JP3053513B2 - Hybridoma, monoclonal antibody produced by the hybridoma, and mouse lymphocyte activating antigen recognized by the monoclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a hybridoma,
The present invention relates to a monoclonal antibody produced by the hybridoma and a mouse lymphocyte activation antigen recognized by the monoclonal antibody. The monoclonal antibody and the activating antigen of the present invention are useful, for example, in the field of basic research on the immune mechanism of a living body, in the medical field of immune system diseases, and the like.

[0002]

[Prior art and its problems] Expression of immune response in living organisms
T cells that play a central role in regulation are known to differentiate from T progenitor cells to mature T cells in the thymus. T progenitor cells rapidly migrate and proliferate after migrating to the thymus, and at the same time, reconstitution of the T cell receptor (TCR) gene occurs, and the gene product is expressed on the cell surface . It is believed that the repertoire of antigens recognized by T cells is largely determined during this thymic differentiation process, since the TCR gene causes few somatic mutations.

[0003] This repertoire selection of T cells is based on CD4 ⁺ 8 ⁺ (doublepositive; hereinafter referred to as DP) in immature thymocytes.
It is known to occur at the cellular stage.
That is, DP cells recognize the major histocompatibility complex of stromal cells via the TCR molecule, and are positively selected or negatively removed. Thus, DP cells are a heterogeneous cell population, but there are few markers that further finely and functionally fractionate this population.

In recent years, it has become clear that an activating antigen expressed when lymphocytes such as CD69 are activated can be an important marker for functionally fractionating DP cells. However, means for identifying such an activation antigen, and separating and extracting such an activation antigen have not been known so far, and the characteristics of the lymphocyte activation antigen itself have not been elucidated at present.

The present invention has been made in view of the above circumstances, and has as its object to provide a monoclonal antibody that specifically recognizes a mouse lymphocyte activating antigen and a hybridoma that produces this antibody. And Another object of the present invention is to make the lymphocyte activating antigen recognized by the above-mentioned monoclonal antibody useful for industrial use by clarifying its characteristics.

[0006]

SUMMARY OF THE INVENTION The present invention provides a hybridoma derived from a fusion cell of hamster spleen cells and mouse myeloma cells immunized with immature mouse thymocytes. I do. The present invention also provides a monoclonal antibody produced by the hybridoma.

Further, the present invention provides a GPI having a molecular weight of about 12 kDa expressed in about 80% of mouse immature thymocytes.
The present invention also provides a mouse lymphocyte activating antigen, which is an anchor protein and is characterized by being recognized by the monoclonal antibody. Hereinafter, the configuration of the present invention will be described in detail. (1) Production of hybridoma The method for producing a hybridoma that produces the monoclonal antibody of the present invention is a known method (Nature, 256, p49).
5, 1975) and the like, for example, as follows. 1) Animal sensitization Mouse thymocytes were used as an antigen, mixed with complete Freund's or incomplete Freund's adjuvant, and injected into hamsters about 1 to 4 times at intervals of one week to several months. Sensitize. 2) Cell fusion The spleen cells excised from the immunized animal and mouse myeloma cells are fused by a conventional method to obtain fused cells. SP2 / 0-Ag14 and the like can be used as mouse myeloma cells. Although several methods for performing cell fusion are known, for example, a method using polyethylene glycol (hereinafter sometimes referred to as PEG), a method using electrofusion, and the like can be used. In the present invention, a fused cell can be obtained by any of these methods. 3) Selection of fused cells Thymocytes were stained using the culture supernatant of the fused cells obtained by the above method, and analyzed using a flow cytometer (flow cytometry). Of antibodies that show a staining pattern different from that of a known antigen among antibodies recognizing the same are selected. Furthermore, considering that many known activating antigens are not expressed on unstimulated peripheral T cells, among the above antibodies, an antibody-producing strain that stained thymocytes and did not stain peripheral T cells was selected. . 4) Cloning In order to make the specific antibody-producing cells selected above into a uniform cell population derived from one clone, a cloning operation is performed using a known ultra-dilution method or the like to stabilize the target monoclonal antibody. To obtain a so-called hybridoma.

[0008] The hybridoma obtained as described above has a functional characteristic of producing a monoclonal antibody that specifically recognizes a conventionally unknown mouse lymphocyte activating antigen. As a typical example, a hybridoma obtained by fusing hamster spleen cells and mouse myeloma cells was named hybridoma PRST1, and was deposited with the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology.
An accession number of RM P-13893 was obtained.

[0009] The hybridoma PRST1 has the following properties. a) Origin: Hybridoma which is a fusion cell of Armenian hamster spleen cells and mouse myeloma cells SP2 / 0-Ag14 b) Morphology: Non-fusiform, almost spherical c) Subculture: possible d) Functional characteristics: Produces monoclonal antibody that specifically recognizes mouse lymphocyte activating antigen e) Cell proliferation: Good in both attachment and suspension f) Storage conditions: Store in liquid nitrogen at -80 ° C or less g) Serum requirement: usually cultured in a medium supplemented with 10% fetal bovine serum. (2) Obtaining antibody The antibody of the present invention is obtained by inoculating the hybridoma from the culture supernatant obtained by culturing the hybridoma or inoculating an animal with the hybridoma.
The protein can be purified from a biological exudate obtained by culturing in vivo by a biochemical technique used for ordinary protein purification.

The antibody of the present invention is a hamster-derived antibody which has been confirmed to specifically react with a mouse lymphocyte activating antigen described later, among several monoclonal antibodies thus obtained. It is characterized by having. The antigen recognized by the monoclonal antibody of the present invention obtained as described above has the following functional and physical properties.

[0011] The monoclonal antibodies recognized antigens are the expression of about 80% of the mouse DP cells is observed, more mature CD4 ⁺ 8 ^- or CD4 ^- 8 ⁺
(Single positive, SP) The expression is rapidly reduced in cells, and the expression is not observed in peripheral T cells. In peripheral T cells, this is
This antigen is an activating antigen for mouse lymphocytes, since its expression is observed when stimulated with nA or the like.

[0012] Since stimulation of mouse B cells with LPS enhances their expression, this antigen is also expressed on mouse B cells. Cells are converted to PI-specific phospholipase C (PI-
This antigen is a GPI-anchored protein since the expression on the cell surface is significantly reduced by PLC) treatment.

[0013] The molecular weight of this antigen, determined by immunoprecipitation using a solubilized hybridoma, is about 12 kDa under reducing conditions. The lymphocyte activating antigen of the present invention, which is recognized by the monoclonal antibody of the present invention and confirmed to have the above properties, can be a good marker for functionally fractionating DP cells. It is believed that many of the resulting findings will increase understanding of immune cell differentiation and activation and will greatly contribute to the establishment of new classification and diagnosis methods for leukemia and the like in the future.

Next, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.

[0015]

【Example】

Example 1 (Production of hybridoma) (1) Immunization of animals Mouse thymocytes 5 × suspended in 1 ml of phosphate buffer
10 ⁷ were administered intraperitoneally to 6-week-old hamsters. Booster immunizations were performed every 2-3 weeks until a sufficiently high antibody titer was observed. (2) Cell fusion The spleen of a hamster with a high antibody titer was isolated. Mouse myeloma cells SP2 / 0-Ag14 and the hamster spleen cells were PEG 1500 at a cell ratio of 1: 2.
Was used for fusion. The fused cells were suspended in a medium, and 96
The mixture was dispensed into each well of a well plate and cultured at 37 ° C. under 5% CO ₂ for 7 days. (3) Screening and Cloning A part of the culture supernatant of each well in which the growth of the hybridoma was completed was collected, and thymocytes were stained using each well, and analyzed by a flow cytometer, and the cell surface of the thymocytes was analyzed. Among the antibodies recognizing the antigen, those showing a staining pattern different from that of the known antigen were selected, and the target hybridoma was screened using this as an index. Next, among the above antibodies, thymocytes were stained and those that did not stain peripheral T cells were selected, and wells of the antibody-producing strain were screened by the limiting dilution method.

Thus, a hybridoma PRST1 producing the monoclonal antibody of the present invention was produced. Example 2 (Acquisition of monoclonal antibody) The hybridoma PRST1 obtained in Example 1 was cultured,
The monoclonal antibody PRST1 was purified from the culture supernatant using a protein A Sepharose column. Example 3 (Isolation of mouse lymphocyte activating antigen) Using the monoclonal antibody PRST1 obtained in Example 2,
Mouse lymphocyte activating antigen was isolated.

That is, the monoclonal antibody PRST1
Sepharose-beads bound to were prepared and used to immunoprecipitate antigen from a T cell hybridoma expressing a lymphocyte activating antigen. When analyzed by electrophoresis, this antigen protein showed a molecular weight of 12 kDa under reducing conditions.

[0018]

As described in detail above, the present invention provides a mouse lymphocyte activating antigen, a monoclonal antibody that specifically recognizes the antigen, and a hybridoma that produces the monoclonal antibody. As a result, immature thymocytes can be finely and functionally fractionated, opening the way to elucidation of mechanisms such as differentiation and activation of immune cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification code FI (C12P 21/08 C12R 1:91) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 5/10 C07K 14 / 705 C07K 16/18 C12N 15/02 C12P 21/08 BIOSIS (DIALOG) JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A hybridoma derived from a fusion cell of a hamster spleen cell and a mouse myeloma cell immunized with a mouse immature thymocyte , comprising a mouse lymphocyte activity.
Producing monoclonal antibodies that recognize sexually active antigens
Hybridoma PRST1 (FERM P-
13893) . 2. The hybridoma PRST1 of claim 1.
Monoclonal produced by (FERM P-13893)
An antibody that recognizes mouse lymphocyte activating antigen
Noclonal antibodies.