GB2140030A - Monoclonal antibody to human urinary bladder cancer - Google Patents

Abstract

Monoclonal antibody specific to a surface antigen of a human urinary bladder cancer cell is produced by preparing a hybridoma between a cell which produces the antibody and a cell which can be permanently maintained in vitro by subculture and recovering the antibody secreted by the hybridoma. The antibody is useful for the detection and treatment of human urinary bladder cancer.

Description

SPECIFICATION Antibody to human urinary bladder cancer The present invention relates to human urinary bladder cancer antibody.

There are many people suffering from cancer and a large number of persons die from cancers, so this is a most significant social problem. In spite of great efforts by many researchers in all fields of science, no primary method for treating a cancer has been found at present. In Japan, a half or more of the total death by cancers are caused by gastric cancer, lung cancer or liver cancer. Other kinds of cancer, however, have tended to increase as the mode of life in Japan changes, for example rectal cancer or malignant tumor of urinary organs including urinary bladder cancer which relates to the subject matter of the present invention.

The treating methods of such cancers consist mainly in surgical operations in combination with radiotherapy, chemotherapy and/or immunotherapy. Such methods, are however, limited to some extent in the treatment of anaphase cancer or progressive cancer. Thus, early diagnosis and early treatment are in any event important. For these purposes, a rapid, highly reliable method of identification and/or diagnosis of the cancer cell and/or cancer tissue is strongly required.

Many extensive researches have been made on a specific antigen relating to a cancer which is present on the surface of the cancer cell. In particular, such a surface antigen has been confirmed in the study using experimental animals. On the contrary, sufficient approach has not been performed for human cancer. From the present knowledge, such a surface antigen of a cancer may comprise for example, (1) antigen present autologously only, (2) antigen present commonly in the same kinds of tumors, or (3) antigen present also in tumors of other organ or even normal cells as well as tumor cells in question.

An immunological method using an antiserum of the same or different species is generally useful for analysis of an antigen. For preparing such an antiserum which can be used for recognizing three aforementioned kinds of antigens it is necessary to repeat an absorption procedure of the antiserum. A decrease of the antibody titer is inevitably accompanied with such a procedure and a resulting antiserum can not be used in practical. Furthermore, if an antiserum to be desired may be obtained, it is almost impossible to reproduce the same antiserum having the same specificity. Under these circumstances, a new means is required for identification of cancer-related antigens and cancer-specific antigens on the surface of a cancer cell.

One of such means was developed by Köhler and Milstein in 1975, Nature, 256, 495 (1975), i.e. cell fusion technique for preparing a monoclonal antibody. Cell fusion technique satisfies such requirements as above-mentioned and subsets of human lymphocyte and monoclonal antibodies to the surface antigen of human leukemic cell or humal melanoma have recently been reported.

Generally, although a specific monoclonal antibody can be obtained by such a cell fusion technique, the antigenicity of a cancer cell scatters between various cancer cells and therefore it is not predictable at present to obtain actually an excellent antibody having the desired specificity. As to human cancer cells, only few, special cancer cells have been reported by now as described above. Particularly, no report has been done on the human urinary bladder cancer.

Of course, any antibody to the bladder cancer is not actually utilizably at present.

The inventors of the present invention have made great efforts to research a method for preparing a monoclonal antibody against the surface antigen of a human urinary bladder cancer cell and attained the invention wherein such antibody with a high specificity, a high titer and a low content of contaminants can be obtained from a hybridoma between a cell capable of producing an antibody against a human bladder cancer cell (hereinafter sometimes referred to as an anti-bladder cancer antibody producing cell" or simply as "an antibody producing cell") and a cell capable of permanent in vitro subculture (hereinafter sometimes referred to as "a subcultured cell"). The present inventors have also discovered that such antibody is useful for classification and/or identification of a human urinary bladder cancer cell.The antibody can also be useful for providing an effective treatment of the human urinary bladder cancer.

It is an object of the present invention to provide a method for preparation of antibody specific to the surface antigen of a human bladder cancer cell.

Another object of the invention is to provide a highly specific anti-bladder cancer antibody and a method of preparation thereof by cell fusion technique.

An object of the invention is to provide a hybridoma capable of producing such an antibody.

A further object is to provide a method of classification and/or identification of the human urinary bladder cancer cells with the aid of the antibody of the invention.

A still further object of the present invention is to provide a pharmaceutical agent for treatment of human bladder cancer, which agent contains the antibody of the invention as an effective component.

The present invention also has an object to provide a useful derivative of such antibody effective in the use for the classification and/or identification of the human bladder cancer cells and the treatment of the human bladder cancer.

Other objects and the remarkable advantages of the present invention will be apparent for those skilled in the art from the following detailed descriptions on non-limitative, specific embodiments.

The method for preparing an anti-bladder cancer anti-body of the present invention comprises preparing a hybridoma from an anti-bladder cancer antibody producing cell and a sub-cultured cell, particularly myeloma, and recovering the antibody secreted by the hybridoma.

The method of the invention will be described in more detail in the followings.

A. PREPARATION OF ANTIBODY PRODUCING CELL In the present invention, the anti-bladder cancer anti-body producing cell may be obtained from any animal species including human. An immunization of the animal is not essential although such preliminary immunization may remarkably improve the collection efficiency of the hybridoma to be desired.

When the origin of such a cell is human, anyone who has a past history of bladder cancer or a high titer value of serum to the bladder cancer cell may be chosen. Alternatively, such cell may be obtained from a living body immunized with an immunogen. The immunogen may be a cancer cell per se, a cell which is treated with glutaraldehyde, mitomycin or heat and thereby can not proliferate, or the surface antigen separated from a cancer cell by an appropriate treatment with e.g. an enzyme and purified.

The immunogen used on the immunization may be mixed with an adjuvant such as Freund's complete or incomplete adjuvant. The immunogen may be administered by any conventional means such as subcutaneous, intraperitoneal, intravenous, intradermal and intramuscular injections. A subcutaneous or intraperitoneal injection may be preferable. One immunization may sufficiently be possible although the immunization can be repeatedly carried out several times with an appropriate interval for example from one to five weeks. By measuring the antibody titer in serum of the immunized animal, the animal whose titer has been sufficiently high may be used to obtain the antibody producing cell, resulting in improvement of the efficiency of the subsequent procedures. The preferable cell is derived from the animal on the 3-5th day after the final immunization.The antibody producing cell is plasmocyte or lymphocyte which is a precursor cell thereof and may be derived from any site of the body, generally spleen, lymph node, peripheral blood or any combination thereof.

B. CELL FUSION A cell which can permanently be maintained by in vitro subculture may be any appropriate cell which can be fused with the antibody producing cell to give a hybridoma capable of producing a desired antibody. Preferable one of such cells is leukemia cell such as myeloma.

Such a cell may be derived from any species such as human, rat, mouse, etc. The cell which is deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or thymidine kinase (TK) is preferable since such parent cells can not grow in a selective medium.

Preferred cell lines, which are available to the public, are GM-1 500-6TG-AI-2 and Rim18226 derived from humans and P3-X63-Ag8, P3/NSl/1-Ag4-1, Sp2/0-Ag14 and X63-Ag8.653 derived from mice.

It is preferable to use an antibody producing cell and a subcultured cell, both of which are derived from the same species, although not essential, from the view points of fusion efficiency, stability of the properties of the fused cells and facility of in vivo culture thereof. Particularly, when the cell line P3-X63-Ag8, P3/NSl/1-Ag4-1, Sp2/0-Ag14 or X63-Ag8.653 is used as the subcultured cell, an inbred BALB/c mouse or its hybrid mouse may be preferably used.

For fusion, an accelerator such as Sendai virus (HVJ) and polyethylene glycol may be used, particularly polyethylene glycol 1000, 1540, 2000, 4000 or 6000 may preferably be used.

The cell fusion may be performed in a solution containing approximately 30-55% of such polyethylene glycol. Further, dimethylsulfoxide may be present in the solution.

C. SELECTION OF HYBRfDOMA In the solution after fusion, there are present, in addition to the fused cell, the remaining parent antibody producing cell and the parent subcultured cell. The former can not survive during the subsequent in vitro culture while the latter may propagate together with the desired hybridoma. It is therefore preferable or even necessary to remove the subcultured cell from the solution containing the mixed cells. For this, HGPRT- or TK-deficient cell is preferably used as the parent subcultured cell and the mixed cells containing the parent cells are cultured in a selection medium containing hypoxanthine, aminopterin and thymidine after the cell fusion, which enables selective growth of the desired hybridoma only. Alternatively, the parent subcultured cell, when this is not such a cell as deficient in HGPRT or TK, may be treated with emetine and actinomycin D prior to the cell fusion. The parent cell can not proliferate by such treatment and therefore the desired hybridoma can be easily selected from the mixed cells.

The thus obtained hybridomas generally contain two or more clones, thus those may not have the completely same properties. For separating them into the individual clones, cloning may be necessary and even desired when a monoclonal antibody may be required. The cloning is also effective in the view point of prevention of the change of population which may often occur in a long-term culture of a system with a number of clones being mixed. The cloning may be performed by limiting dilution culture, soft agar culture or fibrin gel culture. A fluorescent activated cell sortor can be used to sort out the cells in the cloning. The cloning may also be used for separating possible variant cells occurring during the long culture and for retaining the cells having the same properties as the original hybridoma.

The hybridoma of the present invention in the logarithmic growth phase can be maintained for a long period in the freezed form of 1-10 X 106 per ml suspended in a fetal bovine serum containing 5% (V/V) dimethylsulfoxide. The freezing procedure is preferably carried out in a cooling rate of - 1 'C per minute. The storage of the hybridoma is preferable at - 80"C or lower.

The freezed, stored hybridoma is preferably thawed as rapidly as possible. If the cells are washed by a medium to remove dimethylsulfoxide immediately after dissolution, the cells can be suspended in a conventional medium as it is and cultured. When only a small amount of the cells are surviving and growing after thawing, mouse spleen cells etc. should be added.

D. PRODUCTION AND RECOVERY OF ANTIBODY For production of antibody, the hybridoma producing an anti-bladder cancer antibody is cultured in vitro or in vivo.

In in vitro culture an appropriate nutrient broth for the hybridoma of the present invention may be chosen, for example, RPMI 1640 medium containing 10% (V/V) fetal bovine serum, 5 x 10-5 M ssmercaptoethanol, 1 mM sodium pyruvate and antibiotics, or, Dulbecco's modified Eagle's MEM (hereinafter referred to as D-MEM) containing 4.5 g/L glucose instead of RPMI 1 640 medium. An appropriate initial cell concentration for proliferation may generally be approximately 105 per ml while this may depend on each hybridoma, and the cell concentration during the culture is preferably upto 2 X 106 per ml.

In in vivo culture, the hybridoma is transplanted to a living body and grown in the solid form or in ascites. A body fluid, preferably serum or ascites, is taken out of the living body for recovering the antibody secreted by the hybridoma. The obtained crude solution of the antibody may contain as impurities (contaminants) various substances originating from the host living body, nevertheless it is more remarkable than the antibody solution obtained in vitro because of a higher concentration of the desired antibody. When the hybridoma is transplanted intraperitoneally, pristane (2,6,10,1 4-tetramethylpentadecane) can be intraperitoneally administered prior to transplantation, preferably 3-9 weeks before the transplatation. This treatment can enhance the yield of the crude antibody solution but not essential.As a host, an animal of the same species and the same inbreeding as the animal from which the parent cell or cells is/are derived is preferable and in this case the hybridoma can grow in the animal even if not specially treated.

When the serotypes of the histocompatibility of antigens coincide with each other between the hybridoma and the host, the host is necessarily preliminarily treated with for example an administration of anti-lymphocyte antibody or irradiation of X rays. The cells begin to grow from 1-3 weeks after the transplantation.

On the in vitro or in vivo culture of the hybridoma to secrete the antibodies, a radioactive substance such as radio-isotope-labelled leucine and lysine may be added to the medium or administered to the host. Such treatment can give an antibody having the same chemical structure as non-labelled antibody and containing the radioactive substance in a molecule.

The antibodies of the invention can be used as it is in the crude antibody solution, or they can be purified for use by any conventional method for immunoglobulin, for example, ammonium sulfate fractionation or ion exchange chromatography, or by affinity chromatography with protein A or antigen. Such purified antibodies may be used independently or in a mixture thereof.

According to the method for preparation of the anti-bladder cancer antibody of the present invention, the defect attached with the conventional methods can substantially be overcome.

The hybridoma of the invention can be subcultured and substantially permanently proliferated both in vitro and in vivo. Further, the hybridoma can produce an antibody to a specific antigenic determinant. The produced antibody, therefore, may have a monoclonal specificity, is an antibody to the surface antigen of a urinary bladder cancer cell and consists essentially of a single molecular species. The method of the present invention permits the production of a necessary amount of the antibody depending on demand, the avoidance of scatter between lots and further the preparation of a solution containing the antibody with a high titer. In addition, the method does not require a troublesome absorption which has conventionally been inevitable.

On the other hand, non-purified antigen such as a cell per se of the bladder cancer can be used without difficulty in the method and even in such a case the highly specific antibody can be obtained. Such a high reactivity with an antigen of the antibody permits the rapid identification of a bladder cancer cell with a high reliability without the conventional troublesome procedures.

Moreover, since the purity of the antibody is high, allergic reactions due to contaminants inevitably contained in the conventional preparation seldom appear, permitting the use as a remedy for bladder cancer.

The method of classification and/or identification of the present invention can be applied to a subject from any source. For example, clinical materials can be used such as urine, lymphocyte or other bioptic tissue obtained from a patient who may possibly suffer from urinary bladder cancer in the clinical view point.

In the identification, a reagent containing the antibody of the present invention is contacted with a subject containing bladder cancer cells. It may conveniently utilize immunofluorescence microscopy, immunoelectromicroscopy, radioactive binding assay, enzyme immunoassay, etc. In a direct immunofluorescence microscopy, the antibody of the invention may conveniently be used after labeiling it with a fluorescent dye such as fluorescein and Rhodamine. The other convenient form of the antibody of the invention may be one labelled with a marker such as ferritin for an immunoelectromicroscopy, one labelled with a radioisotope such as 1251 and 1311 for a radioactive binding assay or one labelled with an enzyme such as peroxidase, alkaline phosphatase and ss-galactosidase for an enzymeimmunoassay.Of course, an indirect method can be performed with the aid of a secondary antibody or its binding product as a substitute, for example a biotin-labelled anti-human bladder cancer antibody can be used together with avidin as a secondary antibody. Also, a part of the antibody obtained by restrictive cleavage with a chemical and/or enzyme treatment such as F(ab')2 can be used instead of the antibody per se.

Such various derivatives and restriction products are useful for the method of classification and/or identification of the invention and therefore included within the scope of the present invention.

The antibody, its derivatives and restriction products may be mixed for use if necessary.

Furthermore, the reagent for classification and/or identification of the invention may further contain a carrier or a diluent conventionally used.

The present invention also relates to a curing agent (remedy) for treatment of urinary bladder cancer.

The curing mechanism of the antibodies of the invention may be considered to accelerate a complement fixation reaction and attack of macrophage and/or other immunocytes to the cancer cells as the administered antibody binds with the surface antigen of the cancer cell.

The antibody of the present invention may be independently administered per se or in a mixture thereof. When the antibody is chemically combined with an anticancer agent such as mitomycin and doxorubicin hydrochloride or a toxin such as ricin, more advantages may be effected. In this case, the mechanism may be considered that the anticancer agent or the toxin may attack cancer cells as the administered antibody binds with the surface antigen of the cancer cell, resulting in lower toxicity of the anticancer agent or the toxin than used alone. A part of the antibody obtained by restrictive cleavage with a chemical or enzyme treatment, for example F(ab')2, can also be used for the curing agent of the invention instead of the antibody per se. In this case a possibility can be avoided that the tissue of a host may be affected by a damage due to non-specific complement fixation reaction etc.These derivatives (combination products with an anticancer agent or a toxin) and restriction products may be used either independently or in a mixture thereof as the curing agent of the present invention.

For evaluating the acute toxicity of the antibodies, its derivatives or restriction products, the curing agent of the invention was administered to three groups (each of 10 animals) of ICR mice; first in an amount of 2 g/kg perorally, second in an amount of 400 mg/kg intraperitoneally and third in an arnount of 200 mg/kg intravenously, and no death was observed for 14 days. The curing agent of the invention may therefore be safe as a remedy for human urinary bladder cancer.

The curing agent of the invention can be administered subcutaneously, intramuscularly or intravenously, preferably by subcutaneous or intramuscular injection. The curing agent can perorally be administered since a part of the administered agent can be absorbed through the intestinal tract while retaining the structure as an antibody, which has been confirmed by the present inventors.

A preparation for injection can be prepared by for example dissolving or suspending 10 mg of the antibody or its derivative together with 50 mg of mannitol in distilled water to 10 ml, sterilizing by any conventional way, dividing into ampoules of 2 ml in each and freeze-drying.

The obtained product will be dissolved or suspended in saline on use. An injective preparation may contain in addition to the antibody a carrier, a diluent, a buffer, a stabilizer, an isotonic agent, etc., these agents being known to those skilled in the art. The injectable preparation can be prepared in any form of subcutaneous, intramuscular or intravenous injection. An orally administrable agent may be prepared by any conventional method into an enteric drug. A dose rate of the curing agent of the invention may depend mainly on the symptoms, and generally 0.001 mg to 10 g a day per kg of body weight in animal such as mouse and 0.01-3,000 mg a day per kg of body weight in human.

The following Examples illustrate the invention. All five human cell lines in Table 1 are available to the public.

EXAMPLE 1: PREPARATION OF ANTIBODY TO HUMAN BLADDER CANCER CELL A. IMMUNIZATION AND CELL FUSION (1) PREPARATION OF IMMUNOGEN CELL: Human bladder transitional epithelium cancer cell line T24 (2 X 105), subcultured in vitro for a long period, was incubated at 37"C under wet environment containing 5% CO2 in Eagle's MEM Earle's salt medium (hereinafter referred to as MEM) containing 10% (V/V) fetal bovine serum and kanamycin sulfate (final concentration, 60 mg/L) in a 100 mm culture dish (Falcon 3003, Becton-Dickinson, USA). After three days culture, the cells were exfoliated by policeman and the supernatant was removed by centrifugation. The cells were then washed with phosphate buffered saline (PBS, pH 7.2) by centrifugation and suspended in PBS. Approximately 2 X 106 cells were obtained from one culture dish.

(2) CULTURE OF MYELOMA: Mouse myeloma cell line Sp2/0-Ag14 (105 per ml) was cultured in D-MEM containing 10% (V/V) fetal bovine serum, 1 mM pyruvic acid, 2 mM giutamine and 60 mg/L kanamycin sulfate by subculture every three days. The cell was adjusted to a cell concentration of 2.5 X 105 per ml on the day before the subsequent cell fusion and cultured in the aforementioned medium.

(3) IMMUNIZATION WITH T24 CELLS: T24 cells (5 X 105-10 X 106) obtained in (1) above and suspended in 0.4 ml PBS was intraperitoneally injected to female BALB/c mice (NIHON CHARLES LIVER, JAPAN, 9 weeks old) four times approximately every one month for immunization.

(4) CELL FUSION: Cell fusion was performed according to the method of Köhler and Milstein (Immunoassays; Clinical Laboratory Techniques for the 1980's ed. by Nakamura R. M. et al., E.

S. Alan R. Liss, Inc., N. Y., 1980, pp 301-324).

On the fourth day after the final immunization, mice were slaughtered to take out spleen. The spleen was well unfastened, passed through a 1 50 mesh stainless mesh, centrifuged at 400 X g and tris-HCI buffer (0.01 7 M tris(hydroxymethyl)-aminomethane, pH 7.65) containing 0.747% ammonium chloride was added to the precipitated cells to remove erythrocytes and the cells were collected by centrifugation (400 X g). After adding MEM to the cells, the centrifugation was performed at 400 X g and the cells were resuspended in fresh MEM. After repeating this washing procedure three times, the cells were finally suspended in MEM.

Sp2/0-Ag14 cells were exfoliated from the culture vessel by pipetting and transferred to a centrifuge tube. After centrifugation (400 X 9), the collected cells were suspended in MEM, centrifuged (400 x g) to remove serum and resuspended in MEM.

The spleen cell (5 X 107) and Sp2/0-Ag14 (107) were mixed, well pipetted and centrifuged (400 x g). After discarding the supernatant, the precipitate was unfastened by gently tapping the centrifuge tube. To the unfastened cells was added 0.3 ml of a solution of 30% (V/V) polyethylene glycol (PEG 1000) in MEM maintained at 37"C. After stirring gently, the solution was allowed to stand at room temperature for 5 minutes. Centrifugation was performed at 7 X g for 2 minutes and 5 ml of MEM was gradually added. After stirring gently, centrifugation was performed (400 X 9) at room temperature for 5 minutes. The supernatant was discarded, 5 ml MEM was added to the precipitate, centrifuged in a similar manner to those aforementioned and the supernatant was discarded.

To the precipitated cells was added 5 ml of D-MEM medium containing 10% (V/V) fetal bovine serum, 2 mM glutamine, 5 X 10-5 M ssmercaptoethanol, 60 mg/L kanamycin sulfate and further 4.5 g/L glucose (hereinafter referred to as D-MEM-FBS medium). After pipetting, 20 ml of D-MEM-FBS was added to the resultant cell suspension (5 ml). Each 25 ml of the suspension was inoculated in 25 cm2 culture flask (C-25100, Corning, USA) and incubated while standing in wet environment containing 5% CO2 at 37"C overnight, which incubating conditions were always used in the following procedures.

On the next day after inoculation the culture was transferred to a centrifuge tube after slightly pipetting, and centrifuged at 400 X g. After discarding the supernatant, the cell pellet was suspended in 35 ml of D-MEM-FBS containing 1 x 10-4 M hypoxanthine, 4 X 10-7 M aminopterin and 1.6 x 10-5 M thymidine (HAT medium). The suspension (0.1 ml) was placed into each well of 96 well plate (Falcon 3072, Becton-Dickinson, USA) and cultured for a week.

After one week culture in HAT medium, 25 LI of HT medium (HAT medium without aminopterin) was added every 2 or 3 days.

B. SELECTION AND GROWTH OF ANTIBODY PRODUCING HYBRIDOMA A solid-phase enzyme immunoassay was used to examine the production of antibody to T24 cells in the supernatant of each well in the second week after the cell fusion.

T24 cells fixed on a plate (Falcon 3072) were reacted with 40 tl of the supernatant in each well at room temperature for two hours, thoroughly washed with PBS, reacted for two hours with 100 ILl peroxidase-bound anti-mouse immunoglobulin antibody (Catalogue No.

3211-0231, Cappel Lab. Inc., USA) which was diluted 1000-fold with horse serum, and thoroughly washed with PBS. Two hundreds ,ul of citrate buffer (0.1 M, pH 4.5) containing 1 mg/ml of a substrate (o-phenylenediamine) and 0.4,ul/ml of 31% aqueous hydrogen peroxide were added and reacted for 30 minutes to color.

Cloning was performed on three wells containing the antibodies reacting with T24 cell by limiting dilution method as follows: Hundred hybridomas from each of the three wells positive in antibody production were suspended in D-MEM-FBS and mixed with spleen cell obtained from BALB/c mouse in the same manner as used in the cell fusion and adjusted in D-MEM-FBS (1o8 cells). The cell concentration was adjusted to 5 X 106 per ml by adding D-MEM-FBS and the cell mixture was inoculated into each well of 96 well plate (Falcon 3072) in each amount of 0.2 ml and cultured.

On the 13th day after the initiation of the culture, the antibody production was examined on each well by the aforementioned solid-phase enzyme immunoassay. 33 hybridomas producing antibody were obtained including six hybridomas shown in Table 1 below.

EXAMPLE 2: PROPERTIES OF ANTIBODY TO HUMAN BLADDER CANCER CELL A. COMPARISON OF REACTIVITY WITH OTHER CELLS The solid-phase enzyme immunoassay of Example 1 was used to examine the reactivities of the supernatants obtained in Example 1 with other cells subcultured in vitro; Intestine 407 (fetal human intestine cell line), Du 145 (human prostata cancer cell line) and 253J (human renipelvic cancer cell line) as well as T24 (human urinary bladder cancer cell line). As seen from Table 1 below, antibodies secreted by the hybridomas other than T-002 strongly reacted with T24 and slightly or weakly reacted with the other cells, while the antibody secreted by the hybridoma T-002 reacted with T24, weakly reacted with fetal intestine cell and did not react with the other cells.

B. STAINING OF CELL BY FLUORESCENT ANTIBODY TECHNIQUE T24 cells were stained by indirect fluorescent antibody technique using the antibody secreted by the hybridoma T-003. Namely, T24 cells were fixed on a glass slide without fluorescence, reacted with the supernatant (50 it1) at 37 C for 45 minutes under wet circumstance and washed by treating for 3 to 5 minutes with PBS solution containing 1 % bovine serum albumin, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 0.1% sodium azide, this washing procedure being repeated three times.The reaction was further performed in the same conditions for 45 minutes with 50 pl of 20-fold diluted solution of fluorescein-bound antimouse IgG (Miles-Yeda Ltd., Israel, Code No. 65-171) and the washing procedure was carried out in the same manner as aforementioned. After drying, carbonate buffered glycerin solution (0.05 M, pH 9.5, 10% glycerin) was stacked and a cover glass was placed on to observe by a fluorescence microscope (Olympus, Japan, Model AH-RFL-LB). Strong fluorescence was observed on the surface of T24 cell. The fluorescein-bound anti-mouse lgG was used after subjecting to absorption with T24 cell six times.

C. IDENTIFICATION OF CLASSES OF IMMUNOGLOBULIN The classes of the immunoglobulins of the antibodies secreted by the hybridomas T-001 to T-006 were identified by the solid-phase enzyme immunoassay using anti-immunoglobulin (Ig), anti-lgG and anti-lgM (Cappel Lab. Inc., USA, Catalogue Nos. 3211-0231, 3211-0081, 3211-0201) as peroxidase-bound anti-mouse immunoglobulin antibody. As seen from Table 1 below all anti-bodies examined were IgG.

EXAMPLE 3: PREPARATION OF ANTIBODY TO HUMAN BLADDER CANCER CELL A. IMMUNIZATION AND CELL FUSION (1) PREPARATION OF IMMUNOGEN CELL: Human urinary bladder transitional epithelium cancer cell line T24 (2 X 105), subcultured in vitro for a long period, was incubated at 37"C under wet environment containing 5% carbon dioxide in MEM medium containing 10% (V/V) fetal bovine serum and kanamycin sulfate (final concentration, 60 mg/L) in a 100 mm culture dish (Falcon 3003, Becton-Dickinson, USA). After three days culture, the cells were ex-foliated by policeman and the supernatant was removed by centribugation. The cells were then washed with PBS (pH 7.2) by centrifugation and suspended in PBS. Approximately 2 X 106 cells were obtained from one culture dish.

(2) CULTURE OF MYELOMA: Mouse myeloma cell line Sp2/0-Ag14 (105 per ml) was cultured in D-MEM containing 10% (V/V) fetal bovine serum, 1 mM pyruvic acid, 2 mM glutamine and 60 mg/L kanamycin sulfate by subculture every three days. The cell was adjusted to a cell concentration of 2.5 x 105 per ml on the day before the subsequent cell fusion and cultured in the aforementioned medium.

(3) IMMUNIZATION WITH T24 CELLS: T24 cells (5 X 106-10 X 106) obtained in (1) above and suspended in 0.4 ml PBS was intraperitoneally injected to female BALB/c mice (NIHON CHARLES LIVER, JAPAN, 9 weeks old) four times approximately every one month for immunization.

(4) CELL FUSION: Cell fusion was performed according to the method of Köhler and Milstein.

On the fourth day after the final immunization, mice were slaughtered to take out spleen. The spleen was well unfastened, passed through a 1 50 mesh stainless mesh, centrifuged at 400 X g and tris-HCI buffer (pH 7.65) containing 0.747% ammonium chloride was added to the precipitated cells to remove erythrocytes and the cells were collected by centrifugation (400 X 9). After adding MEM to the cells, the centrifugation was performed at 400 X g and the cells were resuspended in fresh MEM. After repeating this washing procedure three times, the cells were finally suspended in MEM.

Sp2/0-Ag14 cells were exfoliated from the culture vessel by pipetting and transferred to a centrifuge tube. After centrifugation (400 X g), the collected cells were suspended in MEM, centrifuged (400 X g) to remove serum and resuspended in MEM.

The spleen cells (2 X 108) and Sp2/0-Ag14 cells (4 X 107) were mixed, well pipetted and centrifuged (400 X g). After discarding the supernatant, the precipitate was unfastened by gently tapping the centrifuge tube. To the unfastened cells was added 1.2 ml of a solution of 30% (V/V) polyethylene glycol (PEG 1000) in MEM maintained at 37"C. After stirring gently, the solution was allowed to stand at room temperature for 5 minutes. Centrifugation was performed at 7 X g for 2 minutes and 20 ml of MEM was gradually added. After stirring gently, centrifugation was performed (400 X g) at room temperature for 5 minutes. The supernatant was then discarded, 20 ml of MEM was added to the precipitate, centrifuged in a similar manner and the supernatant was discarded.

To the precipitated cells was added 1 5 ml of D-MEM-FBS medium. After pipetting, 90 ml of D-MEM-FBS was added to the resultant cell suspension (15 ml). Approximately 25 ml in each of the cell suspension was inoculated in 25 cm2 culture flask (Corning, USA, C-25100) and incubated while standing in wet environment containing 5% CO2 at 37"C overnight, this incubation conditions being used in the following procedures.

On the next day, the culture was slightly pipetted, transferred to a centrifuge tube and centrifuged at 400 X g. After discarding the supernatant, the cell pellet was suspended in 100 ml of HAT medium. Each 0.1 ml of the cell suspension was inoculated in each well of 96 well culture plate (Falcon 3072, Becton-Dickinson, USA) and cultured for one week. After culture in HAT medium for another week, 25 elm of HT medium was added every 2 or 3 days.

B. SELECTION AND GROWTH OF ANTIBODY PRODUCING HYBRIDOMA A solid-phase enzyme immunoassay was used to examine the production of antibody to T24 cells in the supernatant of each well in the second week after the cell fusion.

T24 cells fixed on a plate (Falcon 3072) were reacted with 40 yl of the supernatant in each well at room temperature for two hours, thoroughly washed with PBS, reacted for two hours with 100 elm peroxidase-bound anti-mouse immunoglobulin antibody (Cappel Lab. Inc., USA, Catalogue No. 3211-0231) which was diluted 1000-fold with horse serum, and thoroughly washed with PBS. Two hundreds ,ul of citrate buffer (0.1 M, pH 4.5) containing 1 mg/ml o- phenylenediamine and 0.4,lli/ml aqueous hydrogen peroxide were added and reacted for 30 minutes to color.

Cloning was performed on three wells containing the antibodies reacting with T24 cells by limiting dilution method. Namely, 100 hybridomas from each of the three wells positive in antibody production were suspended in D-MEM-FBS and mixed with spleen cell obtained from BALB/c mouse in the same manner as used in the cell fusion and adjusted in D-MEM-FBS (108 cells). The cell concentration was adjusted to 5 X 106 per ml by adding D-MEM-FBS.

Each 0.2 ml of the cell mixture was inoculated in each well of 96 well culture plate (Falcon 3072) and cultured. On the 14th day after the initiation of the culture, the antibody production was examined on each well by the aforementioned solid-phase enzyme immunoassay. Total 1 6 hybridomas including T-OO9 to T-012 shown in Table 1 below were obtained.

EXAMPLE 4: PROPERTIES OF ANTIBODY TO HUMAN BLADDER CANCER CELL A. COMPARISON OF REACTIVITY WITH OTHER CELLS The solid-phase enzyme immunoassay of Example 1 was used to examine the reactivities of the supernatants obtained in Example 3 with other cells subcultured in vitro; Intestine 407 (fetal human intestine cell line), K-562 (human leukemia cell line) and 253J (human renipelvic cancer cell line) as well as T24 (human urinary bladder cancer cell line). As seen from Table 1 below, the antibody secreted by the hybridoma T-010 was highly specific, i.e. strongly reacted with T24 while scarcely reacted with other cells. The other hybridomas showed similar properties.

B. STAINING OF CELL BY FLUORESCENT ANTIBODY TECHNIQUE T24 cells were stained by indirect fluorescent antibody technique using the antibody secreted by the hybridoma T-010. Namely, T24 cells were fixed on a glass slide without fluorescence, reacted with the supernatant (50 it1) at 37"C for 45 minutes under wet circumstance and washed by treating for 3 to 5 minutes with PBS containing 1% bovine serum albumin, 10 mM HEPES and 0.1% sodium azide, this washing procedure being repeated three times. The reaction was further performed in the same conditions for 45 minutes with 50 yl of 20-fold diluted solution of fluorescein-bound anti-mouse IgG (Miles-Yeda Ltd., Israel, Code No. 65-171) and the washing procedure was carried out in the same manner as aforementioned.After slightly drying, carbonate buffer (0.05 M, pH 9.5) containing 10% glycerin was stacked and a cover glass was placed on to observe by a fluorescence microscope (Olympus, Japan, Model AH-RFL-LB). Strong fluorescence was observed on the surface of T24 cell. The fluoresceinbound anti-mouse IgG was used after subjecting to absorption with T24 cell six times.

C. IDENTIFICATION OF CLASSES OF IMMUNOGLOBULIN The classes of the immunoglobulins of the antibodies secreted by the hybridomas T-OO9 to T-012 were identified by the solid-phase enzyme immunoassay using anti-immunoglobulin (Ig), anti-lgG and Anti-lgM (Cappel Lab. Inc., USA, Catalogue Nos. 3211-0231, 3211-0081, 3211-0201) as peroxidase-bound anti-mouse immunoglobulin antibody. The results are shown in Table 1 below.

EXAMPLE 5: PREPARATION OF ANTIBODY TO HUMAN BLADDER CANCER CELL A. IMMUNIZATION AND CELL FUSION (1) PREPARATION OF IMMUNOGEN CELL: Human urinary bladder transitional epithelium cancer cell line T24 (2 X 105), subcultured in vitro for a long period, was incubated at 37C under wet environment containing 5% carbon dioxide in MEM medium containing 10% (V/V) fetal bovine serum and kanamycin sulfate (final concentration, 60 mg/L) in a 100 mm culture dish (Falcon 3003, Becton-Dickinson, USA). After three days culture, the cells were exfoliated by policeman and the supernatant was removed by centrifugation. The cells were then washed with PBS (pH 7.2) by centrifugation and suspended in PBS. Approximately 2 X 106 cells were obtained from one culture dish.

(2) CULTURE OF MYELOMA: Mouse myeloma cell line Sp2/0-Ag14 (105 per ml) was cultured in D-MEM containing 10% (V/V)fetal bovine serum, 1 mM pyruvic acid, 2 mM glutamine and 60 mg/L kanamycin sulfate by subculture every three days. The cell was adjusted to a cell concentration of 2.5 X 105 per ml on the day before the susequent cell fusion and cultured in the aforementioned medium.

(3) IMMUNIZATION WITH T24 CELLS: T24 cells (5 X 106-10 X 106) obtained in (1) above and suspended in 0.4 ml PBS was intraperitoneally injected to female BALB/c mice (NIHON CHARLES LIVER, JAPAN, 9 weeks old) four times approximately every one month for immunization.

(4) CELL FUSION: Cell fusion was performed according to the method of Köhler and Milstein.

On the fourth day after the final immunization, mice were slaughtered to take out spleen. The spleen was well unfastened, passed through a 1 50 mesh stainless mesh, centrifuged at 400 X 9, and tris-HCI buffer (pH 7.65) containing 0.747% ammonium chloride was added to the precipitated cells to remove erythrocytes and the cells were collected by centrifugation (400 X g). After adding MEM to the cells, the centrifugation was performed at 400 X g and the cells were resuspended in fresh MEM. After repeating this washing procedure three times, the cells were finally suspended in MEM.

Sp2/0-Ag14 cells were exfoliated from the culture vessel by pipetting and transferred to a centrifuge tube. After centrifugation (400 X g), the collected cells were suspended in MEM, centrifuged (400 X g) to remove serum and resuspended in MEM.

The spleen cells (8.5 X 107) and Sp2/0-Ag14 cells (1.7 X 107) were mixed, well pipetted and centrifuged (400 X g). -After discarding the supernatant, the precipitate was unfastened by gently tapping the centrifuge tube. To the unfastened cells was added 0.6 ml of a solution of 30% (V/V) polyethylene glycol (PEG 1000) in MEM maintained at 37"C. After stirring gently, the solution was allowed to stand at room temperature for 5 minutes. Centrifugation was performed at 7 X g for 2 minutes and 5 ml of MEM was gradually added. After stirring gently, centrifugation was performed (400 X g) at room temperature for 5 minutes. The supernatant was discarded, 5 ml of MEM was added to the precipitate, centrifuged in a similar manner and the supernatant was discarded.

To the precipitated cells was added 5 ml of D-MEM-FBS medium. After pipetting, 30 ml of D-MEM-FBS was added to the resultant cell suspension (5 ml). Each 25 ml of the suspension was inoculated in 25 cm2 culture flask (Corning, USA, C-25100) and incubated while standing in 5% CO2 containing wet circumstance at 37"C overnight, this incubation conditions being used in the following procedures; On the next day, the culture was slightly pipetted, transferred to a centrifuge tube and centrifuged at 400 X g. After discarding the supernatant, the pellet was suspended in 35 ml of HAT medium. Each one ml of the suspension was inoculated in each well of 24 well culture plate (Nunc 143982, Nunc, Denmark) and cultured for one week. To each well was added 0.25 ml of HT medium every two or three days.

B. SELECTION AND GROWTH OF ANTIBODY PRODUCING HYBRIDOMA A solid-phase enzyme immunoassay was used to examine the production of antibody to T24 cells in the supernatant of each well on the 1 5th day after the cell fusion.

T24 cells fixed on a plate (Falcon 3072) were reacted with 40 ,ul of the supernatant in each well at room temperature for two hours, thoroughly washed with PBS, reacted for two hours with 100 pl peroxidase-bound anti-mouse immunoglobulin antibody (Cappel Lab. Inc., USA, Catalogue No. 3211-0231) which was diluted 1000-fold with horse serum, and thoroughly washed with PBS. Two hundreds jul of citrate buffer (0.1 M, pH 4.5) containing 1 mg/ml ophenylenediamine as a substrate and 0.4 yI/ml aqueous hydrogen peroxide were added and reacted for 30 minutes to color.

Cloning was performed on two wells containing the antibodies reacting with T24 cells by limiting dilution culture. Namely, 100 hybridomas from each of the two wells positive in antibody production were suspended in D-MEM-FBS and mixed with spleen cell obtained from BALB/c mouse in the same manner as used in the cell fusion and adjusted in D-MEM-FBS (108 cells). The cell concentration was adjusted to 5 x 106 per ml by adding D-MEM-FBS.

Each 0.2 ml of the cell mixture was inoculated in each well of 96 well culture plate (Falcon 3072, Becton-Dickinson, USA) and cultured. On the 1 2th day after the initiation of the culture, the antibody production was examined on each well by the aforementioned solid-phase enzyme immunoassay. Total 10 hybridomas including T-007 and T-008 shown in Table 1 below were obtained.

EXAMPLE 6: PROPERTIES OF ANTIBODY TO HUMAN BLADDER CANCER CELL A. COMPARISON OF REACTIVITY WITH OTHER CELLS The solid-phase enzyme immunoassay of Example 1 was used to examine the reactivities of the supernatants obtained in Example 5 with other cells subcultured in vitro; Intestine 407 (fetal human intestine cell line), Du 145 (human prostata cancer cell line) and 253J (human renipelvic cancer cell line) as well as T24 (human urinary bladder cancer cell line). As seen from Table 1, the antibody secreted by the hybridoma T-007 strongly reacted with T24 and showed a slight or relatively high reactivities with other cells. The antibody secreted by T-008 strongly reacted with T24 and did not react with Intestine 407 nor with 253J.

B. STAINING OF CELL BY FLUORESCENT ANTIBODY TECHNIQUE T24 cells were stained by indirect fluorescent antibody technique using the antibody secreted by the hybridoma T-008. Namely, T24 cells were fixed on a glass slide without fluorescence, reacted with the supernatant (50 ,ul) at 37"C for 45 minutes under wet circumstance and washed by treating for 3 to 5 minutes with PBS containing 1% bovine serum albumin, 10 mM HEPES and 0.1% sodium azide, this washing procedure being repeated three times. The reaction was further performed in the same conditions for 45 minutes with 50 yl of 20-fold diluted solution of fluorescein-bound anti-mouse IgG (Miles-Yeda Ltd., Israel, Code No. 65-171) and the washing procedure was carried out in the same manner as aforementioned.After slightly drying, carbonate buffer (0.05 M, pH 9.5) containing 10% glycerin was stacked and a cover glass was placed on to observe by a fluorescence microscope (Olympus, Japan, Model AH-RFL-LB). Strong fluorescence was observed on the surface of T24 cell. The fluoresceinbound anti-mouse IgG was used after subjecting to absorption with T24 cell six times.

C. IDENTIFICATION OF CLASSES OF IMMUNOGLOBULIN The classes of the immunoglobulins of the antibodies secreted by the hybridomas T-007 and T-008 were identified by the solid-phase enzyme immunoassay using anti-immunoglobulin (Ig), anti-lgG and Anti-lgM (Cappel Lab. Inc., USA, Catalogue Nos. 3211-0231, 3211-0081, 3211-0201) as peroxidase-bound anti-mouse immunoglobulin antibody. The results are shown in Table 1 below.

TABLE 1 SPECIFICITY Hy- Human Human Human Human Human Class bridoma Bladder Fetal Prostata Renipel- Leukemia of Cancer Intes- Cancer vic Immuno tine Cancer globulin T-24 1-407 Du-145 253-J K-562 T-O01 + + + + + - IgG T-002 + + + - lgG T-003 +++ + ++ + IgG T-004 +++ ++ + lgG T-005 +++ ++ + + + ++ IgG T-006 + + + + + - IgG T-007 ++++ ++ ++ +++ IgG T-008 ++++ - ++ - IgG T-0O9 ++++ - + + IgM T-010 + + + 9 + - IgG T-011 ++++ + + + IgG T-0,12 ++++ + ++ - IgG +: positive - : negative EXAMPLE 7: PRODUCTION OF ANTIBODY (IN VITRO CULTURE): the hybridoma T-001, T-002, T-003, T-004, T-005, T-006, T-007, T-008, T-O09, T-010, T-011 or T-012 was suspended in D-MEM containing 20% fetal bovine serum, 2 mM glutamine, 1 mM pyruvic acid, 4.5 g/L glucose, 5 X 10-5 p- mercaptoethanol and 50 mg/L kanamycin sulfate, in a cell concentration of 1 x 105 per ml. The resultant suspension (25 ml) was inoculated in 75 cm2 flask for tissue culture (Corning, USA) and incubated in a 5% CO2 incubator at 37 C.On the fourth day, the supernatant in the stationary growth state was collected from the flask. The numbers of the grown cells were approximately 2 X 106 per ml in each hybridoma and the antibody contents of the supernatants were 1.9 (T-001), 3.3 (T-002), 2.3 (T-003), 2.6 (T-004), 2.7 (T-005), 2.1 (T-006), 3.3 (T-007), 2.0 (T-008), 2.1 (T-0O9), 3.1 (T-010), 4.0 (T-011) and 2.3 ILg/ml (T-012), respectively.

(IN VIVO CULTURE): On the 10th to 30th day after intraperitoneal injection of 0.5 ml of pristane, 5 X 106 of each hybridoma T-001 to T-012, grown in vitro, was intraperitoneally inoculated to the BALB/c mice. After two or three weeks, ascites was collected and centrifuged 1000 X g, 4 C, 15 minutes to obtain the ascitic supernatant in the amount of approximately 30 ml in each hybridoma from ten mice. The antibody contents of the supernatants were 1.1 (T-001), 1.3 (T-002), 2.6 (T-003), 2.3 (T-004), 2.1 (T-005), 1.7 (T-006), 2.0 (T-007), 3.1 (T-008), 2.0 (T-0O9), 1.4 (T-O1 0), 2.2 (T-011) and 3.0 mg/ml (T-012), respectively.

These antibodies were administered to ICR mice (10 animals in each group) in an amount of 2 g/kg perorally, 400 mg/kg intraperitoneally or 200 mg/kg intravenously and the mice were observed for 14 days. No death due to the antibody was observed at all.

On the other hand, the shapes of the hybridomas T-001 to T-012 were approximately spherical, the dimensions thereof were 10-20 IL with almost being approximately 15 , and the hybridomas were floating and weak in adhesion to vessel wall.

EXAMPLE 8: IDENTIFICATION OF HUMAN BLADDER CANCER BY ANTIBODY (1) SUBJECT: A tissue containing human urinary bladder cancer cells obtained in a surgical operation was fixed with 95% alcohol, embedded in paraffin to prepare a sample piece of 4 m.

(2) IDENTIFICATION: The identifications were carried out by a fluorescent antibody technique using antibodies (ascitic supernatant) secreted by the hybridoma T-003.

Namely, O.1 ml of the 100-fold dilution of the aforementioned antibody solution was added to the tissue sample and incubated at room temperature for one hour. After washing thorougly with PBS, 0.1 ml of the 100-fold diluted fluorescein-bound anti-mouse IgG (Miles, USA) was added and incubated at room temperature for one hour. After removing unreacted fluoresceinbound anti-mouse IgG by washing with PBS, fluorescence was observed by a fluorescence microscope (Olympus Vanox, Olympus, Japan) on each subject tissue sample of the slide.

Almost no fluorescence was observed on normal tissue while strong fluorescence was observed on the tissue of bladder cancer. In order to prevent non-specific binding, the fiuorescein-bound anti-mouse IgG had preliminarily treated by absorption with human bladder cancer cell line T24.

EXAMPLE 9: (1) PEROXIDASE LABELLING OF ANTIBODY SECRETED BY HYBRIDOMA T-006 Four mg of horseradish peroxidase (Sigma Type VI, Sigma, USA) was dissolved in 1 ml distilled water and 60 it1 of 0.1 M NalO4 solution, prepared immediately before use, was added and mixed at room temperature for 20 minutes. The resultant solution was dialysed overnight against 1 mM sodium acetate buffer (pH 4.4) and 20 yl of 0.2 M solution carbonate was added. Soon after, the antibody secreted by the hybridoma T-006 (purified ascitic supernatant, protein content of 10 mg) dissolved in 1 ml of 0.01 M carbonate buffer (pH 9.5) was added and maintained at room temperature for two hours while stirring.After reaction, 0.1 ml of freshly prepared aqueous NaBH4 solution (4 mg NaBH4 dissolved in 1 ml distilled water) was added, allowed to stand at 4"C for two hours and dialysed overnight to PBS.

The resultant mixed solution was applied to a column of Sephadex G-1 00(E) (Pharmacia, Sweden), eluted with PBS and first fraction of which absorbances at 280 nm and 403 nm corresponded to each other was collected. To 1 ml of the fraction (enzyme-antibody binding product) was added 10 mg of rabbit serum albumin to dissolve and stored at - 70"C until use.

(2) ALKALINE PHOSPHATASE LABELLING OF ANTIBODY SECRETED BY HYBRIDOMA T-006 Five mg of alkaline phosphatase Type VII (Sigma, USA), preliminarily dialysed to PBS to completely remove ammonium sulfate, and 1 7 mg of antibody secreted by the hybridoma T-006 were dissolved in PBS to a total volume of 1 ml. To the resultant solution was added 10 ,ul of 20% glutaraldehyde solution and stirred at room temperature for two hours. After reaction, the reaction mixture was applied to a Sephadex G-20O column (Pharmacia, Sweden) equilibrated with tris-HCI buffer (pH 7.6) and eluted with the same buffer.A high molecular weight fraction from void volume to the IgG-eluted point was collected, bovine serum albumin was added to 5% (W/V), sterilized by passing a Millipore filter (0.22 , Millipore, USA) and stored at 4"C in dark until use.

(3) ss-GALACTOSlDASE LABELLING OF ANTIBODY SECRETED BY HYBRIDOMA T-006 The antibody secreted by the hybridoma T-006 was labelled with ss-galactosidase according to the method of (2) above. The ss-galactosidase used was Sigma grade IV (Sigma, USA).

(4)1251 LABELLING OF ANTIBODY SECRETED BY HYBRIDOMA T-006 To 10 jul solution of 100 mCi/ml Na'251 (carrier-free, Amersham, USA) was added 50 ,ul of the antibody solution secreted by the hybridoma T-006 (purified ascitic supernatant, protein content of 1.0 mg/ml) and 30 ,ul of 0.5 M phosphate buffer (pH 7.2) containing 0.30 mg/ml chloramine T and well mixed. After 1 5 seconds, 100 ,ul PBS saturated with L-tyrosine was added and immediately mixed. The resultant mixture was chromatographed on a column charged with Amberlite IRA 400 and eluted with PBS containing 1% bovine serum albumin.

The eluted fraction was collected and stored at 4"C until use. The specific activity of the labelled product was 1.0 yCi per mg antibody protein.

(5) FLUORESCEIN LABELLING OF ANTIBODY SECRETED BY HYBRIDOMA T-006 To 1 ml solution of the antibody secreted by the hybridoma T-006 (purified ascitic supernatant) in a concentration of 10 mg/ml, 0.1 ml of 0.5 M carbonate buffer (pH 9.3) was added, 0.1 mg of fluorescein isothiocyanate powder was further added and stirred at 4"C for 6 hours without bubbling. Immediately after reaction, the reaction mixture was applied to a Sephadex G-25 column (Pharmacia, Sweden) to remove unreacted low molecular weight substances and the high molecular weight fraction to be desired was obtained. The obtained product was stored at 4"C in dark until use.

(6) TETRAMETHYLRHODAMINE LABELLING OF ANTIBODY SECRETED BY HYBRIDOMA T-006 To 1 ml of 10 mg/ml solution of the antibody secreted by the hybridoma T-006 (purified ascitic supernatant), 0.1 ml of 0.5 M carbonate buffer (pH 9.3) was added, 0.2 mg tetramethylrhodamine-isothiocyanate powder was further added and stirred at 4"C for 20 hours without bubbling. After reaction the reaction mixture was immediately applied to a Sephadex G-25(g) column (Pharmacia, Sweden) to remove unreacted low molecular weight substances and the high molecular weight fraction to be desired was obtained. The product was stored at 4"C in dark until use.

(7) BIOTIN LABELLING OF ANTIBODY SECRETED BY HYBRIDOMA T-006 One mM d-biotin (244 mg) (Wako Pure Chemical Industries, Ltd., Japan) and 1.5 mM Nhydroxysuccinimide (173 mg) (Eastman Kodak, USA) were dissolved in a mixture of 8 ml dimethylsulfoxide (Wako Pure Chemical Industries, Ltd., Japan) and 5 ml 1,2-dimethoxyethane (Nakarai Chemical, Japan). To the resultant solution was added a solution of 206 mg (1 mM), N,N'-dicyclohexylcarbodimide (Kanto Chemical, Japan) in 0.5 ml 1,2-dimethoxyethane and reacted at 4"C overnight. The resultant precipitate was filtered out to obtain a filtrate.The solvent in the filtrate was removed under vacuum and the residual oily material was dissolved in 10 ml dichloromethane (Wako Pure Chemical Industries, Ltd., Japan) and cooled at 4"C. Ten ml of 0.1 M NaHCO3 solution (4"C) was added and shaked to well mix. The resultant dichloromethane phase was removed, 10 ml of 0.1 M NaHCO3 and then 10 ml of distilled water, 4"C, were added and these procedures were again repeated. The resultant dichloromethane phase was added with anhydrous powder of sodium sulfate (Koizumi Chemical, Japan) to dehydrate. The powder was filtered out and n-hexane was gradually added to the filtrate to be turbid. The solution was cooled to - 20"C, the precipitated crystal was placed in a desiccator to remove the solvent and dry.The product, biotin-N-hydroxysuccinimide ester was obtained.

The biotin-N-hydroxysuccinimide was dissolved in dimethylsulfoxide and the concentration was adjusted to 1 mg/ml. The solution (60it1) was mixed with 1 ml solution of the antibody secreted by the hybridoma T-006 (purified ascitic supernatant, protein content of 1 mg/ml) and reacted at room temperature for 4 hours. After reaction, the solution was dialysed to PBS at 4"C for three days. For this the dialysing liquid was exchanged three times. The dialysate (solution in the dialysis tube) was stored at 4"C until use.

EXAMPLE 10: IDENTIFICATION OF HUMAN BLADDER CANCER BY LABELLED ANTIBODIES (1) SUBJECT: Tissues containing human urinary bladder cancer were obtained in surgical operations. Namely, the raw tissues of 5 X 5 X 2 mm or less in dimensions were fixed while shaking at 4"C for 6 hours in a PLP fixation liquid (0.0375 sodium phosphate buffer, pH 6.2, containing 0.01 M NalO4, 0.075 M lysine and 3% paraformaldehyde).The fixed tissues were washed by treating with pBS containing 10% sucrose (4 C, overnight), PBS containing 15% sucrose (4"C, 6 hours), PBS containing 20% sucrose (4 C, 6 hours) and then PBS containing 20% sucrose and 10% glycerin (4"C, one hour), successively. After washing, the tissue was embedded, freezed in dry ice-ethanol and subjected to a cryostat to obtain a freezed specimen of 10 sum.

(2) IDENTIFICATION METHOD: The freezed specimen was placed on a slide glass coated with bovine serum albumin and dried. After washing with PBS at 4C for 5 minutes three times, the specimen was reacted with 0.005 M periodic acid at room temperature for 10 minutes. After washing three times with PBS maintained at 4"C, the specimen was treated at room temperature for 1Q minutes with PBS containing 10% horse serum. Ten-fold diluted solution of the antibody from T-006 labelled by peroxidase, alkaline phosphatase, fluorescein, rhodamine or biotin prepared in Example 9 was placed on the specimen by a capillary pipette, and reacted at room temperature for 45 minutes and then washed five times with PBS maintained at 4"C.

The obtained sample was identified in the following manner.

(a) FLUORESCENT ANTIBODY METHOD: In the fluorescein- and rhodamine labelled antibodies, the sample was included in glycerin and observed by a fluorescence microscope (Olympus Vanox, Olympus, Japan).

In the biotin labelled antibody, a drop of 5 ig/ml of fluorescein-bound avidin (Funakoshi Yakuhin, Japan) was added on the sample and reacted at 37 C for one hour. After washing five times with PBS, the reacted sample was included in glycerin and observed by a fluorescence microscope (Olympus Vanox, Olympus, Japan).

(b) ENZYME IMMUNOASSQY: In the peroxidase labelled antibody, the sample was reacted with 0.1 M tris-HCI buffer (pH 7.6) containing 0.02.ml/mI of 1% H202 and 0.5 mg/ml of 3,3 diaminobenzidine-HCl at room temperature for 10 minutes. After reaction, the reacted sample was washed with PBS, 4 C, three times and distilled water once. After post-staining with veronal-acetate buffer (pH 4.0) containing 1 % methyl green, the sample was dehydrated by ethanol, dialysed by xylol, included by balsam and the color (brown) was observed by a microscope.

In the alkaline phosphate labelled antibody, the sample was reacted with 0.2 M tris-HCI buffer (pH 8.5, filtered out immediately before use) containing 0.39 M magnesium sulfate, 0.2% lead citrate, 0.6% ssglycerophosphoric acid and 4% sucrose at room temperature for 10 minutes.

After reaction, the reacted sample was washed three times with PBS maintained at 4C and then once with distilled water. The sample was then reacted in 1 % yellow ammonium sulfide solution at room temperature for 5 minutes, washed thoroughly with PBS, included in glycerin and the color (black brown) was observed by a microscope.

These results are shown in Table 2.

Table 2 Reactivity Method Label of Antibody Cancer tissue N.ormal tissue Fluorescent Antibody Method Fluorescein + Rhodamine + Biotin + Peroxidase + 1 Enzyme immuno- Alkaline assay phosphatase + 2 +: positive fluorescence + 1: brown observed + 2: blackish brown observed - : negative EXAMPLE 11: IMAGING OF CANCER CELL BY ANTIBODY Human urinary bladder cancer cell line T24 (1 X 107 cells) was subcutaneously transplanted to the right thigh of female BALB/c nu/nu mice of 8 weeks old (three animals in each group).

After 30 days, the dimension of the tumor was 1 cm2 or more and 1251 labelled antibody secreted by the hybridoma T-006, prepared in Example 9, in a dose amount of 35 juCi per mouse or 1251 labelled mouse IgG as a comparison in a dose amount of 1 0 ILCi per mouse was intravenously injected. After 96 hours, scintigraphy of the whole body was performed by a gamma camera.

A remarkable accumulation of radioactivity on the tumor was observed in all animals administered with '251 labelled antibody of the invention while less accumulation was observed in the control animals.

EXAMPLE 12: MITOMYCIN OR DOXORUBICIN HYDROCHLORIDE LABELLING OF ANTIBODY After adjusting to 10.4 mg/ml with addition of distilled water to the antibody (purified ascitic supernatant) from the hybridoma T-002, T-008 or T-010, 13.0 mg of mitomycin was added, and then hydrochloric acid was added while stirring to adjust pH of the solution to 4.75 and simultaneously 3.7 mg 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. After reacting for 10, 30 or 60 minutes, the reaction was stopped by adding 2 ml acetate buffer (pH 4.70). The reaction mixture was then dialysed to 5 L distilled water at 4"C for 72 hours while exchanging the dialysing solution three times.After concentrating, the dialysate was applied to a column of 1.5 cm in diameter and 55 cm in height charged with Sephadex G-25 (Pharmacia, Sweden) to completely remove low molecular substances. The resultant solution was freeze-dried at - 20"C to obtain the mitomycin-antibody binding product. The binding amounts of mitomycin in the products per mg of the antibody are shown in Table 3.

The binding products of doxorubicin hydrochloride with the antibodies were obtained in the same manner as aforementioned. The binding amounts of doxorubicin hydrochloride in the products per mg of the antibody are shown in Table 3.

On the other hand, these binding products of anticancer agent with the antibody were administered to ICR mice of 10 animals in each group in a dose rate of 400 mg/kg perorally, 100 mg/kg intraperitoneally or 50 mg/kg intravenously. No death was observed for 1 4 days.

TABLE 3 binding ratio (ILg/mg) Anticancer agent antibody 10 minutes 30 minutes 60 minutes mitomycin T-002-derived 4 8 10 T-008 ,, 4 8 9 T-010 ,, 4 7 10 doxorubicin T-002 ,, 4 7 11 hydrochloride T-008 ,, 4 7 10 T-010 ,, 4 7 10 EXAMPLE 13: IN VITRO ANTITUMOR EFFECT OF ANTIBODY In vitro subcultured human urinary bladder cancer cell line T24 was used to examine the antitumor effect of the anti-body or the binding product with the anticancer agent prepared in Example 12. Namely, The cells were adjusted with Eagle's MEM containing 10% fetal bovine serum to a cell concentration of 5 X 104 per ml, placed in a petri dish in each 5 ml, and incubated at 37"C for 24 hours in a 5% CO2 incubator.The antibody from the hybridoma T-008 or the mitomycin bound anti-body from the hybridoma T-008 (60 minutes reaction product) was added in the dish and further cultured. At the time after 24 hours from addition of the drug, 1,uCi/ml of L-[U-'4C] leucine (RCC, USA, specific radioactivity 342 mCi/m mol) was added to the reaction mixture and incubated for two hours. After culture, the culture medium was removed and the cells were washed three times with PBS (pH 7.2) cooled at 0 C, treated with 5% trichloroacetic acid solution and the washed cells were transferred to a filter paper and dried. The radioactivity incorporated into the cellular protein was measured by a liquid scintillation counter (Packard, USA).

The results are shown in Fig. 1, In Fig. 1, the ordinate represents the incorporated amount of radioactivity ('4C-leucine) as percentage of 100 of the amount incorporated by the non-treated control.

As seen from Fig. 1, the concentration showing 50% inhibition of radioactivity incorporation decreased by the binding product of the antibody and mitomycin as compared with the control.

Human urinary bladder cancer cell line T24 (6 X 105 cells per animal) was intraperitoneally transplanted to female BALB/c nu/nu mice of 10 animals in each group. After 5 and 72 hours, the antibody secreted by the hybridoma T-008 or the binding product of the antibody from T-008 with mitomycin (60 minutes reaction product, 50 ILg/ml of mitomycin) was intraperitoneally injected to the mice in a dose rate of 450 pug per mouse (20 y9 of mitomycin per mouse) and the survival ratio was observed for 60 days to estimate the antitumor activity of the administered drug.

The results are shown in Fig. 2. In Fig. 2, the ordinate represents the survival ratio (%) and the abscissa represents the number of day after transplantation of the tumor cells.

As seen from Fig. 2, the number of day for which 50% of animals survived was prolonged by the mitomycin-antigen binding product. Furthermore, the prolongation of life was also observed by the antigen administration alone.

Claims (42)

1. A method for preparing an antibody to a surface antigen of human urinary bladder cancer cells, which method comprises preparing a hybridoma between a cell which produces a said antibody and a cell which can be permanently maintained in vitro by subculture and recovering the said antibody secreted by the hybridoma.

2. A method according to claim 1, wherein the said cell which produces antibody is a spleen cell, lymph node cell, peripheral blood leukocyte or a mixture of two or more thereof.

3. A method according to claim 2, wherein the cell is derived from a mouse.

4. A method according to claim 3, wherein the mouse is a BALB/c mouse or a hybrid mouse thereof.

5. A method according to any one of the preceding claims, wherein the cell which can be permanently maintained in vitro by subculture is a myeloma cell.

6. A method according to any one of the preceding claims, wherein the cell which can be permanently maintained by in vitro subculture is deficient in hypoxanthine-guanine phosphoribosyltransferase or thymidine kinase.

7. A method according to claim 5 or 6, wherein the cell is derived from a mouse.

8. A method according to claim 7, wherein the cell is derived from the cell line P3-X63-Ag8, P3-NS1/1-Ag4-1, Sp2/0-Ag14 or X63-Ag8.653.

9. A method according to any one of the preceding claims, wherein the said cell which produces antibody is derived from an animal immunized with human urinary bladder cancer cells.

10. A method according to any one of the preceding claims, wherein the hybridoma is prepared in the presence of polyethylene glycol.

11. A method according to any one of the preceding claims, wherein the hybridoma is cultured in a medium containing hypoxanthine, aminopterin and thymidine.

1 2. A method according to any one of the preceding claims, wherein the hybridoma is cloned to obtain a single clone.

1 3. A method according to claim 12, wherein the cloning is performed by limiting dilution culture, soft agar culture or fibrin gel culture.

14. A method according to any one of the preceding claims, wherein the hybridoma is cultured in vitro for secretion of the antibody.

1 5. A method according to any one of claims 1 to 13, wherein the hybridoma is transplanted into an animal and cultured in vivo and the secreted antibody is separated from body fluid of the animal.

16. A method according to claim 15, wherein the body fluid is serum or ascites.

17. A method according to claim 1 5 or 16, wherein the animal is a mouse.

18. A method according to claim 17, wherein the mouse is a BALB/c mouse or a hybrid mouse thereof.

19. A method according to any one of the preceding claims, wherein the human urinary bladder cancer is human urinary bladder transitional epithelium cancer cell line T24.

20. A hybridoma which secretes an antibody to a surface antigen of human urinary bladder cancer cells.

21. A hybridoma according to claim 20, which is any one of hybridoma T-001, T-002, T-003, T-004, T-005, T-006, T-007, T-008, T-009, T-010, T-011 and T-012 hereinbefore described.

22. A process for the preparation of a hybridoma which secretes an antibody to a surface antigen of human urinary bladder cancer cells, said process being substantially as hereinbefore described in any one of Examples 1, 3 and 5.

23. An antibody to a surface antigen of human urinary bladder cancer cells, prepared by a method as claimed in any one of claims 1 to 1 9.

24. An antibody to a surface antigen of human urinary bladder cancer cells and which has been produced by a hybridoma as claimed in claim 20 or 21.

25. A derivative or restriction product of an antibody as claimed in claim 23 or 24.

26. A restriction product according to claim 25, which has been obtained by restrictive cleavage of the antibody by a chemical or enzyme treatment.

27. A derivative according to claim 25 comprising the antibody to which is bound a radioactive label, a fluorescent dye, an enzyme, a marker for electron microscopy or a residual group containing a structure to which a further substance is capable of binding.

28. A derivative according to claim 27, in which the radioactive label is 1251.

29. A derivative according to claim 27, in which the fluorescent dye is fluorescein or rhodamine.

30. A derivative according to claim 27, in which the enzyme is a peroxidase, alkaline phosphatase or fi-galactosidase.

31. A derivative according to claim 27, in which biotin is bound to the antibody.

32. A derivative according to claim 27 substantially as hereinbefore described in any one of sections (1) to (7) of Example 7.

33. A reagent suitable for use in the detection of a human urinary bladder cancer, which reagent comprises an antibody as claimed in claim 23 or 24 or a derivative or restriction product as claimed in any one of claims 25 to 32, together with a carrier or a diluent therefor.

34. A method of detection of a human urinary bladder cancer in which an antibody as claimed in claim 23 or 24 or a derivative or restriction product as claimed in any one of claims 25 to 32 is used.

35. A method according to claim 34, wherein a sample containing the cancer cells is brought into contact with the antibody, derivative or restriction product and the antigen-antibody reaction, if any, which occurs is utilized to detect the cancer.

36. A method according to claim 35, wherein the sample is derived from human or animal.

37. A method for the detection of a human urinary bladder cancer, said method being substantially as hereinbefore described in any one of Examples 8, 10(2)(a) and 10(2)(b).

38. An antibody as claimed in claim 23 or 24 to which an anticancer agent is bound.

39. An antibody according to claim 38, in which the anticancer agent is mitomycin or doxorubicin hydrochloride.

40. An antibody according to claim 38, which is any one of the binding products hereinbefore described in Example 1 2.

41. A pharmaceutical composition suitable for use in the treatment of human urinary bladder cancer, which composition comprises as active ingredient an antibody as claimed in claim 23 or 24 or any one of claims 25 to 32 together with a pharmaceutically acceptable carrier or diluent.

42. A pharmaceutical composition according to claim 41 which is in the form of an injectable preparation.