JP3472664B2 - Anti-fibroblast growth factor 5 monoclonal antibody - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-FGF5 monoclonal antibody useful for detecting fibroblast growth factor 5 (hereinafter referred to as “FGF5”) and a method for producing the same.

[0002]

2. Description of the Related Art Fibroblast growth factor (hereinafter referred to as "FGF")
Say. ) Regarding the family, FGF1 cloned for the first time in 1985, although up to the present number 9 among the members belonging to the family has been cloned.
It has been clarified that (aFGF) and FGF2 (bFGF) are involved in very important life phenomena such as development, cell differentiation, cell proliferation, morphogenesis, and carcinogenesis in vivo (Toru Imamura et al. , Clinical Science 25, 1012-1021 (1989); Yoji Mitsui et al., Protein Nucleic Acid Enzyme 36 (7), 1237-1246 (1991)). In addition, although the most recent 10 th member has been found, the details of which are not yet or Akira et al. In recent years, other members have been gradually elucidating important functions in vivo (RF Tutrone et al., J. Urol. 149, 633-639 (1
993); B. Feldman, Science 267, 246-249 (1995); Jea
n M. Hebert et al., Cell 78, 1017-1025 (1994); F.
Coulier et al., Prog. Growth Factor Res. 5, 1-14 (1
994); S. Werner et al., J. Invest. Dermatol. 105, 5
79-584 (1995); H. Ohuchi et al., Biochem. Biophys.
Res. Commun. 204 (2), 882-888 (1994)).

Among them, FGF5, which is the fifth member of the FGF family, has a function as a regulator of the hair cycle (Jean M. Hebert et al., Cell, 7
8, 1017-1025 (1994)), and that it is induced at the gene level in skin regeneration (S. Werner et al., Proc. Natl. Acad. Sci. USA 89,
6896-6900 (1992)).

On the other hand, very recently, it is becoming clear at the gene level that FGF5 has some function in the developmental process of the brain and is constantly expressed in the brain (Kazuo Ozawa et al. Chemistry 67 (7), 569 (199
Five)). These facts suggest that the abnormality of FGF5 in the body may cause various diseases at the gene level. Therefore, it can be said that grasping the behavior of FGF5 at the protein level and analyzing it is important for elucidating the causes of these diseases and making a diagnosis.

The FGF family has about 30% homology among members, and when an antibody is produced using a recombinant protein of FGF5 as an antigen, the produced antibody is FGF.
Likely to cross-react with other members of the family. Furthermore, since FGF5 has about 93% homology with mouse FGF5, the recombinant protein of FGF5 has very weak antigenicity in the mouse to be immunized, and does not form an antibody. It is also possible that it will cause shock death.

Although a polyclonal antibody that specifically recognizes FGF5 has already been prepared (B. Bates et al., Mol.
Cel. Biol. 11 (4), 1840-1845 (1991)), polyclonal antibodies must be immunized from the animal each time and must be prepared from the blood, resulting in large variations in antibody titers from lot to lot and reproducibility. There is a shortcoming that it is scarce. These drawbacks can be solved by producing a monoclonal antibody, but at present, there is no monoclonal antibody that specifically recognizes FGF5.

[0007]

The present invention does not recognize other members of the FGF family as antigens, and
It is an object of the present invention to provide a monoclonal antibody that specifically recognizes, a method for producing the same, and a method for detecting FGF5 using this monoclonal antibody.

[0008]

As a result of intensive studies to solve the above problems, the present inventors have found that the amino acid sequence of FGF5 has a low homology with the sequences of other members of the FGF family. The inventors have found that the use of a peptide having as a immunogen yields a monoclonal antibody that binds to FGF5 without binding to other members of the FGF family, and completed the present invention.

That is, the present invention provides an anti-fibroblast growth factor 5 monoclonal antibody having the following properties. (A) It binds to fibroblast growth factor 5. (B) a member other than the fibroblast growth factor 5 which is a member of the fibroblast growth factor family,
Do not combine.

The present invention also relates to a member of the fibroblast growth factor family of the amino acid sequences of fibroblast growth factor 5 and said fibroblast growth factor 5
Provided is the above-mentioned anti-fibroblast growth factor 5 monoclonal antibody, which recognizes, as an antigen, a region having a low homology with the amino acid sequences of other than the above.

The present invention also provides the anti-fibroblast growth factor 5 monoclonal antibody, wherein the region is at least a part of the amino acid sequence shown in SEQ ID NO: 1 in Sequence Listing.

The present invention also relates to a member of the fibroblast growth factor family of the amino acid sequence of fibroblast growth factor 5 and said fibroblast growth factor 5
After immunizing a mammal with a conjugate of a peptide having at least a part of the amino acid sequence having a low homology with the amino acid sequence of other than the carrier, the splenocytes of the mammal are taken out and cultured with the splenocytes. 2. A method comprising the steps of producing a hybridoma by fusing with cells, culturing the hybridoma, and collecting an antibody protein from the culture.
A method for producing the described monoclonal antibody is provided.

The present invention also provides a method for detecting fibroblast growth factor 5 by an immunological method using a monoclonal antibody, which method uses the above-mentioned monoclonal antibody, preferably a method for evaluating hair or a method for evaluating brain function. I will provide a.

In the present specification, "specifically binds to FGF5" means that the FG reacts with human FGF5.
It means that F5 is specifically recognized as an antigen and does not bind to members other than the above-mentioned FGF5 belonging to the FGF family.

The present invention will be described in detail below. FGF5
The cDNA sequence of the FGF family including is known (M. Jaye et al., Science, 251, (1986); JA Ab
raham et al., EMBO J., 5, 2523-2528 (1986); R. Moor
e et al., EMBO J., 5, 919-924 (1986); P. Delli-Bovi
et al., Cell, 50, 729-737 (1987); X. Zhan et al.,
Mol Cel Biol., 8, 3487-3495 (1988); I. Marics et
al., Oncogene, 4, 335-340 (1989); JS Rubinet a
L., Proc. Natl. Acad. Sci. USA, 86, 802-806 (198
9); A. Tanaka et al., FEBS Lett., 363 (3), 226-230.
(1995); M. Miyamoto et al., Mol. Cel. Biol., 13, 4
251-4259 (1993)), the putative amino acid sequence is already known.

The present inventors translated these cDNA sequences into amino acid sequences and compared them (FIGS. 1 to 8). As a result, among the amino acid sequences of FGF5, SEQ ID NO: 1 in the sequence listing
It was found that the portion having the amino acid sequence shown in 1) is a member belonging to the FGF family and has low homology with members other than FGF5 (other members of the FGF family). In addition, there is a putative sequence to which a sugar chain is added in the vicinity of this part (Fig. 1), and this part is FGF5.
It was also found that there is a high possibility that the protein is present on the protein surface. In addition, in FIGS. 1 to 8, “*” indicates a portion where the amino acids match, and the underline indicates a portion selected as an antigen. In each figure, the upper row shows the amino acid sequence of FGF5, and the lower row shows the FGF family. The amino acid sequences of other members of are shown.

The present inventors have paid attention to these points, and the synthesis of a peptide having the above sequence (hereinafter, also referred to as "a peptide specific for FGF5") is synthesized by TANA LABORATORIES, LC.
(Texas, USA). After immunizing a mammal with the obtained peptide, spleen cells were taken out and fused with cultured cells to prepare a hybridoma, which successfully produced a monoclonal antibody that specifically binds to FGF5. did.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below, focusing on a monoclonal antibody specific for FGF5 and a method for producing the same, in accordance with production procedures.

<1> Antigen The peptide specific for FGF5 used for immunizing a mammal includes the sequence shown in SEQ ID NO: 1 in the Sequence Listing as its amino acid sequence. It can be used in the present invention as long as it has at least a part of the amino acid sequence of a region having low homology with the amino acid sequences of other members. Such a peptide can be partially obtained, for example, by isolating it from an enzymatic degradation product of FGF5, but it is preferably chemically synthesized by a conventional method. For example, the above peptide can be obtained by inputting the above sequence using a commercially available peptide synthesizer. Alternatively, the peptide may be obtained by entrusting it to a company that provides a peptide synthesis service.

The peptide thus obtained (hereinafter, also referred to as "synthetic peptide") can be used as an antigen as it is, and it can be used as a keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA: Bovine). SerumAlbumin), ovalbumin (OVA: Ovalbumin) and the like can be used in combination with a carrier. When such a carrier is used, the antigenicity can be enhanced even if the peptide alone has low or no antigenicity. In this case, antibodies against the carrier can be prepared, but they can be removed at the stage of selecting the hybridoma.

<2> Immunization of mammals The mammals to be immunized with the above-mentioned antigens are not particularly limited as long as they are mammals usually used in immunization experiments, and examples thereof include mice, rats and rabbits. In addition, the method of immunization is
It may be carried out according to a usual immunization method. For example, synthetic peptide or a synthetic peptide bound to a carrier is repeatedly administered with Freund's complete or incomplete adjuvant every 1 to 2 weeks, and the final administration is repeated. Two
A method in which only a synthetic peptide is administered after a week or more and final immunization is carried out can be mentioned.

The dose of the synthetic peptide or the synthetic peptide bound to the carrier at this time is 0.5 m per dose.
About g / kg to 50 mg / kg is suitable. If the dose is less than 0.5 mg / kg, the antibody may not be sufficiently produced. Further, even if it exceeds 50 mg / kg, no further immune effect can be expected, and further, immunosuppression occurs in vivo, and the desired antibody may not be obtained.

<3> Preparation of hybridoma The animal immunized as described above is sacrificed 3 to 4 days after the final immunization, the spleen is excised, and splenocytes are taken out. A splenocyte-cultured cell hybridoma can be obtained by fusing this cell with a cultured cell that is a fusion partner in the presence of a fusion promoter and selecting the resulting fused cell.

The cultured cells to be fused are not particularly limited as long as they can be fused with splenocytes, but myeloma cells (myeloma cells) are generally suitable. Further, in order to be able to distinguish unfused cells and fused cells after cell fusion, those having a specific drug marker for selection are preferable.

Examples include hypoxanthine / guanine / phosphoribosyl transferase deficient. Such cells cannot grow in a medium (HAT medium) supplemented with hypoxanthine aminopterin and thymidine, but fused cells of these cells and normal cells are HAT.
It can grow in the medium and can be distinguished from unfused cells. Specifically, P3X63Ag8.65
3 strains, P3 / NSI / 1-AG4-1 strain, FO strain, SP
Commercially available strains of myeloma cells such as the 2 / 0-Ag14 strain are mentioned, but are not limited thereto.

Cell fusion is usually RPMI1640, MEM
The antibody-producing cells (spleen cells) and myeloma cells are mixed with a fusion promoter in a medium such as the above at a mixing ratio of 10: 1 to 2: 1. As a fusion accelerator,
There is no particular limitation as long as it is a fusion agent that is commonly used in cell fusion experiments, and specific examples thereof include Sendai virus and polyethylene glycol having an average molecular weight of 500 to 7,000. Alternatively, they may be fused by electric pulses. The cells that have completed cell fusion are diluted with, for example, RPMI1640 or MEM medium, the cells washed by centrifugation are suspended in a selective medium such as HAT medium, and the cells are dispensed into a multiplate or the like and cultured, and only the hybridomas are cultivated. Let it grow.

<4> Selection of hybridoma The hybridoma obtained as described above is a mixture of hybridoma strains that produce monoclonal antibodies against different antigenic determinant sites of peptides used as antigens or monoclonal antibodies against proteins used as carriers. Since it is the body, the synthetic peptide, namely FGF, is selected from these.
Hybridomas producing a monoclonal antibody that binds to a peptide specific for 5 are selected.

As a method of this selection, an enzyme immunoassay method (ELISA method) using a synthetic peptide used as an antigen is used.
Is preferred. For example, a synthetic peptide is immobilized on a plastic plate or the like, and a hybridoma culture supernatant is further immobilized on this plate, which is then labeled with an enzyme, a fluorescent substance, or a luminescent substance .
The amount of the antibody that binds to the synthetic peptide can be known from the amount of the bound label by adding the antibody. Alternatively, the antibody produced by the hybridoma may be solid-phased and then sequentially incubated with a second antibody labeled with a synthetic peptide, an enzyme or the like.

Further, from the hybridoma producing a monoclonal antibody which binds to the synthetic peptide, a hybridoma which binds to FGF5 and produces a monoclonal antibody which is a member of the FGF family and does not bind to anything other than the FGF5 is selected.

One strain of the hybridoma thus obtained is from the Institute of Biotechnology, Institute of Industrial Science and Technology (Postal Code 305, 1-3-1 Higashi Tsukuba City, Ibaraki Prefecture, Japan) since June 7, 1996. to, it has been deposited as FE R M P-15676.

<5> Preparation of monoclonal antibody that specifically binds to FGF5 FGF5 was prepared from the hybridoma obtained as described above.
In order to obtain a monoclonal antibody that specifically binds to, for example, the culture supernatant of the antibody-producing hybridoma cultured in an appropriate medium, or the ascites obtained by culturing the antibody-producing hybridoma in the mouse abdominal cavity is subjected to a conventional method. It may be purified by ammonium sulfate fractionation, gel filtration, affinity chromatography and the like.

<6> Utilization of monoclonal antibody which specifically binds to FGF5 Since the monoclonal antibody which specifically binds to FGF5 of the present invention specifically reacts with human FGF5, a usual antigen-antibody reaction was used. FGF5 can be detected by applying the antigen detection method. For example, it is used as an antibody for staining in Western blotting or microautoradiography, an antibody for staining in immunostaining of human-derived cultured cells, skin tissues, etc., and as a neutralizing antibody that suppresses the action of FGF5 by adding it to the culture system. It is possible, and FGF5 can be detected by using it as a detection reagent for qualitative or quantitative determination of FGF5. Therefore, by using the anti-FGF5 monoclonal antibody of the present invention, FGF in hair growth is
We can explore the role of G5. For example, the hair can be evaluated by detecting FGF5 in the hair. Furthermore, by staining the brain or the like with the monoclonal antibody of the present invention and detecting FGF5 in brain cells, for example, the brain function can be evaluated and the role of FGF5 in the brain can be explored.

[0033]

EXAMPLES Examples of the present invention will be described below.

<1> Preparation of hybridoma Synthesis of peptides and binding to a carrier (KLH) were carried out by TANA.
We requested LABORATORIES, LC (Texas, USA).

100 mg of the above carrier-bonded synthetic peptide was dissolved in 100 ml of physiological saline and mixed and emulsified with 100 ml of complete Freund's adjuvant (manufactured by SIGMA), and BALB / c mouse of 8 weeks old (CLEA Japan).
Was administered subcutaneously. 200 mg of the carrier-bonded synthetic peptide-incomplete Freund's adjuvant equivalent mixture emulsion was intraperitoneally administered 1 week and 2 weeks thereafter. Furthermore, 3
Two weeks after the second administration, 50 ml of physiological saline in which 50 mg of the synthetic peptide was dissolved was intravenously injected.

After 3 days, the spleen was removed from the mouse, and the splenocytes were suspended in RPMI1640 medium and washed.
On the other hand, mouse myeloma cell line SP2 / 0-Ag14
(Purchased from Dainippon Pharmaceutical Co., Ltd.) was cultured at the logarithmic growth phase in accordance with cell fusion and collected by centrifugation. 10
For 8 splenocytes, 2 × 10 7 myeloma were mixed in the above medium and the cells were pelleted by centrifugation. After removing the supernatant, 50% PEG4 kept at 37 ℃
1 ml of RPMI1640 medium containing 000 (manufactured by Sigma: polyethylene glycol) is gradually added dropwise over 1 minute,
Stir gently for 1 minute. Furthermore, RPMI16 at 37 ℃
2 ml of 40 medium was added dropwise over 2 minutes, and 8 ml was added dropwise over 3 minutes with stirring.

After completion of the dropping, the supernatant was removed by centrifugation and the cell pellet was added to 40 ml of GIT medium (manufactured by Wako Pure Chemical Industries).
100 ml per well was dispensed into four 96-well plates (Sumitomo Bakelite Co., Ltd.). The next day, 25 ml of HAT medium was added, and 4 days later, 25 ml of HAT medium was added. After culturing for 1 week,
The culture supernatant was removed in half and 100 ml of GIT medium was added.

Approximately 2 weeks after cell fusion, only hybridomas in which myeloma and spleen cells were fused formed colonies, and the culture was continued until the diameter of the colonies became approximately 1 mm. At this point, the antibody secreted in the culture supernatant was
The above-mentioned synthetic peptide and a commercially available secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG Fc fragment antibody; manufactured by CHEMICON) were assayed by a sandwich ELISA method. Of these, the culture supernatant in the well with the highest antibody titer was further assayed by the same sandwich ELISA method as described above, except that the commercially available FGF5 recombinant protein (manufactured by Sigma) was used as the antigen. Finally, the hybridoma in the well having a high antibody titer was cloned by the limiting dilution method to obtain a monoclonal antibody-producing hybridoma strain. This strain, in June 1996 7 Agency of Life Institute of Advanced Industrial Science and Technology than days (zip code 305, Japan Higashi, Tsukuba, Ibaraki chome No. 1 No. 3), is deposited as FE R M P-15676 There is.

<2> Preparation of monoclonal antibody that specifically binds to FGF5 The hybridoma cell line obtained above was cultured to produce FGF.
A monoclonal antibody that specifically binds to 5 was collected.

The above hybridoma was cultured in GIT medium, and when the cell concentration reached about 5 × 10 6 cells / ml, the culture supernatant was collected by centrifugation, filtered with a filter having a pore size of 0.22 mm, and the filtrate was obtained. Was purified with a protein A column kit (manufactured by Amersham Japan) to obtain anti-FGF5 monoclonal antibody.

<3> Evaluation of Anti-FGF5 Monoclonal Antibody (1) Reactivity of Anti-FGF5 Monoclonal Antibody with FGF5 Regarding the monoclonal antibody obtained above, commercially available F
Specificity was evaluated by the enzyme immunoblotting method using recombinant proteins of GF5, FGF6, and FGF7 (manufactured by Sigma). Electrophoresis was performed according to a conventional method using a 5-20% density gradient polyacrylamide gel (Pagel: manufactured by Ato). The amount of applied was 100 ng of FGF per lane. After completion of electrophoresis, one lane of the gel was cut out and subjected to CBB staining to detect total protein. After immersing the rest of the gel in a transfer buffer (100 mM Tris; 192 mM glycine), a blotting apparatus (semi-dry blotting apparatus: manufactured by ATTO) was used, and the electrophoretic material was immersed in the transfer buffer in advance from the gel (immobilon). (Immobilon); manufactured by Millipore).

The membrane on which the electrophoretic material was transferred was transferred to a TBS buffer (20 mM Tris; 500 mM NaCl;
After soaking in pH 7.5 for 15 minutes, blocking buffer (5% skim milk or 1% BSA and 0.
Soak in 1% Tween20 in TBS buffer),
It was gently shaken at 25 ° C. for 1 hour.

Thereafter, the membrane was replaced with 0.1% Tween.
After washing with a TBS buffer containing 20 (TTBS buffer) (15 minutes once, 5 minutes twice), the monoclonal antibody solution obtained in the above <2> (10-fold diluted solution to about 2
ml) and gently shaken at 25 ° C. for about 1 hour.
After washing the membrane, a commercially available secondary antibody solution (horseradish peroxidase (HRP) -labeled goat anti-mouse IgG Fc fragment antibody; CHEMICON) was used.
N), dip 20,000 times diluted solution in about 2 ml),
It was gently shaken at 25 ° C for about 1 hour.

After washing the membrane with TTBS buffer (15 minutes once, 5 minutes 4 times), a commercially available HRP detection kit (Amersham Co .; ECL kit) was used.
A band of the electrophoretic material bound with the anti-FGF5 monoclonal antibody was detected. The band to which HRP was bound was 4-
It can also be detected using chloro-1-naphthol as a substrate.

FIG. 9 shows the results of the enzyme immunoblotting method.
Shown in. As is clear from this result, although the monoclonal antibody obtained above binds to FGF5,
And does not bind to FGF7. In addition, the synthetic peptide used as an antigen is a portion having no homology with other FGF families and does not react with other members of the FGF family. That is, the present monoclonal antibody was found to have a high homology with FGF5 among the members of the FGF family.
Was found to react specifically with.

(2) Determination of isotype of anti-FGF5 monoclonal antibody The membrane attached to a commercially available isotyping kit (manufactured by Amersham) was used as a mixed solution of anti-human FGF5 monoclonal antibody (hybridoma culture supernatant) and a secondary antibody attached to the kit. And incubated for 15 minutes at room temperature.
After washing the membrane with PBS (phosphate buffer) containing 0.1% Tween 20 for 5 minutes twice, the membrane was immersed in a coloring agent included in the kit and incubated at room temperature for 15 minutes or longer to detect the isotype. As a result, the isotype of the anti-FGF5 monoclonal antibody was
It was found to be IgG1 with a k light chain.

[0047]

The anti-FGF5 monoclonal antibody of the present invention specifically binds to FGF5.
It can be used to detect hair, and can be used for hair evaluation, brain function evaluation, and the like.

[0048]

[Sequence list]

SEQ ID NO: 1 Sequence length: 13 Sequence type: Amino acid Topology: linear Sequence type: Peptide Array Ala Ser Ala Ile His Arg Thr Glu Lys Thr Lys Thr Gly 1 5 10

1

[Brief description of drawings]

FIG. 1 is a diagram showing the homology of the amino acid sequence of FGF1 with FGF5.

FIG. 2 is a diagram showing the homology of the amino acid sequence of FGF2 to FGF5.

FIG. 3 is a diagram showing the homology of the amino acid sequence of FGF3 to FGF5.

FIG. 4 is a diagram showing the homology of the amino acid sequence of FGF4 to FGF5.

FIG. 5 is a diagram showing the homology of the amino acid sequence of FGF6 to FGF5.

FIG. 6 is a diagram showing the homology of the amino acid sequence of FGF7 to FGF5.

FIG. 7 shows the homology of the amino acid sequence of FGF8 with FGF5.

FIG. 8 is a diagram showing the homology of the amino acid sequence of FGF9 to FGF5.

FIG. 9 is a photograph showing the results of electrophoresis of FGF5 by enzyme immunoblotting.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI G01N 33/53 G01N 33/577 B 33/577 A61K 39/395 N // A61K 39/395 C12N 15/00 ZNAA (72) Inventor Tomomi Kato 560 Kashio-cho, Totsuka-ku, Yokohama-shi, Kanagawa Pola Chemical Industry Co., Ltd., Totsuka Laboratory (56) Reference JP-A-7-149797 (JP, A) JP-A-6-125784 (JP, A) Special Table 1- 503518 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 16/22 C12N 15/09 ZNA C12P 21/08 G01N 33/50 G01N 33/53 G01N 33/577 A61K 39 / 395 CAOLD (STN) CAPLUS (STN) SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / MEDLINE / WPID S (STN)

Claims (6)

(57) [Claims] 1. An amino acid sequence represented by SEQ ID NO: 1.
Mammalian lymphocytes and mammalian lymphocytes immunized with peptides
A hybridoma obtained by fusing with Roman cells
A monoclonal antibody that binds to FGF5 and is produced by. 2. The hybridoma has an accession number of FERM.
 The hybridoma of P-15676, according to claim 1.
Monoclonal antibody. 3. A hive with an accession number of FERM P-15676.
Redoma. 4. Use of the antibody according to claim 1 or 2.
And a method for detecting FGF5. 5. Using the antibody according to claim 1 or 2.
A method for detecting FGF5 in hair. 6. Using the antibody according to claim 1 or 2.
A method for detecting FGF5 in brain cells.