Cytotechnology 3: 31-37, 1990. 9 1990 Kluwer Academic Publishers. Printed in the Netherlands.
A human-human hybridoma secreting anti-Pseudomonas aeruginosa exotoxin-A monoclonal antibody with highly potent neutralizing activity
Masato Kuriyama, Yuzo Ichimori, Susumu Iwasa and Kyozo Tsukamoto Central Research Division, Takeda Chemical Industries, Ltd., 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532, Japan Received 25 January 1989; accepted in revised form 31 May 1989
Key words: human-human hybridoma, monoclonal antibody, Pseudomonas aeruginosa exotoxin Abstract
A hybridoma secreting human monoclonal antibody (MAB) against Pseudomonas aeruginosa exotoxin A (PEA) was constructed by fusing Epstein-Ban- virus-transformed peripheral blood lymphocytes with human B lymphoblastoid cell line TAW-925. The human-human hybridoma stably produced human IgG2 MAB at the rate of 0.4-0.5 gg/ml per 106 cells per day for more than six months, and the MAB was capable of neutralizing the in vitro cytotoxic and in vivo lethal effects of PEA with approximately 100and 70-fold, respectively, higher activity than serum polyclonal antibody preparations. Abbreviations: MAB - Monoclonal Antibody, PEA - Pseudomonas aeruginosa exotoxin A, LPS Lipopolysaccharides, OMP - Outer Membrane Proteins, P. - Pseudomonas, EBV - Epstein-Ban" Virus, PEG - Polyethylene Glycol
Introduction
Human monoclonal antibodies (MABs) are useful for prophylactic and therapeutic applications against tumors and infectious diseases, and several methods have been developed to produce cell lines secreting such human MABs (Olsson and Kaplan, 1980; Croce et al., 1980; Kozbor and Roder, 1981; Sikora and Wright, 1981; Edwards et al., 1982; Glassy and Ferrone, 1982; Cote et al., 1983; Butler et at., 1983). We also reported the establishment of the excellent parental cell lines HM-5 and TAW-925 giving high fusion frequency, and the subsequent production of (mouse-human) -human and human-human hy-
bridomas secreting antibodies against tetanus toxoid and hepatitis B viral surface antigen (Ichimoil et al., 1985; Ichimori et al. 1987). These hybridomas stably produced human MABs with high affinity to the antigens. Here we applied the method to the construction of human-human hybridomas secreting highly potent neutralizing antibody of Pseudomonas aeruginosa exotoxin A (PEA), using a 6-thioguanine and ouabain resistant human B lymphoblastoid cell line TAW-925 as parental cells. PEA inhibits protein synthesis by ADP-ribosylating elongation factor 2 as in the case of diphtheria toxin or ricin (Iglewski and Kabat, 1975), and exerted harmful effects upon mammalian cells.
32 Murine and human MABs against lipopolysaccharides (LPS) or outer membrane proteins (OMP) extracted from Pseudomonas (P.) aeruginosa were already reported elsewhere and some have been used in passive immunotherapy for patients with cystic fibrosis, thermal burns and cancer (Mutharia et at. 1984; Bogard etal., 1985; Homberger et al. 1985; Sawada et al., 1987). As for the preparation of anti-PEA MABs, several papers have appeared in recent years on murine MABs (Klinger and Shuster, 1984; Galloway et al., 1984; Chia et al., 1986), but few papers have dealt with human ones. Human anti-PEA MABs, however, are expected to be successfully used in combination with anti-LPS or OMP MABs in order to confer complete protection against infection with P. aeruginosa. In this study we have constructed a human-human hybridoma secreting such an anti-PEA MAB with high neutralizing activity.
Materials and methods
Preparation of EBV solution The EBV solution was prepared from the B95-8 marmoset cell line. These cells were seeded at 2 x 105/ml in growth medium [RPMI-1640 medium (Flow Labs. Ltd., VA, USA) containing 10% fetal calf serum (FCS), kanamycin (100 gg/ml), NaHCO 3 (1.8 mg/ml)] and cultured for 8 days in a humidified atmosphere of 5% CO 2 in air. The culture supematant was harvested and filtered through a 0.45 gm membrane filter, and then stored in liquid nitrogen until used.
Transformation of human PBL with EBV Human PBL were prepared from the heparinized blood of healthy donors by centrifugation on Ficoll-Hypaque solution. The PBL were suspended in growth medium at a cell density of 2 x 107/ml and the PBL suspension was incubated with 10 fold volume of the above EBV solution for 1.5 hr at 37~ The PBL suspension was
diluted with the growth medium containing 20% FCS to 2 x 105 cells/ml and seeded in a volume of 100 gl to each well of 96-well microculture plate (Nunc Intermed, Denmark), and then cultured for 18 days.
Fusion procedures The HAT (hypoxanthine, aminopterin, thymidine) sensitive and ouabain resistant B lymphoblastoid cell line TAW-925 was used as a parent cell line. 1 x 107 EBV transformed cells and 1 x 107 TAW-925 cells were mixed, precipitated by centrifugation, and then suspended in 0.3 ml of 45 % polyethylene glycol 6000 (Koch-Light Labs., Ltd., Colnbrook Berks, UK) in basal medium (a mixture consisting of an equal volume of Iscove's medium and Ham F12 medium). After the suspension was incubated for 7 min at 37~ with gentle shaking, 12 ml of prewarmed (37~ basal medium was added at a speed of 2 ml/min. Cells were precipitated again, suspended at 2 x 105 tumor cells/ml in basal medium supplemented with hypoxanthine 10.4 M, aminopterin 4 x 10 -7 M, thymidine 1.6 x 10-5 M, ouabain 5 x 10.7 M, and 10% FCS and seeded in a volume of 100 gl to each well of 96-well microculture plates. The culture media were screened for anti-PEA antibody. Antibody titers were determined by ELISA employing PEA-coated microplates and anti-human IgG-horseradish peroxidase conjugates.
Purification of human MAB An established hybridoma clone P7E9C7, stably secreting anti-PEA MAB, was transferred into and propagated in the basal medium containing 2 gg/ml insulin, 2 gg/ml transferrin, 2 x 10.6 M ethanolamine, 2.5 x 10.8 M sodium selenite and 3 mg/ml GFS (a 55% to 70% ammonium sulphate fraction of serum from adult cattle) (Kitano et al., 1986). The culture supematant harvested was salted-out with 47% saturated ammonium sulphate and the resultant precipitate was dialyzed
33 against 20 mM phosphate buffer (pH 8.0), followed by sequential chromatographies using a DEAE-cellulose column and a Protein A column. The MAB P7E9C7 was determined to be IgG2 isotype by using subclass-specific antisera. /
In vitro neutralizing assay Various concentrations of PEA were mixed with an equal volume of antibody-containing solutions, followed by incubation at 37~ for 60 min. Then the 100 ~tl portions of the mixed solutions were added to each well of a 96-well microculture plate containing 5 • 10 4 BALL-1 cells (human B !ymphoblastoid cell line) in 100 gl and further incubated at 37~ for 5 days in a CO 2 incubator. The number of viable BALL-1 cells was measured by a conventional colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Tada et al., 1986).
In vivo neutralizing assay
mants were then fused with TAW-925 cells using PEG and seeded at 2 x 10 4 cells/well on microplates. The growth of hybridomas was obtained in 744 wells (70%) out of 1056 wells seeded, and 200 wells (19%) gave positive results for antiPEA antibody. One of them showing the highest antibody titer was cloned by a conventional limiting dilution method using mouse thymocytes as a feeder, and a hybridoma, designated P7E9C7, was obtained. The human-human hybridoma gave stable antibody production (0.4-0.5 gg/ml per 10 6 cells per clay) for more than six months.
Neutralizing activity In an in vitro assay BALL-1 cells were used as the target of PEA, and to the target cells (5 • 104/well) were added various amounts of PEA in the absence and the presence of 25 ng of the MAB P7E9C7. As shown in Fig. 1, 25 ng (0.17 pmol) of the MAB almost completely neutralized 20 ng (0.30 pmol) of PEA, and the results suggested that one antibody molecule is capable of neutralizing approximately two toxin molecules.
Serum from a healthy donor infected with P.
aeruginosa was used as PEA-specific polyclonal antibody. Concentration of PEA-specific polyclonal antibody in serum was determined by ELISA using purified anti-PEA MAB as a standard. Various concentrations of antibody solutions were mixed with an equal volume of 0.6 gg/ml PEA and incubated at 37~ for 60 rain. Then 500 gl portions of the preincubated solutions were intraperitoneally injected into ddY mice. Neutralizing activity of the antibodies was evaluated 7 days after the injection in terms of the survival ratio of the recipient mice.
100
75 Q
o
o
50
o\~
25
tl
Results
i
2
i
20
{ 200
P E A (.g~'woH~
Antibody-producing human-human hybridoma Peripheral blood lymphocytes from antibodypositive donors were first transformed with EBV and screened for anti-PEA antibody. Transfor-
Fig. 1. Cytotoxicity of Pseudomonas aeruginosa exotoxin A (PEA) against human B lymphoblastoid cell line BALL-I (5 • 104 cells/well) in the absence (0) and the presence (O) of 25 ng/well human monoclonal antibody P7E9C7.
34 100
/
75 -
o" 50 Z
25
/ tl
0
I
./"
I
I
1
1
10
100
Antibody
,I 1000
(ng/well)
Fig. 2. Neutralizing activity of human monoclonal antibody PTEgC7 (O) and serum polyclonal antibody preparation ( 0 ) against the cytotoxic effect of Pseudomonas aeruginosa exotoxin A (20 ng/well) upon human B lymphoblastoid cell line BALL-1 (5 x 104 cells/well). The concentrations of the serum antibody preparation was expressed as the quantities equivalent to the MAB PTE9CT.
equivalent to the MAB PTE9C7 by ELISA. Various amounts of the sample antibodies were incubated with 20 ng/weI1 of PEA and added to the target BALL-1 cells. The human MAB showed approximately 100-fold higher neutralizing activity than the serum polyclonal antibody preparation tested. In an in vivo study PEA preincubated with antibodies was injected into mice and the survival rates of the recipient mice were estimated (Table 1). Both of the MAB P7E9C7 and the serum polyclonal antibodies significantly neutralized the toxic effect of PEA upon the mice. In these in vivo experiments the LDs0 values were calculated by Probit analysis using SAS procedure (SAS Institute Inc., 1985). The LDs0 of the MAB is 0.08 gg/mouse, and the LDs0 of the serum polyclonal antibodies is 6.0 gg/mouse. Therefore the MAB showed a 70-fold higher activity than the serum polyclonal antibodies.
Chromosomal analyses Another in vitro study was to compare the human MAB with serum polyclonal antibodies obtained from healthy donors infected with P. aeruginosa (Fig. 2). Anti-PEA antibody concentrations in serum were expressed as the quantities
The chromosomes of parental TAW-925 and P7E9C7 were analyzed by the Giemsa and trypsin-Giemsa methods at 6 months after the isolation of the hybridoma. Representative patterns
Table 1. Blocking effects of antibody on the lethal toxicity of Pseudomonas aeruginosa exotoxin A against mice Antibody
Dose b (gg/mouse)
Survivors c at 7 days
Control
0
0/ 4
Monoclonal antibody P7E9C7
0.03 0.O6 0.12 0.60
0/4 1 /4 4/ 4 4/ 4
Serum polyclonal antibodya
3.0 6.0 12.0
0/4 2/4 4/ 4
aConcentration of anti-PEA specific polyclonal antibody in serum was determined by ELISA using purified anti-PEA MAB as standard bVarious amounts of antibodies were incubated with Pseudomonas aeruginosa exotoxin A (0.15 gg/mouse) at 37~ for 60 rain and then injected intraperitoneally into ddY mice. Each group consisted of 4 mice. CNeutralizing activity of the antibodies was evaluated from the number of survivors at 7 days after injection.
35
:i: ¸
Fig. 3. Representative chromosomal patterns of TAW-925 (A) and P7Egc7 (B). The ckromosomes were an',ilysed by the Giemsa and tryp~in-Giemsa me.thods_ TAW-925 c~11had abn0rmaal chromosomes (marker chromosomes) indicated by the ano~s: 3q-, 9q-, 17p-, t(13:?) ~md t(21 "?). P7Egc7 cell also had all these marker chromosomes (indicated by arrows).
36 are shown in Fig. 3. TAW-925 was a diploid cell line with abnormal chromosomes (marker chromosomes) 3q-, 9q-, 17p-, t(13:?) and t(21:?). The P7E9C7 cell was tetraploid and contained all of the marker chromosomes present in TAW-925 cells even after prolonged passage of the cell for as long as 6 months.
Discussion Murine MAB against PEA have been reported before (Chia et aI., 1986; Galloway et al., 1984; Klinger et al., 1984). Those MABs were able to neutralize the cytotoxic activity of PEA in vitro and in vivo. But Murine MABs were recognized by the human immune system as foreign antigens. Therefore human MABs are more useful than murine MABs for prophylactic and therapeutic application against tumors and infectious diseases. Human B lymphoblastoid cell line TAW-925 was used as a parental cell line and fused with EBV-transformed lymphocytes which are capable of secreting antibody. This method gave stable human-human hybridomas with a high fusion frequency as described before (Ichimori et al., 1987). The hybridoma P7E9C7 obtained here stably secreted anti-PEA MAB with good productivity, and the MAB showed strong neutralizing activity in vitro and in vivo, which is approximately 100- and 70-fold, respectively, higher than that of the serum polyclonal antibody preparatio n. Ohtsuka et al. reported an EBVtransformant FK-001 secreting human monoclonal IgM antibody against PEA (Ohtsuka et al., 1985). The FK-001 MAB, however, showed only a slight neutralizing activity in an in vitro cytotoxicity test. Our human MAB P7E9C7, which can be supplied in a large amount, are useful for prophylactic and therapeutic applications as a substitute for serum polyclonal antibody preparations. Anti-PEA human MAB (P7E9C7) and antiLPS human MAB (Sawada et al., 1987) have different effects against P. aeruginosa disease. Anti-PEA MAB neutralizes PEA secreted by P. aeruginosa, in contrast anti-LPS MAB recognizes surface antigen of P. aeruginosa. Therefore
if these MABs are used in a cocktail, they may exert synergistic effect on P. aeruginosa infection.
Acknowledgements We wish to thank Dr. A. Kakinuma of our laboratory for his encouragement and helpful discussions throughout this study. We are also grateful to Miss K. Harada and Mr. Y. Toyoda for their technical assistance. This work was partly supported by Research and Development Project of Basic Technologies for Future Industries from the Ministry of Intemational Trade and Industry, Japan.
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Address for offprints: M. Kuriyama, Central Research Division, Takeda Chemical Industries, Ltd., 17-85, Jusohonmachi 2chome, Yodogawa-ku, Osaka 532, Japan