JP2001354699A - Antibody binding to angiotensin ii - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a monoclonal antibody binding to angiotensin 11 useful for detection and measurement of angiotensin 11 and diagnosis based on the detection and measurement and further for treatment of cardiovascular diseases. SOLUTION: This monoclonal antibody binds to angiotensin 11 produced from a hybridoma cell obtained by cell fusion of lymphocyte obtained by immunizing angiotensinogen knock-out animal with angiotensin 11 with a myeloma. An animal cell produces the above monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to angiotensin II
The present invention relates to a monoclonal antibody that binds to angiotensin II, a system for detecting and measuring angiotensin II using such an antibody, and a diagnostic agent.

[0002]

_2. Description of the Related Art Angiotensin I is a peptide consisting of 10 amino acids (NH ₂ -Asp-Arg-Val-Ty).
r-Ile-His-Pro-Phe-His-Leu
-COOH), which is produced from angiotensinogen, a precursor protein, by processing with renin. Furthermore, angiotensin I is processed by angiotensin converting enzyme (ACE), and angiotensin II (NH ₂ -Asp-) comprising 8 amino acids is processed.
Arg-Val-Tyr-Ile-His-Pro-P
he-COOH). Angiotensin II is mediated by specific receptors and regulates blood pressure such as vascular smooth muscle contraction, contraction of renal glomerular mesangial cells, contraction of renal glomerular import / export arterioles, promotion of sodium reabsorption of proximal tubule, increase of adrenal aldosterone secretion, etc. It has been shown to have a direct effect on regulation and water / electrolyte metabolism. In recent years, it has become clear that angiotensin II has an action as a growth factor separately from the above-mentioned action. In other words, based on studies using cultured cells and pharmacological studies using angiotensin receptor antagonists and angiotensin converting enzyme inhibitors, angiotensin II is a specific receptor and intracellular signal transduction for constituent cells of cardiovascular and renal tissues. It has been shown through the system that it has various biological activities such as growth, promotion of extracellular matrix synthesis, and hypertrophy (Science, 245, 186).
(1989), Journal of Crdiovascular Pharmacology, 19
(suppl.1), S51 (1992), Kidney International, 40, 58
3 (1991)). From these findings, it is considered that angiotensin II is involved not only in hypertension but also in circulatory proliferative diseases such as cardiac hypertrophy, arteriosclerosis, myocardial infarction, and glomerulosclerosis.

As described above, angiotensin II is a physiologically and pathophysiologically important factor, and its detection and measurement system in living tissue is important in medical diagnosis and medical research. Currently, angiotensin II is measured by radioisotope radioimmunoassay using polyclonal and monoclonal antibodies (Journalof
Cardiovascular Pharmacology, 8 Suppl 10: S23-8 (198
6), Biochemcal Biophysical Research Communication
143 (1): 133-9 (1987)) and an enzyme immunoassay using a polyclonal antibody (Peninsula, EIA for
Angiotensin II (Human), EIAH-7002) is common.
However, since these methods use radioisotopes and are expensive, a simpler and cheaper measuring system is desired.

[0004]

An object of the present invention is to obtain an anti-angiotensin monoclonal antibody required for a simpler angiotensin measuring system. By using such an antibody, it is expected that a large amount, short time, and inexpensive measurement of a specimen sample containing angiotensin can be performed. Antibodies that neutralize the physiological activity of angiotensin II by binding to angiotensin II are considered to be caused by excessive angiotensin II, such as hypertension, cardiac hypertrophy, arteriosclerosis, myocardial infarction, and glomeruli It is expected to be effective for improvement and treatment of circulatory proliferative diseases such as sclerosis.

[0005]

Means for Solving the Problems The present inventors have obtained a monoclonal antibody that binds to angiotensin II using KLH-conjugated angiotensin II as an immunogen using an angiotensinogen knockout animal, and completed the present invention.

[0006] That is, the present invention is an antibody that binds to angiotensin II, and is a monoclonal antibody capable of detecting angiotensin II in a living body by an enzyme immunoassay.

[0007] The present invention is also a monoclonal antibody capable of neutralizing angiotensin II activity.

Further, the present invention relates to an animal cell such as a hybridoma cell producing the above monoclonal antibody.

For example, in the production of monoclonal antibodies in mice, non-mouse protein-derived molecules are used as antigen molecules. This is because, when a mouse-derived antigen is used, immunological tolerance has already been established for the molecule, and thus it is difficult to recognize the molecule as an antigen molecule, and it is difficult to produce antibodies.

Incidentally, it is known that the primary structure of angiotensin II is widely common to mammals. Therefore, even if a mouse is immunized with human angiotensin II, it cannot be considered as an "antigen derived from a non-mouse protein".

In order to solve this problem, the present inventors have conceived to use a mouse which is considered to have no tolerance to angiotensin, ie, an angiotensinogen knockout mouse (Tsukuba hypotensive mouse), as an immunized animal. did. It was thought that this could be used to produce a highly specific anti-angiotensin monoclonal antibody.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
For example, it can be prepared by the method described below. That is, basically, Galfre and Milstein, Methods in
Enzymology, Vol. 73, Academic Press, New York (19
81); James W. Goding, Monoclonal Antibodies: Princi
ples and Practice, Academic Press, Orlando, Florid
a (1986); Current Protocols in Molecular Biology,
FM Ausubel et al., WileyInterscience, New York (1
987).

To prepare a monoclonal antibody,
It is better to use purified antigen peptides. This purified peptide is also used for antibody titer assays. The method for preparing the antigen peptide used for preparing the monoclonal antibody of the present invention will be described in detail in Examples.

Next, knockout animals are repeatedly injected intraperitoneally or subcutaneously with KLH-conjugated angiotensin II together with an adjuvant to generate an immune response. Subsequently, B lymphocytes are removed from the immunized animal, and cell fusion is performed with an animal cell having infinite proliferation ability by the method described in the above-mentioned literature. As the knockout animal, a knockout mouse is preferable. A preferred cell line for fusion is mouse myeloma cells, and P3-X
63-Ag8-U1 myeloma cells (ATCC CRL1
597) are particularly preferred.

After cell fusion, culturing is continued in an appropriate selection medium according to a conventional method, and angiotensin is selected from surviving cells.
Cell lines producing antibodies that bind to II are selected by ELISA or the like. Further, it is desirable to perform cloning several times on the obtained monoclonal antibody-producing cells to select a strain that is monoclonal and has stable antibody-producing ability. As the cloning method, a limiting dilution method, a soft agarose method, a selection method using a cell sorter, or the like can be used.

When the monoclonal antibody of the present invention is used for treating humans, it is desirable to use a human monoclonal antibody. Human monoclonal antibodies include:
In addition to human monoclonal antibodies, so-called chimeric antibodies, that is, those in which only the variable region is composed of an amino acid sequence derived from an animal such as a mouse, and the constant region uses any other sequence. In addition, a monoclonal antibody by a so-called CDR grafting technique, that is, one in which only the complementarity-determining region is composed of an amino acid sequence derived from an animal and all other sequences are derived from a human, the human-type monoclonal antibody referred to herein. included.

According to the current recombination technology, these chimeric antibodies or monoclonal antibodies produced by CDR grafting are prepared by extracting the corresponding gene from a mouse hybridoma producing the monoclonal antibody of the present invention, and regenerating the human antibody gene. And can be easily prepared by introducing the gene into an animal cell and expressing it. For example, Int. J. Cancer, 44, 424-433.
(1989), Pro Natl AcadSci USA, 87, 8095-8099 (199
0). Also, for the purpose of improving the production efficiency of monoclonal antibodies without changing the amino acid sequence,
The gene corresponding to the antibody of the present invention taken out from the hybridoma can be expressed in other animal cells. The monoclonal antibodies of the present invention also include monoclonal antibodies obtained by these genetic recombination techniques and animal cells producing the same.

As a method for producing a humanized monoclonal antibody, in addition to the above, there is also a method of cell fusion of human B lymphocytes with human or mouse myeloma or heteromyeloma. Methods for cell fusion with human myeloma include, for example, J. Immunol Methods, 61, 17-32 (198
3) As a method of cell fusion with mouse myeloma, for example, Proc Natl Acad Sci USA, 77, 6841-6845
(1980), as a method for cell fusion with heteromyeloma, for example, Proc Natl Acad Sci USA, 81, 3214 (1984)
And so on. In performing these methods, human B lymphocytes are fused in vivo before cell fusion.
It is better to stimulate with tro. As a method,
For example, Biochem Biophys Rec Commun, 135, 495 (1986)
May be used.

[0019]

The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the invention thereto.

Example 1 Preparation of Antigen Peptide for Immunization A peptide having cysteine at the N-terminal of angiotensin II (SEQ ID NO: 1) was chemically synthesized using a peptide synthesizer (Applied Biosystems Model 431A). This synthetic peptide was added to a 10 mM phosphate buffer (p
H7.5), and incubated with 10 mg of maleimide-activated hemocyanin (Boehringer Mannheim Biochemica) at 25 ° C. for 2 hours. Then, the reaction solution was diluted with 10 mM phosphate buffer (pH 7.5).
Was dialyzed to obtain an antigen peptide solution for immunization.

Example 2 Preparation of Antigen Peptide for Serum Antibody Titer Measurement A peptide having cysteine at the N-terminal of angiotensin II (SEQ ID NO: 1) was chemically synthesized using a peptide synthesizer (Applied Biosystems Model 431A). This synthetic peptide was added to a 10 mM phosphate buffer (p
H7.5) (10 mg / mL) and incubated with 10 mg of maleimide-activated bovine serum albumin (Boehringer Mannheim Biochemical) at 25 ° C. for 2 hours, and then the reaction solution was diluted with 10 mM phosphate buffer (pH
7.5) was dialyzed to obtain an antigen peptide solution for measuring serum antibody titer.

Example 3 Monoclonal Antibody Reacting with Angiotensin Peptide
Preparation of body 10 g of the antigen peptide prepared in Example 1 was added to Freund's complete adjuvant (BACTO, 1:
In addition to 1), angiotensinogen knockout mice (6 weeks old, male) are irrigated every 2 weeks with i. p. Was administered.
After performing this four times, the fifth time, 3 days before the cell fusion, 50 μl of the above mixture was applied to i. v. Was administered.

Immediately before cell fusion, the mouse was killed, spleen cells were homogenized in PBS, the residue was filtered through a nylon mesh, and washed once by centrifugation with PBS. 2 × 10 ^{7 of} these splenocytes were used for mouse myeloma (P3-X63-Ag
8-U1) 4 × 10 ⁷ and ordinary method (Kohler, Milstein; Natur
e, 256, 495-497 (1975)).

That is, both cells were mixed and spun down, and 1 ml
50% polyethylene glycol (Wako Pure Chemicals, 200
0) HAT medium (GIT medium, Wako Pure Chemical Industries) was added dropwise over 2 minutes, polyethylene glycol was diluted with 10 ml of HAT medium over 5 minutes, and finally 100 ml with the same medium, then 200 μl / well And dispensed into five 96-well plates. After culturing at 37 ° C. in 5% CO ₂ for one week, half (100 μl / well) was removed,
A medium (GIT medium, Wako Pure Chemical Industries) was added (100 μl /
Well). Two weeks later, screening was performed using a plate coated with angiotensin II bound to BSA described below.

That is, BSA-bound angiotensin II
Of a 50 mM NaCO ₃ buffer solution (pH 9.5) (1
μg / ml) in a 96-well plate (Falcon,
50 μl per well at 37 ° C.
For 1 hour. After washing, 200 μl of 3% BSA / PBS was added to each well, and blocking was performed at 37 ° C. for 1 hour. After washing again, 50 μl of the culture supernatant was added to each well, and the mixture was allowed to stand at room temperature for 1 hour, and then 0.05% Tween / P was added.
Washed three times with BS.

Next, a goat anti-mouse IgG-alkaline phosphatase conjugate (Ta) diluted 2000-fold with PBS containing 3% BSA and 0.2% skim milk
go) (50 μl per well) and left at room temperature for 1 hour. After washing again, a 1 mg / ml solution of p-nitrophenyl phosphate disodium salt (Wako Pure Chemical Industries, Ltd.) dissolved in 1 M diethanolamine buffer (pH 9.8) containing 0.25 mM magnesium chloride was added at 100 μl / well.
l was added and reacted at room temperature for 30 minutes. The absorbance at 405 nm was examined using an ELISA reader for a 96-well plate (Molecular Davice Vmax), and hybridomas secreting a monoclonal antibody that binds to angiotensin II were selected.

The hybridoma thus selected was cloned twice by the limiting dilution method and established. Specifically, mouse peritoneal exudate cells were cultured in HT medium for 10 hours.
What was prepared at a rate of ⁶ / ml was dispensed into each well,
Hybridoma cells suspended in HT medium were seeded at a rate of 0.5 cells per well. The cells were cultured in a 5% CO ₂ incubator at 37 ° C. for 2 weeks, and the culture supernatant was screened by the above-mentioned ELISA method to pick up a single colony, thereby establishing a cell line.

[Example 4] Culture of hybridoma and purification of antibody Hybridoma 4 producing antibodies obtained by the above-described operations
Clones (CFK10C9, CFK11B5, CFK1
2A3, CFK15E101) was cultured in an eRDF medium (Gibco BRL), which is a serum-free medium containing insulin, transferrin, ethanolamine, and selenite, and the culture supernatant was collected. The antibody in the culture supernatant was purified by a conventional method using a protein G column.

Example 5 Detection of Specificity of Anti-Angiotensin II Monoclonal Antibody
Discussion The antigen specificity of the anti-angiotensin monoclonal antibody selected in Example 4 was examined using the BSA-bound angiotensin II prepared in Example 2. That is, 50 mM NaC of BSA-bound angiotensin II
96 solutions of O ₃ buffer (pH 9.5) (1 μg / ml)
50 μl per well was dispensed into a well plate (Falcon, manufactured by PVC) and left at 37 ° C. for 1 hour.
After washing, 200 μl of 3% BSA / PBS per well
In addition, blocking was performed at 37 ° C. for 1 hour. After washing again,
50 μl per well of the culture supernatant (× 1, × 1/3, × 1/9, × 1/27, × 1/8) diluted at a 3-fold ratio
1, x 1/243, x 1/729), allowed to stand at room temperature for 1 hour, and washed three times with 0.05% Tween / PBS.

Next, a goat anti-mouse IgG-alkaline phosphatase conjugate (Ta) diluted 2000-fold with PBS containing 3% BSA and 0.2% skim milk
go) (50 μl per well) and left at room temperature for 1 hour. After washing again, a 1 mg / ml solution of p-nitrophenyl phosphate disodium salt (Wako Pure Chemical Industries, Ltd.) dissolved in 1 M diethanolamine buffer (pH 9.8) containing 0.25 mM magnesium chloride was added at 100 μl / well.
l was added and reacted at room temperature for 30 minutes. The absorbance at 405 nm was examined using an ELISA reader for 96-well plate (Molecular Davice Vmax) (see FIG. 1).

[0031]

According to the diagnostic agent and the diagnostic kit using the monoclonal antibody of the present invention, for example, the blood angiotensin II concentration can be measured. For example, if one of the monoclonal antibodies of the present invention is bound to a 96-well plate and a sandwich enzyme immunoassay is performed with a commercially available rabbit anti-angiotensin II polyclonal antibody, the detection and concentration of angiotensin II can be easily determined. It can be carried out. In addition, by using the monoclonal antibody of the present invention as a therapeutic agent, it can be used for improving and treating pathological conditions of circulatory proliferative diseases such as hypertension, cardiac hypertrophy, arteriosclerosis, myocardial infarction, and glomerulosclerosis. .

[0032]

[Sequence list]

SEQ ID NO: 1 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Cys Asp Arg Val Tyr Ile His Pro Phe 15

[Brief description of the drawings]

FIG. 1 shows the binding specificity of monoclonal antibody CFK10C9. The horizontal axis of the graph represents the amount of the added hybridoma culture supernatant, and the vertical axis represents BSA-bound angiotensin.
Indicates the amount of angiotensin II monoclonal antibody reacted with the II binding plate (the same applies hereinafter).

FIG. 2 shows the binding specificity of monoclonal antibody CFK12A3.

FIG. 3 shows the binding specificity of monoclonal antibody CFK11B5.

FIG. 4 shows the binding specificity of monoclonal antibody CFK15E10.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12P 21/08 (C12P 21/08 (C12N 5/10 C12R 1:91) C12R 1:91) C12N 5/00 B (C12P 21/08 15/00 C C12R 1:91) C12R 1:91) (72) Inventor Akira Zanma 4-2-2 Asahigaoka, Hino-shi, Tokyo Teijin Co., Ltd. Tokyo Research Center (72 Inventor Takashi Uemura 4-2-2 Asahigaoka, Hino-shi, Tokyo F-term in Tokyo Research Center, Teijin Limited 4B024 AA01 AA11 BA43 GA03 GA18 HA15 4B064 AG27 CA10 CA20 CC24 CE12 DA01 DA13 4B065 AA91X AA92X AB05 AC14 BA08 BB32 CA25 CA44 CA46 4H045 AA11 AA20 CA40 DA76 EA23 EA50 FA72 GA26

Claims (4)

[Claims] 1. A monoclonal antibody that binds to angiotensin II produced from a hybridoma cell obtained by cell fusion of a lymphocyte obtained by immunizing an angiotensinogen knockout animal with angiotensin II and myeloma. 2. An animal cell that produces the monoclonal antibody according to claim 1. 3. The animal cell according to claim 2, which is a hybridoma cell or a cell derived from a hybridoma cell. 4. A monoclonal antibody that binds to the knockout gene product and is produced from a hybridoma cell obtained by cell fusion of a myeloma and a lymphocyte obtained by immunizing the knockout animal with the knockout gene product.