JP3029635B2 - Methods for obtaining antibody-producing mutants - Google Patents

Description

The present invention relates to a method for obtaining an antibody-producing variant of a class other than the original class from an antibody-producing cell line.

`` Prior art '' Antibodies are IgA, based on the amino acid sequence of the heavy chain constant region.
It is classified into five classes of IgD, IgE, IgG and IgM,
Each class of antibodies has different physiological functions. For example, IgA is in the form of a monomer or a dimer, and is present in secretions such as milk and saliva. In the case of infants, IgA is characterized in that it can be absorbed into the body from the intestine. IgE is also deeply involved in allergic reactions. IgM is a pentamer
Appears early in the primary immune response and causes complement activation. In addition, the secondary response replaces IgM with IgG. IgG is the only antibody that can pass from the mother to the fetus through the placenta during pregnancy and has the highest concentration in the blood. That is, these classes of antibodies have their own unique properties and play their own role in living organisms.

On the other hand, in recent years, attempts have been made to obtain a hybridoma by fusing antibody-producing cells with a myeloma cell line, and cloning the hybridoma to obtain a monoclonal antibody-producing cell line. Monoclonal antibody-producing cell lines can repeatedly produce a large number of uniform antibody populations that recognize one antigenic determinant. The monoclonal antibody thus obtained is used for various applications such as application to the medical field, such as diagnosis or treatment of disease, and research on cell differentiation, antigen analysis and functional analysis of viral glycoproteins.

"Problems to be Solved by the Invention" However, since antibodies produced by antibody-producing cell lines are antibodies of one of these classes,
Depending on the purpose of use, it may be necessary to obtain another class of antibodies that recognize the same antigenic determinant.

In this case, there is a method of preparing a new antibody-producing cell line of a different class, but it is almost impossible to prepare antibodies having the same specificity. Therefore,
It would be desirable to develop a method for producing a mutant strain that produces another class of antibodies recognizing the same antigenic determinant from an existing antibody-producing cell line.

As such a method, a method of selecting a naturally mutated cell line from an antibody-producing cell line is also conceivable, but a naturally mutated cell line appears only at a low ratio of 1 in 10 ⁴ to 10 ⁶ cells. However, obtaining the desired mutant strain from them requires a great deal of labor.

The present invention has been made to solve such a problem, and an object thereof is to provide a method for efficiently obtaining a class-switched antibody-producing mutant from an antibody-producing cell line. is there.

“Means for Solving the Problems” The present inventors have established a method for searching for an inducer that causes a class-switched antibody-producing mutant to appear from an antibody-producing cell line, and based on this method, various substances have been established. As a result of the search, it was found that interleukins and antibody production enhancing factors were suitable for this purpose, and based on this, the present invention was completed.

That is, the method for obtaining an antibody-producing mutant strain of the present invention comprises, as an antibody-producing cell line, at least one selected from the group consisting of interleukin 2, italleukin 3, italleukin 4, italleukin 6, and an antibody production enhancer. It is characterized by culturing in an added medium and isolating a class-switched antibody-producing mutant.

 Hereinafter, the present invention will be described in detail.

(1) Antibody-producing cell lines Various antibody-producing cell lines such as human-human hybridoma, mouse-mouse hybridoma, and cells transformed with EB virus can be used. These antibody-producing cell lines include, for example, MPC-11 (IgG secreting myeloma cell line), ST
K-1 (mouse hybridoma), Namalva cell line (human Burkitt's lymphoma, IgM secretion type) and the like. All of these cell lines can be obtained from Dainippon Pharmaceutical Co., Ltd.

Although not a cell line, human B lymphocytes can also be used. Human B lymphocytes can be easily separated from blood by an ordinary method using Ficoll.

(2) Medium used for cell culture A commonly used serum-free medium or serum medium can be used. As the serum medium, a medium in which 10% fetal calf serum (FCS) is added to a basic synthetic medium can be excited. As a serum-free medium, serum albumin (or egg yolk lipoprotein), insulin, transferrin,
A medium to which factors such as ethanolamine and selenium are added is preferably used. As the basic synthetic medium, Dulbecco's MEM (DMEM) medium, Ham F-12 medium, RDF medium (RPMI
A medium in which 1640, DMEM and Ham F-12 medium are mixed at a ratio of 2: 1: 1), an E-RDF medium (a medium in which amino acids and vitamins of the RDF medium are enriched) and the like are used. RDF medium is used.

(3) Mutation inducer At least one selected from the group consisting of interleukin 2, interleukin 3, interleukin 4, interleukin 6, and an antibody production enhancing factor is used.
Here, the antibody production-enhancing factor means an autocrine substance derived from Namalba cells, and has a molecular weight of about 110 K daltons, pH
It is stable at pH 6 to pH 12, and consists of proteins that are inactivated by heating. These interleukins and antibody production enhancing factors are already known substances and can be easily obtained. Interleukins can be obtained from, for example, Zenzyme Co. (Cosmo Bio Co., Ltd.) or Sigma.

Antibody production enhancing factors include, for example, in vitro cellular development biology (In Vitro
Cell. Develop. Biol.), Vol. 25, pages 243-247, 1989. In the examples of the present invention, an antibody production enhancing factor purified by the following method was used.

After culturing Namalba cells in 10% FCS / RDF medium, the cells were collected and suspended in phosphate buffered saline to a cell density of 1 × 10 ⁶ cells / ml. After crushing the cells with a sonicator, the supernatant was collected by centrifugation. The protein concentration in the supernatant is about 100
μg / ml. This supernatant was equilibrated with a 10 mM sodium phosphate buffer (pH 7.4) and the DEAE-column (volume: 60 ml)
Was served. Elution of the enhancing factor was performed by a gradient in which 0 to 1 M salt was added to the same buffer, and the factor was eluted at a salt concentration of about 0.4 M.

The eluted factor fraction was applied to a hydroxyapatite column (volume: 5 ml) equilibrated with a 10 mM sodium phosphate buffer (pH 6.7), and eluted with a gradient of 10 to 600 mM of the same buffer (pH 6.7). The factor eluted at a concentration of about 400 mM. The eluted same factor fraction was purified again by the above-mentioned hydroxyapatite column. Further, the same factor fraction purified again by the hydroxyapatite column was applied to a Superose 6HR10 / 30 column (manufactured by Pharmacia) and purified to high purity to obtain an antibody production enhancing factor.

(4) Method for obtaining mutant strain The antibody-producing cell strain is cultured in the above-described serum medium or serum-free medium, and the mutant inducer is added to the culture medium from 2 to 5 days before isolating the mutant strain under normal conditions. Perform culture. The mutant strain obtained by culturing in the above-mentioned inducer is subjected to a fluorescence activated cell sorter (fluorescence activated cell sort).
er; FACS) or cloning.

The fluorescence-excited cell sorter is an apparatus that binds an antigen present on the surface of an antibody-producing cell to a fluorescently-labeled antibody, and fractionates cells to which the fluorescently-labeled antibody has bound in an electric field. As the fluorescence excitation cell sorter, for example, “EPICS” (trade name) manufactured by CoulterCorp can be used.

"Action and Effect" According to the method for obtaining an antibody-producing mutant of the present invention, the mutagenic agent was selected from the group consisting of interleukin 2, interleukin 3, interleukin 4, interleukin 6, and an antibody production enhancing factor. By culturing at least one of them, the appearance rate of the specific antibody-producing mutant can be increased to 10 or more. Furthermore, for mutants that have been class-switched to various classes other than the original class, an increase in their appearance rate is observed, so that specific antibody-producing mutants of any class can be obtained at high frequency. .

"Example" Hereinafter, the present invention will be described in more detail with reference to examples.

As an antibody producing cell line, human-human hybridoma HB
This hybridoma HB4C5 was cultured in various mutagenic agents using 4C5 (a cell line that produces an IgM class human monoclonal antibody, No. 1879 of Microtechnical Research Laboratories, Inc.).
The appearance rate of the switched mutant strain was measured.

As the mutagen, interleukin 2, interleukin 3, interleukin 4, interleukin 6, and an antibody production enhancing factor were used alone. That is, hybridoma HB4C5 was prepared in a 10% FCS / E-RDF medium containing 5 and 25 μg / ml of an antibody production enhancer or 1 to 100 U / ml of interleukins by a conventional method.
Cultured for days.

After culturing in this manner, a cell sorter (trade name “EPICS
752 ", manufactured by Coulter Corp.).

To isolate the mutant strain, first, the HB4C5 cells cultured in the above-described mutagenic agent were transformed with FITC-labeled anti-human IgG,
It was reacted with A, IgD or IgE antibody at 4 ° C. for 1 hour.

HB4C5 mutants that produce a certain class of antibodies have that class of antibodies on the cell surface. So, for example,
Since the mutant class switched to IgG has IgG on the cell surface, by reacting with FITC-labeled anti-human IgG, the FITC-labeled anti-human IgG antibody reacts with IgG on the cell surface, and Cells. In this way,
An IgG-producing mutant can be obtained by collecting cells emitting fluorescence reacted with the FITC-labeled anti-human IgG antibody using a cell sorter. Similarly, FITC-labeled anti-human IgA
, And collecting cells that emit fluorescence, an IGA-producing mutant can be obtained.

Table 1 shows the appearance rates of the mutant strains obtained by such a method.

For comparison, the appearance rate of the mutant strain was similarly determined for cells cultured in a medium to which no mutagenic substance was added.

As is evident from Table 1, by culturing in a medium supplemented with an antibody production enhancing factor or an interleukin, mutants class-switched into various classes can be obtained with high frequency.

Continuation of the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 5/00, 15/00 CA (STN) BIOSIS (DIALOG) WPIDS (STN)

Claims (3)

(57) [Claims] 1. An antibody-producing cell line comprising interleukin 2,
Culturing in a medium to which at least one selected from the group consisting of interleukin 3, interleukin 4, interleukin 6, and an antibody production enhancing factor is added, and isolating a class-switched antibody-producing mutant strain; A method for obtaining an antibody-producing mutant. 2. The method according to claim 1, wherein said culturing is performed for 2 to 5 days. 3. The method for obtaining an antibody-producing mutant according to claim 1, wherein the antibody-producing mutant is separated using a fluorescence-excited cell sorter.