JPH06141852A - Antibody and hybridoma producing the same - Google Patents

Abstract

PURPOSE:To obtain a new hybridoma capable of producing a monoclonal antibody specifically reactive with human c-kit proto-oncogene products. CONSTITUTION:The objective hybridoma can produce a monoclonal antibody positively reactive with human c-kit proto-oncogene products in biological specimens directly obtained from humans [e.g. hybridoma L6 (FERM P-13224). Each of c-DNAs of a c-kit proto-oncogene isolated from the c-DNA library of leukemia cell strain UT-7 is transfected into mouse fibroblast L-cell and BALB/3T3 cell to produce transfected cells. Mice are then respectively immunized with the stably manifestative strains obtained above, and a hybridoma between mouse spenocyte and mouse myeloma cell is produced, and such a clone is selected as to produce an antibody specifically reactive with a c-kit proto-oncogene-incorporated transfectant, thus obtaining the objective hybridoma L6 or L15 (FERM P-13225).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention produces a monoclonal antibody having specificity for a human c-kit protooncogene product and recognizing two different antigenic determinants (hereinafter referred to as epitopes). The present invention relates to a hybridoma, a method for producing the monoclonal antibody using the hybridoma, and the monoclonal antibody obtained thereby.

Human c-kit protooncogene product is
It has been known from studies investigating the presence or absence of RNA that many are expressed in undifferentiated hematopoietic stem cells, and abnormal expression of the c-kit protooncogene product affects tumorigenesis at the stem cell level and hematopoietic tumors. Is believed to cause. Therefore, the present antibody is useful as a diagnostic agent and therapeutic agent for hematopoietic tumors such as leukemia.

[0003]

2. Description of the Prior Art v-kit is a retroviral oncogene obtained from feline fibrosarcoma. A protein encoded by c-kit corresponding to v-kit (c-kit
The protooncogene product) is a receptor-type tyrosine kinase whose ligand molecule has recently been identified, and is called stem cell factor (SCF). The W mouse, which has an abnormality in the c-kit protooncogene, has been studied for a long time as a model of anemia and pigment abnormality, but recent studies have shown that this abnormality is involved in signal transduction by the c-kit protooncogene product and stem cell factor. It has been clarified that it is a related abnormality. ACK2 (J. Exp. Me), which has been an antibody against mouse c-kit protooncogene product
d. , Vol. 174, p. 63, 1991), a human c-kit protooncogene product has been reported to be detected on leukemia cells. However, it is not possible to directly detect the product on a human biological sample (for example, human bone marrow cells). could not.

It can be said that hematopoietic tumors such as leukemia and lymphoma are diseases in which cells in an undifferentiated stage have turned into tumors, and some cytokines are also involved in the proliferation of tumorous blood cells. It is clear. c-
It has also been reported that stem cell factor, which is a ligand of the kit protooncogene product, stimulates the proliferation of cells of some acute myeloid leukemias.

As described above, the c-kit protooncogene product and its ligand, stem cell factor, play an important role in the hematopoietic mechanism, and not a few abnormalities related to its signal transduction system affect the normal hematopoietic mechanism. Conceivable. However, until now, the expression of the c-kit protooncogene product in hematopoietic tumors and the correlation with their pathological conditions have not been examined in detail. C-kit protooncogene mR slightly in hematopoietic tumor cells
There are only studies that investigated the presence or absence of NA.

[0006]

DISCLOSURE OF THE INVENTION The present invention requires that the presence or amount of the human c-kit protooncogene product in hematopoietic tumors be simply measured, or the human c-kit protooncogene product itself. It was made for the purpose of meeting the needs of the industry such as the need to separate and purify.

[0007]

Means for Solving the Problems The present invention has been made to achieve the above objects, and as a result of investigations from various aspects, an antibody having specificity for a human c-kit protooncogene product, particularly We came to pay attention to the monoclonal antibody. Then, further studies were conducted, and a fused cell (hybridoma) capable of producing the monoclonal antibody was successfully created, resulting in completion of the present invention.

That is, the present invention relates to human c in a biological sample.
-Kit relates to a monoclonal antibody that specifically reacts with a protooncogene product and its production system.

In order to create a hybridoma for producing the desired monoclonal antibody, the present invention not only focuses on the human megakaryoblastic cell line (UT-7) as an immunogen, but also further improves the efficiency. For this purpose, the cDNA of the target gene was isolated from the cDNA of the donor, and the transduced strain obtained by transducing this into the recipient was used. That is, the present inventors first of all, human c
-Kit leukemia cell line UT highly expressing protooncogene product
CDNA of the proto-oncogene c-kit isolated from the cDNA library of -7, mouse fibroblast L-cell,
Transduced cells (transfectants) transduced into Balb / 3T3cell and Balb / 3T3cell were prepared (HCK / L, HCK / 3T3).

Then, the thus obtained stable expression strain is immunized into, for example, a mouse as an immunogen, and its spleen cells, lymph node cells or B lymphocytes are obtained as antibody-producing cells. The antibody-producing cells and mouse, human or rat myeloma cells are used to form fused cells (hybridomas) by the so-called cell fusion method.
A clone producing an antibody that specifically reacts with a transfectant into which the it protooncogene is introduced is selected. Antibody-producing cells (hybridomas) thus obtained
Were designated as L6 and L15. All of these have been deposited at the Institute of Microbial Engineering, Institute of Industrial Science (L-
6: Micro-Technology Research Institute 13224; L-15: Micro-Technology Research Institute 13225).

Each of these clones is cultured according to a conventional method, and monoclonal antibodies L6 and L15 are separated and collected. The obtained monoclonal antibody was confirmed to be the desired anti-c-kit protein antibody by immunoprecipitation method, Western-blotting method, or the like.

The anti-c-kit protooncogene product monoclonal antibody thus obtained is a cell using flow cytometry, such as a method of directly labeling these with a fluorescent dye or a method of using a secondary antibody labeled with a fluorescent dye in advance. It can be used for the measurement of the c-kit protooncogene product expressed on the surface and other uses.

[0013]

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.

[0014]

Example 1 Preparation of monoclonal antibody

(1) Preparation of immunogen cDNA of c-kit protooncogene isolated from cDNA library of leukemia cell line UT-7 known to highly express c-kit protooncogene product After incorporating into the animal cell expression vector BCMGSNeo, mouse fibroblast L-cell and Balb / 3T3ce
The transduced cells (transfectants) transduced into 11 were prepared (HCK / L, HCK / 3T).
3) This was used as an immunogen.

(2) Preparation of fused cells

(A) Immunization Three 8-week-old Balb / c mice (female) were fed with HCK / 3.
T3, HCK / L, and CD34 / L transfectants were intraperitoneally administered at 2-week intervals. After completion of immunization, HCK / 3T3, using serum collected from the tail vein of the mouse,
The reactivity to HCK / L, CD34 / L transfectants and leukemia cell line UT-7cell was checked. It was confirmed that a significant difference was obtained in the reactivity with a transfectant different from the immunogen, and final immunization and cell fusion were performed.

(B) Cell fusion 4 days after the final immunization, spleen cells of mice immunized with HCK / 3T3 and HCK / L transfectants and mouse myeloma-derived cells P3-X63-Ag8-U1 (P3).
U1) Using the cells, cell fusion was performed according to a conventional method.

(C) Screening of hybridoma

As a primary screening of hybridomas reactive with leukemia cell line, positive clones were selected by reactivity of hybridoma culture supernatant by indirect fluorescent antibody method using leukemia cell line UT-7cell as an antigen. The hybridoma culture supernatant and the UT-7 cell suspension were reacted for 30 minutes with ice cooling and washed, and then a FITC-labeled mouse IgG antibody solution was added (ice cooling, 30 minutes). After washing, the fluorescence intensity was measured by a flow cytometer.

Reactivity with transduced cells As a secondary screening, an indirect fluorescent antibody method using live cells as an antigen was used, and positive clones were selected based on the reactivity of the hybridoma culture supernatant to transduced cells (transfectants). did.

The hybridoma culture supernatant obtained by immunization with HCK / 3T3 transfectant was HCK /
The hybridoma culture supernatant obtained by screening using L and CD34 / L transfectants and immunizing with HCK / L transfectants was HCK / 3T3.
Screened with transfectant, Balb / 3T3. The screening of the hybridoma culture supernatant was performed by immunizing HCK / 3T3 and HCK / L transfectants with 945 well and 707 w, respectively.
I went to ell. Judgment is HCK / L, CD34
The clones with positive / negative reactivity to / L were selected, and the clones with positive / negative reactivity to HCK / 3T3 and Balb / 3T3 were selected.

As a result of the screening, 2 clones (L
6, L15) was obtained (FIG. 1). Each of these hybridomas has been deposited at the Institute of Microbial Science and Technology (L-6: FERM P-13224; L-15: FE).
RM P-13225).

(D) Purification of antibody After the culture supernatant was concentrated by ultrafiltration, binding buffer (Bior) was added.
ad, ProteinA MAPSII Buffer)
Mix the same amount with and use it as a sample. Protein A-Se
A phase CL-4B column (Pharmacia) was equilibrated with the binding buffer, the sample was passed through the column to bind the antibody, and then the column was washed with the binding buffer. 0.
Elution with 2M Gly-HCI (pH 3.0) gave fractions of monoclonal antibody L6 and monoclonal antibody L15, respectively.

(E) Labeling of antibody For each purified antibody, biotin labeling and FITC
(Fluorescein isothiocyanate isomer) was labeled.

Biotin labeling (Biorad Bioti
Nylation Kit) antibody solution 0.05M borate buffer (pH 8.6)
After changing to 1 mg / ml, the biotinylated reagent (Amersham, Biotinylation Kit) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution is Sephadex
After applying unreacted biotin on a G-25 column, biotinylated antibodies were obtained.

The FITC-labeled antibody solution was replaced with 0.05 M carbonate buffer (pH 9.5) to prepare 1 mg / ml, and then FITC (Becton)
Dickinson, Fluorescein Is
DMSO for thiocyyanate Isomer
(1-2 mg / ml) and added to the antibody solution with gentle stirring. After stirring at 4 ° C. overnight (in the dark), the reaction solution was applied to a Sephadex G-25 column to remove unreacted FITC, and FITC-labeled antibodies were obtained.

[0028]

Example 2 Properties of antibody

(1) Reactivity with human bone marrow cells The reactivity with bone marrow cells was examined by the fluorescent antibody method using human bone marrow cells as an antigen. Bone marrow of healthy subjects was collected and 170
mM NH ₄ Cl was added to hemolyze and then washed. The FITC-labeled anti-c-kit antibody was added to this solution, and the solution was allowed to stand for 30 minutes under ice cooling, washed, and the fluorescence intensity was measured by a flow cytometer.

As a result, the monoclonal antibodies L6 and L1
All 5 were positive for human bone marrow cells (Fig. 2).

(2) Reactivity with various leukemia cell lines The reactivity of each antibody was examined by the indirect fluorescent antibody method using various leukemia cell lines as antigens. The obtained results are shown in Table 1 below.

[0032]

[Table 1]

(3) Immunoprecipitation The obtained antibody was immunoprecipitated.

The method will be described below. HCK / 3T
3 transfectants (positive cells), Balb / 3T
Plate 3 (negative cells) into the plate and incubate at 37 ° C until the cells adhere. After washing with a methionine- and cysteine-free medium (DME (-Met, -Cys), ³⁵ S radiolabeling medium (Trans- ³⁵ S) was added, and the mixture was incubated at 37 ° C for 1 hour.
Cultured for 6-18 hours. After washing the cells, Lysis B
Add well and dissolve well. After leaving it on ice for 30 minutes, transfer to Tube and centrifuge (800 rpm, 5 mi
n), the supernatant was collected and used as Lysate. Lysate
Non-specific binding substances in Protein G-Sepha
After removal using ose 4Fast Flow (Pharmacia) (4 ° C., 30 minutes), centrifugation (15000 r
(pm, 1 min) and the supernatant was collected. Reaction with antibody (4
(C, 30 minutes), and then Protein G-Sepharo
se was added and slowly rotated at 4 ° C. for 30 minutes. P
After washing the protein G-Sepharose, SDS
Add sample buffer, boil for 3 minutes, centrifuge (150
(00 rpm, 1 min) to prepare a sample for electrophoresis. After running this on SDS-PAGE, 25% EtOH
After fixing (20 minutes, shaking), gel drying, autoradiography was performed for detection. As a result, this antibody was c-
Kit proto-oncogene product, molecular weight 120-140 KD
Specifically bound to the protein (Fig. 3). In the figure, Mo
Among Abs (monoclonal antibodies), Negative refers to a negative antibody, that is, an antibody that reacts with a different one, and ACK2 is an antibody against a mouse c-kit protooncogene product.

(4) Blocking test A blocking test was performed between the obtained antibody and ACK2.

HCK / 3T3 transfectant cell suspension, various unlabeled antibodies and small test tubes in ice-cooled 3
After reacting for 0 minutes to block the antigen, wash and centrifuge (1
After 400 rpm for 3 min), the supernatant was removed. F
Add various ITC-labeled antibodies and react on ice for 30 minutes,
After washing, the fluorescence intensity was measured by a flow cytometer.

From the results of the blocking test, the monoclonal antibodies L6 and L15 recognized different epitopes from ACK2, and L6 and 15 also recognized different epitopes.

(5) Determination of Subclass Mouse Monoclonal Tying Kit (THE BI
NDING SITE). The antibody subclass was IgG ₁ for both L6 and L15.

[0039]

According to the present invention, the human c-ki is first used.
Monoclonal antibodies that react specifically with the t protooncogene product were generated. Since this monoclonal antibody has high specificity and specificity to the human c-kit protooncogene product present in human bone marrow cells and the like, by measuring this, it can be used as a diagnostic agent for hematopoietic tumors such as leukemia. Not only is it useful, it can also be used as a therapeutic drug for them,
Further, it can be used for isolation and purification of human c-kit protooncogene product.

Furthermore, according to the present invention, an excellent hybridoma can be obtained by adopting the transduction method,
It also has a remarkable effect that the desired monoclonal antibody can be efficiently produced.

[Brief description of drawings]

FIG. 1 HCK / 3T3 of hybridomas L6 and L15
It is the figure which showed the reactivity with a transfectant.

FIG. 2 is a drawing showing the reactivity of monoclonal antibodies L6 and L15 with human bone marrow cells. B112 is a negative antibody.

FIG. 3: Four kinds of monoclonal antibodies (Negative)
FIG. 2 is a drawing showing the results of immunoprecipitation by autoradiography of an antibody, ACK2 that is an antibody against a mouse c-kit protooncogene product, and antibodies L6 and L15 according to the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12P 21/08 8214-4B G01N 33/574 D 9015-2J 33/577 B 9015-2J // ( C72P 21/08 C12R 1:91) (72) Inventor Mitsuru Nakamura 3599-1, Chikamachi, Kumamoto City, Kumamoto Prefecture 102 Hidaka Heights 102 (72) Inventor, Kayoko Hayakawa 3-27 Minamikoshiya, Koshigaya City, Saitama Prefecture −31

Claims (6)

[Claims] 1. A hybridoma capable of producing a monoclonal antibody characterized by reacting positively with a human c-kit protooncogene product in a biological sample obtained directly from human. 2. The hybridoma is hybridoma L6.
Alternatively, the hybridoma according to claim 1, which is hybridoma L15. 3. A method for producing a specific monoclonal antibody against a human c-kit protooncogene product, which comprises using the hybridoma according to claim 1. 4. A method for producing a specific monoclonal antibody L6 or L15 against a human c-kit protooncogene product, which comprises using the hybridoma according to claim 2. 5. A specific monoclonal antibody against a human c-kit protooncogene product produced by the method according to claim 3. 6. A specific monoclonal antibody L6 or L15 directed against a human c-kit protooncogene product produced by the method according to claim 4.