Cytotechnology 4: 139-143, 1990. 9 1990Kluwer Academic Publishers. Printed in the Netherlands.
A new human fusion partner, HK-128, for making human-human hybridomas producing monoclonal IgG antibodies Hiroharu Kawahara 1, Koji Yamada 1, Sanetaka Shirahata 2 and Hiroki Murakami 2 1Department of Food Science and Technology; 2Graduate School of Genetic Resources Technology, Faculty of Agriculture, Kyushu University 46-09, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan Received28 December 1989; accepted in revisedform 20 March 1990
Key words: human fusion partner, human-human hybridomas, IgG producer, human monoclonal IgG antibodies Abstract
A hypoxanthine-aminopterin-thymidine (HAT) sensitive human fusion partner cell line, HK-128 was established from a human plasmacytoma line, LICR-LON-HMy2 (HMy2). The HK-128 cells showed a 100% cloning efficiency. Fusion efficiency of HK-128 was so high that one hybridoma cell was produced by fusion of 105 cells of HK-128 with lymphocytes, obtained from lymph nodes of breast cancer patients. About 90% of the resulted hybridomas were IgG producers. The remainder revealed IgM producing activity, which was lost by long term culture. This result indicates that the HK-128 cell line has an advantage for making hybridoma cells producing IgG. Among ca. 7,000 hybridomas obtained by fusion of HK-128 with lymphocytes of a breast cancer patient, we could establish a hybridoma cell line which produced IgG specifically reacting to a human breast cancer cell line, MCF-7.
Introduction
Human monoclonal antibody (MoAb) is useful not only for diagnosis but also for therapy of various human diseases. To produce human-human hybridomas only a small number of human fusion partners are available. Among them, NAT30 (Murakami et al., 1985) and HO-323 (Ohashi et al., 1986) are excellent fusion parmers, because of their high hybridoma acquisition efficiency. Those fusion partner cell lines are HAT sensitive and synthesize a small amount of gchain of IgM molecule, but do not secrete the immunoglobulin. Most stable hybridomas derived from them were IgM producers (Murakami et al., 1985; Hashizume et al., 1987; Aihara et al.,
1988). This result suggested that the gene of immunoglobulin class expressed by the fusion partner cells regulated the immunoglobulin class stably produced in the resultant hybridomas. Thus, we established a new human fusion partner cell, HK-128, from an IgG producing human plasmacytoma cell line (HMy2) for efficient acquisition of IgG producing hybridomas.
Materials and methods
Cells and cell culture A HAT-sensitive human plasmacytoma, LICRLON-HMy2 (HMy2) cells (O'Hare et al., 1982)
140 were used as a starting cell line for establishing a new human fusion partner for acquisition of human-human hybridomas producing IgG. HMy2 cells were inoculated to 96 well culture plates (Nunc, Denmark) at a cell density of 1 cell/well, and wells having single cell were marked 2-3 h after inoculation. The clone that proliferated most quickly was recloned as above. After 5 sequential clonings, a clone having a hundred percent cloning efficiency was isolated and named as HK128, though the original HMy2 gave only 25% of clonally growing cells. The HK-128 ceils were fused with human lymphocytes obtained from either regional lymph node of cancer tissue o r peripheral blood. Reactivity, of the MoAbs produced by the resultant hybridomas was tested against human breast adenocarcinoma line MCF7 (Soule et al., 1973), human stomach adenocarcinoma line MKN-45 (Hojo, 1977), human lung adenocarcinoma line PC-8 (Kuga et al,, 1975) and human normal lung fibroblast WI-38 cells (Hayflick, 1965). These cells were cultured in ERDF medium (Kyokuto Seiyaku, Japan) (Murakami, 1989) supplemented with 10% fetal bovine serum (FBS) (M.A. Bioproduct, ML), under humidified 5% CO2/95% air atmosphere at 37~
Preparation of lymphocytes Human lymphocytes were isolated from lymph nodes of breast cancer patients. The lymph nodes were cut into small pieces and squeezed between two slide glasses to make lymphocytes in suspension. The cells obtained from lymph nodes were washed three times with ERDF, and then used for fusion experiments. Peripheral blood lymphocytes were separated by density gradient centrifugation (Boyum, 1968) using a lymphocyte separation medium, LSM (Organon Teknika Corp, NC).
Fusion procedure After mixing the fusion partner cell line, H K - 1 2 8 (107 cells) with 1 x 107 or 2 x 107 lymphocytes,
the mixture was centrifuged at 200 xg for 5 min. After removing the supematant, 1 ml of 50% (w/v) polyethylene glycol M.W.4000 (PEG) (Merck, West Germany) dissolved in ERDF was added to the cell pellet for 1 min. Then, 9 ml of ERDF was added to the cell suspension t ~ dilute PEG solution, and centrifuged at 200 xg for 5 min. The cells were resuspended in 50 ml of 15% FBS-ERDF, and then 100 ~tl aliquots were inoculated into each well of 96 well culture plates. The fused cells were incubated at 37~ under humidified 5% CO2/7% 02/88% N2 atmosphere, The cells were maintained in 15% FBS-ERDF containing 100 I.tM hypoxanthine, 0.044 I.tM aminopterin, 16 ~tM thymidine (HAT medium) to select hybridomas.
Reactivity of monoclonal antibody (IgG) to cancer cells Reactivity of monoclonal IgG produced by the hybridomas obtained above was tested against cancer cell lines by a modified ELISA (Douillard et al., 1983). Three human cancer cell lines and a normal fibroblast cell line were cultured to confluency in 96 well culture plates, and then fixed with 0.05% glutaraldehyde dissolved in a phosphate-buffered saline (PBS) for 15 min. After washing twice with PBS containing 0.05% tween 20 (TPBS), the fixed cells were incubated with PBS containing 1% bovine serum albumin (BSA) for 1 h to prevent non-specific adsorption of MoAbs. After washing three times with TPBS, those cells were incubated with 50 ~tl each of spent media of hybridomas for 1 h, and then washed three times with TPBS. These cells were incubated with 100 ~tl each of second antibody solution (peroxidase conjugated anti-human IgG; 1: 2000) (TAGO,CA) for 1 h. After washing three times with TPBS, cells were reacted with substrate solution containing 0.003% H202, and 0.3 mg/ml p-2.2'-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in 0.1 M citrate buffer (pH 4.0) for 30 min. The enzyme reaction was stopped by the addition of 100 ].tl of 1.5% succinic acid, and absorbency at 405 nm
141 Table 1. Fusion efficiencies of various parent cells with lymphocytes isolated from lymph nodes or peripheral blood
Parent cells
Origin of lymphocytes
Efficiency (clone/105 parent cells)
HK-128
Breast cancer Thyroid cancer Stomach cancer*
3.8 3.6 2.0
HMy2
Breast cancer
0.1
HO-323
Breast cancer
2.0
Stomach cancer*
1.0
* Lymphocytes separated from peripheral blood of a stomach cancer patient were used for the experiment.
(A405) of the reaction mixture was measured with an EAR400 microplate photometer (SLR-Lab. Austria).
Results
Isolation of ilK-128 cells To'enhance cloning efficiency of HMy2 cells, the cells were cloned repeatedly by the limiting dilution method. Cloning efficiency of HMy2 cells was only 4% in the first cloning. One of the clones obtained by the first cloning was used for the secopd cloning. The cloning efficiency was increased to 10% in the second cloning, 28% in the third, 42% in the fourth, and finally 100% in the fifth cloning. Among the clones having 100% cloning efficiency, the highest proliferating clone was selected and designated as HK-128.
Fusion efficiency of ilK-128 cells Fusion efficiencies of three human fusion part-
ners, HK-128, HMy2, and HO-323 were compared when these parmer cell lines were fused with lymphocytes obtained from lymph nodes or peripheral blood of several cancer patients. As shown in Table 1, fusion efficiency of HK-128 ceils was much higher than that of HMy2, when they were fused with lymph node lymphocytes of breast cancer patients. The fusion efficiency of HK-128 cells was even higher than that of HO323 cells. Similar results were obtained when HK-128 and HO-323 cells were fused with peripheral blood lymphocytes of stomach cancer patients. In this case, fusion efficiency of peripheral blood lymphocytes was half of that of lymph node lymphocytes, both in HK-128 and HO-323 cells.
Ig classes secreted by hybridomas Ig classes of hybridomas derived from two fusion partners (HK-128 and HO-323) are shown in Table 2. Most hybridomas derived from HK-128 cells produced IgG and only 10% of the hybridomas produced both IgG and IgM. Most of the
Table 2. Ig class produced by hybridomas derived from HK-128 and H0-323 cells
Percent of hybridomas secreting Ig Parent cells
HK-128 HO-323
IgG
IgM
IgG +IgM
None
90 0
0 90
10 2
0 8
142 latter clones lost IgM productivity later, but maintained IgG productivity. On the other hand, 90% of the hybridomas derived from HO-323 cells produced solely IgM and only 2% of the hybridomas produced both IgG and IgM. However, IgG productivity of the latter clones was lost by repeated culture.
Screening of hybridomas producing IgG reactive to breast cancer cells
A total of 7000 hybridomas produced by fusion of HK-128 cells and lymph node lymphocytes of breast cancer patients were screened to isolate hybridomas secreting IgG reactive to a human breast cancer cell line, MCF-7 cells. Three clones secreted IgG reactive to MCF-7 cells, and finally one stable clone (HBll-17) was obtained. As shown in Fig. 1, the MoAb reacted with a breast
0.2
(a)
(b)
0
I
0.2
(c)
I
I
'
0.1
I (d}
' i
0 0
i
2
3
4
0
I
D i l u t i o n of a n t i b o d y
2
3
4
( 1 / 2 n)
Fig. 1. Reactivityof HB11-17against three human cancerand a normal fibroblastcell line. Culture supematantof HB11-17 (O) and human serum IgG (O) containing 1 ~tg/ml of IgG were diluted 2 times with PBS containing 1% BSA sequentially,and reacted with (a) MCF-7, (b) PC-8, (c) MKN-45, and (d) WI-38 cells.
cancer cell line, MCF-7 dose-dependently, but did not react with a lung cancer PC-8, a stomach cancer MKN-45, or a normal fibroblast WI-38 cell lines.
Discussion Some human parent cell lines for producing human-human hybridomas have been established since first reported by Olsson et al., (1980), e.g., GM1500-6TG-2 cell line (Croce et al., 1980), UC729-6 cell line (Glassy et al., 1983)9 Recently, Clark et al., (1988) established TK6TGr.p1 cells to generate human-human hybridomas producing anti-idiotype antibodies. Generally, t h e s e cells have low fusion efficiencies of about 0.1-1.0 clone per 105 parent cells9 To solve this problem, such new cell lines as NAT-30 and HO-323 cells (Murakami et al., 1985; Hashizume et al., 1986; Aihara et al., 1988) were established9 However, these two fusion partners originally were IgM producers and most hybridomas derived from them were also IgM secretors. These results suggested that establishment of a new human fusion partner cell line was necessary for efficient acquisition of hybridomas secreting IgG. Since the high hybridoma acquisition efficiency of NAT30 and HO-323 cells seemed to be due to their high cloning efficiency, we tried to enhance cloning efficiency of an IgG-producing human fusion partner HMy2 cells by sequential cloning using the limiting dilution method. The clone obtained after 5 sequential clonings was named HK-128. The HK-128 cells gave 100% cloning efficiency and 40 times higher fusion efficiency than HMy2 cells. This indicates that this procedure is useful for obtaining a fusion partner cell line giving high fusion efficiency9 When HK-128 and HO-323 were fused with human lymphocytes, most hybridomas derived from HK-128 were IgG secretors and those derived from HO-323 were IgM secretors. Small number of clones secreted both IgG and IgM in both cases, but IgM secretion was unstable in hybridomas derived from HK-128 and IgG secretion in those derived from HO-323. Thomas et
143 al., (1988) reported that the hybridomas obtained by fusion of lymphocytes from breast cancer patients with a parent cell line secreting IgG stably produced human IgG MoAbs. These results suggest that Ig class expressed in the fusion partner cell lines and lymphocytes should be matched so that resultant hybridomas can stably maintain their Ig productivity. The MoAb I g G secreted by the hybridoma, HB11-17, was reactive to a breast cancer cell line MCF-7, but not reactive to a lung cancer PC-8, a stomach c a n c e r MKN-45, or a normal fibroblast WI-38. It is now verified that the newly established fusion partner cell line HK-128 can efficiently give hybridomas stably secreting IgG when fused with human lymphocytes. This cell line is also applicable for the acquisition of human hybridomas secreting monoclonal IgG specifically reactive to breast cancer cell line.
References 1. Aihara K, Yamada K, Murakami H, Nomura Y and Omura H (1988) Production of human-human hybridomas secreting monoclonal antibodies reactive to breast cancer cell lines. In Vitro Cell. Develop. Biol. 24: 959-962. 2. Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. (Suppl.) 97: 77-87. 3. Clark SA, James SE and Fitchett M (1988) A new parental cell line for human-human hybridoma production. Cytotechnology 1: 359-363. 4. Croce CM, Linnenbach A, Hall W, Steplewski Z and Koprowski H (1980) Production of human hybridomas secreting antibodies to measles virus. Nature 288: 488489. 5. Douillard JY and Hoffman T (1983) Enzyme-linked immunosorbent assay for screening monoclonal antibody production using enzyme labeled second antibody. Methods in Enzymology 92: 168-174. Academic Press, Inc.
6. Glassy MC, Handley HH, Hagiwara H and Royston I (1983) UC729-6, a human lymphoblastoid B-cell line useful for generating antibodies-secreting human-human hybridomas. Proc. Natl. Acad. Sci. USA 80: 6327-6331. 7. Hashizume S, Murakami H, Kamei M, Hirose S, Shirai T, Yamada K and Omura H (1987) Specificity of anti-polynucleotide monoclonal antibodies from human-human hybridomas. In Vitro Cell. Develop. Biol. 23: 53-56. 8. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37: 614-636. 9. Hojo H (1977) Establishment of cultured cell lines o f human stomach cancer origin and their morphological characteristics. (In Japanese) Niigata Med. J. 91: 737-752. 10. Kuga N, Yoshida K, Seido T, Oboshi S, Koide T, Shimosato Y and Nomura T (1975) Heterotransplantation of cultured human cancer cells and human cancer tissues into nude mice. Gann 66: 547-560. 11. Murakami H, Hashizume S, Ohashi H, Shinohara K, Yasumoto K, Nomoto K and Omura H (1985) Human-human hybridoma secreting antibodies specific to human lung carcinoma. In Vitro Cell. Develop. Biol. 21: 593-596. 12. Murakami H (1989) Serum-free media used for cultivation of hybridomas. Monoclonal Antibodies: Production and Application, pp. 107-141. Alan R. Liss, Inc. 13. O'Hare MJ, Smith CM and Edwards PAW (1982) A new human hybridoma system (LICR-LON-HMy2) and its use in the production of human monoclonal antibodies. In: Peeters H (ed). Protides of Biological Fluids. Colloquium 30, pp. 265-268. Pergamon Press, Oxford. 14. Ohashi H, Hashizume S, Murakami H, Aihara K, Shinohara K and Omura H (1986) HO-323, A human B-lymphoblastoid cell line useful for making human-human hybridomas. Cell Biol. Intl. Rep. 10: 77-83. 15. Olsson L and Kaplan HS (1980) Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity. Proc. Natl. Acad. Sci. USA 77: 54295431. 16. Soule AD, Vazquez J, Long A, Albert S and Brennan M (1973) A human cell line from a pteural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51: 14091416. 17. Thomas BK, Birgitte BR, Carsten R and Jespe r Z (1988) Human-human hybridomas and human monoclonal antibodies obtained by fusion of lymph node lymphocytes from breast cancer patients. Cancer Res. 48: 3208-3214.
Address for offprints: H. Kawahara, Department of Food Science and Technology, Faculty of Agriculture, Kyusyu University, 46-09, 6-10-1 Hakozaki, Higashiku, Fukuoka 812, Japan
A new human fusion partner, HK-128, for making human-human hybridomas producing monoclonal IgG antibodies Hiroharu Kawahara 1, Koji Yamada 1, Sanetaka Shirahata 2 and Hiroki Murakami 2 1Department of Food Science and Technology; 2Graduate School of Genetic Resources Technology, Faculty of Agriculture, Kyushu University 46-09, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan Received28 December 1989; accepted in revisedform 20 March 1990
Key words: human fusion partner, human-human hybridomas, IgG producer, human monoclonal IgG antibodies Abstract
A hypoxanthine-aminopterin-thymidine (HAT) sensitive human fusion partner cell line, HK-128 was established from a human plasmacytoma line, LICR-LON-HMy2 (HMy2). The HK-128 cells showed a 100% cloning efficiency. Fusion efficiency of HK-128 was so high that one hybridoma cell was produced by fusion of 105 cells of HK-128 with lymphocytes, obtained from lymph nodes of breast cancer patients. About 90% of the resulted hybridomas were IgG producers. The remainder revealed IgM producing activity, which was lost by long term culture. This result indicates that the HK-128 cell line has an advantage for making hybridoma cells producing IgG. Among ca. 7,000 hybridomas obtained by fusion of HK-128 with lymphocytes of a breast cancer patient, we could establish a hybridoma cell line which produced IgG specifically reacting to a human breast cancer cell line, MCF-7.
Introduction
Human monoclonal antibody (MoAb) is useful not only for diagnosis but also for therapy of various human diseases. To produce human-human hybridomas only a small number of human fusion partners are available. Among them, NAT30 (Murakami et al., 1985) and HO-323 (Ohashi et al., 1986) are excellent fusion parmers, because of their high hybridoma acquisition efficiency. Those fusion partner cell lines are HAT sensitive and synthesize a small amount of gchain of IgM molecule, but do not secrete the immunoglobulin. Most stable hybridomas derived from them were IgM producers (Murakami et al., 1985; Hashizume et al., 1987; Aihara et al.,
1988). This result suggested that the gene of immunoglobulin class expressed by the fusion partner cells regulated the immunoglobulin class stably produced in the resultant hybridomas. Thus, we established a new human fusion partner cell, HK-128, from an IgG producing human plasmacytoma cell line (HMy2) for efficient acquisition of IgG producing hybridomas.
Materials and methods
Cells and cell culture A HAT-sensitive human plasmacytoma, LICRLON-HMy2 (HMy2) cells (O'Hare et al., 1982)
140 were used as a starting cell line for establishing a new human fusion partner for acquisition of human-human hybridomas producing IgG. HMy2 cells were inoculated to 96 well culture plates (Nunc, Denmark) at a cell density of 1 cell/well, and wells having single cell were marked 2-3 h after inoculation. The clone that proliferated most quickly was recloned as above. After 5 sequential clonings, a clone having a hundred percent cloning efficiency was isolated and named as HK128, though the original HMy2 gave only 25% of clonally growing cells. The HK-128 ceils were fused with human lymphocytes obtained from either regional lymph node of cancer tissue o r peripheral blood. Reactivity, of the MoAbs produced by the resultant hybridomas was tested against human breast adenocarcinoma line MCF7 (Soule et al., 1973), human stomach adenocarcinoma line MKN-45 (Hojo, 1977), human lung adenocarcinoma line PC-8 (Kuga et al,, 1975) and human normal lung fibroblast WI-38 cells (Hayflick, 1965). These cells were cultured in ERDF medium (Kyokuto Seiyaku, Japan) (Murakami, 1989) supplemented with 10% fetal bovine serum (FBS) (M.A. Bioproduct, ML), under humidified 5% CO2/95% air atmosphere at 37~
Preparation of lymphocytes Human lymphocytes were isolated from lymph nodes of breast cancer patients. The lymph nodes were cut into small pieces and squeezed between two slide glasses to make lymphocytes in suspension. The cells obtained from lymph nodes were washed three times with ERDF, and then used for fusion experiments. Peripheral blood lymphocytes were separated by density gradient centrifugation (Boyum, 1968) using a lymphocyte separation medium, LSM (Organon Teknika Corp, NC).
Fusion procedure After mixing the fusion partner cell line, H K - 1 2 8 (107 cells) with 1 x 107 or 2 x 107 lymphocytes,
the mixture was centrifuged at 200 xg for 5 min. After removing the supematant, 1 ml of 50% (w/v) polyethylene glycol M.W.4000 (PEG) (Merck, West Germany) dissolved in ERDF was added to the cell pellet for 1 min. Then, 9 ml of ERDF was added to the cell suspension t ~ dilute PEG solution, and centrifuged at 200 xg for 5 min. The cells were resuspended in 50 ml of 15% FBS-ERDF, and then 100 ~tl aliquots were inoculated into each well of 96 well culture plates. The fused cells were incubated at 37~ under humidified 5% CO2/7% 02/88% N2 atmosphere, The cells were maintained in 15% FBS-ERDF containing 100 I.tM hypoxanthine, 0.044 I.tM aminopterin, 16 ~tM thymidine (HAT medium) to select hybridomas.
Reactivity of monoclonal antibody (IgG) to cancer cells Reactivity of monoclonal IgG produced by the hybridomas obtained above was tested against cancer cell lines by a modified ELISA (Douillard et al., 1983). Three human cancer cell lines and a normal fibroblast cell line were cultured to confluency in 96 well culture plates, and then fixed with 0.05% glutaraldehyde dissolved in a phosphate-buffered saline (PBS) for 15 min. After washing twice with PBS containing 0.05% tween 20 (TPBS), the fixed cells were incubated with PBS containing 1% bovine serum albumin (BSA) for 1 h to prevent non-specific adsorption of MoAbs. After washing three times with TPBS, those cells were incubated with 50 ~tl each of spent media of hybridomas for 1 h, and then washed three times with TPBS. These cells were incubated with 100 ~tl each of second antibody solution (peroxidase conjugated anti-human IgG; 1: 2000) (TAGO,CA) for 1 h. After washing three times with TPBS, cells were reacted with substrate solution containing 0.003% H202, and 0.3 mg/ml p-2.2'-azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt in 0.1 M citrate buffer (pH 4.0) for 30 min. The enzyme reaction was stopped by the addition of 100 ].tl of 1.5% succinic acid, and absorbency at 405 nm
141 Table 1. Fusion efficiencies of various parent cells with lymphocytes isolated from lymph nodes or peripheral blood
Parent cells
Origin of lymphocytes
Efficiency (clone/105 parent cells)
HK-128
Breast cancer Thyroid cancer Stomach cancer*
3.8 3.6 2.0
HMy2
Breast cancer
0.1
HO-323
Breast cancer
2.0
Stomach cancer*
1.0
* Lymphocytes separated from peripheral blood of a stomach cancer patient were used for the experiment.
(A405) of the reaction mixture was measured with an EAR400 microplate photometer (SLR-Lab. Austria).
Results
Isolation of ilK-128 cells To'enhance cloning efficiency of HMy2 cells, the cells were cloned repeatedly by the limiting dilution method. Cloning efficiency of HMy2 cells was only 4% in the first cloning. One of the clones obtained by the first cloning was used for the secopd cloning. The cloning efficiency was increased to 10% in the second cloning, 28% in the third, 42% in the fourth, and finally 100% in the fifth cloning. Among the clones having 100% cloning efficiency, the highest proliferating clone was selected and designated as HK-128.
Fusion efficiency of ilK-128 cells Fusion efficiencies of three human fusion part-
ners, HK-128, HMy2, and HO-323 were compared when these parmer cell lines were fused with lymphocytes obtained from lymph nodes or peripheral blood of several cancer patients. As shown in Table 1, fusion efficiency of HK-128 ceils was much higher than that of HMy2, when they were fused with lymph node lymphocytes of breast cancer patients. The fusion efficiency of HK-128 cells was even higher than that of HO323 cells. Similar results were obtained when HK-128 and HO-323 cells were fused with peripheral blood lymphocytes of stomach cancer patients. In this case, fusion efficiency of peripheral blood lymphocytes was half of that of lymph node lymphocytes, both in HK-128 and HO-323 cells.
Ig classes secreted by hybridomas Ig classes of hybridomas derived from two fusion partners (HK-128 and HO-323) are shown in Table 2. Most hybridomas derived from HK-128 cells produced IgG and only 10% of the hybridomas produced both IgG and IgM. Most of the
Table 2. Ig class produced by hybridomas derived from HK-128 and H0-323 cells
Percent of hybridomas secreting Ig Parent cells
HK-128 HO-323
IgG
IgM
IgG +IgM
None
90 0
0 90
10 2
0 8
142 latter clones lost IgM productivity later, but maintained IgG productivity. On the other hand, 90% of the hybridomas derived from HO-323 cells produced solely IgM and only 2% of the hybridomas produced both IgG and IgM. However, IgG productivity of the latter clones was lost by repeated culture.
Screening of hybridomas producing IgG reactive to breast cancer cells
A total of 7000 hybridomas produced by fusion of HK-128 cells and lymph node lymphocytes of breast cancer patients were screened to isolate hybridomas secreting IgG reactive to a human breast cancer cell line, MCF-7 cells. Three clones secreted IgG reactive to MCF-7 cells, and finally one stable clone (HBll-17) was obtained. As shown in Fig. 1, the MoAb reacted with a breast
0.2
(a)
(b)
0
I
0.2
(c)
I
I
'
0.1
I (d}
' i
0 0
i
2
3
4
0
I
D i l u t i o n of a n t i b o d y
2
3
4
( 1 / 2 n)
Fig. 1. Reactivityof HB11-17against three human cancerand a normal fibroblastcell line. Culture supematantof HB11-17 (O) and human serum IgG (O) containing 1 ~tg/ml of IgG were diluted 2 times with PBS containing 1% BSA sequentially,and reacted with (a) MCF-7, (b) PC-8, (c) MKN-45, and (d) WI-38 cells.
cancer cell line, MCF-7 dose-dependently, but did not react with a lung cancer PC-8, a stomach cancer MKN-45, or a normal fibroblast WI-38 cell lines.
Discussion Some human parent cell lines for producing human-human hybridomas have been established since first reported by Olsson et al., (1980), e.g., GM1500-6TG-2 cell line (Croce et al., 1980), UC729-6 cell line (Glassy et al., 1983)9 Recently, Clark et al., (1988) established TK6TGr.p1 cells to generate human-human hybridomas producing anti-idiotype antibodies. Generally, t h e s e cells have low fusion efficiencies of about 0.1-1.0 clone per 105 parent cells9 To solve this problem, such new cell lines as NAT-30 and HO-323 cells (Murakami et al., 1985; Hashizume et al., 1986; Aihara et al., 1988) were established9 However, these two fusion partners originally were IgM producers and most hybridomas derived from them were also IgM secretors. These results suggested that establishment of a new human fusion partner cell line was necessary for efficient acquisition of hybridomas secreting IgG. Since the high hybridoma acquisition efficiency of NAT30 and HO-323 cells seemed to be due to their high cloning efficiency, we tried to enhance cloning efficiency of an IgG-producing human fusion partner HMy2 cells by sequential cloning using the limiting dilution method. The clone obtained after 5 sequential clonings was named HK-128. The HK-128 cells gave 100% cloning efficiency and 40 times higher fusion efficiency than HMy2 cells. This indicates that this procedure is useful for obtaining a fusion partner cell line giving high fusion efficiency9 When HK-128 and HO-323 were fused with human lymphocytes, most hybridomas derived from HK-128 were IgG secretors and those derived from HO-323 were IgM secretors. Small number of clones secreted both IgG and IgM in both cases, but IgM secretion was unstable in hybridomas derived from HK-128 and IgG secretion in those derived from HO-323. Thomas et
143 al., (1988) reported that the hybridomas obtained by fusion of lymphocytes from breast cancer patients with a parent cell line secreting IgG stably produced human IgG MoAbs. These results suggest that Ig class expressed in the fusion partner cell lines and lymphocytes should be matched so that resultant hybridomas can stably maintain their Ig productivity. The MoAb I g G secreted by the hybridoma, HB11-17, was reactive to a breast cancer cell line MCF-7, but not reactive to a lung cancer PC-8, a stomach c a n c e r MKN-45, or a normal fibroblast WI-38. It is now verified that the newly established fusion partner cell line HK-128 can efficiently give hybridomas stably secreting IgG when fused with human lymphocytes. This cell line is also applicable for the acquisition of human hybridomas secreting monoclonal IgG specifically reactive to breast cancer cell line.
References 1. Aihara K, Yamada K, Murakami H, Nomura Y and Omura H (1988) Production of human-human hybridomas secreting monoclonal antibodies reactive to breast cancer cell lines. In Vitro Cell. Develop. Biol. 24: 959-962. 2. Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. (Suppl.) 97: 77-87. 3. Clark SA, James SE and Fitchett M (1988) A new parental cell line for human-human hybridoma production. Cytotechnology 1: 359-363. 4. Croce CM, Linnenbach A, Hall W, Steplewski Z and Koprowski H (1980) Production of human hybridomas secreting antibodies to measles virus. Nature 288: 488489. 5. Douillard JY and Hoffman T (1983) Enzyme-linked immunosorbent assay for screening monoclonal antibody production using enzyme labeled second antibody. Methods in Enzymology 92: 168-174. Academic Press, Inc.
6. Glassy MC, Handley HH, Hagiwara H and Royston I (1983) UC729-6, a human lymphoblastoid B-cell line useful for generating antibodies-secreting human-human hybridomas. Proc. Natl. Acad. Sci. USA 80: 6327-6331. 7. Hashizume S, Murakami H, Kamei M, Hirose S, Shirai T, Yamada K and Omura H (1987) Specificity of anti-polynucleotide monoclonal antibodies from human-human hybridomas. In Vitro Cell. Develop. Biol. 23: 53-56. 8. Hayflick L (1965) The limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37: 614-636. 9. Hojo H (1977) Establishment of cultured cell lines o f human stomach cancer origin and their morphological characteristics. (In Japanese) Niigata Med. J. 91: 737-752. 10. Kuga N, Yoshida K, Seido T, Oboshi S, Koide T, Shimosato Y and Nomura T (1975) Heterotransplantation of cultured human cancer cells and human cancer tissues into nude mice. Gann 66: 547-560. 11. Murakami H, Hashizume S, Ohashi H, Shinohara K, Yasumoto K, Nomoto K and Omura H (1985) Human-human hybridoma secreting antibodies specific to human lung carcinoma. In Vitro Cell. Develop. Biol. 21: 593-596. 12. Murakami H (1989) Serum-free media used for cultivation of hybridomas. Monoclonal Antibodies: Production and Application, pp. 107-141. Alan R. Liss, Inc. 13. O'Hare MJ, Smith CM and Edwards PAW (1982) A new human hybridoma system (LICR-LON-HMy2) and its use in the production of human monoclonal antibodies. In: Peeters H (ed). Protides of Biological Fluids. Colloquium 30, pp. 265-268. Pergamon Press, Oxford. 14. Ohashi H, Hashizume S, Murakami H, Aihara K, Shinohara K and Omura H (1986) HO-323, A human B-lymphoblastoid cell line useful for making human-human hybridomas. Cell Biol. Intl. Rep. 10: 77-83. 15. Olsson L and Kaplan HS (1980) Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity. Proc. Natl. Acad. Sci. USA 77: 54295431. 16. Soule AD, Vazquez J, Long A, Albert S and Brennan M (1973) A human cell line from a pteural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51: 14091416. 17. Thomas BK, Birgitte BR, Carsten R and Jespe r Z (1988) Human-human hybridomas and human monoclonal antibodies obtained by fusion of lymph node lymphocytes from breast cancer patients. Cancer Res. 48: 3208-3214.
Address for offprints: H. Kawahara, Department of Food Science and Technology, Faculty of Agriculture, Kyusyu University, 46-09, 6-10-1 Hakozaki, Higashiku, Fukuoka 812, Japan
