JPH0440891A - Production of human monoclonal antibody-productive hybridoma using il-6 - Google Patents

Abstract

PURPOSE:To obtain the title hybridoma in higher probability along with in creased antibody productivity by adding IL-6 to a medium in fusing antibody- productive human B-cell strain and myeloma or in cloning. CONSTITUTION:When a cell fusion between antibody-productive human B-cell or cell derived therefrom and another cell is to be made, in culture immediately after their mixing and/or cloning, IL-6 is added to the culture solution. The IL-6 will be used for growing more clones in hybridoma formation and cloning by e.g. the limiting dilution technique after combining the two kinds of cell. Although the concentration of the IL-6 in its addition seems to be appropriate at about several nanogram/ml, the optimal concentration may be checked and used for each kind of the cells. The IL-6 to be used is e.g. recombinant IL-6 of Escherichia coli origin; however, may be another recombinant IL-6 derived from other bacteria or animal cells, or IL-6 for animal species other than human being.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a method for producing a hybridoma that continuously produces human monoclonal antibodies, and a method for producing a monoclonal antibody using the hybridoma [Prior Art] When applied clinically, monoclonal antibodies derived from mouse cells have antigenicity to humans, and there is a risk that immune complexes may be formed in the body, causing organ damage to the patient. Because the immunoglobulin antibody neutralizes the administered mouse antibody, it cannot be administered for a long period of time. Therefore, a chimeric antibody (S, L, Morrison et al., Pr.
oc, Natl, Acad.

Sci, [ISA, vol. 81: p6851.1984
), or not only the Fc region but also the Fab region.
Humanized antibodies (P, T, Jones et al., Na
ture, vol. 321: p522゜1986)
are produced using genetic engineering techniques, and clinical efficacy has been reported for anti-IL-2 receptor antibodies and the like. on the other hand,
The production of human monoclonal antibodies using lymph nodes and peripheral blood lymphocytes derived from melanoma patients and their clinical effectiveness on melanoma patients have been reported (RoF, Ir1
et al., Proc. Natl.

^cad, Sci, USA, vol. 83: p8694.
(see 1986).

Methods for obtaining human monoclonal antibodies include the hybridoma method of human lymphocytes and human myeloma, the hybridoma method of human lymphocytes and mouse myeloma, and the Epste 1n-Baarvirus (E) method of human B cells.
A transformation method using B virus) has been used, but the hybridoma method has poor cell fusion efficiency and unstable antibody production from hybridomas, and when cells are cloned by limiting dilution method, growth is extremely slow. For these reasons, it has been difficult to obtain hybridomas that stably produce the desired antibodies.

Interleukin 6 (IL-6) is a protein widely involved in cell proliferation and differentiation (T, Kishimo
to.

Blood, vol. 74: pl, 1989).

In recent years, it has become possible to produce recombinant IL-6 using genetic engineering (Y.

(See Asagoe et al., Biotechnology, Vol. 6: p. 806, 1988), and its applications are being developed widely. The effect of IL-6 on mouse hybridoma proliferation has already been reported (J, V, Damme
et al., J. EXIToMed, vol. 165: p91
4゜1987), but its effect on human hybridomas has not been reported, and human hybridomas that produce the desired human monoclonal antibodies were created from human lymphocytes, and IL-6 was used to develop antibodies. No known applications.

[Problem to be solved by the invention]

The present invention aims to provide a novel method for efficiently producing hybridomas that produce human monoclonal antibodies, and further provides a method for producing human monoclonal antibodies using the hybridomas.

[Means to solve the problem]

The present inventor has conducted intensive research on the effect of IL-6 when producing human monoclonal antibody-producing hybridomas from human lymphocytes. 6 increases the hybridoma formation rate, and furthermore, it is possible to increase the hybridoma formation rate by adding IL to the medium during cloning by limiting dilution method etc.
They found that addition of IL-6 increased the growth rate of cloned cell lines, and discovered that IL-6 is effective in producing hybridomas that produce human monoclonal antibodies.

That is, the present invention provides a method for producing a hybridoma that produces human monoclonal antibodies by cell fusion of antibody-producing B cells or cells derived therefrom with other cells, during culture immediately after mixing both cells; The present invention provides a method for producing a hybridoma characterized by adding IL-6 to the culture medium during cloning, and a method for producing a monoclonal antibody characterized by culturing the hybridoma, and the following: Explain in detail.

[Detailed description of the invention]

1. Human monoclonal antibody: A human monoclonal antibody refers to an antibody secreted from a single human B cell population (including human B cell lines and hybridomas derived from human B cells).

2. A hybridoma that produces human monoclonal antibodies: A hybridoma that produces human monoclonal antibodies is a hybridoma that is a fusion of biologically derived human cells and myeloma cells, or a hybridoma that is a fusion of human cells and myeloma cells, or a cell that is infected with, for example, EB virus. It refers to a hybridoma obtained by fusing established human lymphocytes and myeloma cells, and furthermore, a hybridoma obtained by fusing a hybridoma prepared by the above method with myeloma cells. The myeloma cells used here mean, for example, human myeloma cells represented by U266 and mouse myeloma cells represented by P3111, but may also be other human myeloma cells or mouse myeloma cells, and furthermore, other animal species. Also refers to myeloma cells of origin.

3. Method of using IL-6 to produce hybridomas producing human monoclonal antibodies: IL-6 is
After bringing the two types of cells together, they can be used to grow more clones during hybridoma formation and cloning, such as by limiting dilution. IL
It is thought that the appropriate concentration of -6 to be added is several nanograms/-, but the optimum concentration may be determined depending on each cell.

The IL-6 used is recombinant human I derived from Escherichia coli.
An example is L-6, but it may also be produced from other bacteria or animal cells, recombinant human IL-6, or IL-6 from animal species other than humans.

〔Example〕

Examples will be shown below to explain the present invention in more detail, but the present invention is not limited to these Examples.

Effect of IL-6 on the fusion of B cell lines and myeloma was investigated.

EB virus-transformed human B cell line T that continuously produces IgG type monoclonal antibodies against hepatitis virus
APC-301 (Human Ce1l Vol. 1 No. 1 p.
31 (see 1988) and human myeloma 6
JCT (Human Ce1l Volume 1 No. 1 p3119
RPMI 1 containing 10% FC3 (see 1988)
640 medium, and IXIO' TAPC-301 and 2X1 (
16 6JCTs were fused. After fusion, 0, 0.
2 or suspended in HAT selection medium containing 2 ng/ml of IL-6 and plated in a 96-well microculture plate.
TAPC-3013XIO' cells were divided into wells and cultured, and 10 days later, the wells in which hybridomas had grown were counted, and the antibodies in the medium were detected by a conventional EIA method.

The results are shown in Table 1. This table shows EB virus-transformed B cell line TAPC-301 and human myelomalo JC.
When T is fused and cultured in 300 wells, the number of wells in which hybridomas have grown and the proportion of hybridomas producing antibodies among the grown hybridomas are shown.

The above results in Table 1 show that the appearance rate of human hybridomas increases in proportion to the IL-6 concentration, and that all grown hybridomas retain the ability to produce antibodies. That is, it is shown that the addition of IL-6 increases the fusion efficiency of human hybridomas.

After 10 days, the wells in which hybridomas had grown were counted, and the antibodies in the culture medium were detected by standard EIA.

The results are shown in Table 2. This table shows the number of wells in which hybridomas grew when the human hybridomas obtained in Example 1 were cloned by the limiting dilution method, and the proportion of hybridomas producing antibodies among the grown hybridomas.

Table 2 The human hybridoma established in Example 1 was incubated at 0°0.2
Or cloned by limiting dilution method to 1 cell per well in RPM11640 medium containing 21 g/rnl IL-6 and 10% FC3, and the above results were proportional to the concentration of IL-6. This shows that the growth rate of human hybridomas has increased, and that all grown hybridomas retain the ability to produce antibodies. That is, the addition of IL-6 increases the cloning efficiency of human hybridomas.

〔Effect of the invention〕

The hybridoma production method for producing human monoclonal antibodies using IL-6 provided by the present invention increases the probability of producing the desired human hybridoma and improves antibody productivity. Human monoclonal antibodies produced by this method are useful for clinical applications and diagnosis.

Claims (1)

[Claims] 1. Immediately after mixing both cells in a method for producing a hybridoma that continuously produces human monoclonal antibodies by cell fusion of antibody-producing human B cells or cells derived therefrom and other cells. 1. A method for producing a hybridoma, which comprises adding IL-6 to a culture solution during culturing and/or cloning. 2. A method for producing a monoclonal antibody, which comprises culturing a hybridoma produced by the method according to claim 1.