CA1288074C - Monoclonal antibody, process for preparation thereof and diagnostic drugcontaining the same - Google Patents

Abstract

ABSTRACT

Monoclonal antibody which specifically binds to a membrane protein encoded by human chromosome No. 21 having aberration in number, process for preparation thereof utilizing cell fusion technique, and a diagnostic drug comprising the same are provided.

This monoclonal antibody is useful for diagnosis of Down's disease caused by 21-trisomy.

Description

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Monoclonal antibodY, process for Preparation thereof and diagnostic drug containinq the same BACKGROUND OF THE INVENTION

1. Field of the invention The present invention relates to a monoclonal antibody which specifically binds to a me~brane protein encoded by human chromosome No. 21 having aberration in number, a process for preparation thereof, and a diagnostic drug for Down's syndrome containing the antibody.

2. Description of the prior art Recently, hereditary and nonhereditary congenital chromosomal aberration diseases have been increased, and this tendency has become a worldwide phenomenon. Medical approaches against these aberration diseases mainly depend on analysis of exanthrope up to recently, and analysis and test method thereof on hereditary factors, particularly chromosomal aberration have been extremely limited. This is because since chromosome cannot be observed in ordinal cells and can only be observed in ,;~, ,, ' ~88074 cells in mitotic period, special techniques are required for fixing method thereof. Therefore, early diagnosis, particularly prenatal diagnosis of chromosomal aberrations is a social demand, and establishment of easy and highly confidential diagnostic methods therefor has been desired.

Though many of fertilized eggs having any chromosomal aberration are naturally selected during early phase of pregnancy, about one percent of born children are born in a state having the aberration, and thus many of them have malformations and/or intensive disturbances in anima development. Kind of chromosomal aberration is classified into aberration in number and aberration in lS structure. Triploid formed by fertilizing one egg with two sperms does not grow even if it is born, whereas since a patient of trisomy wherein the same chromosome excessively exists in the nucleus grows accompanying mental and physical aberrations, wrestling therewith from whole society has been desired. 21-Trisomy having the chromosome No. 21 in an excess number of one causes Down's ~yndrome (hereinafter simply referred to as "Down's disease"). Natality of children of Down's disease tends to increase in accordance with recent increase of marriage in high age, and thus methods for early diagnosis prior to childbirth have been desired.

SUMMARY OF THE INVENTI~N

It is a feature of one embodiment of the present invention to provide novel monoclonal antibodies useful for diagnosing, in simple and high confidence, Down's disease caused by chromosome No. 21 having aberration in number, a process fcr preparation thereof, and a diagnostic drug containing said monoclonal antibody.

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The present inventors have succeeded in obtaining mono-clonal antibodies capable of recognizing in a high sensitivity the aberration of human chromosome No. 21 in number by apply-ing known cell fusion techniques. Furthermore, they have developed a diagnostic drug useful for diagnosis of Down's disease by utilization of the monoclonal antibodies.
In accordance with an embodiment of the present invention there is provided a monoclonal antibody that has been produced by a hybridoma obtained by a process comprising the steps of immunizing an animal with a Chinese hamster ovary cell into which human chromosome No. 21 has been introduced and which has a membrane protein encoded by human chromosome No. 21 and fusing an antibody-producing cell taken from said animal with a myeloma cell to form hybridomas, that specifically binds lS said Chinese hamster ovary cell and that binds a human fibro-blast cell that is trisomic for human chromosome No. 21 but does not bind a normal human fibroblast cell.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides a monoclonal antibody which can specifically bind to a membrane protein encoded by human chromosome No. 21 having aberration in number.
It has been discovered that at least one membrane pro-tein, among the many such proteins encoded by genes located on human chromosome No. 21, is produced by normal human fibroblasts, in substantially lesser amounts than by human fibroblasts containing an aberrant number of chromosome No.
21, as is the case for Down's disease patients. It has also been found that, in accordance with the present invention, monoclonal antibodies can be reproducibly obtained that recognize chromosome 21-polysomic human fibroblast cells by virtue of binding a membrane protein that is present, at detectable levels, in such polysomic fibroblast cells but not in normal fibroblast cells.
Monoclonal antibodies of the present invention are useful for diagnosis of Down's disease caused by 21-trisomy because 0~

they speci~ically bind to the membrane protein encoded by human chromosome No. 21 having aberration in number.
A monoclonal antibody of the present invention can be obt:ained, for example, by a process comprising steps of immunizing an animal with a Chinese hamster ovary cell into which human chromosome No. 21 has been introduced and which has a membrane protein encoded by the human chromosome No. 21;
fusing antibody-producing cells taken out from said animal with a myeloma cell to form hybridomas; subjecting the hybri-lo domas to selection, screening and cloning to obtain a hybri-doma capable of producing a monoclonal antibody which speci-fically binds to the membrane protein encoded by human chro-mosome No. 21 having aberration in number; and culturing the hybridoma.
The Chinese hamster ovary cell (hereinafter referred to as CHO cell) into which human chromosome No. 21 has been introduced and which has a membrane protein encoded by human chromosome No. 21 used in the above process may be obtained by known cell fusion techniques. An example of such CH0 cell includes so-called 2Fur [see D. Patterson et al., Som. Cell Genet., 1, 91-110 (1975)]. Sample cultures of 2Fur cell are available from FRI (Fermentation Research Institute Agency of Industrial Science and Technology), Ibaraki, Japan under the deposit No. FERM BP-2492.
Examples of the animals to be immunized as used in the above process include mouse, rat and the like, and mouse is usually used.
Route and schedule of administration of the antigen into the animal may variously be varied. For example, according to a preferred administration method, the antigen is first treated with mitomycin and intraperitoneally administered by several portions to the animal. Antibodies are produced in the spleen of the animal by administration of the antigen.
As antibody-producing cells, spleen cells are usually used.
As the myeloma cell to be fused with the thus obtained .,. ~,.~

~ ~88~74 - 4a -antibody-producing cells, a cell induced from BALB/C mouse MOPC 21 myeloma (as referred to as NS-l) is, for example, used. This NS-l has resistance to 8-azaguanine, and dies in a medium containing hypoxanthine-aminopterinthymidine (HAT
medium) since it lacks the enzyme hypoxanthine guanine phos-phoribosyltransferase. Therefore, use of NS-l is convenient for selective culture of hybridomas to be obtained. As a fusing agent for cell fusion, polyethylene glycol is usually used. The above-mentioned cell fusion techniques, myeloma lo cells to be used, and the like are known, per se [myeloma cells; Kohler et al., Fur. J. Immunol., 6, 292-295 (1976), and fusing method, Kohler et al., ibid., 6, 511-516 (1976)].
Resulting hybridomas are in a conventional manner screened for an antibody-producing ability, and then ~ ~8~074 cloned, whereby progenies of respective hybridomas from respective phyletic lines are obtained. Monoclonal antibodies which specifically bind to the membrane protein encoded by human chromosome No. 21 having aberration in number can successively be synthesized and secreted by respectively infinitely growing these cell lines, i.e. clones in a cell culture medium (in vitro culture) or in uivo. Each of monoclonal antibodies thus produced can be recovered according to a method known in the art.

The monoclonal antibody thus obtained specifically binds, when a fibroblast cell is targeted, for example, to the membrane protein encoded by chromosome No. 21 having aberration in number.

The present invention also provides a diagnostic drug for Down's disease owing to 21-trisomy containing the above-mentioned monoclonal antibody. Aberration of chromosome No. 21 in number can readily be determined by applying this diagnostic drug, for example, to human fibroblasts or the like. Therefore, diagnosis of Down's disease can readily be carried out by use of this diagnostic drug. The diagnostic drug of the present invention can practically be used through any immunological test methods, for example, immunofluorescence, an immunoadhesionhemocyte-agglutinating reaction method, radioimmunoassay or the like. For example, in utilization of immunofluorescence, human cells are treated with the monoclonal antibody of the present invention, then reacted with an anti-mouse IgG labeled with a fluorescent dye such as FITC, and set in a fluorescence microscope, and the state of luminescence is checked, whereby presence or absence of chromosomal aberration can readily be diagnosed.

~88074 Monoclonal antibodies and diagnostic drug of the present i~vention are further explained below according to non-restricting examples.

Examples Example 1 (1) Production of antibodies CHO cells (2Fur) into which human chromosome No. 21 had been introduced and which had a me~brane protein encoded by the chromosome No. 21 was treated with mitomycin C, and washed with PBS ~phosphate buffer). A
2Fur suspension of 5 x 106 cells/0.5 cc in PBS was intraperitoneally injected into a female BALB/C mouse (immuni~ation). Two weeks thereafter, a 2Fur suspension of 1 x 10 cells/0.5 cc in PBS, and further two weeks thereafter, a 2Fur suspension of 1 x 107 cells/0.5 cc in PBS were injected in the same manner as above. Three days thereafter, the spleen of the mouse was taken out, finely pulverized in RPMI-1640 medium, subjected three times to centrifugation (1500 rpm) for washing, and resuspended in the medium.
(2) Cell fusion 1.1 x 108 cells of the spleen cell obtained in the above procedure and 1.0 x 10 cells of BALB/C mouse myeloma cell (NS-l) once washed in advance with RPMI-1640 medium were mixed, and centrifuged at 1500 rpm for 5 minutes to prepare a pellet. Then, 1 ml of a solution obtained by dissolving polyethylene glycol (1540) in RPMI-1640 medium to 50 wt~ was added to the pellet with stirring, RPMI-1640 medium was added thereto to the total volume of 10 ml, and the mixture was stirred for 6 minutes. The ,~. ' .

~288074 mi~ture was subjected to centrifugation at 1000 rpm for 5 minutes to remove polyethylene glycol, and the solid matters were resuspended in 40 ml of RPMI-1640 medium containing 20 vol% of fetal calf serum (FCS).

(3) Selection, screening and cloning 0.2 ml (5 x 106 cells) of the above suspension was placed in each of 192 wells of a 96 wells-plate for tissue culture, and half amount of the culture supernatant was replaced every 3 to 4 days with HAT medium (136.1 mg/ml hypoxanthine, 1.76 mg/ml aminopterine and 38.75 mg/ml thymidine). The culturing condition was a temperature of 37C, a humidity of 100% and a CO2 gas concentration of 7%. 10 days after the start of the culturing, hybridomas were observed in 119 wells (emergence rate: 62~).
Culture supernatants in the 119 wells, which revealed positive results, were subjected to screening by checking according to enzyme-labeled antibody technique (ELISA) 20 _using 2Fur whether or not the supernatants contain~
antibodies, and the results revealed 20 wells to be positive (10.6% of the total wells).

Cells in each of the 20 wells were subjected to limiting dilution using HAT medium with supplementation of BALB/C
mouse thymocyte of 1.0 x 107 cellstml, 0.2 ml portions of the resulting cell suspensions were placed in each well of a 96 wells-plate for tissue culture, and culturing was carried out. In this connection, hybridoma number per well and well number were 48 wells of 1 cell/well, 45 wells of 5 cells/well and 3 cells of 20 cells/well.
Culturing conditions were the same as above. The culture supernatants were respectively subjected to the same ELISA as above-mentioned to screen those which are negative for a normal CHO cell and positive for 2Fur and as the result desired clones were ~l~8~3074 obtained from three wells. The cells in each of the three wells were recloned two times according to the limiting dilution method, and the resulting hybridomas were res-pectively named OK-l, OK-2 and OK-3. Sample cultures of hybridomas OK-2 and OK-3 are available from FRI (Fermentation Research Institute Agency of Industrial Science and Technol-ogy), Ibraki, Japan under the deposit Nos. FERM BP-1802 and FERM BP-1803, respectively.
It has been found by an enzyme-labelled antibody tech-nique using an anti-mouse antibody that monoclonal antibodies produced from hybridomas OK-l, OK-2 and OK-3 belong to the classes IgM, IgG and IgM, respectively.
Furthermore, proteins to which the monoclonal antibodies produced by OK-l, OK-2 and OK-3 respectively recognize and bind are as follows.
- Monoclonal antibody produced by OK-1 This antibody recognizes four bands, 10, 68.9 and 86.6 kilodaltones in Western blotting of human Leukemia T cell (TALL-1) protein.
- Monoclonal antibody produced by OK-2 This antibody recognizes bands of 40 and 90 kilodaltons in Western blotting of TALL-1 protein.
- Monoclonal antibody produced by OK-3 This antibody recognizes four bands of 86.4, 74.2, 61.4 and 36.4 kilodaltons in Western blotting of TALL-1 protein, and recognizes three bands of 89, 82 and 45 kilodaltons in Western blotting of 2FUr protein. *Note, TALL-1 cell line:
see MAMMALIAN CELL CULTURE TECHNOLOGY, edited by M. Shikita et al. SOFT SCIENCE PUBLICATIONS, Tokyo, pp. 141-162, 1985.
Example 2 Diaqnosis examle by detecting the protein accordinq to immunofluorescence q~;307~

Fibroblasts of a normal human being and a patient of Down's disease were placed together with a cover glass (12 x 12 mm) for a microscope in a Petri dish having a diameter of 35 mm, and cultured. As the medium 5 was used a ~ixture wherein 10% nonessential amino acid (NEAA), 10 mM sodium pyruvate, 0.1% lactoalbumin hydrolyzate (LAH) and 10% FCS were contained in MEM. The cells were cultured at room temperature for 5 days, fixed in a 3.7% formaldehyde solution in PBS
immediately after washing the cover glass 2 to 3 times with PBS, washed with distilled water, and further fixed in acetone at -20C.

15 ~Q of the monoclonal antibody obtained in Example l (antibody derived from hybridoma OK-l, OK-2 or OK-3) was placed on the fixed cells, and the mixture was allowed to react at room temperature for one hour. After washing the mixture three times with PBS, lS ~ of antimouse IgG
antibody or an antimouse IgM antibody, as a second antibody, labelled with FITC was placed thereon, the mixture was allowed to react at room temperature for one hour. The mixture was washed three times with PBS, once washed with distilled water in order to remove salts, placed on a slide glass at a state of the surface fixing the cells down, and sealed with *Gelvatol to obtain a test sample piece.

The test sample was tested about presence of luminescence on the surface layer using a fluorescence microscope.
The resulting results are shown in Table 1. Fibroblasts of the patient of Down's disease emitted fluorescence, whereas those of the normal human being did not emit.
Thus, it will be appreciated that presence of chromosomal aberration can readily be diagnosed in a high sensitivity without using a special technique according to the present invention.

*Trade ~ark ~lX8~3Q74 In order to confirm the existence of the protein on the membrane, detection by membrane immunofluorescence wherein the cells are not fixed and reacted with a fluorescent dye was also carried out as paralleled to the above method, and the resulting results were the same as those obtained above.

Table 1 Presence or absence of luminescence by immunofluorescence Monoclonal Fibroblast antibody Normal human Patient of bein Down's disease g Produced by OK-l - +
Produced by OK-2 - +
Produced by OK-3 - +
Note +: Luminescence was observed -: Luminescence was not observed

Claims (5)

1. A monoclonal antibody that has been produced by a hybridoma obtained by a process comprising the steps of immunizing an animal with a Chinese hamster ovary cell into which human chromosome No. 21 has been introduced and which has a membrane protein encoded by human chromosome No. 21 and fusing an antibody-producing cell taken from said animal with a myeloma cell to form hybridomas, that specifically binds said Chinese hamster ovary cell and that binds a human fibroblast cell that is trisomic for human chromosome No. 21 but does not bind a normal human fibroblast cell.

2. The monoclonal antibody as claimed in claim 1, where-in said Chinese hamster ovary cell is of the 2Fur cell line.

3. The monoclonal antibody as claimed in claim 1, where-in said hybridoma which produces said monoclonal antibody is the OK-2 or OK-3 cell line.

4. Use of the monoclonal antibody of claim 1 for diagno-sing Down's disease.

5. A method of diagnosing Down's disease which comprises the steps of:
applying to a human fibroblast cell, the monoclonal anti-body as claimed in claim 1, and observing whether or not said monoclonal antibody binds said human fibroblast cell.