JPS62104574A - Novel hybridoma - Google Patents

Abstract

PURPOSE:A hybridoma that is obtained by fusing a hybridoma from human lymphoid cells and different animal's lymphoic cell strain with human lymphoid cell, thus being a cloned hybridoma not forming antibodies. CONSTITUTION:Human lymphoid cells are fused with a different animal's lymphoid cell strain such as mouse myeloma cell strain using polyethylene glycol as a fusing agent to prepare a hybridoma. Then, the resultant hybridoma is fused with human lymphoid cells to give a cloned hybridoma which does not form antibodies by itself. The use of the hybridoma as a mother strain for cell fusion makes the cell fusion high efficient to give useful hybridomas forming human antibodies in high frequency.

Description

[Detailed description of the invention] Industrial applications The present invention relates to novel hybridomas.

Conventional technology The method for producing monoclonal antibodies using hybridomas, which was developed by Koehler and Milstein and has become popular in recent years, is capable of producing antibodies that exhibit monospecificity for each antigenic determinant, and that has become popular in recent years. It has many advantages, such as being able to freely multiplex it depending on the situation and always obtaining homogeneous specimens with good reproducibility. In this sense, the method of obtaining monoclonal antibodies (hereinafter sometimes abbreviated as MoAb) using hybridomas has been highly evaluated for its effectiveness in many fields. In addition to the simple detection of antigens, its use has been developed to purify trace components and apply it to diagnostic drugs, and further applications to preventive and therapeutic drugs are being considered.

Among these uses, mouse MoAb can be used in cases other than direct administration to humans such as prophylactic and therapeutic drugs, but in the case of prophylactic and therapeutic drugs, mouse MoAb is a foreign protein to humans. It is not appropriate to use mouse M o Ab. This is because when mouse proteins are administered, antibodies are induced by the mouse proteins entering the human body.
This is because not only the effect is reduced, but also there is a risk of allergic reactions caused by foreign proteins.

In general, research on human MoAbs is much larger than that on mice.
1] is late. The reason for this is that the method for producing human M o Ab-producing strains is more difficult than that for mice;
This is because there are few successful cases.

Typical methods for producing human MoAb-producing strains include methods using human-human hybridomas, methods using human-mouse heterohybridomas, and Epstein-Barr virus (Epstein-Barr virus; hereinafter referred to as Er).
There is a method using established cells transformed with 3V (abbreviated as 3V). Although many research results using these methods have been reported recently, it has been pointed out that each method has practical drawbacks.

Human-human hybridomas generally have low fusion efficiency;
In addition, mouse-human hybridomas tend to shed human chromosomes and have poor human antibody production stability. Furthermore, it is difficult to clone the antibody-producing cells obtained from EBV transformants. For this reason, it is said that when the proliferation rate of cells that do not produce the target antibody is rapid, the stability of antibody production decreases.

In contrast to these experimental systems, a method has recently been proposed in which a mouse/human heteromyeloma is used as a parent strain and is fused with antibody-producing cells to obtain a hybridoma that produces the desired human antibody. It has been reported as a stock acquisition method [Proc.
tl, Acad, Sci,), 80°730g-
7312 (1983)]. The present inventors also investigated mouse myeloma cells (P3U1) and human peripheral blood lymphocytes (hereinafter referred to as P3U1).
The heterohybridoma (hybrid cell) HM-5 (abbreviated as BL) has been recognized to be superior in fusion efficiency with human PBL to the parent strains of mice and humans, and a patent application filed in 1975
No. 9-246247 (filed on November 22, 1982) Specification]. However, if a parent strain with even higher fusion efficiency than this is established, the frequency of obtaining hybridomas that produce the desired human antibody will likely further increase, and the establishment of an excellent parent strain will lead to the practical application of the hybridoma method. This is the key.

Problems to be Solved by the Invention The present inventors have recognized that human monoclonal antibody-producing hybridomas can be advantageously obtained using the above-described hybrid cell mouse-human hybridoma strain 8M-5 as a parent strain (the above-mentioned characteristics). Application No. 59-246247) is
In this case, we speculate that the reason why the results are much more advantageous than when using mouse cells as the parent strain is that some human chromosomes are present in the hybrid cells that are the parent strain. .

The present inventors pushed forward this idea and created a new parent strain with even more chromosomes than the hybrid cell by using a hybrid cell fused with some human cells as a parent strain. In the hope that it will be possible, the above mentioned HM-
The present invention was completed based on the recognition that hybridomas obtained by re-fusion with human lymphoid cells using strain 5 as a parent strain would be a useful parent strain for obtaining human monoclonal antibody Jodo hybridomas.

Means for Solving Field Problems The present invention is a hybridoma of a human lymphoid cell and a lymphoid cell line of a xenogeneic animal, and a hybridoma of a human lymphoid cell, which itself is cloned and does not produce antibodies. The present invention provides hybridomas that have been developed.

” Concerning hybridomas that are fusions of bipedal human lymphoid cells and lymphoid cell lines from xenogeneic animals, and human lymphoid cells, normal lymphocytes obtained from healthy individuals and human lymphoid cells are used as human lymphoid cells. Name a human lymphocyte cell line. Normal lymphocytes may be from any of the spleen, lymph nodes, endocrine plates, etc., but peripheral blood lymphocytes (PBL) are advantageously used because of their ease of availability.

Although these lymphoid cells may be used as they are, it is preferable to activate them in advance with a polyclonal mitogen (such as Hawklide mitogen).

Examples of hybrid cells include heterohybridomas of cell lines derived from lymphocytes of mammals other than humans (for example, experimental animals such as mice and rats) and human lymphoid cells similar to those described above. Among these, mammalian lymphocyte-derived cell lines have the best track record in fusion experiments, and are mouse myeloma cell lines with excellent cell fusion efficiency and proliferation, especially hyboxanthin-guanine-phosphoribonortransferase-deficient 1o-rcpRT- Advantageously, cells with genetic markers such as A. In order to produce a hybridoma of a hybrid cell, that is, a human lymphoid cell and a lymphoid cell line of a xenogeneic animal, these cells can be combined with a fusion agent such as Sendai virus or polyethylene glycol (PEG), or subjected to electrical stimulation, etc. It can be fused in this way. An example in which PEG is used will be given, but of course the method is not limited to this method.

The degree of polymerization of PEG is usually between 1000 and 6000. The treatment time is 0.5 to 30 minutes, and the degree of a is 10 to 80%, etc., but as an example of preferable conditions, PEG 6000 is treated at 35 to 55% for 4 to IO minutes to achieve efficient fusion. I can do it. Various combinations are possible as a medium for selective growth of fused cells, and one example that is advantageously used is when HGPRT-mouse myeloma cell line and human PBL are fused, the fused cells include, for example, hypoxanthine, aminopterin.

It can be selectively grown using demidine medium (HAT medium), azaserine medium, hyboxanthin medium (AH medium), or the like.

What are the hybrid cells thus obtained? 1 GP RT+, but a marker such as HGP RT- can be inserted. For this purpose, for example 8-azaguanine (8-
The objective can be achieved by adding AZG) etc. to the medium and increasing its concentration sequentially.

In order to produce a hybridoma of the hybrid cell thus obtained and a human lymphoid cell, both cells can be efficiently fused by the same method as described above. Regarding the hybrid cells used here, it is very effective to check in advance the fusion efficiency with human PBL, the proliferation ability of the obtained hybridoma, the ease of cloning, etc., and select the optimal hybrid cell line. be.

Further, during this selection, it is preferable to select hybrid cells that do not secrete antibodies. Selective propagation of the resulting hybridoma of hybrid cells and human lymphoid cells can also be carried out by using HAT medium or AH medium in the same manner as described above.

The hybridoma thus obtained is HG P RT+
However, by adding 8-AZG to the medium and increasing its concentration sequentially, HG P RT-
It is possible to insert genetic markers such as Hybridomas into which genetic markers have been introduced in this manner can be used as viable parent strains for cell fusion with antibody-producing cells.

``Bipedal antibody-producing cells may be lymphocytes derived from normal humans, or, for example, ``if the target antibody is an infectious substance, human lymphocytes infected with the substance.''

If it is directed against cancer, cancer-bearing human lymphocytes can be used. These lymphocytes are in vitro
After stimulation with the relevant antigen or polyclonal lymphocyte mi[-diene (Hawk Leid mitonen, Staphylococcus aureus Cowan 1, etc.), or Epstein-Barr virus (Epstcin-Barr virus).
By using the hybridoma in a fusion experiment after transforming it with r virus, it becomes possible to obtain the desired antibody-producing hybridoma more efficiently.

Effects and Examples Hereinafter, the mouse-human-human hybridoma MHH-II, which further discloses the present invention by reference examples and examples, has been submitted to the Fermentation Research Institute (IFO).
It has been deposited as O-50063.

Reference Example 1 Method for Measuring Human Antibodies As a method for measuring human antibodies, an enzyme-linked immunosorbent assay (ELISA method) was used. That is, goat anti-human IgG or IgM at 15 μg/m
0,01 containing O, OIM NaCl so as to be l
Suspended in M phosphate buffer (pH 8,0) and dispensed 100μρ into each well of a 96-well microplate.
The reaction was carried out at 4°C for 24 hours. After the reaction, 100 μ2 of phosphate buffer containing 2% bovine serum albumin (BSA) was dispensed into the wells to block excess binding sites in the wells, and treated at 4° C. for 24 hours to prepare a plate for use in ELISA.

100μ2 of the culture supernatant was added to the plate thus prepared, and the reaction was allowed to proceed at 24°C for 3 hours.

After the reaction, wash well with physiological saline and add goat anti-human Ig labeled with horseradish perokindase (HRP).
G or IgM Fi to each well at 100 μ9 min, 10 Fg I+, 0. 100μ of enzyme substrate solution added with
2 was added to the well, the enzymatic reaction was carried out at room temperature for 15 minutes, and the reaction was stopped with normal sulfuric acid. After stopping the reaction, the wavelength of 49
The amount of colored pigment was measured at 2 nm, and the human l.
The amount of antibody was quantified by comparison with gM or IgG (Miles).

Reference Example 2 Production of mouse-human heterohybridoma (hybrid cells) and production of 8-azaguanine-resistant strain (1) Production of hybrid cells Ficol 1-Hypaqu from human terminal blood of healthy individuals
Lymphocytes (PBL) were separated by specific gravity centrifugation using e and mixed with a mixture of Iskot's medium containing 10% non-mobilized beef tallow serum and Ham's F-12 medium (■・r-i) medium. (■
・1-1-10F) at a concentration of 2×10”/ml.

This lymphocyte suspension was dispensed into a tissue culture flask (Corning F-150 flask), and 32 μg/m
Hawk Lid Mytone En (P
WM) and place it at 37°C in a carbon dioxide gas bubbler.
ics, Microbiol, Immunol,)
, 81. 1-7 (1978)] at a cell ratio of 2.1, and treated with 45% PEG 6000 (manufactured by Kotzholite) for 7 minutes to perform cell fusion. After fusion, the tumor cells were suspended in l-H-10F at 2XIO5/ml, seeded in 1 ml portions in a Linpro 24-well multi-day tube, and cultured at 37°C in a carbon dioxide atmosphere. After 24 hours of culture, I-I
A T (hyboxanthin: l X to'M, aminopterin, 4 X 10-'M, thymidine 1.
6X 10-'M) containing I.H-LOF (HAT medium)
HAT selective culture was started by adding 1 ml of HAT. Furthermore, HAT selective culture was continued by discarding 1 ml of the old solution and adding 1 ml of new HAT medium at odd-numbered days of 3, 5, and 7 from the day of initial addition. Hybridoma proliferation was observed 10-14 days after cell fusion.

(2) Obtaining an 8-azaguanine-resistant strain In order to convert the hybrid cell obtained in (1), which has excellent proliferation ability and does not produce antibodies, into an I-I A T-sensitive strain, 8-azaguanin ( 8-AZG) supplemented medium - culture was continued. The strains that became resistant to 100 μM 8-AZG were examined for I (AT sensitivity), and 6 susceptible strains (1
-IM-1 to 6) were obtained.

Reference Example 3 Comparison of fusion between 6 types of hybrid cell lines and human PBL To compare and examine the cell fusion efficiency of each of the 6 types of I-IAT sensitive hybrid cell lines obtained in Reference Example 2 with that of P3Ul. , 5-7 in the presence of 32 μg/ml PWM
Cell fusion was performed with PBL that had been cultured for days.

Table 1 shows the results. When P3Ul was used as the parent strain, although the fusion efficiency was high, no hybridoma capable of stably producing antibodies was obtained.

On the other hand, when hybrid cells were used as the parent strain, especially in HM-3 to HM-5, the fusion efficiency was higher than that of P3U1, and the antibody production of the obtained hybridomas was also stable.

(Cis bottom margin) Reference Example 4 Chromosome analysis of hybrid cells I-I AT-sensitive hybrid cells HM obtained in Reference Example 2
-5 chromosome was analyzed.

Approximately 2XIO'' cells were suspended in LOML growth medium containing 2 μg/ml colchicine (Wako Pure Chemical Industries, Ltd.) at 37°.
After culturing for 1°5 hours at
It was left standing at ℃ for 15 minutes. Then, by centrifugation, the fixative solution (
After washing the cells twice with acetic acid: methanol (1.3), they were suspended in a few drops of the same fixative solution, placed on a slide glass, and air-dried. The specimen thus obtained was stained with Giemsa staining solution and observed under a microscope (Giemsa method). The stained specimen was destained using the same fixative solution, which was added to a phosphate buffer solution (PBS, pH 5, containing 0.02% trypsin (Gibco)).
8) for 0.6 minutes at 0°, washed with PF35, stained with Giemsa solution again, and observed under a microscope (G-band method)
.

As a result, it was found that at least the 17th human chromosome exists in HM-5.

Example 1 Production of a hybridoma consisting of a hybrid cell and human PBL and production of an 8-azaguanine-resistant strain (1) Production of a hybridoma consisting of a hybrid cell and human PBL Hybrid with the highest fusion efficiency shown in Reference Example 3 Cell HM-
5 strains and human PI3L produced by the same method as in Reference Example 2 were mixed to a cell ratio of 2.1, and 45% P.E.
Cell fusion was carried out by treatment with G 6000 (manufactured by Kotzholite) for 7 minutes. After fusion, the tumor cells were grown in I-I-r-10F at a concentration of 2 x 105/ml, seeded in 1 ml aliquots in a 24-well tube, and cultured at 37°C in a carbon dioxide atmosphere. did. After 24 hours of culture, HAT selective culture was started by adding 1 ml of HAT medium. Furthermore, LIAT selective culture was continued by discarding 1ml of the old solution and adding 1ml of new I-IAT medium at 3, 5, and 7 odd days after the initial addition. Hybridoma proliferation was observed 10-14 days after cell fusion.

(2) Obtaining an 8-azaguanine-resistant strain A hybridoma (MHI hybridoma) of the I-IM-5 strain and human PBL obtained in (1) that has poor growth ability and no antibody production is obtained. For HAT-sensitive strains, culture was performed in a medium supplemented with 8-azaguanine (8-AZG). That is, 1 μ■ concentration (7) 8-A
Culture was started with l-H-10F containing ZG, and 8-A
The ZG concentration was successively increased by 2 times starting from 1 μι and culture was continued. I-r AT sensitivity was examined for strains that became resistant to 100 μM of 8-AZG, and 12 susceptible strains (
MI-11-1-1 to 12) were obtained.

For these 12 strains, the amount of antibodies produced was measured by the method described in Reference Example 1, but no antibodies were detected in any of them.

Example 2 MHI-(hypuridoma strain and human P
Comparison of fusion between P L 12 species obtained in Example 1) II A T sensitive M T
- I H hybridoma line cell fusion efficiency of each
-3271g for comparison with that of the I M-5 strain.
Humans cultured for 5 to 7 days in the presence of PWM/ml)
Cell fusion with PBL was performed. Table 2 shows the results, so mouse/human/human hybridoma Ni
The fusion efficiency of the l-I H-11 strain was higher than that of any other M H1 strain.
The acquisition rate of human antibody-producing hybridomas was higher than that of the HM-5 strain or the control strain HM-5, and twice as high as that of the 8M5 strain (Table 2).

Also, mouse-human-human hybridoma M I-[)1
-9 strain, the cell fusion efficiency itself was ■(M-
Although it was lower than the I-IM-5 strain, the IJ rate of human antibody-producing cells was higher than that of the I-IM-5 strain, indicating that this younger sister also had a high efficiency (Table 2).

Table 2 M HH-283/192 (43,2) [1,08]
7/ 83(8,4)[0,09]MI[I(385/
168 (50,6) [1,26] 13/85 (15
J) [0,19]MH1,I-4124/2+2(58
,,5)[1,46] 10/+24(8,1)[0
,12] M 11-1-5 44/16
8(26,2)[0,6512/44(4,5)[0,
03]M [(H-621/144(14,6)[0
, 36 pieces l/21 (4,8) [0,02]
MHI (-7206/252 (81,7) [2,04]
19/206 (9,2) [0,193MHH-814
2/165 (86,1) [2,15 pieces 14/14
2(9,9)[0,21]MIII-9192/38
2 (50,3) [1,26] 61/192 (31,8
)[0,40]Ml(H-101,32/282(46
,8)[1,17] 26/132(19,7)[0
,23] MHH11238/240(99,2)[2,
48] 62/238(26,1)[0,65]Iv
N(I(-12271/333(81,4)[2,04
141/271(15,1)[0,31,]]1 Cell fusion efficiency - Growth well failure/seeding well dispersion"2 antibody positive rate -
Number of antibody-positive wells/number of proliferating wells (measured after 3 passages of proliferating cells).

“3 [ ] indicates the ratio to 106 normal lymphocytes.

Example 3 Chromosomal analysis of MHH hybridoma Chromosome analysis of mouse-human-human hybridoma MHH-11, which has a high cell fusion efficiency and a high rate of obtaining human antibody-producing cells shown in Example 2, was carried out using the Giemsa method described in Reference Example 4, Two methods were used: the G-band method.

As a result, MHI-11 has the first
It was discovered that in addition to human chromosome number 7, there are two other human chromosomes, numbers 16 and 19.

Effects of the Invention The hybridoma of the present invention can be used as a parent strain for cell fusion, and useful human antibody-producing hybridomas can be obtained with high frequency.

Claims (2)

[Claims] (1) A hybridoma of a human lymphoid cell, a lymphoid cell line of a xenogeneic animal, and a human lymphoid cell is fused and is a cloned hybridoma that itself does not produce antibodies. (2) The hybridoma according to claim 1, which has a genetic marker introduced therein.