JP2000041675A - Antibody against human tek (tunica interna endothelialcell kinase) - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibody against human TEK, specifically binding with the TEK, and useful for diagnosis or the like of disease accompanying vascularization, leukemia, solid cancer or the like by the detection of the presence of the TEK in various tissues and cells. SOLUTION: This antibody is the new one specifically binding with TEK, usable for detecting the presence or absence of the TEK in various tissues and cells and useful for diagnosis or the like of disease accompanying vascularization, leukemia, solid cancer or the like. The antibody is obtained by immunizing an animal by the TEK or the like as an immunogen to allow the animal to produce a polyclonal antibody having the reactivity to the TEK in the serum thereof, and separating and purifying the produced polyclonal antibody, or extracting the spleen of the immunized animal, fusing the antibody- producing cell included in the spleen with a myeloma cell to provide a hybridoma, cloning the obtained hybridoma, and culturing the cloned hybridoma to provide a uniform monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to TEK (tuni).
ca interna endothelialcel
1kinase: vascular endothelial cell kinase) and its use.

[0002] TEK (also called TIE-2) is a transmembrane receptor tyrosine kinase present in hematopoietic progenitor cells and vascular endothelial cells. It has been clarified that TEK present in vascular endothelial cells plays an important role in angiogenesis during fetal angiogenesis and solid tumor growth. On the other hand, although the physiological significance of TEK present in hematopoietic progenitor cells has not been elucidated yet, there is a possibility that TEK acts on proliferation or differentiation of hematopoietic progenitor cells, proliferation of leukemia cells, and the like. Therefore, the antibody against TEK according to the present invention can be used for examining the presence or absence of TEK in various organs, tissues, and cells to find diseases involving angiogenesis, leukemia,
Alternatively, it contributes to diagnosis of solid cancer and the like.

[0003]

2. Description of the Related Art Conventionally, hematopoietic progenitor cells have often been defined as CD34 antibody-positive bone marrow cells.
The activity of the D34 antigen is not yet clear. In recent years, attempts have been made to define hematopoietic progenitor cells based on the presence or absence of functional molecules. One of them is a transmembrane receptor tyrosine kinase. This type of tyrosine kinase activates the tyrosine kinase in the intracellular part by binding to a soluble or membrane-bound ligand molecule, and DNA is mediated by several signal transduction molecules.
It is a molecule that can send proliferation or differentiation signals to So far, it has been confirmed that tyrosine kinases such as c-kit and F1t3 are present in hematopoietic progenitor cells, and that hematopoietic progenitor cells are actually proliferated or differentiated by respective ligand molecules.

[0004]

On the other hand, TEK is a receptor tyrosine kinase present in vascular endothelial cells,
encoded by the ie-2 gene. Also, TE
The ligands for K have been found to be angiopoietin 1 and angiopoietin 2. T in the vascular endothelium
EK is also known to be essential for the growth of vascular smooth muscle around the vascular endothelium. Analyzing at a cellular level what organs, tissues and cells in a living body this TEK is present in can greatly contribute to the diagnosis or treatment of diseases involving angiogenesis. In addition to investigating the pathogenesis of hematopoietic diseases by examining the distribution of hematopoietic cells, not only can the presence or absence of TEK in leukemia cells, etc. It is thought to contribute to the diagnosis and treatment of the disease.

[0005]

Means for Solving the Problems The present invention has been made to achieve the above object, and as a result of earnest research,
This led to the creation of an antibody capable of analyzing the presence or absence of TEK at the cell level, and completed the present invention.

[0006] The production of the desired antibody can be carried out as follows. That is, an animal is immunized with at least one selected from TEK serving as an immunogen, its extracellular domain, its intracellular domain, and a peptide having a partial amino acid sequence thereof, and the serum thereof reacts with TEK. To obtain a polyclonal antibody having specificity. In order to obtain a highly specific purified polyclonal antibody, the antiserum obtained as described above may be purified by affinity chromatography or the like. Alternatively, the spleen of the immunized animal is removed, and the antibody-producing cells contained therein are fused with myeloma cells to produce a hybridoma, which is then cloned to obtain a large amount of a uniform monoclonal antibody.

As the immunogen, in addition to the above-mentioned human TEK or a fragment thereof, for example, the following can be used: The cDNA of human TEK can be used in an animal cell expression vector pBCMGS-neo or other known expression vectors. A transduced cell line (e.g., BaF / TEK described below) transduced with various cells such as a mouse IL-3-dependent cell line Ba / F3 after appropriately incorporating the same, a culture thereof, a cell lysate, A human TEK-producing cell; a culture thereof, a cell lysate, a peptide separated / purified therefrom; at least a part of which is synthesized by peptide synthesis based on the amino acid sequence information of TEK. Alternatively, it may be prepared by genetic manipulation and used.

The following may also be used as immunogens: human TEK cDNA fragments (extracellular domain, intracellular domain, etc.) and tag proteins (part of immunoglobulin, for example, Fc fragment (crystal)).
isable fragment); GST (Glut)
aths s transfer); MBP
(Maltose Binding Protein)
Fusion protein expressed by a host bacterium (such as Escherichia coli) in which a gene obtained by fusing a gene encoding the gene encoding the gene is fused with a vector; the host bacterium obtained as described above, that is, the transformant itself, a culture thereof, and a bacterium. Fragmented body, it (ra)
Fusion protein isolated (purified) from human; TEK (fragment)
And a fusion protein comprising a tag protein.

More specifically, for example, the human fetal cD
TEK cDN cloned from NA library
A extracellular domain and part of mouse immunoglobulin (F
Escherichia coli is transformed using a fusion gene with the gene encoding c) together with a commercially available vector for Escherichia coli, and the fusion protein obtained by expressing this fusion gene in Escherichia coli can be used as an immunogen. . Thus, an E. coli transformant (E. coli) capable of stably producing a fusion protein by expressing the fusion gene is obtained.
TEK-Fc) was obtained and deposited at the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (FERM P-16919).

The mouse IL-3-dependent cell line Ba
A transduced cell line (hereinafter sometimes referred to as a transfectant) BaF / TEK in which / H3 cDNA was transduced with human TEK cDNA was prepared and used as an antigen for screening an anti-TEK antibody-producing hybridoma. It can also be used as an immunogen as described above.

[0011] Examples of animals used for immunization include goats, sheep, horses, rabbits, rats, mice, and the like. However, in order to produce monoclonal antibodies, there is a problem with compatibility with the myeloma cells used for fusion. Usually, mice or rats can be used. Upon immunization, an immunogen may be used in admixture with various adjuvants to promote an immune response.

The preparation of hybridomas producing monoclonal antibodies is described in (Koehler, G. and Mil.
Stein, C.I. , Nature 256, 49
5 (1975)). That is, myeloma cells are mixed with antibody-producing cells selected from spleen cells, lymph node cells or B lymphocytes isolated from an immunized animal and fused in the presence of polyethylene glycol.
HAT medium (1 × 10- ⁴ M hypoxanthine, 4 × 10
- ⁷ M aminopterin, 1.6 × 10- ³ M thymidine-containing) Hybridoma selectively grown by, secretion of the antibody was observed to the transfectants specifically binding to the culture supernatant Cells are cloned by limiting dilution.

The hybridoma thus produced can produce a monoclonal antibody from its culture supernatant or ascites obtained by transplantation into an animal derived from myeloma cells. As a method for purifying the antibody from the antiserum of the immunized animal, the culture supernatant of the hybridoma, or ascites, known purification methods such as salting out with ammonium sulfate, ion exchange chromatography, and affinity chromatography can be used.

Thus, a hybridoma capable of stably producing an anti-TEK antibody capable of specifically reacting with human TEK was obtained. The hybridomas thus obtained were named C9C3 and A1F9 and deposited with the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology (deposit numbers are FERM P-16918 and FERM P-, respectively).
16917). These clones are cultured according to a conventional method, and the monoclonal antibody is separated and collected. The obtained monoclonal antibody was confirmed to be the desired anti-TEK antibody by immunoprecipitation or the like.

Detection of cells expressing the receptor can be easily measured by flow cytometry or the like using an antibody against the extracellular domain of the receptor type protein, and a TEK-specific antibody of the intracellular domain can be detected. Can be measured by immunohistological staining or the like.

In recent years, it has been found that the extracellular domain of many receptor proteins deviates as a soluble receptor in body fluids by some mechanism. This soluble receptor is present in a very small amount in body fluids, and can be detected by a high-sensitivity immunodetection method such as a sandwich-type enzyme immunoassay using at least two types of antibodies, particularly monoclonal antibodies.

The anti-human TEK antibody according to the present invention (for example,
A polyclonal antibody that uses a fusion protein of human TEK and Fc as an immunogen has a TEK ligand-like activity, and when TEK, its receptor (receptor), is present, binds to this to generate a signal, A new finding of inducing phosphorylation was also obtained. Therefore, this antibody, which is an antibody against the extracellular domain of the receptor and induces autophosphorylation by binding to the receptor, that is, the present antibody having ligand-like activity, has potential for treatment as well as diagnosis as an angiopoietin agonist. It also has

The present invention also relates to human TE as described above.
In addition to newly providing a method for measuring K, and thus a method for detecting / measuring cells that express and produce it, and a method for inducing tyrosine phosphorylation, anti-human TEK antibodies, buffers, standard activating reagents, and other necessary TEK containing various substances
(Producing cells) A kit for measurement is also provided.

Hereinafter, embodiments of the present invention will be described.

[0020]

Example 1 Preparation of Monoclonal Antibody A monoclonal antibody that specifically binds to human TEK was prepared as follows.

(1) Preparation of Immunogen An Escherichia coli expression vector was prepared by fusing an extracellular domain of TEK cDNA cloned from a human placenta cDNA library with a gene encoding a part of mouse immunoglobulin (Fc). E. coli (transformant Escherichia coli TE)
K-Fc: FERM P-16919), and the obtained fusion protein was used as an immunogen.

(2) Preparation of Hybridoma Hybridomas (fused cells) were prepared as follows.

A) Immunization and cell fusion After administering the above fusion protein to Balb / c mice several times, spleen cells of immunized mice and mouse myeloma-derived cell line (P3-X63-Ag8-U1: P3U1) Was subjected to cell fusion according to a conventional method.

B) Screening of anti-TEK antibody-producing hybridomas As a method for screening anti-TEK antibody-producing hybridomas, a transfectant BaF / TEK obtained by transducing human TEK cDNA into a mouse IL-3-dependent cell line Ba / F3 was used. The indirect antibody method using as an antigen was used. Selecting a hybridoma that produces an antibody that binds to the transfectant and does not bind to the parent cell line (Ba / F3);
Cloned. As a result, clone C9C3 (FE
RM P-16918) and clone A1F9 (FER
MP-16917) was obtained. (Fig. 1)

C) The culture supernatant of C9C3 was subjected to ultrafiltration and concentration, followed by binding buffer (BioRad Protein M).
(APS buffer). Protei
nA-Sepharose CL-4B (Pharmacia) was equilibrated with a binding buffer, and the mixture was allowed to flow through the column to bind the antibody. Then, the column was washed with the binding buffer. 0.2M Gly-HCL buffer (p
H3.0) was passed through the column for elution to obtain an antibody fraction.

[0026]

Example 2: Binding experiment Before reacting the C9C3 antibody with the transfectant, the TEK-Fc fusion protein, which was 100 times the amount of the antibody, was previously reacted with the antibody.
The binding between the C9C3 antibody and the hybridoma was completely blocked, confirming that the C9C3 antibody is an antibody that can specifically bind to TEK. (Fig. 2)

[0027]

Example 3 Properties of Antibody C9C3 Antibody and A1F9
When the subclass of the antibody was assayed using the Ouchterlony method, all were IgG1.

[0028]

Example 4: Immunoprecipitation Transfectants and human umbilical cord vascular endothelial cells (HUVEC)
After reacting the cell lysate with C9C3 antibody, Pro
tein G-Sepharose (Pharmaci
Immunoprecipitation was performed using a). Immunoprecipitation SDS
-After subjected to PAGE, the DNA was transferred to a PVDF membrane and detected by Western blotting using a polyclonal antibody against the C-terminus of TEK. The molecules immunoprecipitated from the transfectants by C9C3 are 145 kDa and 136 kDa molecules, which are stained by the anti-TEK polyclonal antibody, and whose staining is completely blocked by the peptide which is the antigen of the polyclonal antibody. Was. As a result, the C3C9 antibody can be expressed not only in transfectants but also in vascular endothelial cells.
It was confirmed that the antibody could also react with K. (Fig. 3)

[0029]

[Example 5: Reactivity with bone marrow cells] Biotin-labeled C
After simultaneously reacting the 9C3 antibody and various anti-human leukocyte antibodies with bone marrow cells, they were reacted with streptavidin-FITC and analyzed by flow cytometry. The C9C3 antibody reacted with 20% of CD19-positive cells as B lymphocytes, but hardly reacted with CD3-positive cells as T lymphocytes. In addition, a lineage (Lin) marker (CD) known as a fraction containing hematopoietic stem cells in bone marrow cells
3, CD4, CD8, CD13, CD14, CD19,
Glycophorin A) Differentiating lineage marker-negative CD34-negative CD34-positive cells that reacted with 27% of the negative CD34-positive cells and also reacted with 20% of the lineage-negative c-kit-positive cells and considered to contain the most undifferentiated hematopoietic cells 26%. (Figure 4) This is C9C3
It has been proved that hematopoietic progenitor cells can be analyzed in detail by using antibodies.

[0030]

Example 6: Polyclonal antibody having TEK ligand-like activity Escherichia coli in which a gene obtained by fusing the extracellular domain of human TEK cDNA with a gene encoding a part of mouse immunoglobulin (Fc fragment) is incorporated together with a vector. (E. coli TEK-Fc: FE
RMP-16919) was used as an immunogen, and the fusion protein was administered intraperitoneally to mice to immunize the mice. After the immunization, the IgG fraction of the antiserum collected from the mouse was used as an anti-TEK polyclonal antibody and subjected to the following reaction.

Ba cultivated for 12 hours in an IL-3-independent manner
F / TEK cells were reacted with 5 μg / ml anti-TEK polyclonal antibody at 37 ° C. for 10 minutes. As a control,
The BaF / TEK cells were reacted with IgG having non-specific reactivity.

After the reaction, the cells were dissolved in a lysis buffer (1% Trit).
ox X-100, 50 mM HEPES, pH7.
4, 10% glycerol, 10 mM sodiu
m pyrophosphate, 10 mM sodi
um fluoride, 4Mm EDTA, 2mM
sodium orthovanate, 50mg
/ Ml aprotinin, 1 mM PMSF, 1
00 mM leupeptin, 25 mM pepst
atin A). The lysate was immunoprecipitated and tyrosine autophosphorylation of the intracellular domain of TEK was determined by anti-phosphotyrosine antibody (4G10, Upsta
te Biotechnology, LakePlac
(id, NY) using Western blotting. (Fig. 5)

[0033]

According to the present invention, an antibody that specifically binds to human TEK has been produced for the first time. Since this antibody has high specificity and reacts specifically with human TEK, not only can it be accurately measured, but also an assay of the expression cells has become possible. Therefore, the present invention makes it possible for the first time to clarify the distribution of TEK-expressing cells in the bone marrow, and as a result, it has become possible to analyze the presence or absence of leukemia cells and the like.

The antibody according to the present invention induces autophosphorylation by binding to its receptor (TEK),
Since it has a ligand-like activity for EK, it is expected to have a therapeutic potential as well as a diagnosis as an angiopoietin agonist.

Further, according to the present invention, since an excellent hybridoma was successfully prepared, a remarkable effect that a desired monoclonal antibody can be efficiently produced is exhibited.

[Brief description of the drawings]

FIG. 1 is a drawing in which a transfectant (BaF / TEK) and its parent strain (Ba / F3) were stained with a C3C9 antibody and analyzed using flow cytometry.

FIG. 2 is a drawing in which transfectants after pre-reaction of a C9C3 antibody and a TEK-Fc fusion protein were stained and analyzed using flow cytometry.

FIG. 3 is a diagram in which a molecule immunoprecipitated from a cell lysate of a transfectant with a C9C3 antibody is stained by Western blotting.

FIG. 4 is a drawing in which bone marrow cells are stained with a C9C3 antibody and various anti-human leukocyte antibodies and analyzed by flow cytometry.

FIG. 5 is a diagram showing the results of Western blotting of a cell lysate after reacting a polyclonal antibody having TEK ligand-like activity with BaF / TEK cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 39/395 AED C12P 21/08 C12P 21/08 C12N 5/00 B (C12P 21/08 C12R 1:91 ) F-term (reference) 4B024 AA01 AA11 BA43 BA44 BA46 BA53 GA03 GA08 GA18 GA27 HA03 4B064 CA10 CA20 CC01 CC24 CE06 CE12 DA01 DA13 4B065 AA91X AB05 AC14 AC15 BA08 BA21 BD14 CA25 CA44 CA46 4C085 AA11 AA34 DDA19 DD AA11 AA20 AA30 CA46 CA50 DA75 DA76 EA24 EA50 FA71 FA72 GA10 GA26

Claims (10)

[Claims] 1. An antibody that specifically binds to human TEK. 2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody. 3. The monoclonal antibody according to claim 1, which binds to an extracellular domain of human TEK. A hybridoma that produces the monoclonal antibody according to claim 2 or 3. 5. The hybridoma A1F9 or C9C3. 6. The polyclonal antibody according to claim 1, which binds to an extracellular domain or an intracellular domain of human TEK. 7. The polyclonal antibody according to claim 1, wherein a fusion protein of the extracellular domain of human TEK and a crystalline fragment of immunoglobulin (Fc) is used as an immunogen. 8. A method for inducing tyrosine phosphorylation of an intracellular domain TEK, comprising binding the polyclonal antibody according to claim 1, 2, 6 or 7 to an extracellular domain of human TEK. 9. An immunoassay for human TEK, which comprises using an antibody that specifically binds to human TEK. 10. A kit for measuring human TEK, comprising an antibody that specifically binds to human TEK.