JPH05184383A - Bispecific antibody - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody having bispecific property, capable of carrying out label by a labeling agent or cytoxin activating agent without chemically treating an antibody and capable of freely changing the labeling agent, etc. CONSTITUTION:The objective bispecific antibody being an antibody having specificity to two different antigens, in which either one of these antigens is biotin or a biotin derivative and the other is antigen different from the first antigen is obtained from a fused cell obtain by cell fusion of an anti-biotin monoclonal antibody producing cell having specificity to biotin or the biotin derivative and monoclonal antibody producing cell having specificity to antigen other than biotin or a biotin derivative.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bispecific antibody, and more specifically, to a biospecific antibody having specificity for biotin or a biotin derivative and an antigen different from the biotin or biotin derivative. Regarding antibodies.

[0002]

2. Description of the Related Art As a method for highly sensitively measuring a trace substance in a body fluid such as blood or urine, an immunological measurement method is used in the medical field,
Especially, it is widely used in the field of diagnosis. Especially 1
In 975, Milstein et al. Announced a method for producing a monoclonal antibody by cell fusion of mouse myeloma cells and antibody-producing cells in the spleen, and since the monoclonal antibody by the hybridoma method has high specificity, Since it has advantages such as the fact that it is possible to supply an antibody in a relatively simple manner and in a large amount, it has attracted much attention and has been extensively studied and used.

When this monoclonal antibody is used as a tracer antibody, a radioisotope, an enzyme or a fluorescent substance is chemically treated and bound to the antibody molecule. However, such chemical treatment of the antibody molecule damages the antibody molecule, reduces the specific activity of the antibody, and affects the stability of the antibody.
These cause the stability and sensitivity of the assay system to decrease.

On the other hand, a method for expanding the range of use of an antibody by making the antibody molecule have two different antigen recognizing properties by utilizing the high specificity of the monoclonal antibody has been studied. It has already been reported that an antibody in which the binding affinity of one of the multispecific antibodies directly recognizes the enzyme. However, since this antibody directly recognizes an enzyme, when used as a labeled antibody in the field of immunological diagnosis, it is possible to bring about diversity in the type of enzyme, that is, the type of labeling agent. could not.

Further, in the field of medicine, a method of binding an antitumor agent to an antibody to selectively deliver the drug to the tumor site has been studied. In this case as well, the compound as the antitumor agent and the tumor site are studied. Since it is necessary to modify both of the antibodies recognizing., The anti-tumor drug compound and the antibody are adversely affected, and it has been difficult to easily create various combinations.

[0006]

In view of the above, it is applicable to a wide range of labeling agents or cytotoxic activators,
It has been demanded to provide an antibody having excellent stability.

[0007]

The object of the present invention is to label an antibody with a labeling agent or a cytotoxic activator without chemically treating the antibody. Furthermore, the labeling agent in the assay system can be changed, or the cytotoxic activity in the treatment of cancer can be improved. The present invention provides a monoclonal antibody having bispecificity, which can be widely applied even if the agent is changed.

[0008]

The bispecific antibody according to the present invention is a monoclonal antibody having specificity for both a certain antigen and biotin or a biotin derivative. That is, by combining this bispecific antibody with a biotinylated enzyme, a fluorescent substance or a cytotoxic active substance, it is possible to use the antibody molecule as a tracer or a targeting agent without chemically treating it. It is possible to increase the stability of the antibody and further to use biotinylated enzymes and the like in different antigen measurement systems and to deliver various anticancer agents by using biotinylated anticancer agents.

The bispecific antibody according to the present invention can be produced according to the following procedure.

First, a monoclonal antibody-producing hybridoma for the antigen to be measured and a monoclonal antibody-producing hybridoma for biotin are prepared according to ordinary methods. Next, different enzymes in the salvage pathway of nucleic acid biosynthesis in these hybridomas are deleted. That is, the hybridoma producing a monoclonal antibody against the antigen is a hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficient cell, and the monoclonal antibody producing biotin is a thymidine kinase (TK) deficient cell. After fusion of these two different enzyme-deficient cells by the polyethylene glycol method, HAT medium (hypoxanthine (10 ⁻⁴ M) and aminopterin (4 × 10 ⁻⁷ M) were added to normal medium).
And a medium containing thymidine (1.6 × 10 ⁻⁵ M))
Use to select only fused cells. From the hybrid hybridoma thus obtained, a cell line producing an antibody that simultaneously recognizes the antigen and biotin by cloning was selected, and the obtained monoclonal antibody-producing strain was administered intraperitoneally to a mouse to contain the antibody. Make ascites,
The target antibody is obtained from this ascites. This antibody is a bispecific antibody having a binding affinity for an antigen and a binding affinity for biotin, and an antibody measurement system is prepared by using this antibody as a tracer. This antibody can also be used as a targeting agent for delivering a compound having a cytotoxic activity to a tumor site or the like. The above-mentioned monoclonal antibody-producing strain has been deposited at the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology, and is referred to as Microtechnology Research Institute No. 11531 (TB
HF2H6) or Microtechnical Research Institute No. 11532 (TB
The antibody produced by the hybrid hybridoma that is HF1H50) is a bispecific antibody having specificity for biotin and human serum albumin, and by using it, biotin-HRPO as a labeling agent,
Human serum albumin can be measured by EIA.

In addition, Micromachine Research Institute, No. 12287 (TB
The antibody produced by the hybrid hybridoma, CF1H3), is a bispecific antibody with specificity for biotin and carcinoembryonic antigen (CEA).

The hybridoma producing a monoclonal antibody against an antigen (second antigen) different from the biotin or biotin derivative is a hybridoma producing a monoclonal antibody against human serum albumin HSA.
F6H1C11, HSAF6H33C11, hybridoma CE producing monoclonal antibody against carcinoembryonic antigen
AF22H5, CEA3519, Elastase 1 monoclonal antibody producing hybridoma ELSF1
3H4, hybridoma IGEF3H22 for producing monoclonal antibody against IgE, hybridoma INSF9H2 for producing monoclonal antibody against insulin, hybridoma SCCF1H3 for producing monoclonal antibody against squamous cell carcinoma-associated antigen SCC, hybridoma TSHF8H4 for producing monoclonal antibody against TSH, and the like. The monoclonal antibody-producing hybridoma against the antigen can be used without particular limitation as long as it can be treated as described above to produce a bispecific antibody.

Examples of the second antigen in this case include proteins, polypeptides, polysaccharides, nucleic acids, and combinations thereof. Among these are phosphoproteins, lipoproteins, glycoproteins,
Mucoproteins, bacteria, viruses, tumor cells, rickettsia or their surface antigens are included.

Among such antigens, proteins and polypeptides include somatotropin, prolactin,
Insulin, LH-RH, hormones such as gonadotrophin, thyroxine-binding globulin, α ₁ -antitrypsin, α ₁ -acid glycoprotein, α ₂ -macroglobulin, plasma proteins such as albumin, immunoglobulins or fragments thereof, complement Factors, blood coagulation factors, tissue hormones such as secretin, gastrin and the like can be mentioned.

In addition to these, those known as cancer-related antigens,
For example, CEA, AFP, SCC, Span-1, CA1
9-9, CA125, B72.3 antigen (TAG-7
2), T antigen and the like.

Further, a receptor on the cell surface and the like can also be mentioned.

Furthermore, examples of the second antigen include various antigens present in Gram-positive bacteria and Gram-negative bacteria, various viruses such as rickettsia, mycoplasma or hepatitis virus, tumor virus, and AIDS-causing virus, and their produced antigens. .. Also among these antigens are:
Various alkaloids, various steroids, penicillin and cephalosporin antibiotics, gentamicin, kanamycin, erythromycin and other antibiotics, barbituric acid drugs, catecholamines, ephedrine and other drugs,
Examples include various drugs and drugs such as chlorpromazine, azepines, vitamins, prostaglandins. These antigens can be selected without particular limitation as long as the object of the present invention can be achieved, and can be selected from those known by literatures in the field.

The bispecific antibody thus obtained is used in an immunological measurement system such as a sandwich method for measuring an antigen in a body fluid such as blood or a tissue staining method for measuring an antigen in a tissue. can do.

Furthermore, a compound having cytotoxic activity such as an anticancer drug or ricin or diphtheria toxin is biotinylated, and in particular, a bispecific antibody that recognizes biotin and a cancer-associated antigen is reacted with the biotinylated cytotoxic compound. Antibody-drug conjugates can be formed.
The antibody-drug conjugate thus obtained allows the delivery of the drug to specific sites recognized by the bispecific antibody, eg tumor cells. By using the bispecific antibody thus obtained, it becomes possible to carry various drugs using only one kind of antibody.

The measurement of the antigen by the sandwich method can be carried out as follows.

After reacting a sample such as serum or urine with a solid-phased antibody obtained by binding an antibody against the antigen to be measured to an insoluble carrier, the unreacted liquid is removed by washing. Next, a bispecific antibody solution having a binding affinity for the measurement antigen and biotin is reacted. Further, after washing the solid phase, a biotin solution having an enzyme bound thereto is mixed and the reaction is carried out. In this reaction, the bispecific antibody and the biotinylated enzyme may be mixed in advance, and this mixed solution may be reacted with the solid phase after the reaction of the sample. The solid phase that has reacted with this biotinylated enzyme is washed and a substrate solution is added to carry out an enzymatic reaction. After stopping the enzymatic reaction, the absorbance of the reaction solution is measured, and the concentration of the antigen to be measured can be determined from the standard curve prepared by using the standard antigen instead of the sample.

In the bispecific antibody of the present invention, one of the antigen recognition sites recognizes biotin. Since biotin can bind to proteins, nucleic acids and various other chemical substances, it can be used as an enzyme as a labeling agent. It is possible to use various compounds such as a fluorescent substance without being limited to
Various drugs such as antitumor agents can also be used. Furthermore, biotin can be used in a system with avidin to increase the sensitivity of the measurement system.

[0023]

EXAMPLES The present invention will be described below based on examples.

Example 1 Preparation of anti-human serum albumin (HSA) antibody-producing hybridoma 50 μg of HSA (Miles Inc.) was prepared as an emulsion with Freund's complete adjuvant (Yatron), and this was subcutaneously injected into BALB / c mice. .. This was repeated 4 times every 2 weeks, and after 2 weeks, 50 μg of HSA was dissolved in physiological saline and intraperitoneally administered. The spleen cells of this mouse were extracted 3 days later and fused with mouse myeloma cells (P3 × 63Ag8 · U1: P3U1) using the polyethylene glycol method. After selecting the fused cells in HAT medium, the anti-HSA of the culture supernatant was selected.
The antibody activity was examined using ¹²⁵ I-labeled HSA, and further cloned to obtain anti-HSA monoclonal antibody-producing hybridomas (HSAF6H1C11, HSAF6
H33C11) was established.

The anti-HSA monoclonal antibody-producing hybridoma was intraperitoneally administered to mice treated with 2,6,10,14-tetramethylpentadecane (Pristane: Aldrich) to prepare antibody-containing ascites.

The subclass of the monoclonal antibody produced was IgG ₁ . Example 2 Preparation of Monoclonal Antibody Against Biotin 100 μg of biotin (VECTER Lab.) Bound to rabbit IgG as a carrier protein was freunded.
An emulsion was prepared with complete adjuvant (Yatron), and this was subcutaneously administered to BALB / c mice. This was performed twice every two weeks, and after two more weeks, 100 μg of biotin bound to rabbit IgG was dissolved in physiological saline and intraperitoneally administered. The spleen cells of this mouse were extracted 3 days later, and cell fusion was performed with mouse myeloma cells (P3U1) using the polyethylene glycol method.
After selecting the fused cells in HAT medium, the anti-biotin antibody activity of the culture supernatant was determined by adding biotin labeled with horseradish peroxidase [biotin-HRPO] (VECT
ER Lab. ), And further cloned to establish an anti-biotin / monoclonal antibody-producing hybridoma.

The monoclonal antibody BRIF2H15C23 secreted from these hybridomas was selected. The subclass of this monoclonal antibody was IgG ₁ .

Example 3 Preparation of salvage pathway enzyme-deficient hybridoma (a) HGP of anti-HSA / monoclonal antibody producing cells
RT deficiency logarithmic growth anti-HSA / monoclonal antibody producing hybridoma (HSAF6H1C11, HSAF6H33C
11) was cultured in a medium containing 1 μg / ml of MNNG (N-methyl-N′-nitro-N-nitrosoguanidine) for 3 hours to perform mutagen treatment. After the mutagen treatment, cells were grown for 2 days in a normal medium, and then 8-azaguanine was added to 20 times.
Culture was performed in a medium supplemented with μg / ml. A part of the cells (8-azaguanine-resistant cells) capable of growing in this 8-azaguanine-added medium was cultured in HAT medium and cells that did not grow (cells in which the salvage pathway did not work) were selected. Further, cells having an antibody activity against HSA were selected by the ELISA method, and the cells were fused with the HGPRT-deficient mouse myeloma cells (P3U1) by the polyethylene glycol method. Among these fused cells, cells that cannot grow in HAT medium (HSAF6H1G, HSAF6
H33J) was selected. 8-azaguanine resistant cell line is H
It was confirmed that these 8-azaguanine-resistant cells are HGPRT-deficient cells because they cannot grow in AT medium and they cannot grow in HAT medium even when fused with P3U1 cells.

(B) TK deficiency of anti-biotin / monoclonal antibody-producing cells Anti-biotin / monoclonal antibody-producing hybridoma (BRIF2H15C23) in the logarithmic growth phase was MNNG1μ.
After culturing for 3 hours in a medium containing g / ml, mutagen treatment was performed. After the mutagen treatment, the cells were grown in a normal medium for 2 days, and then treated with 5-bromo-2'-deoxyuridine (Bud).
R: Sigma B-5002) was cultured in a medium supplemented with 30 μg / ml. A part of the cells (BudR resistant cells) capable of growing in this BudR-added medium was cultured in HAT medium and cells that did not grow (cells in which the salvage pathway did not work) were selected. Furthermore, cells that have antibody activity against biotin were selected by the ELISA method, and this and P3U1 cells were treated with PE.
Cells were fused by the G method and cells (BRIF2H15B1) capable of growing in HAT medium were selected. If the BudR resistant cell is a TK deficient cell, P that is an HGPRT deficient cell
The fused cells with 3U1 cells can complement each other's defective enzymes and grow in HAT medium. The obtained fused cells are H
Proliferation was confirmed in AT medium, and it was confirmed that the BudR resistant cells were TK-deficient cells. Example 4 Preparation of Bispecific Antibody (TBHF1) Hybridoma HSAF6H prepared in Example 3 (a)
1G (HGPRT-) and 1.1 x 10 ⁷ cells each of the hybridoma BRIF2H15B1 (TK-) prepared in Example 3 (b) were subjected to cell fusion by the polyethylene glycol method. After the fusion, the fused cells which were cultured in HAT medium and proliferated were selected, and the antibody activity of the culture supernatant of the fused cells to HSA and biotin was examined by the ELISA method. Next, this antibody was reacted with an HSA-immobilized ELISA plate, and this was reacted with biotin-HR.
PO was reacted. The HRPO activity on the solid phase was measured using o- phenylenediamine and _H 2 _{O 2.} After measuring the antibody activity, the fused cells having bispecific antibody activity were cloned to establish hybrid hybridomas (Table 1).

This hybrid hybridoma was intraperitoneally administered to BALB / c mice treated with pristane,
Antibody-containing ascites was prepared. The IgG ₁ concentration in this ascites was 2.4 to 13 mg / ml.

[0031]

[Table 1] Example 5 Preparation of bispecific antibody (TBHF2) HSAF6H33J (HGPRT-) and BRIF2H1
3.2 × 10 ⁵ cells each of 5B1 (TK−) were subjected to cell fusion by the polyethylene glycol method in the same manner as in the production of the bispecific antibody (TBHF1) of Example 4. After selecting the fused cells in HAT medium, the bispecific antibody activity was measured and further cloned to establish hybrid hybridomas (Table 2).

This hybrid hybridoma was intraperitoneally administered to pristane-treated BALB / c mice,
Antibody-containing ascites was prepared. The IgG ₁ concentration in this ascites was 1.6 to 11 mg / ml.

[0033]

[Table 2] Example 6 HSA sandwich method using bispecific antibody EIA anti-HSA. HSA diluted solution (1000, 300, 100, 30, 10 ng / ml) was added to beads immobilized with monoclonal antibody HSAF6H1 (2 μg / bead).
100 μl and 200 μl of 50 mM phosphate buffer were reacted at room temperature for 3 hours. After washing this twice with purified water,
Bispecific antibody TBHF2H6 ascites diluted solution 100 μl
And 200 μl of 50 mM phosphate buffer were reacted at 4 ° C. overnight. After washing twice with purified water, further biotin-HRP
300 μl of O diluted solution (1 × 10 ⁶ ) was reacted at room temperature for 3 hours. After washing twice with purified water, the beads were transferred to a reaction test tube and the substrate solution (o-phenylenediamine, H
₂ O ₂ ) (300 μl) was added, the mixture was incubated at room temperature for 15 minutes, the reaction was stopped with 1 N sulfuric acid (1000 μl), and the absorbance of the reaction solution at 490 nm was measured. The results thus obtained are shown in FIG.

Example 7 HGPRT deficiency of anti-CEA / monoclonal antibody producing cells The anti-CEA / monoclonal antibody producing hybridoma strain CEA3519 was treated in the same manner as in Example 3 (a) to produce hypoxanthine / guanine / phosphoribosyl transferase. A (HGPRT) -deficient strain was prepared. CEA
3519 strain was used for resistance to 8-azaguanine and HA
20 kinds of T-sensitive cells were obtained.

Example 8 Preparation of Anti-CEA Bispecific Antibody HA prepared according to Example 7 from CEA3519 strain
One of the T-sensitive cells, A2 strain, and the TK-deficient BRIF2H15B1 cells prepared in Example 3 (b) were treated in the same manner as in Example 4 or 5. Each cell is 2.
Using 8 × 10 ⁶ cells each, they were fused 1: 1 and seeded in 400 wells. As a result, cell proliferation was observed in 18 wells. Those which have antibody activity against both CEA and biotin in the HAT screen are selected and subjected to two additional screening treatments to establish four clones, namely, H3, H4, H5, H6 clones (here The thus obtained cells were referred to as TBCF1 cells), and the H3, H4 and H5 clones were subjected to ascites treatment in mice. This operation was also performed according to Examples 4 and 5.

Example 9 TBCF1H3 antibody obtained from the TBCF1H3 clone obtained in Example 8 (diluted solution of mouse ascites, SRI
CEA using an antibody concentration of 200 μg / ml)
The sandwich method EIA was performed.

Bead 1 immobilized with CEA4230 antibody
0, 4, 10, 40, 200 and 500 ng / ml
100 μl CEA solution and 200 μl buffer in the dilution series of
The mixture was placed in a solution consisting of and and reacted at room temperature for 3 hours with stirring. After washing three times with purified water, 100 μl of the above TBCF1H3 antibody (200 μg / ml) and 200 μl of buffer solution
1 was added. The reaction was carried out at 4 ° C for 15 hours, and the product was washed 3 times with purified water.

Next, 300 μl of biotin-HRPO solution
In addition, the mixture was reacted at room temperature for 3 hours with stirring and then washed three times with purified water. The beads were transferred to a test tube for reaction, 300 μl of a substrate solution containing o-phenylenediamine was added, and the mixture was reacted at room temperature for 30 minutes, and 1000 μl of 1N sulfuric acid was added.
Was added to stop the reaction, and the absorbance at 492 nm was measured. The buffer used here was 50 mM phosphate buffer (1% BSA, pH 7.4).

The standard curve of CEA obtained here is shown in FIG.

[0040]

INDUSTRIAL APPLICABILITY The bispecific antibody of the present invention can be used in place of a labeled antibody in which an enzyme or a fluorescent substance is bound by a conventional chemical method by combining it with a biotinylated enzyme or a fluorescent substance. Is possible,
Since antibody molecules can be used as tracers without chemical treatment, production efficiency can be improved, stability can be increased, and biotinylated enzymes can be used as common reagents in assay systems for different antigens. It has advantages such as

[Brief description of drawings]

FIG. 1 shows a standard curve of the sandwich method EIA of HSA prepared using the monoclonal antibody secreted by the hybrid hybridoma of the present invention.

2 is a sandwich method E of CEA obtained in Example 9. FIG.
The standard curve of IA is shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical indication location G01N 33/53 U 8310-2J 33/531 A 8310-2J 33/574 E 9015-2J 33/577 B 9015-2J (C12P 21/08 C12R 1:91)

Claims (6)

[Claims] 1. An antibody having specificity for two different antigens, wherein one of the antigens is biotin or a biotin derivative and the other is an antigen different from the first antigen. Bispecific antibody. 2. A fusion cell of an anti-biotin / monoclonal antibody-producing cell having specificity for biotin or a biotin derivative and a monoclonal antibody-producing cell having specificity for an antigen other than biotin or a biotin derivative. The bispecific antibody according to claim 1, wherein 3. The antigen different from the first antigen is a protein, a polysaccharide, a nucleic acid and a combination thereof.
Or the bispecific antibody according to 2. 4. The antigen different from the first antigen is human serum albumin, carcinoembryonic antigen, elastase-1, Ig.
E, insulin, squamous cell carcinoma-related antigen (SCC), TS
The bispecific antibody of claim 1 or 2, which is H. 5. The fused cells TBHF1H50 [(Accession number) Microtechnology Research Institute No. 11532], TBHF2H6
[(Contract number) Microtechnology Research Institute, No. 11531] or T
BCF1H3 [(Consignment number)
No.], the bispecific antibody according to claim 1 or 2. 6. The bispecific antibody according to claim 1, wherein the biotin derivative is a biotinylated enzyme or a biotinylated fluorescent substance.