JPH0246297A - Production of monoclonal antibody, antibody-producing hybridoma and antibody - Google Patents

Abstract

PURPOSE:To produce a versatile antibody specific to T cell-type tumor by producing a monoclonal anti-human T cell receptor idiotype antibody using a specific hybridoma. CONSTITUTION:An antibody-producing cell collected from the spleen of a mouse immunized with a human T cell-type leukemia cell strain is fused with a mouse myeloma cell to obtain an antibody-producing hybridoma. The obtained hybridoma is selectively grown on a HAT-containing medium and screened to obtain a hybridoma capable of producing human T cell receptor idiotype monoclonal antibody (anti-hTcR-MoAb). The hydridoma is cultured in a liquid medium or in the abdominal cavity of a thymectomized nude mouse. The antibody-containing liquid is centrifuged and the MoAb is separated from the supernatant liquid and purified. The obtained MoAb is suitable as a biopharmaceutical which can be administered to human as a remedy or missile therapeutic agent.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a monoclonal antibody (hereinafter abbreviated as MoAb) that has the ability to bind to human T cell receptor idiotype (hereinafter sometimes abbreviated as hTcR-Id). The present invention relates to a hybridoma producing the antibody and a method for producing the antibody. The MOAb of the present invention is useful as a therapeutic agent for T-cell tumors to which chemotherapy is not effective.

Problems to be Solved by Conventional Technologies and Inventions Many chemotherapeutic agents are used as anticancer drugs, but these may react with normal tissues and cells in addition to cancer cells and cause serious side effects. In recent years, in order to compensate for this drawback, layer-selective methods have been developed that eliminate only cancer cells by binding chemotherapy agents or toxins to cancer cell-specific antibodies or other ligands. Drugs, so-called missile therapy agents, have been created and are being applied clinically.

Especially Köhler and Milstein [Natur]
e), 256, 495 (1975)], advances in MoAb production technology have enabled the development of excellent anti-cancer antibodies, and these M
oAbs have come to clinical application.

Recently, the idea of idiotype antibodies has been introduced,
For example, the idiotype (Id) of immunoglobulin on the tumor cell membrane in B-cell lymphoma is regarded as a type of tumor-specific antigen, and anti-Id-MoAb is applied in actual treatment [New England Journal of Medicine ( New Engl.

J. Med.), 306. 517 (1982)
]. Similarly, hTcR-1d, which is expressed in T-cell tumors such as T-cell lymphoma, can also be considered as a type of ll1rIK antigen, and it is expected that these anti-TcR-1d-MoAbs will be applied to therapy. However, although these anti-TcR-Id-MoAbs have excellent tumor specificity, they have to be prepared for each individual cancer patient, making them less versatile, and due to their diversity, they may vary depending on the case. In some cases, it may be difficult to produce antibodies and the time for treatment may be lost.

Means to solve the deep type Based on this technical background, the present inventors have conducted intensive research with the aim of producing antibodies that are specific to T-cell tumors and have versatility in tumor treatment. As a result, by fusing murine murine cells obtained by immunization with a human T-cell leukemia cell line and murine myeloma cells, the hTcR-1d structure expressed in T-cell tumors such as T-cell lymphoma was detected. MoAb that recognizes the relatively public part of
The present invention was completed as a result of obtaining a hybridoma that stably produces and conducting further research on the hybridoma.

Specifically, the present invention provides: (1) a monoclonal anti-human T cell receptor idiotype antibody having the following properties;

■Positive for at least two types of human T-cell leukemia cell lines.

■Exhibits cross-reactivity with approximately 2-6% of normal human peripheral blood lymphocytes.

(2) A hybridoma producing the above antibody obtained by immunizing an animal with a human T-cell leukemia cell line and produced by fusion with a myeloma cell.

(3) A method for producing the antibody described in (1) above, which comprises culturing a hybridoma producing the antibody in a liquid medium to obtain the antibody, and (4) producing the antibody described in (1) above. This method of producing the antibody is characterized by transplanting the producing hybridoma into a tissue-compatible animal or a thymectomized nude mouse, and obtaining the antibody from the produced ascites.

Monoclonal anti-human T cell receptor idiotype antibody of the present invention (anti-hT cR-1d-MoAb)
is positive for at least two types of human T-cell leukemia cell lines, and about 2% of normal human peripheral blood lymphocytes.
6% cross-reactivity, for example, the mouse monoclonal anti-human T cell receptor idiotype antibody KT38 (hereinafter referred to as MoAb KT3 B) obtained in the Examples below.
It is sometimes abbreviated as. ) is a human T-cell leukemia cell line TALL-1 cell or JOr (T cell on KAT cell).
It is an IgG1 class anti-hTcR-1d antibody that reacts with cell receptors (TcR), but does not recognize the common region of TcR consisting of α and β chains, but recognizes its variable region. be. Furthermore, since the antibody binds to a relatively public part of the variable region, it can also be used for human T-cell leukemia cell lines (neoplastic T cells) different from the immunogen, or for about 2 to 30 minutes. It also reacts with 6% of normal human peripheral blood lymphocytes. For these reasons, the anti-hTcR-rd-MoAb of the present invention has both high specificity and relatively versatile properties, and has the potential to be applied to immunotherapy for T-cell tumors. In this case, in addition to the use of antibodies alone, substances with appropriate antitumor activity, such as methotrexate, daunomycin, anticancer chemotherapeutics such as vincristine or cisplatin, or ricin, abrin, diphtheria toxin, neocarzinostatin, etc. Toxin proteins such as, and even tumor necrosis factor.

When combined with cytokines such as lymphotoxin, interferon, and interleukin, it can be used as a so-called missile therapy agent, an anticancer agent that specifically eliminates only cancer cells.

In the present invention, antibody-producing cells derived from an animal immunized with a human T-cell leukemia cell line were fused with myeloma cells to create antibody-producing hybridomas, and mice are preferably used as such animals.

In addition, the parent myeloma cell line is hypoxanthine-aminobuterin-thymidine (HAT)-sensitive mouse myeloma cell X63-Ag8 [J, F, Kearn
ey et al.; Journal of Immunology (J.

Immunol, ), 123,154 g (197
9)], 5p210 [M, Shulman et al.: Naci+-(Nature).

276.269 (1978)] and the like are preferably used.

The above antibody-producing cells are human T-cell leukemia cell line TA
LL-1 cells [J, Kal) pler et al.: Cells (C
ell), 35, 295 (1983)] or JU
RKAT cells [S, Carrel et al.: European Journal of Immunology (Eur, J.

lmll1uno1. ), 16,649 (1986
)] may be obtained from any animal species immunized with
In particular, it is preferably obtained from mouse pancreas. Next, these antibody-producing cells are fused with myeloma cells to create a hybridoma. Examples of fusion promoters include polyethylene glycol (hereinafter sometimes abbreviated as PEG),
Sendai virus etc. are used, and PEG is particularly preferably used. In the method using PEG, the degree of polymerization of PEG is generally about too-eooo, the incubation time is about 0.5-30 minutes, and the concentration of PEG is about 10-
80% or the like is used. Preferably about 35-55%
using PEG4000 or 6000 at about 37℃
It is best to treat the cells for 1-10 minutes.

The generated hybridomas are selected and grown in a medium supplemented with IAT, and antibodies in the culture are assayed by, for example, radioimmunoassay, enzyme immunoassay, immunofluorescence, immunoadhesion method, or mixed blood cell adhesion method. be able to. In addition, as a secondary screening method, for example, target cell T
Anti-hTcR-1d-
MoAb-producing hybridomas can also be selected.

Hybridomas positive for antibody activity are immediately subjected to cloning, which is usually done using the limiting dilution method [Experimental Cell Research (Exp', Ce11Res, )].
, I 21,309 (197B)]. Regarding the culture supernatant of the cloned hybridoma, antibody activity can be measured using the method described above, hybridomas that stably produce antibodies with high titer can be selected, and the desired monoclonal hybridoma can be obtained. .

Anti-hTcR-1d-MoAb is produced and accumulated by culturing the hybridoma of the present invention in a liquid medium or intraperitoneally in tissue-compatible animals or thymectomized nude mice. List. As a liquid medium, for example, a basal medium for animal cell culture [Iskotsu and Ham's F12 equidistant combination medium (■/H medium), RPMI-1
640 medium, etc.] with fetal bovine serum (hereinafter sometimes abbreviated as F'C9), etc., or GIT
Examples include culture medium (Wako Pure Chemical Industries, Ltd.) (see JP-A-60-145088). Cultivation is typically carried out for 3-60 days, preferably about 5-10 days, at about 30-38°C, preferably about 37°C. For intraperitoneal implantation in animals (mouses), approximately 5 x l'0'2xl per animal.
A sufficient amount of antibody-containing ascites fluid 2-2-10 can be obtained by intraperitoneally injecting O', preferably about 2-5 x 10'' antibody-producing hybridomas and raising them for about 10-40 days.

Antibodies in a liquid medium or ascites fluid can be purified using a combination of known biochemical techniques. For example, after centrifuging the antibody-containing solution, the supernatant is salted out (usually using ammonium sulfate or sodium sulfate), the resulting protein precipitate is dissolved in an appropriate buffer solution, and then dialysis is performed, followed by column chromatography ( (ion exchange column, gel filtration column, etc.) to separate and purify the antibody of interest. By the above separation and purification operation, for example, a protein weight ratio of 9 is obtained from a 500-
Approximately 5-30 mg of MoAb with purity greater than 0% can be obtained. In addition, similar antibodies were detected from 10 samples of ascites fluid.
0mg obtained. These purified antibody preparations contain about 0.1% or less of foreign proteins such as bovine or mouse-derived serum albumin, immunoglobulins, or other impure proteins, making them convenient for administration to humans as pharmaceuticals. used.

The MoAb obtained as described above is treated with a proteolytic enzyme (pahain, hepsin, etc.) and a reducing agent, etc., thereby retaining its binding ability to hTcR-1d.
Fragments of ab, F ab' and F (ab') can be obtained and used for the same purpose as the MoAb of the present invention.

Anti-hT cR-1d-MoA produced according to the present invention
b specifically recognizes the variable region of the tumor T cell receptor and binds to some tumor T cells but not to most normal cells. Moreover, because it is homogeneous and has a high titer,
It is highly suitable for use as a biologic. For example, after filtration and sterilization using a membrane filter,
It can be formulated as an injection or the like by itself or by mixing with appropriate pharmacologically acceptable carriers, excipients, diluents, etc.

The MoAb preparation obtained as described above can be administered to humans as a therapeutic agent for T-cell tumors, and can be used not only as an antitumor effect of the antibody itself but also as a carrier for appropriate anticancer drugs, that is, as a missile therapy agent for cancer cancer. It is expected that the drug will be administered to patients to completely cure cancer.

Furthermore, monoclonal antibodies of the present invention (e.g., MoAb
KT38) can be used, for example, as a detection reagent for human T-cell leukemia cell lines (tumor T cells) TALL-1 cells and JURKAT cells; in the case of q, MoAbKT
By pre-labeling 38 with a fluorescent substance (e.g., fluorescein isothiocyanate) or an enzyme (e.g., horseradish peroxidase), tumorigenic T.
Cells can be identified. It is also possible to indirectly detect cells using a fluorescent or enzyme-labeled anti-mouse IgG antibody.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference examples and examples, but the present invention is not limited thereto.

The mouse-mouse hybridoma KT38 disclosed in Example 1 has been deposited with the Fermentation Research Institute (IFO) since July 21, 1985 under the number IFO50170.
Also, since August 4, 1988, the deposit number FERM BP-199 has been deposited with the Microbial Research Institute (FRI) of the Ministry of International Trade and Industry.
It has been deposited as No. 3.

Reference Example 1 Indirect Fluorescent Antibody Method A test antibody solution containing a hybridoma culture supernatant was added to about 5×10' target cells and allowed to react at 4° C. for 30 minutes.

After washing twice with phosphate saline buffer (hereinafter sometimes abbreviated as PBS), FITC-labeled rabbit anti-mouse immunoglobulin (DAKO, Denmark) was added as a secondary antibody and reacted at 4°C for 30 minutes. . After further washing twice with PBS, fluorescence microscopy or FACS (rluo
Rescein-activated cell
The presence or absence of reactivity was determined using 5-orter).

Reference Example 2 Co-Modulation Experimental 5 x 10' TALL-1 cells or JU/RKA
10% F'C9-rLPMII640 medium containing test antibody solution (hybridoma culture supernatant, etc.) for T cells 40
0 μQ was added and cultured at about 37° C. for 20 hours. PBS
After washing three times with , anti-CD3 antibody was added and incubated at 4°C for 30 minutes, and then FITC-labeled rabbit anti-mouse immunoglobulin was added as a secondary antibody. After reacting at 4°C for 30 minutes, the plate was washed again and the development of CD3 antigen was analyzed by flow cytometry.

Example 1 Establishment of anti-hTcR-1d producing hybridoma! RPMI 1640 medium containing 0% FC9 (GIB
Human T-cell leukemia cell line TAL maintained in culture at 37°C, 5% GO, 8°C in
Approximately 07 L-1 cells were collected in 7-week-old female BAL13/
c Mice were intraperitoneally immunized four times at one-week intervals, the pancreas was removed 3 days after the final immunization, and pancreatic cells and mouse myeloma cells X63-Ag3 were fused according to a conventional method [Journal of National・Cancer Institute (J, Natl, CancerInst, 6
6,489 (1981)].

The fused cells were selectively cultured in HAT medium, and the wells in which proliferation was observed were used as reference examples! Screening was performed using the indirect fluorescent antibody method described in . In other words, the culture supernatant of each well was added to target cells TALL-1, and after staining with a fluorescently labeled antibody, judgment was made using a fluorescence microscope. As a result, positive reactions were observed in 42 types of hybridoma culture supernatants.

Next, as a secondary screening, the co
-modulation experiment was carried out and TALL-1
Hybridomas were selected that reduced CD3 antigen on cells. As a result, 191 hybridi species of 42 species that were positive in the primary screening showed positive results, which were subjected to cloning and the mouse-mouse hybridoma KT of the present invention was tested.
I got 38.

The obtained mouse-mouse hybridoma KT38 was
LB/c mice were grown intraperitoneally, and the ascites was reduced to 50%.
After salting out ammonium sulfate, DE-52 (Whatman
, USA) column chromatography to separate and purify the IgG antibody fraction. The obtained mouse monoclonal anti-human T cell receptor idiotype antibody KT 38 (
MoAb KT 38) was prepared using the ophthalony method [0,0uc
hterlony, L, ^, N1Isson: Handbook 1 of Experimental Immunology (I
Landbook of Experimenta
Immunology), 2nd ed.
0xford: BlackvellSctenttf
4c Publications, chapter
r 19 (1973)] and determined to be IgG1.

Example 2 Reactivity with various cultured cells MoAb KT3 B described in Example I was reacted with various blood system cultured cell lines according to Reference Example 1 together with anti-CD3 antibody T3 and anti-TCR (α chain and β chain) antibody WT31. After reacting and labeling with FITC, it was analyzed by flow cytometry.

The results were as shown in Table 1.

MoAb KT3 B of the present invention is an immunogen TALL
In addition to -1 cells, it also reacted with JURKAT cells, which are T cell lines, but did not react at all with other five types of T cell lines, four types of B cell mulberry lines, and two types of bone marrow cell lines.

(Hereafter, extra white) Table 1 Monoclonal antibody T3 (anti-CD3 antibody).

Reactivity of WT31 (anti-α/β TcR antibody) and KT38 (anti-α/β TcR-[d antibody) to human hematological tumor cell lines Cell line Monoclonal antibody T
3 WT31 KT38 Tcells TALL-1+ +
+JURKAT + + +
11PB-ALL + +PEER+ CCRF -CEM 0LT4 p12- Ichikava B calls RPMl 1788 aj 1 Daudi・BALL-1 Example 3 Reactivity with normal human peripheral blood lymphocytes Mononuclear cells from heparinized peripheral blood of 12 healthy individuals Separate the cells and use monoclonal antibody K according to the method described in Reference Example ① below.
Reacted with T38, T3 and WT31, FITCfi
The positive rate was measured by post-diagnosis flow cytometry. The results were as shown in Table 2.

Anti-CD3 antibody T3 and anti-α/β・TcR antibody WT31
MoAb KT38, which is different from
Approximately 1.9-6.1%) were positive.

(Hereafter, extra white) Myeloid calls 1L60 Table 2 Monoclonal antibody T3 (anti-CD3 antibody).

WT31 (anti-α/β TcR antibody) and KT38 (anti-α/β TcR-1 antibody) reactive lymphocyte monoclonal antibodies against human peripheral blood-derived lymphocytes 1M, P.

Y, F.

T.E.

S.G.

S.O.

A, S.

T, T.

A.J.

A, 0° Y,M.

M,N.

60.0 71.8 87.4 80.6 95.9 76.3 74.9 76.3 T2.0 75.1 72.7 58.2 69.4 84.3 77.3 93.2 74. 1 72.0 73.9 68.7 F 1.9 68.5 "Positive rate (%) Example 4 MoAb KT approximately 5x by radioimmunoprecipitation method
1 mci Na''J (Aa+e
rsham, UK) and 100 μg of lactoperoxidase (Calbiochem, USA), then 0.03% hydrogen peroxide was added at 10μρ for 3 minutes at 5 min intervals.
The cells were washed after each addition. 1″I drifting cells are 0.5%
Nonidet P40.5mM EDTA.

1o+M Phenylmethylsulfonyl fluoride.

20mM iodoacetamide, 0.14M NaCl
and 50mM containing ID/m12 pepstatin A)
Cell membranes were dissolved in Lis-HCl buffer (pH 7.3) on water for 30 minutes, and then centrifuged at 27,000xg for 30 minutes. A test antibody solution was added to the supernatant, incubated overnight, and rabbit anti-mouse immunoglobulin binding^fffig
Add el 10 beads (Biorad, USA) 4
Time ink evolved. After thoroughly washing the beads, add Laemmli's sample lysis buffer [Nature,
227.680 (1970)] and dissolve in 5D.
It was subjected to S-PAGE. Weber and Osborn's method [Journal of Biological Chemistry (
J. Biol. Chell.) 244.

4406 (1969)], and after the electrophoresis was completed, the gel was dried, and the XAR-5 film (
(Kodak, USA) at -80° C. and the chromatograms were analyzed. The results were as shown in Figure 1.

When JtJRKAT cells were used as antigen, anti-CD3
In antibody Leu4, a Dalton band was observed at molecular weight 20-26 under reduction, but MoAb KT3 of the present invention
In No. 8, two Dalton bands were observed at about 40 and 50 under reduction, and one Dalton band at about 90 under non-reduction. Almost similar results were obtained in the case of TALL-1 cells.

However, in the case of peripheral blood mononuclear cells, a clear Dalton band was observed at approximately 20-26 under reduction with Leu4 antibody;
No band was observed with MoAb KT38 of the present invention.

[Brief explanation of the drawing]

Figure 1 is 11! Three types of labeled cells, JURKAT (A
), TALL-1 (Figure 8) and human peripheral blood mononuclear cells (
The membrane-solubilized components of the test antibody [Mslgl: non-specific mouse IgGI, Leu4: anti-CD3 antibody (IgGl
), KT38: anti-α/βTcR-1d antibody (1gG1)
], and the obtained immunoprecipitate was subjected to 5DS-PAGE
This shows the results of the analysis. In figure A, 12.5% 5OS-PAGE, 13°0
In the figure, 1005% 5DS-PAGE was used. For JUrtKAT cells, mercaptoethanol (
The analysis was performed both in the presence (+) and absence (-) of ME). Agent Patent Attorney Iwa 1) Hiroshi 31 - 22 - 22 - 31 -

Claims (1)

[Scope of Claims] (1) A monoclonal anti-human T cell receptor idiotype antibody having the following properties. [1] Positive for at least two or more types of human T-cell leukemia cell lines. [2] Shows cross-reactivity with approximately 2-6% of normal human peripheral blood lymphocytes. (2) Mouse monoclonal anti-human T with the following properties
The antibody according to claim 1, which is a cell receptor idiotype antibody KT38. [1] Ig class and subclass: IgG1 [2] Recognition antigen: variable region of T cell receptor formed by α chain and β chain [3] Reactivity with tumor cells: TALL of human T cell leukemia cell line -1 cells and JURKAT cells [4] Reactivity with normal human peripheral blood lymphocytes: approximately 2-6%
shows cross-reactivity. (3) A hybridoma producing the antibody, which is created by fusion of a cell producing the antibody according to claim 1 obtained by immunizing an animal with a human T-cell leukemia cell line, and a myeloma cell. (4) Human T-cell leukemia cell line is TALL
4. The hybridoma according to claim 3, which is a -1 cell. (5) Myeloma cells contain hypoxanthine, aminobuterin,
The hybridoma according to claim 3, which is a thymidine-sensitive mouse myeloma cell X63-Ag8. (6) The antibody according to claim 2, which is produced by fusion of mouse myeloma cells X63-Ag8 with spleen cells that produce the antibody according to claim 2 obtained by immunizing a mouse with TALL-1 cells. The hybridoma according to claim 3, which is the mouse-mouse hybridoma KT38. (7) A method for producing the antibody according to claim 1, which comprises culturing a hybridoma that produces the antibody in a liquid medium to obtain the antibody. (8) The production method according to claim 7, wherein the hybridoma is the hybridoma according to claim 6. (9) A method for producing the antibody according to claim 1, which comprises transplanting a hybridoma producing the antibody into a tissue-compatible animal or a thymectomized nude mouse, and obtaining the antibody from the ascites produced. (10) The hybridoma according to claim 6 is used as BALB/c
10. The method according to claim 9, which comprises intraperitoneally implanting the mouse.