JP2688824B2 - Monoclonal antibody NKY13 - Google Patents

Description

TECHNICAL FIELD The present invention relates to a monoclonal antibody NKY13 that recognizes sugar chains of glycoproteins secreted by cancer cells and a hybridoma that secretes them. More specifically, the present invention stably reacts with human intestinal cancer and normal intestinal epithelial cells, and also with a sialic acid-containing sugar chain, particularly a monoclonal antibody NKY13 that reacts with a sugar chain having the following structure and rat peritoneal cavity. It relates to a hybridoma that grows and secretes NKY13.

NeuAcα2 → 3Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glc Conventional technology Glycoproteins, glycolipids, and proteoglycans are present on the cell surface, and when normal cells transform into cancer cells, these It is known that a sugar chain of a substance is mutated. Conventionally, sugar chain mutations have been studied mainly by isolation and analysis of sugar chains. However, since the technology for producing a monoclonal antibody was developed by Köhler and Milcyutine in 1975, attempts to produce a monoclonal antibody that specifically reacts with a sugar chain mutated on the surface of a cancer cell have been popular. It was started. In addition, the importance of monoclonal antibodies that recognize sugar chain antigens has been pointed out since the experimental results that most of the monoclonal antibodies that recognize cancer-associated antigens recognize sugar chains have been reported. The sugar chain structures of several sugar chain antigens recognized by these antibodies have also been determined. It is considered that such a monoclonal antibody plays an extremely important role not only in cancer research but also in clinical fields such as cancer diagnosis, examination, and treatment. In fact, one of the cancer-associated sugar chain antigens, CA19-9, is present in the serum of cancer patients, and its level is a monoclonal antibody NS19-9 (J.
ani) et al., The Journal of Biological Chemistry (J.Biol.chem.) 257 , 17365 (1982)), and is effective in diagnosing pancreatic cancer. .

Problems to be Solved by the Invention Monoclonal antibodies obtained to date are selected using cancer cells as immunogens, and antibodies produced are bound to cancer cells and not to corresponding normal cells as an index. Usually, it is prepared, or glycolipids extracted from cancer cells are mixed with an appropriate immune aid to prepare it as an immunogen. However, since the cancer-associated sugar chain antigen that appears in the serum of cancer patients is a mucin-type glycoprotein that is synthesized and secreted from cancer cells, it has been desired to prepare antibodies against such sugar chains of glycoprotein. .

The present inventor has previously invented a method using a solid-phased glycopeptide for measuring the titer of an antibody against a glycoprotein sugar chain (Japanese Patent Application No. 61-57772). By using this method, it is possible to effectively select, from a large number of monoclonal antibodies prepared by using cancer cells as an immunogen, those that recognize sugar chains. Antibodies that identify mucin-type glycoprotein sugar chains were selected from these monoclonal antibodies, and their usefulness was shown.

Means for Solving the Problems The monoclonal antibody NKY13 is
To select a hybridoma that produces a monoclonal antibody reactive with a mucin-type glycopeptide prepared from cells (ATCC CCL233) among many hybridomas obtained by fusing splenocytes of immunized mice with myeloma cells It was obtained. That is, one of the features of the present invention
The first point is that the antibody is a monoclonal antibody that reacts with glycoprotein sugar chains, which have been difficult to obtain in the past, particularly mucin-type glycoprotein sugar chains.

The antibody of the present invention reacts not only with human intestinal cancer tissue but also with normal tissue, but the antigen in the normal tissue does not appear in the serum of healthy subjects, and only the antigen in the cancer tissue appears in the serum of various cancer patients.

The antibodies of the present invention include SW1116 (ATCC CCL233) and LS180 (A
TCC CL-180) and glycolipids extracted from human meconium slightly react. This suggests that the antigen recognized by the antibody is mainly glycolipid on the cell surface and mucin-type glycoprotein in the serum of cancer patients. The antibody reacts with normal cells because the antigen exists as a glycolipid in normal cells, and most of the sugar chains are bound to the polypeptide chains due to canceration to become mucin-type glycoproteins, and It is thought that it will be secreted extracellularly as it is covered.

The antibodies of the present invention are NS19-9 (supra), CA50 (JE
Monsson et al., Biochim.et Biophys.Acta 834 , 110, (19
85)), FH9 (Y. Fukushi et al., Biochemistry 25 , 2859 (1986)), MLS102 (A.
Kurosaka et al., Fuebs Letters (FEBS L)
etters) 215 , 137 (1987) and Japanese Patent Application No. 61-179757), and has different antigen specificity from any known antibody that recognizes a cancer-associated sugar chain antigen.

The antibody of the present invention also reacts with glycoproteins contained in LS180 (ATCC CL-180), human meconium, human colostrum, human umbilical cord blood, human amniotic fluid, human semen, human saliva, etc., and oligosaccharides of human colostrum. Have sex. This corresponds well to what is called carcinoembryonic antigen because the cancer-associated antigen is transiently expressed in the fetal period, and like CA19-9, the mucin-type glycoprotein that serves as the antigen is limited to cancer cells. It also shows that it appears in the secretion fluid of normal subjects.

Furthermore, the antibody of the present invention is NeuAcα2 → 3Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glc It has reactivity with sugar chains having the structure. In addition, oligosaccharide alcohols isolated by treating mucin-type glycoprotein with an alkali in the presence of a reducing agent also react with the antibody. This property is in contrast to the fact that when CA19-9 exists as a mucin-type glycoprotein sugar chain, the oligosaccharide alcohol prepared by the alkali treatment does not react with NS19-9.

The hybridoma secreting the antibody is a human colon cancer cell.
Spleen cells obtained from mice immunized intraperitoneally with SW1116 and myeloma cells were cell-fused according to a conventional method, and clones producing antibodies having reactivity with the sugar chain of the mucin-type glycopeptide prepared in advance from SW1116 cells were obtained. It was obtained by selecting. The hybridoma was prepared in 10% dimethyl sulfoxide, 90% fetal calf serum (FCS) under liquid nitrogen (-1).
It can be stored in a stable state at 98 ℃ for a long time. If necessary, the hybridoma is partially thawed and RPMI-1640 containing 20% FCS or Dulbecco's minimum essential medium (Dulb
After culturing and proliferating in ecco's MEM), pristine was preliminarily injected into the abdominal cavity about 2 m to 3 months before 0.5 m per mouse.
l By administering to the abdominal cavity of previously administered mice,
A large amount of monoclonal antibody NKY13 from ascites 2 days after 0 days
Can be obtained. At this time, it is possible to stably obtain 7 to 14 mg of the antibody from about 5 ml of ascites per mouse. The antibody in this ascites fluid can be easily purified to a uniform monoclonal antibody by fractionating with ammonium sulfate, dialyzing it, and passing it through an affinity column such as protein A-sepharose 4B. Furthermore, the monoclonal antibody NKY13 is secreted in the culture medium of the hybridoma at a concentration of 20 to 40 μg / ml in the culture for about 3 days,
It is also possible to use the antibody in this culture medium.

The hybridoma of the present invention has been used in the United Kingdom since May 26, 1987.
ECACC for Porton Down, Salisbury, Wiltshire SP4 OJG
(European Collection of Animal Cell Cultures) under the Budapest Treaty with deposit number 87052601.

[Examples] Hereinafter, the antibody will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

(Example 1) Method for producing hybridoma SW1116 cells as immunogen and Balb as immunized mouse
/ c was used. SW1116 cells cultured in Eagle's medium containing 10% fetal bovine serum in Balb / c were suspended at 1 to 2 × 10 ⁶ cells / mouse in 0.5 ml of physiological saline.
It was administered intraperitoneally 9 times over a month. Then 1 per animal
About 2 × 10 ⁶ cells are suspended in 0.5 ml of physiological saline and intravenously injected into the same mouse, and 3 days later, splenocytes are obtained from the same mouse. 1.5 × 10 ⁸ spleen cells and 2 × 10 ⁷ myeloma cells (SP2 / 0-Ag14) were mixed in Dulbecco's culture medium containing no fetal serum, and the cells were washed by centrifugation (1,000 rpm, 5 minutes). After that, 0.5 ml of a solution containing 35% polyethylene glycol (PEG, # 1000) was added to Dulbecco's culture medium containing no fetal bovine serum, and after mixing, centrifugation was performed at 1,000 rpm for about 5 minutes to remove splenocytes and myeloma cells. Fused. The PEG used for fusion was 5 ml of Dulbecco's culture solution containing no fetal bovine serum.
Diluted with 5 ml of Dulbecco's culture medium containing 20% fetal bovine serum and removed by centrifugation (1,000 rpm, 5 minutes). PE
G-depleted cells are 72% Dulbecco's medium, 8% NCTC109
Suspend in 50 ml of culture medium (HT medium), drop by drop (about 0.1 ml) into a 96-well tissue culture plate, and culture for 24 hours. twenty four
After 1 hour, 1 HT medium containing 0.8 μM aminobuterin in each well
It was added drop by drop to obtain a HAT medium. The hybridoma obtained by the fusion was selected from unfused splenocytes and myeloma cells by culturing in HAT medium. Approximately 10 days after cell fusion, the culture supernatant was taken from each well and the G-50I adsorbed to the well was prepared by the method described in Example 4.
The reactivity with (see Reference Example) was examined. The wells containing the clones that reacted with G-50I were further cloned twice by the dilution method, and finally 14 hybridoma clones were obtained. Of the 14 hybridoma clones, a clone that strongly reacts with G-50I, does not react with G-50II, and hardly reacts with glycolipid is selected,
It was named NKY13.

(Example 2) Preparation and purification method of monoclonal antibody 0.5 ml of pristene is intraperitoneally administered per Balb / c mouse. Approximately one month later, 10 ⁷ cells of NKY13-secreting hybridomas were cultured in a hybridoma growth medium containing 10% Dulbecco's medium, 10% NCTC109 medium, and 20% fetal bovine serum. The mice are suspended in Hanks' physiological saline solution and intraperitoneally administered to the mice. Ascites fluid is obtained from the mice after about 10 days. At this time, about 10 mg of NKY13 was contained in about 5 to 7 ml of ascites per mouse. Ammonium sulfate is added to this ascites to make it 50% saturated. This is centrifuged, the precipitate is dissolved in 0.1 M borate buffer (pH 8.2) (buffer A), and further dialyzed against the same buffer. Next, the sample is divided into several times and applied to a column of protein A-sepharose CL-4B, and the column is washed with buffer solution A. This column was previously equilibrated with buffer A and contains 6 ml of resin. About 30 mg of IgG can bind to this column. 0.1 bound antibody to column
Elute with 0.1M acetic acid containing 5M sodium chloride. The eluate is
Neutralize with 1M Tris. 50% ammonium sulfate in the eluate
The precipitate obtained by centrifuging and adding to saturation is dissolved in buffer A and dialyzed against buffer A to obtain monoclonal antibody NKY1.
Get three.

Example 3 Reactivity of Monoclonal Antibody NKY13 with Human Intestinal Cancer Tissue Formalin-fixed human intestinal cancer tissue is dehydrated and embedded in paraffin. Next, a thin section is cut out from the tissue, and then paraffin is removed from the section using xylene. Further, it is washed with alcohol to remove xylene, and finally the tissue section is washed with phosphate buffered saline (PBS) to immerse the section in PBS. The section is reacted with the monoclonal antibody NKY13 overnight. After washing the section, an anti-mouse IgG antibody fluorescently labeled with Fluorescein isothiocyanate (FITC) was reacted and unbound FITC-I
After washing and removing gG, the area where NKY13 reacted is observed by a fluorescence microscope. NKY13, unlike MLS102, reacted with both cancerous and normal areas.

Example 4 Reactivity of Monoclonal Antibody NKY13 with Mucin-type Glycopeptide A mucin-type glycopeptide (G-50I) isolated from human intestinal cancer cells SW1116 or LS180 was bound via polylysine and glutaraldehyde. After adding 1% bovine serum albumin to a vinyl chloride well and allowing it to stand for 1 hour, the well is washed, and the monoclonal antibody NKY13 is further added and reacted overnight. After washing the well, protein A labeled with ¹²⁵ I was added and reacted for 2 hours. After washing the well, the binding activity of NKY13 was measured using the radioactivity of ¹²⁵ I bound to the well as an index, and NKY13 bound to −50 I. . Even if the same operation is performed using G-50I with sialic acid removed, NKY13
Since it did not show binding activity, it was revealed that NKY13 reacts with the sugar chain containing sialic acid of the mucin-type glycopeptide.

The NKY13 binding activity can also be measured by a method using NKY13-bound polystyrene beads. Polystyrene beads (EP-03, # 80, Sekisui) are washed with phosphate buffered saline (pH 7.4) (PBS) containing 0.15 M sodium chloride. The beads are left to stand in PBS containing NKY13 at a concentration of 0.1 mg / ml at 4 ° C. overnight to coat the beads with antibody. The beads are washed 3 times with a PBS solution containing 5% bovine serum albumin (BSA), further immersed in the same solution and left for 1 hour. After removing the solution by suction, 100 μl of the sample aqueous solution (SW1116 G-5
(Including 0I), 50 μl of PBS containing 0.2% Tween 20, 50 μl
^12.5 ng of ¹²⁵ I-NKY13 and 0.1% BSA in PBS solution (about
Add 200,000 counts, mix well, and leave at 4 ° C overnight. Next, the beads were washed 3 times with PBS, and the radioactivity bound to the beads was measured with a γ-counter to determine the antibody binding activity (Fig. 1).

Example 5 Reactivity of Monoclonal Antibody NKY13 with Various Mucin-type Glycoproteins Reactivity of normal human amniotic fluid, human umbilical cord blood, and human maternal blood with NKY13 using polystyrene beads according to Example 4 Upon examination, the results shown in FIG. 1 were obtained.

RMG (Rectal Mucin-type Glycoprotein) is methine prepared from intestinal cancer tissue by the method of (H. Nakajima et al. (J. Biochem.) 93 , 651 (1983)). Most of the sugar chain structure of the type Glycoprotein has been clarified (A. kurosaka et al., The Journal of Biological Chemistry (J. Biol. Chem.) 258 , 11594 (198).
3)).

Human meconium glycoprotein was prepared as follows.
Human meconium (1.9 g) is suspended in 10 ml of water, then 90 ml of a chloroform / methanol mixture (2: 1, v / v) is added, and the mixture is well stirred and left to separate into a lower organic layer and an upper aqueous layer. The upper layer is dried and extracted with the same chloroform / methanol mixed solution three times. The extract and the lower layer of the first extraction were combined and dried to obtain a glycolipid fraction of human meconium. Water was added to the extraction residue, and the mixture was stirred and then centrifuged to remove insolubles, and the supernatant was used as a human meconium glycoprotein fraction. This glycoprotein fraction was used in Example 4
According to the above, the reactivity with NKY13 was investigated using polystyrene beads, and the results shown in Fig. 1 were obtained.

Similarly, the reactivity of human colostrum, human semen and human saliva with NKY13 was examined and the results shown in Table 1 were obtained.

Example 6 Reactivity of Monoclonal Antibody NKY13 with Various Glycolipids Bovine and porcine brains, SW1116 cells and LS180 cells are homogenized in water at 4 ° C. with a Dounce homogenizer. Add methanol and chloroform to the homogenate so that the final concentration becomes chloroform / methanol / water (2.7; 5.4; 2, v / v), stir at room temperature for 30 minutes, extract glycolipids and centrifuge. After homogenizing by adding 2 volumes of water to the precipitate, 8
Add a volume of chloroform / methanol mixture (1: 2, v / v), extract glycolipid with stirring, and centrifuge to obtain an extract. 2
The two extracts are filtered through Celite 535, mixed in a separatory funnel and water is added to make chloroform / methanol / water (1: 2: 2.4, v / v). After gently stirring, the mixture is allowed to stand and the upper layer is removed. To the lower layer, 3 times the amount of methanol of water is added and well stirred. After combining with the previous upper layer, isobutanol is added and then dried under reduced pressure. After stirring the dried sample in a chloroform / methanol / water mixture (60: 30: 4.5, v / v) overnight, centrifugation is performed to remove the insoluble matter and the supernatant is dried. The dry preparation is dialyzed against a small amount of water. After dialysis, the dried sample was dissolved in methanol and centrifuged to remove insoluble matter, and the supernatant was used as a glycolipid fraction.

As the glycolipid fraction of human meconium, the one obtained in Example 5 was used.

Methanol is added to each glycolipid fraction so that the glycolipid contained in 1 mg of starting material (wet weight) is dissolved in 20 μl of methanol. Then 20 μl of the methanol solution is added to the polyvinyl chloride well. Evaporate the methanol under reduced pressure to coat the surface of the wells with glycolipids. Add 1% bovine serum albumin, incubate for 30 minutes, wash the wells, then add monoclonal antibody NKY13 and allow to react for 3 hours at room temperature. After washing, add ¹²⁵ I protein A and allow to react for 2 hours. Were washed and the antibody binding activity was measured by the radioactivity bound to the wells. As a result, it reacts with glycolipids of SW1116 cells and LS180 cells,
It reacted slightly with human meconium glycolipids but not with other glycolipids (Table 1).

(Example 7) Sugar chain structure recognized by the monoclonal antibody NKY13 According to the above method, the reactivity of the following sugar chains with NKY13 was examined from the inhibitory activity against the binding of G-50I and NKY13. The same experiment was conducted for NS19-9. The results are shown in Table 2.

(Example 8) Measurement of NKY13 antigen level in serum of cancer patients In the same manner as in Example 4, NKY13 antigen levels in serum of normal subjects and various cancer patients were measured using polystyrene beads, and the results shown in Fig. 3 were obtained. Got Particularly, the sera of patients with colorectal cancer, gastric cancer, and pancreatic cancer have high NKY13 antigen levels, indicating that NKY13 is effective in diagnosing these cancers.

Reference Example Preparation of G-50I G-50I is human intestinal cancer cell SW1116 (ATCC CCL233) or LS1.
80 (ATCC CL-187) cell membrane (plasma membrane)
It is a glycopeptide prepared from a mucin-type glycoprotein that is secreted extracellularly at the same time, and is prepared by the following method.

Human intestinal cancer cells SW1116 (ATCC CCL233) were cultured in a Dulbecco's culture medium containing 10% fetal bovine serum for 7 days, and then collected.
Phosphate buffered saline (sodium chloride 137 mM, potassium chloride 2.7 mM, sodium monohydrogen phosphate 8.1 mM, potassium dihydrogen phosphate 1.5 mM, potassium chloride 1.0 mM, magnesium chloride
After repeatedly washing with 0.5 mM, abbreviated as PBS hereinafter), PBS containing 1% Triton X-100 (manufactured by Rohm and Haas) was added, stirred under ice cooling, and the supernatant obtained by centrifugation was dialyzed. Lyophilized. Add this to chloroform / methanol (2:
1, v / v), defatted and suspended in an acetate buffer containing 0.01 M calcium acetate, and 1/50 of Pronase P (Kaken Pharmaceutical Co., Ltd.) was added to the dried product, and a small amount of toluene was added. After being left at 37 ° C. for 3 days, sufficient protein degradation was performed. Add an equal volume of 10% trichloroacetic acid aqueous solution to the almost clarified solubilized cell digestion solution, stir it, leave it to stand and centrifuge the resulting insoluble matter, and add an equal volume of ether to the resulting supernatant. After mixing well, the mixture was centrifuged to remove the supernatant. Ether was added to the aqueous layer to extract trichloroacetic acid, and the procedure of centrifugation was repeated 3 times. After confirming that the aqueous layer had reached pH 5, equilibrate with 0.5M pyridine-acetic acid buffer (pH 5.0) in advance. Column packed with Sephadex G-25 (1.3 x 6
0 cm), developed with the same buffer, and the effluent was collected with a fraction collector. Fractions positive for the orcinol-acetic acid reaction were collected and lyophilized. The obtained dry powder was dissolved in 0.5M pyridine-acetic acid buffer (pH 5.0), gel filtered using Sephadex G-50 in the same manner as above, and fractionated in 5 ml fractions with a fraction collector. The fraction (G-50I) passed through at this time contains glycoprotein derived from mucin type (O-glycoside type) glycoprotein as the main component. FIG. 2 shows the relationship between the fraction number (horizontal axis) and [ ¹⁴ C] glucosamine radioactivity (dpm × 10 ⁻² / 8 μl, vertical axis).

Fractions with fraction numbers 20 to 29 were collected and the solvent was removed by freeze-drying to obtain mucin-type glycopeptide G-50I.

Incidentally, the fractions with fraction numbers 30 to 56 are glycopeptide fractions derived from serotype (N-glycoside type) glycoprotein, which was designated as serotype glycopeptide G-50II.

Further, G-50I and G-50II can be similarly prepared from intestinal cancer cell LS180 (ATCC CL-187).

EFFECTS OF THE INVENTION As described above in detail, the monoclonal antibody NKY13 of the present invention enables highly sensitive determination in serodiagnosis of intestinal cancer, gastric cancer, pancreatic cancer and the like. Furthermore, since the hybridoma that secretes the antibody can be stored for a long period of time and stably grows in the abdominal cavity of mice, it has become possible to supply the monoclonal antibody by a conventional method, if necessary. Therefore, the monoclonal antibody NKY13 of the present invention and the hybridoma secreting it exhibit a very remarkable effect in the diagnosis and treatment of cancer.

[Brief description of the drawings]

Figure 1 shows S for NKY13 adsorbed on polystyrene beads.
It shows the binding activity of G-50I of W1116, human amniotic fluid glycoprotein, human meconium glycoprotein, RMG, human cord blood glycoprotein, human maternal blood glycoprotein.
The vertical axis shows the ¹²⁵ I-NKY13 binding value, and the horizontal axis shows the amount of the added sample as the amount of sialic acid. FIG. 2 shows the relationship between the fraction number (horizontal axis) of the G-50I and G-50II eluted fractions of SW1116 and the [ ¹⁴ C] glucosamine radioactivity (dpm × 10 ⁻² / 8 μl, vertical axis). [ ¹⁴ C]
Glucosamine was added to the culture solution during SW1116 culture and incorporated into the amino sugar and sialic acid of the cell glycoprotein. Figure 3 shows NKY13 in serum of normal subjects and various cancer patients.
The antigen value is shown.

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Claims (3)

(57) [Claims] 1. A monoclonal antibody which reacts specifically with human intestinal cancer cells and with all sugar chains having the following structures. NeuAcα2 → 3Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glc 2. The monoclonal antibody is the hybridoma NKY13.
Monoclonal antibody NKY13 produced by (ECACC 87052601), The monoclonal antibody according to claim 1. 3. A hybridoma producing the monoclonal antibody according to any one of claims 1 and 2.
NKY13 (ECACC 87052601).