JPH07118298A - Monoclonal antibody to pdgf-a and its production - Google Patents

Abstract

PURPOSE:To provide the monoclonal antibody, an active fragment thereof, and a hybridoma capable of producing said monoc lonal antibody. CONSTITUTION:Of an amino acid sequence coded by. the gene of human PDGF- A, a peptide rich in hydrophilicity and having 14 amino acids from the 176th amino acid to 189th amino acid low in homology with PDGF-B (Glu-Cys-Ala-Cys- Ala-Thr-Thr-Ser-Leu-Asn-Pro-Asp-Tyr-Arg) is synthesized and then immunized to obtain the monoclonal antibody reactive with PDGF-A protein itself.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody and a method for producing the same, more specifically, PDGF-A.
The present invention relates to a monoclonal antibody that specifically reacts with, an active fragment thereof, a method for producing the monoclonal antibody, and a hybridoma producing the monoclonal antibody.
INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention is useful in basic research and diagnosis of diseases such as arteriosclerosis, myelofibrosis, interstitial pneumonia, and wound treatment, and industrial fields related thereto.

[0002] In the present invention, the "active fragment" of a monoclonal antibody refers to a fragment having antigen-antibody reaction activity, specifically, F (ab ').
₂ , Fab ′, Fab, Fv, and recombinant Fv form.

[0003]

2. Description of the Related Art PDGF (Platelet-deri)
ved growth factor) is a growth factor mainly for cells derived from mesoderm, and is a combination of PDGF-A chain and PDGF-B chain, and three isomers of PDGF-AA, -BB, and -AB. Composed of. PDGF is mediated by PDGF receptors on the surface of smooth muscle cells and fibroblasts.
Promotes cell growth. The PDGF receptor has α-type (= A
Type) and β type (= B type) are found, and both types of receptors have a tyrosine kinase domain.

PDGF was found in human platelets as a major factor in serum responsible for the growth-promoting activity of fibroblasts and smooth muscle cells, but PDGF-B was subsequently detected by the sarcoma virus (simian sarcoma virus).
s; SSV) transform gene (transf)
Since it has a high homology with v-sis, which is an activating gene), it is famous that the oncogene attracted attention as the first example of the cell growth factor itself.

PDGF is present in α granules in platelets and is produced by its precursor cells, bone marrow megakaryocytes. Production and secretion from various cancer cells including osteosarcoma and glioma have been reported. But,
Recently, it has been revealed that it is also produced and secreted from various normal cells such as activated macrophages, vascular endothelial cells, vascular smooth muscles, fibroblasts and the like. Thus, PDGF produced from various cells is not only autocrine proliferation of tumor cells, but also arteriosclerosis, myelofibrosis, interstitial pneumonia, wound healing, mesenchymal cell development and differentiation, pregnant uterine smoothness. It is involved in a wide range of pathological and physiological processes such as hypertrophy of muscles.

[0006] PDGF causes various metabolic changes in the cells that act, induces cells in the G ₀ phase to the G ₁ phase, and
It is a receptive factor that causes a change in preparation for entering the period. P
After binding to the DGF receptor, a PI response occurs within minutes within the cell membrane, resulting in activation of protein kinase C, elevation of Ca ²⁺ , Na ⁺ / H ⁺ ion exchange, and the like. With these changes, c-myc, c-fos, c-j in the nucleus
Expression of gene groups such as un is observed.

In human bone marrow megakaryocytes, PDGF-A and PD
GF-B mRNA is expressed almost 1: 1. When a wound occurs, the platelet aggregation locally occurs due to the exposure of the subendothelium and the blood coagulation reaction.
Is released. PDGF locally migrates neutrophils and monocytes and activates them. Then, in the vascular endothelial cells, macrophages are activated to produce / secrete PDGF, which stimulates local migration / proliferation of fibroblasts. Fibroblasts themselves also secrete PDGF-AA and proliferate to repair wounds.

[0008] Monocytes do not produce PDGF, but when differentiated into macrophages, PDGF-A and PD are responsive to stimulation.
Produces and secretes GF-B. In interstitial pneumonia, macrophages in bronchoalveolar lavage fluid (BALF) contain large amounts of P.
It produces and secretes DGF-B and is involved in the migration / proliferation of fibroblasts, which is the essential form of lung fibrosis. Vascular endothelial cells are stimulated by thrombin, blood coagulation factor X, hypoxic exposure, etc.
Secretes DGF and causes hypertrophy / proliferation of vascular smooth muscle. Endothelial cells have a pool of PDGF,
When the pool is exhausted, it will be newly synthesized and secreted.

Normal smooth muscle cells do not express the PDGF gene, whereas cultured smooth muscle cells and arteriosclerotic smooth muscle cells express mainly the PDGF-A chain gene,
Secretes PDGF-AA. Pathological smooth muscle cells (different in phenotype from normal vascular wall smooth muscle cells) in the indurated plaque
It also expresses the DGF-α receptor, which causes the development of arteriosclerotic lesions.
In order to progress, autocrine proliferation of smooth muscle cells by PDGF-AA in addition to PDGF-B produced by macrophages is important for vicious cycle formation.

In osteosarcoma and glioma, expression / production of PDGF-B chain is observed, suggesting tumor growth by autocrine. In human glioma, PDG was found in most cell lines.
Although it expresses the F-A chain, many express the B chain, and the PDGF-β receptor is simultaneously expressed in many of these cell lines. On the other hand, in human tumor cell lines such as gastric cancer, colon cancer, hepatocellular carcinoma, lung cancer, breast cancer, ovarian cancer, bladder cancer, leukemia, PDGF expression and production are observed, but the receptor is not expressed. These stimulate fibroblasts and smooth muscle cells around the cancer tissue to cause an increase in connective tissue, and especially in the scirrhous type found in gastric cancer, lung cancer and breast cancer,
It is considered to be greatly involved in its formation.

In the pregnant uterus, which is developed and maintained by a delicate hormonal environment such as estrogen and progesterone, smooth muscle cell hypertrophy / proliferation is required in the process. Uterine smooth muscle cells showed PDGF-A
It has been clarified that A and PDGF-α receptors are expressed and produced and proliferate by an autocrine mechanism.

Hamartomatous pulmonary vascular myomatosis (Pulmonar)
y lymphangiomyomatosis; LA
M) is a respiratory disease observed only in women of reproductive age, and diffuse smooth muscle hyperplasia is observed in the lungs, which causes respiratory failure due to destruction of the alveolar wall. Since an estrogen receptor, which is not normally found in proliferated abnormal smooth muscle cells, is detected only in women of reproductive age, interest in the relationship between its onset and female hormones, hormone therapy has been performed. It is being appreciated. CDNA was synthesized using total RNA collected from LAM lung tissue to generate P
When CR is performed, almost no PDGF-B or PDGF-β receptor is detected, but the PDGF-A or PDGF-α receptor has a band having almost the same intensity as that of the late-gestation uterine smooth muscle cells. That is, the involvement of the autocrine mechanism by PDGF-A is considered.

[0013]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a PD
In order to contribute to the functional analysis of GF-A, the analysis of various diseases in which PDGF-A is involved, etc., it is to provide a monoclonal antibody against PDGF-A, an active fragment thereof, and a hybridoma producing the monoclonal antibody.

The present inventor has analyzed the involvement of PDGF-A in many diseases mentioned above by using PDGF-A.
It was found that it is important to produce a monoclonal antibody against A. cerevisiae, and as a result of earnest research, it was found that the amino acid sequence encoded by the human PDGF-A gene was rich in hydrophilicity and was homologous to PDGF-B. Low amino acid 176 to amino acid 189 14
By synthesizing a peptide having an individual and immunizing the peptide, a hybridoma producing a monoclonal antibody that reacts with the PDGF-A protein itself was successfully established, and the present invention was completed.

Since the monoclonal antibody of the present invention recognizes the PDGF-A molecule in paraffin-fixed tissues, that is, it reacts with formalin-fixed paraffin sections, it can be used for daily pathological work requiring safe and rapid processing. Can also be applied. The present invention has been completed based on these findings.

[0016]

The present invention thus provides a monoclonal antibody against PDGF-A or an active fragment thereof. According to the present invention, (1) a rodent is a synthetic peptide Glu-Cys-Ala-Cys-Ala-T containing amino acids 176 to 189 of the amino acid sequence of human PDGF-A protein.
hr-Thr-Ser-Leu-Asn-Pro-As
Immunizing with p-Tyr-Arg, (2) removing the spleen from the immunized rodent to form a spleen cell suspension, and (3) adding the spleen cell suspension Antibody-producing cells are mixed with mouse myeloma cells in the presence of a fusion promoter to fuse both cells, and (4) the fused cells are diluted and cultured in a medium that does not support unfused myeloma cells. Hybridomas that are fused with myeloma cells are selected,
(5) In the supernatant of each culture well containing the hybridoma, the presence of the antibody is confirmed using the index as to whether or not it reacts with the synthetic peptide, and (6) after selecting the hybridoma that produces the desired antibody PDG characterized in that it is made into a single clone by the limiting dilution method, and (7) the antibody is recovered from the culture supernatant of the hybridoma of the single clone.
A method for producing a monoclonal antibody that reacts with F-A is provided.

Further, according to the present invention, there is provided a hybridoma producing a monoclonal antibody which reacts with PDGF-A. The hybridoma of the present invention can be obtained, for example, from the Institute of Biotechnology, National Institute of Bioscience and Technology, under the contract number FERM.
Hybridoma deposited as P-13878
SE-2H10 can be mentioned.

The present invention will be described in detail below. The monoclonal antibody against PDGF-A in the present invention and the hybridoma producing the monoclonal antibody can be prepared as follows.

(1) A peptide (Glu-Cys-Ala-Cys-Ala-Thr-) containing amino acids 176 to 189 of the PDGF-A protein molecule.
Thr-Ser-Leu-Asn-Pro-Asp-T
yr-Arg) is synthesized and then purified. This peptide is
Since it is a low molecular weight substance (hapten), it is usually bound to the carrier protein KLH and then immunized to a rodent to induce an immune response. In addition, it is desirable to use an adjuvant in combination in order to enhance the immune responsiveness to rodent antigens.

(2) Antibody-producing cells are prepared from the immunized rodent. Specifically, the spleen is removed from the immunized rodent to form a suspension of spleen cells.

(3) The previously cultured myeloma cells and antibody-producing cells are fused with each other in the presence of a fusion promoter such as polyethylene glycol. Specifically, spleen cells are fused with mouse myeloma cells. As myeloma cells, those that can be distinguished from antibody-producing cells in the following selective culture (for example, α-azaguanine resistant strain) are used.

(4) Antibody-producing cells are viable, but myeloma cells are killed. The cells are diluted and cultured in a selective medium (a medium that does not support unfused myeloma cells) to fuse the antibody-producing cells with the myeloma cells. Select hybridomas.

(5) The presence of the antibody in the supernatant of each culture well containing the hybridoma is confirmed by using the index as to whether or not it reacts with the synthetic peptide. Methods such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay are used to detect the antibody.

(6) After selecting a hybridoma which produces the desired antibody, it is made into a single clone by the limiting dilution method. Specifically, the produced hybridomas are screened based on the ELISA which shows reactivity with synthetic peptides. In the limiting dilution method, cell suspension is serially diluted,
Finally, each hybridoma strain is purely cultured so that only one hybridoma cell exists in the culture well (hole). Finally react with PDGF-A molecule,
Cells that produce antibodies that do not react with the GF-B molecule are selected.

(7) The antibody is recovered from the culture supernatant of the hybridoma of the single clone. The present monoclonal antibody can be prepared by purifying the culture supernatant of the hybridoma or the ascites obtained by administering the hybridoma into the abdominal cavity of the mouse.

The monoclonal antibody against PDGF-A of the present invention has the specificity shown in the following examples.
For example, the monoclonal antibody reacts with PDGF-A protein. The monoclonal antibody also recognizes the PDGF-A molecule in paraffin-fixed tissue.

[0027]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 (1) Preparation of PDGE-A Peptide (i) Synthesis of Peptide Among the amino acid sequences encoded by the PDGF-A gene, it is highly hydrophilic and PDGF-B. Was synthesized with low homology from the 176th amino acid to the 189th amino acid 14 amino acids using a peptide synthesizer 431A (manufactured by Applied Biosystems).

(Ii) Purification of peptide The synthesized peptide is first cleaved from the resin. As the cleavage liquid, 0.75 g of crystalline phenol, 0.25 ml of 1,2-ethanediol, 0.5 ml of thioanisole, 0.5 ml of distilled water, and 10 m of trifluoroacetic acid (TFA).
1 was used. The dried resin with peptide and a stir bar are placed in a flask, stoppered, and cooled in an ice bath for 30 minutes. Put the above cutting solution in another container and cool in an ice bath (30
Minutes). The cleavage solution is placed in the flask containing the resin with peptide. After that, leave it to reach room temperature, then
Stir for 1.5 hours with a stir bar. The reaction solution obtained was 6
Filter through a μm membrane filter and pool the filtrate in a separate flask. Add 1-2 ml of TFA to the flask used for cutting and wash the solution on the wall, which is also filtered through the filter and the filtrate is pooled. The filtrates are combined, 50 ml of ice-cooled diethyl ether is added, and the mixture is left for about 30 minutes (the precipitation of the peptide is sufficiently confirmed). The precipitate (peptide) is collected by filtration with a 3 μm membrane filter. The crude peptide is dried by vacuum drying. 20 mg of the obtained crude peptide was dissolved in 2 ml of distilled water,
It is filtered through an 8 μm filter, and the obtained filtrate is FPL
Purified at C. The column used was PepRPC10 / 10 (Pharmacia) and was eluted with a gradient of 70% acetonitrile against 0.1% TFA. The elution fraction of the peptide was collected and purified peptide was freeze-dried.
3 mg was obtained.

(Iii) Carrier protein (KL
H: Binding with keyhole limpet hemocyanin, manufactured by Pierce Co., Ltd. The protocol of the super carrier system manufactured by Pierce Co. was followed. Peptide 2m obtained by the above (ii)
g was dissolved in 200 μl of binding buffer. 2 mg of maleimide-activated KLH was dissolved in 200 μl of distilled water. Peptide and maleimide activated KLH are mixed and allowed to stand at room temperature for 2
Reacted for hours. The reaction was carried out by stirring using a rotator.

(2) Immunization BALB / c mice 6 weeks old (female) were injected with 100 μg of PDGF-A-KLH prepared in (1) together with Freund's complete adjuvant in the footpad of the mouse. Then, after 1 week, the mice were immunized by injecting PDGF-A-KLH and Freund's incomplete adjuvant intraperitoneally once a week for a total of 5 times.

[0032] (3) fusion 3 days after the final immunization, the spleens were removed from the mice.
The spleen taken out is shredded, filtered through a mesh, and RP
The spleen cells were suspended in M11640 medium to give 1 × 10 ⁸ cells.
I got it. This spleen cell was mixed with a mouse-derived α-azaguanine resistant strain (hypoxanthine-guanine phosphoribosyl transferase deficient strain) P3U1 (2 × 10 ⁷ cells) at a ratio of about 5: 1 and centrifuged (1500 rpm, 5
Minutes). To the obtained cell pellet, 2 ml of 50% polyethylene glycol 4000 (manufactured by Merck) / RPM11640 solution was added in warm water at 37 ° C. with stirring for 1 minute. To this, 15 ml of RPM11640 solution was added with stirring for 6 minutes to perform cell fusion. After fusion, add a large amount (about 40 ml) of RPM11640 solution,
The supernatant was removed by centrifugation (1500 rpm, 5 minutes). Then containing hypoxanthine (100 μM), aminopterin (0.4 μM), thymidine (10 μM) 1
It was prepared in 0% FCS-RPM11640 medium (HAT medium) so that the spleen cells would be 1 × 10 ⁴ ml.

(4) Selection of hybridoma 200 μl of the cell suspension prepared in the above (3) was dispensed into five 96-well microplates at 37 ° C., 5% C
The cells were cultured in a CO ₂ incubator under O ₂ . 1
After a week, only hybridomas form colonies,
It was confirmed that they were proliferating.

(5) Detection of antibody Since it was confirmed that the hybridoma was proliferating sufficiently, the culture supernatant was used to detect the antibody by the enzyme-linked immunosorbent assay (ELISA) described below. PDGF above
The -A partial peptide was dispensed at a concentration of 10 µg / ml in 50 µl aliquots onto an assay plate (Dynatech Immunol 2) and antigen-fixed (4 ° C / overnight). Then, the antigen solution was removed, and a blocking solution (Block Ace, manufactured by Dainippon Pharmaceutical Co., Ltd.) was added for blocking (room temperature, 1 hour). Then, the blocking solution was removed, and 50 μl of the hybridoma culture supernatant was dispensed and reacted at room temperature for 1 hour. P
After washing the plate with BS, 50 μl of a 1000-fold diluted peroxidase-labeled anti-mouse Ig antibody (manufactured by Kappel) was added and allowed to bind (room temperature, 1 hour). Then, wash again with PBS, and then
Phosphoric acid-citrate buffer solution (pH containing phenylenediamine (0.4 mg / ml) and 0.012% hydrogen peroxide solution)
5.0) 100 μl was added to these plates and antibodies were detected by color reaction. As a result, 11 wells reacting with the PDGF-A partial peptide were selected.

(6) Cloning The hybridoma growing in the well selected in (5) above was cloned by the limiting dilution method. 10% FCS-R so that the cell concentration after dilution is 0.5 cells / well
It diluted with PM11640 culture medium and 200 microliters was dispensed to the 96 well microplate. After culturing at 37 ° C. in the presence of 5% CO _2, whether or not the antibody against the PDGF-A partial peptide has been produced after the colony has grown to a certain size, the enzyme immunoassay of the above (5) is performed again. I checked by law. Thus, it was possible to obtain two types of clonal cells that react with the PDGF-A partial peptide.

(7) Determination of subclass Anti-mouse antibody [anti-IgG ₁ , anti-IgG _2a , anti-IgG _2b ,
Anti-IgG ₃ , anti-IgM, anti-IgA], and anti-mouse light chain antibody (anti-k, λ) (MAB Isotyping Kit, Pharmingen) were reacted with anti-PDGF-A antibody of the culture supernatant water of the hybridoma. By the ELISA
The subclass of the antibody produced by the two hybridomas obtained was determined. As a result, both were found to be IgM, k.

(8) Reactivity with protein The antibody that reacts with the obtained PDGF-A peptide is PDG.
It was examined whether it would react with the F-A protein. PDG
F-A protein was dispensed at a concentration of 1 μg / ml into an assay plate in an amount of 50 μl to fix the antigen (4 ° C./night).
Then, the antigen solution was removed, and blocking solution (Block Ace: manufactured by Dainippon Pharmaceutical Co., Ltd.) was added for blocking (room temperature, 1 hour). After that, the blocking solution was removed, 200 μl of the hybridoma culture supernatant was dispensed, and
Reacted for hours. After washing the plate with PBS, 50 μl of a 1000-fold diluted peroxidase-labeled anti-mouse Ig antibody (manufactured by Kappel) was added and allowed to bind (room temperature, 1 hour). Then, after washing with PBS again, ortho-phenylenediamine (0.4 mg / m 2
l), 100 μl of a phosphate-citrate buffer (pH 5.0) containing 0.012% hydrogen peroxide was added to these plates, and the antibody was detected by a color reaction. as a result,
One clone was obtained that reacted with the PDGF-A protein, and this clone was named SE-2H10.

(9) Reactivity with tissue It was examined whether the obtained monoclonal antibody against the PDGF-A molecule reacts with tissue of a living body. The material used was tissue of uterine muscle and hamartomatous angiomyoma at 31 weeks of gestation.
Each tissue was fixed with cold acetone or 10% formalin solution and then stained. For staining, after blocking by adding a blocking solution, 0.5 µg of an antibody produced by the SE-2H10 clone was added and reacted (room temperature, 1 hour). Fifty
mM Tris-HCl (Tris-HCl) buffer pH 7.
Wash at 6 for 5 minutes. Then biotinylated anti-mouse Ig
A solution prepared by diluting (Capel) 100 times was added and reacted (room temperature, 1 hour). After washing with Tris buffer, AB solution (manufactured by Vector Co.) was added and the reaction was continued for 30 minutes. After washing with the same buffer, diaminobenzidine (0.25 mg / ml),
Color was developed with Tris buffer containing 0.02% hydrogen peroxide. As a result, as shown in Table 1, PD was detected in smooth muscle cells.
Expression of GF-A was observed.

[0039]

[Table 1]

The hybridoma was deposited under the contract number FERM P-1387 at the Institute of Biotechnology, Institute of Industrial Science and Technology.
Hybridoma SE-2H1 entrusted as No. 8
It is 0.

[0041]

INDUSTRIAL APPLICABILITY Since the monoclonal antibody against PDGF-A of the present invention reacts with the PDGF-A protein, basic research and diagnosis of diseases such as arteriosclerosis, myelofibrosis, interstitial pneumonia, and wound treatment. , And their related industrial fields. Further, the monoclonal antibody of the present invention is used for PDGF-in tissues fixed with paraffin.
Since it recognizes the A molecule, it can be applied to daily pathological work requiring safe and rapid processing.

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Claims (6)

[Claims] 1. A monoclonal antibody against PDGF-A or an active fragment thereof. 2. PDGF in paraffin-fixed tissue
-The monoclonal antibody or an active fragment thereof according to claim 1, which recognizes an A molecule. 3. A synthetic peptide Glu-Cys-Ala-Cys-Ala-Thr-T containing amino acids 176 to 189 of the human PDGF-A protein.
hr-Ser-Leu-Asn-Pro-Asp-Ty
The monoclonal antibody or an active fragment thereof according to claim 1, which is a monoclonal antibody against r-Arg or an active fragment thereof and reacts with the human PDGF-A molecule itself. 4. (1) A synthetic peptide Glu-Cys-Ala-Cys-containing a rodent animal containing the amino acids 176 to 189 of the amino acid sequence of human PDGF-A protein.
Ala-Thr-Thr-Ser-Leu-Asn-P
(2) Immunization with ro-Asp-Tyr-Arg and removing the spleen from the immunized rodent to form a suspension of spleen cells (3) Suspension of the spleen cells The solution is mixed with mouse myeloma cells in the presence of a fusion promoter to fuse both cells, and (4) the fused cells are diluted and cultured in a medium that does not support unfused myeloma cells, and the antibody Hybridomas in which the producing cells and myeloma cells are fused are selected, and (5) the presence of the antibody in the supernatant of each culture well containing the hybridomas is confirmed by using the index as to whether or not it reacts with the synthetic peptide, 6) After selecting a hybridoma that produces a desired antibody, the clone is made into a single clone by the limiting dilution method, and (7) the antibody is recovered from the culture supernatant of the hybridoma of the single clone.
A method for producing a monoclonal antibody that reacts with DGF-A. 5. The method according to claim 4, wherein the rodent is a BALB / c mouse, and the myeloma cell is P3U1. 6. A hybridoma that produces a monoclonal antibody that reacts with PDGF-A.