WO2001079266A1

WO2001079266A1 - Antibody against human kgfr

Info

Publication number: WO2001079266A1
Application number: PCT/JP2000/008736
Authority: WO
Inventors: Takehiko Koji; Mitsuru Nakamura
Original assignee: Nichirei Corporation
Priority date: 2000-04-17
Filing date: 2000-12-11
Publication date: 2001-10-25
Also published as: AU1735801A; JP2001302699A

Abstract

A polyclonal antibody specifically binding to human keratinocyte growth factor receptor (KGFR). Because of having a high specificity to KGFR, this antibody makes it possible to accurately and quickly detect and determine KGFR in various organs, tissues and cells. Thus, it is usable in detecting and immunoassaying various tumors such as pearl tumor.

Description

Antibodies to human KGFR

The present invention relates to an antibody that specifically binds to KGFR (Keratinocyte Growth Factor Receptor: epidermal growth factor receptor) and its use.

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. KGFR, or FGFR2 (FGF receptor 1-2), is one of the J3 transmembrane receptor tyrosine kinases, and its extracellular domain is called KGF (Keratinocyte Growth Factor). Is reported to transmit cell proliferation signals to intracellular signal transmitters. In addition, 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. Conventional technology

Conventionally, to examine the distribution of KGFR in tissues, the in situ hybridization method--staining of frozen tissue sections using a monoclonal antibody or a polyclonal antibody--has only been performed. Be No antibody has been used that can clearly stain formalin-fixed, paraffin-embedded tissue sections from other FGFR-family cell growth factors. Therefore, we will contribute to the diagnosis of various tumors, solid cancers, etc. by preparing antibodies that specifically recognize KGFR and can stain thin sections of tissues embedded in formalin-fixed paraffin. At the same time, they came to believe that they could contribute to the development of such treatments.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1

2 shows the amino acid sequence of the antigen peptide (1).

Figure 2

2 shows the amino acid sequence of the antigen peptide (2).

Fig. 3

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. 4

Drawing substitute photographs, in which formalin-fixed and paraffin-embedded cholesteatoma tissue sliced specimens are stained with polyclonal antibody (A), and a polyclonal antibody is used as an immunogen, beptide. 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.

Fig 5

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. Problems to be solved by the invention

Given the need for early diagnosis of various tumors and cancers in the current state of the art, in view of the fact that antibodies that stain formalin-fixed paraffin sections are particularly important in the pathological area, such sections are treated with FGFR2 (i.e. for the purpose of producing an antibody capable of dyeing clearly distinguish such as cell growth factors belonging to another KGP ¹ R family one KGFR) than, the present invention has been made. Means for solving the problem

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. We conducted intensive screening for peptides that can be used. As a result, we succeeded for the first time to screen the target peptide.

Then, a complex of the peptide having the specific amino acid sequence and the carrier protein was used as an immunogen, and this was used to immunize rabbits. 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.

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. In addition, 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 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.

That is, 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.

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 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. If necessary, 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. As an animal to be injected, 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. In order to obtain the antibody of the present invention from the obtained antiserum, any conventionally known method may be used. For example, 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.

As mentioned above, immunoassays include radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay, chemiluminescence immunoassay, immunohistochemical staining, immunoprecipitation, and others. For example, the present antibody may be used as a first antibody and / or a second antibody labeled with an enzyme.

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 (KGFR) to be detected on a tissue section is reacted. In this case, if an enzyme is bound to the first antibody, KGFR (or cells that produce or contain KGFR) can be detected by developing a colored substrate by the enzyme activity. . That is, staining is performed by the following operations: 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 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. 3) A solution containing a specific substance (KGFR) is added and reacted for a certain period of time. 4) An antibody (enzyme-labeled anti-KGFR antibody: second antibody) that binds to an enzyme-labeled specific substance is added. In addition, 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.

These two types of antibodies need to recognize different epitopes, but in the present invention, 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.

In the immunoassay according to the present invention, in addition to using an enzyme-labeled antibody, 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. As the 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. In the case of alkaline phosphatase, p-nitrophenyl phosphat Riferyl phosphate is used. In the case of ^ -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. In addition to newly provided kits, kits for measuring human KGFR (or cells producing or containing cells) containing anti-human KGFR antibodies, labeled anti-human KGFR antibodies, enzyme substrates, coloring reagents, buffers, and other necessary substances To provide.

Hereinafter, examples of the present invention will be described.

Example 1

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

(1) Preparation of immunogen

The synthesis of 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. That is, 1 Omg of antigen peptide and 3 mg of thyroglobulin are dissolved in 1 ml of distilled water, 1-ethyl-3- (3-dimethylaminopropyl) carboximide hydrochloride of 301111 is added, and the mixture is shielded from light overnight at room temperature. After the reaction, it was dialyzed sufficiently against distilled water. Antigen chip Whether or not the sunset and the carrier-protein were bound was confirmed by SDS-PAGE.

(2) Serum preparation

The resulting carrier-protein and peptide conjugate (equivalent to 100 μg 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.

In the former case, 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. Next, elution was performed with a 0.1 M glycine solution (pH 3.0) to obtain an IgG fraction. The purity of this purified antibody was determined by SDS-PAGE electrophoresis. Similarly, an antibody against the peptide (1) was prepared.

Example 2

(Immunohistochemical staining and inhibition test of cholesteatoma tissue) After formalin fixation, the paraffin-embedded cholesteatoma tissue block was sliced, attached to a slide glass, and fixed. Thereafter, deparaffinization treatment and deperoxidase treatment were performed. On the other hand, 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.

As a result, as shown in Fig. 3 (a photograph substituted for a drawing), the cells constituting the connective tissue were stained (a positive image is shown at the site of the arrow). In addition, this staining was not observed with the anti-KGFR polyclonal antibody that had previously been reacted with the peptide as the immunogen, as shown in 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.

Example 3

(Positive rate of multiple cases)

When staining was performed on 32 pearl tumor tissues in the same manner as in Example 2, it was confirmed that the positive rate was 72%.

The invention's effect

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. In addition, 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. For example, 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.

Claims

The scope of the claims

1. An antigen comprising the 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 paragraph 2, wherein the antibody according to paragraph 2 is a polyclonal antibody against human KGFR.

4. Polyclonal antibodies that specifically bind to human KGFR.

5. An immunoassay for human KGFR, comprising using the antibody according to any one of items 2 to 4.

6. A human, comprising the antibody according to any one of Items 2 to 4, which is a kit for measuring KGFR.