JPS59175896A - Preparation of human antibody - Google Patents

Abstract

PURPOSE:To prepare immunoglobulin easily from a hybrid cell obtained with suppressing cell disorder in cell fusion, by using a cell strain produced by subjecting human B cell to tumorigenesis. CONSTITUTION:Human B cell is subjected to tumorigenesis, to give a cell strain containing immunoglobulin of IgA type on the surface of the cell, preferably a cell strain not capable of living in a medium containing aminopterin. This cell strain and an uncancerated human antibody producing cell are subjected to cell fusion by a conventional procedure. The prepared hybrid cell is cultivated, and immunoglobulin derived from the uncancerated antibody producing cell is collected from the supernatant liquid of the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing human antibodies using a cell line formed by tumorigenic human B cells.

In recent years, cell fusion technology has developed rapidly, and expectations for its application to human antibody production are increasing.

Conventionally, research on hybrid cells capable of producing human antibodies has been conducted using the following two methods.

(1) Using myeloma cell lines from mice, rats, etc.
A method for creating hybrid cells with human antibody-producing cells.

(2) A method for creating hybrid cells using human myeloma.

However, in the hybrid cells of human cells and xenobiotic animal cell lines produced by the first method, human chromosomes rapidly disappear, making it extremely difficult to obtain hybrid cell lines that stably produce antibodies over a long period of time. there were. Also, in the second method,
Cell lines die rapidly in polyethylene glycol solution, which is the easiest to use as a cell fusion agent, and myeloma cell lines secrete the entire IgG type antibody, so it is only secreted by forming hybrid cells. There were problems such as the inability to screen for IgG anti-type whole stool, which is a major immunoglobulin.

Therefore, the present inventors investigated various cell lines that can be effectively fused with human antibody-producing cells and that can be easily screened for all IgG antibody types among various immunoglobulins secreted from hybrid cells. As a result, the B cells turned into tumors, and by using a cell line that has IgA type immunoglobulin on the cell surface, there is less cell damage caused by cell fusion agents such as polyethylene glycol, and cell fusion can be carried out more effectively. is possible, and the resulting hybrid cells are IgG type and I type derived from non-cancerous antibody-producing cells.
Is it possible to easily detect major immunoglobulins such as gM type? This discovery led to the completion of the present invention.

That is, the present invention provides a cell line (hereinafter referred to as
This hybrid cell line (referred to as IgA type B cell line) and non-cancerous human antibody-producing cells are fused to form hybrid cells, and the hybrid cell line is used to generate antibodies derived from non-cancerous antibody-producing cells. This is a method for producing immunoglobulin.

In general, cell lines in which B cells have become tumors are identified as floating cell lines that have at least one of complement C3 receptors, IgGFc receptors, surface immunoglobulins, etc. on the surface of their cell membranes. The present inventors conducted a comparative study of numerous B cell lines, and found that when hybrid cells were formed using IgA type B cell lines, scales produced immunoglobulin derived from non-cancerous antibody-producing cells; And Ig
It has been found that the production amount of immunoglobulins belonging to classes such as G and IgM can be easily screened.

The IgA and WB cell lines used in the present invention differ from human myeloma in that they have IgA type immunoglobulin on their cell surfaces. -! In addition, in terms of cell morphology, IgA type B cell lines have more protrusions on the surface and have stronger cohesion among cell lines.

A more preferred IgA type B cell line used in the present invention is that when present in a culture solution, IgA type B cell lines contain IgA from the cell line in the culture solution.
This strain has been confirmed to secrete gA. Confirmation of gA can be performed by detecting the supernatant of the liquid using an enzyme immunoassay method or the like.

The faster the time required for doubling of the IgA type B cell line in the logarithmic growth phase, the easier it is to use for the purpose of the present invention;
Preferably, the time is in the range of 2 to 36 hours.

Cell lines whose growth rate has been reduced due to mycoplasma infection or the like are not suitable for the purposes of the present invention.

Examples of IgA type B cell lines used in the present invention include Nature, Vol. 294, p. 173 (1)
0M1056A shown in 981), and the construction: "
Monoclonal Antibodies and T Cell Hyperdomas” edited by Hamerlingula, Elspear/Northern Netherlands Biochemistry A/
, Press (Monoclonal Antibody
esand 'l' Ce1l Hybridomas
, edited by G.J.

HWmmerling etal,, Elsevie
r/North-Holland Biomedical
Press, Amsterdam, 1981)
Examples include GM923 shown on pages 432 to 444 of .

It can also generally be obtained by long-term culture of peripheral blood lymphocytes or by viral oncogenesis with Epstein-Barr virus.

The IgA type B cell line is treated with the cell fusion agent Polyethyl~5.
- It exhibits strong resistance to contact with lengelicol, and hybrid cell lines can be obtained more effectively than human myeloma.

The basic composition of the medium used for culturing the IgA type B cell line is not particularly limited, but a medium RPMI-1640 containing 10 to 20% fetal bovine serum is commonly used.

The non-cancerous human antibody-producing cells used in the present invention have r=J ability acquired from peripheral lymphocytes, lymph nodes, pancreas, etc., and although there are no particular restrictions, they are usually peripheral blood lymphocytes due to ease of acquisition. is used. Cell fusion may be carried out by mixing cells to be fused at room temperature in the presence of a cell fusion agent.

Various cell fusion agents are known, such as polyethylene glycol solutions and inactivated Sendai virus, and although there are no particular limitations, polyethylene glycol solutions are easy to use because they are easy to prepare. After cell fusion, the non-cancerous antibody-producing cells that have been fused gradually die, and there is no particular problem in obtaining a hybrid cell line.
Type B cell lines do not die, so separation from them requires some ingenuity.

Although it is possible in principle to separate hybrid cell lines from unfused 'fcIgA type B cell lines by comparing the properties of individual fused cell colonies, an easier separation method is to separate them from IgA type B cell lines. There is a method in which an enzyme-deficient strain that dies in a medium containing aminopterin is selected in advance and used as a cell line for cell fusion. According to this method, only hybrid cells can be selectively grown by culturing in a medium containing aminopterin after cell fusion.

In order to select a strain deficient in this enzyme, for example, if a strain deficient in hypoxanthine guanine phosphoribosyltransferase (hereinafter abbreviated as HGPRT) is obtained, drugs such as 6-thioguanine or 8-azaguanine must be used. To select a cell line that can grow in a medium containing
- This is possible by selecting cell lines that can grow in a medium containing bromodeoxyuridine.

It is known that the action of aminopterin mainly inhibits the de novo nucleic acid synthesis pathway, but a similar effect is caused by azaserine, which has an inhibitory effect on glutamyl transferase.
It can also be obtained with 6-diacy5-oxo-L-norleucine.

In order to obtain cells that are sensitive to aminopterin, the required drug concentration varies depending on the drug sensitivity of the IgA type B cell line to be used, so first, culture the cells in a medium containing various concentrations of the drug. , determine the drug concentration that exhibits 1/2 the growth rate when no drug is added. Next, 20~
Proliferated cell lines -+m may be selected by continuing cell culture while gradually increasing the drug concentration over 2 to 6 months up to a 100-fold concentration. Preferred aminopterin-sensitive IgA type B cell lines include the above-mentioned IgA type B cell lines.
Type A B cell line GM1056A was selected in a medium containing 6-thioguanine, and G was made into an aminopterin-sensitive cell line.
There is M1056A-TGR. This cell line has IgA type immunoglobulin on the cell surface, and IgA is also secreted into the culture supernatant.

An attempt was made to deposit the cell line at the Institute of Fine Technology, but the request was not accepted. However, it is stored frozen at liquid nitrogen temperature.
It is always available for distribution.

Details of cell fusion will be described below.

When obtaining human antibody-producing cells from peripheral blood, layer them on top of Ficoll-Conray solution, 400G, 3
Centrifugation is performed for 0 minutes, and the peripheral blood lymphocyte fraction (hereinafter abbreviated as PBL) obtained as an intermediate layer is used. If you want to obtain all antibody-producing cells from other tissues, cut the tissue into small pieces and use a stainless steel mesh to separate the solid components and lymphocytes. Contaminated red blood cells should be hemolyzed with a 0.14M ammonium chloride solution. and remove it.

The thus obtained suspension containing human antibody-producing cells is mixed with an aminopterin-sensitive IgA type B cell line, and protein components such as fetal bovine serum are completely removed by centrifugation.

The mixing ratio of both cells is particularly limited. From a cell ratio of 1:1, one of the cells may be added in excess of about 100 times.

Subsequently, a cell fusion promoter with a molecular weight of 1500 to 600
0 polyethylene glycol dissolved in a protein-free medium or balanced salt solution to a concentration of 35 to 55%,
The mixture is slowly dripped at a rate of 0.1 to 1-1 per 107 cells, left for 2 to 3 minutes, washed with a protein-free medium, and the polyethylene glycol is sufficiently diluted.

Subsequently, O7 minopterin at a minimum to 100-fold concentration that completely kills aminopterin-sensitive IgA type B cell lines;
The cells were then dispersed in a tr medium containing hypoxanthine and thymidine, which are substrates for the salvage circuit of nucleic acid synthesis, and dispensed into a 96-well microtest plate, and the fused cells were grown in a carbon dioxide incubator. , allowing the antibody to be released into the culture supernatant. The concentrations of hypoxanthine and thymidine are not particularly limited as long as they do not cause cell damage;
-5M or so is desirable.

The advantage of using the IgA type B cells of the present invention is that = 1
0- (1) There is extremely little cell damage when in contact with a 45% polyethylene glycol solution for about 10 minutes.

(2) Propagation of hybrid cells is easy.

(3) The production ability of human antibody-producing cells can be easily transferred to cell lines.

(4) It is extremely easy to detect hybrid cells capable of producing antibodies such as IgG type and IgM type.

etc.

Human antibodies can generally be obtained from the culture supernatant of fused cells. In order to obtain only specific antibodies,
For example, it is possible to select a specific strain from among the fused cell lines, incubate it, and isolate specific antibodies from it. In addition, this hybrid cell line was transplanted into the peritoneal cavity of a mammal in which rejection reactions and natural killer activity were suppressed.
It is possible to make them multiply. As the mammal used here, athymic animals such as nude mice are easily used.

Example H) Isolation of aminopterin-sensitive (HGPRT-deficient) B cell line from B IJ bulb-derived cell line GM1056A
640 medium was grown, and then culture was continued for 10 days in the same medium supplemented with 1 μ2/− of 6-thioguanine, and then 2 μf/1 nl, 5
The cells were cultured for 15 days each in a medium containing 6-thioguanine at tn/ml, 10 tit/ml, and 20 μg/ml, and the viable cell population was transferred using Myflopiperatra. A total of 25 cell rounds obtained from different wells were finally obtained.

When a portion of the cell population from each well was separated and cultured in RPM11640 medium containing various concentrations of aminopterin and 10 μl of fetal bovine serum, 4×1
When cultured for 10 days in a medium containing 0-'M or more aminopterin, 2 out of 25 wells died. Based on this fact, these two wells were determined to be enzyme-deficient cell lines and aminopterin-sensitive cell lines. All of these cell populations were re-dispersed and cultured to obtain a cell population that is thought to have originated from a single cell, and the aforementioned aminopterin sensitivity was confirmed again, and this cell line was subjected to the following cell fusion. provided.

Resistance test to polyethylene glycol Prior to carrying out cell fusion, the degree of cell damage caused to the cell line by polyethylene glycol was examined.

After preparing Hank's solution containing all 45 units of polyethylene glycol with a molecular weight of 2000 and adjusting the pH to 8.2,
Filtered and sterilized. Washed with Yo〈Nox liquid 2
The polyethylene glycol solution was gradually added dropwise to 10 pieces of GM1056A-TGR over 2 minutes and 30 seconds, and then left at 37° C. for 1 minute. The cell viability of the cell line GM1056A-TGR used at this time was measured by the exclusion ability of the dye Trivan blue, and it was found that 96
It was calculated that

Next, the medium RPM11640, which does not contain protein components, was added for 2 hours.
Polyethylene glycol = 13- was gradually diluted using 〇-. This cell suspension was gently centrifuged,
The cells were resuspended in medium RPM11640 containing 10 g of fetal bovine serum. The culture medium containing the polyethylene glycol-treated cells was dispensed into each well of a 24-well culture plastic plate, and the carbon dioxide concentration was 5 cm and the temperature was 37 cm.
After culturing for 6 days in a carbon dioxide incubator set at 0.degree. C., the survival rate of the cell line was measured and found to be 92%.

Cell fusion Healthy subjects' peripheral blood 207-(z) Blood was collected using heparin as an anticoagulant, and Ficoll-Conrey solution (specific gravity 1.07
7) fresh lymphocytes by specific gravity centrifugation using 2X
A suspension containing 107 cells was obtained.

Then, the lymphocytes were transferred to the same number of cell lines GM1056A-T.
The mixture was mixed with GR, centrifuged again with Hank's solution, washed three times, and the washing solution was thoroughly removed.

As a cell fusion agent, Hank's solution containing 45 parts of polyethylene glycol with a molecular weight of 2000 was prepared, and after adjusting the pH to 8.2, it was sterilized by filtration. This fusion promoter solution was gradually added dropwise to the washed cell mixture-14 over a period of 2 minutes and 30 seconds, and then left at 57°C for 1 minute.

Subsequently, the polyethylene glycol was gradually diluted using 20% of the protein-free medium RPMI 1640. This dilution was centrifuged gently and 10-7 M aminopterin, 10-'M hypoxanthine and 1
．． The medium was replaced with a medium (hereinafter referred to as I (abbreviated as AT medium) 80-) containing 5×IQ""M of thymidine.

Four 96-sized tissue culture microtest plates were prepared, and 200 μt was dispensed into each hole. Half of the medium was replaced with fresh HAT medium every 4 days, and after 20 days, proliferation of the cell population was observed under an inverted microscope. When the presence of human IgG and IgM in the culture supernatant was analyzed using an enzyme immunoassay method using peroxidase-conjugated antibodies, human IgG was present in 76 culture wells, and human IgM was present in 34% of the culture wells. It was confirmed that each was produced.

Comparative Example Human myeloma strain U-266 (C1inicalE
Experimental Immunology Vo
1, 7 (1970), pages 477 to 489], aminopterin-sensitive cells were isolated and established in the same manner as in the Examples.

The sensitivity of this strain to polyethylene glycol was measured in the same manner as in Example 1, and as a result, the survival rate before the experiment was 9.
Among the top 3, the survival rate was 23% after polyethylene glycol treatment and culture for 3 days.

Next, cell fusion with lymphocytes from a healthy individual was performed in the same manner as in the example. After culturing for 20 days, no viable cells were detected using a microscope, and neither IgG nor IgM was detected in the strained culture supernatant.

Claims (2)

[Claims] (1) h) The use of hybrid cells obtained by cell fusion of a cell line in which B cells have turned into tumors and have IgA-type immunoglobulin on the cell surface, and human antibody-producing cells that have not turned into cancer. A method for producing human antibodies. (2) H) A cell line in which B cells have turned into tumors and has IgA-type immunoglobulin gold on the cell surface cannot survive in a medium containing aminopterin. How to produce human antibodies.