JPH0429357B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Fields The present inventors created a human-human hybridoma that produces a human monoclonal antibody specific to human lung cancer cells, and were able to obtain the antibody in large quantities and with high purity. . The new hybridomas are stably subcultured and produce antibodies specific for lung cancer cells. This lung cancer-specific antibody can be used in medical fields such as cancer immunological research, clinical diagnosis, and immunotherapy, and as a means for purifying cancer-specific antigens. Prior Art Kohler, G. and Milstein, K. produced mouse monoclonal antibodies by cell fusion of mouse splenocytes and mouse myeloma cells (Nature, 256
Vol. 495-497, 1975 and Eur.J.Immunol.6
Vol. 511-519, 1976). A feature of the method for producing monoclonal antibodies using hybridomas is that antibodies that react only with a single antigenic determinant can be repeatedly obtained in large quantities outside of the subject. Therefore, using this method, monoclonal antibodies against antigens such as tumors and viruses can be produced, and the antibodies can be used to infect living organisms.
Attempts have been made to apply it to medical research, and numerous reports have been made, but most of them concern mouse monoclonal antibodies. Steinity, M. produced antibodies by culturing antibody-producing human B-lymphocytes obtained by transformation with Epstein-Barr virus (Nature, 269
Vol. 420-422, 1977). Problem to be Solved by the Invention Since the presentation of the basic cell fusion technique by Kohler and Milstein, much research has been done on the creation of various hybridomas and the basic or applied research on monoclonal antibodies produced by these hybridomas. efforts have been made (for example,
Annual Rev.Biochem., vol. 50, pp. 657-680.
1981). This document, as well as the documents cited in this document, demonstrate the many advantages obtained by producing monoclonal antibodies from hybridomas, as well as the complexities of its operation and its consequences. The general technique, although conceptually well understood, presents many difficulties in each particular case, thus requiring modifications and uncertainties to resolve it. In fact, when creating a given hybridoma, there is no question whether the desired hybridoma will be obtained, whether it will produce antibodies if it is obtained, or whether the antibodies so produced will have the desired specificity. It is completely difficult to predict whether it will have . The degree of success is primarily influenced by how many viable antibody-producing lymphoid cells are obtained and the selection of tumor cells as parent cells for fusion and the viability of those cells. However, there are still many unknown and uncertain factors that affect success, and it is extremely difficult to create hybridomas that produce monoclonal antibodies with the desired specificity. Particularly in the case of human-human hybridomas, it is difficult to even obtain human antibody-producing fusion cells, and it can be said that creating human-human hybridomas that produce monoclonal antibodies with desired specificity is extremely difficult. B Several approaches have been attempted to produce human monoclonal antibodies against human cancers in vitro, but they have so far been largely unsuccessful. These include, for example: (a) A method using the Epstein-Barr virus to transform antibody-producing lymphocytes of cancer patients. This method has had little success as establishing the transformed cells requires a long and tedious process and is very difficult to clone. (b) Cell fusion of mouse myeloma and antibody-producing lymphocytes from cancer patients. Although this method produces mouse-human hybridomas with excellent proliferative properties similar to mouse-mouse hybridomas, it also has the major disadvantage that this hybrid cell has inherent genetic instability. . In other words, the mouse myeloma sheds the human chromosome, which contains the gene for forming the Katsupar chain, which is important for antibodies, out of the fused cells. Therefore, the possibility of producing a hybridoma that produces human monoclonal antibodies is extremely low. C. Many mouse monoclonal antibodies against human cancer produced by mouse-mouse hybridomas have been created, but since these are foreign to humans, for example, repeated injections into humans may cause shock. There is a risk. Therefore, it is effective to use human-human hybridomas formed by fusing human tumor cells with cells that produce antibodies against human cancer. That is, this hybridoma is capable of producing monoclonal antibodies against human cancers with little risk of shock. D When mass culturing hybridomas in vitro to produce monoclonal antibodies, a medium supplemented with serum (hereinafter referred to as serum medium) such as fetal calf serum (hereinafter referred to as FCS) is generally used as a medium. . However, serum media are not suitable for large-scale culture because serum is expensive and there is variation between lots. Furthermore, since serum consists of dozens or more components and is added in large amounts, it is extremely difficult to purify antibodies secreted into the culture medium. Means and Effects for Solving the Problems The present invention provides antibody-producing cells of lung cancer patients, such as cells isolated from lymph nodes of lung cancer patients, and cells from human tumor cell lines incapable of producing antibodies. The present invention relates to antibodies produced by hybridomas obtained by fusion of Specifically, when measured by indirect fluorescent antibody method from hybridomas obtained by cell fusion, select those that produce antibodies having the characteristics (a) to (c), and compare the antibodies produced by the hybridomas. It is something. (b) Specific fluorescence is observed for lung cancer cells. (b) No specific fluorescence is observed for cells of stomach cancer, kidney cancer, melanoma, and bladder cancer. (c) Does not react with normal cells and no specific fluorescence is observed. In other words, for the problems to be solved mentioned above,
The present invention was made possible by taking the following measures. A Regarding problem A. (a) In order to obtain many viable, desired human antibody-producing lymphocytes, lymphocytes were prepared from lymph nodes removed from lung cancer patients that were particularly contaminated with cancer cells. . The lymphocytes were quickly cultured with the patient's cancer cells, lymphocyte inducing factors, etc., and transformed into blast cells. (b) NAT-30, which has high fusion efficiency as tumor cells used as parent cells for fusion (Patent application No. 58-247772)
No. 1, "Parental cell line for human hybridoma production") and its sublines were used. NAT-30 and its substrains are hypoxanthine-guanine-phosphoribosyltransferase (HGPRT)-deficient mutant cells of Namalva cells, which are human Burkitt lymphoma cells. In addition, in order to make the cells viable, 6-thioguanine was removed from the subculture medium (medium containing 30 μg/ml 6-thioguanine).
Two days before fusion, half of the medium was replaced with fresh medium every day. A human-human hybridoma that produces human monoclonal antibodies specific to desired lung cancer cells was created by (a), (b), etc. B Regarding problem B. We found that cells from stable human tumor cell lines deficient in HGPRT have particularly high fusion efficiency.
We created human-human hybridomas using NAT-30 and its substrains and solved the problems. C Regarding problem C. The present inventors decided to create human-human hybridomas, and (a) used antibody-producing cells of lung cancer patients to
(b) We solved this problem by creating a human-human hybridoma that stably produces human monoclonal antibodies against human lung cancer by fusion with cells from a stable human tumor cell line deficient in HGPRT. . D Regarding problem D. In order to create a system in which hybridomas can proliferate even in a medium to which no serum is added (hereinafter referred to as serum-free medium) and produce monoclonal antibodies, the present inventors developed human tumor cells that proliferate even in serum-free medium. NAT-30 and its sublines were used as parental cells. Hybridomas using these parental cells were grown in serum-free medium in the same manner as the parental cells. And the hybridoma produced monoclonal antibodies in serum-free medium. Examples of serum-free media include (a) Dulbecco's modified medium containing four components: insulin, lactoferrin, ethanolamine, and selenium (hereinafter Dulbecco's modified medium is referred to as DME), (b) lactoferrin-transferred medium. There is one that replaces Erin, and one that adds ECGS to (c), (a), and (b). By using a serum-free medium, it has become possible to culture hybridomas at low cost, and furthermore, it has become possible to purify them very easily. As described above, the lung cancer cell antibody of the present invention is produced using viable human antibody-producing lymphocytes obtained from the lymph nodes of lung cancer patients and NAT-30, which is a human tumor-derived cell with high fusion efficiency. and its substrain, and the resulting hybridoma is produced. Moreover, this hybridoma proliferated not only in serum medium but also in serum-free medium, and continued to stably produce anti-lung cancer cell antibodies. Example 1 Preparation of human antibody-producing lymphocytes Human lymphocytes were selected from lymph nodes removed from lung cancer patients that were particularly contaminated with cancer cells, and immediately added with 10% FCS.
The tissue was cut into pieces in DME, and then the cut tissue was sandwiched and pressed between two glass slides to suspend lymphocytes, cancer cells, and the like. The thus suspended cells were prepared to have 1 to 2 x 10 ⁶ lymphocytes per ml of the above medium, and 10 μg/ml of pokeweed mitogen was added thereto. This was stored in a chemist at 37°C for 2 to 3 days.
% carbon dioxide gas in an incubator. After culturing, lymphocytes were prepared in a conventional manner and used as a human antibody-producing lymphocyte cell fraction. Preparation of NAT-30 cells and sublines thereof NAT-30 cells were prepared as follows. First, Namarpa cells (Dainippon Pharmaceutical) were incubated at 45°C.
Float the cells at a concentration of 1 x 10 ³ cells/ml in a DME + 10% FCS supplemented serum medium containing 0.25% agar, take 5 ml of the cells in a 5 cm dish, and incubate at 37°C in an incubator with 5% carbon dioxide gas and 95% air. The cells were cultured for 3 weeks. Thereafter, 90 clones were grown in a 96-well plate (Nunc) (200μ per well).
culture medium) and cultured in the same manner for 2 weeks in the above serum medium containing no agar. For the purpose of selecting strains incapable of producing antibodies, the culture supernatant from each well was taken and the amount of antibody in the supernatant was measured using an enzyme immunoassay (Katsupel).
As a result, seven cell lines for which no antibodies were detected were selected and sufficiently grown in 96 wells. 24 wells each of the proliferated cell lines (per well
The volume of culture solution was increased by using 1.5 ml of culture medium) and 5 cm shear plates (5 ml of culture medium per plate) to obtain approximately 5 x 10 ⁶ cells each. Leave 3 x 10 ⁴ cells for each strain and suspend the other cells in 15 ml of freshly prepared serum medium, freeze at -5°C and thaw at room temperature, repeating the freezing/thawing process twice to kill the cells. I let it happen. 5 ml of the medium containing the dead cells was transferred to three 5 cm petri dishes, and 1 x 10 ⁴ cells of the remaining unkilled cells were transplanted into each plate. When cultured for 5 days in the same manner as above, most of the cells died, but the surviving cells were collected by centrifugation and each strain was transplanted into 30 wells of a 96-well plate. As a result of culturing for 3 weeks while replacing the medium every 4 to 6 days, 2 out of the 7 strains were found to proliferate. For the two strains, the cells in each of the five wells with the highest growth rate were increased in culture medium volume in the order described above, and each was cultured at approximately 1.
×10 ⁶ cells were obtained. Each cell was collected separately by centrifugation, washed once with DME, and placed in a serum-free medium (ITES medium, Proc. Natl. Acad. Sci. USA, Vol. 79,
1158-1162, 1982), each was suspended in 5 ml. Culture was carried out in the same manner as above for 4 weeks, changing the medium every 4 to 6 days. Three types of cells were selected from those with the highest growth rate when cultured in this serum-free medium, and were allowed to sufficiently proliferate to obtain approximately 1 x 10 ⁷ cells each. Each of these was suspended in 10 ml of the above serum medium containing 3 μg/ml of 6-thioguanine, and cultured for 4 weeks while replacing the medium every 4 days. Thereafter, viable cells were removed and suspended in the same medium containing 30 μg/ml of 6-thioguanine at a concentration of 1×10 ⁵ cells/ml.
The cells were cultured for 4 weeks in the same manner as above. In this way 6
- Obtained a thioguanine-resistant strain. NAT this stock
Named 30. A sub-line of NAT-30 cells is 0.25 NAT-30 cells.
% agar (DME supplemented with 30 μg/ml 6-thioguanine and 10% FCS) at a concentration of 10 ³ cells/ml, and cultured in a 5% carbon dioxide gas incubator at 37° C. for 3 weeks. Among the clones that had proliferated during that time, the one with the best growth was selected and named NAT30-8. NAT-30 and NAT30-8 are subcultured in the above serum medium containing 30 μ/ml 6-thioguanine. Ten days before the fusion, the medium was changed to a serum medium without the addition of 6-thioguanine, and two days before the fusion, half of the medium was replaced with fresh medium every day for culturing. Creation of hybridoma 3x of the above NAT-30 or NAT30-8 cells
10 ⁷ cells and the above human antibody-producing lymphocytes 3-10×
¹⁰⁷ cells were used for cell fusion. Each cell was treated with DME 2
The mixture was washed twice, mixed in a 50 ml centrifuge tube, and spun down at 1200 rpm for 7 minutes. The supernatant was completely removed and the resulting cell pellet was heated to 42.5°C, pre-warmed to 37°C.
% polyethylene glycol (average molecular weight 1500) and 1 ml of DME supplemented with 15% dimethyl sulfoxide was added portionwise over 1 minute. Furthermore, after leaving it at 37℃ for 1 minute, it was preheated to 37℃.
9 ml of DME was gradually added over 5 minutes. 1500rpm
The mixture was centrifuged for 10 minutes and the supernatant was removed. Add 100 ml of DME supplemented with 15% FCS to the obtained cell pellet,
100μ of each was dispensed into well plates. twenty four
After an hour, 100μ of 2x HAT medium (medium prepared by adding 2 x 10 ^-4 M of hypoxanthine, 2 x 10 ^-7 M of aminopterin and 3.2 x 10 ^-5 M of thymidine to the above 15% FCS medium) was added to each well. added to. Thereafter, discard half of the medium every 4 days, add 100μ of HAT medium to each well, and incubate for 4-6 weeks in an incubator containing 7% oxygen and 5% carbon dioxide.
Cultured at ℃. Hybridomas that grew during this period were cultured in HT medium (HAT medium without aminopterin) for another week, and then
Cultured in serum-free and serum media. Although the hybridoma has now passed 7 months, it is still stably producing antibodies. Indirect fluorescent antibody method The following method was used to determine specificity. Cancer cells and normal diploid cells were cultured and allowed to adhere to a 96-well plate or a tissue culture chamber (Lab-Tech) for 1 to 5 days in a 5% carbon dioxide gas incubator at 37°C. After culturing, fix with 0.05% glutaraldehyde or 4% formaldehyde. After fixation, phosphate buffered saline (hereinafter referred to as
Wash three times with PBS (referred to as PBS), add PBS containing 3% bovine serum albumin, and leave at room temperature for 30 minutes.
After leaving to stand, wash 5 times with PBS, add 100μ of culture medium from wells where hybridomas are growing,
Leave in a 5% carbon dioxide incubator at 37°C for 30 minutes. After this, wash 5 times with PBS and add fluorescently labeled (FITC) antibody against goat human antibody (e.g. Wako Pure Chemical Industries, Ltd., Tago) to 20-50 times the amount in PBS.
Dilute with water, add, and leave in a 5% carbon dioxide incubator at 37°C for 30 minutes. After being left to stand, it was washed five times with PBS and immediately observed under a fluorescence microscope. Judgment is given when specific fluorescence is observed in 50% or more of the cells, 25-50% is judged as +, less than 25% is judged as +, and no specific fluorescence is observed in most of the cells. If it is not found, it is marked -. Table 1 shows the results of culturing hybridomas in serum medium and examining the specificity of the monoclonal antibodies produced. As shown in Table 1, Hyb-10-
7 reacted particularly strongly with the lung cancer cell line PC-8, and with other lung cancer cell lines QG-56 and QG-
90 also reacted. There was almost no reaction with other cancer cell lines. It did not react with normal diploid cells. Hyb-4-7 reacted strongly with lung cancer cell lines PC-8, QG-56, and QG-90, but did not react with other cells. Hyb-10-7 and
Hyb-4-7 also reacted with primary cultured lung cancer cells. Hyb-S60, Hyb-7 and Hyb-9 did not react at all in any of the cells. Example 2 The hybridoma of Example 1 was cultured in a serum-free medium, and the specificity of the medium was determined as described in Example 1. The composition of the serum-free medium is insulin-free.
DME contains four components: 10 μg/ml, 25 μg/ml lactoferrin, 10 μM ethanolamine, and 2.5×10 ⁻⁸ M selenium. As is clear from Table 2, the specificity of each monoclonal antibody does not change at all even when cultured in a serum-free medium.

【table】

[Table] Example 3 Hyb-10-7 was cultured in serum and serum-free medium, and the monoclonal antibodies thereof were purified. As shown in Table 3, when cultured in serum-free medium, 0-50% saturated ammonium sulfate fraction and Sepharose
A highly pure monoclonal antibody could be produced only by gel filtration using CL-4B (Pharmacia).

[Table] Effects of the invention We were able to create a human-human hybridoma that produces human monoclonal antibodies that specifically react with lung cancer cells. In addition, since this hybridoma can produce antibodies in serum-free medium,
It was possible to easily obtain inexpensive and highly pure antibodies. The production of such lung cancer-specific human monoclonal antibodies is expected to be useful in medical fields such as cancer immunological research, clinical diagnosis, and immunotherapy, and as a means for purifying cancer-specific antigens. It will be done.

Claims (1)

[Scope of Claims] 1. Produced by a hybridoma formed by the fusion of antibody-producing cells of a lung cancer patient and cells from a human tumor cell line, and measured by indirect fluorescent antibody method, the following: A human monoclonal anti-lung cancer cell antibody exhibiting the reactions of (a), (b), and (c). (b) Specific fluorescence is observed in lung cancer cells. (b) No specific fluorescence is observed for cells of stomach cancer, kidney cancer, melanoma, and bladder cancer. (c) Does not react with normal cells and no specific fluorescence is observed. 2. Claim 1, wherein the cells from the human tumor cell line are hypoxanthine-guanine-phosphoribosyltransferase-deficient mutant cells of Namalva cells, which are human Burkittlin's lymphoma cells.
The human monoclonal anti-lung cancer cell antibody described in Section 1.