DE3341367A1 - MELANOMIC DIAGNOSIS CONTAINING MONOCLON-SPECIFIC ANTIBODY - Google Patents

Description

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The present invention relates to a reagent for use in melanoma diagnosis, the reagent being a Contains monoclonal anti-melanomic antibody derived from a mammal recognizes its own melanoma antigen. The invention also relates to diagnostic methods for the detection of melanoma (Melanoma carcinoma, sarcoma}, * where the said reagent is used.

According to the invention, on a T cell level the existence of a melanoma-specific antigen, the different Animal species, found that is formed on the surface of melanoma cells (Nature, 294, 748-750, 1981).

The present invention is based on the object to provide a diagnostic reagent that is monotonous Contains monoclonal anti-melanomic antibodies.

The object of the invention is also to provide a monoclonal To provide anti-melanomantibodies that are not species-specific.

Another object is to provide diagnostic equipment (diagnostic kit) that acts as a reagent contains a monoclonal anti-melanoma antibody.

Another object is to provide diagnostic methods for finding melanomas in mammals, in particular People who use a non-species-dependent monoclonal anti-melanoma antibody will.

Other preferred embodiments of the present invention emerge from the following description.

According to the present invention, (1) a reagent for use in the diagnosis of melanoma in humans that contains a monoclonal antibody that contains is capable of recognizing a mammal's own melanoma antigen.

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nen; furthermore, (2) diagnostic equipment that contains said reagent, and (3) immunoassays for the detection of human melanoma antigens are available posed. The reagent can be the monoclonal antibody and as a further part associated therewith, for example contain a radioactive element or an enzyme or other carrier component.

The melanoma antibody according to the invention is preferably an M2590 antibody.

The invention is explained in more detail with reference to the following figures explained.

Fig. 1 shows schematically a method according to the invention

to diagnose melanoma again; 15th

Figure 2 is a plot of the discovery of

Melanoma antigens using a radioimmunoassay according to the invention;

Figure 3 graphically depicts the specificity of the present invention in locating human Melanoma antigens again by radioimmunoassay.

The previously found melanoma-specific antigenic properties, which is peculiar to different animal species is also recognized by an antibody that is produced by isoimmunization is obtained; accordingly it was shown that the antigen is a melanoma-specific antigen. In detail Melanoma cells from C57BL / 6 mice are treated with mitomycin and C57BL / 6 mice intraperitoneally several times administered for immunization. The blood cells of the animals with P3U1 myeloma cells using polyethylene glycol fused (fused.) to produce a monoclonal antibody. This antibody is a IgM class antibodies against mice; but defies his heart

He reacts by immunization with mouse melanoma not only with mouse melanoma but also with melanoma from other living things such as human and hamster melanoma. However, it has been found that the monoclonal antibody is a has such a specificity that it is in no case associated with neuroblastoma, myeloma, fibrosarcoma, scaly cell carcinoni / Acanthoma, cervical arc inoma and lymphoma or reacts with normal tissues (from the skin, eyes, cerebrum, etc.) of humans and mice.

Biochemical analysis has shown that the human or mouse melanoma antigen is a glycoprotein with a Molecular weight is 31,000. In particular, experiments with treatment, with exoglycosidase and treatment with tunicamycin led to the discovery that the antigenic action exerted by the monoclonal antimelanomantibody according to comparative example (as "M2590 antibody" is recognized, is asparagine-bound sugar chains with terminal salivary acids (sialic acid). ■ In general, a monoclonal antibody recognizes only an antigen determinant of antigen molecules. Is a If a specified antigen determinant is a protein, only one such determinant is present on a molecule. Therefore only one molecule of the monoclonal antibody can be combined with one antigen molecule. However, recognizes the invention monoclonal antibody a tumor antigen that is composed of sugar chains. That means that many Sugar chains are present on an antigen molecule, and that therefore many antibody molecules can combine with one antigen molecule. Accordingly, the monoclonal Antibodies according to the invention with its highly specific reactivity have high sensitivity when used as diagnostic or therapeutic agent is used. Since, as mentioned above, the monoclonal of the invention Antibodies, such as the M2590 antibody, are equivalent to non-human animal melanoma cells and human

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Melanoma lines can react, are the results . of animal experiments can be directly transferred to humans. Taking these relationships into account, the above is Antibodies an ideal means of diagnosing melanoma. In particular, one can expect an immunoassay at that of the monoclonal antibody in combination with a label such as an isotope or enzyme is an excellent one Diagnostic agent is.

The production of M2590 antibody as a melanoma antibody preferred according to the invention is shown in the following comparative example described.

Comparative example

(1) sensitizing the mouse with antigen.

In a culture fluid RPMI-1640 (containing 4% bovine fetal serum, manufactured by Gibco Co.), 1 × 10 / ml of mouse B-16 melanoma cells (J. Natl. Cancer Inst., 32, 535-545, 1964) were added 100 μg / ml mitomycin C ^{reacted at 37 °} C. for 45 minutes. The B16 melanoma cells (1 × 10) treated with mitomycin C were administered intraperitoneally to C57BL / 6 mice once a week; this immunization was repeated for 10 weeks. In addition, three days before the

fusion, 1 × 10 Bi6 melanoma cells were treated with Mitomycin C, injected intravenously into the animals. Three or four days later, the spleens were removed from the mice and broken into pieces to obtain an RPMI-1640 cell suspension =

(2) hybridization

10 ml of an RPMI-1640 suspension with a content of 1 χ 10 Myeloma cells P3U1 (Curr. Top. Microbiol. Immunoil., 81, 1-7,

1978), and 10 ml of a suspension containing 1 × 10 spleen cells, prepared according to (1), (if necessary, both

be used in numbers of 1x10; in other Words: the mixing ratio of myeloma cells to miIz cells can be 1:10 to 1: 1) were in a 50 ml

Entered centrifuge tube and centrifuged for 10 minutes at room temperature and 400 χ g. The supernatant was completely removed and the residue in a constant temperature bath at 37 ⁰ C is introduced. While slowly mixing the cells with the tip of a pipette, 1 ml of a heated 50% polyethylene glycol (PEG) solution was slowly added over a period of 1 minute. The suspension was stirred with the same pipette for a further minute. Then 2 ml of heated RPMI-1640 was slowly added with stirring over a period of 2 minutes. In addition, 7 ml of RPMI-1640 were added over 2 to 3 minutes. The mixture was centrifuged at 400 χ g for 10 minutes at room temperature. The supernatant was removed and 10 ml of RPMI-1640 containing 10% calf serum was added.

The mixture was gently stirred. The cell suspension was gradually increased in an amount of 0.1 ml per well A flat-bottomed micro test plate with 96 wells was applied and cultivated in a carbon dioxide incubator. (3) Investigations

The next day after the start of the cultivation, 0.1 ml HAT (hypoxanthine, aminopterin, thymidine) culture fluid added; after 2, 3, 5, 8 and 11 days, half of the supernatant in each well was peeled off and 0.1 ml of fresh HAT culture fluid added. Then it became Culture medium with HT (hypoxanthine, thymidine) culture fluid exchanged every 3 or 4 days.

The target cells used were (1) B16 mouse melanoma cells, (2) human melanoma cells and (3) EL-4 lymphoma cells from C57BL / 6 mice. 5 χ 10 target cells were treated with 0 50 μΐ the culture supernatant of the hybridoma (including the antibody) at 0 ⁰ C for 50 minutes implemented. The reaction was carried out on a 96-well polystyrene microtiter plate (manufactured by Dynatech Lab. Co.). The cells were then washed by centrifugation and treated with 50 μl anti-mouse Ig antibodies

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(50,000 cpm / well), reacted with J at 0 ⁰ C for 1 hour. The reaction product was washed well and dried; then, its radioactivity was measured with a gamma counter (manufactured by Dynaboard Co.). The antibody which reacted with (1) but not with the target cells (2) and (3) was tentatively regarded as a mouse melanoma-specific antibody (M562 antibody), and then the next step was carried out. The antibody that reacted with (1) and (2) but not with (3) was called an antibody (M2590 antibody) that recognizes a melanoma antigen peculiar to various species. Those antibodies which reacted with all of the target cells (1), (2) and (3) or did not react with any of the target cells were excluded as non-specific antibodies.

(4) Obtaining cells capable of producing the desired antibody

The limit dilution was used. When with examinations a recess was found in which there were hydridoma cells that had a specific antibody produced, cloning of this recess was carried out by the limiting dilution method. When pulling up of the cells, the cells were adjusted so that their concentration was 30 cells / 20 ml. The cell suspension was divided into 0.2 ml portions in wells of a flat-bottomed microtiter plate with 96 wells. 5 days after the growing, 0.1 ml of an HA (hypoxanthine, aminoptenin) culture fluid was added to each well; 12 days later, half of the culture medium was used

3Ό fresh HA culture fluid exchanged. Then the culture medium every 3 or 4 days exchanged with fresh HA culture fluid.

Cells M562 produced antibodies were produced with D ~ and cells M2590 antibody, with D ₁₀

designated.

(5) Purification of the antibody

1x10 hybridoma cells were intraperitoneally administered to each of (BALB / C χ C57BL / 6) F ₁ mice; after 10 days the ascites containing the antibody was collected. 10 ml ascites were centrifuged, placed in a dialysis tube and dialyzed against 0.001M phosphate buffer (pH 6.5) for 48 hours. In this way all monoclonal antibodies could be separated. The separated monoclonal antibody was dissolved in a small amount of 3% NaCl solution and dialyzed against 0.1 M phosphate / sodium chloride buffer (pH 7.2). As a result, most of the other proteins could be removed along with the supernatant. By repeating this process two to three times, the monoclonal antibody had a purity of more than 95%. The immunoglobulin class of the M2590 and M562 antibodies was IgM; in addition, they were euglobulins.

The melanoma antibodies obtained as previously described can be used as a melanoma diagnostic agent, for example, in the form of a solution.

The following example describes a method for diagnosing melanoma using the invention Melanoma diagnostic agent.

example

(1) Solid phase immunoassay

A solution of purified M2590 antibody (purified by isoelectric deposition) at a concentration of 2 mg / ml using 0.1M phosphate / sodium salt buffer (pH 7.2). 60 μΐ of the antibody solution were per recess on a polystyrene plate containing 96 recesses (manufactured by Dynatech Lab. Co.) was added in form and reacted at room temperature over a period of one hour. Then became 0.5% bovine serum albumin (BSA) was added and the plate three-

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times with phosphate / sodium chloride buffer (BSA-PBS) for removal of the antibody not bound to the plate was washed. In addition, 100 μΐ BSA-PBS were added and run the reaction at room temperature for one hour to complete the protein ^ reaction groups (Step 1). After careful washing, 40 μΐ were one TttKiorantigenextrakt or a supernatant from a culture of Cancer cells, which were prepared as described below, were added to the recess and reacted for four hours at 4 ° C (Step 2) ο Four hours later the plate was washed three times with STW buffer (pH 7.6) (containing 20 mM Tris, 0.15M Sodium Chloride, 0.2% Sodium Nitride, 5mM EDTA, 2mM Phenylmethylsulfonyl Fluoride and 1% NP-40 surfactant) and then washed with 50 μΐ (10,000 cpm) M2590

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Antibodies labeled with J were reacted according to the method (step 3) described in Biochemical Journal, 108, 611-618, (1968). After performing this reaction for one hour at 40 ⁰ C the reaction product was its radioactivity measured by well gewaschen.und gamma counter (Step 4). This operation ttf is shown in FIG.

The aforementioned tumor antigen extract and the cancer cell culture supernatant were made as follows:

Tumor cells (corresponding to 2x10), extracted from tissues or tissue fragments that have been surgically resected and corresponded to 100 mg weight, were reacted with 1 ml STN buffer (Iph-isoelectric pH 7.6) for 1 hour at 4 ⁰ C. The reaction mixture was centrifuged at 10,000 rpm for 5 minutes to obtain the supernatant. A cancer antigen was extracted and contained in the supernatant. '

Cancer cells (2 × 10 ⁶ ) were cultured in RPMI-1640 containing 4% calf serum, and the supernatant of the culture broth was obtained.

(2) Preparation of the calibration curve and the accuracy of the immunoassay in solid phase:

There were tumor antigens of varying numbers _from C57BL / 6

Melanoma (B16) cells or human melanoma cells (P-39) according to the method as described / prepared in the last two paragraphs of section (1) above. The tumor cell extracts formed from different antigen concentrates were added to the immunoassaysy system described in (1) and tested. The result, as shown in FIG. 2, is such that when an extract corresponding to 10 / ml of tumor cells was gradually diluted to a ratio of 1:10, the detection fell almost linearly at a concentration of 10 / ml and more , the minimum for detection being 10 -10 ² / ml.

These experimental results show that the solid phase immunoassay using this antibody (M2590) is very accurate and enables the detection of very low levels of melanoma antigen.

(3) Specificity of the solid phase immunoassay In order to increase the specificity of the solid phase immunoassay method test, tumor antigen extracts according to (1) from three types of human melanoma cells (P-36, P-39, P-22), hamster melanoma cells, two types of mouse melanoma cells (B-16, S-91) human cervical carcinoma (Heia), surgically resected Fragments from a melanoma patient (human, two cases), human skin cancer (flaky cell carcinoma) and prepared resected specimens of basalioma. These tumor antigen extracts were solid phase immunoassay subject to section (1). The result according to FIG. 3 was that extracts of melanoma cells which from humans, hamsters and mice, whether they were cultured cell strands or surgically resected specimens were identified by immunoassay; however, the antibody did not react at all cancer cells other than melanoma cells, such as human cervical carcinoma, flaky cell carcinoma, and acanthoma. The results are shown in FIG.

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The following conclusions can be drawn from the disclosed results:

(1) Human melanoma can be specifically diagnosed using a cell or tissue extract. (2) immunoassays using the monoclonal anti-melanoma antibody, like M2590 antibodies, have high sensitivity and can be a melanoma antigen in a human Melanoma cell extract in which 1000 to 500 melanoma cells exist, find.

According to the invention, for example, various immunoassay methods can be used, for example at those of the monoclonal antibodies (labeled or unlabelled) with other conventional solid supports such as glass beads / or where other markings such as an enzyme, a fluorescent compound, etc. are used will; an indirect method using secondary antibodies is also suitable. Accordingly, the monoclonal antibodies according to the invention on various conventional carriers or materials such as a test tube, Carrier beads, polymer particles and the like can be applied.

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Claims (16)

EUROPEAN PATENT ATTORNEYS D ex mLImN PATENT LAWYERS EUROPEAN PATENT REPRESENTATIVES MANDATAIRES EN BREVETS EUROHEENS DR. ROLF E. WILHELMS DR. HELMUT KILIAN EDUARD-SCHMID-STRASSfc 2 8000 MUNICH 90 TELEPHONE (0B9) 65 20 91 'TELEX 52 34 67 (WiIp-CfJ TELEGRAMS OF PATRANS MUNICH TELECOPIER size 2 (089) 222 P 1769-DE Masaru Taniguchi
Chiba / Japan Melanoma diagnostic agent containing specific monoclonus antibody Priority: November 16, 1982 - JAPAN - No. 200730/82 Claims (IJ reagent for use in the diagnosis of human melanoma, characterized in that the reagent contains a monoclonal antibody which is able to recognize a mammalian melanoma antigen, as well as a label or a carrier portion or a material that contains this antibody is associated. 2. Reagent according to claim 1, characterized in that g e k e η η, that the monoclonal antibody is M2590 antibody. 3. Components of diagnostic equipment for detection of human melanoma, which is in the form of a solid phase to which an anti-melanoma monoclonal antibody is attached which is capable of recognizing a mammalian melanoma antigen. 4. Component according to claim 3, in which the solid phase has a carrier on or in which an immunoassay can be executed. 5. The component of claim 3, wherein the monoclonal antibody is M2590 antibody. 6. The component of claim 4, wherein the monoclonal antibody is M2590 antibody. 7. The reagent of claim 1, wherein the marker portion is a marker component used in immunoassays. 8. The reagent of claim 7, wherein the label portion is a radioactive element. 9. The reagent of claim 8, in which the labeling portion is an enzyme. 10. Diagnostic procedures for finding presence of melanoma in a human having an anti-melanoma monoclonal antibody, capable of recognizing an acid animal's own melanoma antigen, with an extract of tissue or cells of the person concerned, in which melanoma cells are suspected, hatches and the amount of said Antibody is determined, which reacts with antigens present in said extract. BAD ORIG / NAL Taniguchi: ""'- - '"'■-"·' · P 1769-DE 11. Diagnostic method according to claim 10, wherein as a monoclonal Antibody M2590 antibody is used. 12. The diagnostic method of claim 11, wherein at least part of the monoclonal antibody used carries a label / which allows the amount of labeled Measure antibodies that react with antigens present in the extract. 13. The diagnostic method of claim 12, wherein the label is a radioactive isotope. 14. Diagnostic method according to claim 12, wherein the Label is an enzyme. 15. Diagnostic method according to claim 10, wherein a "Sandwich" assay is carried out, initially unlabeled monoclonal anti-melanoma antibodies only in the extract present melanoma antigen reacts, and then being labeled Antimelanomantibodies react with said antigen, taking the amount of labeled and affecting this Way converting antibody is measured. 16. Diagnostic equipment for finding human Melanoma, which contains a monoclonal antibody that is able to recognize acid animals own melanoma antigen, and a substrate for performing an immunoassay.