JPH04187096A - Hybrid monoclonal antibody having double specificity - Google Patents

Abstract

PURPOSE:To provide the subject antibody having bonding affinity to both of a specific compound or its salt and a target antigen, capable of extremely specifically bonding to the target antigen and, at the same time, having an activity to kill cancer cell by cytotoxic action. CONSTITUTION:The objective antibody has bonding affinity to both of a compound of formula (X is OH or H) or its salt and a target antibody (preferably a tumor-relating antigen, especially human transferrin receptor). It is preferable to get an immune complex by bonding the compound of formula or its salt to the antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a hybrid monoclonal antibody with bispecificity. More specifically, one of the bispecifics is anthracycline compounds such as TAN-1120 (e.g., TAN-1120 and its reduced form, etc.) or its salts, while the other is a target antigen, especially on the cancer cell surface membrane. The present invention relates to a hybrid monoclonal antibody (hereinafter sometimes abbreviated as hybrid MoAb) against a tumor-associated antigen, and a polydoma that produces the same.

The present invention also relates to a cancer treatment method that uses the hybrid MoAb described above to specifically bind TAN-1120 or a salt thereof to, for example, cancer cells, thereby making it possible to exert a lethal effect on cancer cells.

[Prior art and problems to be solved by the invention] Anthracycline compounds such as adriamycin (hereinafter sometimes abbreviated as AM) and taunomycin (hereinafter sometimes abbreviated as DM) were developed in the 1960s. Since their discovery, some have now been widely applied clinically. In particular, DM is effective in treating acute leukemia and neuroblastoma, and AM is effective in treating some solid cancers, and their wide anticancer spectrum is effectively utilized clinically. However, side effects of these compounds include bone marrow suppression, alopecia, cardiotoxicity, and stomatitis. AM in particular has a broad anticancer spectrum, but its side effects have made it difficult to use. Therefore, various studies have been made to reduce the side effects specific to these anthracyclines. Among these efforts, aclanomycin A was developed as a compound of a family with relatively few side effects.
was developed. This compound is used for the treatment of malignant lymphoma, acute leukemia, gastric cancer, and other solid cancers, and has also been reported to cause hair loss and low toxicity.

Recently, a new bacterial species of the genus Streptomyces [Streptomyces
ptomyces triangulatus 5ub
sp, angiostaticus S-14519 (
IFO14801, FERMBP-2199, etc.] is the following one-ship formula [where X is a hydroxyl group (TAN-1120)]
It was discovered that *tCt hydrogen atom (TAN-1120 reduced form) produces a strong anti-tumor substance with few side effects. This compound is a physiologically active substance that has a strong angiogenesis inhibitory effect on neovascularization induced during the proliferation of various tumor cells, and is expected to be a new anticancer agent [Physicochemical properties of TAN-1120 class and manufacturing method, respectively, EP publication number 0.
Publication No. 376177, page 9, line 12 to page 10, line 7 and 11
See page 54, line 54 to page 12, line 52. However, these anthracycline compounds still have serious side effects, and the current situation is that the development of anticancer agents with even fewer side effects is awaited for clinical application.

On the other hand, anti-tumor immune complexes, which combine anti-cancer antibodies with chemotherapeutic agents or biological toxins, are used as drugs that selectively kill tumor cells.
A so-called "missile therapy agent" has been developed. These recognize and bind to tumor-specific antigens or tumor-related antigens on tumor cells, and they also bind to the DNA of these tumor cells.
It has the property of inhibiting protein synthesis, protein synthesis, or microtubule formation, leading to death. Therefore, tumor organ
It is tissue/cell specific and has few side effects on normal cells.

Some antibody-drug or antibody-toxin conjugates have already been used clinically, with some good results. [Y,
Tsukada et al.: Proceedings of the National Academy of Sciences, Ninisny (
Proc, Natl, ^cad, Sic, U
SA), 79, 621 (1982, l, M, V, P
imm et al.: Cancer Immunology and Immunotherapy (Cancer Immunol, 1mm
unother, )+12+125(198
2)].

However, chemical bonding reactions between antibodies and drugs or antibodies and toxin proteins often impair antibody activity or pharmacological activity of drugs or toxins, and the emergence of even more powerful selective anticancer agents is expected.

In particular, in the case of low-molecular anticancer drugs, (1) an appropriate functional group that can be used for binding with antibodies is required; (2) chemical bonding with antibodies usually reduces pharmacological activity to 1/10 to 1/100 or less; There have been problems such as the fact that the effect is greater than that of toxin proteins, and therefore (1) conditions must be provided to allow the antibody-drug linker to be cleaved within tumor cells.

Based on this technical background, in order to make TAN-1120, which has a strong cytotoxic effect, tumor-specific and enable clinical application, the present inventors combined it with an anti-cancer antibody and developed an unprecedented antibody-drug. This research was carried out with the aim of developing a complex. In the production of such antibody-drug conjugates,
As mentioned above, several problems were considered. In other words, with conventional methods that utilize chemical bonding reactions, 1) the antitumor activity of the drug is significantly reduced, and furthermore, the antigen binding ability of the antibody is also reduced; 2) the use of homopolymers of antibodies that are inactive as selective anticancer agents or easily metabolized; High molecular weight polymers lacking in usefulness in vivo are produced as by-products.■ It is difficult to control the number of drug molecules that bind to antibodies, making it impossible to obtain antibody-drug complexes of consistent quality.■ Drugs that can be used for binding are limited to those with appropriate functional groups, and problems include the difficulty of using a drug that shows the best therapeutic index.

In contrast, recent advances in protein binding technology or hybridoma production technology have made it possible to produce bispecific antibodies with new functions. Particularly in the latter field of cell fusion, it has become relatively easy to create bispecific antibody-producing triomas and tetraomas [C, M
Ilstein et al.: Nature, 30
5, 537 (1983): M.

R, 5uresh et al. Nibron Tinges of National Academy of Sciences, Ninis
(Proc, Notl, Acad, Sci, U
, S, ^, ), 83°7989 (1986)].

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have applied and developed this new technology, and as a result, the present inventors have succeeded in solving the above-mentioned problems. No need for chemical bonding with drug molecules, and 100% biological activity of antibodies and drugs
Hybrid M allows for the creation of retained antibody-drug immune complexes.

We developed an Ab and created an anti-human cancer immune complex using it. That is, the present invention provides a hybrid MoAb with bispecificity, one of which is TAN-1.
120 or its salts, the other relates to antibodies against target antigens, such as tumor-associated antigens on cancer cell membranes, and polydomas that produce them.

The present invention also provides hybridomas that produce antibodies against TAN-1120s or their salts (hereinafter sometimes abbreviated as anti-ATC antibodies) and target antigens, such as the human transferrin receptor (hereinafter referred to as anti-ATC antibody), which is abundantly expressed on cancer cell membranes. , sometimes abbreviated as hTfR), resulting in the production of a bispecific hybrid MoAb that produces a resulting tetraoma.
furthermore, this hybrid MoAb
The present invention relates to a selective anti-human cancer immune complex obtained using the method.

Bispecific hybrid M of the invention.

When creating Ab-producing polydomas, anti-ATC antibody-producing hybridomas are used, and such hybridomas can be created, for example, by the method described below.

That is, first, anthracyclines such as AM, D
M, TAN-1120, etc. are inoculated into animals to promote the production of anti-ATC antibodies. In this case, if these compounds are used as immunogens as they are, it will not be possible to induce high-titer antibody production. It is used as an immunogen by binding to serum albumin (hereinafter sometimes abbreviated as BSA) or thyroglobulin.

As the inoculated animal, for example, rabbits, horses, horses, mice, guinea pigs, etc. are used, and mice are particularly preferably used in the case of MoAb production.

As for the inoculation method, it is sufficient to follow the commonly practiced method.
For example, 1 to 100 μg per mouse, preferably 10 to
25 μg is emulsified with an equal volume (0.1 m) of physiological saline and complete Freund's adjuvant and injected subcutaneously in the back or abdomen, or intraperitoneally, 3 to 6 times every 2 to 3 weeks.

Individuals with high antibody titers are selected from these immunized animals, such as mice, and their lungs and/or lymph nodes are collected 3 to 5 days after the final immunization, and the antibody-producing cells contained therein are fused with myeloma cells. The fusion operation can be carried out according to a known method, and examples of the fusion promoter include polyethylene glycol (hereinafter sometimes abbreviated as PEG), Sendai virus, etc., and PEG is preferably used.

Myeloma cells include NS-1, P3U 1.5p210
etc., and P3U1 is particularly preferably used.

For example, the preferred ratio of lung cells to myeloma cells is 1:1.
~10:1, to which PEG with a molecular weight of 1°000 to 9,000 was added at a concentration of 10% to 80%, and the mixture was heated at 20°C to 37°C.
, preferably 3 to 10 minutes at 30 to 37°C.

Various methods can be used to screen for hybridomas producing anti-A and C antibodies, such as anthracyclines (e.g. TAN-1'120.AM) - human serum albumin (hereinafter sometimes abbreviated as H3A). Hybridoma culture supernatant was added to the microplate on which the complex had been adsorbed, and then horseradish peroxidase (hereinafter sometimes abbreviated as HRP)-labeled anti-mouse immunoglobulin antibody was added to remove the anti-mouse immunoglobulin antibody bound to the solid phase of the plate. A
Examples include the EIA method for detecting TC monoclonal antibodies. Hybridomas positive for antibody activity that are selected and bred in a HAT (hypoxanthine, aminopterin, and thymidine) supplemented medium are immediately subjected to cloning, which is usually easily carried out by the limiting dilution method or the like. The antibody titer of the culture supernatant of the cloned hybridoma is measured by the method described above, and hybridomas that stably produce antibodies with high titers are selected to obtain the desired monoclonal anti-A and C antibody-producing hybridomas. be able to.

Anti-ATC antibody (I
As an example of a hybridoma producing gG l + λ chain, the mouse hybridoma ADH2 obtained in the Examples below is used.
-357 (I FO50258, FERM BP-3
142), etc.

Further, as a tumor-related antigen that is a target antigen, for example, hT fR, which is relatively abundantly expressed in many tumor cells, can be considered. hT fR is purified from human placental tissue according to known methods [P, A.

Seligman et al.: Journal of Biological Chemistry (J, Biol, Chem,), 2
54, 9943 (1979)], a highly pure specimen can usually be obtained by the following method. ■4% human placental tissue
Triton-X-100 in pH 7.5 phosphate saline buffer (20mM disodium phosphate, 0.15M Na
C1: Homogenized in C1 (hereinafter sometimes abbreviated as PBS), further subjected to ultrasonication, and then centrifuged.

After salting out the obtained supernatant with ammonium sulfate, it was applied to a column bound with an antibody against human transferrin (hT f>) and added to a phosphate buffer (20 mM disodium phosphate; , sometimes abbreviated as PB). Then, wash thoroughly with 0.5
hT f in 0.02 M glycine buffer (pH 10,0) containing MNaC1 and 0.5% Triton-X-100.
Elute the R fraction. ■Add the above hT fR fraction to hT
PB (pH
After washing with 7,5), 1M NaCl and 1% Triton-
0.5M glycine buffer containing X-100 (pHIo,
0) to obtain a purified hT fR preparation. Or hT fH
It is also possible to elute and isolate in one step using a column bound with an antibody to the target.

Immunization of animals with hT fR and fusion of anti-hT fR antibody-producing cells with myeloma cells can be carried out in the same manner as described for anthracyclines. anti-hTf
Various methods can be used to screen R antibody-producing hybridomas, but for example, hybridoma culture supernatant is added to a microplate to which anti-mouse IgG antibody has been adsorbed, and then an HRP-labeled hTfR purified preparation is added to fix the plate. The EIA method detects anti-hT fR monoclonal antibodies bound to the phase, or the 562 cell line (leukemia cells), which expresses a large amount of TfR on the cell surface membrane, is immobilized on a microplate and hybridoma culture supernatant is added to it. Examples include cell-EIA method in which HRP-labeled anti-mouse IgG antibody is added.

As an example of a hybridoma producing an anti-hT fR antibody (IgG+, 2-chain) produced according to the above production method, mouse hybridoma 22C6 obtained in the reference example described below is used.
(IFO50172, FERMBP-2054), etc.

Hybrid M with bispecificity of the invention.

There are several ways to make Abs. One is a chemical method, in which anti-ATCMoAb and anti-TfRMo
The anti-cancer MoAb is covalently bonded to the Ab. Another method is to fuse two types of hybridomas that produce anti-ATC MoAb and anti-cancer MoAb, respectively, to produce a hybrid hybridoma (eg, tetraoma) and to produce the desired bispecific antibody. The latter hybrid hybridoma method is preferably used as a means to obtain antibodies of consistent high quality in large quantities and with good yield.

In order to chemically bond two types of MoAbs, substituents present in the antibody molecule, such as an amine 7 group, a carboxyl group, a hydroxyl group, or a sulfhydryl group, can be utilized. For example, (1) the reactive amine 7 group of one antibody and the reactive carboxyl group of the other antibody are converted to a water-soluble carbodiimide reagent [e.g., l-ethyl-3-(3-dimethylami/propyl)-carbodiimide, 1-cyclo Kinru 3-(2-morpholinoethyl)-carbodiimide-p-toluenesulfonate, etc.) in an aqueous solvent. Esters [e.g., p-
After maleimidation by reacting maleimidomethylcyclo with 1-carboxylic N-hydroxysuccimide ester, N-(ε-maleimidocabroyl)succimide ester, etc., i) dithiolyze the other antibody. An antibody reduced with threitol (hereinafter sometimes abbreviated as DTT), or ii) the other antibody reduced with N-sufucimidyl-3-(2-pyridyldithio)propionate (hereinafter sometimes abbreviated as 5PDP). An antibody into which a sulfhydryl group has been introduced, or iii) thioether bonding with the sulfhydryl group of the Fab' fraction obtained by reducing the other antibody after treatment with pepsin, (3) Scraping the reactive amino groups of both of the two antibodies. (4) Reduce the two antibodies with DTT or 5PD.
A sulfhydryl group is introduced with P, and a heterodimer is produced by reoxidation. (5) Both antibodies are treated with pepsin [originally, F a
There are methods such as creating a b' heterodimer. There are also reports of combining various of these methods to efficiently produce the desired heterodimeric bispecific antibody without impairing the activities of the two types of antibodies [M, J,
Glennie et al.: Journal of Immunology, 139.2367 (
1987); Tsunehiro Kitayo: Organic Synthetic Chemistry, 42, 283 (
1984)], the bispecific hybrid MoA of the present invention
It can be used to prepare b.

After completion of the binding reaction as described above, the bispecific antibody conjugate is prepared using Sephadex G100 or G200, Sepharose 6B or 4B, Ultrogel AcA44 or 34. Gels such as Sephacryl 3200 can be purified and fractionated by perchromatography. Alternatively, selective fractionation is also possible by combining affinity chromatography using an antigen-binding column.

Bispecific hybrid M of the invention.

There are several methods for creating polydomas that produce Ab [Hiroshi Niimoto et al.: Proteins, Nucleic Acids, and Enzymes.

33.217 (1988), any method may be used, but for example: A thymidine kinase-deficient strain is cloned and made HAT-sensitive by stepwise acclimatization to the culture medium.

Similarly, a HAT-resistant anti-hT fR antibody-producing hybridoma was made resistant to 8-azaguanine (hereinafter sometimes abbreviated as AZG), and a hypoxanthine-guanine-phosphoribosyltransferase-deficient strain was cloned.
It is assumed to be T-sensitive. Next, the tetraomas obtained by fusing the two in a conventional manner are selected using a HAT-supplemented medium, and then TAN-
Clone a tetraoma that secretes a hybrid antibody that has the ability to bind to both Type 1120 or a salt thereof and hT fR.
), the other anti-hTfR
After labeling the antibody-producing hybridoma with tetramethyl rhodamine inthiocyanate (hereinafter sometimes abbreviated as TRITC), the two are fused according to a conventional method. The resulting cell suspension was treated with fluorescein activirad.
Cell sorter (hereinafter sometimes abbreviated as FACS)
to select and clone tetraomas that simultaneously exhibit green fluorescence of FTTC and red fluorescence of TRITC.
Examples of methods include: It is also possible to select and clone tetraomas by using completely opposite markers of the parent strains.

For cell fusion in these operations, methods such as Sendai virus, fusion promoters such as PEG, or electrical stimulation are used. Preferably, PEG is used, and an example thereof is given below, but of course the method is not limited to this. That is, the molecular weight is approximately 1,000 to 6,
000, with a concentration of about 10 to 80%, and the processing time is about 0.5 to 30 minutes. As an example of preferable conditions, PEG 6,000 with a concentration of about 35 to 55% is used, and the processing time is about 0.5 to 30 minutes.
Efficient fusion can be achieved by contacting cells at 37°C for 10 minutes.

Selection of polydomas can be carried out using the above-mentioned HAT-added medium, and for this purpose, drug-resistant strains are obtained by drug acclimation methods such as 8-AZG, 6-thioguanine, or 5-BrdU. Additionally, various selective media can be used depending on the introduction of new markers into the fused cells.

Examples of this include media supplemented with neomycin and hygromycin B [B, Sugden, et al.: Molecular and Cellular Biology (M.
o1. Ce11. Biol.

), 5,410 (1985). Furthermore, as mentioned above, hybridomas labeled with different fluorescent dyes are fused,
Another method is to sort double-labeled hybrid hybridomas by FACS [L, Karawa
Jew et al.: Journal Oop Immunological Methods (J, Immunol, Methods), 9
6°265 (1987)].

Various methods can be used to screen for hybrid antibody-producing polydomas. For example, ■anti-ATC mentioned above
EI for screening antibody-producing hybridomas
A, ■ EIA in which polydoma culture supernatant is added to a microplate with anti-mouse immunoglobulin antibody adsorbed to the solid phase, and then HRP-labeled hT fR is added to detect the anti-hT fR antibody bound to the plate solid phase. , and ■ Add the test culture supernatant to a microplate on which the anthracycline-H3A complex has been adsorbed to the solid phase, and then add HRP.
When using EIA for detecting a hybrid antibody with bispecificity by adding labeled hT fR, or when using an anti-hT fR antibody (two chains) having a light chain different from the anti-ATC antibody (lambda chain), Anthracyclines-H in phase
EIA, in which a test culture supernatant is added to a microplate on which the 3A complex has been adsorbed, and then a chain-specific antibody is added to the HRP- or biotin-labeled anti-mouse IgG to detect bispecific antibodies; Modified methods of can be used in combination as appropriate.

Polydomas positive for hybrid antibody activity are subjected to cloning, which is usually easily accomplished by limiting dilution. The antibody titer of the culture supernatant of the cloned polydoma is measured using the method described above, and polydomas that stably produce high titer antibodies are selected to obtain the desired monoclonal hybrid antibody-producing polydoma. can be obtained.

The polydoma of the present invention described above can usually be cultured in a liquid medium or intraperitoneally in an animal (for example, intraperitoneally in a mammal such as a mouse) by a known method. Purification of antibodies in culture fluid and ascites is performed using a combination of known biochemical techniques. For example, centrifuge the cell culture fluid or ascites, remove the supernatant, and salt out (
(usually using ammonium sulfate or sodium sulfate). The obtained protein precipitate is dissolved in an appropriate solution, and after dialysis, it is subjected to column chromatography (ion exchange column, gel filtration column, protein A column, hydroxyapatite column, etc.) to separate and purify the target antibody. I can do it. Alternatively, a column in which two different antigens are insolubilized can be used to separate and separate them in one step.
Can be purified.

By the separation and purification operations as described above, about 1 to 5 mg of a hybrid MoAb with a purity of 90% or more in protein weight ratio can be obtained from, for example, the IQ culture supernatant. Also, 2
3 to 10 mg of the same antibody can be obtained from the ascites fluid of 0-.

The hybrid MoAb obtained as described above is homogeneous as a protein, and is treated with a proteolytic enzyme (such as pepsin) to obtain F-1120, which retains the ability to bind to cancer-related antigens such as TAN-1120s or their salts and hT fR. (ab'), fragments, etc., which can be used for similar purposes as the hybrid MoAb of the present invention.

As an example of a hybrid antibody-producing polydoma produced according to the above production method, tetraoma ADTFI-157 (IF○ 50259.
FERM BP-3143).

In addition, as polydomas producing the hybrid MoAb of the present invention, anti-ATC MoAb producing hybridomas and anti-h
I gave an example of a tetraoma with a TfRMoAb-producing hybridoma, or a trioma with a hybridoma producing one MoAb and a cell producing the other MoAb, or a cell producing each MoAb after immortalization with Epstein-Barr virus etc. Even hybridomas obtained by cell fusion can be used for the same purposes as the tetraomas described above, as long as they produce the hybrid MoAb of the present invention.

In addition, these polydomas are mouse IgG.

When producing Ab, the bispecific hybrid M
DN encoding the variable region containing the antigen recognition site of oAb
A, and genetic manipulation technology [Z.

Steplewski et al.: Bros.
National Academy of Sciences Ninisny (
Proc, Natl, Acad, Sci, USA
), 85+4852 (1988)].
A mouse-human chimeric antibody can also be produced by linking genes encoding the constant region of the antibody. Such chimeric antibodies are advantageously used when administered to humans because of their low antigenicity.

The bispecific antibody or anticancer agent of the present invention (e.g., TAN-
Several methods are used for cancer treatment using an anti-human cancer immune complex prepared from the bispecific antibody and the bispecific antibody. For example, ■Hybrid M of the present invention.

Administering Ab to a cancer-bearing patient in advance, and administering TAN-1120 or a salt thereof after sufficient time has elapsed for binding to cancer tissues/cells; (i) administering the hybrid MoAb and TAN-
1120 or a salt thereof to a cancer-bearing patient at the same time, or
120 or its salt, unreacted TAN-
Examples of such methods include separating Class 1120 or a salt thereof and then administering the obtained anti-human cancer immune complex to a cancer-bearing patient. The anticancer agent used in this case may be any agent as long as it has antitumor activity and reacts with the anti-ATC antibody described above. Examples of such a compound include a compound represented by the general formula [wherein X represents a hydroxyl group (TAN-1120) or a hydrogen atom (TAN-1120 reduced product]] or a salt thereof; Palminomycin [T, Ueda: Journal;
of Antibiotics (J.

Antibiotics), 41, 404 (19
88) Ko, palminomycin-DNA complex [K, K
imura et al.: Agricultural and Biological Chemistry (＾gric, Biol,
Chem, ), 53 + 1797 (1989);
JP-A-2-145598], barbylated anthracycline (FCE21424゜24366.2436
7) [G, Ca5sinelli et al.: Journal
of antibiotics (J, An-tibio
tics), 43.19 (1990);
2-26529] and morpholinylated anthracyclines (MRA-CN) [E, M, Acton et al.:
Journal of Medicinal Chemistry (J.

Med, Chem, ), 27,638 (1984), etc. may also be used.

Various target antigens can be considered in the present invention, and typical examples include tumor-related antigens, which are cancer cell membrane surface antigens, immunocompetent cell surface receptors, and virus-infected cell surface antigens. Among these, hT fR is often used as a tumor-related antigen, but several cancer-related sugar chain antigens including colic embryonic antigen, CEA, α-fetoprotein, and CA19-9 [ S
.

Hakomori: Cancer 0 Research (Canc
er Res,).

Earthworks, 2405 (1985) Membrane immunoglobulin idiotype of B-cell lymphoma [R,A.

Miller et al. New England Journal of Medicine.
), 306°517 (1982)] and the receptor idiotype of T cell lymphoma [L, L, Lan1
er et al.: Journal of Immunology (J, Is
munol, ), 137°2286 (1986)].

Immune complexes of the invention (eg, anti-cancer immune complexes, etc.).

The hybrid MoAb or TAN-1120 or its salt may be used as such or mixed with an appropriate pharmacologically acceptable carrier, excipient, diluent, etc., after sterilization by filtration using a membrane filter, etc., if necessary. , formulated as an injection, etc., and administered to mammals (mice, mice, etc.)
It can be administered to rats, cats, dogs, pigs, cows, monkeys, humans, etc., and used to treat various tumors.

The dosage of the anti-cancer immune complex of the present invention is determined based on the target disease,
Although it varies depending on the symptoms and administration route, for example, when administering intravenously to adult cancer patients, hybrid MoA
b as about 0602-1 mg/kg per day, preferably about 0.04-0.4 mg/kg, TAN-1120
Approximately 0.1 to 5 μs/kg per day as a compound or its salt
Preferably it is about 0.2-2 μg.

As described above, the hybrid MoAb of the present invention can bind to the target antigen with high specificity, and at the same time effectively kills cancer cells through the cytotoxic effect of the bound TAN-1120 or its salt. cancer treatment can be carried out selectively and effectively.

The present invention will be specifically explained below using reference examples and examples, but it goes without saying that these do not limit the scope of the present invention.

IFOFRI Animal Cell Mouse/Mouse Hybridoma 22C650172BP
-2054FRI: Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Institute of Microbial Technology Reference Example I Preparation of TAN-1120 (1) Culture of producing bacteria Glucose 2%, soluble starch 3%, corn stew.
1% liquor, 1% raw soybean flour, 0.5% peptone, 0.3% sodium chloride, 0.5% precipitated calcium carbonate, (
After dispensing 500 d of seed culture medium consisting of (pH 7.0) into a 2Q Yosaka flask and sterilizing it at 120°C for 20 minutes,
Streptomyces trianginillatus subs. angiostaticus strain S-14519 (IFO1
4801°FERM BP-2199) and 2
Culture was carried out on a reciprocating shaker for 40 hours at 8°C. 500 d of the thus obtained seed culture was used as seed culture medium 3 having the same composition as above.
Transplant into a 50C tank containing 0Q and culture with aeration at 28℃ for 48 hours [Aeration rate 100% (30C/min), stirring:
280 revolutions per minute].

(5 g of the seed culture obtained by adding glucose 5.5%,
Main culture medium 100 consisting of 3.5% corn gluten meal and 0.7% precipitated calcium carbonate (pH 7.0)
Transplanted into a 20012 volume tank containing Q and incubated at 28°C for 90
0 hour aeration agitation culture aeration rate 100% (100ff/min)
, stirring: 150 revolutions per minute] to obtain 95 g of culture.

(2) The culture solution (90Q) obtained in purification (1) was adjusted to pH 6.6,
After adding 180 g of methanol and stirring for 30 minutes, Hyflo Supercell (Johns-Manvi)
-11e, USA) as a filter aid.

This furnace solution (241) was concentrated under reduced pressure, methanol was distilled off, and the pH was adjusted to 5.0 with 2Off of ethyl acetate.
After washing twice, the remaining aqueous layer was adjusted to pH 7.0 and extracted twice with 7.5 g of i-butanol.

The i-butanol layer (13Q) was dissolved in 2% NaHCOs water (3
After sequentially washing twice with Q) and twice with water (6 g), the mixture was concentrated to dryness under reduced pressure, and the remaining oily residue was washed with diethyl ether (50011). After repeating extraction three times by adding chloroform (5'0On) to the ether-insoluble residue, the chloroform extracts were combined and concentrated to obtain 2.3 g of an oily residue.

This residue was dissolved in chloroform and adsorbed on a column of Nlicagel (Merck & Co., West Germany). M, chloroform (3I2) containing 2% methanol, 1% acetic acid.

lllt with chloroform (IQ) containing 5% methanol.
The i-th expansion was performed.

The active fractions were collected, washed with 2% aqueous NaHCOs, and concentrated to give a powdery residue (430+ng).

Of this, 150 mg was divided into three portions and purified by high performance liquid chromatography. The column is YMC-pac
k 5H-3430DS (Yamamura Chemical.

Japan), mobile phase was 40% acetonitrile-0.05M.
Phosphate buffer (pH 3,0), flow rate 10 d/min
d fraction, each fraction N.

12 and 13 (60 d in total) were collected and concentrated. Concentrate (2
Adjust the pH of 5d) to 7.2 and add chloroform (15d) to 7.2.
), the chloroform layers were combined and washed with water (15
1112), then add methanol and concentrate to obtain T
AN 1120 (14.5 mg) was obtained as a dark red powder.

(3) Preparation of reduced product TAN-1120 (20
mg) in acetonitrile-0.05M phosphate buffer (p
The mixture was dissolved in a mixture of H3,0) (2:1,5), and sodium cyanoborate (5 mg) was added while cooling with water and stirring.

After stirring for an additional 15 minutes under water cooling, the reaction mixture was added with water (10 m
) was added to adjust the pH to 9.5, and chloroform (2512
) was extracted. After washing the chloroform layer with water, the resulting residue was subjected to silica gel thin layer chromatography (Me
RCK, PLC plate, silica gel 60pts
4. The film was coated to a thickness of 2 llIm) and developed with a mixed solution of formalm-methanol (9:1). Rfo.

The band 65 was scraped off, eluted with a chloroform-methanol mixture (2:1), and concentrated to yield the derivative (9,
511+g) were obtained as a red powder.

AM maleimidated with N-(γ-maleimido butyryloxy)-sufushiimide was added to H3A that had been modified and reduced with N-succimidyl pyridyl dithiopropionate to prepare an AM-ISA complex with a thioether bond. Next, this protein complex was heated at 96 μs/m at 50 μs/m.
A solid-phase antigen was prepared by adding it to a well microplate at a rate of 100 μQ/well.

(2) Assay method 100 μC of the test hybridoma culture supernatant was added to the above antigen-sensitized plate and allowed to react at room temperature for 2 hours. 0.0
20mM phosphate saline buffer containing 5% Tveen20 (p
After thoroughly washing the plate with H7,3 (hereinafter abbreviated as PBS-Tw), HRP-labeled rabbit anti-mouse IgG antibody was added, and the reaction was further allowed to proceed at room temperature for 2 hours.

After washing, 0.1M citrate buffer containing ortho-phenylenediamine and H,O, as enzyme substrates was added to each well, and the enzyme reaction was carried out at room temperature. After stopping the reaction with IN sulfuric acid, the amount of colored dye was measured at a wavelength of 492 nm using Multiscan (manufactured by Flow).

Reference Example 3 Anti-hT fR anti-antibody production/Vibridoma creation (1) Purification of hTfR Human placental tissue of 1.5 kg was cut into small pieces and soaked in PBS (p
After blending in H7,5), the mixture was centrifuged.

The obtained precipitate was homogenized in PBS containing 4% Triton X-100, and further centrifuged after sonication. Next, about 32g of ammonium sulfate was added per 100ml of supernatant, and after salting out, it was applied to an anti-hTf antibody binding column with 0.5g of ammonium sulfate per 100ml of supernatant.
MNaC1-containing PB (pH 7).

5) and thoroughly washed. 0.5M NaCl and 0°5
0.02M glycine buffer containing % Triton X-100 (
The hTfR fraction eluted at pH 10,0) was further treated with hT
After applying to f binding column and washing with PB containing 1M Nacl, 1
0.05 containing MNacI and 1% Triton X-100
Approximately 1.5 mg of hTfR purified sample was obtained by elution with M glycine buffer (pH 10,0).

(2) Elephant Ring After adding an equal volume of Freund's complete adjuvant to 200 μg of the above hT fR purified sample in physiological saline and thoroughly emulsifying it, prepare BALB/c mice (♀, n=10=
(20 μg/WIl/mouse) was administered intraperitoneally and subcutaneously to the back, and booster immunizations were performed at 3-week intervals. After four booster immunizations, the same hT fR antigen solution (30 μg 10.1 d physiological saline/
mice) were administered intravenously.

(3) Cell fusion Three days after the final immunization, the lungs were removed and a lung cell suspension was prepared using a conventional method (approximately 108 cells). Next, 2X 10' mouse myeloma cells (P3U1) were added, and PEG600
Köhler and Milstein's method [Naech+-
(Nature), 256, 495 (1975)].

After completion of the fusion, the cell mixture was suspended in so-called HAT medium containing hypoxanthine, aminopterin, and thymidine, and cultured for 10 days. Thereafter, as soon as the selection of parent cells was completed, the HAT medium was replaced with an HT medium in which aminopterin was removed, and culture was continued.

Commercially available rabbit anti-mouse IgG antibody solution 20μg/m2
Dispense 100 μg into a 6-well microplate and leave it at 4°C overnight, then add 2% BSA-containing PBS (pH 7.3).
A sensitized plate was prepared by adding

In addition, the hTfR purified sample obtained in (1) was subjected to HRP according to the conventional method.
Used for EIA after labeling [Tsunehiro Kitayo: Organic Synthetic Chemistry, 1λ
, 283 (1984)]. That is, add the hybridoma culture supernatant to the second antibody-sensitized plate and incubate at room temperature for 2 hours.
After reacting for an hour, it was washed with PBS.

Next, HRP-labeled hTfR was added, and the mixture was further reacted at room temperature for 2 hours. Hereinafter, an enzyme reaction was carried out by the method described in Reference Example 2-(2), and the antibody titer was measured.

A hybridoma with particularly strong binding ability was cloned by limiting dilution method to obtain anti-hT fRR body-producing mouse hybridoma 22C6. The subclass of this antibody is IgG+ (Ni chain), and it showed high affinity for human tumor cell lines 562 (leukemia cells) and A431 (epitheloid carcinoma cells).

Reference Example 4 Cytotoxicity Test The cytotoxic activity of TAN-1120 was determined by a known method [H,T
ada et al.; Journal of Immunological Methods, Ehiki, 257 (1984)].

That is, a 96-well tissue culture microplate (flow
10% fetal bovine serum (F
Add 50 μg of target cells at a concentration of 2X I05 cells/〆 to 50 μa of a sample serially diluted 2 times with RPMI 1640 medium containing e2), and incubate at 37°C in 5% carbon dioxide gas for 48
Cultured for hours. After culturing, live cells were stained with dimethylazoyl diphenyltetrazolium hydrochloride (MTT) and dissolved in 10% 5DS-0.01N HCg.
The absorbance at 590 nm was measured using Titertic Multiscan (Flow Laboratory). The absorbance obtained is proportional to the number of viable cells.

Example 1 Creation of anti-ATC antibody-producing hybridoma (1) Immunization The maleimidized AM described in Reference Example 2-() was converted into 5PD
An immunogen was prepared by adding it to BSA sulfhydrylated using P. This AM-BSA complex 200μg/d
After adding an equal amount of complete Freund's adjuvant to a physiological saline solution and thoroughly emulsifying it, the solution was administered intraperitoneally and subcutaneously to the back of BAL B/C7 mice (female, 20 ug 10.2 m/mouse) at intervals of 2 to 3 weeks. A booster immunization was performed. After three booster immunizations, AM-BSA complex solution (50 μg 10.1% physiological saline/mouse) was intravenously administered to the individual that showed the maximum serum antibody titer 10 days later.

(2) Cell fusion reference example 3 - Cell fusion was performed according to the method described in (3).

(3) Selection and cloning of hybridomas Hybridomas were screened by EIA using the AM-ISA binding microplate described in Reference Example 2, and the anti-ATC MoAb-producing mouse hybridoma ADH2-357 was created using the same method as in Reference Example 3-(4). Obtained.

(4) Purification of antibodies B A L B pre-injected with 0.5t12 mineral oil intraperitoneally
/c Six mice were inoculated intraperitoneally with 5×10”m/mouse of mouse hybridoma ADH2-357.

Approximately 14 to 18 days later, accumulation of ascites was observed, so it was collected and salted out with 45 to 50% saturated ammonium sulfate.
gG@min was obtained. Furthermore, purified 1gG (antibody ADR2-357) was purified using a DEAE-cellulose column according to a conventional method.
obtained. The immunoglobulin class, subclass, and light chain type of this antibody were determined to be Igc by the Aucteronie method and the EIA method.

−λ chain.

(5) Antibody properties ■ Mouse hybridoma ADR2-35 obtained in (3)
TAN- obtained in Reference Example 1-(2) was added to the culture supernatant of No. 7.
An equal amount of 10 μg/m solution of 1120 was added and reacted at room temperature for 1 hour. Then, the mixed solution was mixed with AM described in Reference Example 2.
- When subjected to EIA using a HSA-sensitized microplate, the results shown in FIG. 1 were obtained.

Antibody ADR2-357 showed a strong reaction with free TAN-1120, and its binding to AM-H3A@ production plates was blocked.

(6) ■Saoni Mamebushi 1 Purified antibody ADR2-357 and TAN obtained in (4)
-1120 (250 μg/l112) was subjected to the cytotoxicity test described in Reference Example 4, and the ability of antibody ADR2-357 to neutralize the cytotoxic activity of TAN-1120 was measured.

The results were as shown in Figure 2. Antibody ADH2-3
57 inhibited the cytotoxic activity of TAN-1120 against murine leukemia cells P388 and human epithelioid carcinoma cells A431 by 50% at approximately 15- to 30-fold molar doses.

(1) (1) Anti-ATC antibody-producing mouse hybridoma ADR2-357 obtained in Cell Fusion Example 1 and anti-h obtained in Reference Example 3
T fR antibody-producing hybridoma 22C6 was incubated at 0.5 μg/mFJTc and 1°5 μg/mTRlT, respectively.
The cells were incubated for 30 minutes at 37° C. in Iscohuham's F12 mixed medium containing C, and fluorescently stained. Next, after adding LSM solution (sold by Wako Pure Chemical Industries, Ltd.) to remove dead cells, both hybridomas were mixed at a ratio of 1=1, and P
Cell fusion was performed using EG6000.

After incubation at 37°C for 2 hours, 25,000 cells double-stained with fluorescein and rhodamine were collected by subjecting to FAC3, and then 5 x 10 cells/well of mouse thymocytes were seeded as feeders9.
The double-stained cells described above were seeded at a rate of 10 cells/well in a 6-well microplate and cultured.

(2) Tetraoma selection and cloning fusion 1~
Two weeks later, the culture supernatants of wells in which cell proliferation was observed were subjected to EIA for measuring bispecific antibodies described below to measure antibody activity. In other words, A created in Reference Example 2-(1)
The culture supernatant of the test tetraoma was added to the M-H3A sensitized microplate, and after reacting at room temperature for 2 hours, it was washed with PBS-Tw.

Next, a chain-specific antibody was added to the biotin-labeled anti-mouse IgG, and after further reaction for 2 hours at room temperature, HRP-labeled avidin was added and washed, and the enzyme activity bound to the solid phase was determined according to Reference Example 2-(2). Measured using the method described.

Cloning by limiting dilution method was performed on the wells showing high hybrid antibody activity, and the desired bispecific antibody-producing mouse tetraoma ADTFI-157 was obtained. FIG. 3 shows the antibody dilution curve of ADTFI-157 culture soil serum.

(3) Purification of hybrid antibodies 5 x 106 mouse tetraomas/mouse AD in 6 BALB/C mice intraperitoneally administered with 0.5 m mineral oil in advance
TFI-157 was inoculated intraperitoneally.

Approximately 14 to 18 days later, an accumulation of ascites was observed, which was collected and ammonium sulfate was added to a final concentration of 1.3M. This antibody solution was prepared in advance at 0.1M PH (
pH 7,0) - Ether Toyo/X1l-R650M [Tosoh
Co., Ltd.] column (5 x 8 cm) for adsorption. After thorough washing with equilibration buffer, 0.1 MPB (pH 7,0)-
1,3M ammonium sulfate solution 500m and 0.1M
The IgG antibody was eluted by applying a linear concentration gradient (flow rate 60 d/hour) to PB (pH 7,0) 500 m.

Next, the fractions showing bispecific antibody activity were collected and 10mM
Dialyzed against PB (pH 6,8). The obtained antibody solution was applied to a hydroxyapatite column (7.6X100+n+a) equilibrated with 10mM PB (pH 6.8).
) and applied a linear concentration gradient (flow rate 1 d/min) between 10 and 300 mM potassium phosphate buffer to
Antibodies were eluted.

The results obtained were as shown in FIG.

The main peak was a bispecific antibody and some monospecific antibodies were eluted. When a purified sample of the bispecific antibody obtained from this main peak was subjected to 5DS-polyacrylamide gel electrophoresis (under DTT reducing conditions), single bands were obtained for each of the H chain and L chain. Ta.

Purified bispecific antibody ADTF 1-1 obtained in (3)
57 (20 μg/l112) was added to four types of human tumor cell lines: 562 (leukemia cells), A431 (epitheloid carcinoma cells),
NUGC4 (gastric cancer cells) and AM-RC-6 (kidney cancer cells) 2 x 106 cells/jtI2 were added and reacted at 37°C for 1 hour. After washing cells by centrifugation, FI
Add TC-labeled rabbit and mouse IgG antibodies and incubate at 4°C.
Allowed time to react. After washing the cells again, suspend them in PBS and
Analyzed by AC3.

The results obtained were as shown in FIG.

As a result, the bispecific antibody ADTFI-157 was found to be 56
It was shown that it strongly bound to 2 and A431 cells, weakly to NUGC4 cells, but hardly bound to AM-RC-6.

(2) (1) Maleimidation of anti-ATC antibody Anti-ATC antibody ADH2-357 1 obtained in Example 1
After dissolving 0 mg in 5IIM acetate buffer (pH 5,0) 2-, 2 times the mole of N-(ε-maleimidocaproyloxy)
50 μl of dimethylformamide solution of succimide ester
g was added and reacted at 30°C for 20 minutes. Reaction mixture to 0
．． The binding reagent was removed by applying to a Sephadex G-25 column equilibrated with 1M'J acid buffer (pH 6.5).

(2) Sulfhydrylation of anti-hTfR antibody 610 mg of anti-hTfR antibody 22C obtained in Reference Example 3 was
After dissolving in 0.05M PBS (pH 7,3), 50 μg of a 2-fold molar methanol solution of SPDP was added.

After reacting at 30°C for 30 minutes, 0.1M DTT aqueous solution 5
After reduction by adding 0 μg, the mixture was applied to the Sephadex G-25 column described in (1) to remove excess reagent.

(3) Preparation of bispecific antibody Add (2
8 mg of sulfhydrylated anti-hT fR antibody prepared in
was slowly added while stirring under water cooling to allow post-reaction. The reaction mixture was applied to a Sephacryl S-200 column,
As a result of separating and removing the unreacted antibody from the chemically bound bispecific antibody, approximately Tmg of anti-ATC-anti-hT fR bispecific hybrid antibody was obtained.

(4) Binding ability of bispecific antibodies (3) Example 2-(2)
The antibody was subjected to EIA as described in , and an antibody dilution curve was prepared.

The results were as shown in FIG.

Human epithelioid carcinoma cell A431 hybrid nude-7
Subcutaneously implanted into 15 mice (Jcl: AF-nu).
After 2 days, they were divided into 3 groups (5 mice per group) and treated with PBS (control) and TA.
N-1120 single agent (2 μg), and TAN-1120
(2 μg) and the purified bispecific antibody (0.5 mg) obtained in Example 2-(3) were administered intravenously and the progress was observed.

The results were as shown in FIG. TAN-1120
The single-drug administration group tended to slightly suppress cancer growth, but there was no significant difference from the control group.

However, in the group in which the same amount of TAN-1120 was administered as an immune complex with the bispecific antibody of the present invention at a 1:1 molar ratio, a remarkable cancer growth suppressive effect was observed.

[Brief explanation of drawings]

Figure 1 shows the anti-ATC antibody-producing mouse produced in Example 1.
The culture supernatant of hybridoma ADR2-357 was mixed with TA
It is an antibody dilution curve obtained by subjecting it to EIA described in Reference Example 2 in the presence (◯) and absence (·) of N-1120 (final concentration 5 μg/〆). Figure 2 shows the antibody ADH2 obtained in Example 1-(4).
-357 TAN -1120 (250pg/j!I2
) is shown. As target cells, mouse leukemia cell P388 (○) and human epithelioid carcinoma cell A
431(·) was used. Figure 3 shows a hybrid obtained by subjecting the culture supernatant of the bispecific antibody-producing mouse tetraoma ADTFI-157 prepared in Example 2-(2) to EIA as described in Example 2-(2). Antibody dilution curve. Figure 4 shows the bispecific antibody ADTF1- obtained using the ether-Toyovar column described in Example 2-(3).
Figure 2 shows the hydroxy annotite column chromatography pattern of the 157-containing fraction. Figure 5 shows that the bispecific antibody ADTFI-157 purified in Example 2-(3) was applied to four human tumor cell lines, K56
2. A431. After adding to NUGC4 and AM-RC-6 and reacting at 37°C for 1 hour, FITC-labeled second
Results of antibody binding and analysis by FAC3 (dashed line: antibody ADTFI-157 added, solid line: antibody ADTFI-1
57 (no additives). FIG. 6 is a hybrid antibody dilution curve obtained by subjecting the bispecific antibody produced in Example 3-(3) to EIA as described in Example 2-(2). FIG. 7 shows the results of observing the targeting effect of the bispecific antibody produced in Example 2-(3) in nude mice transplanted with human epithelioid carcinoma cells. Control group (PBS administration:・) and human TAN
-TA compared to the 1120 single agent (2 μg) administration group (○)
N-1120 (2 μg)/bispecific antibody (0,5++
+g) Significant inhibition of cancer growth was observed in the complex administration group (mu). Agent Patent attorney Iwa 1) Hiroshi (and 4 others) Figure 1 22 21 2 @ Antibody dilution factor Antibody concentration (μg/m7) Figure 3 Antibody dilution factor 17, 5 35.0 52.5
Elution volume (Cml) Figure 6 Antibody concentration (ng/ml) %f Tip strength

Claims (5)

[Claims] (1) i) Binding affinity for both the compound represented by the formula ▲ mathematical formula, chemical formula, table, etc. ▼ [in the formula, X represents a hydroxyl group or a hydrogen atom] or a salt thereof, and ii) the target antigen A hybrid monoclonal antibody with (2) The hybrid monoclonal antibody according to claim 1, wherein the target antigen is a tumor-associated antigen. (3) The hybrid monoclonal antibody according to claim 2, wherein the tumor-associated antigen is human transferrin receptor. (4) The hybrid monoclonal antibody according to claim 1 is bound to a compound represented by the formula ▲a mathematical formula, a chemical formula, a table, etc.▼ [wherein, X represents a hydroxyl group or a hydrogen atom] or a salt thereof. immune complex. (5) A polydoma producing the hybrid monoclonal antibody according to claim 1.