JP3583214B2 - Monoclonal antibody against human SCF - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a monoclonal antibody having specificity for human Stem cell factor (hereinafter, also referred to as SCF), a hybridoma producing the same, and a method for producing the monoclonal antibody using the same.
[0002]
SCF is a molecule which plays an important role in hematopoiesis and the like. Measuring its blood concentration has important significance in diagnosis of various diseases including blood diseases, and the present invention greatly contributes to it. Can be done. In addition, the monoclonal antibody capable of inhibiting the binding of the SCF to the human c-kit gene product in the present invention has use not only as a diagnostic agent but also as a therapeutic agent.
[0003]
[Prior art]
Human SCF is a ligand for the c-kit receptor expressed on the surface of hematopoietic stem cells, and its precursor is composed of 273 amino acids, and finally has a signal sequence of 25 amino acids at the N-terminus and is finally composed of 248 amino acids. It is a membrane-bound hematopoietic factor containing a sugar chain and having a molecular weight of about 30 kDa.
[0004]
Thus, since the SCF was cloned in 1990, research on its action in vitro and in vivo has been advanced. As a result, it has become clear that SCF is involved in many physiological phenomena, including a role in constitutive hematopoiesis and a role in germ cell maturation. Then, it was found that SCF exhibits a synergistic effect with cytokines such as IL-3 and IL-6 and growth factors such as b-FGF in hematopoiesis, and promotes proliferation and differentiation of hematopoietic cells at various stages. Therefore, SCF is important not only as a growth factor for hematopoietic stem cells but also as a growth factor for erythrocytes, platelets, granulocytes, and lymphocytes, and clinical application is greatly expected.
[0005]
SCF is expressed as a cell membrane-bound form in bone marrow stromal cells and vascular endothelial cells.
It is presumed that a part thereof is produced as a soluble form by a decomposition reaction by a protease. That is, the SCF includes a membrane-bound SCF and a soluble SCF (secretory SCF), and the soluble SCF has a physiological activity.
In fact, there is a report that the serum SCF of healthy humans has an average of 3.3 ± 1.1 ng / ml of soluble SCF. (Langley et al., Blood 81, 656, 1993) However, whether soluble SCF measured as a soluble SCF by a conventional method in this way can show its activity in a living body or not can be determined. The following problems are shown. In other words, there is no guarantee that the soluble SCF measured by the conventional method is a true soluble SCF having biological activity.
[0006]
[Problems to be solved by the invention]
As described above, the SCF receptor is a c-kit proto-oncogene product (hereinafter sometimes referred to as C-KIT) expressed on the cell membrane surface of hematopoietic stem cells, hematopoietic progenitor cells, or vascular endothelial cells. I know. This C-KIT is also released into the blood as a soluble molecule, and its concentration is estimated to be 10 times or more that of the soluble SCF in the blood.
Therefore, there is a possibility that a part of the soluble SCF in the blood binds to the soluble C-KIT and cannot show any activity.
[0007]
However, in the conventional method for measuring soluble SCF in blood, the presence or absence of binding to soluble C-KIT cannot be detected separately.
There is a need for a new technique capable of distinguishing both of them, that is, soluble SCF bound to soluble C-KIT and soluble SCF not bound, and measuring only SCF that is truly effective in vivo. .
[0008]
The present invention has been made to meet such needs in the art. As a result of various studies, the present invention specifically binds to human SCF and blocks the binding between human SCF and human C-KIT. If an antibody with high specificity that can be created, it is possible to construct a measurement system using it, and as a result, for the first time, focusing on achieving the above-mentioned object, high specificity that matches this object From the viewpoint that the creation of antibodies, especially monoclonal antibodies, is the basis, the establishment of a novel monoclonal antibody production system has been newly established for the purpose of the present invention.
[0009]
[Means for Solving the Problems]
The present invention has been made in order to achieve the above object, and as a result of earnest research, an anti-SCF antibody, particularly a monoclonal antibody and an anti-SCF antibody capable of measuring soluble SCF in blood that has truly activity The production total system has been completed, and the present invention has been completed.
Hereinafter, the present invention will be described in detail.
[0010]
In order to create a fusion cell (hereinafter sometimes referred to as a hybridoma) for producing a desired monoclonal antibody, the present invention not only focuses on human HeLa cells as an immunogen, but also aims to further improve efficiency. Then, a transduced cell (hereinafter sometimes referred to as a transfectant) obtained by isolating a cDNA of a target gene from a donor cDNA and transducing the cDNA into a recipient was used. That is, the present inventors firstly transduced a mouse fibroblast Balb / 3T3 cell with SCF cDNA isolated from a cDNA library of HeLa cells that highly express SCF (transfectant cell line). ) Was prepared.
[0011]
Next, the transfectant is used as an immunogen to immunize a mouse, for example, and spleen cells, lymph node cells or B lymphocytes are obtained as antibody-producing cells.
A fusion cell (hybridoma) is formed between the antibody-producing cells and mouse, human or rat myeloma cells using a so-called cell fusion method, and a clone producing an antibody that specifically binds to the above transfectant is selected. By doing so, a monoclonal antibody can be obtained.
The monoclonal antibody thus obtained is previously bound to, for example, biotin-labeled SCF, and the binding ability of this biotin-labeled SCF to a C-KIT-positive cell line is determined by flow cytometry by further reacting, for example, AVIDIN-FITC. Monoclonal antibodies capable of detecting and blocking the binding can be selectively obtained.
[0012]
Thus, a hybridoma capable of stably producing an anti-SCF antibody capable of inhibiting the binding between SCF and C-KIT was obtained. The hybridoma thus obtained was named SCF235, and was deposited at the National Institute of Bioscience and Biotechnology, National Institute of Advanced Industrial Science and Technology (FERM P-15295).
[0013]
Each of these clones is cultured according to a conventional method, and a monoclonal antibody is separated and collected. The obtained monoclonal antibody was confirmed to be the target anti-SCF antibody by immunoprecipitation, Western blotting, or the like.
[0014]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but it is needless to say that the present invention is not limited to only these Examples.
[0015]
[Example 1: Preparation of antibody]
[0016]
(1) Preparation of immunogen The SCF cDNA isolated from the cDNA library of HeLa cells that highly expresses SCF was incorporated into an animal cell expression vector pBCMGS-neo, which was then used as a mouse fibroblast cell line Balb / 3T3 cell. And the resulting transfectants were used as immunogens.
[0017]
(2) Preparation of hybridoma
(A) The above transfectants were intraperitoneally administered to Balb / c mice (females) of 8 weeks of immunity at 2-week intervals. The effect of immunization was evaluated by the reactivity of serum of peripheral blood collected from the tail vein of the mouse with the immunogen. After confirming the effect, final immunization and cell fusion were performed.
[0019]
(B) Cell fusion Four days after the final immunization, spleen cells of the immunized mouse were fused with a mouse myeloma-derived cell line SP-2 according to a conventional method.
[0020]
(C) Screening of anti-SCF antibody-producing hybridoma As an anti-SCF antibody-producing hybridoma screening method, an indirect antibody method using a transfectant and its parent cell line (Balb / 3T3) as an antigen was used.
A hybridoma producing an antibody that binds to the transfectant but does not bind to the parent cell line (Balb / 3T3) was selected and cloned. As a result, a clone (SCF235) was obtained. (Fig. 1)
[0021]
(D) Purification of antibody The culture supernatant of SCF235 was subjected to ultrafiltration and concentration, and then mixed with an equal amount of a binding buffer (BioRad Protein MAPS buffer). Protein A-Sepharose CL-4B (Pharmacia) was equilibrated with a binding buffer, and the above mixture was allowed to flow through the column to bind the antibody. Then, the column was washed with the binding buffer. Elution was performed by flowing 0.2 M Gly-HCl buffer (pH 3.0) through the column to obtain an antibody fraction.
[0022]
[Example 2: Binding experiment]
After allowing biotin-labeled SCF (R & D Systems) to react with SCF235 antibody for 15 minutes at room temperature, C-KIT positive cell line (HCK / 3T3: C-KIT obtained by transducing c-kit gene into Balb / 3T3 cells). (KIT transfectant) at 4 ° C. for 60 minutes.
Thereafter, avidin-FITC (AVIDIN-Fluorescein isothiocyanate isomer) was added, and the mixture was further reacted at 4 ° C. for 30 minutes in a dark place.
[0023]
The cells were used as a sample for flow cytometry according to a conventional method, and the binding of biotin-labeled SCF to C-KIT was measured. As a result, the binding was completely inhibited as compared with the case where the antibody was not reacted with the SCF235 antibody in advance. (Fig. 2)
That is, it was confirmed that the SCF235 antibody was a monoclonal antibody capable of inhibiting the binding of SCF to C-KIT.
[0024]
Example 3: Properties of Antibody
When the subclass of the SCF235 antibody was assayed using a mouse monoclonal antibody isotyping kit (Amersham), it was found to be IgG1.
[0025]
After 5 μg of the obtained SCF235 antibody was subjected to SDS-PAGE in a reduced state, the gel was subjected to Coomassie staining. As a result, two 50 kDa and 25 kDa bands specific to mouse IgG were detected. (FIG. 3) As the gel, a gradient gel having a polyacrylamide concentration of 4 to 20% was used.
[0026]
【The invention's effect】
For the first time, a monoclonal antibody that specifically binds to human Stem cell factor has been produced by the present invention. This monoclonal antibody is capable of blocking the binding of the human c-kit gene product to human Stem cell factor.
[0027]
By utilizing this property and using a measurement method using the antibody, it is possible to selectively measure only free soluble SCF not bound to C-KIT in blood.
[0028]
For example, if the SCF antibody is immobilized, only free SCF (secreted SCF) selectively binds to the antibody, and SCF bound to free C-KIT coexisting in the blood will Does not combine with Therefore, if the SCF captured by the immobilized SCF antibody is measured according to a method such as using a labeled secondary antibody, a free SCF bioassay can be performed.
[0029]
In this way, not only can the free soluble SCF in the patient's own blood be measured by using the measurement method using the antibody, but also the metabolism of the artificially administered soluble SCF can be monitored. it can.
It is clear that this greatly contributes to the development of diagnostics and therapeutics for diseases such as blood diseases.
[Brief description of the drawings]
FIG. 1 is a drawing in which a transfectant was stained with an SCF235 antibody and analyzed using flow cytometry.
FIG. 2 is a drawing in which a transfectant of C-KIT and biotin-labeled SCF were reacted after reaction with SCF235, and analyzed using flow cytometry.
FIG. 3 is a drawing obtained by subjecting SCF235 antibody to SDS-PAGE under reducing conditions, followed by Coomassie staining.

Claims (3)

It is characterized by using hybridoma SCF235 (FERM P-15295), wherein the subclass is IgG, which specifically binds to human Stem cell factor (SCF), and which binds human Stem cell factor and human c-kit gene product. A method for producing a monoclonal antibody capable of inhibiting binding. The method according to claim 1, wherein the subclass is IgG, which specifically binds to human Stem cell factor (SCF), and prevents binding between human Stem cell factor and human c-kit gene product. A monoclonal antibody derived from hybridoma SCF235 (FERM P-15295). Hybridoma SCF235 (FERM P-15295).

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