JPS6011889B2 - Passageable lymphokine-producing human T cell fusion line and method for obtaining the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a fusion line of human T cells capable of producing lymphokine and a method for obtaining the same. The present invention relates to a passable lymphokine-producing human T cell fusion line induced by fusing a leukemia cell line with mitogen- or antigen-stimulated human T cells in the presence of a fusion promoter, and a method for obtaining the same.

The phenomenon of cell fusion was discovered by Okada in 1958 using Sendavirus [HVJ], resulting in a dramatic development in the field of somatic cell genetics.

[Y. 0ka ship, BikenJ. , 1 103 (195
8)] In 1975, K6hier and Milstein successfully applied this cell fusion technology to the field of immunology. In other words, by fusion of lumbar cells obtained from immunized mice with HAT (hypoxanthine, aminopterin, thymidine) sensitive mouse myeloma cells, monoclonal antigen-specific antibodies are produced permanently. This was the first proof that cell fusion strains could be obtained. [
GK6hie andC. [Mistein, Natcho E 256 495 (1975)] Lymphocytes included in the immune system of humans and animals are broadly classified into cells derived from the thymus (T cells) and cells derived from the bone marrow (B cells). B cells are cells that secrete antibodies, and the cell fusion of Kohler et al. was performed between mouse-derived B cells and HAT-sensitive mouse myeloma cells.

On the other hand, T cells are stem cells in the bone marrow.
differentiates and matures within the thymus and develops into peripheral organs (e.g. lymph nodes,
wisteria viscera) and circulates in the dish stream. T cells play an important role in regulating responses in living organisms. It is well known that this regulatory control function of T cells is promoted by soluble mediators, collectively called lymphokines, released by T cells.

〔Day. ○． Kunkel and F. J. Dixon
,Advances mlmmunology29 5
6 (1980) Published by Academic Press] Conventionally, attempts have been made to treat various diseases such as cancer, allergies, and infectious diseases by controlling the body's immune response. Lymphokines are medically extremely important substances, as they can be applied as more effective immunotherapeutic agents and are expected to be widely applied in the medical field as clinical diagnostic agents.

However, with conventional methods, lymphokines cannot be obtained in sufficient quantities, and their low purity is a major impediment to their application in the medical field. Lymphokines are a group of non-antibody protein factors produced by lymphocytes in response to antigen-specific stimulation, mitogen stimulation, etc., and among lymphocytes, they are mainly produced by T cells. The main lymphocytes and their functions are shown below. '11 Lymphokine that acts on macrophage■ Migrationinh
ibito factor (hereinafter abbreviated as MIF).

Macrophage migration inhibitory factor) Action: Prevents macrophage migration in vitro.

■ Macropha group activatingfac
tor (hereinafter abbreviated as MAF).

Macrophage activator) Action: Promotes phagocytosis, bactericidal action, etc. of macrophages. ■ Monoc Macrophage Chemotact (MCF, monocyte-macrophage chemotactic factor)
Action: Chemotaxis of monocytes and macrophages in vitro (
chemone×is).

'21 Polymorphonuclear leukocytes
Lymphokine that acts on realle (this ocy)■
Leukocyte (migration) inhibitory factor (leucocyte-Mig)
rationi oil ibitoryfactor. LIF
) Action: Prevents migration of polymorphonuclear leukocytes in vitro.

■ Chemotactic factor
Action: induces chemotaxis of neutrophils, eosinophils, and basophils in vitro.

Tsuyoshi Lymphokines that act on lymphocytes■ Interleukinro (Interleukin0,
IL-□) Action: Promotes division and proliferation of T cells stimulated by antigen or mitogen.

'41 Lymphokine that acts on other cells■ Lymphotoxin (LT) action: j
Injury and detach L cells and Hera cells with nvMo.

■ y-interface (heron, m to y-1n)
Fy) Action: Interfering with virus pathogenesis.

■ Colony Stimulating vector (
CSF) Action: Acts on bone marrow leukocyte progenitor cells (CPU-C) to promote differentiation and proliferation of these cells into forehead granulocytes or macrophages.

The activity of the above-mentioned lymphokines is measured in vitro (in vitro), but lymphokines that are found to correspond to in vivo (in vivo) activities have also been reported.

For example, in the tuberculin reaction, chemical
In addition to vasodilatory edema due to media r, accumulation of numerous macrophages is observed. This is a macrophage migrated and assembled by sensitized T cell-derived chemotactic factor (MCF), which is further immobilized by MIF.
It has been shown that MIF is involved in biological defense that efficiently accumulates and activates macrophages and effectively promotes the disposal of foreign substances. In addition to MCF and MIF, lymphokines that are expected to be used as medicines in the future include MAP and lymphotoxin.
Examples include interleukinro, WF-y, and CSF.
As a method for producing these lymphokines, '11 antigen-sensitized peripheral blood lymphocytes are cultured together with the antigen.

[21. Method for culturing peripheral blood LJ lymphocytes or Fuji viscera cells with mitogen.

A method of establishing and culturing antigen-specific T cell clones using t31 T cell growth factor (IL-0).

Q), Method 2' requires a large amount of blood and the amount of lymphokine produced is extremely small, making it difficult to obtain large quantities of highly purified products. In the method of '3-, specific lymphokines can be produced in the presence of IL-1D, but the productivity from T cells is poor, and it is difficult (and expensive) to obtain human antibodies. It has problems such as being These are caused by the fact that lymphokine-producing T cells cannot be passaged, have low proliferative properties, and have low proliferative properties even when cultured in the presence of growth factors such as IL-0.

For the above reasons, it is currently extremely difficult to secure the amount of IJ protein necessary for clinical application. Cell fusion technology is used as one way to solve these problems, and T cell fusion lines have already been established in mice, making it possible to analyze the lymphokines produced by these cells.

However, lymphokines produced by mouse T cell fusion lines cannot be clinically applied to humans. Due to the necessity of human immunology and clinical applications, it is expected to establish a fusion line of lymphokine-producing human T cells that can be passaged. It is highly conceivable that human T cell fusion may be carried out in the same manner as mouse T cell fusion.

namely, lymphokine-producing T cells (which cannot be passaged) and HATs (hypoxanthine, aminopterin, thymidine).
T cell fusion involves fusion of sensitive T-lineage tumor cells (which can be passaged) in the presence of a fusion promoter, and then selecting only cells that can proliferate in HAT cells, which are then cloned to produce the target lymphokine. This is a method of selecting stocks.

[○． Cathenne et al. “Natme” No. 292 Reed,
p. 842, 1981] However, H
Providing AT sensitivity requires extremely complicated operations and is difficult to establish. The present inventors investigated various methods for obtaining a passable, lymphokine-producing human T cell fusion line, and found that human T cells treated with a protein synthesis inhibitor or a protein synthesis inhibitor and an RNA synthesis inhibitor By fusing an acute leukemia cell line with human T cells stimulated with mitogen or antigen in the presence of a fusion promoter, we succeeded in obtaining a ``passageable lymphokine-producing human T cell fusion line,'' and the present invention I was able to complete it.

The invention generally consists of the following steps.

A. After stimulating lymphocytes isolated from human peripheral blood and the cornea or thymus aseptically removed by surgery with mitogen or antigen, the mitogen or antigen bound to the cells is removed as much as possible.

The antigen or mitogen used may be any substance that induces transformation of human T cells, and is appropriately selected and used depending on the target lymphokine.

As a mitogen, phytohemagglutinin-P (
(abbreviated as PHA-P), connacabalin A (ConA), and the like. B. After treating human acute leukemia cells with an inhibitor of protein synthesis or both an inhibitor of RNA synthesis, the inhibitor in the culture medium is removed by centrifugation.

As human acute leukemia cell lines, T-cell tumor cells such as CEM, TALL, and MOLT-14 strains can be used.

Known eukaryotic protein synthesis inhibitors are used as protein synthesis inhibitors, including emetine hydrochloride,
Irreversible protein synthesis inhibitors such as pactamycin are preferred.

In addition to using only these protein synthesis inhibitors, it is desirable to use RNA synthesis inhibitors in combination. Actinomycin D is preferred as an inhibitor of RNA synthesis, but other examples include Q-amantine, ethidium bromide, rifampicin, and adriamycin.

Conditions for treatment with these inhibitors are selected from conditions that can completely prevent division and proliferation of human acute leukemia cell lines. For example, when CEM (2×1 pi)/secretion is treated with emetine hydrochloride alone, the concentration is 10-4 to 10-5M, and when actinomycin D is used together, the concentration is 10-4 to 10-5M.
10-5M, actinomycin DO. A range of 0.05 to 0.2 meta/iso (processing temperature: 37°C, processing time: 2 hours) is desirable. C. A human acute leukemia cell line whose proliferation has been stopped by adjusting as above or by combining lymphokine-producing human T cells with a protein synthesis inhibitor or a protein synthesis inhibitor and an RNA synthesis inhibitor is treated with an appropriate fusion promoter. merge in the presence of

Mixing ratio of human T cells and human acute leukemia cell line is 1:1
The range is from 2:1 to 15:1, preferably from 2:1 to 15:1.

As a fusion promoter, polyethylene glycol (hereinafter referred to as P
(abbreviated as EG), polyvinyl alcohol, viruses with cell fusion complexes, especially paramyxovlms to which Sendai virus (HVJ) belongs and its activated products, but generally PEG (molecular weight 1000-4
000) is used. D The live cell concentration of the fused cell line is 1
The cells were cultured in 96-well culture plates containing nutrient medium supplemented with feeder layers. Human cells whose growth has been stopped by antibiotics or X-ray irradiation are used as the feeder layer.

Any medium that can proliferate human acute leukemia cell lines can be used as the nutrient medium.

For example, RPMI-1640 with 10 ml of fetal calf serum (FCS)
%, 2-mercaptoethanol, 5 x 10-5 mL, and a medium supplemented with 2 mM glutamine is preferably used. One week after culturing, the human acute leukemia cell line and feeder layer treated with these inhibitors were completely killed, and only the fused cells proliferated.

It should be noted that confirmation that the fusion strain was obtained was carried out by (1) analysis of cell surface antigen and (2) analysis of karyotype.

E. Analyzing the culture supernatant of the wells in which the fused cells grew,
Confirm target lymphokine production. The fused cell line obtained by the above method can be passaged for a long period of time while retaining lymphokine activity, and furthermore, by cloning, a subline that efficiently produces lymphokines can be obtained. By culturing the subline in vitro or in vivo, lymphokines can be produced.
For these culturing methods, known methods may be used. For example, in vitro culture methods include the fly azure culture method and the static pupil culture method in a Petri dish or Lou bottle. For the in vivo culture method, the fused cells are inoculated into nude mice, nude rats, or non-human animals to which human Koshito cells can be transplanted, solid tumors or ascites are formed, and after an appropriate growth period,
Examples include a method of collecting ascitic fluid and/or blood using a conventional method in the field, and a method of using hamsters with weakened immune responses as described in JP-A-54-98307.

The passageable lymphokine-producing human T cell fusion line is not only useful for mass production of lymphokines, but also enables the acquisition of large quantities of target lymphokine-producing cells.
) can also be used as an extraction source. DNA (c) is complementary to the extracted messenger RNA using reverse transcriptase.
After producing DNA), it is also possible to make lymphokines in microorganisms (bacteria, yeast, actinobacteria, molds) using conventional genetic recombination methods. Next, the present invention will be specifically described with reference to examples.

Example 1 ○' Preparation of lymphokine-producing human T cell fusion line Human peripheral blood lymphocytes (hereinafter abbreviated as PBL) (HLA-A2, -A
w24, 1B7, 1Bw35, 1Cw included -Cw7) 1
RPMI-164 evacuation site [10% fetal bovine serum, 5 x 10-5 M2-mercaptoethanol, 2 m
M glutamine-containing] (hereinafter abbreviated as RPMI medium), P
After stimulation with HA-P5 for 2 days, PHA bound to cells by N-acetylgalactosamine 0.1 M
- P was removed as much as possible.

On the other hand, human acute leukemia cell line CEM (HLA-
Emetine hydrochloride (105 M) was added to 2 x 1 cells/field of AI, -Aw30, -Aw30, -B8, -B40, and after treatment for 37,002 hours, emetine hydrochloride in the culture solution was removed by centrifugation.
After mixing the PBL and CEM prepared above at a ratio of 10:1, the cell pellet obtained by centrifugation was added with 0.5 to 46% polyethylene glycol (PEG-1540).
, 15% dimethyl sulfoxide, 5-yamata' poly-L
-Add arginine and centrifuge. After fusion, the number of living cells in the fused cells was adjusted to 2 × 1 cells, and the feeder layer was treated with mitomycin C (mitomycin C concentration 25%).
C25% in a 96-well culture plate containing CEM8
The cells were cultured at 37q0 in an atmosphere of 95% air.

(Unless otherwise specified, the culture conditions shown in the following examples are 37q0 in an atmosphere of 25% CO and 95% air.
) For one week after culturing, half of the culture medium was replaced every day to alleviate the effects of drugs released from dying CEM. Lymphokine activity was measured using the cells in the wells in which proliferation of the fused cells was observed by the method shown in "t2'.

During this period, the CEM treated with Etchimen and the CEM treated with mitomycin C, which were cultured as a control, were completely killed.

Culture of t2' fused cells Lymphotoxin activity The fused cells obtained by '1' were cultured in RPMI medium for 1 day in the presence of PHA-P20, and the lymphotoxin activity of the culture supernatant was determined by L-P3 ( As a result of measuring the target cells (sub-line of L cell), activity was observed in two fusion strains (B-10 and F-8).

This activity is maintained for more than 3 months. Lysophotoxin was measured according to the method of Kobayashi et al. by Manji Seikoku. That is,
L-P3, 5 formed in advance on a microplate
Add 25 samples of test substance and actinomycin D (4 meta′ above) to 0 samples (2 x 1 cells), add 37 samples.
After culturing between 00 and 20 to 2 capes, cells were fixed and stained with glutaraldehyde, and the number of morphologically normal cells was counted. [Y. Kobayashietal-,
J. lmmunology 122 791 (1979
)]t3- Characteristics of fusion strains E-10 and F-8 [1-
Analysis of cell surface antigens■ CEM is performed using a fluorescent cell sorter (FluorescenCe Activa).
It is known that the monoclonal antibody OKT3 does not react with the monoclonal antibody OKT3, based on the analysis conducted by Ill Soner. OKT3 reactivity to 1
pbi to two-s using 251-ProteinA
ndingassay and lmmunoHuoresc
I checked with ence.

As a result, while there was no reactivity with CEM, the reactivity of E-10 was found to be similar to that of PBL-T cells, and the reactivity of F-8 was found to be about 50%.

■ two-s using 1251-Protein A
HLA antigens of E-10 and F-18 were examined by tepbinding assay.

PBL is HLA-A2, -BW35pOS; tiVeC
While EM is HLA-AI, -B8 positive, both E-10 and F-8 are HLA-AI, -A2,
It was one B8 positive. ■ Karyotype analysis CEM, E-10, and F-8 color washings number of 50 to 8 each.
The metaphase nucleus of No. 0 was investigated.

As a result, CEM is 84.3 Sat 0.9 (average value S.E.
) [85] (median), whereas E-10
is 95. Sat 1.7 [94], F-8 91.5 ± 1.6
[93], and approximately 10 chromosome gains were observed. Example 2 Inhokine-producing cell fusion strain E-1 obtained in Example 1
The MIF activity of a 4-fold dilution of the culture obtained by culturing for 1 day in an RPMI column containing 1 pg of 0 was determined by Harrington's method [J. Thanington, Jr. et al., J.
．． lmmunologyll0 752, (1983)
], the macrophage migration inhibition rate was 29.9%, and no activity was observed in the culture supernatant of CEM cultured under the same conditions.

In addition, 07 PBLI / ConA (10A evening / desk)
The culture supernatant obtained by culturing for 1 day in HEPES-containing MEM medium showed a macrophage migration inhibition rate of 25.2%. The MIF activity of E-10 is maintained for more than 6 months. Example 3 Echimene hydrochloride 5x10-5M, actinomycin DO. 1 Muta' was added and treated at 37°C for 2 hours, which was fused with PBL prepared in the same manner as in Example 1, and cultured according to the method of Example 1. As a result, Emetine hydrochloride and actinomycin cultured as controls The D-treated CEM was completely killed, and only the fused cells were observed to proliferate.

Lymphotoxin activity was measured in the culture supernatant obtained by culturing this fusion strain in the same manner as in Example 1, '2). As a result, two fusion strains (C-5 and D-9) were found to have activity. Ta.

Example 4 Using mitomycin C as a feeder layer, the lymphokine-producing human T cell fusion strain E-10 obtained in Example 1 was used.
R containing treated (processing conditions are the same as in Example 1) CEM2×1 culture/secretion and 20% ConA bulk sexed PBL
The subline EIO-20 was obtained by cloning by limiting dilution method (0 cells - milk solids/well) in PMI medium.

Then, 1 piece of EIO-20 was added to the RP skin medium containing secretion.
The culture supernatant obtained by culturing for days was subjected to ammonium sulfate fractionation (50-100%
As a result of measuring the skin F activity of the sample obtained by dialyzing against P (saturated), the macrophage migration inhibition rate was 32%.
Met.

Furthermore, MAF activity was determined on a daily basis. W. The method of Mmray et al. [J. E
xp. Med. , 153 1690 (1981)].

That is, human macrophage-like cell lines U 937 and M
Intracellular superoxide anion (○ summer) generated by the reaction with AP was detected by color change of nitroflutetrazolium (cells turn blue due to the presence of ○ summer).
As a result, 30% of the total number of cells changed color and MAF activity was observed. The MIF activity was measured on samples obtained by culturing the CEM strain in the same manner and dialyzing the culture conditions against ammonium sulfate fraction (50-100% saturation) and P. 9.9%, and 21% of the total cells were discolored by nitroflutetrazolium. Example 5 The culture supernatant obtained by culturing the subline EIO-20 obtained in Example 4 for 1 day in RPMI medium containing 1 piece/table was subjected to ammonium sulfate fractionation (50 to 10% saturation) and to PBS. For samples obtained by dialysis, MAF activity was measured using the method of Cameron et al.
,D. J. andW. Day. Churchill; Jou
malofClinicalInvestigatio
n 63 977 (1979)] was modified.

[MAF activity using increased tumor cell damage as an indicator-M
AF-C- measurement method] PBL separated from human peripheral blood by the Fico-urographin method was adjusted to 2.5 x 1 PBL/secretion in RPMI-164 containing 20% human serum, and then Macrophages were added to a 96-well culture plate, incubated at 37°C for 0.5 to 2 hours, and washed three times with RPMI medium, and the macrophages were attached to the bottom of the plate.

Next, 200 ml of RPMI 164 Sekenchi containing 20% human serum was added to each well, and after washing for 1 to 7 days, 20
200 samples diluted 1,000,000 times in RPMI-164 containing % human serum were added and cultured for 4 to 2 hours to activate macrophages. The number of macrophages in one well was 2 to 10×1.

On the other hand, target Hititoyu cells (human leukemic T cell line TALL, human proerythroblast cell line K562, etc.) were cultured for 2 hours in thymidine-containing RPMI medium for 3 days to obtain labeled target tumor cells for 3 days. Ta.

Next, in wells containing 2 to 10 x 1 activated macrophages, the ratio (E/T) of the number of activated macrophages (E) to 3-day labeled tumor cells (T) is 5 to 10. 3-day labeled target tumor cells and RPMI medium were added and incubated at 37°C for 2 days.

The supernatant and cells were separated using a plate centrifuge, and the radioactivity (cpm) of the supernatant was measured using a liquid scintillation counter. The MAF-C activity value was calculated using the following formula. %.

fMAF-C: Kamigumo Kirimitsuken species≧word≧≧word presentation rhombus crab Kensho sex (Note 2) Control radioactivity: Radioactivity (cpm) of the supernatant obtained by centrifugation after culturing the RPMI tower containing one 3-day labeled target tumor cell at 3700 nm for 2 days. “Sample (
MAF-C activity (100 dilution) was 88.1%.

Claims (1)

[Scope of Claims] 1. A passageable lymphokine-producing human T cell fusion obtained by fusing a human acute leukemia cell line with a mitogen- or antigen-stimulated human T cell and isolating only the fused cells. KK. 2. The passageable lymphokine-producing human T cell fusion line according to claim 1, which uses a human acute leukemia cell line treated with a protein synthesis inhibitor or a combination of a protein synthesis inhibitor and an RNA synthesis inhibitor. 3 Human acute leukemia cell lines are treated with a protein synthesis inhibitor or a combination of a protein synthesis inhibitor and a PNA synthesis inhibitor, while human T cells are stimulated with mitogenes or antigens, and both are treated in the presence of a fusion promoter. After merging with
A method for obtaining a passable lymphokine-producing human T cell fusion line by isolating only fused cells.