WO2001079266A1 - Anticorps anti-recepteur du facteur de croissance du keratinocyte humain - Google Patents

Anticorps anti-recepteur du facteur de croissance du keratinocyte humain Download PDF

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Publication number
WO2001079266A1
WO2001079266A1 PCT/JP2000/008736 JP0008736W WO0179266A1 WO 2001079266 A1 WO2001079266 A1 WO 2001079266A1 JP 0008736 W JP0008736 W JP 0008736W WO 0179266 A1 WO0179266 A1 WO 0179266A1
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WO
WIPO (PCT)
Prior art keywords
kgfr
antibody
peptide
human
immunogen
Prior art date
Application number
PCT/JP2000/008736
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English (en)
Japanese (ja)
Inventor
Takehiko Koji
Mitsuru Nakamura
Original Assignee
Nichirei Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nichirei Corporation filed Critical Nichirei Corporation
Priority to AU17358/01A priority Critical patent/AU1735801A/en
Publication of WO2001079266A1 publication Critical patent/WO2001079266A1/fr

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Classifications

    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to an antibody that specifically binds to KGFR (Keratinocyte Growth Factor Receptor: epidermal growth factor receptor) and its use.
  • KGFR Keratinocyte Growth Factor Receptor: epidermal growth factor receptor
  • KGFR is a cell growth factor receptor that is classified into FGFR 2 among the fibroblast growth factor receptors (FGFR) family (FGFRs 1-4) due to the characteristics of the sequence of the mRNA that encodes it.
  • FGFR fibroblast growth factor receptors
  • FGF receptor 1-2 FGFR2
  • KGF Keratinocyte Growth Factor
  • KGF may not only contribute to the development and growth of normal epithelial cells, but may also be involved in the growth of tumors in various organs derived from epithelial tissue. Therefore, the antibody against KGFR, which is its receptor according to the present invention, contributes to the diagnosis of various tumors and solid cancers by examining the presence or absence of KGF I in various organs, tissues and cells.
  • FIG. 4 is a photograph as a substitute for a drawing, in which a sliced specimen of cholesteatoma tissue embedded in formalin and embedded in paraffin is stained with a polyclonal antibody.
  • FIG. 4B is a photograph (B) as a substitute for a drawing, in which a sliced sample of choleroma tissue embedded in formalin-fixed paraffin was stained after pre-reaction.
  • 17 is a photograph substituted for a drawing, showing that staining at the site indicated by the arrow was lost by reaction with the peptide.
  • Drawing substitute photograph formalin-fixed, paraffin-embedded cholesteatoma
  • Drawing substitute photograph (A) in which a sliced specimen of tissue was stained with a polyclonal antibody, and a section of cholesteatoma tissue embedded in formalin-fixed paraffin after the polyclonal antibody was allowed to react with a peptide as an immunogen in advance.
  • This is a drawing substitute photograph (B) that stained the specimen.
  • 17 is a photograph substituted for a drawing, showing that staining at the site indicated by the arrow was lost by reaction with the peptide.
  • the present invention has been made to achieve the above-mentioned object, and as a result of studies from various directions, focused on immunogens. It not only produces antibodies that specifically bind to KGFR, but also has an amino acid sequence that is as short as possible from the industrial point of view of ease of production, especially from an industrial standpoint. Each of these requirements was fulfilled from the viewpoint that good binding was also required, and that it was necessary to smoothly immunize animals and efficiently produce antisera or antibodies.
  • the present invention succeeded in creating an antibody capable of analyzing and measuring the presence or absence and the amount of KGFR at the cell level by using a peptide having a specific amino acid sequence as a novel immunogen.
  • the antibody according to the present invention is an antibody that specifically recognizes KGFR, and is a novel antibody that has hitherto been unknown.
  • human KGFR is sometimes simply referred to as KGFR.Immunoassay includes detection of KGFR, confirmation and analysis of its presence, and qualitative or quantitative analysis of KGFR. , Immunoprecipitation and all other immunoassays are included.
  • the present invention relates to a novel immunogen consisting of a specific peptide, a novel antibody obtained using the same, a specific binding property of the novel antibody to KGFR, and the construction of a novel Atsushi system using the same. In addition, they have very useful features.
  • the immunogen used to prepare the novel antibody according to the present invention includes a peptide (1) having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing (FIG. 1), and / or SEQ ID NO: 2. (2) (FIG. 2) having a different amino acid sequence.
  • These peptides (1) and (2) are novel as immunogens for producing anti-KGFR antibodies, but are appropriately produced by a conventionally known method such as using a peptide synthesizer.
  • Immunization may be performed according to a conventional method.
  • the peptide is conjugated to BSA, KLH, thyroglobulin and other known carrier proteins, and this conjugate (complex) is used as an immunogen.
  • Freund's complete adjuvant or non-adjuvant is used.
  • Complete horse mackerel This is done by injecting the animal as many times as necessary with various adjuvants such as subjuvant.
  • a mouse, a heron, a rat, a sheep and the like can be used, and the injection can be performed according to a known method. Then, blood is collected from the injected animal to obtain antiserum.
  • any conventionally known method may be used.
  • a protein purification method such as affinity chromatography, ammonium sulfate salting out, or ion exchange chromatography is appropriately employed.
  • the antibody produced in this way has very high specificity for human KGFR, and has the property of specifically and selectively recognizing and reacting with KGFR and binding to it.
  • Human KGFR immunoassay can be performed by various methods.
  • immunoassays include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay, chemiluminescence immunoassay, immunohistochemical staining, immunoprecipitation, and others.
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • fluorescence immunoassay fluorescence immunoassay
  • chemiluminescence immunoassay chemiluminescence immunoassay
  • immunohistochemical staining immunoprecipitation, and others.
  • the present antibody may be used as a first antibody and / or a second antibody labeled with an enzyme.
  • an antibody an anti-KGFR antibody according to the present invention: a first antibody
  • KGFR an enzyme that recognizes a substance (KGFR) to be detected on a tissue section
  • an enzyme is bound to the first antibody
  • KGFR or cells that produce or contain KGFR
  • staining is performed by the following operations: 1) reaction with the labeled first antibody, 2) reaction with the enzyme reagent, and 3) color development.
  • the first antibody may be reacted with a second antibody (labeled) that specifically binds.
  • EIA EIA by the sandwich method
  • An antibody that binds to the substance (KGFR) (the anti-KGFR antibody of the present invention: the first antibody) is immobilized on a microtiter plate or polystyrene beads, etc. 2) Then, the plate or beads are blocked with a protein such as albumin.
  • a solution containing a specific substance (KGFR) is added and reacted for a certain period of time.
  • An antibody (enzyme-labeled anti-KGFR antibody: second antibody) that binds to an enzyme-labeled specific substance is added.
  • EIA is performed by reacting for a certain period of time, 5) adding the enzyme substrate, and measuring the degree of color development with a spectrophotometer.
  • an antibody obtained using the peptide (1) as an immunogen is obtained as the first antibody, and a peptide (2) is obtained as the immunogen.
  • the second antibody can be used as the second antibody, and vice versa.
  • an antibody labeled with biotin instead of an enzyme-labeled antibody using an avidin-biotin system is used, and an avidin-labeled enzyme is used as an enzyme.
  • the system may be assembled.
  • avidin any of avidin derived from egg white, avidin derived from microorganisms (for example, streptavidin) and the like can be used.
  • Labeling enzymes include horseradish peroxidase (HRP), small intestine alkaline phosphatase, 5-galactosidase, peroxidase, and glucose immunoassay (EIA). Enzymes commonly used in EIA are used as appropriate, and chromogenic substrates that are compatible with these enzymes and commonly used in EIA are used as appropriate. For example, in the case of HRP, 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), TMBZ ⁇ HC1, TMBZ ⁇ PS, ABTS, o-phenylenediamine, p-hydroxyphenyl Acetic acid or the like is used.
  • HRP horseradish peroxidase
  • EIA glucose immunoassay
  • alkaline phosphatase p-nitrophenyl phosphat Riferyl phosphate is used.
  • ⁇ -galactosidase O- 12-nitrophen- ⁇ -D-galactobilanoside, 4-methylumberiferyl- ⁇ -D-galactovyranoside Etc. are used.
  • the present invention also provides a novel immunogen as described above, a novel antibody obtained using the same, a method for detecting and measuring KGFR using the same, and a method for staining, detecting and measuring cells expressing and producing the same.
  • An immunogen was prepared as described below, and a polyclonal antibody was prepared using the immunogen.
  • peptide (2) having the amino acid sequence shown in SEQ ID NO: 2 was obtained by a solid phase method using an automatic peptide synthesizer (manufactured by Beckman), and then purified by high-speed chromatography.
  • the amino acid composition of the obtained peptide was hydrolyzed with 1 N hydrochloric acid at 120 ° C; overnight, and then measured using an amino acid analyzer. It was confirmed that the amino acid composition agreed with the theoretical value.
  • Peptide (1) was prepared in the same manner, and a carrier protein was bound to this peptide as follows.
  • the resulting carrier-protein and peptide conjugate (equivalent to 100 ⁇ g of peptide) were injected into the back of a egret with Freund's complete adjuvant. Thereafter, the antigen protein equivalent to 10 peptides each week was mixed with Freund's incomplete adjuvant and immunized a total of 8 times in the same manner. On the 10th day after the last immunization, whole blood was collected to obtain about 8 Oml of antiserum. This antiserum could be used as an anti-KGFR antibody by directly diluting it at an appropriate magnification. This antiserum could also be used after purifying the immunoglobulin fraction or purifying it using a column on which an immunogen peptide was immobilized in accordance with a conventional method.
  • the antiserum obtained as described above was treated as follows to obtain a purified antibody. That is, an IgG fraction was isolated from this antiserum by affinity chromatography using protein A. That is, 2 ml of protein A Sepharose CL4B (Pharmacia) was packed in a column, and equilibrated with a 1.5M glycine solution of 101111 (PH8.7). Dilute the serum with the same volume of 1.5 M glycine solution (pH 8.7), apply it to the column to bind IgG to protein A, and then use 3.0 ml of 1.5 M glycine solution (pH 8.7) Was washed.
  • Example 1 Immunohistochemical staining and inhibition test of cholesteatoma tissue
  • the paraffin-embedded cholesteatoma tissue block was sliced, attached to a slide glass, and fixed. Thereafter, deparaffinization treatment and deperoxidase treatment were performed.
  • the antibody against peptide (2) produced in Example 1 was labeled with peroxidase according to a commonly used labeling method. After adding the peroxidase-labeled anti-KGFR polyclonal antibody to the above and reacting for 5 minutes, wash well, react with the substrate solution (diaminopentidine, hydrogen peroxide) dropwise, and wash with purified water. Then, the cells were sealed and examined under a microscope.
  • FIGS. 4 and 5 both of which are photographs substituted for drawings.
  • Fig. 4 (B), Fig. 5 (B) As is evident from the above results, it has been clarified that the formation or growth of cholesteatoma is related to the cell proliferation mechanism mediated by KGF-KGFR. Similar results were obtained when an antibody against peptide (1) was used.
  • the present invention has created an anti-KGFR antibody.
  • This antibody is a novel antibody that has the property of specifically binding to KGFR, and has a high property of recognizing KGFR.Thus, the use of this antibody makes it possible for the first time to perform excellent KGFR immunoassay. Was.
  • This assay provides reproducible results and is very useful for diagnosing cancer and other diseases It is.
  • examples of the assay or detection method using the antibody include EIA, RIA, immunohistochemical staining, immunoprecipitation, and Western blotting.By comprehensively utilizing these methods, It has become possible to more accurately diagnose cancer and other diseases such as prostate cancer.
  • the present invention has made it possible for the first time to clarify the distribution of KGFR-expressing cells in tumor tissue. With such a method, it is possible to analyze the presence or absence in various tumor tissues and the like, and the present invention plays an important role also in the pathology field.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un anticorps polyclonal se liant spécifiquement au récepteur du facteur de croissance du kératinocyte humain. En raison d'une spécificité élevée vis-à-vis du récepteur du facteur de croissance du kératinocyte, cet anticorps permet la détection et la détermination précises et rapides du récepteur du facteur de croissance du kératinocyte dans divers organes, tissus et cellules. Ainsi, on peut l'utiliser pour détecter et doser sur le plan immunologique diverses tumeurs telles que le cholestéatome.
PCT/JP2000/008736 2000-04-17 2000-12-11 Anticorps anti-recepteur du facteur de croissance du keratinocyte humain WO2001079266A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU17358/01A AU1735801A (en) 2000-04-17 2000-12-11 Antibody against human kgfr

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000-115253 2000-04-17
JP2000115253A JP2001302699A (ja) 2000-04-17 2000-04-17 ヒトｋｇｆｒに対する抗体

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Publication Number Publication Date
WO2001079266A1 true WO2001079266A1 (fr) 2001-10-25

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AU (1) AU1735801A (fr)
WO (1) WO2001079266A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8101723B2 (en) 2008-11-07 2012-01-24 Galaxy Biotech, Llc Monoclonal antibodies to fibroblast growth factor receptor 2
WO2014138364A2 (fr) 2013-03-06 2014-09-12 Genentech, Inc. Méthodes de traitement et de prévention de la résistance à un médicament d'un cancer
WO2015191986A1 (fr) 2014-06-13 2015-12-17 Genentech, Inc. Méthodes de traitement et de prévention de la résistance du cancer aux médicaments
US10172937B2 (en) 2013-08-01 2019-01-08 Five Prime Therapeutics, Inc. Method of treatment of malignant solid tumors with afucosylated anti-FGFR2IIIb antibodies
US11091555B2 (en) 2017-05-16 2021-08-17 Five Prime Therapeutics, Inc. Method of treating gastric cancer with anti-FGFR2-IIIb antibodies and modified FOLFOX6 chemotherapy
CN115028728A (zh) * 2016-09-27 2022-09-09 高地和群岛大学 抗原生物标志物
US11447553B2 (en) 2015-11-23 2022-09-20 Five Prime Therapeutics, Inc. FGFR2 inhibitors alone or in combination with immune stimulating agents in cancer treatment

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BOTTARO DONALD P. ET AL.: "A keratinocyte growth factor receptor-derived peptide antagonist identifies part of the ligand binding site", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 13, May 1993 (1993-05-01), pages 9180 - 9183, XP002937849 *
ISHIWATA TOSHIYUKI ET AL.: "Characterization of keratinocyte growth factor and receptor expression in human pancreatic cancer", AMERICAN JOURNAL OF PATHOLOGY, vol. 153, no. 1, July 1998 (1998-07-01), pages 213 - 222, XP002937853 *
MIKI TORU ET AL.: "Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene", PROC. NATL. ACAD. SCI. USA, vol. 89, no. 1, January 1992 (1992-01-01), pages 246 - 250, XP002937851 *
MIKI TORU ET AL.: "Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop", SCIENCE, vol. 251, 4 January 1991 (1991-01-04), pages 72 - 75, XP002937850 *
ZHOU JIE ET AL.: "Keratinocyte growth factor down-regulates expression of the sucrase-isomaltase gene in Caro-2 intestinal epithelial cells", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 50, 11 December 1998 (1998-12-11), pages 33367 - 33373, XP002937852 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8603987B2 (en) 2008-11-07 2013-12-10 Galaxy Biotech, Llc Monoclonal antibodies to fibroblast growth factor receptor 2
US8101723B2 (en) 2008-11-07 2012-01-24 Galaxy Biotech, Llc Monoclonal antibodies to fibroblast growth factor receptor 2
US9382324B2 (en) 2008-11-07 2016-07-05 Galaxy Biotech, Llc Monoclonal antibodies to fibroblast growth factor receptor 2
US9834609B2 (en) 2008-11-07 2017-12-05 Galaxy Biotech, Llc Methods of detecting a tumor expressing fibroblast growth factor receptor 2
US10138301B2 (en) 2008-11-07 2018-11-27 Galaxy Biotech, Llc Monoclonal antibodies to fibroblast growth factor receptor 2
US10689448B2 (en) 2008-11-07 2020-06-23 Galaxy Biotech, Llc Monoclonal antibodies to fibroblast growth factor receptor 2
WO2014138364A2 (fr) 2013-03-06 2014-09-12 Genentech, Inc. Méthodes de traitement et de prévention de la résistance à un médicament d'un cancer
US11235059B2 (en) 2013-08-01 2022-02-01 Five Prime Therapeutics, Inc. Afucosylated anti-FGFR2IIIB antibodies
US10172937B2 (en) 2013-08-01 2019-01-08 Five Prime Therapeutics, Inc. Method of treatment of malignant solid tumors with afucosylated anti-FGFR2IIIb antibodies
WO2015191986A1 (fr) 2014-06-13 2015-12-17 Genentech, Inc. Méthodes de traitement et de prévention de la résistance du cancer aux médicaments
US11447553B2 (en) 2015-11-23 2022-09-20 Five Prime Therapeutics, Inc. FGFR2 inhibitors alone or in combination with immune stimulating agents in cancer treatment
CN115028728A (zh) * 2016-09-27 2022-09-09 高地和群岛大学 抗原生物标志物
US11091555B2 (en) 2017-05-16 2021-08-17 Five Prime Therapeutics, Inc. Method of treating gastric cancer with anti-FGFR2-IIIb antibodies and modified FOLFOX6 chemotherapy

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JP2001302699A (ja) 2001-10-31
AU1735801A (en) 2001-10-30

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