JP2001302699A - Antibody against human kgfr - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibody which acts against human keratinocyte growth factor receptor(KGFR), can be used for the accurate and rapid detection and assay of KGFR in various kinds of organs, tissues and cells, and can be utilized for the detection and immunoassay of cholesteatoma and other various tumors, because of having high specificity to KGFR. SOLUTION: The polyclonal antibody specifically binding to the human keratinocyte growth factor receptor(KGFR).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a KGFR (Kerati
The present invention relates to an antibody that specifically binds to an epidermal growth factor receptor (nocyte growth factor receptor) and its use.

[0002] KGFR is characterized by the characteristics of the sequence of the mRNA that encodes it, and the GFFR is a member of the fibroblast growth factor receptor (FGFR) family (FGFR1-4).
It is a cell growth factor receptor classified as R2. KGF
R, that is, FGFR2 (FGF receptor 2) is one of the transmembrane receptor tyrosine kinases, and has an extracellular domain in its epidermal cell growth factor (Keratinocyte Growt).
h Factor: hereinafter also referred to as KGF), which transmits a signal for cell proliferation and propagation to an intracellular signal transducer. In addition, KGF may not only contribute to the development and proliferation 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 KGFR in various organs, tissues and cells.

[0003]

2. Description of the Related Art Conventionally, in order to examine the distribution of KGFR in tissues, only in situ hybridization and staining of frozen tissue sections using monoclonal antibodies or polyclonal antibodies have been carried out. No antibody has been used that can clearly stain the formalin-fixed, paraffin-embedded tissue section from other FGFR family cell growth factors and the like. Therefore, we have contributed to the diagnosis of various tumors, solid cancers, etc. by preparing antibodies that specifically recognize KGFR and can stain thin sections of formalin-fixed, paraffin-embedded tissues. And thought that it could contribute to the development of such treatments.

[0004]

In the current state of the art, in view of the necessity of early diagnosis of various types of tumors and cancers, in view of the fact that antibodies for staining formalin-fixed paraffin sections are particularly important in the pathological region, this technique has been considered. FG
The present invention has been made for the purpose of producing an antibody that can be clearly distinguished from other cell growth factors belonging to the KGFR family other than FR2 (that is, KGFR) and stained.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made to achieve the above-mentioned object, and as a result of studies from various fields, focused on immunogens. It not only produces an antibody that specifically binds to KGFR, but also has an amino acid sequence that is as short as possible, particularly from the industrial point of view of ease of production, and therefore has good binding to a carrier protein. Peptides that can satisfy each of these requirements, from the viewpoint that it is necessary that the immunity to animals be performed smoothly and that antiserum or antibodies be efficiently produced. An intensive screening was performed. As a result, we succeeded for the first time to screen the target peptide.

[0006] Then, a complex of the peptide having the specific amino acid sequence and the carrier protein is used as an immunogen, the rabbit is immunized, and the obtained antiserum is KGFR.
The present invention has been completed as a result of confirming, for the first time, that it specifically binds to, and conducting further studies based on these useful new findings.

[0007] The present invention has 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. In the present invention, human KGFR may be simply referred to as KGFR, and immunoassay includes qualitative or quantitative analysis of KGFR, in addition to detection and confirmation of KGFR, and immunohistological staining, Sedimentation and all other immunoassays are included.

That is, the present invention relates to a novel immunogen comprising a specific peptide, a novel antibody obtained by using the same, a specific binding property of the novel antibody to KGFR, and the construction of a novel assay system using the same. Moreover, it has a very useful feature. Hereinafter, the present invention will be described in detail.

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
Alternatively, a peptide (2) having the amino acid sequence shown in SEQ ID NO: 2 (FIG. 2) can be mentioned. These peptides (1) and (2) are novel as immunogens for preparing anti-KGFR antibodies, but are appropriately produced by a conventionally known method such as using a peptide synthesizer.

[0010] Immunization may be carried out according to a conventional method. The peptide is bound to BSA, KLH, bovine thyroglobulin and other known carrier proteins, and this conjugate (complex) is used as an immunogen, and if necessary, Freund's complete adjuvant. Alternatively, it is carried out by injecting the animal as many times as necessary with various adjuvants such as an incomplete adjuvant. As the animal to be injected, mice, rabbits, rats, 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. In order to obtain the antibody of the present invention from the obtained antiserum, any conventionally known method may be used. For example, protein purification methods such as affinity chromatography, ammonium sulfate salting out, and ion exchange chromatography are appropriately employed.

[0011] The antibody thus prepared is human KG.
It has very high specificity for FR, and has the property of specifically and selectively recognizing and reacting with KGFR and binding to it.
An immunoassay for FR can be performed.

As described above, the immunoassay includes a radioimmunoassay (RIA) and an enzyme immunoassay (EI).
A), fluorescence immunoassay, chemiluminescence immunoassay, immunohistochemical staining, immunoprecipitation, and the like are exemplified, and the present antibody may be used as a first antibody and / or an enzyme-labeled second antibody.

For example, in the case of immunohistochemical staining, an antibody (an anti-KGFR antibody according to the present invention: a first antibody) that specifically recognizes a substance to be detected (KGFR) is reacted on a tissue section. In this case, if the enzyme is bound to the first antibody,
By developing a colored substrate by its enzymatic activity,
KGFR (or cells that produce or contain KGFR) can be detected. That is, staining is performed by the operations of 1) reaction with the labeled first antibody, 2) reaction with the enzyme reagent, and 3) color development. If necessary, after the reaction with the first antibody (unlabeled), the first antibody may be reacted with a second antibody (labeled) that specifically binds.

In the case of EIA, for example, EIA by the sandwich method, 1) an antibody that binds to a specific substance (KGFR) (anti-KGFR antibody of the present invention: first antibody)
Is immobilized on a microtiter plate or polystyrene beads or the like, 2) the plate or beads are then blocked with a protein such as albumin, and 3) a specific substance (KGF
R) is added thereto and reacted for a certain period of time. 4) An antibody (enzyme-labeled anti-K
GFR antibody: second antibody) and react for a certain period of time.
5) EIA is performed by adding an enzyme substrate and measuring the degree of color development with a spectrophotometer.

Although these two types of antibodies need to recognize different epitopes, in the present invention, an antibody obtained by using peptide (1) as an immunogen is used as a first antibody, and peptide (2) is used as an immunogen. The obtained antibody can be used as the second antibody, and vice versa.

In the immunoassay according to the present invention, in addition to using an enzyme-labeled antibody, an avidin-biotin system is used, an antibody labeled with biotin is used instead of the enzyme-labeled antibody, and an avidin-labeled enzyme is used as the enzyme. To assemble the assay system. As avidin,
Avidin derived from egg white, avidin derived from microorganisms (for example, streptavidin) and the like can be used.

As the labeling enzyme, an enzyme commonly used in an enzyme immunoassay (EIA) such as horseradish peroxidase (HRP), bovine small intestine alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc. is appropriately used. A chromogenic substrate commonly used in EIA is used as appropriate. For example, in the case of HRP, 3,3 ′, 5,5′-tetramethylbenzidine (TMBZ), TMBZ · HCl,
TMBZ PS, ABTS, o-phenylenediamine,
p-Hydroxyphenylacetic acid and the like are used, in the case of alkaline phosphatase, p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate and the like are used, and in the case of β-galactosidase, o-nitrophenyl-β- D-galactopyranoside, 4-methylumbelliferyl-β-D-galactopyranoside and the like 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. Not only newly provided, but also a kit for measuring human KGFR (or a cell producing or containing the same) containing an anti-human KGFR antibody, a labeled anti-human KGFR antibody, an enzyme substrate, a coloring reagent, a buffer, and other necessary substances Is also provided.

Hereinafter, embodiments of the present invention will be described.

[0020]

Example 1 An immunogen was prepared as described below, and a polyclonal antibody was prepared using the immunogen.

(1) Preparation of immunogen Peptide having the amino acid sequence shown in SEQ ID NO: 2 (2)
Is synthesized by an automatic peptide synthesizer (Beckman)
And purified by high-performance chromatography. The amino acid composition of the obtained peptide was hydrolyzed with 1N hydrochloric acid at 120 ° C. overnight, and then measured using an amino acid analyzer, and it was confirmed that the amino acid composition agreed with the theoretical value. Peptide (1) was similarly prepared.

A carrier-protein was bound to this peptide as follows. That is, 10 mg of antigen peptide and 3 mg of bovine thyroglobulin were dissolved in 1 ml of distilled water,
After 0 mg of 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride was added and the reaction was allowed to proceed overnight at room temperature while protecting from light, the mixture was dialyzed sufficiently against distilled water. Whether or not the antigen peptide and the carrier protein were bound was determined by SDS-PAGE.
Confirmed by

(2) Preparation of serum The obtained carrier protein and peptide conjugate (100
(corresponding to μg of peptide) was injected into the back of the rabbit with Freund's complete adjuvant. Thereafter, every week
00 μg of the peptide-equivalent antigen protein was mixed with Freund's incomplete adjuvant and immunized a total of eight times. On the 10th day after the last immunization, whole blood was collected to obtain about 80 ml of antiserum. This antiserum could be used as an anti-KGFR antibody by directly diluting it at an appropriate magnification. In addition, this antiserum could be used after purifying the immunoglobulin fraction or purifying it using a column on which a peptide as an immunogen was immobilized according to a conventional method.

In the former case, the antiserum obtained 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 10 ml of a 1.5 M glycine solution (pH 8.7). The serum was diluted with the same volume of a 1.5 M glycine solution (pH 8.7) and passed through a column to bind IgG to protein A, and then 3.0 ml of 1.
The column was washed with a 5M glycine solution (pH 8.7). Next, elution with a 0.1 M glycine solution (pH 3.0)
A gG fraction was obtained. The purity of this purified antibody was determined by SDS-PAG.
The assay was performed by E electrophoresis. Similarly, peptide (1)
Was prepared.

[0025]

Example 2 (Immunohistochemical staining and inhibition test of cholesteatoma tissue) After fixing with formalin, the cholesteatoma tissue block embedded in paraffin was sliced, attached to a slide glass and fixed. Thereafter, deparaffinization treatment and deperoxidase treatment were performed. On the other hand, the antibody against the peptide (2) produced in Example 1 was labeled with peroxidase according to a commonly used labeling method. This peroxidase-labeled anti-KGFR polyclonal antibody was added to
After reacting for a minute, the well was washed well, and a substrate solution (diaminobenzidine, hydrogen peroxide) was added dropwise to react, washed with purified water, sealed, and observed under a microscope.

As a result, as shown in FIG. 3 (a photograph as a substitute for a drawing), cells constituting the connective tissue were stained (a positive image is shown at the site of the arrow). In addition, this staining is shown in FIGS.
As shown in (all of the photographs as substitutes for drawings), no anti-KGFR polyclonal antibody was preliminarily reacted with the peptide as the immunogen, and was not observed. (FIG. 4 (B), FIG. 5
(B)). As is clear from the above results, it was revealed 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.

[0027]

Example 3 (Positive rate of multiple cases) The same staining as in Example 2 was performed on 32 pearl tumor tissues, and it was confirmed that the positive rate was 72%.

[0028]

According to the present invention, an anti-KGFR antibody has been created. The present antibody is a novel antibody having the property of specifically binding to KGFR, and has a high property of recognizing KGFR.
An immunoassay for FR has become possible for the first time.

This measurement method provides reproducible results and is very effective in diagnosing cancer and other diseases. The present assay or detection method using the present antibody includes EIA, RI
A, immunohistochemical staining, immunoprecipitation, Western blotting, etc., and by comprehensively utilizing these methods, more accurate diagnosis of cancer and other diseases such as prostate cancer has become possible. For example, for the first time according to the present invention, K
It has become possible to clarify the distribution of GFR-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.

[0030]

[Sequence List] SEQUENCE LISTING <110> Nichirei Corporation <120> Antibody against Human KGFR <130> 6289 <141> 2000-4-17 <160> 2 <210> 1 <211> 15 <212> PRT <213> Artificial sequence <400> 1 His Ser Gly Ile Asn Ser Ser Asn Ala Glu Val Leu Ala Leu Phe 1 5 10 15 <210> 2 <211> 12 <212> PRT <213> Artificial sequence <400> 2 Cys Lys Val Ser Asn Tyr Ile Gly Gln Ala Asn Gln 1 5 10

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of antigenic peptide (1).

FIG. 2 shows the amino acid sequence of antigenic peptide (2).

FIG. 3 is a drawing-substitute photograph of a sectioned sample of cholesteatoma tissue fixed in formalin and embedded in paraffin, stained with a polyclonal antibody.

FIG. 4 is a drawing substitute photograph, in which a sliced specimen of cholesteatoma tissue embedded in formalin and then embedded in paraffin is stained with a polyclonal antibody (A), and the polyclonal antibody is an immunogen peptide FIG. 4B is a photograph (B) as a substitute for a drawing, in which a sliced specimen of cholesteatoma tissue embedded in formalin-fixed paraffin was preliminarily reacted and then stained. It is a photograph substituted for a drawing which shows that the staining of the site of the arrow was lost by the reaction with the peptide.

FIG. 5 is a drawing substitute photograph, in which a thin section of a cholesteatoma tissue embedded in formalin and fixed in paraffin is stained with a polyclonal antibody (A), and the polyclonal antibody is an immunogen peptide FIG. 4B is a photograph (B) as a substitute for a drawing, in which a sliced specimen of cholesteatoma tissue embedded in formalin-fixed paraffin was preliminarily reacted and then stained. It is a photograph substituted for a drawing which shows that the dyeing | staining of the site | part of an arrow was lost by reaction with the peptide.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 15/09 ZNA (C12P 21/08 C12R 1:91) C12R 1:91) (C12P 21/08 C12N 15/00 ZNAA C12R 1:91) C12R 1:91)

Claims (6)

[Claims] An antigen comprising a peptide (1) or (2) having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing. 2. An antibody obtained using the peptide (1) or (2) having the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing as an antigen. 3. The antibody according to claim 2, wherein the antibody according to claim 2 is an antibody against human KGFR. 4. An antibody that specifically binds to human KGFR. 5. An immunoassay for human KGFR, comprising using the antibody according to any one of claims 2 to 4. 6. A kit for measuring human KGFR, comprising the antibody according to any one of claims 2 to 4.