WO1994000591A1 - Anticorps monoclonal, son procede d'obtention, cellule productrice de cet anticorps, et methode de diagnostic - Google Patents

Anticorps monoclonal, son procede d'obtention, cellule productrice de cet anticorps, et methode de diagnostic Download PDF

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Publication number
WO1994000591A1
WO1994000591A1 PCT/JP1993/000854 JP9300854W WO9400591A1 WO 1994000591 A1 WO1994000591 A1 WO 1994000591A1 JP 9300854 W JP9300854 W JP 9300854W WO 9400591 A1 WO9400591 A1 WO 9400591A1
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WIPO (PCT)
Prior art keywords
antibody
monoclonal antibody
src
cells
cancer
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PCT/JP1993/000854
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English (en)
Japanese (ja)
Inventor
Hisaaki Kawakatsu
Yumiko Takagaki
Junichi Yano
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Nippon Shinyaku Co., Ltd.
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Publication date
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Publication of WO1994000591A1 publication Critical patent/WO1994000591A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • Monoclonal antibodies manufacturing methods, antibody-producing cells, and diagnostic techniques
  • the present invention relates to a monoclonal antibody specifically recognizing a site present at the C-terminus of a src family gene product and having a function of controlling the activity of a phosphatase.
  • the src gene was first known as an oncogene of Rous sarcoma virus (v-src). The corresponding oncogene precursor in cells of higher organisms was called the c-src gene.
  • the src gene is located at human chromosome locus 20ql3.3.
  • the src gene has an analog called the src family. For example, the yes gene is located at human chromosome 18q21.3, the fgr gene is located at 1P36.1, and the fyn gene is located at 6q21.
  • tyrosine kinase belonging to src family are known, and most of them are activated by phosphorylation at C-terminal domain. Has a tyrosine residue that decreases. It has been found that c-src, obtained by substituting this tyrosine with another amino acid by genetic engineering techniques, has a carcinogenic potential.Research on the src gene has only just begun, From the study of the amino acid sequence and the study of deficiency, the potential for application to pharmaceuticals and diagnostics is expected.
  • extracellular signals are transmitted via receptors to the src family, which hangs inside the cell membrane, and the information is transmitted by the SH2-containing protein. Propagated to signaling in the tall (IP), diglyceride (DG), and GTPase systems, causing a variety of responses.
  • IP tall
  • DG diglyceride
  • GTPase GTPase
  • crk has only the SH2 region without tyrosine phosphatase activity. crk is thought to cause cell carcinogenesis by binding to the product of the src family, which has thi-sin lin oxidase activity, using the SH2 region (Mayer et al. Nature, 332, 272-275 (1988)).
  • c-src has been reported to be high in colorectal cancer, neuroblastoma, retinoblastoma, small cell lung cancer, colon cancer, breast cancer, and rhabdomyosarcoma (S Pahlman and U. Hammerling, Am. Rev.
  • C-fyn, c-lyn, c-lck, c-hck, c-blk, etc. which are mainly expressed in immunocompetent cells, are deeply involved in lymphocyte-specific signaling.
  • C-lck is said to be highly expressed in T lymphomas and colon cancer in the-part
  • C-lyn is highly expressed in T cell leukemias.o
  • the binding site of this antibody has been identified as the SH2 region of src, but this has not been confirmed (Lipsiuli cill. J. vii ul. 8, 352-360 (1983)).
  • Kempridge has marketed a polyclonal antibody using the C-src (4-1 to 485) peptide, it lacks specificity and binding ability, and it is also SH2. It has nothing to do with the region or the C-terminus of the src family.
  • the present inventors have made intensive studies and as a result, have discovered a monoclonal antibody that specifically exists at the end of the src family and has a function of controlling the phosphorylation of the enzyme. Successful acquisition resulted in the completion of the present invention.
  • the gist of the present invention is as follows: 1 The phosphatase present at the end of the src family A monoclonal antibody itself that specifically recognizes a site having a function of controlling the activity; (2) the peptide of SEQ ID NO: 1 to obtain the monoclonal antibody; (3) an antibody-producing cell that produces the monoclonal antibody 4 The monoclonal antibody is used for pathological diagnosis.
  • the monoclonal antibody according to the present invention can be obtained as follows.
  • the C-terminal peptide of the above C-src that serves as an antigen is chemically synthesized.
  • peptides having an amino acid sequence as described in Sequence Listing 1 described below can be synthesized.
  • the peptide as shown in SEQ ID NO: 1 is a novel compound, and the present inventors are the first to obtain a monoclonal antibody of the c-src gene using this peptide as an antigen.
  • the peptide obtained as described above is converted into an appropriate carrier, for example, a keyhole limpet hemocyanin or the like. Can be immunized by binding to
  • Suitable chemical reagents such as glutaraldehyde, carbodiimide, MBS (N- (m-maleimide benzoyloxy) succinimide) And SMCC (sulfosuccinimidyl 4- (N-maleimide) cyclohexan-1-carboxylate) and the like.
  • the present invention is used as an antigen, A method for obtaining a monoclonal ⁇ -null antibody according to the above is described.
  • an animal for example, an animal such as a mouse which has been subjected to an experiment as a conventional method can be used. It is desirable to administer the antigen into the abdominal cavity or the like.
  • the administration may be repeated several times at intervals of several weeks after administration. preferable. An administration interval of 2 weeks is sufficient, and the number of administrations is preferably 2 to 4 times. Thereafter, the animal is sacrificed, for example, an organ such as a spleen is removed, and the cells are removed according to a conventional method.
  • the myeloma cells used here for example, the SP2 / 0-AgI4 strain or the like is cultured in a medium containing, for example, PCS (fetal calf serum), and preferably cells in a logarithmic growth phase are used.
  • PCS fetal calf serum
  • the cell fusion method involves the ratio of immunized cells to myeloma cells in a 1: 1 ratio of cell numbers.
  • the mixture may be mixed in the range of 10 to 1 and a fusion agent such as polyethylene glycol or a method such as electric stimulation may be used.
  • cells are immediately or after preculture in a normal medium, and hypoxan.
  • HAT medium supplemented with tin, ryminopterin and thymidine
  • tin, ryminopterin and thymidine By culturing in a so-called HAT medium supplemented with tin, ryminopterin and thymidine, only cells fused with a combination of immunized cells and myeloma cells can be selected.
  • antibody-producing cells can be selected while confirming the antibody titer by, for example, enzyme immunoassay (ELISA).
  • ELISA enzyme immunoassay
  • the cells for which the antibody titer could be confirmed were repeatedly cultured in a well plate and a plate according to a conventional method such as an ultra-dilution method or a soft agar method. Screening can be performed by conducting an immunochemical test of the antibody, an antigen specificity test, or the like. In addition, sorting can be performed using a micro-manipulator or a cell sorter.
  • the antibody producing cell according to the present invention can continuously produce the monoclonal antibody according to the present invention in a normal state. Therefore, when the monoclonal antibody according to the present invention is used, the supernatant of the culture solution of the cell according to the present invention can be directly used as the monoclonal antibody solution according to the present invention as it is. .
  • the monoclonal antibody according to the present invention is obtained by administering antibody-producing cells to an animal body (for example, intraperitoneally), which is usually subjected to an experiment, such as a mouse, and transforming an animal body fluid such as ascites. It can also be produced by sampling.
  • an animal body for example, intraperitoneally
  • an experiment such as a mouse
  • transforming an animal body fluid such as ascites. It can also be produced by sampling.
  • the monoclonal antibody D-null according to the present invention In order to purify and obtain the monoclonal antibody D-null according to the present invention from the culture solution or ascites fluid of the cell according to the present invention, it can be obtained by, for example, a method such as ammonium sulfate precipitation, ion exchange chromatography, or the like. .
  • the monoclonal antibody thus obtained is stored as a stable substance by adding various buffers, salts and, if necessary, azide, or the like, or by freeze-drying, for example. can do. Identification of each class
  • Identification of each class of immunogenic n-purine of these antibodies can be performed by the BLISA method using class-specific antibodies.
  • the monoclonal antibody obtained in this manner can be confirmed to specifically bind to each protein by the following immunobiochemical technique.
  • This technique can be applied to the identification and quantification of such peptides in biological specimens. That is, a sample derived from a living organism under the peptide or containing the peptide is fractionated by SDS-polyacrylamide gel electrophoresis according to a conventional method, and then subjected to nylon membrane or nitrose cellulose measurement. Plot into a blank. To bind the peptide was transferred onto main assembler emissions and the mono-click b Naru antibody, then, 35 S or '25 1 is combined with-labeled was Puroti down A (protein A). At this time, excess monoclonal antibody and protein A should be thoroughly removed by washing each time.
  • the position of the base petit de depending on the specificity of the mono-click polyclonal antibody used will be the 3 S S or 125 I present in accordance with the amount, 35 S or on main assembler down 125 Identify and quantify peptides by detecting 1 with RI scanner or autoradiography. Can be.
  • protein A-Sepharose or the like is added according to a conventional method, and centrifugation and washing with a buffer solution are repeated to remove the contaminants other than the peptide of the present invention. After that, electrophoresis is performed according to a conventional method to detect radioisotope. If the peptide is present in the sample cells, a band corresponding to the specificity of the monoclonal antibody used will appear.
  • the cells can be fixed with an appropriate fixing solution such as a solution of formaldehyde phosphate buffer (PBS) and, if necessary, subjected to a triton treatment to prepare a sample.
  • PBS formaldehyde phosphate buffer
  • the sample is thoroughly washed with a phosphate buffer.
  • piotin-conjugated anti-immunoglobulin as a secondary antibody, wash thoroughly. Fluorescence labeling of the resulting peptide-monoclonal antibody-piotin-conjugated secondary antibody complex was performed. By reacting vidin and observing the fluorescence with a fluorescence microscope, peptides in the cells can be detected.
  • the monoclonal antibody according to the present invention By using the monoclonal antibody according to the present invention, a human or other mammalian src family gene product can be identified with high sensitivity. Therefore, the monoclonal antibody according to the present invention can be used for pathological diagnosis of human or other mammalian src family gene-derived diseases.
  • Examples of the disease derived from the src gene include colorectal cancer, lung cancer, breast cancer, neuroblastoma, rhabdomyosarcoma, retinoblastoma, and other cancers, osteolithiasis, and the like.
  • a general pathological diagnostic method using a monoclonal D-nal antibody can be applied as it is. Specifically, for example, Western blotting
  • tissues such as colon and lung obtained by biopsy or surgery are fixed in formalin and paraffin-embedded sections are used in the present invention.
  • pathological diagnosis can be performed.
  • lung cancer can be diagnosed by performing a sputum test. Specifically, sputum is collected from the subject, spread on a slide glass by smearing, and then immersed in, for example, -20: acetone or methanol. It can be diagnosed by fixing, reacting with the monoclonal antibody of the present invention, staining by a conventional method, and detecting activation of the c-src family. Confirmation of activated c-src along with morphological observation will facilitate definitive diagnosis.
  • a peptide having a sequence consisting of 19 amino acids was chemically synthesized according to a conventional method.
  • the amino acid sequence of this peptide is as shown in SEQ ID NO: 1.
  • the peptide and the carrier conjugate were purified using a disposable gel filtration column (manufactured by Biorad). In this way, 12 mg of the peptide-KLH conjugate was obtained.
  • a 10-week-old female BALB / C mouse 0.1 ml of a solution containing 100 mg of the substance obtained in (1) and complete adjuvant of Freund (DIFC0) were intraperitoneally D A mixture with 1 ml was administered. Thereafter, a mixed solution of the above solution and incomplete adjuvant (manufactured by DIPC0) was intraperitoneally administered twice at two-week intervals. Thereafter, the mouse was killed by cervical dislocation and the spleen was aseptically probed.
  • DIFC0 complete adjuvant of Freund
  • D-MBM Dulbecco's modified MBM
  • Spleen cells suspended in D-MBM were obtained. This was centrifuged at 100 G for 5 minutes to collect spleen cells.
  • the cells were put into 1 ml of a solution obtained by adding 20 mM HBPES buffer (PH 7.4) to a 0.84% ammonium chloride solution and hemolyzed. Cells were collected by centrifugation at 1,000 G for 5 minutes. It was suspended again in D-MBM medium.
  • FCS ⁇ shea calf serum
  • the cells in the wells confirmed by the BLISA method were spread on a soft agar medium in a 60 mm petri dish. The cells were cultured for 2 weeks in a 10% CO 2 ⁇ 37t incubator to form colonies.
  • the resulting colonies were picked and placed on 24-well culture plates. Again, the antibody activity was confirmed by the BLISA method. Cells in the well in which the antibody activity was confirmed were seeded on a soft agar medium in a 60 mm0 dish. The cells were cultured in an incubator with 10% C ⁇ 2 ⁇ 37 for 2 weeks to form colonies. The resulting colonies were picked and placed on 24-well culture plates. Again, antibody activity was confirmed by the BLISA method, and useful cells were selected.
  • Antibody No. 28 are from the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology (1-3 Tsukuba, Higashi, Ibaraki, Japan (zip code: 2005)). International Deposit (Accession No .: PBRM BP-4253. Deposit date: March 30, 1993).
  • the ascites was diluted approximately 10-fold with an aqueous solution containing Tris 50 mM (pH 8.2) salt 150 mM NaN 3 0.02%. This solution was diluted with monoclonal anti-mouse immunoglobulin (available from Ricoh Corning Rex, Inc.) if the subclass of the antibody was IgG. Those with a subclass of IgG 2 were purified using a brotin A-Sepharose CL-4B column (manufactured by Pharma Ryo). Elution was performed with IgGJ ⁇ glycine monohydrochloride 0.1M (pH2.8).
  • IgG 3 was carried out with an Imuno View Buffer (Pierce).
  • each class is class specific antibodies (IgA, IgG ,, IgG 2a IgG 2b, IgG 3, IgM has had use those Baiorai de Co.) was added, 37 After washing for 2 hours, the plate was washed thoroughly with PBS. To this, peroxidase-conjugated anti-mouse whole mouse antibody was added, and the plate was allowed to stand for 1 hour in a 371 incubator. After washing with PBS, the color former ( (ABTS) was added for 15 minutes for color development, and each class was judged. Table 1 shows some of the results. table 1
  • the cells were cultured in a medium free from phosphoric acid or methionine for 2 to 3 hours (starvation). After this, the medium was replaced again, and 32 P-orthophosphate (NBX-053 370 MBq / ml manufactured by NB) or 35 S-methionine (SJ1015 370 MBq / ral manufactured by Amersham) was added. Culture was continued for another 2 to 4 hours.
  • 200 1 was prepared by suspending protein A-Galose (manufactured by Zymed) at 10% in RIPA buffer, and the mixture was reacted at 4: 1 for 1 hour. After the reaction, the mixture was centrifuged at 15,000 rptn for 1 minute to separate from the supernatant. RIPA buffer to wash precipitated antigen-antibody-protein A-sepharose complex 0.75 ml of the buffer solution was added, and the mixture was stirred well, centrifuged at 15, OOO rpm for 1 minute, and separated from the supernatant again. This operation was repeated five times. to this
  • the sample buffer 30/1 for SDS-PAGB electrophoresis was added, and the mixture was heated with lOOt for 2 minutes to denature the protein.
  • the electrophoresis was performed on a 10% poly-middle gel. After electrophoresis, the proteins in the gel 5% METHANOL, after fixing with a solution containing 7.5% acetic acid, 35 S sensitizer when the label (NBN Co., ENLIGHTIUNG) were sensitized with .
  • the gel was dried, exposed to X-ray film or an imaging plate (for Fuji, BAS2000), and the band specifically recognized by the antibody was detected.
  • Figure 1 shows the protein bands detected using these antibodies.
  • lane numbers 1 to 3 and 7 to 9 are neuroblastoma NB-1
  • 4 to 6 and 10 to 12 are acute lymphoblastic leukemia cells SKW3.
  • Monoclonal antibodies used antibody numbers 28, 2, 5, 8.11 for antibody numbers 1, 4, 7, and 10, antibody number 29 for antibody numbers 3, and antibody number 40 for 3, 6, 9, and 12.
  • antibody ban Nos. 28 and 29 had particularly good detection sensitivity in the immunoprecipitation method.
  • Antibody No. 28 showed a band that was not detected in the 32 P lapel at the 35 S label, and recognized that it recognized the vicinity of the thiacin residue, which is a phosphorylated site.
  • Antibody No. 29 showed the same pattern in both 32 ° and 3 s S, and was proved to be an antibody recognizing other than phosphorylated sites.
  • antibody No. 40 detected a band different from the above two antibodies, and by these, it was possible to analyze the difference in information transmission due to the presence or absence of phosphorylation.
  • the result is shown in figure 2.
  • the antibody used was antibody number 29.
  • the cell lines that produced lysate were as follows: lane number 1 is acute lymphoblastic leukemia cell ⁇ 3, lane number 2 is S3, and lane number 3 is monocyte leukemia cell.
  • lane number 6 is neuroblastoma SC-N-RT, and lane number 7 is NB-1.
  • Antibodies Nos. 28 and 29 were used as antibodies. Lane No. 1 indicates the whole protein solubilized rat brain. Lane No. 2 shows the No. 28 affinity column, and Lane No. 3 shows the protein eluted with 3M KSCN from the affinity column of 29 Ban.
  • Diagnosis example 1 Pathological diagnosis of colorectal cancer ''
  • Each colon section obtained by biopsy or surgery from 76 subjects was used as a paraffin-embedded pathological section, which was immersed in xylene to remove paraffin. These were then treated with 0.1% NaN 3 sodium, 0.3% hydrogen peroxide methanol for 30 minutes, then 5% normal goat serum, 1% bovine serum albumin (BSA) Z PBS Blocking was performed with (-) for 20 minutes, and the mixture was allowed to react overnight with a monoclonal antibody (antibody number 28) of the present invention (antibody number 28) diluted 1500-fold at 4 in a moist chamber (moist chamber).
  • the monoclonal antibody according to the present invention can be used particularly for pathological diagnosis of precancerous conditions.
  • most of the adenoma stained enteric cases have mosaic-like staining, and the presence or absence of mosaic-like can be used as an indicator of a precancerous condition.
  • Each of the lung sections obtained by biopsy or surgery from nine subjects was used as a paraffin-embedded pathological section, which was immersed in xylene to remove the paraffin.
  • These were then treated with 0.1% NaN 3 sodium, 0.3% hydrogen peroxide Z-metal for 30 minutes, followed by 5% normal goat serum, ⁇ bovine serum albumin
  • the monoclonal antibody according to the present invention can be sufficiently used for diagnosis of lung cancer.
  • Figure 1 shows the detection band by immunoprecipitation.
  • the horizontal axis represents the lane number, rate NShigerugo 1-6 lapel by 32 P, lanes trial No. 7 - the label 1 2 by 35 S, represents respectively.
  • the vertical axis indicates the direction of electrophoresis, the upper part indicates a higher molecular weight, and the lower part indicates a lower molecular weight.
  • Lane number 1 molecular weight Ma - representing the position of the car ( ⁇ c).
  • Fig. 2 shows an example of use in the dust plotting method.
  • the ⁇ axis indicates the lane number, and the ordinate indicates the electrophoresis direction.
  • Lane number 8 represents the position of the molecular weight marker ( 14 C).
  • FIG. 3 shows an example of separation using an affinity column.
  • the horizontal axis represents the lane number, and the vertical axis represents the direction of the electric swing.
  • Lane number 4 indicates the position of molecular weight markers ( '4 C).

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Abstract

Anticorps monoclonal qui reconnaît spécifiquement le site permettant de contrôler l'activité des phospho-enzymes présentes à la terminaison C d'un produit génique de la famille des virus du sarcome de ROUS agissant comme précurseur génique carcinogène, ledit anticorps s'obtenant par utilisation, comme antigène, d'un peptide spécifique présent à la terminaison C dudit virus; l'invention porte également sur une cellule produisant ledit anticorps, le processus d'obtention de cet anticorps et la méthode de diagnostic pathologique du cancer ou d'un précancer avec ledit anticorps.
PCT/JP1993/000854 1992-06-26 1993-06-24 Anticorps monoclonal, son procede d'obtention, cellule productrice de cet anticorps, et methode de diagnostic WO1994000591A1 (fr)

Applications Claiming Priority (4)

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JP19321392 1992-06-26
JP4/193213 1992-06-26
JP5/103609 1993-04-05
JP10360993 1993-04-05

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WO1994000591A1 true WO1994000591A1 (fr) 1994-01-06

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008513793A (ja) * 2004-09-22 2008-05-01 トリパス イメージング， インコーポレイテッド ガンの予後のためのマーカー候補を分析および最適化するための方法およびコンピュータープログラム製品

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Vol. 170, No. 2, (1990), S. SHOJI et al., "Myristoylated src-Oncogene Products are Potently Detected by the Monoclonal Antibody to the Amino-Terminal Myristoyl-Gly-Ser-Ser-Lys src-Peptide", p. 657-664. *
EMBO JOURNAL, Vol. 1, No. 12, (1982), T. TAMURA AND H. BAUER, "Monoclonal Antibody Against the Carboxy Terminal Peptide of pp60src of Rous Sarcoma Virus Reacts with Native pp60src", p. 1479-1485. *
JOURNAL OF VIROLOGY, Vol. 51, No. 2, (1984), S.J. PARSONS et al., "Monoclonal Antibodies to Rous Sarcoma Virus pp60src React with Enzymatically Active Cellular pp60src of Avian and Mammalian Origin", p. 272-282. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008513793A (ja) * 2004-09-22 2008-05-01 トリパス イメージング， インコーポレイテッド ガンの予後のためのマーカー候補を分析および最適化するための方法およびコンピュータープログラム製品

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