JPS58146512A - Human c-virus (leukemia virus)-producing cell strain (mt-2) and method for establishing its cultivation - Google Patents

Abstract

PURPOSE:To provide the titled cell strain extremely useful for the diagnosis by antigen-antibody reaction, the prevention of diseases by the preparation of vaccine, and the remedy of diseases by the preparation of monoclonal antibody, by establishing the cultivation using a material derived from the adult patient of T-cell leukemia. CONSTITUTION:The leukemia cells are separated from the material derived from the adult patient of T-cell leukemia, and normal leucocytes are separated from the blood of healthy man. The leukemia cell and the leucocyte are mixed together and cultured to cause the proliferation of the cells. The objective human C-virus-producing cell strain (MT-2) is obtained by separating the cell strain derived from normal human lymphocyte, capable of producing the human C-virus in a large amount, containing the antigen (ATLA) reacting specifically with the serum of the adult patient of T-cell leukemia, and exhibiting almost completely positive reaction to ATLA. The strain can produce C-virus continuously, and the virus is expected to be applicable to the diagnosis, prevention and remedy of leukemia.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides C
The present invention relates to a type virus-producing cell line (MT-2) and a method for establishing a culture of this cell line.

Adult T-cell leukemia is a relatively new disease concept with epidemiological characteristics that occur frequently in the Western and Shikoku regions (high B
et al., Blood 50: 481.197? ) In clinically severe cases, adults are affected, and peripheral T cells exhibiting nuclear irregularities and division of labor appear, accompanied by enlarged lymph nodes and liver/splenic tumors.
In most cases, it is a disease that progresses subacutely, and because it often causes skin lesions, there is concern about whether it is the same as mycosis fungoides.

Recently, American researchers succeeded in establishing type C virus-producing cells from patients with mycosis fungoides (Pro
c, Nat, Acad+ 8ci, 77:7
415, 1980).

However, this disease is clinically different from adult T-cell leukemia, and the present inventors have been conducting research to establish type C virus-producing cell lines using a method different from the method used by them to establish cultured cells. Ta. As a result, the present inventors succeeded in establishing a cell line (MT-1) that continuously and abundantly produces type C virus using adult T-cell leukemia patient materials (Miyoshi et al.).

Nature 294: 770, 1981).

Therefore, the purpose of the present invention is to provide a human type C virus (leukemia virus) producing cell line (MT-2) that can continuously produce type C virus, and also to provide a method for establishing a culture of this cell line. The purpose is to provide. In order to control human leukemia, it is necessary to clarify its etiology, and by achieving the purpose of the present invention,
This virus can be used in the diagnosis, prevention, and treatment of leukemia in the future, and can also be applied to vaccines and monoclonal antibody products.

The type C virus-producing cell line of the present invention is obtained by a mixed culture of leukemia cells isolated from adult T-cell leukemia patient materials and leukocytes isolated from neonatal blood or normal human blood.

A cell line (M
T-2) is derived from ligament leukocytes. It was previously known that lymphocytes from ligament or normal individuals never become a proliferative system by themselves unless infected with EB virus. Therefore, by mixed culture of white blood cells and normal human leukocytes, FB virus nuclear antigen (EBNA) negative normal human
The reproducible establishment of a cell line derived from IJ bulbs can be said to be a breakthrough.

Hereinafter, the present invention will be explained in detail based on examples.

I) Method for establishing type C virus-producing cell line Separate leukemic cells from 10-20 ml of peripheral blood of an adult T-cell leukemia patient by Ficoll-Conray gravity centrifugation or dextran method. On the other hand, neonatal plaster blood or normal human peripheral blood 10
-20- Separate leukocytes by the same method as above. Both leukocytes are mixed and cultured at a concentration of about I x 10 /lt using a petri dish with a diameter of δ. In this case, the cells are mixed at the same time.

Alternatively, one may be added to the other after 1-2 weeks. As a culture solution, RPMI1
640 medium to which 10% fetal bovine serum, 10% human cord serum, and antibiotics are added is used. Thereafter, change the culture medium twice a week. Cell proliferation occurs after about 1-1 months of mixed culture. By this method, a δ cell line was established, and the 1st line (MT-1) was derived from leukemia cells (Miyoshi et al., Gann 71: 15 Is, 1980).
,.

The other two lines (MT-1, MT-8) were confirmed by chromosome analysis to be derived from normal human leukocytes (Miyoshi et al.
Gann 72: 978, 1981; Miyoshi et al.
Pathophysiology January 1957 issue).

h) Characteristics of type C virus producing cells MT-1, MT-2, and M'I'-8 all proliferate in suspension while forming cell clusters, and the doubling time of the cells is 4
0-60 hours. The cells are mainly composed of morphologically FC9C9 lymphoid cells, with multinucleated giant cells mixed therein. As shown in Table 1, all cell lines form rosettes with sheep red blood cells.
It is Leu-1 positive, Ia antigen positive, and gB virus nuclear antigen (EBNA) negative. Chromosome composition is MT
Only -1 has a complex abnormality involving 14q+ (Miyoshi et al., Cancer Genet, Cytogen
etc.

8:21S1, 1981), MT-2 and MT-3 are both 46, XY. These eight systems possess an antigen (ATLA) that specifically reacts with the serum of adult T-cell leukemia patients and continuously produce type C virus (Yanuma et al., P.
roc, Nat, Acad.

8ci, 78: 6476, 1981; Miyoshi et al., Nature 294: 770, 1981
). Among these, iT-2 produces a particularly large amount of type C virus particles, and almost 100% of the cells are ATLA positive.

Therefore, MT-2 is considered to be extremely useful for future research and clinical applications. As a result of recent biochemical analysis of type C virus particles produced from MT-2, it has become clear that this virus has the characteristics of a so-called retrovirus (Kichihi et al., Proc. Nat.
Acad, Sci, in press). In this way, more E rosettes were obtained from adult T-cell leukemia patients.
+Leu -1+ + +Leu-g
a --- teu-aa --- 0KIal + + +A
TLA + + 10 ATLV + + and RBN
A --- Xenotransplantation + + Basic and clinical applications of 111C virus-producing cells (
The biological activity of ATLV produced from MT-2 transformed normal human lymphocytes with IIATLV was investigated. First, we attempted to transform MT-2 cells into hydrin I spheres with cell-free p-fluid, but so far we have not been successful.

Next, when MT-2 cells were irradiated with a lethal dose of X-rays and cultured in a mixed culture with human lymphocytes, normal lymphocytes were transformed into approximately 100 ml of mitotic cells and began to continuously produce ATLM ( Miyoshi et al., Gann 72: 99
7, 1981).

Therefore, the biological activity of human TLV derived from MT-2 has been demonstrated, and we believe that by using such a mixed culture method, we will be able to freely establish human T cell lines with moderate functionality. This will bring about a great deal of knowledge in the fields of medicine and biology.

(2) Diagnostic significance of ATLA antibodies Taking advantage of the fact that almost 100% of MT-2 cells are ATLA positive, we investigated the presence or absence of antibodies against ATLA in the serum of various leukemia/lymphoma patients and control subjects. indirect fluorescent antibody method (Yanuma et al., Proc, Nat, Acad,
Bci, 78:6476.1981).

- As the next serum, test serum (10-fold dilution) was heated to 87°C for 80 min.
After incubation for 1 minute, use FITC-labeled anti-human 19 as secondary serum.
Goat serum G (20 times diluted) was reacted at 87° C. for 80 minutes, and observed using a Nikon epifluorescence microscope F. For antibody-positive cases, serum dilution and antibody titer measurements were also performed. As an antigen, a smear of MT-2 cells was fixed with acetone for a month, but since almost 100% of M'r-2 cells are ATLA positive, it is possible to very clearly determine the presence or absence of ATLA antibodies. did it. As shown in Table 2,
All 7 cases of adult T-cell leukemia and 2 cases of mycosis fungoides were ATL.
The patient was A-antibody positive.

Among other leukemia patients, 8 out of 88 were antibody dominant, but the reason for this is currently under investigation. on the other hand,
Fifteen lymphoma/bone marrow shield patients and 41 healthy control subjects were negative for gold-side antibodies. This result was confirmed by Yasunuma et al. (Proc, N
at.

Acad, 8ci, ) 8':6476.19
Similar to the results of 81), it has been shown that the search for ATL human antibodies is useful for the diagnosis of adult T-cell leukemia and related diseases, and its clinical significance is enormous.

Adult T-cell leukemia F 7. Mycosis 22 Other leukemias 888 Lymphoma/myelopathy 150 Healthy control subjects 410 (8) Medical and biological effects expected from MT-2 in the future (a) Vaccine using ATLV Fabrication of and AT based on it
Prevention of LV infection Although the route of A'l'LV infection is still unclear, this problem will be clarified in due course. In areas where the incidence of adult T-cell leukemia is high, prophylactic administration of a vaccine prepared using AT5/ produced from MT-2 is considered.

(b) Production MT of heterospecific antibodies against human'rLv
A'l'LV produced from -2 in monkeys.

Specific antibodies against ATL'V are generated by injecting them into goats, sheep, cows, horses, and rabbits.

(C) Preparation of monoclonal antibody against human 'I'LVK An antibody against A'I'LV produced from M'['-1it is produced using a hybridoma.

(bt t Using the antibody prepared as in (a), AT
It will be possible to detect L, A, and ATLV, and furthermore, it will be possible to treat adult T-cell leukemia with these antibodies.

((1) Cloning of human TLv gene M'I'
By using molecular biological techniques to isolate the genes that regulate carcinogenesis from ATI and V produced by ATI and V-1, and clarifying their nucleotide sequences, it is possible to conduct research on cancer control at the genetic level.

As described above, according to the MT-2 of the present invention, not only can it contribute to a wide range of basic medical and biological research, but it can also be used for diagnosis by antigen-antibody reactions, prevention by vaccine production, and treatment by monoclonal antibody production. , which has a very wide range of industrial applications.

Patent applicant: President of Kochi Medical University

Claims (1)

[Claims] L. Leukemia cells isolated from adult T-cell leukemia patient material and leukocytes isolated from normal human blood are mixed and cultured,
Among the cell lines obtained, they produce a large amount of human type C fils and possess an antigen (ATLA) that specifically reacts with the serum of adult T-cell leukemia patients, with almost 100% of the cells containing ATLA.
Human type C virus producing cell line (MT-2) derived from positive normal human lymphocytes. (ix) Leukemia cells are separated from adult Ta-positive leukemia patient materials, leukocytes are separated from normal human blood, and the leukemia cells and leukocytes are mixedly cultured to cause cell proliferation, and among the obtained cell lines, H)C The antigen (AT
A method for establishing a culture of a type C virus producing cell line (Mr-g), which consists of the steps of extracting a cell line derived from normal Hidori 77 cells, which possess LA) and almost 100% of the cells are ATLA positive.