FR2543971A1 - ANTIBODIES AGAINST HUMAN BLADDER CANCER - Google Patents

Abstract

A MONOCLONAL ANTIBODY SPECIFIC TO THE SURFACE ANTIGEN OF A HUMAN BLADDER CANCER CELL IS PRODUCED BY A HYBRIDOMA BETWEEN AN ANTIBODY PRODUCING CELL AND A MYELOMA. THE ANTIBODY IS USEFUL FOR THE IDENTIFICATION AND / OR CLASSIFICATION OF HUMAN URINARY BLADDER CANCER AND AS AN AGENT FOR THE TREATMENT OF HUMAN URINARY BLADDER CANCER.

Description

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  Antibodies to human bladder cancer.

  The present invention relates to human cancer of the bladder, more particularly a specific antibody

  bladder cancer that can be produced by a cell-

  obtained by fusion between a cell capable of producing such an antibody and a cell capable of proliferating permanently in vitro by subculturing, the cell obtained

  by merger being subsequently referred to as "hy-

  bridome ". There are a lot of people who suffer from cancer and a large number of people die from cancer, so this is a very important social problem Despite great efforts on the part of many researchers in all fields of science, no primary cancer treatment method has been found so far In Japan, half or more of all cancer deaths are caused by gastric cancer, lung cancer or cancer liver, other kinds of cancer, however, tend to increase as lifestyle changes in Japan, for example rectal cancer or malignant tumor of the urinary organs including cancer of the

  urinary bladder which relates to the subject of this invention

tion.

  The methods of treating such cancers are

  are mainly used in surgical operations

  pairing with radiotherapy, chemotherapy

  and / or immunotherapy Such procedures are, however

  dant, limited to a certain extent to the treatment of

  anaphase cancer or progressive cancer So a diagnosis

  early tic and early treatment are important in

  all cases Under these conditions, a rapid, high

  highly reliable for the identification and / or diagnosis of

  the cancer cell and / or cancer tissue is strong-

ment required.

  Numerous and extensive research has been done on a specific cancer antigen which

  is present on the surface of the cancer cell

  particular, such a surface antigen was confirmed in the study using experimental animals On the contrary, a sufficient approach was not made for cancer

  human According to present knowledge, such an anti

  surface gene for cancer includes, for example, (1) a

  antigen present only autologously (even

  che), (2) an antigen usually present in the same kinds of tumors, or (3) an antigen also present in tumors of other organs or even cells

  normal as well as tumor cells in question.

  An immunological procedure using an antiserum of the same or different types is generally useful

  for the analysis of an antigen To prepare such an anti-

  serum which can be used to recognize the three kinds of antigens mentioned above, it is necessary

  to repeat the antiserum absorption process A di-

  antibody titer inevitably follows with such a procedure and the resulting antiserum cannot be used in practice In addition, if one can obtain the desirable antiserum, it is almost impossible

  to reproduce the same antiserum having the same specificity.

  In these circumstances, a new means is required to

  identification of cancer-related antigens and anti-

  specific cancer genes on the surface of a cancer cell. One of these means was developed by Kbhler and Milstein in 1975, Nature, 256, 495 (1975), i.e.

  the technique of cell fusion to prepare an anti

  monoclonal body The cell fusion technique

  meets requirements such as those mentioned above

  on it and there have been reports recently of subgroups of anti

  body of human lymphocytes and monoclonals against

  surface gene for human leukemia cells or

human melanoma.

  Generally, although one can get an anti

  specific monoclonal body by such a technique

  cell fusion, the antigenicity of a cancer cell

  reuse varies according to the various cancer cells and therefore we cannot currently predict whether we will actually obtain an excellent antibody having the

  specificity desired With regard to cells can-

  human cereals, so far only few special cancer cells have been reported as described above In particular, no publications have been made on human urinary bladder cancer Of course, any

  antibody to bladder cancer is not real-

currently usable.

  The inventors of the present invention have made great efforts to research a process for preparing a monoclonal antibody against the surface antigen of a

  human urinary bladder cancer cell and have resulted

  ti to the invention according to which one can obtain such an antibody with a high specificity, a high titer and a low concentration of contaminants from a

  hybridoma between a cell capable of producing an anti

  body against a human bladder cancer cell (sometimes later referred to as a "cell

  producing anti-bladder cancer antibodies ", or sim-

  also "antibody producing cell" and a cell

  capable of permanent in vitro subculturing (designated some-

  thereafter under the name of "transplanted cell")

  present inventors have also discovered that such an an-

  body is useful for classification and / or identification

  cation of a urinary bladder cancer cell

  main Antibodies can also be useful in providing effective treatment for human urinary bladder cancer. It is an object of the present invention to provide a process for the preparation of an antibody specific for the surface antigen of a cancer cell.

human bladder.

  Another object of the invention is to provide a highly specific anti-bladder cancer antibody and a process for its preparation by the technique of

cell fusion.

  An object of the invention is to provide a hybrid

  dome capable of producing such an antibody.

  Another object of the invention is to provide a

  classification and / or identification process for these

  Human urinary bladder cancer using

the antibody of the invention.

  Yet another object of the present invention is to provide a pharmaceutical agent for the treatment

  human bladder cancer, which agent contains the an-

  antibody of the invention as an effective component.

  The present invention also aims to provide a useful derivative of such an antibody effective in

  use for classification and / or identification

  tion of human bladder cancer cells and the

  treatment of human bladder cancer.

  Other objects and the remarkable advantages of the present invention will be obvious to experts

  in art from the following detailed descriptions

  on non-limiting, specific embodiments.

  The process for preparing an anti-

  bladder cancer of the present invention is to prepare a hybridoma from a cell producing the anti-bladder cancer antibody and a cell

  transplanted, especially myeloma, and recovering the anti

body secreted by the hybridoma.

  The process of the invention will be described in

suite with more details.

  PREPARING THE ANTIBODY PRODUCING CELL.

  In the present invention, the production cell

  this anti bladder cancer antibody can be obtained from any animal species including humans Immunization of animals is not essential

  although such preliminary immunization may notice

  significantly improve the recovery efficiency of the hy-

bridome desirable.

  When the origin of such a cell is human-

  ne, anyone with a history of bladder cancer or a high titer of serum against the bladder cancer cell can be chosen or alternatively, one can obtain such a cell from a living body immunized with an immunogen The immunogen may be a per cell, a cell which is treated with glutaraldehyde, mitomycin or heat and therefore cannot proliferate, or the surface antigen separated from a cancer cell by a treatment

  suitable with for example an enzyme and purified.

  The immunogen used in immunization can be mixed with an adjuvant such as Freund's complete or incomplete adjuvant. The immunogen can be administered

  by any conventional means such as subcutaneous injections

  born, intraperitoneal, intravenous, intradermal

  and intramuscular - A subcutaneous or intra injection

  peritoneal may be preferable It is possible that only one immunization is possible although the immunization can be carried out repeatedly several times with an appropriate time interval for example from one to five

  weeks By measuring the titer of the antibody in the se-

  rum of the immunized animal, the animal whose titer has been high enough to obtain the antibody-producing cell can be used, which has the effect of improving

  the effectiveness of the following operations The preferred cell

  maple is extracted from the animal on the 3rd-5th day after the final immunization The cell producing the anti

  body is a plasma cell or a lymphocyte which is a

  The precursor to it and can come from anywhere

  which site of the body, usually the spleen, a lymph node

  phatic, peripheral blood or any combination of

these.

B MELTING OF CELLS.

  A cell which can be permanently stored by subculturing in vitro can be any cell

  which can be merged with the production cell

  antibody to give a hybridoma capable of pro-

  to make a desired antibody One of these cells which is preferable is a leukemic cell such as a myeloma cell. Such a cell can come from any species such as man, rat, mouse, etc.

  The cell that is devoid of hypoxanthine-guanine phos-

  phoribosyltransferase (HGPRT) or thymidine kinase (TK) is preferable because such parent cells do not

  cannot develop in a selective medium.

  An example of a preferable cell line is GM-1500-6 TG-A 1-2 or RPMI 8226 derived from humans or P 3-X 63-Ag 8, P 3 / NSI / 1-Ag 4-1 , Sp 2-0-Ag 14, X 63-Ag 8 653,

etc, derived from the mouse.

  It is preferable to use a production cell.

  antibody and a transplanted cell, both from

  from the same species, although this is not essential, in terms of the efficiency of fusion, the stability of the properties of cells obtained by fusion and the ease of in vivo culture thereof. In particular, when the cell line P 3-X 63-Ag 8, P 3 / NSI / 1-Ag 4-1, Sp 2/0-Ag 14 or X 63-Ag 8 653 is used as subcultured cell, it is preferably possible to use a BALB / c mouse

inbred or its hybrid mouse.

  For the fusion, an accelerator such as the Sendai virus (HVJ) and polyethylenereglyco can be used; in particular, polyethylene 1000, 1540, 2000, 4000 or 6000 may be preferably used. The fusion of the cells can be carried out in a solution containing about 30 to 55% of such a polyethylene glycol In addition, dimethyl sulfoxide

  may be present in the solution.

C SELECTION OF THE HYBRIDOME.

  In the solution after the merger, are present,

  in addition to the cell obtained by fusion, the cell pa-

  remaining antibody-producing rent and the pa-

  rent transplanted The former cannot survive during subsequent in vitro culture while the latter can spread with the desired hybridoma It is therefore

  preferable or even necessary to eliminate the repi-

  quée of the solution containing the mixed cells For this, one preferably uses the cell deprived of

  HGPRT or TK as a transplanted parent cell and those

  mixed cells containing the parent cells are cultured

  born in a selection medium containing hypoxan-

  thine, aminopterin and thymidine after cell fusion, allowing selective growth of the desired-hybridoma only Or alternatively, the cell

  parent transplanted, when it is not a poor cell

  as for HGPRT or TK, can be treated with eme

  tine and actinomycin D before cell fusion.

  The parent cell cannot proliferate with such treatment.

  and therefore one can easily choose the hy-

  desired bridome from mixed cells.

  The hybridomas thus obtained generally contain

  two or more clones, therefore these

  may not have quite the same properties.

  To separate them into individual clones, cloning may be necessary and even desired when an antibody is

  likely to be required Cloning is also effective

  cace from the point of view of preventing change of po-

  pulation which can often occur in a long-term culture of a system with a number of clones

  mixed Cloning can be carried out by dilution culture

  Limiting tion, by culture on soft agar or by culture on fibrin gel It is also possible to use a fluorescent activated cell sorter to sort the cells in cloning. Cloning can also be used to separate the possible aberrant cells appearing 2 during the long culture. and to keep the cells

  which have the same properties as the original hybridoma.

  The hybridoma of the present invention in the logarithmic growth phase can be maintained in

  suspension for a long time in a con- form

  frozen and at the rate of 1 10 × 10 6 per ml suspended in

  a fetal bovine serum containing 5% (v / v) of dimethylsul-

  foxide The freezing process is preferably carried out at a cooling rate of 10 C per minute. It is preferable to keep the hybridoma at 800 C or lower. The frozen hybridoma, preserved, is preferably thawed as quickly as possible If the cells

  are washed with a medium intended to remove dimethyl-

  sulfoxide immediately after dissolution, the cells can be suspended in a conventional medium and can be cultured. After thawing, only a small quantity of surviving and developing cells remains, it is necessary to add mouse spleen cells, etc.

  D PREPARATION AND RECOVERY OF THE ANTIBODY.

  For the preparation of the antibody, the hybridoma producing an anti-bladder cancer antibody is

grown in vitro or in vivo.

  In in vitro culture, a nutritive broth suitable for the hybridoma of the present invention can be chosen, for example, RPMI 1640 medium which contains% (v / v) fetal bovine serum, 5 x 10 M e-mercaptoethanol , 1 m M sodium pyruvate and antibiotics,

  or else the modified Eagle's MEM medium from Dulbecco (desi-

  gne below by D-MEM) which contains 4.5 g / l of glucose instead of RPMI 1640 medium An initial concentration of cells suitable for proliferation can be

  general of the order of 10 per ml while being able to depend-

  of each hybridoma, and the concentration of cells

  in the culture is preferably up to 2 x 10 6 per ml.

  In in vivo culture, the hybridoma is trans-

  planted in a living body and developed in the form

  solid or in ascites We take from the body vi-

  a body fluid, preferably serum or

  ascites, to recover the antibody secreted by the hybrid

  dome The crude solution of the antibody obtained may contain

  identify various substances as impurities (contaminants)

  from the host living body, however it is more

  marked as the antibody solution obtained in vitro due to the higher concentration of the antibody

  desired When the hybridoma is transplanted intraperitoneally

  ideally, pristane can be administered (2,6,10,14-

  tetramethylpentadecane) intraperitoneally before transplantation, preferably 3 to 9 weeks before transplantation This treatment may increase the yield

  of the crude antibody solution but is not essential.

  As host, an animal of the same species and of the same blood

  that the animal from which the parent cell (s) pro-

  comes (comes) is preferable and in this case the hybrid-

  dome can grow in the animal even if it does not have

  been treated specially When the serotypes of histo-

  compatibility of antigens coincide with each other between the hybridoma and the host, the host is necessarily

Z 2543971

  treated beforehand by administering for example an anti-lymphocyte antibody or by irradiation with X-rays The cells begin to grow at

  from 1 to 3 weeks after transplantation.

  In the in vitro or in vivo culture of hybrid

  dome for the secretion of antibodies, a radioactive substance

  active such as leucine and lysine labeled with a radioisotope can be added to the medium or administered to the host. Such treatment can give an antibody having the same chemical structure as the non-mar

  qué and containing the radioactive substance in a molecule.

  The antibodies of the present invention can be used as such in the crude antibody solution, or they can be purified for use by any conventional method for immunoglobulin, for example, by sulfate fractionation. 'ammonium

  or by ion exchange chromatography, or by chroma-

  affinity tography with protein A or the antigen.

  Such purified antibodies can be used independently

in particular or as a mixture.

  According to the method of preparation of the antibody

  anti-bladder cancer of the present invention, the

  convenience associated with conventional methods can be clearly overcome The hybridoma of the invention can be transplanted and can be proliferated substantially so

  permanent both in vitro and in vivo In addition, the hy-

  bridoma can produce an antibody against a determinant

  specific antigen The antibody produced, therefore

  quent, may have monoclonal specificity, is an antibody to the cell surface antigen

  of urinary bladder cancer and consists of essential-

  ment of a single molecular species The process of the pre-

  sente invention allows the production of a necessary quantity

  demand-dependent antibody, suppress it

  dispersion of the batches and moreover the preparation

  he ration of a solution containing the antibody having a titer

  In addition, the process does not require diffuse absorption.

  ficility which has been conventionally inevitable Furthermore, one can use an unpurified antigen such as a cell per se without difficulty in the process and even one can

  in this case obtain the highly specific antibody.

  Such a high reactivity with an antibody antigen allows rapid identification of a bladder cancer cell with high reliability without going through difficult conventional methods In addition, given the high purity of the antibody, the reactions allergic

  due to the contaminants inevitably contained in the pre-.

  classic paration rarely occur, allowing its

  use as a remedy for bladder cancer.

  The classification and / or identification process

  tion of the present invention can be applied to a subject

  of any origin For example, one can use materials

  clinical rials such as urine, a lymphocyte or other bioptic tissue obtained from a patient possibly suffering from urinary bladder cancer of the point

from a clinical point of view.

  In identification, a reagent containing the an-

  the antibody of the present invention is contacted with

  a subject containing bladder cancer cells.

  She can conveniently use immuno microscopy

  fluorescence, immunoelectromicroscopy, binding test

  its radioactive, enzyme immunoassay, etc. In microsco-

  direct immunofluorescence pie, it can be used conveniently

  the antibody of the invention after having marked it with

  a fluorescent dye such as la-fluorescein and rho-

  damine The other convenient form of the antibody of the invention may be a form labeled with a labeling substance such as ferritin for immunoelectromicroscopy, a form labeled with a radioisotope such as 12 I and I for a radioactive bond or a mar form

  quée with an enzyme such as peroxidase, phospha-

  alkaline tase and 0-galactosidase for an immunoassay

  zymatic Of course, one can carry out an in-

  direct using a secondary antibody or its binding product as a substitute, for example an anti-human bladder cancer antibody can be used with avidin

  as a secondary antibody Also, part of the an-

  tibody obtained by restrictive cleavage with treatment

  chemical and / or enzymatic such as F (ab ') 2 can be used

  read in place of the antibody per se Such diverse derivatives and restriction products are useful for

* classification and / or identification process

  vention and therefore are included as part of the

present invention.

  The antibody, its derivatives and its res-

  triction can be mixed for use if necessary In addition, the reagent for classification and / or identification of the invention may still contain a

  support or diluent conventionally used.

  The present invention also relates to a curative agent (remedy) for the treatment of urinary bladder cancer. It can be considered that the curative mechanism of the antibodies of the present invention accelerates a reaction of

  complementary fixation and attack of the macrophage and / or-

  other immunocytes against cancer cells when

  that the antibody being administered binds with the sur-

face of the cancer cell.

  The antibody of the present invention can be administered per se independently or in admixture When

  the antibody is chemically combined with an anti

  cancer such as mitomycin hydrochloride and doxo-

  rubicin or a toxin such as ricin, we can get

  nir more advantages In this case, we can consider that the mechanism is such as the anti-cancer agent or the toxin

  can attack cancer cells when the anti

  administered body binds to the cell surface antigen

  cancerous lule, resulting in lower toxicity

  cited anti-cancer agent or toxin only when used alone A fraction of the antibody obtained by

  restrictive division by chemical or enzymatic treatment

  tick, for example F (ab ') 2, can also be used

  for the healing agent of the invention instead of the an-

  antibody per se In this case, we can avoid the possibility that the tissue of a host could be affected by deterioration due to a non-specific complementary binding reaction, etc. These derivatives (combination products

  with an anti-cancer agent or with a toxin) and these pro-

  restriction duits can be used either independently

  either as a mixture as a curative agent of the present invention. To assess the acute toxicity of antibodies, its derivatives and restriction products,

  the curative agent of the invention has three groups (from 10

  each) of IRC mice; first in an amount of 2 g / kg orally, secondly in an amount of 400 mg / kg intraperitoneally and thirdly in an amount of 200 mg / kg intravenously, and no deaths were observed for 14 days The healing agent of the invention can therefore be used without danger as

  remedy for human urinary bladder cancer.

  The healing agent of the invention may be ad-

  administered by subcutaneous, intramuscular or intra-

  venous, preferably by subcutaneous or indirect injection

  the healing agent can be administered

  orally since part of the agent

  can be absorbed through the intestinal tract while

  retaining the structure as an antibody, which has been con-

  affirmed by the present inventors.

  A preparation for injection can be prepared by dissolving or suspending for example 10 mg of the antibody or of its derivative with 50 mg of mannitol in 10 ml of distilled water, by sterilization in any conventional manner, dividing it into fractions of 2 ml each to put it in ampoules and lyo-

  philisant The product obtained will be dissolved or added to

  board in saline at the time of use.

  An injection may contain in addition to the anti-

  body a support, a diluent, a buffer, a stabilizer, an isotonic agent, etc., these agents being given by those who

  are specialists in the art We can prepare the preparation

  injection for any form of subcutaneous, intramuscular or intravenous injection

  prepare an agent which can be administered orally by

  wear what conventional process and introduce it into a

  enteric cament A unit quantity of the foraging agent

  ri.son of the invention may depend mainly on the symp-

  tomes, and generally ranges from 0.001 mg to 10 g per day per kg of body weight in an animal such as mice and from 0.01 to 3000 mg per day per kg of body weight in humans. The present invention will be illustrated by the

following non-limiting examples -

  EXAMPLE 1: PREPARATION OF AN ANTIBODY AGAINST A CELL

OF HUMAN BLADDER CANCER.

  IMMUNIZATION AND FUSION OF CELLS.

  (1) PREPARATION OF THE IMMUNOGENIC CELL: Incubation was made

  ber a T 24 human bladder epithelium cancer cell line (2 x 105), obtained by subculturing in

  in vitro for a long period, at 370 C in a humid environment containing 5% of C 02 in a salt medium, Earle from MEM of Eagle (hereinafter referred to as MEM) containing 10% (v / v) of bovine serum fetal and kanamycin sulfate (final concentration 60 mg / l) in a

  culture dish- (Falcon 3003, Becton-Dickinson, USA).

  After three days of culture, the cells were exfoliated

  force and the supernatant was removed by centrifugation

  The cells were then washed with a solution.

  phosphate buffered saline (PBS, p H 7.2) centrifuged

  giant and were suspended in PBS medium. About 2 x 10 6 cells were obtained in the culture vessel.

  (2) CULTURE OF MYELOMA: -We cultivated a cell line-

  mouse myeloma Sp 2 / O-Ag 14 (105 per ml) in D-MEM containing 10% (v / v) fetal bovine serum, 1 M 4 of pyruvic acid, 2 m M of glutamine and 60 mg / l kanamycin sulfate by subculturing every three days The cell was adjusted to a cell concentration of 2.5

  x 105 per ml the day before cell fusion follows

  and we cultivated it in the environment mentioned above

  above. (3) IMMUNIZATION WITH T 24 CELLS: The T 24 cells (5 x 10 6 10 x 10 6) obtained in (1) above and suspended in 0.4 ml of PBS were injected intraperitoneally into mice BALB / c females (NIHON CHARLES LIVER, JAPAN, 9 weeks old) four times

  approximately per month for immunization.

  (4) MELTING OF CELLS: The fusion of the cells

  lules according to the method of Kbhler and Mistein (Immunotests; Clinical Laboratory Techniques for 1980 ed by Nakamura R M et al, E S Alan R Liss, Inc, N Y 1980,

p 301-324).

  On the fourth day after the final immunization, the mice were killed to extract the spleen The spleen was

  detached, passed through a 150 mesh sieve, cen-

  trifugated at 400 x g and tris-H Cl buffer (tris-

  (hydroxymethyl) aminomethane 0.017 M, p H 7.65) containing 0.747% of ammonium chloride to the precipitated cells

  to remove the erythrocites and the cells were recovered

  centrifugation (400 xg) After adding MEM to cells, centrifugation was performed at 400 xg and cells were further suspended in fresh MEM After repeating this washing process three times, finally cells suspended in MEM. Sp 2/0-Ag 14 cells were exfoliated from the culture vessel by pipetting and were transferred to a centrifuge tube. After centrifugation (400 xg), the recovered cells were suspended in MEM, centrifuged (400 xg ) to remove the serum

  and again suspended in MEM.

  The spleen cell (5 x 107) and the Sp 2 / O-Ag 14 line (107) were mixed, carefully pipetted and centrifuged (400 x g) After rejecting the liquid

  supernatant, the precipitate was detached by striking gently

  the centrifuge tube To the detached cells, 0.3 ml of a polyethylene glycol solution (PEG 1000) at 30% (v / v) in MEM maintained at 37 ° C. was added.

  stirred gently, the solution was allowed to stand at room temperature

  ambient temperature for 5 minutes The centrifugation was carried out 4 th at 7 × g for 2 minutes and 5 ml of MEM were gradually added After stirring gently, the centrifugation (400 × g) was carried out at temperature

  room temperature for 5 minutes We removed the supernatant liquid

  Giant, 5 ml of MEM was added to the precipitate, centrifuged in the same manner as mentioned above and the

supernatant.

  To the precipitated cells, 5 ml of D-MEM medium containing 10% fetal bovine serum, 2 m M of glutamine, 5 × 10 M e-mercaptol, 60 mg / l of kanamycin sulfate and still glucose were added. 4.5 g / l (hereinafter referred to as D-MEM-FBS medium) After pipetting, 20 ml of DMEM-FBS were added to the resulting cell suspension (5 ml). Each 25 ml fraction was inoculated in a 25 cm 2 culture vessel (C-25100, Corning, USA) and allowed to incubate in a humid atmosphere containing 5% C 02 at 37 C overnight, conditions

  that have always been used in pro-

following cedures.

  The day after inoculation, the culture was transferred to a centrifuge tube after having

  pipette gently, and centrifuged at 400 x g.

  After removing the supernatant, the dumpling

  of cells was suspended in 35 ml of D-MEM-

  FBS containing 1 x 10-4 M hypoxanthine, aminoptera-

  rine 4 x 10-7 M and thymidine 1.6 x 10-5 M (HAT medium).

  The suspension (0.1 ml) was placed in each well of a 96-well plate (Falcon 3072, Becton-Dickinson, USA) and cultured for one week After culturing for one week in HAT medium, added 25 µl of HT medium (HAT medium without aminopterin) every 2 or 3 days.

  B SELECTION AND GROWTH OF THE PRODUCING ANTIBODY

THE HYBRIDOME.

  A solid phase enzyme immunoassay was used to examine the production of the T 24 antibody in the supernatant of each well

  the second week after cell fusion.

  The fixed T 24 cells were reacted on a plate (Falcon 3072) with 40 µl of the supernatant liquid in each well at room temperature for two hours, washed with PBS, allowed to react for two hours with 100 µl of immunoglobulin antibody linked to peroxidase (catalog N 3211-0231, Cappel Lab.

  Inc, USA) which has been diluted 1000 times with hair serum

  val, and washed entirely with PBS 200 41 citrate buffer (0.1 M, p H 4.5) containing 1 mg / ml of a substrate (o-phenylenediamine) and 0.4 μl / ml of a 31% aqueous oxygenated water solution was added and allowed to react

  for 30 minutes until colored.

  Cloning was carried out in three wells

  holding the antibodies reacting with the T 24 cells by the limiting dilution method as follows: hybridomas from each of the three wells positive for the production of the antibody were put

  suspended in D-MEM-FBS and mixed with cells

  spleen cells obtained from BALB / c mice in the same manner as that used in the fusion of

  cells and adjusted in D-MEM-FBS (108 cells).

  The cell concentration was adjusted to 5 x 10 per ml by adding DMEM-FBS and the cell mixture was inoculated into each well of the 96-well plate (Falcon 3072) in an amount of 0.2 ml each and it was cultured The thirteenth day after the initiation of the culture, the production of the antibody in each well was examined using the above-mentioned solid phase enzyme immunoassay 33 antibody-producing hybridomas were obtained including 6 hybridomas indicated in the

table 1 below.

  EXAMPLE 2 PROPERTIES OF THE ANTIBODY AGAINST THE CELL

OF CANCER OF THE HUMAN BLADDER.

  COMPARED TO REACTIVITY WITH OTHER CELLS.

  The solid phase enzyme immunoassay of Example 1 was used to examine the reactivities of the

  supernatant liquids obtained in Example 1 with other

  very cells transplanted in vitro; Intestine 407 (cell line of the fetal human intestine), Du 145 (line

  of human prostate cancer cells) and 235 J (li-

  human renopelvic cancer cell) as well as T 24 (human urinary bladder cancer cell line) As seen in Table 1 below, antibodies secreted by hybridomas other than T-002

  reacted strongly with the T 24 line and reacted slightly

  slightly or weakly with the other cells, while the antibody secreted by the T-002 hybridoma reacted with the

  line T 24, reacted weakly with the cells of the

  testin fetal and did not react with the other cells.

  B COLORING OF THE CELL BY ANTIBODY TECHNIQUE

FLUORESCENT.

  The T 24 cells were stained by the technique

  than indirect fluorescent antibody using the an-

  antibody secreted by hybridoma T-003 Namely, cells

  the T 24s were fixed on a glass strip without fluorescent

  rescence, reacted with the supernatant (50 p) at 37 C for 45 minutes in a humid atmosphere and were

  washed by treating them for 3 to 5 minutes with a solution

  tion of PBS containing 1% bovine serum albumin,

  m M of HEPES (N-2-hydroxyethylpiperazine-N'-2- acid

  ethanesulfonique) and 0.1% sodium nitride, this washing process being repeated three times The reaction was again carried out under the same conditions for 45 minutes with p 1 of a 20-fold diluted solution of bound anti-mouse Ig G

  to fluorescein (Miles-Yoda Ltd, Israel, Code N 65-

  171) and the washing process was carried out in the same manner

  re as mentioned above After drying, the carbonate-buffered glycerin solution (0.05 M, w H 9.5, 10% glycerin) was set aside and a glass cover was placed over it to make a observation by means of

  fluorescence microscope (Olympus, Japan, Model AH-RFL-

  LB) Strong fluorescence was observed on the surface of the T cell 24 Anti-mouse IgG linked to fluorescence was used after being subjected to absorption six times

with T cell 24.

  C IDENTIFICATION OF IMMUNOGLOBULIN CLASSES.

  The immunoglobulin classes of the antibodies secreted by hybridomas T-001 to T-OO 6 have been identified by the solid phase enzyme immunoassay which uses anti-immunoglobulin (Ig), anti-Ig C and anti-Ig M ( Cappel

  Lab Inc, USA, Catalog N 3211-0231, 3211-0081, 3211-

  0201) as an anti-mouse immunoglobulin antibody linked to peroxidase As seen in table 1 below, all the antibodies examined were Ig G.

  EXAMPLE 3 PREPARATION OF ANTIBODIES AGAINST A CELL

BLADDER CANCER HUMAN.

  IMMUNIZATION AND FUSION OF CELLS.

  (1) PREPARATION OF IMMUNOGENIC CELLS: We incubated

  the transition epithelium cancer cell line

  nel of the human urinary bladder T 24 (2 x 105) obtained by subculturing in vitro for a long period, at 37 C in a humid atmosphere containing 5% carbon dioxide in the MEM medium which contains 10% (v / v) bovine fetal serum and kanamycin sulfate (final concentration, mg / l) in a 100 mm culture vessel (Falcon 3003, BectonDickinson, USA) After three days of culture,

  the cells were exfoliated in force and the li-

  quid supernatant by centrifugation Cells have

  then washed with PBS (p H 7.2) by centrifugation and suspended in PBS Approximately

  2 x 106 cells from a culture vessel.

  (2) CULTURE OF MYELOMAS: The cell line was cultivated

  mouse myeloma Sp 2 / O-Ag 14 (105 per ml) in D-MEM containing 10% (v / v) fetal bovine serum, 1 m M pyruvic acid, 2 m M glutamine and 60 mg / 1 sulfate

  of kanamycin by transplanting every three days The cell

  lule was adjusted to a cell concentration of 2.5 x 105 per ml the day before cell fusion

  following and cultivated in the medium mentioned above.

  (3) IMMUNIZATION WITH T 24 CELLS: T 24 cells (5 x 10 6 10 x 10 6) obtained in (1) above and suspended in 0.4 ml of PBS were injected intraperitoneally into mice BALB / c females (NIHON CHARLES LIVER JAPAN, 9 weeks old) four times

  approximately every month for immunization.

  (4) CELL MERGER: The cell fusion was carried out

  iules according to the method of-Kbhler and Milstein.

  On the fourth day after the final immunization, the mice were killed to remove the spleen. The spleen was carefully detached, passed through a 150 mesh steel cloth, centrifuged at 400 xg, and tris buffer was added. H Cl (p H 7.65) containing 0.747% of ammonium chloride to the cells precipitated to eliminate the

  erythrocytes and the cells were recovered by centrifugation

  tion (400 x g) After adding MEM to the cells, centrifugation was carried out at 400 x g and a

  again the cells suspended in fresh MEM.

  After repeating this washing process three times, we

  finally suspended the cells in MEM.

  Sp 2/0-Ag 14 cells were exfoliated from the culture vessel by pipetting and transferred to a centrifuge tube. After centrifugation (400 xg), the recovered cells were suspended in MEM, and were centrifuged (400 xg) to remove the serum and resuspended in MEM. Spleen cells (2 x 10) and Sp 2/0-Agl 4 cells (4 x 10) were mixed, pipetted with

  care and centrifuged (400 x g) After evacuating the li-

  as the supernatant, the precipitate was gently detached by gently hitting the cell centrifuge tube

  1.2 ml of a polyethylene solution

  lene glycol (PEG 1000) at 30 X (v / v) in MEM maintained at 370 c After having stirred gently, it was left to stand

  the solution at room temperature for 5 minutes.

  Centrifugation was carried out at 7 xg for 2 minutes and 20 ml of MEM were gradually added. After stirring gently, centrifugation (400 xg) was carried out at room temperature for 5 minutes. The supernatant was then removed, added 20 ml of MEM

  to the precipitate, centrifuged in the same way and

mined the supernatant.

  To the precipitated cells, 15 ml of D-MEM-FBS medium were added. After pipetting, 90 ml of D-MEM-FBS were added to the resulting cell suspension (15

  ml) About 25 ml of the cell suspension was inoculated

  them in each 25 cm 2 culture container (Corning, USA, C-25100) and they were incubated while leaving them to stand in a humid atmosphere containing 5% C 02 to 370 C overnight, these conditions being incubated

  used in the following procedures.

  The next day, the fresh culture was pipetted-

  it was transferred to a centrifuge tube and

  centrifuged at 400 x g After removing the excess liquid

  swimming, the cell pellet was suspended in 100 ml of HAT medium Each 0.1 ml fraction of the cell suspension was inoculated into each well of

  the 96-well culture plate (Falcon 3072, Becton-

  Dickinson, USA) and cultivated for one week After cultivation

  ture in HAT medium for another week, we have

  added 25 µl of HT medium every 2 or 3 days.

  B SELECTION AND GROWTH OF THE PRODUCING HYBRIDOME

THE ANTIBODY.

  A solid phase enzyme immunoassay was used to examine the production of the antibody against

  the T 24 cells in the supernatant of each go-

  det during the second week after cell fusion. The T 24 cells fixed on the plate were reacted with 40 μl of the supernatant liquid in

  each scoop at room temperature for two hours

  res, washed thoroughly with PBS, reacted for two hours with 100 µl of peroxidase-bound anti-mouse immunoglobulin antibody (Cappel Lab Inc, USA, Catalog No. 3211-0231) which was diluted 1000 times with horse serum, and they were washed completely with PBS- 200 μl of citrate buffer (0.1 M, p H 4.5) containing 1 mg / ml of o-phenylenedimain and

  0.4 µl / ml of aqueous hydrogen peroxide solution was added

  and we made them react for 30 minutes to get

coloring.

  Cloning was carried out in three wells containing the antibodies which react with T cells 24 by

  the limiting dilution process, i.e. 100 hybrido-

  mes of each of the three positive buckets in year production

  antibodies were suspended in D-MEM-FBS and mixed

  dipped with spleen cells obtained from -from-

  ris BALB / c in the same way as that used in cell fusion and adjusted in D-MEM-FBS (108 cells) The cell concentration was adjusted to x 10 per ml by adding D-MEM-FBS Each 0.2 ml fraction of the cell mixture was inoculated into each well of the 96-well culture plate (Falcon 3072) and cultured On the 14th day after initiation of the culture, the production of antibody-a been examined in each well

  using the solid phase enzyme immunoassay

  mentioned above A total of 16 hybridomas were obtained.

  including T-009 to T-012 indicated in table 1 below.

  EXAMPLE 4 PROPERTIES OF THE ANTIBODY AGAINST THE CELL

OF HUMAN BLADDER CANCER.

  COMPARED TO REACTIVITY WITH OTHER CELLS.

  The solid phase enzyme immunoassay of Example 1 was used to examine the reactivities of the supernatant liquids obtained in Example 3 with other cells obtained by subculturing in vitro; Intestine 407 (human fetal intestine cell line), K-562 (human leukemia cell line) and 253 J (human renopelvic cancer cell line) as well as T 24 (bladder cancer cell line human urinary) As seen in Table 1 below, the antibody secreted by the TO 10 hybridoma was highly specific, i.e. reacted strongly with T 24 while it reacted to trouble with the other cells

  other hybridomas have shown similar properties.

  B COLORING OF THE CELL BY ANTIBODY TECHNIQUE

FLUORESCENT.

  The T 24 cells were stained by the technique

  than indirect fluorescent antibody using the an-

  antibody secreted by the hybridoma T-O 10 Namely, the cells

  the T 24s were fixed on a glass strip without fluorescent

  rescence, reacted with the supernatant (50 1) at 37 C for 45 minutes in a humid atmosphere and were

  washed by treatment for 3 to 5 minutes with a solution

  tion of PBS containing 1% bovine serum albumin, 10 m M

  HEPES and 0.1% sodium nitride, this washing process

  ge being repeated three times The reaction was still reali-

  seated under the same conditions for 45 minutes with 50 μl of a 20-fold diluted solution of anti-mouse Ig G bound to fluorescein (Miles-Yeda Ltd, Israel, Code N 65-171) and the process was carried out. wash in the same way as

  mentioned above After having dried slightly, the drum

  carbonate pon (0.05 M, p H 9.5) containing 10% glycerol

  rine was put aside and a glass cover was placed over it to make an observation using the fluorescence microscope (Olympus, Japan, Model AH-RFL-LB) o Strong fluorescence was observed on the surface of T-cell 24 We used anti-mouse Ig G linked to fluorescein

  after having subjected it six times to absorption with the

lule T 24.

  C IDENTIFICATION OF IMMUNOGLOBULIN CLASSES.

  The immunoglobulin classes of the antibodies secreted by hybridomas T-OO 9 to T-012 have been identified by the solid phase enzyme immunoassay which uses anti-immunoglobulin (Ig), anti-Ig C and anti -Ig M (Cappel Lab Inc, USA, Catalog N 3211-0231, 3211-0081, 3211-0201) as bound anti-mouse immunoglobulin antibody

  with peroxidase The results are given in the table

beau 1 below.

  EXAMPLE 5 PREPARATION OF THE ANTIBODY AGAINST THE CELL

HUMAN BLADDER CANCER.

  IMMUNIZATION AND FUSION OF CELLS.

  (1) PREPARATION OF IMMUNOGENIC CELLS: Incu-

  ber the transi- epithelium cancer cell line

  of the human urinary bladder T 24 (2 x 105) obtained by subculturing in vitro for a long period, at 370 C

  in a humid atmosphere containing 5% carbon dioxide

  nick in MEM medium which contains 10% (v / v) fetal bovine serum and kanamycin sulfate (final concentration, 60 mg / l) in a 100 mm culture vessel (Falcon 3003, Becton-Dickinson, USA) After three days of culture, the cells were exfoliated in force and

  removed supernatant by centrifugation.

  The cells were then washed with PBS.

* approximately 2 x 106 cells per culture vessel.

  (2) CULTURE OF MYELOMAS: We cultivated the line of cel-

  Sp 2 / O-Ag 14 mouse myeloma lules (105 per ml) in D-MEM containing 10% (v / v) fetal bovine serum, 1 m M pyruvic acid, 2 m M glutamine and 60 mg / l sulfate

  of kanamycin by transplanting every three days The cell

  lule was adjusted to a cell concentration of 2.5 x 105 per ml the day before cell fusion

  following and cultivated in the medium mentioned above.

  (3) IMMUNIZATION WITH T 24 CELLS: T 24 cells (5 x 10 6 10 x 10 6) obtained in (1) above and suspended in 0.4 ml of PBS were injected intraperitoneally into mice BALB / c females (NIHON CHARLES LIVER, JAPAN, 9 weeks old) four times

  approximately every month for immunization.

  (4) CELL MERGER: The cell fusion was carried out

  lules according to the method of Kbhler and Milstein.

  On the fourth day after the final immunization, the mice were killed to remove the spleen. The spleen was carefully detached, passed through a steel mesh, centrifuged at 400 x g and added to

  cells precipitated from tris-H Cl buffer (p H 7.65)

  holding 0.747% ammonium chloride to remove erythrocytes and the cells were recovered by centrifugation (400 x g) After adding MEM to the cells, the centrifugation was carried out at 400 x g and a

  again the cells suspended in fresh MEM.

  After repeating this washing process three times, we

  finally suspended the cells in MEM.

  The Sp 2/0-Agl 4 cells were exfoliated from the culture vessel by pipetting and transferred to a centrifuge tube. After centrifugation (400 xg), the recovered cells were suspended in MEM, they were centrifuged (400 xg) to remove the serum and

  they were again suspended in MEM.

  The spleen cells (8.5 x 107) and Sp 2/0-Agl 4 cells (1.7 x 107) were mixed, carefully pipetted and centrifuged (400 xg). After removing the supernatant, detached the precipitate by gently hitting the centrifuge tube To the detached cells, 0.6 ml of a solution of polyethylene glycol (PEG 1000) at 30 lo (v / v) was added in MEM maintained at 37 WC After having gently stirred , the solution was allowed to stand at room temperature for 5 minutes. Centrifugation was carried out at 7 xg for 2 minutes and 5 ml of MEM were gradually added. After stirring gently, centrifugation (400 xg) was carried out at temperature

  ambient for 5 minutes The supernatant liquid has been removed

  Giant, 5 ml of MEM was added to the centrifuged precipitate of

  the same way and the supernatant liquid was removed.

  To the precipitated cells, 5 ml of D-MEM-FBS medium was added. After pipetting, 30 ml were added.

  D-MEM-FBS to the resulting cell suspension (5 ml).

  About 25 ml of the suspension was inoculated into each 25 cm culture container (Corning, USA, C-25100) and incubated while allowing them to stand in a humid atmosphere containing 5% C 02 to 370 C overnight, these incubation conditions being used in the following procedures.

  The next day, the fresh culture was pipetted-

  it was transferred to a centrifuge tube and

  centrifuged at 400 x g After removing the excess liquid

  swimming, the cell pellet was suspended in 35 ml HAT medium Each 1 ml fraction of the suspension was inoculated into each well of the 24-well culture plate (Nunc 143982, Nunc, Denmark) and cultured for one week To each scoop, we added

  0.25 ml of HT medium every two or three days.

  B SELECTION AND GROWTH OF THE PRODUCING HYBRIDOME

THE ANTIBODY.

  A solid phase enzyme immunoassay was used to examine the production of the T 24 antibody in the supernatant of each well

  the 15th day after the fusion of the cells.

  The T 24 cells fixed on a plate (Falcon 3072) were reacted with 40 μl of the supernatant liquid in

  each scoop at room temperature for two hours

  res, washed thoroughly with PBS, reacted for two hours with 100 µl of peroxidase-bound anti-mouse immunoglobulin antibody (Cappel Lab Inc USA, Catalog No. 3211-0231) which was diluted 1000 times with horse serum, and washed thoroughly with PBS 200 µl of buffer was added

  citrate (0.1 M, p H 4.5) containing 1 mg / ml of o-phenylene-

  diamine as substrate and 0.4 µl / ml and were made

  gir for 30 minutes to obtain coloring.

  Cloning was carried out in two wells containing

  antibodies that react with T cells 24

  by limiting dilution culture, i.e. 100 hybri-

  domes from each of two positive wells in antibody production were suspended in D-MEM-FBS and mixed with spleen cells obtained from BALB / c mice in the same manner as that used in the fusion and adjusted in D-MEM-FBS (10 cells). The cell concentration was adjusted to 5 x 10 per ml by adding DMEM-FBS. Each 0.2 ml fraction of the cell mixture was inoculated into each well of the

  96-well culture plate (Falcon 3072, Becton-Dickin-

  bran, USA) and cultured On the 12th day after the initiation of the culture, the production of the antibody in each well was examined by means of the enzyme immunoassay in phase

  above mentioned solid We obtained a total of 10 hybri-

  domes including T-007 and T-008 shown in Table 1

below.

  EXAMPLE 6 PROPERTIES OF THE ANTIBODY AGAINST THE CELL

OF CANCER OF THE HUMAN BLADDER.

  COMPARED TO 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 obtained by subculturing in vitro; Intestine 407 (human fetal intestine cell line), Du 145 (human prostate cancer cell line) and 253 J (human renopelvic cancer cell line) as well as T 24 (bladder cancer cell line

  human urinary) As seen in Table 1, the an-

  antibody secreted by T-007 hybridoma reacted strongly

  with the T-24 and showed slight or relatively reactive

  high with other cells The antibody secreted

  T-008 reacted strongly with T 24 and did not react with Intestinal 407 or 253 J.

  B COLORING OF THE CELL BY ANTIBODY TECHNIQUE

FLUORESCENT.

  The T 24 cells were stained by the technique

  than indirect fluorescent antibody using the an-

  antibody secreted by hybridoma T-008 Namely, cells

  T 24 cells were fixed on a glass slide without fluorescence, reacted with the supernatant (50 μl) at 370 ° C. for 45 minutes in a human atmosphere and were

  washed by treatment for 3 to 5 minutes with a solution

  tion of PBS containing 1 X bovine serum albumin, m M HEPES and 0.1% sodium nitride, this process

  of washing being repeated three times The reaction was carried out

  read again under the same conditions for 45 minutes with 50 μl of a 20-fold diluted solution of anti-mouse Ig G bound to fluorescein (Miles-Yeda, Ltd, Israel, Code N 65-171) and the washing process of the same

  way as mentioned above After drying lightly-

  the carbonate pad (0.05 M, w H 9.5) containing X glycerin was set aside and a glass cover slip was placed over it for observation.

  fluorescence microscope (Olympus, Japan, model AH-RPL-

  LB) Strong fluorescence was observed on the surface of T cells 24 Anti-mouse IgG linked to fluorescence was used after being subjected to absorption six times

with T cells 24.

  (C) IDENTIFICATION OF THE CLASSES OF IMMUNOGLOBULIN Eo The classes of antibody immunoglobulins

  secreted by hybridomas T-007 and T-008 have been identified

  relied on by the solid phase enzyme immunoassay used

  using anti-immunoglobulin (Ig), anti-Ig G and anti-Ig M (Cappel Lab Inc USA, Catalog N 3211-0231, 3211-0081, 3211-0201) as bound anti-mouse immunoglobulin antibody

  with peroxidase The results are given in the table

bleau below.

TABLE 1

  SPECIFICITY Intestine Cancer Cancer Cancer Leukemia class 1 '

  Human Hybri Human Fetal Human Human Immuno-

  renal globulin human dome bladder prostate pelt T-24 I407 Du-145 253-J K-562 T-001 + + + + Ig G T-002 ++ + Ig G T-003 +++ + ++ + Ig G T-004 +++ ++ + Ig G T-005 +++ ++ + ++ Ig G T-006 +++ + + Ig G T-007 ++++ ++ ++ ++ + Ig G T-008 ++++ ++ Ig G T-009 ++++ + + Ig M T-010 +++ + Ig G T-0-11 ++++ + + + Ig G T- 012 ++++ + ++ Ig G +: positive: negative EXAMPLE 7: PRODUCTION OF ANTIBODIES (IN VITRO CULTURE): The hybridoma T-OO 1, T-002, T-003, T was suspended -004, T-005, T-006, T-007, T-008, T009, T-010, T-Oll or T-012 in D-MEM containing 20 X fetal bovine serum, 2 m M glutamine , 1 m M pyruvic acid, 4.5 g / l glucose,

  5-mercaptoethanol 5 x 10-5 and 50 mg / 1 ka sulfate

  namycin at a cell concentration of 1 x 105 per ml.

  The resulting suspension (25 ml) was inoculated into a container.

  75 cm 2 pient for tissue culture (Corning, USA) and

  incubated in a 5% C 02 to 37 C incubator

  On the fourth day, the supernatant was recovered from the container in the stationary growth state. The number of cells developed was approximately 2 x 10 6 per

  ml in each hybridoma and the antibody content of the li-

  quides supernatants was respectively 1.9 (T-O Ol),

  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-OO 9), 3.1 (T-O 10),

4.0 (T-Ol) and 2.3 µg / ml (T-012).

  IN VIVO CULTURE: On the 10 th to 30 th day after intraperitoneal injection of 0.5 ml of pristane, BALB / c mice were inoculated intraperitoneally, 5 × 10 6 of each T-OO 1 hybridoma at T-012, developed in vitro Two or three weeks later, the ascites were recovered and centrifuged

  at 100 x g, 4 C, 15 minutes to obtain the supernatant liquid

  giant ascites in an amount of about 30 ml for each hybridoma from 10 mice The antibody content of the supernatants was respectively

  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-OO 9),

  1.4 (T-O 10), 2.2 (T-Oil) and 3.0 mg / ml (T-012).

  These antibodies were administered to ICR mice (10 animals in each group) in an amount of 2 g / kg orally, 400 mg / kg intraperitoneally or 200 mg / kg intravenously and the mice were observed for 14 days No deaths due to the antibody were observed. Furthermore, the forms of the T-OO 1 to

  T-012 were approximately spherical, their dimensions

  sions were 10-20 V, most being about 15 p, and the hybridomas floated and adhered weakly to the

container wall.

  EXAMPLE 8: IDENTIFICATION OF HUMAN BLADDER CANCER

BY ANTIBODY.

  (1) SUBJECT: A tissue containing cancer cells from

  the human urinary bladder obtained by a surgical operation

  gicale was coagulated with 95% alcohol coated in

  paraffin to prepare a 4 µm sample.

  (2) IDENTIFICATION: The identifications were carried out by a fluorescent antibody technique using antibodies (ascitic supernatant) secreted by

the T-003 hybridoma.

  Namely, we added to the fabric sample

  0.1 ml of the solution, diluted 100 times, of the above antibody

  mentioned and incubated at room temperature for one hour After washing thoroughly with PBS, 0.1 ml of fluorescein-bound anti-mouse Ig G (Miles, USA) diluted 10 O was added and incubated at room temperature for 1 hour After removing unreacted fluorescein-bound anti-mouse Ig G by washing with PBS, fluorescence was observed on each tissue sample subject of

  the glass coverslip using a fluorescence microscope

  cence (Olympus Vanox, Olympus, Japan) Almost no fluorescence was observed on normal tissue while strong fluorescence was observed on bladder cancer tissue In order to avoid non-specific binding; anti-mouse Ig G linked to fluorescein was treated beforehand by absorption with the

  T 24 cells of human bladder cancer.

EXAMPLE 9:

  (1) PEROXIDASE LABELING OF THE ANTIBODY SECRETED BY

THE HYBRIDOME T-006.

  4 mg of horseradish peroxidase (Sigma Type VI, Sigma, USA) were dissolved in 1 ml of distilled water and

  added 60 l of a 0.1 M solution of Na I 04 prepared immediately

  diat before use, and mixed at temperature

  room for 20 minutes The solution was dialyzed

  sultante all night long against a buffer at 1 m M

  sodium acetate (p H 4.4) and 20 µm of solution was added

  0.2 M sodium carbonate immediately after,

  the antibody secreted by the T-006 hybridoma (li-

  quid purified ascites supernatant, protein content of mg) dissolved in 1 ml of 0.01 M carbonate buffer (p H 9.5) and kept at room temperature for two hours while stirring. After reaction, added 0.1 ml of freshly prepared aqueous Na BH 4 solution (4 mg Na BH 4 dissolved in 1 ml of distilled water) and allowed to stand at 40 ° C for two hours and dialyzed

overnight against PBS.

  The resulting mixed solution was sent (R) to a column of Sephadex G-100 (R) (Pharmacia, Sweden), eluted with PBS and the first fraction was recovered, the absorbances at 280 nm and 403 nm of which corresponded to each other. To 1 ml of the fraction (antibody-enzyme binding product), 10 mg of rabbit serum albumin was added for dissolution and was stored at -70 ° C.

until use.

  (2) LABELING OF THE ALKALINE PHOSPHATASE OF THE ANTIBODY

SECRETED BY THE HYBRIDOME T-006.

  5 mg of alkaline phosphatase type VII (Sigma, USA), dialyzed before against PBS to completely eliminate ammonium sulfate, and 17 mg of antibody secreted by the hybridoma T-006 were dissolved in reduced PBS to a total volume of 1 ml To the resulting solution was added 10 µl of a 20% glutaraldehyde solution

  and stirred at room temperature for two hours

  res After reaction, the reaction mixture was sent to a column of Sephadex G-200 (R) (Pharmacia, Sweden) supplemented with tris-H Cl buffer (p H 7.6) and eluted with water. using the same buffer A high molecular weight fraction was collected, from the empty volume at the point of Ic G elution, up to 5% (v / v) of bovine serum albumin was added thereto, it was sterilized by passage through a millipore filter (0.22, Millipore, USA) and kept at 40 C

in the dark until use.

  (3) LABELING BY THE e-GALACTOSIDASE OF THE SECRET ANTIBODY

BY THE HYBRIDOME T-006.

  The antibody secreted by the T-006 hybridoma was labeled with $ galactosidase according to the method of (2) above. The e-galactosidase used was of the Sigma type.

quality IV (Sigma, USA).

  (4) I MARKING OF THE ANTIBODY SECRETED BY

THE HYBRIDOME T-006.

  To 10 μl of Na I solution at 100 m Ci / ml (free of support, Amersham, USA), 50 μl of the antibody solution secreted by hybridoma T-006 (purified ascites supernatant liquid, content protein of 1.0 mg / ml) and 30 µl of 0.5 M phosphate buffer (p H 7.2) containing 0.30 mg / ml of chloramine T and carefully mixed 15 seconds later, added 100 1 µl of PBS saturated with L-tyrosine and mixed immediately

  chromatographed the resulting mixture on a char column

  gated with Amberlite IRA 400 and eluted with PBS containing 1% bovine serum albumin The fraction eluted

  was collected and kept at 4 C until use.

  The specific activity of the labeled product was 1.0 p Ci

per mg of antibody protein.

  (5) FLUORESCEIN LABELING OF THE SECRET ANTIBODY

BY THE HYBRIDOME T-006.

  To 1 ml of the solution of the antibody secreted by the hybridoma T-006 (purified ascites supernatant liquid) at a concentration of 10 mg / ml, 0.1 ml of 0.5 M carbonate buffer (p H 9 was added) , 3), a further 0.1 mg of fluorescein isothiocyanate powder was added and the mixture was stirred at 4 ° C. for 6 hours without bubbling. Immediately after reaction, the reaction mixture was sent to a Sephadex G-25 column ( R) (Pharmacia, Sweden) to remove unreacted low molecular weight substances

  and we got the high molecular weight fraction

  sirée The product obtained was stored at 4 C in the dark

until use.

  (6) TETRAMETHYLRHODAMINE LABELING OF THE ANTIBODY

SECRETED BY THE HYBRIDOME T-006.

  At 1 ml of a 10 mg / ml solution of the antibody se-

  created by T-006 hybridoma (ascites supernatant

  purified), 0.1 ml of tetra carbonate buffer was added

  methylrhodamine-isothiocyanate and the mixture was stirred at 40 ° C. for hours without bubbling. After reaction, the reaction mixture was immediately sent to a Sephadex G-20 (R) column (Pharmacia, Sweden) to remove the low molecular weight substances having no not reacted and

  the desired high molecular weight fraction was obtained.

  The product was stored at 4 ° C. in the dark before use.

tion.

  (7) BIOTIN MARKING OF THE ANTIBODY SECRETED BY

THE HYBRIDOME T-006.

  1 m M d-biotin (244 mg) (Walko Pure Chemical Industries, Ltd, Japan) and 1.5 m M N-hydroxysuccinimide

  (173 mg) (Eastam Kodak, USA) were dissolved in a mixture

  8 ml of dimethyl sulfoxide (Walko Pure Chemical Indus-

  tries, Ltd, Japan) and 5 ml of 1,2-dimethoxyethane (Nakarai Chemical, Japan) To the resulting solution,

  added a solution of 206 mg (1 m M), of N, N ') dicyclohexyl-

  carbodiimide (Kanto Chemical, Japan) in 0.5 ml of 1,2-

  dimethoxyethane and reacted at 4 C overnight.

  The resulting precipitate was separated by filtration to obtain a filtrate. The solvent contained in the filtrate was removed in vacuo and the residual oily material was dissolved in 10 ml of dichloromethane (Walko Pure Chemical Industries, Ltd, Japan) and cooled. at 4 C 0.1 ml of 0.1 M Na HCO 3 solution (4 C) was added and stirred to mix well. The resulting dichloromethane phase was removed, 10 ml of 0.1 H Na 3 HCO was added. M and then 10 ml

  distilled water at 4 ° C, and these procedures were repeated again.

  The resulting dichloromethane phase was added to anhydrous sodium sulfate powder (Koisumi Chemical,

  Japan) to dehydrate The powder was separated by wire

  tration and m-hexane was gradually added to the fil-

  trat to make cloudy The solution was cooled to -200 C, the precipitated crystal was placed in a desiccator to

  remove the solvent and allow it to dry.

  duit, the ester of biotin-N-hydroxysuccinimide.

  The biotin-N-hydroxysuccinimide was dissolved in dimethyl sulfoxide and the concentration was adjusted to 1 mg / ml The solution (60 μl) was mixed with 1 ml of the solution of the antibody secreted by the T hybridoma -006 (purified ascites supernatant, protein content of 1 mg / ml) and it was reacted at room temperature for 4 hours. After reaction, the solution was dialyzed against PBS at 4 ° C. for three days. , the dialysis fluid has been changed three times The dialysate (solution

  in the dialysis tube) was kept at 4 ° C until needed.

station.

  EXAMPLE 10: IDENTIFICATION OF HUMAN BLADDER CANCER

BY MARKED ANTIBODIES.

  (1) TOPIC: Tissues containing bladder cancer

  human urinary tract were obtained during surgical operations

  ie, the raw fabrics 5 x 5 x 2 mm in diameter

  mensions or less are set while stirring at 4 C for

  6 hours in a PLP fixing liquid (phos buffer)

  sodium phate, p H 6.2, containing 0.01 M Na IO 4, 0.075 M lysine and 3 X paraformaldehyde) The fixed tissues have

  been washed by treating them with PBS containing 10% su-

  crose (4 C, overnight), PBS containing 15% su-

  crose (40 ° C., 6 hours), PBS containing 20% sucrose (4 ° C., 6 hours), and then in PBS containing 20% of

  sucrose and 10% glycerin (4 C, 1 hour) successively.

  After being washed, the fabric was coated, frozen in dry ethanol and subjected to a cryostat to obtain

a 10 Nm frozen specimen.

  (2) IDENTIFICATION METHOD: The frozen sample was placed on a microscope slide coated with bovine serum albumin and dried after having been washed three times with PBS at 40 ° C. for 5 minutes, we reacted

  the sample with 0.005 M periodic acid at time

  room temperature for 10 minutes after washing

  three times with PBS maintained at 4 C, the water was treated

  sample at room temperature for 10 minutes with PBS containing 10% horse serum The sample was placed on the sample using a capillary pipette.

  antibody solution diluted 10-fold, from hybrid

  dome T-006 labeled with peroxidase, alkaline phosphatase, fluorescein, rhodamine or biotin prepared in Example 9 and reacted at room temperature for 45 minutes and then washed five times with PBS at 4 ° C. The sample obtained has

  was identified as follows.

  (a) FLUORESCENT ANTIBODY METHOD: In antibodies

  labeled with rhodamine and fluorescein, the sample

  tillon was drowned in glycerin and observed under a

  fluorescence microscope (Olympus Vanox, Olympus, Japan).

  In the antibody labeled with biotin, a drop of avidin at 5 μg / ml linked to fluorescein (Funakoshi Yakuhin, Japan) was added to the sample and it was reacted at 37 ° C. for 1 hour After washing five times with PBS, the reacted sample was drowned in glycerin and examined under a microscope.

  fluorescence (Olympus Vanox, Olympus, Japan).

  (b) ENZYMATIC IMMUNOTEST: In the peroxidase-labeled antibody, the sample was reacted with a 0.1 M tris-H Cl buffer (p H 7.6) containing 0.02 ml / ml H 202

  at 1% and 0.5 mg / ml of 3,3-diaminobenzidine-H Cl at the time-

  room temperature for 10 minutes After reaction, the reacted sample was washed with PBS, at 4 ° C., three times, and with distilled water once After having been colored with veronalacetate buffer (p H 4 , 0) containing 1% methyl green, the sample was dehydrated with ethanol, dialysed with xylol, coated in balm and

  observed the color (brown) under the microscope.

  In the antibody labeled by the alca- phosphatase

  line, we reacted the sample with buffer

  tris-H Cl 0.2 M (p H 8.5, filtered immediately after use

  sation) containing 0.39 M magnesium sulfate, 0.2 X lead citrate, 0.6% 0-glycerophosphoric acid and 4 X sucrose at room temperature for 10 minutes After reaction, the sample having reacted was washed three times with PBS maintained at 40 C and then once

  with distilled water The sample was then reacted

  in a 1% solution of yellow ammonium sulfide at room temperature for 5 minutes, it was washed completely with PBS, incorporated into glycerin

  and the color (black-brown) was observed under the microscope.

  These results are shown in Table 2.

TABLE 2

  Method Reactivity labeling the antibody Tissue Normal cancerous tissue Fluorescein method + the fluorescent antibody Rhodamine + Biotin + Immunotest Peroxidase + 1 enzymatic Phosphatase + 2 alkaline +: positive fluorescence + 1: brown observed + 2: brown not observed: negative

  EXAMPLE 11 SCINTIGRAPHIC IMAGE OF THE CANCER CELL

BY ANTIBODY.

  A T 24 cell line (1 x 10 cells) of human urinary bladder cancer was transplanted subcutaneously into the right thigh of 8-week-old female BALB / c nu / nu mice (three animals in each group ) 30 days later, the size of the tumor

  was 1 cm 2 or more and intravenous injection

  the antibody labeled with I 125 secreted by the hybridoma T-006, prepared in Example 9, in a unit quantity of 35 p Ci per mouse or the mouse Ig G labeled with u 12 for comparison in a unit quantity of 10 p Ci per mouse 96 hours later, it was carried out using a camera

  gamma whole body scintigraphy.

  A remarkable increase in the tumor was observed.

  radioactive mulation in all animals to which

  the I 125 labeled antibody of the invention was administered

  tion while less accumulation was observed in

control animals.

  EXAMPLE 12 MARKING OF THE ANTIBODY WITH HYDROCHLORIDE

OF DOXORUBIN OR MITOMYCIN.

  After adjusting to 10.4 mg / ml by addition

  distilled water, added to the antibody (liquid supernatant

  giant purified ascites) from the hybridoma T-002, T-008 or T-O 10, 13.0 mg of mitomycin, and then hydrochloric acid was added while stirring in order to adjust the p H

  of the 4.75 solution and simultaneously 3.7 mg of chlorhy-

  1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide drate.

* After having reacted for 10, 30 or 60 minutes, the reaction was stopped by adding 2 ml of acetate buffer (p H 4.70) The reaction mixture was then dialyzed against 5 liters of distilled water at 4 C for 72 hours while changing the dialysis solution three times After concentrating, the dialysate was sent to a column 1.5 cm in diameter and 55 cm in height loaded with

  Sephadex G-25 (R) (Pharmacia, Sweden) to eliminate com-

  completely low molecular weight substances In order to obtain the mitomycin-antibody binding product, the

  resulting solution was lyophilized at -20 o C The propor-

  mitomycin binding in products by

  mg of antibodies are shown in Table 3.

  The binding products of doxo- hydrochloride

  rubicin with the antibodies were obtained in the same way as mentioned above The binding proportions of doxorubicin hydrochloride in the products per mg

  of antibodies are shown in Table 3.

  On the other hand, these anti-cancer agent binding products were administered to IRC mice of groups of 10 animals each in a unit quantity of 400 mg / kg orally, 100 mg / kg intraperitoneally. or 50 mg / kg intravenously None

  death was observed for 14 days.

TABLE 3

  Anti-Antibody agent Binding rate (pg / mg) cancer min 30 min 60 min. derivative of T-002 4 8 10 mitomycin "T-008 4 8 9

"T-O 10 4 7 10

  hydrochloride derived from T-002 4 7 11 from doxo "T-008 4 7 10 rubicin" T010 4 7 10

  EXAMPLE 13: IN VITRO ANTI-TUMOR EFFECT OF THE ANTIBODY.

  To examine the anti-tumor effect of the antibody, or of the binding product with the anticancer agent prepared in Example 12, a T 24 cell line was used.

  human urinary bladder cancer transplanted in vitro.

  That is, the cells were adjusted with Eagle MEM containing 10% fetal bovine serum to a cell concentration of 5 x 104 per ml, placed in a petri dish in fractions of 5 ml each, and incubated them at 370 C for 24 hours in an incubator at 5% of C 02 The antibody derived from the hybridoma T-008 or the antibody linked to the mitomycin derived from the hybridoma T-008 (product d '' reaction of 60 minutes) was added in

  the box and cultivated later 24 hours after the addi-

  1 p Ci / ml of L- (U-C 14) leu-

  cine (RCC, USA, specific radioactivity of 342 m Ci / m mole) to the reaction mixture and incubated for two hours. After culture, the culture medium was eliminated and the cells were washed three times with PBS (p H 7.2) and cooled to 00 C, treated with a solution

  5% trichloroacetic acid and the cells were transferred

  washed on a paper filter and dried

  measured the radioactivity incorporated into the protein cel-

  using a liquid scintillation counter

(Packard, USA).

  The results are shown in Figure 1.

  In Figure 1, the ordinate represents the amount of radioactivity (C leucine) as a percentage of the amount

  incorporated by the untreated witness.

  As shown in Figure 1, the concentration

  tion indicating 50% inhibition of the incorporation of radioactivity was decreased by the binding product

  the antibody and initomycin compared to the control.

  EXAMPLE 14: IN VIVO ANTI-TUMOR EFFECT OF THE ANTIGEN.

  A T 24 cell line (6 x 10 cells per animal) of human urinary bladder cancer was transplanted intraperitoneally to female BALB / c nu / nu mice in groups of 10 animals each 5 and 72 hours afterwards. antibody secreted by hybridoma T-008 or the binding product with mitomycin of the antibody derived from T-008 (60-minute reaction product, 50 μg / ml of mitomycin) was injected intraperitoneally into mice by a unit quantity of 450 pg per mouse (20 pg of mitomycin per mouse) and the proportion of survivors has

  observed for 60 days to estimate anti-

tumor of the administered drug.

  The results are shown in Figure 2.

  In Figure 2, the ordinate represents the survival rate

  vant (%) and the abscissa represents the number of days after

  transplant of tumor cells.

  As can be seen from Figure 2, the number of days for which 50% of the animals survived

  was extended by the mitomycin-anti-

  In addition, the prolongation of the

  shelf life by administration of the antigen alone.

Claims (37)

  1 Method for preparing an antibody against the surface antigen of a human bladder cancer cell, characterized in that it comprises the preparation   -5 of a hybridoma between a cell capable of producing an-   antibody and a cell which can be permanently preserved by in vitro subculturing and td recovery of   the antibody secreted by the hybridoma.   2 The method of claim 1, wherein the cell capable of producing the antibody is a cell   spleen, a lymph node cell, a cell -   peripheral blood leukocyte or a mixture thereof.   3 The method of claim 2, wherein   the cell is derived from a mouse.   The method of claim 3, wherein the mouse is a BALB / c mouse or a hybrid mouse thereof. The method of claim 1, wherein the cell which can be permanently stored by in vitro subculturing is a myeloma cell 6 The method of claim 1, wherein the cell which can be permanently stored   by subculturing in vitro is deficient in hypoxanthine-   guanine phosphoribosyltransferase or thymidine kinase.   7 Method according to claim 5 or 6, in   which the cell is derived from a mouse.   8 Method according to claim 7, in which the cell is derived from the P 3-X 63-Ag 8 cell line,   P 3 / NSI / 1-Ag 4-1, Sp 2/0-Agl 4 or X 63-Ag 8 653.   9. The method of claim 1, wherein the cell capable of producing the antibody is derived from an animal immunized with bladder cancer cells. human urinary.   Method according to claim 1, in the-   which hybridoma is prepared in the presence of polyethylene- glycol.   11 Method according to claim 1, in the-   which hybridoma is grown in a medium containing hypoxanthine, aminopterin and thymidine for selection.   12 Method according to claim 1, in the-   which hybridoma is cloned to obtain a unique clone.   13 The method of claim 12, in the-   which cloning is carried out by limited dilution culture   aunt, culture on soft agar or culture on gel brine.   14 The method of claim 1, in the-   which hybridoma is cultured in vitro for secretion of the antibody.   15 The method of claim 1, in the-   which hybridoma is transplanted into an animal and grown   in vitro and the secreted antibody is separated from a cor- animal porel.   16 The method of claim 15, in the   which body fluid is serum or ascites.   17 The method of claim 15 or 16, in which the animal is a mouse.   18 The method of claim 17, in the-   which mouse is a BALB / c mouse or a hybrid mouse of these.   19 Method according to any one of the claims   1 to 18, in which urinary bladder cancer   human is a T 24 cancer cell line of the epi-   transitional thelium of the human urinary bladder.   20 Hybridoma producing an antibody against the surface antigen of a human urinary bladder cancer cell, prepared from a cell capable of producing the antibody and a cell which can be stored   permanently by subculturing in vitro.   21 Hybridoma T-001, T-002, T-003, T-004, T-005,   T-006, T-007 i T-008, T-009, T-010, T-011 or T-012.   22 Antibodies to the surface antigen of a human urinary bladder cancer cell, prepared   according to the method of claim 1.   23 Antibody according to claim 22 which is secreted by the hybridoma T001, T-002, T-003, T-004, T-005,   T-006, T-007, T-008, T-009, T-010, T-011 or T-012.   24 Reagent for classification and / or identification   tificat :: on of cancer of the human urinary bladder, which   contains one or more antibodies according to claim- 22.   A reagent according to claim 24 which   also holds a support or diluent.   26 Reagent according to claim 24 or 25, wherein the cancer of the human urinary bladder is the   trans 24 epithelium cancer T cell line of the urinary bladder.   27 Reagent for cancer identification   the urinary bladder of humans or animals.   28 Anti-restriction derivative or product body according to claim 22.   29 A derivative according to claim 28, in which a radioactive substance, a fluorescent dye, an enzyme, a labeling substance for electron microscopy or a residual group containing a structure   for the secondary combination of it is related (e) chi- even to the antibody.   Restriction product according to claim 28, which is obtained by restrictive cleavage of the antibody   by chemical treatment or enzymatic treatment.   31 Derivative according to Claim 28, in which the radioactive substance is 1 I. 32 Derivative according to Claim 29, in which   the fluorescent dye is fluorescein or rhodamine.   33 The derivative according to claim 29, wherein   the enzyme is peroxidase or alkaline phosphatase.   34 The derivative according to claim 29, wherein the structure is biotin.   Reagent for classification and / or identification   tification of human urinary bladder cancer, which contains one or more derivatives or restriction products according to claim 28.   36 Reagent according to claim 35 which   also holds a support or diluent.   37 Classification and / or identification process   cation of human urinary bladder cancer with utility   The reagent according to claim 24 or 35.   38 The method of claim 37, in the-   what a subject containing the bladder cancer cell   human urine is in contact with the reagent and   is the antigen-antibody reaction.   39 The method of claim 38, in the-   what the subject is derived from humans or animals.   Pharmaceutical agent in the form of quan-   unitary unit for the treatment of human urinary bladder cancer containing one or more antibodies according to claim 22.   41 The agent of claim 40 which contains more a support or a diluent.   42 Antibody binding product according to re-   vendication 22 with an anti-cancer agent.   43 Product according to claim 42, in the-   which anti-cancer agent is mitomycin hydrochloride or doxorubicin.   44 Pharmaceutical agent for the treatment of   human urinary bladder cancer containing one or more   several binding products according to claim 42.   Pharmaceutical agent according to claim   or 44, which is in injectable form.   46 Method for the treatment of human urinary bladder cancer with the use of the agent according to claim 40 or 44.