JPS59187796A - Antihuman prostatic cancer antibody - Google Patents

Abstract

PURPOSE:An antihuman prostatic cancer antibody, obtained by collecting an antibody secreted from a hybridoma of a cell capable of producing an antihuman prostatic cancer antibody and a cell capable of in vitro subculture, and having improved specificity for the human prostatic cancer cells. CONSTITUTION:A cell capable of producing an antihuman prostatic cancer antibody, e.g. a splenic cell, is subjected to the cell fusion with a cell capable of in vitro subculture, e.g. a myelomatous cell, deficient in hypoxanthine-guanine phosphoribosyltransferase, in a solution containing 30-35% fusion accelerator, e.g. Sendai virus or polyethylene glycol, and the fused cell is then cultivated in a culture medium containing hypoxanthine, aminopterin and thymidine to give a hybridoma, which is then transplanted to a living body and multiplied in a solid or ascitic culture medium. Blood serum or ascites of the living body are then collected to afford the aimed antihuman prostatic cancer antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing an antibody against human prostate cancer, the antibody, and a fused cell (hereinafter referred to as a hybridoma) that secretes the antibody.

In recent years, cancer has become a disease with the highest mortality rate among all diseases and has become a social problem.

Countermeasures have been investigated from all six aspects, but no fundamental solution has yet been found. In Japan, stomach cancer, lung cancer, and liver cancer are common, accounting for more than 50% of all cancer deaths, but on the other hand, due to changes in lifestyle, types of cancer that were previously rare are also on the rise. Among these, urinary system malignant tumors such as prostate cancer are a typical example, which is increasing year by year. Treatment methods include surgery, radiation therapy, chemotherapy, and immunotherapy, but they have limitations in terminal cancers and advanced diseases.
Early diagnosis and early treatment are considered the best methods. Therefore, there is currently a need for a method for identifying and diagnosing cancer at an early stage and for a treatment method different from conventional methods.

On the other hand, a huge amount of research has been conducted on cancer-related antigens that exist on the surface of cancer cells, and their existence has been proven in experimental animal diseases, but research has not been sufficient in human cancers, and their existence has been proven. It is unclear.

Research on such antigens has been carried out using allogeneic antisera and xenogeneic antisera, but as the absorption procedure is repeated to increase specificity, the antibody titer decreases, making it difficult to find antisera that can withstand practical use. I couldn't get it easily.

In recent years, with the advent and development of monoclonal antibody production technology using cell fusion methods, it has become possible to produce antibodies with high titer and excellent specificity. It has been reported that monoclonal antibodies that react with human cell surface antigens such as human colon cancer have been obtained. Regarding human prostate cancer, this is also the case (if 4r antibodies can be obtained with excellent specific 1'11 antibodies, they will be useful in identifying, diagnosing, and treating cancer cells).

However, although it is said that it is generally possible to produce specific antibodies using monoclonal antibody production techniques using cell fusion methods, the antigenicity of cancer cells varies, making it difficult to produce the desired specific FI. It is currently impossible to predict whether or not fc antibodies will be obtained. Furthermore, there are almost no reports regarding human prostate cancer, and even less there are no antibodies that have been put to practical use.

The present invention was achieved by producing antibodies against human prostate cancer by cell fusion method, and by confirming that it is possible to produce antibodies with excellent specificity for cancer cells. The antibody of the present invention, which is jqed in this way, is useful as an identifying agent, a diagnostic agent, and a therapeutic agent for human prostate cancer.

A method for producing antibodies against human prostate cancer cells generally consists of the following steps.

A. Step of preparing antibody-producing cells: Antibody-producing cells for human prostate cancer cells may be obtained from any animal species including humans, and although it is not essential to perform immunization in advance, it is possible to perform this. Therefore, the efficiency of collecting the desired hybridoma can be significantly increased.

When using human cells, those with a history of prostate cancer or those with high serum antibody titers against the cells can be selected. For immunization of cancer cells, cancer cells themselves or cells that have lost their proliferative ability by treatment with glutaraldehyde or mitomycin may be used, or the surface antigens may be isolated and purified from the cells by an appropriate method. Good too. For additional immunization, auxiliary agents such as 70 in 1 to complete or incomplete azicobant can be mixed into the immunogen. The immunogen administration method during immunization may be subcutaneous injection, intraperitoneal injection, intravenous injection, intra-agency injection, intramuscular injection, etc., but subcutaneous injection or intraperitoneal injection is preferable. Immunization may be carried out once or repeatedly at appropriate intervals, preferably 1 to 5 weeks. By measuring the antibody titer against the cells in the serum of the immunized animal and using animals with sufficiently high antibody titers as a source of antibody-producing cells, the efficiency of subsequent operations can be increased. For fusion, 3 days after the final immunization
It is preferable to use antibody-producing cells derived from animals after ~5 days. The antibody-producing cells are plasma cells and their progenitor cells, lymphocytes, which can be absorbed by IF from any part of the individual.
, 1, but generally can be obtained from tiles, lymph nodes, peripheral surfaces, or any suitable combination thereof.

[3. Process of cosmetic fusion: The other parent cell, which can be subcultivated for a long time in vitro, may be any cell that can be fused with antibody-producing cells to produce a hybridoma that meets the purpose. Cells with a high probability of this are leukemia cells such as myeloma.

The species of origin may be human, rat, mouse, etc.

As will be described later, in order to remove mixed parental cells after fusion, it is preferable to use cell lines deficient in hybokinindenguanine phosphoribosyltransferase or cells deficient in thymidine kinase. For example, human-derived GM 1500
-6T G -△12. RP M T 8226. Mouse-derived P3-X63-Ao8. P3-NS I/
1-Ag4-1.5p210-ΔΩ14. X 63-
Ag8,653 etc. can be used.

Although it is not essential that the species of duplicate antibody-producing cells and the cells capable of long-term subculturing be the same, the efficiency of the fusion, the stability of the properties of the cells after fusion, and the in vivo In terms of ease of culturing (2i), it is generally advantageous to use the same strain in many cases. In particular, mouse-derived P3. -X63-
Δgf1. P3-N S T/1-A (14-1, S
When using 11210-Ag14 or X63-Δ98.653, it is preferable to use syngeneic BALB/c or BALB/B mouse as the animal from which antibody-producing cells are obtained. For fusion, it is preferable to use a fusion accelerator such as Sendai virus or poly-T-ethylene glycol, particularly poly-1-ethylene glycol 1000. +54
0.2000.40001! or 60002'i: It is preferable to use. Fusion is carried out in a solution containing about 30 to 51% of this. J) Dimedyl sulfoxide may be further added as an auxiliary agent.

C, Hybridoma Town W: In the mixture after fusion, hybridoma Sen, parent 111
One cell of antibody-producing cells and cells that can be subcultured in vitro for a long period of time remain. The former is usually a long-term in-1
There is no problem since the latter cannot withstand one or more cultures, but it is desirable to exclude the latter as they may grow together with the target hybridoma. Therefore, as the latter,
After fusion using hypoxanthine guanine phosphoribosyltransferase or thymidine kinase-deficient cells, the cells are cultured in a medium containing hypoxanthine, aminopterin, and demidine. This allows only hybridomas to grow selectively. When hypoxanthine guanine phosphoribosyltransferase or thymidine kinase-deficient cells are not used as parent cells, the I vesicles are treated with emedin and actinomycin D prior to fusion to cause the cells to lose their proliferative ability. Hybridomas are selected from a mixture with parental cells.
3- You may choose.

Hybridomas obtained in this manner generally contain two or more types of cancer cells and are not a population of cells with completely identical properties. If it is desired to isolate individual clones, it is necessary to perform cloning.

Cloning is of course used to produce antibodies with a single specificity, but it is also used to eliminate changes in bovicolecidin that often occur during long-term culture in a system where many types of antibodies coexist. This is the right column from the viewpoint of prevention, and it is desirable to do so. As a cloning method, a limiting dilution method, a soft agar method, or a fibrin gel method can be used. It is also possible to separate cells during []-gin using a fluorescence-activated cell sorting device. Also,
In response to the appearance of mutant strains that occur during long-term culture, it is possible to preserve cells with the characteristics of the original cells by performing cloning from time to time. Examples of hybridomas secreting antibodies against human prostate cancer produced according to the production method described above include P-001 and P-00 as shown in the Examples below.
2, P-003 and HaP-004.

In the logarithmic growth phase, the hybridoma of the present invention is grown in live fetal oil containing 5% (V/V) dimethyl sulfoxide as a frost protection substance at 1 to 10 x 10 cells/m+.
It can be stored for a long period of time by suspending and freezing it. In that case, the cooling rate during freezing is preferably -1°C/min, and storage is preferably carried out at -80°C or lower.

It is best to thaw as quickly as possible, and if the cells are immediately washed with a medium to remove dimethyl sulfoxide after thawing, they can be suspended in a normal medium and culture can be resumed. However, if the survival rate of the cells at the time of thawing is poor and the proliferation activity is extremely low, it is necessary to add mouse tile cells or the like as appropriate.

D. Production of Kisui: In producing antibodies, hybridomas that produce antibodies against human prostate cancer cells are cultured in vitro or in vivo.

In the case of in vitro cultivation, a suitable nutrient medium for propagation of the hybridomas according to the invention, 1 = e.g. 10% (V
/V) (7) Live fetal blood m, 5xlOM(f)B
- R PM I 1640 containing mercarzotoethanol, 1mM sodium birhinate and antibiotics
A culture medium can be used. Also, RPM I 164
D containing 4,5a/l-glucose instead of 0 medium
ulbecco's modified Eagle
's MEM (hereinafter abbreviated as D-MFM) may be used. The appropriate initial concentration for cell proliferation varies depending on each hybridoma, but is generally about 109 cells per cell.
ml, and the cell concentration during culture is 2X 10' cells/m+
It is desirable not to exceed.

Furthermore, by transplanting the hybridoma of the present invention into a living body and growing it in a solid or ascites form, and collecting body fluid, preferably serum or ascites, from the living body, antibodies secreted by the hybridoma can be produced. The crude antibody solution obtained by this method has the disadvantage that it contains various substances derived from the host organism as impurities.
It is superior in that it contains a significantly higher concentration of the target antibody than antibody solutions obtained by in vitro culture. When hybridomas are transplanted into the peritoneal cavity and allowed to proliferate, bristan (2,6,10,14-tetramedylpentadecane) is administered intraperitoneally before transplantation, preferably 3 to 9 weeks before. In particular, this procedure is not essential, although it can increase the yield of crude antibody solution. The living organism used as the host is preferably an animal of the same species and generation as the parent cell 17- of the hybridoma to be transplanted. In this case, the hybridoma usually proliferates within the cow's body without any special treatment, but if the histocompatibility antigen types of the hybridoma and the host do not match, it is generally necessary to administer an anti-lymphocyte antibody to the host organism. It is necessary to perform treatments such as X-ray irradiation in advance. After transplantation, it usually takes one to three weeks for the cells to grow.

When culturing Hybridone in vitro or in the body of a cow to produce and secrete antibodies, a radioactive substance such as radioactive isotope-labeled leucine or lysine is added to the medium or administered to the host, so that the inside of the molecule is secreted. It is possible to produce antibodies that contain a radioactive substance and whose chemical structure is completely the same as that of an unlabeled antibody.

Examples of antibodies against human prostate cancer produced according to the production method of the present invention include antibodies secreted by hybridomas shown in Table 1 below. Its -1Q- characteristics and immunoglobulin class are as shown in Table 2 below.

The antibody of the present invention may be used as a crude antibody solution, but
According to conventional immunoglobulin purification methods such as ammonium sulfate fractionation method and ion exchange chromatography, or p
It can be purified and used by affinity chromatography using rOtein A or antigen.

Furthermore, these antibodies can be used in combination as necessary.

Specific examples will be described below.

Long-term in vitro passage culture human prostate cancer cells 8PC932
x10 cells were placed in a 100 mm culture dish (Falcon
3003), Eagles MEM Earl Salt (hereinafter MF
(abbreviated as M), 10% (V/V) beef tallow serum and J, and the antibiotic kanamycin sulfate (final concentration 60111 (1/l-
) was cultured at 37° C. in a humid atmosphere containing 5% carbon dioxide gas. [31] After that, the cells were treated with Policeman C.
"Detach and culture" - Remove the supernatant by centrifugation, and then wash with phosphate buffered saline (pl-17,2, hereinafter abbreviated as PBS) for 1111 minutes, and then transfer to PBS! ! ! Made it muddy. Approximately 2 x 10 cells were obtained from one culture plate.

(2) Culture of myeloma cells Mouse myeloma cells 511210-ΔCJ1410 cells/ml were added to D-MFM with fetal bovine serum 10% (V/V).
The cells were cultured in a medium supplemented with 1 mM pyruvate, 2 mM glutamine, and 60 ma/L of kanamycin sulfate, and subcultured every 3 days. For S r12/O-A <11/l, the cell concentration was adjusted to 2.5×10 cells/m+ on the day before cell fusion, and the cells were cultured in the above medium.

(3) Immunization with 8PC93 cells 10 8PC93 cells from (1) above suspended in 0.5 ml of PBS were injected into female BAL, l'3/c mice (Nippon Charles River Co., Ltd., 9 weeks old). times, about 3
Immunization was performed by intraperitoneal injection every other week.

(/l) Cell fusion Cell fusion is 1
It was carried out using the method of (I mmunoassay).
s: C1inicalL laboratory
Techniques for the 1980'
s ed.

by Nakamura R,M, et at.
E, S, AlanR, Ltss, Inc, N.
Y., 1980. pp301-324).

On the 4th day after the final immunization, the mice were sacrificed, the tiles were taken out, loosened well, passed through a 150 mesh stainless steel mesh, and centrifuged at 400xg.
Tris-HCl buffer containing 47% ammonium chloride (0
, 017M tris(hydroxymethyl)aminomethane, 11-47,65) was added to remove the red spheres, and the tile cells were collected by centrifugation (400xg).

The cells were washed by adding MEM, centrifuging at 111 ft (400×g), suspending the cells in fresh MEM three times, and finally suspending them in MEM.

5t) 210-Δg14 was peeled off by ribpetuffing into a culture container and transferred to a centrifuge tube. Centrifugation (400X!J
), MEM was added to the collected cells, suspended, and centrifuged (4).
After removing the baijiu by washing (00×g), it was resuspended in MEM. The above tiles are 10x107 pieces 5p210-A
After mixing 107 2X 0.014 and pipetting well, centrifuge at 11t (400xg) L. After removing the supernatant, the precipitate was loosened by gently tapping the centrifuge tube. MFM with 30% (V/V) polyethylene glycol (P F G 1000) added at 37°C
0.6 ml was added to the loosened cells. After stirring gently, the mixture was allowed to warm to room temperature for 5 minutes. After centrifuging at 7Xg for 2 minutes, slowly add 5ml of MEM.
)'l)- added. After gentle stirring, the mixture was centrifuged at 400×g for 5 minutes at room temperature. The supernatant was discarded, 5 ml of MEM was added to the precipitate, centrifuged in the same manner, and the supernatant was removed. 5 ml of the following medium was added to the precipitated cells and pipetted. The medium was ll-MEM, 10% (V/V) fetal bovine serum, 2mM glutamine, 5x S 10 M β-mercaptoethanol, 60mg/
Add kanamycin sulfate of I-, and add 4 glucose.
．． 5(+/l-) was used.

Note that this medium is abbreviated as D-MFM-FBS.

-, 40 ml of cell suspension in 10 ml of r)-ME
Add M-FBS, dispense 25 ml each into 25 cm culture flasks (Corning, C-25100), and incubate overnight at 37°C in a humid atmosphere containing 5% carbon dioxide. did. The cultivation was carried out under the same conditions as described below. The next day, after being lightly pipetted, the mixture was transferred to a centrifuge tube and centrifuged at 40+'IX G. After removing the supernatant, add 1 x 10~'M hyboyazanthine, 4 x 10
M azeta nobuterin and 1.6X10” M thymidy
7- IT (S (hereinafter referred to as l-
(abbreviated as 1'AT medium), suspended in 60ml, and
ml in a 96-well culture plate ([2 alcon 3
072) and cultured for one week. after that,
25 μm of 1-JT medium was added every 20 or 30 minutes. The LIT medium used was 1-IAT medium with aminopraline removed.

B. Selection of antibody-producing hybridomas and proliferation Two weeks after cell fusion, the presence or absence of antibody production against 8PC93 cells in each sea urchin root oil was examined by enzyme-linked in-phase immunoassay.

Play RPC931111 cells 1- (Falcon
30?2) and culture supernatant 4 in each tube.
After reacting with 0 μm for 2 hours at room temperature,
Pel A:t washed and diluted 1000 times with horse serum.
Sidase-conjugated anti-mouse immunoglobulin antibody (Cappe
l Lab, Inc.

USA, catalog number 3211-0231) 10
0 μm for 2 hours, and then washed with PBS.

Que>R buffer (0.1M, 1)H4.5) disubstrate (0-phenylenediamine) at 11H/m! and 3
A solution containing 0.4 μm/ml of 1% hydrogen peroxide was added to the
A color reaction was carried out for 30 minutes. 8PC93
Cloning was performed on two wells containing antibodies that reacted with cells. Cloning was performed using the limiting dilution method as follows.

100 antibody-producing hybridomas in two wells were each suspended in D-MEM-FBS, while the above A (
Using the same method as used for fusion in step 4), 10 tile cells from BAIB/C mice were prepared in D-MFM-FBS, and the two types of cells were mixed. D-MFM-FBS was added so that the cell density was 5 x 106 cells/ml, and 0.2
ml was placed in each well of a 96-well culture plate (Falcon 3072) and cultured. One day after the start of culture, each hybridoma was examined for the presence or absence of antibody production using the enzyme-linked solid-phase immunoassay described in -. As a result, a total of 38 hybridoma strains were obtained, including the 4 strains shown in Table 1.
.

Production of C0 antibody = (Produced by in vitro culture) Hybridoma P-001, P-002, P-003 or p-004, 20% live fetal serum, 2mM glutamine.

1mM pyruvyl, 4.5g/L glucose,
D-ME containing kanamycin sulfate of 5X10 M β~Mercap 1~■ Tsukinol Onibi 50111Q/L
25 ml of this cell suspension was placed in a 75 cm i silk fabric culture flask].
(]-Ning Co., USA) and cultured at 37°C in a carbon dioxide incubator containing 5% carbon dioxide gas. The culture supernatant was collected at the 4th day when proliferation reached almost steady state.

-RiR- The number of cells at this time was approximately 2 x 10 cells/m! and the antibody content of the supernatant was 3.1μ (J/ml,
2.8μ0/ml, 2.8μq/ml, 2.
It was 5μ (J/ml).

(Produced by in vivo culture) Nibrisukun (2.6°1
0.14-tetramethylpentadecane) 0.5ml
Hybridomas P-001, P-002, p-003 or P-00 grown in vitro were intraperitoneally administered to BALB/c mice 10 to 30 days after intraperitoneal administration.
5 x 106 cells/l were inoculated. Collect ascitic fluid 2 to 3 weeks after inoculation and centrifuge @ (1000Xσ, 4℃, 15
Ascites supernatant was obtained in 1 minute). Approximately 30 ml of ascites supernatant was obtained from 10 mice for each hybridoma;
Its antibody content is 2.5 ing/ml and 2.0 ml, respectively.
m (1/ml, 1.5mg/ml, 1.8m
It was Mm1.

Table 1 shows the shape, size, and properties of these four strains.

These antibodies were administered to 1 group of 10 ICR mice at 2 (1
/ ko orally, 400 mg/k (I ip or 20
0m! +/k() venous and venous infiltration, and after 14 days of observation, no death due to these antibodies was observed.

Table-1 29-Example 2 Hi-1~Characteristics of Imayomura Kogi against prostate cancer cells
The following cells, which had been passaged 1 in. 1 [ ], were used.

lntesl:ine 407 (human fetal small intestine cell line), K-562 (human leukemia cell line), Br17 (
Hi1~peripheral lymphocyte cell line) and 8PC93 (Hi1~prostate cancer cell line).

Using the culture supernatant obtained in Example 1, the reactivity with the 2L cells was examined by enzyme-linked in-phase immunoassay. Table -
As shown in Figure 2, an extremely specific antibody for <8PC93 was obtained.

B,! Cell staining by photoantibody method 8PC93 cells were stained by indirect fluorescence antibody method using an antibody secreted by p-002, one of the hybridomas.

8PC93 cells were fixed on a non-fluorescent glass slide, reacted with 50 μm of culture supernatant at 37°C for 45 minutes in a humid atmosphere, and then added with 1% bovine serum albumin and 10 mM in PBS.
1-IFPFs (N-2-L nido D-xyethylhiberazine-N-2-ethanesulfonic acid) and 0.1
Cleaning was carried out three times by treating for 3 to 5 minutes in a solution containing 1 to 1% lithium azide. Fluorescein-conjugated anti-mouse I (JG (M1les-Yed)
aLtd, Israel, coat number g 65-171
) was further reacted with 50 μm of a 20-fold diluted solution for 45 minutes under the same conditions as above. Washing was also performed in the same manner. After drying, carbonate buffer/glycerin solution (0.05
M.

I) I-19, containing 5, 10% glycerin) was overlaid, a cover glass was placed thereon, and it was examined under a fluorescence microscope (Olympus, Model A I-f-RF L-LB). As a result, strong fluorescence was observed on the surface of 8PC93 cells. The fluorescein-conjugated anti-mouse TaG used was
The absorption procedure was performed 6 times using 8PC93 cells in advance.

Identification of the immunoglobulin class of the C0 immunoglobulin class ζ antibody was performed by enzyme-linked in-phase immunoassay. Peroxidase-conjugated anti-mouse immunoglobulin antibodies include anti-immunoglobulin (Io), anti-1 (lG), and anti-1 (IM) (both CaIlpel).
l-ah, Inc., USA, catalog number 3241-0231.3211-0081.3211-
0201) was used.

All four types of antibodies examined were ■gG as shown in Table 2.

31- Table-2 +: positive reaction, -2 negative reactions (Kumijin dialect! Shima Mura Moto) 32- Continuation of page 1 0 shots by Ken Saito 3-26-1 Hyakunincho, Shinjuku-ku, Tokyo 204 ■Initiated by Hatahiko Kobayashi 3-26-1 Hyakunincho, Shinjuku-ku, Tokyo 102 0 shots Tomoko Chiku Nagareyama Civil Engineering 1410-2 ■Initiator: Ken Matsunaga 3-26-1 Hyakunincho, Shinjuku-ku, Tokyo -303 0 shots clear person Takami Fujii 5-11-21 Towa, Adachi-ku, Tokyo 0 shots Akio Yoshikumi 2-19-46 Higashi, Kunitachi City

Claims (1)

[Scope of Claims] (1) Human prostate cancer cell surface, which involves preparing hybridomas from anti-human prostate cancer antibody-producing cells and cells that can be subcultured for long periods in vitro, and collecting antibodies secreted by the hybridomas. Method for producing antibodies against antigens. (2) The method according to claim 1, wherein the anti-human prostate cancer antibody-producing cells are tile cells, lymph node cells, peripheral leukocytes, or a mixture thereof. (3) The method according to claim 1 or 2, wherein the anti-human prostate cancer antibody-producing cells are mouse-derived cells. <4) The anti-human prostate cancer antibody producing cells are BAI-B/
4. The method according to claim 3, wherein the cell is a C71 mouse cell or its hybrid mouse cell. (5) Claims 1 to 4, characterized in that the 111 cells capable of long-term subculturing are myeloma cells.
The method described in any of the above. (6) The method according to claim 5, wherein the cells capable of long-term subculturing are myeloma cells deficient in thymidine kinase or guanine phosphoribosyltransferase. (7) The method according to any one of claims 1 to 6, characterized in that the cells that can be subcultured are mouse-derived cells. (8) The long-term subculture Cells that can be subcultured are P 3-X 63
-Ag3, P 3-N S I / 1-A g4-1
．． 5D210-Δ(71,'I or X63-ΔQ 8
, 6.53 cells. (9) The anti-human prostate cancer antibody-producing cells are cells derived from an animal immunized with human prostate cancer cells, according to any one of claims 1 to 8. Method. (10) Claims 1 to 1, characterized in that polyethylene glycol is used as a fusion promoter in the process of fusing prostate cancer antibody-producing cells to the anti-human 1 and the cells capable of long-term subculturing. The method described in any of Item 9. (11) Claims 1 to 10, characterized in that the hybridoma is grown selectively in a medium containing hypoxanthine, aminopterin, and demidine. The method described in any of the paragraphs. (12) The method according to any one of claims 1 to 11, characterized in that the hybridoma is cloned and separated into single clones. <13) The method according to claim 12 is characterized in that the hybridoma is cloned using a limiting dilution method, a soft agar method, or a fibrin ring method.
(14) The method according to any one of claims 1 to 1310, characterized in that the hybridoma is cultured in vitro to secrete antibodies. <15) The method 1 according to any one of claims 1 to 13, characterized in that the hybridoma is transplanted into a living body and the antibody is separated from the body fluid of the animal. 16. The method according to claim 15, wherein the body fluid is serum or ascites. (17) The method according to claim 15 or 16, wherein the animal to which the hybridoma is transplanted is a mouse. (18) The animal to which the hybridoma is transplanted is transferred to B.L.
18. The method according to claim 17, wherein the mouse is a B/c mouse or a crossbred mouse thereof. (19) The method according to any one of claims 1 to 18, wherein the human prostate cancer cell line is human prostate cancer cell line 8PC93. (20) An antibody against human pre-Q adenocarcinoma cell surface antigen produced by the method according to any one of claims 1 to 19. (21) Hybridoma P-001, P-002,
21. The antibody according to claim 20, which is secreted from P-003 or p-oo4. (22) A hybridoma that secretes an antibody against a human metastatic adenocarcinoma cell surface antigen, which is produced from anti-human prostate cancer antibody-producing cells and cells that can be subcultured for long periods in vitro. (23) Hyfrit-vP-001, P-002,
The hybridoma according to claim 22m, which is P-0035- or P-00/l.