharmaceutical agents, isolated human tumor-associated antigen, diagnostic agent and method for in vitro diagnosis - Google Patents

Description

Field of the invention

The present invention relates to an isolated, human carcinoma-associated antigen, which is in substantially pure form, an antibody directed against the antigen for the diagnosis of human carcinoma or as a carrier of diagnostic agents, a diagnostic agent comprising the antigen or antibody, and a method for in vitro - diagnosis of human carcinoma using diagnostic agents containing the antigen or antibody.

Background for the invention

The possibility of developing more accurate methods for the detection and diagnosis of cancer by identifying and characterizing tumor-associated antigens (ie antigens expressed by tumors) is of great medical interest.

Monoclonal antibodies against tumor-associated antigens can play an important role in the detection of cancer due to their high specificity. To date, most of the monoclonal antibodies raised against cancer-associated antigens have been of mouse origin and have been expressed by hybridomas resulting from a fusion of spleen cells from a mouse immunized with a human cancer cell line or cells from a cancer patient with a mouse myeloma cell line. Immunogenicity in the mouse is a requirement 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, as patients recognize these antibodies as foreign proteins and may therefore develop a hostile immune response against the murine antibody. The result can 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 agents for administration to patients with cancer 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 murine monoclonal anti-tumor antibodies are illustrated by the colon adenocarcinoma antibody 17-A1, which has been used in diagnosis and therapy but has now been found to

react with normal as well as tumor tissue (Hybridoma 5 Suppl. 1, 1986, special publication regarding Ca-17-Al, editor Z. Steplewski).

The immune response in patients to administered murine monoclonal antibody has been described by numerous investigators (eg, 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).

Colorectal cancer is one of the most frequently occurring cancers and one of the main causes of death from cancer. The prognosis for this type of cancer has not improved for a long time, and new methods for detecting colo-rectal cancer and adjuvant therapy accompanied by surgery are therefore necessary.

There is therefore a need for a human carcinoma tumor-associated antigen, in particular one that is not predominantly expressed by normal tissue, and antibodies against such an antigen for diagnostic and therapeutic purposes.

Description of the invention

The present invention thus concerns an isolated, human carcinoma-associated antigen for non-medicinal use, which antigen is characterized by the fact that it is in essentially pure form, and has an apparent molecular weight of 43 kD and an isoelectric point in the range 5.4-6, 2, and that it has at least one epitope that is reactive with a monoclonal antibody produced by the human hybridoma cell line B9165 (ECACC 87040201) or an analog thereof.

The term "analog" is used in the present context to indicate a protein or polypeptide of similar amino acid composition or sequence to the present antigen, allowing for minor variations that do not adversely affect the immunogenicity of the analog. The analogous polypeptide or protein may be derived from a source other than carcinoma tissue, such as from a recombinant organism, or may be partly or wholly of synthetic origin. The term is further intended to denote 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 indicates the presence of the novel antigen in human colon carcinoma and breast carcinoma tissue, but not in duodenal adenocarcinoma tissue, melanoma or Burkitt's lymphoma tissue or human peripheral blood leukocytes (see Table I in the following ).

Immunohistochemical analysis by standard procedures using a monoclonal antibody with specific reactivity to the antigen indicates the presence of the novel antigen in colon adenocarcinoma, ovarian adenocarcinoma, renal adenocarcinoma, breast adenocarcinoma, lung adenocarcinoma and non-seminomal testicular carcinoma tissue, but not in lung epithelial carcinoma, sarcoma, malignant melanoma, B-lymphoma or thymoma tissue, or in normal tissue other than mammary tubules, mammary ductuli or prostatic epithelium (see Tables IIA and IIB below).

It is therefore concluded that the new antigen is

one that is largely absent from normal human tissue and has been found to be expressed by carcinoma tissue, particularly adenocarcinoma tissue, but not by other malignant tissue as determined by standard immunocytochemical and immunohistochemical analysis. In particular, the new antigen appears to be one associated with colon adenocarcinoma.

A tumor-associated antigen expressed by colon carcinoma is described in EP 199,586. However, this antigen differs from the antigen according to the invention in characteristics such as molecular weight and isoelectric point, and it is indicated to be present in both normal and malignant colon tissue (ie it does not appear to be tumor-specific), while the antigen according to the invention have been shown to be present in several different malignant tissues, as indicated above.

The antigen according to the invention can, for example, be obtained by isolation from extracts of carcinoma cells or extracts of carcinoma tumors by affinity chromatography on an insoluble antibody formed against the antigen, according to procedures well known in the field (cf. Johnstone, A. & Thorpe, R., Immunochemistry in Practice, Blackwell, Oxford 1987). Further purification by one or more additional protein purification procedures (eg, size exclusion chromatography), ion exchange chromatography, ligand affinity chromatography, hydrophobic interaction chromatography, electrophoresis in gels with or without denaturation, and various precipitation procedures) may be necessary to obtain the antigen in sufficiently pure form for detailed molecular characterization and for use for diagnostic purposes.

However, to ensure a sufficient supply of the antigen, it may be advantageous to produce the antigen by recombinant DNA techniques. These may include (a) isolation of a nucleotide sequence encoding the antigen, for example by establishing a cDNA or gene library of human carcinoma cells and screening for positive clones according to conventional methods;

(b) introducing said sequence into a suitable replication-competent expression vector; (c) transformation of a

suitable microorganism host with the vector prepared in step (b); (d) culturing the microorganism prepared in step (c) under suitable conditions for expression of the antigen and (e) harvesting the antigen from the culture.

Steps (a)-(e) of the method can be carried out according to standard methods, for example as described in 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 establish 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 a subsequence thereof can then be introduced by conventional peptide synthesis,

e.g. liquid or solid phase peptide synthesis where solid phase peptide synthesis is the preferred procedure (see R.B. 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 a first amino acid to a solid carrier, after which the other amino acids are added in order by peptide bonding until the desired length has been obtained.

According to another feature, the present invention relates to an antibody or binding fragment thereof for the diagnosis of human carcinoma, or as a carrier of pharmaceutical agents, which antibody is characterized in that it binds to colon adenocarcinoma, ovarian adenocarcinoma, renal adenocarcinoma by immunohistochemical analysis on frozen, acetone-fixed tissue sections , breast carcinoma, lung adenocarcinoma, non-seminomal testicular 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, renal glomeruli, renal tubules, lung alveoli, bronchial epithelium, testis, epidermis, tonsillar lymphatic tissue, tonsillar epithelium, smooth muscle or blood vessels, and which binds to a human carcinoma-associated cytoplasmic antigen having an apparent molecular weight of 43,000 and an isoelectric point in the range of 5.4-6.2, and which has at least one epitope which is reactive with a monoclonal antibody produced by d a human hybridoma cell line B9165 (ECACC 87040201).

The antibody is advantageously a monoclonal antibody as these tend to have higher specificity than polyclonal antibodies, which makes them more useful for accurate diagnostic determinations. Due to the considerations stated above, the antibody, when intended for injection into patients, should preferably be of

human origin, i.e. one produced by a fusion product

of a human lymphocyte and a human cell line or a fusion product of a human lymphocyte and e.g. a murine

myeloma cell line, rather than a fusion product of a murine lymphocyte and a murine myeloma cell line which has hitherto been common for the production of monoclonal antibodies against human tumor-associated antigens. Such monoclonal antibodies can be formed against different epitopes on the antigen. An example of a useful monoclonal antibody is that designated C-OU1, produced by the human hybridoma cell line B9165 deposited on April 2, 1987 at the European Collection of Animal Cell Cultures (ECACC), Center for Applied Microbiology and Research , Porton Down, Salisbury, Wiltshire, United Kingdom, under deposit no. ECACC 87040201.

The monoclonal antibody according to the invention can be produced by a method comprising a) isolation of antibody-producing cells from a cancer patient, b) fusion of cells that produce the antibody with cells of a suitable human fusion cell line, and selection

and cloning the resulting hybridoma cells which

produces specified antibody, and

c) culturing the cells from step b) in a suitable medium while producing the indicated antibody, and harvesting the antibody

from the growth medium.

The antibody-producing cells used for fusion to 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 Kohler and Milstein, Nature 256, 1975, p. 495, or Kohler, Immunological Methods vol. II, Academic Press, 1981, pp. 285-298, i.e. preferably in the presence of a fusion promoter such as polyethylene glycol. The human fusion cell line used is preferably of a type that is unable to survive in selective medium; and one type of cell line frequently used for cell fusions is one lacking the enzyme hypoxanthine-guanine-phosphoribosyltransferase, and consequently unable to grow in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium).

The selection of hybridoma cells that produce an antibody to the carcinoma antigen can then be performed by culturing unfused antibody-producing cells, unfused human fusion cells, and putatively fused cells in a selective medium (such as HAT) in which the unfused human fusion cells do not can grow and eventually die out. The unfused antibody-producing cells can only survive for a limited period of time, after which they also die out. On the other hand, successfully fused cells continue to grow as they have inherited permanent growth properties from the human parental fusion cells and the ability to survive in the selective medium of the parental antibody-producing cells. The resulting antibody-producing cells can be cultured in vitro after cloning in a suitable medium such as RPMI 1640. This results in the production of monoclonal antibodies of very high purity as these are secreted into the culture supernatant of 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 monoclonality, the antibody may be a polyclonal antibody. This can be produced 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 suitable intervals (eg two weeks to one month) up to six months before the first blood draw. During maintenance of this established immunization system, the animals are then bled approx. 1 week after each booster immunization, and antibody is isolated from the serum by conventional means, e.g. as described in Harboe and Ingild,

Scand. J. Immun. 2 (Suppl. 1), 1973, pp. 161-164.

For certain purposes, it may be advantageous for the antibody to be a hybrid antibody which contains a binding site directed specifically to an epitope of the antigen according to the invention, and another directed specifically to another epitope of the same antigen, an epitope of another antigen or a pharmaceutical agent. The term "binding site" is to be understood to indicate the antigen recognition structure in the variable region of the antibody molecule. Hybrid antibodies enable special procedures for detecting the antigen in a sample and for directing a pharmaceutical agent or other biologically active molecule or other antigen to the site of the tumor where the reagent has the greatest effect. 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 agent with which the hybrid antibody may be reactive is preferably selected from cytotoxic or antineoplastic agents (cf. Collier, R.J. and Kaplan, D.A., Scientific American 251, 1984, p. 44).

The hybrid antibody can be produced by hybrids

between two monoclonal cell lines that produce the two relevant antibodies, or can be produced by chemical binding of fragments of the two antibodies.

The antibody can for different purposes, see below-

for, be an anti-idiotypic antibody, ie an antibody directed against the site of an antibody reactive with the epitope on the antigen. The anti-idiotypic antibody is directed against an antibody that is reactive with the antigen according to the invention. The anti-idiotypic antibody can be prepared in a similar manner to that indicated above for the monoclonal or polyclonal antibody. The antibody can also be an anti-anti-idiotypic antibody directed against the anti-idiotypic antibody defined above.

According to another important feature, the present invention relates to a diagnostic agent comprising an antibody according to the invention as described above, an anti-idiotypic antibody as described above, or an anti-anti-idiotypic antibody as described above, or a binding fragment of antibody.

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 linked agglutinate in the presence of a carcinoma antigen in the sample being tested, where no labeling of the antibody is necessary, it is preferred for most purposes providing the antibody with a label to detect bound antibody. In a double antibody ("sandwich") determination, at least one of the antibodies can be equipped with a marker. Substances usable as markers in the present context can be selected from enzymes, fluorescent substances, radioactive isotopes and ligands such as biotin.

Examples of enzymes that can be used as marker substances are peroxidases, e.g. horseradish peroxidase, or phosphatases, e.g. alkaline phosphatases. As enzymes are not directly detectable, they must be combined with a substrate to form a detectable reaction product such as e.g. can be fluorescent or colored. Examples of usable substrates are H₂C₂/o-phenylenediamine, I₂C₂/azinodiethylbenzthiazoline sulfonic acid, H₂C₂/diaminobenzidine and p-nitrophenyl phosphate. Such reaction products can be detected with the naked eye as

a color loss or change, or by means of a spectrophotometer.

Examples of fluorescent substances which can be used as marker substances are B^C^/p-hydroxyphenylacetic acid and methylumbelliferyl phosphate. Such substances can be detected using a fluorescence spectrophotometer in a manner known per se.

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 agent comprises at least one antibody linked to a solid carrier. This can be used in a double antibody determination, in which case the antibody bound to the solid carrier is not labeled, but the unbound antibody is labeled. The solid support may be composed of a polymer or may comprise a matrix on which the polymer is applied. The solid carrier can be selected from a plastic, e.g. latex, polystyrene, polyvinyl chloride, nylon, polyvinylidene difluoride, or cellulose, e.g. nitrocellulose, silicon, silica and a polysaccharide such as agarose or dextran.

For use in a diagnostic determination can

the solid support can be of any suitable shape. It can thus be in the form of a plate, e.g. a thin layer or preferably a microtiter plate, a strip, film, et

paper or micro-particles such as latex beads or the like.

Instead of being linked directly to the solid support, the monoclonal antibody can be linked to a spacer compound immobilized to a solid support. Examples of spacer compounds include protein A.

It should be noted that practically all methods or applications based on intact antibodies can instead be carried out using fragments of the antibodies, for example F(ab')2 or Fab fragments (cf. Delaloye, B.

et al., J. Clin. Invest. 87, 1986, p. 301).

For use in a Sandwich determination, the diagnostic agent may additionally comprise another antibody. This second antibody may be labeled and/or linked

a solid carrier as described above. In this embodiment, either one or both antibodies can be a monoclonal antibody as described above.

Alternatively, the diagnostic agent according to 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 otherwise exhibit any of the features described above for diagnostic agents, including an antibody according to the invention, although they will detect bound antibody rather than binding of an antibody to the antigen.

According to a further feature, the invention relates to a method for in vitro diagnosis of human carcinoma expressing the antigen according to the invention, comprising bringing a sample of a body fluid from a patient suspected of having cancer into contact with a diagnostic agent according to the invention comprising an antibody or a binding fragment of an antibody or an isolated antigen according to the invention, and determine the presence of bound antigen, antibody or fragment of antibody. The antibody can be labeled and/or bound to a solid support as exemplified above. The body fluid may be selected from blood, plasma, serum, lymph, pulmonary sputum, urine and gastrointestinal fluids.

In a preferred embodiment of the method, the sample is incubated with a first monoclonal antibody coupled to a solid carrier and then with a second monoclonal or polyclonal antibody equipped with a marker. An example of this embodiment of the sandwich ELISA (enzyme-linked immunosorbent assay) described in Voller, A. et al.,

Bull. World Health Organization. 53, 1976, p. 55.

In an alternative embodiment (a so-called competitive binding determination), the sample can be incubated with a monoclonal antibody coupled to a solid carrier, and then with a labeled carcinoma antigen, where the latter competes with regard to binding sites on the antibody with any carcinoma antigen present in the sample.

An alternative embodiment relates to a method for

in vitro diagnosis of human carcinoma comprising bringing a tissue sample from a patient suspected of having cancer into contact with a diagnostic agent according to the invention comprising an antibody according to the invention, and determining the sites on the sample to which the antibody is bound. Such an 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 using an antibody or a fragment of an antibody according to the invention concerns a method for locating tumors (in particular carcinomas) in vivo. This method comprises administering a diagnostically effective amount of an antibody according to the invention which is labeled to enable its detection, and determining the location of bound antibody. The antibody can be labeled using a radioactive isotope and then injected and localized according to known methods, e.g. gamma ray detector of suitable configuration (cf. Mach, J.-P. et al., Nature 248, 1974, p. 704).

A further embodiment relates to a method for the in vitro diagnosis of human carcinoma, comprising bringing a sample of a body fluid from a patient suspected of having cancer into contact with a diagnostic agent according to the invention comprising the antigen or the anti-idiotypic antibody according to the invention, and determining the presence of bound antibody against the antigen according to the invention present in said body fluid.

According to a further feature, the invention relates to an antibody according to the invention or a binding fragment thereof as a carrier of a pharmaceutical preparation for the treatment of human carcinoma. The preparation comprises an antigen according to the invention or an antibody according to the invention, a pharmaceutical agent and a pharmaceutically acceptable excipient.

The excipient used in the preparation may be any pharmaceutically acceptable carrier.

This carrier can be any carrier that is usually used in the preparation of injectable preparations, for example a diluent, suspending agent, etc., such as isotonic or buffered saline. The preparation can be prepared by mixing a diagnostically effective amount of the antibody with the carrier in an amount resulting in the desired concentration of the antigen or antibody in the preparation.

In some cases, it may be advantageous to link the antigen or antibody to a carrier, in particular a macromolecular carrier. The carrier is typically a polymer to which the toxin is bound by hydrophobic, non-covalent interaction, such as a plastic, e.g. polystyrene, or a polymer to which the antigen or antibody is covalently bound, such as a polysaccharide or a polypeptide, for example bovine serum albumin, ovalbumin or albumin hemocyanin. Furthermore, the carrier may advantageously be selected from a pharmaceutical agent, e.g. a cytotoxic or anti-neoplastic agent to which the antigen or antibody is linked. The carrier can also be another antibody directed against a cytotoxic effector mechanism, for example cytotoxic cells. The carrier should preferably be non-toxic and non-allergenic. The antigen or antibody can be multivalently linked to the macromolecular carrier as this can provide an increased effectiveness of the preparation.

For oral administration, the preparation may be in form

of a tablet, capsule, granule, paste, gel, mixture or suspension, optionally equipped with a coating with extended release, or a coating that protects the antigen or antibody from passage through the stomach.

Solid formulations, i.e. granules, tablets and capsules, may contain fillers, e.g. sugars, sorbitol, mannitol and silicic acid; binders, e.g. cellulose derivatives such as carboxymethylcellulose and polyvinylpyrrolidone; breaking agents, e.g. starch, sodium bicarbonate and calcium carbonate; lubricants, e.g. magnesium stearate, talc and calcium stearate. Semi-solid formulations, i.e. pastes or gels, may comprise a gelling agent such as an alginate, gelatin, carrageenan, gum tragacanth 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 such as starch, gum, gelatin, etc. liquid formulations, i.e. 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 antigen according to the invention can be suspended in the liquid carrier according to common practice.

The coating with extended release can, for example

be an enteric coating which can be selected from shellac, cellulose acetate esters such as cellulose acetate phthalate, hydroxypropyl methyl cellulose esters such as hydroxypropyl methyl cellulose phthalate, polyvinyl acetate esters such as polyvinyl acetate phthalate, and polymers of methacrylic acid and (meth)acrylic acid esters.

The preparation can also be adapted for rectal administration, for example as a suppository. Such a suppository may contain conventional excipients such as cocoa butter and other glycerides.

Finally, the invention relates to the use of the antigen according to the invention or an anti-idiotypic antibody according to the invention for the preparation of an agent for the diagnosis of human adenocarcinoma, or the use of an antibody or anti-anti-idiotypic antibody according to the invention and for the preparation of an agent for the diagnosis of human carcinoma.

Treatment of cancer, in particular carcinomas,

which expresses the carcinoma antigen according to the invention, can be carried out by a number of procedures well known in the field. An antibody against the antigen (in particular a monoclonal antibody for the reasons stated above) which is coupled as a transporter to a pharmaceutical agent, can be injected into cancer patients to fight the tumor directly, or via different effector mechanisms, for example complement-mediated cytotoxicity or antibody-dependent, cell-mediated cytotoxicity. The antibody can be modified for injection into the patient as indicated above, for example by linking to pharmaceuticals (and thus transporting these to their site of activity), or to other antibodies directed against a cytotoxic effector mechanism such as antigens on cytotoxic T cells or on other cytotoxic cells. It is taken into account that hybrid antibodies containing a binding site for the antigen and another against another antigen or a pharmaceutical agent as described above, also

can advantageously be used to provide a two-way attack on the tumor in question.

The antibody according to the invention can be used in extracorporeal devices for the removal of circulating tumor antigen or immune complexes containing tumor antigen, and thereby reconstitute the anti-cancer immune response by enabling the immune system to be recovered from paralysis caused by specified tumor antigen or immunocomplexes.

The carcinoma antigen according to the invention can be used for immunization to trigger an anti-cancer immune response in the body. The antigen can further be used in vitro to form effector cells against cancer by culturing the antigen with leukocytes from a cancer patient.

The carcinoma antigen can be used in extracorporeal devices for the removal of anti-tumour antibody or immunocomplexes in a similar way as when using the antibody.

The antibody against the carcinoma antigen can further be administered to elicit an anti-idiotypic or anti-anti-idiotypic immune response. An anti-idiotypic antibody (either monoclonal or polyclonal or a fragment thereof) raised against an antibody which reacts with the carcinoma antigen of the invention may express epitopes similar to those of the antigen, and may therefore be used in a similar manner for immunization to form an anti -carcinoma immune response. Such antibodies can further be used in extracorporeal devices as described above, for the antigen and primary antibody.

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

Brief description of the drawings

The invention is further described in detail in the following examples and with reference to the accompanying drawings, in which

fig. 1 shows immunocytochemical staining with C-0U1 of COLO 201 cells (colonic adenocarcinoma cells, positive) and staining of the same cells with a non-reactive human hybridoma IgM (negative);

fig. 2A and B show immunohistochemical staining with C-0U1 of a frozen tissue sample from colonic adenocarcinoma (2A) and normal, tonsillar tissue (2B);

fig. 3A and B show an electron microscopy analysis of the reaction of C-0U1 with Colon 137 cells (colonic 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

fig. 5 shows an analysis of carcinoma antigen by SDS-PAGE blotting labeled with C-0U1. Extract of Colon 137

(colon adenocarcinoma cells, positive) (5A) and extract of HUTU 80 (duodenal adenocarcinoma cells, negative) (5B).

Example 1

Preparation of human monoclonal antibody (C-0U1)

Mesenteric lymph nodes draining the tumor area in patients with colo-rectal cancer were sampled under sterile conditions. Cell debris was removed by filtration through cotton, and the lymphocytes were purified by centrifugation on "Ficoll-Isopaque" (Boehringer-Mannheim, Mannheim, BRD).

The lymphocytes were fused with the human fusion cell line W1-L2-729-HF2 (hereinafter 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 between HF2 and lymphocytes (IO<7>) 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 a period of 1 minute with constant shaking. Before dilution of PEG with RPMI-1640, the cells were incubated for an additional 2 min. The fusion product was then washed and resuspended in dissolution medium (RPMI-1640, 10% FCS (calf fetus--4 -7

serum) supplemented with HAT (2x10 M hypoxanthine, 4x10 M aminopterin, 3.2x10 ^ M thymidine). The cells were plated at 2x10^ cells in 200 µl per well of 96-well microtiter plates. The cells were maintained in selective medium for two weeks. Further cultivation was performed in RPMI-1640 + 10% FCS supplemented with hypoxanthine and thymidine. Growing hybrids emerged 10 days to 4 weeks after fusion. Cloning was performed by limiting dilution without precursor cells.

Supernatants from wells with 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 bicarbonate, pH 9.6. Coated wells were washed with PBS-"Tween" (phosphate-buffered saline - 0.05% "Tween" 20) and were incubated for 2 hours at room temperature with supernatants diluted 1:10 in PBS-"Tween". Development was done with alkaline phosphatase (AP)-linked 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-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 incubation at 37°C. Standard curves for quantification were constructed with dilutions of IgM (Cappel) or IgG (Kabi AB, Stockholm, Sweden). Hybrids that produced immunoglobulin (Ig) determined by ELISA were propagated by transfer to 24-well macroplates (Nunc A/S, Denmark), and the supernatants were further analyzed by immunocytochemical analysis with regard to turnover with tumor cells or by immunohistochemical analysis with consideration of reactions with tumor tissue 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 yg of IgM per ml when it was allowed to grow for two weeks without any change of medium.

Example 2

Antigen characterization

a) Immunocytochemical analysis

The analysis with regard to anti-tumor reactivity was

performed by immunocytochemical analysis and by immunohistochemical analysis. Immunocytochemical analysis was performed on cell films prepared from various human tumor cell lines and peripheral human blood leukocytes. The cells were fixed on slides by treatment with formol-acetone (9.5% formaldehyde, 43% acetone in 86 mM phosphate buffer, pH 7.2). About. 50 µl C-0U1 supernatant (from hybridoma B9165;

ECACC 87040201) was placed on the film of fixed cells and was incubated overnight at 4°C in a humidified chamber before rinsing and incubation for 1 h at room temperature with horseradish peroxidase (HRP)-labeled rabbit anti-human IgM (Dakopatts) diluted 1 :80 on PBS-"Tween". Finally, peroxidase substrate (0.01% H 2 O 2 and diaminobenzidine to 0.6 µg/ml in PBS) was added. The films were lightly counterstained with hematoxylin and mounted. Table I shows the results obtained by analyzing C-0U1 on films of different cells.

Reactivity of C-0U1 determined by immunocytochemistry

A selective reactivity is evident. Alternatively, the live cells were incubated with the hybridoma antibody at 4°C, followed by the enzyme-labeled anti-Ig antibody. The cells were then spread on slides, fixed with glutaraldehyde (0.17% in PBS) and incubated with substrate.

Fig. 1A shows the staining of live COLO 201 cells (colonic adenocarcinoma cells) with C-0U1, while Fig. IB shows a lack of staining using a non-reactive human hybridoma IgM in the first layer. The method used is the accepted standard procedure for labeling molecules exposed on the cell surface, and the labeling shown in fig. 1, is in accordance with 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-u chain antibody (supplied from Dakopatts, Copenhagen, Denmark) prior to incubation with the hybridoma antibody C-0U1 (0.5 µg/ml) (the Fab' fragments were prepared according to 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 for the immunocytochemical analysis. In this case, the same antibody preparation was labeled with peroxidase as that used for the preparation of the Fab' fragments. Fig. 2A shows that after application of C-0U1, only the tumor cells were stained in a section of colon adenocarcinoma. Fig. 2B shows lack of staining of tonsillar tissue. Tables IIA and B summarize the reactivity as analyzed on a variety of tissues, with Table IIa showing the results from malignant tissue, and Table IIB showing the results from non-malignant tissue.

Normal colonic epithelium showed binding of all analysed, human IgM, monoclonal antibodies, myeloma IgM as well as normal, polyclonal, human IgM. This general binding of IgM to normal colonic epithelium was thus judged to be non-specific, an interpretation that was further supported by analysis by electron microscopy and isoelectric focusing (see below). This non-specific binding of antibody is also in accordance with the knowledge of the person skilled in the art.

c) Electron microscopy

For electron microscopy, 10 5/ml adenocarcinoma cells were grown in RPMI-1640 medium, 10% FCS, in tubes with plastic coverslips for 3 days until a monolayer had been obtained. The cells were fixed in 0.1% (v/v) glutaraldehyde in 0.1 M PBS,

pH 7.2, for 30-60 min at 4°C and was then washed in PBS supplemented with bovine serum albumin (BSA) and lysine-HCl overnight at 4°C. The cells were dehydrated in from 30% to 90% ethanol at progressively lower temperatures to -20°C, and were then infiltrated in "Lowicryl" K4M (Chemische Werke Lowi, BRD) at -35°C and were polymerized overnight at -35 °C under ultraviolet light and then for a further 2 days at ambient temperature.

Ultrathin (50-60 nm) sections of cellular "Lowicryl" mounted on coated nickel grids were used. The immunolabeling procedure consisting of floating the grids with the incisions directed downwards on top of different solutions included the following steps: 1) the grids were placed on drops of 1% (w/v) NaBH4 in PBS for 10 min; 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-0U1 antibody for 1 hour at room temperature; 4) the grids were washed in 0.1% BSA-Tris for 2x5 minutes; 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 washing in 0.1% BSA-Tris for 2x5 minutes, the grids were placed on drops of goat anti-rabbit IgG labeled with gold probes (Janssen Pharmaceuticals) of 15 nm, dissolved in 3% BSA-Tris, incubated for 30 minutes at room temperature; 7) the grids were washed in 0.1% BSA-Tris for 2x5 minutes and in redistilled water for 2x5 minutes and dried; and 8) the ultrathin sections were stained with 1% 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 electron microscope operated at 80 kV.

Attempts to determine the specificity of the immunocytochemical reactions included exclusion of the primary antibody and use of a human IgM (Cappel) in place of the primary antibody.

Fig. 3A shows the distinct pattern of labeling of colon adenocarcinoma cells (Colon 137) in regions around the ends of intermediate filaments, and Fig. 3B shows lack of labeling (using a similar procedure) of duodenal adenocarcinoma cells (HUTU 80). Sections of colon adenocarcinoma cancer tissue showed labeling only of the tumor cells, again associated with intermediate filaments (not shown). Normal colonic epithelium showed no labeling. Electron microscopy thus shows 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 cancer tissue and normal tissue were dissolved by ultrasound treatment (4x30 sec-on ice) in extraction buffer (75 mM NaCl, 75 mM KC1, 10 mM Hepes, 5 mM EDTA, 5% 2-mercaptoethanol, 5 mg/l "Trasylol" , 0.01 mM "Lenpeptin", 0.01 mM "Pepstatin"). After sonication, urea was added to 6 M together with 80 mg of sucrose, and the homogenate was incubated for 30 minutes at 37°C. Insoluble material was removed by centrifugation (5 minutes at 15,000 x g).

Isoelectric focusing was performed with the extracts placed on 1% agarose thin layer gel ("Agarose" IEF, Pharma-cia) containing 6 M urea, 3% (v/v) servalytes 3-10 and 1%

(w/v) servalytes 4-6 (Serva). Gel-based film (LKB) was used as support. Focusing was performed at 1500 V/h before electrophoretic transfer of the proteins onto nitrocellulose. Remaining binding sites were blocked by incubating the nitrocellulose in 0.1 M Tris-HCl, pH 7.5, containing 0.1 M NaCl, 2 mM MgCl2 and 0.05% "Tween" 20. The nitrocellulose was cut into strips, incubated for 2 hours at room temperature

trip with the hybridoma antibody C-0U1 diluted in PBS-"Tween" to approx. 200 ng/ml. After washing with PBS-"Tween", the nitro-cellulose strips were incubated with alkaline phosphatase-conjugated F(ab')2-anti IgM antibody (Jackson Immunoresearch), followed by a substrate (a solution of 5-bromo-4-chloroindoxyl-phosphate and nitro blue tetrazolium).

Fig. 4, left panel, shows the staining of extracts of a colon adenocarcinoma tumor, and fig. 4, right field, an extract of normal colonic epithelium. I. staining of total protein with India ink; II. pickling with C-0U1; and III. staining with a different hybridoma IgM. C-0U1 clearly shows distinct staining of acidic proteins (pl 5.4-6.2) in the tumor extract, but not in the extract of normal colon.

e) SDS-PAGE and Western blotting

Extracts of Colon 137 (colon adenocarcinoma cells) and HUTU 80 (duodenal adenocarcinoma cells) were prepared by dissolving with detergents (2% SDS, 4M urea,

10 mM iodoacetamide) and was incubated for 15 min 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-0U1, followed by incubation for 2 hours with AP rabbit anti-human IgM (Sigma). The prints 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. The molecular weight was calculated from the mobility of the following pre-stained molecular weight markers: 8-galactosidase (116 K), fructose-6-phosphatase kinase (84 K), pyruvate kinase (58 K), fumarase

(48.5 K), lactic dehydrogenase (36.5 K), triose phosphate-ase isonerase (26.6 K).

The results in fig. 5 shows that C-0U1 reacts with

a protein with a molecular weight of 43,000 found in extracts of colon adenocarcinoma, and not in extracts of duodenal adenocarcinoma.

Claims (13)

1. Antibody or binding fragment thereof for the diagnosis of human carcinoma, or as a carrier of pharmaceutical agents, characterized by the fact that, by immunohistochemical analysis on frozen, acetone-fixed tissue sections, it binds to colon adenocarcinoma, ovarian adenocarcinoma, renal adenocarcinoma, breast carcinoma, lung adenocarcinoma, non-seminomal testicular 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, renal glomeruli, renal tubules, pulmonary alveoli, bronchial epithelium, testis, epidermis, tonsillar lymphatic tissue, tonsillar epithelium, smooth muscle or blood vessels, and which binds to a human carcinoma-associated cytoplasmic antigen having an apparent molecular weight of 43,000 and an isoelectric point in the range 5.4-6.2, and having at least one epitope reactive with a monoclonal antibody produced by the human hybridoma cell line B9165 (ECACC 87040201). 2. Antibody according to claim 1, characterized in that it is a monoclonal antibody. 3. Antibody according to claim 1, characterized in that it is produced by a human lymphocyte. 4. Antibody according to claim 1, characterized in that it is a polyclonal antibody. 5. Antibody according to claim 3, characterized in that it is produced from a human-human hybridoma cell line. 6. Antibody according to claim 5, characterized in that the human-human hybridoma cell line is B9165 (ECACC 87040201). 7. Isolated human carcinoma-associated antigen for non-medicinal use, characterized in that it is in substantially pure form, and has an apparent molecular weight of 43 kD and an isoelectric point in the range of 5.4-6.2, and that it has at least one epitope that is reactive with a monoclonal antibody produced by it human hybridoma cell line B9165 (ECACC 87040201) or an analog thereof. 8. Antibody according to claim 1, characterized in that it is a hybrid antibody that contains a combination site that is specifically directed to an epitope on an antigen according to claim 7 or 8, and another that is specifically directed to another epitope on the same antigen, an epitope on another antigen, e.g. a differentiation antigen of cytotoxic T cells, or a pharmaceutical such as e.g. is selected from cytotoxic and antineoplastic agents. 9. Anti-idiotypic antibody for diagnostics of human carcinoma, or as a carrier of pharmaceutical agents, characterized in that it is directed against an antibody according to any of claims 1-6 and 8. 10. Anti-anti-idiotypic antibody for the diagnosis of human carcinoma, or as a carrier of pharmaceutical agents, characterized in that it is directed against an anti-idiotypic antibody according to claim 9. 11. Diagnostic means, characterized in that it comprises an antibody according to any of claims 1-6 or 9-10, or a binding fragment of an antibody according to claims 1-6 or 9-10. 12. Diagnostic means, characterized in that it comprises an antigen according to claim 7. 13. Method for in vitro diagnosis of human carcinoma, characterized in that a tissue sample or a sample of a body fluid from a suspected cancer patient is brought into contact with a diagnostic agent according to claim 11 or 12, which agent comprises an antibody or a binding fragment of an antibody according to any of claims 1-6 or 9-11, or an antigen according to claim 7, or an anti-idiotypic antibody according to claim 9, after which the places on the tissue sample are determined to which antibody or fragment of antibody is bound, or the presence of antigen from the sample of body fluid bound to the antibody or fragment of the antibody or the presence of bound antibody against an antigen according to claim 7 found in the body fluid is determined.