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Antibody production by human × human hybridomas in serum-free medium
Journal of Immunological Methods, 78 (1985) 271-278
271
Elsevier JIM 03455
Antibody Production by Human × Human Hybridomas in Serum-Free Medium S.P.C. Cole 1,2, E.H. Vreeken and J.C. Roder 3 Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6. Canada
(Received 12 November 1984, accepted 19 December 1984)
Four human x human hybridomas were adapted to growth in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin (BSA/Tf medium). Production of specific monoclonal antibodies was maintained for more than 2 months. Although the maximal cell density achieved was lower than that in serum-supplemented medium, immunoglobulin production was similar or higher when results were expressed on a per viable cell basis. Thus it is feasible to grow human × human hybridomas in serum-free culture and it is possible that this will become the method of choice for large scale production of human monoclonal antibodies. Key words: serum-free medium - human bybridomas - albumin - transferrin
Intrmiuction O n e of the p r o b l e m s r e m a i n i n g in h u m a n m o n o c l o n a l a n t i b o d y t e c h n o l o g y is that o f b u l k p r o d u c t i o n . M u r i n e h y b r i d o m a s m a y be r e a d i l y g r o w n as ascites t u m o r s in syngeneic mice, increasing the yield of a n t i b o d y u p to 1000-fold. W i t h h u m a n h y b r i d o m a s , the use of i m m u n o d e f i c i e n t mice is required to avoid x e n o g r a f t rejection. A b r a m s et al. (1984) have r e p o r t e d that m u r i n e × h u m a n h y b r i d o m a s secreting h u m a n i m m u n o g l o b u l i n grow r e a d i l y in d o u b l y p r i s t a n e - p r i m e d a t h y m i c (nude) mice. I n o u r hands, this a p p r o a c h has not b e e n successful with h u m a n × h u m a n h y b r i d o m a s (Cole et al., 1985; R o d e r et al., 1985). Instead, we a n d others (Truitt et al., 1984) have f o u n d it necessary to passage h u m a n h y b r i d o m a s in n u d e mice as s u b c u t a n e o u s solid tumors, followed b y culture in vitro, b e f o r e i n t r a p e r i t o n e a l i n o c u l a t i o n of the cells in a second n u d e m o u s e to grow as ascites. U n f o r t u n a t e l y , I To whom correspondence should be addressed. 2 Recipient of grants from the Queen's University Dean's Medical Research Council Fund, Ontario Respiratory Disease Foundation and the Ontario Cancer Foundation. 3 Recipient of grants from the Terry Fox Special Initiatives Program of the National Cancer Institute, the Natural Sciences and Engineering Research Council and the Medical Research Council. Abbreviations: FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; RIA, radioimmunoassay; Tf, transferrin; Ig, immunoglobulin; TT, tetanus toxoid; IMDM, Iscove's modified Dulbecco's medium. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
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the volume of ascites fluid per mouse is usually small ( < 2 ml) and the fraction of mice developing ascites is only about one-third. Furthermore, this is rather an expensive and time-consuming procedure and the maintenance of an adequate animal care facility for immunodeficient mice is beyond the resources of many laboratories. Thus ascites production appears limited as a routine method for production of large quantities of human monoclonal antibodies. As an alternative to ascites production, hybridomas may be grown in large scale tissue culture systems. However, to be practical and economically feasible, a serumfree culture system is essential. Elimination of serum would also facilitate purification of human immunoglobulins from spent culture medium. In the present study we describe the adaptation of several human X human hybridomas to growth in serumfree culture. In our system human immunoglobulin production and specific antibody production were maintained for more than 2 months in culture. Thus it is feasible to grow human hybrids in serum-free culture and it is possible that this will be the method of choice for large scale production of human monoclonal antibodies.
Materials and Methods
Source of materials Cells were routinely cultured in the absence of antibiotics in RPMI 1640 medium in 25 cm 2 flasks in a 5% CO 2 atmosphere at 37°C. All cells were checked monthly for mycoplasma contamination using the DAPI (4',6-diamidino-2-phenylindole) DNA-binding assay (Russell et al., 1975) and found to be negative. Bovine serum albumin (RIA grade) (BSA) was obtained from Sigma Chemical Co. and iron-saturated transferrin (Tf) was obtained from Boehringer, Mannheim. Tetanus toxoid and standardized anti-tetanus toxoid antisera were obtained from Connaught Laboratories and prepared as described (Kozbor and Roder, 1981). Human X human hybridomas Human hybridomas B5C and A1H are 2 subclones of a hybridoma obtained from the fusion of Epstein-Barr virus (EBV)-transformed peripheral blood B lymphocytes of a patient with small cell lung cancer and KR-4, our thioguanine- and ouabain-resistant human B lymphoblastoid cell line (Kozbor et al., 1982; Cole et al., 1984). These clones had been in culture for more than a year and had maintained stable antibody production. XY134-1 and XY134-2 are 2 subclones of a human x human hybridoma secreting anti-tetanus toxoid (TT) antibody obtained as described elsewhere (Kozbor et al., 1982). The original anti-TT-producing hybrid was cloned at 1 cell per well and 5 clones producing 0.5 /xg/ml to 5 /xg/ml human anti-TT antibody were obtained. Two clones were selected for adaptation to serum-free culture. The 4 human hybrids were adapted to serum-free culture in a step-wise fashion over a period of 4 - 6 weeks. At the time of testing, the cells had been cultured in 0.5% BSA and 10 /~g/ml transferrin in RPMI 1640 medium for 6-12 weeks. The clones were compared to their counterparts grown in RPMI 1640 supplemented with
273
10% fetal bovine serum (FBS) for cell growth, human immunoglobulin production and specific antibody production. In the case of B5C and A1H, supernatants were tested for reactivity with lung tumor cells and, in the case of XY134-1 and XY134-2, for anti-TT antibody production. f I
Cell growth curves Cultures were seeded at 5 × 104 cells/well in 24-well Costar plates and were counted by trypan blue exclusion using a hemocytometer 3, 4, 6 and 7 days after plating. Triplicate wells were counted on each day. Human immunogiobulin assay For quantitation of human immunoglobulin, polyvinylchloride plates (Falcon 3912) were coated with F(ab')2 fragments of goat anti-human immunoglobulin (Cappel) (10 /~g/ml in 50 mM bicarbonate buffer, pH 9.6) for 1½ h at room temperature. After washing 4 times with phosphate-buffered saline (PBS), RPMI/10% FBS was added to the wells for 1 h to block non-specific binding. After washing, hybridoma supernatants and standards were added and antibodies that bound were detected with heavy chain-specific goat anti-human IgG or anti-IgM conjugated to alkaline phosphatase (1/1000) (Sigma). Color was developed with p-nitrophenylphosphate (Sigma) (1 mg/ml in 50 mM bicarbonate buffer, pH 9.6 and 1 mM MgC12). The enzyme reaction was stopped by the addition of 2 M NaOH. Amounts of immunoglobulin were calculated by interpolation of standard curves (IgG range: 70 ng/ml to 8.6 lag/ml; IgM range: 50 ng/ml to 1 /~g/ml) obtained with affinity-purified human IgG and IgM (Cappel). Tissue culture medium was used as a negative control. In addition, IgG standards were included in anti-/~ chain assays while IgM standards were included in anti-~ chain assays in each experiment to test for cross-reactivity. All sample supernatants were tested in duplicate and at several dilutions. All determinations were performed at least twice in separate assays. Assay for anti-tetanus toxoid monoclonal antibodies For quantitation of anti-TT antibody from hybrids XY134-1 and XY134-2, polystyrene microtiter plates (Dynatech) were coated with 1 t~g/well of "IT in 50 rrrM bicarbonate pH 9.6 overnight at room temperature. After washing the plates 4 times with PBS/0.05% Tween 20, RPMI/10% FBS was added to the wells for 2 h at room temperature to block non-specific binding. After washing, standard anti-TT antibodies (range: 10 ng/ml to 1 btg/ml) and XY134-1 and XY134-2 hybridoma supernatants were added to the wells and incubated overnight at 4°C. The following day the plates were washed and Tr-specific antibody bound was detected by addition of alkaline phosphatase-conjugated heavy chain-specific anti-human immunoglobulin antibody (Sigma) diluted 1/1000 in 0.5% BSA/PBS for 3 h at room temperature. Color was developed with p-nitrophenylphosphate as described above, Negative controls included wells with no antigen and wells with antigen and tissue culture medium. Amounts of anti-'IT antibody were calculated by interpolation of the standard curve and corrected by multiplication by 0.8 since only 80% of the standard antibody is specific for "IT (Kozbor and Roder, 1981).
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Reactivity of hybridoma supernatants with tumor cells S u p e r n a t a n t s from h y b r i d o m a s B5C a n d A I H were screened using an enzymelinked i m m u n o s o r b e n t assay (ELISA) against glutaraldehyde-fixed l u n g t u m o r cells as described previously (Cole et al., 1984). Negative controls i n c l u d e d K R - 4 supern a t a n t (contains IgG) a n d purified IgM ( 1 0 0 / t g / m l ) .
Results Human hybridomas were initially cultured in RPMI 1640 medium containing 10% FBS. The cells were subsequently fed with medium containing decreasing amounts of FBS. The cells were allowed 1 - 2 weeks at a given percentage of FBS before decreasing the FBS once again. At 2½% FBS, the cells generally did very
XYI34-1
BSC
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60
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I 3
I 5
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i I
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I 5
I 7
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80
60
40
20
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Fig. 1. Growth curves of human ×human hybridomas in serum-free and serum-supplemented media. Cultures were set up in 24-well plates at 5 × 104 cells/ml in RPMI 1640 medium supplemented with (a) 0.5% BSA and 10 #g/ml transferrin (BSA/Tf) (O ©) or (b) 10% FBS (e -e). Viable cells were counted using a hemocytometer. Each point represents the mean of cell counts from 3 replicate wells.
275 TABLE I VIABILITY O F H U M A N × H U M A N MENTED MEDIA Days a
Medium b
H Y B R I D O M A S IN S E R U M - F R E E A N D S E R U M - S U P P L E -
% viability ~ ( + SD; n = 3) XY134-1
XY134-2
AIH
B5C
3
BSA/Tf 10% FBS
72 + 1 86+4
68 + 3 97_+2
71 + 2 94+3
86 5:2 95+1
4
BSA/Tf 10% FBS
75 + 1 88 + 2
73 + 5 91 5-1
76 + 4 94 5-1
76 + 2 92 + 3
6
BSA/Tf 10% FBS
58 5:5 84 5- 7
65 5- 3 73 5- 0
65 5-1 85 5:6
65 __3 91 + 3
7
BSA/Tf 10% FBS
41 + 6 795-5
56 + 3 69-t-3
48 5- 2 805-6
48 _+1 84+1
Cultures were set up in 24-well Costar plates at 5 × 10 4 cells/ml per well and tested on the specified days after seeding cultures. b The media compared were 0.5% BSA in RPMI 1640 with 10 ~tg/ml transferrin (BSA/Tf) and RPMI 1640 supplemented with 10% FBS (10% FBS). Viability of triplicate cultures was determined by trypan blue exclusion using a hemocytometer. a
T A B L E II H U M A N I M M U N O G L O B U L I N P R O D U C T I O N A N D REACTIVITY O F A N T I B O D I E S F R O M H U M A N × H U M A N H Y B R I D O M A S IN S E R U M - F R E E A N D S E R U M - S U P P L E M E N T E D M E D I A Clone a
Medium b
ELISA A 4 9 2 n m c Cell line tested d A549
KD-EBV
Immunoglobulin level e IgM (/t g / m l )
IgG (/t g / m l )
Cells/ml f ( × 10 - 6 )
IgM (pg/rrd per 106 cells)
AIH
BSA/Tf 10% FBS
0.359 0.286
0.578 0.431
7.4 7.8
< 0.07 < 0.07
0.52 0,83
14.2 9.4
B5C
BSA/Tf 10% FBS
0.655 0.598
0.861 0.643
14.1 16.5
< 0.07 < 0.07
0,65 1,00
21.7 16.5
a H u m a n hybridomas A I H and B5C are 2 subclones of a hybridoma obtained from the fusion of EBV-transformed B lymphocytes of a small cell lung cancer patient and KR-4. b The media compared are 0.5% BSA in RPMI 1640 with 10 ~tg/ml Tf (BSA/Tf) and R P M I 1640 supplemented with 10% FBS (10% FBS). c Hybridoma supernatants were collected 7 days after plating and tested in an ELISA on glutaraldehyde-fixed cells in 96-well plates. Negative controls (KR-4 supematant, myeloma IgM) gave absorbances of less than 0.030. Values shown are the m e a n of duplicate determinations. d A549 is a h u m a n lung adenocarcinoma cell line and KD-EBV is a cell line derived from peripheral blood lymphoeytes of a small cell lung cancer patient. e Hybridoma s u p e m a t a n t s were collected 7 days after plating, diluted 1 : 10 and 1 : 50 and tested in an ELISA for IgM and IgG content by comparison to affinity-purified standards. The values shown are the mean of duplicate determinations. f The density of viable cells was determined by trypan blue exclusion. The values shown are t h e mean of triplicate cultures.
276 p o o r l y . This effect was not transient since viability c o n t i n u e d to be very low even after 4 weeks in this m e d i u m . By contrast, when transferred to m e d i u m c o n t a i n i n g 1% BSA a n d 10 f f g / m l transferrin, the cells flourished. Even after r e d u c i n g the a m o u n t of BSA to 0.5%, g o o d cell g r o w t h was observed. A f t e r growing the h y b r i d s in B S A / T f m e d i u m for 6 - 1 2 weeks, g r o w t h curves were d e t e r m i n e d a n d the results are shown in Fig. 1. H y b r i d s grew to a m a x i m a l cell d e n s i t y of a b o u t 0.8-1.1 x 10 6 c e l l s / m l in R P M I / 1 0 % F B S while in B S A / T f , the m a x i m a l cell d e n s i t y achieved was generally lower a n d r a n g e d f r o m 4.6 x 105 c e l l s / m l to 6.5 x 10 5 c e l l s / m l . Cell viability was also d e t e r m i n e d a n d the d a t a are s u m m a r i z e d in T a b l e I. It can be seen that, in a d d i t i o n to cessation of growth, cell v i a b i l i t y b e g a n to d i m i n i s h once m a x i m a l cell d e n s i t y was reached. T h e decrease in cell viability was m o r e p r o n o u n c e d in serum-free cultures than in s e r u m - s u p p l e m e n t e d cultures. However, the differences b e t w e e n cell viability in 10% F B S a n d B S A / T f were n o t consistent a m o n g the 4 h y b r i d s tested. P r o d u c t i o n of h u m a n I g G a n d I g M of h y b r i d s in s e r u m - s u p p l e m e n t e d m e d i u m a n d B S A / T f m e d i u m was q u a n t i t a t e d b y E L I S A . T h e c o n c e n t r a t i o n s of h u m a n i m ~ u n o g l o b u l i n s (Igs) were d e t e r m i n e d in culture s u p e r n a t a n t s h a r v e s t e d 6 or 7 d a y s after i n i t i a t i n g fresh cultures. T h e s u p e r n a t a n t s were stored at - 2 0 ° C a n d then tested in an E L I S A . T h e results are p r e s e n t e d in T a b l e s II a n d III. As i n d i c a t e d , Ig p r o d u c t i o n of all 4 h y b r i d o m a s in serum-free m e d i u m was c o m p a r a b l e to o r in the case of XY134-2 higher t h a n that o b t a i n e d in s e r u m - s u p p l e m e n t e d m e d i u m . Specific a n t i b o d y p r o d u c t i o n was also tested in serum-free cultures. S u p e r n a t a n t s f r o m h y b r i d s XY134-1 a n d XY134-2 were tested for a n t i - T T activity a n d the results are shown in T a b l e III. N o significant decreases in anti-q-T a n t i b o d y p r o d u c t i o n in serum-free a n d s e r u m - s u p p l e m e n t e d cultures were found. In fact, w h e n expressed on a p e r viable cell basis, p r o d u c t i o n of a n t i - T T b y XY134-2 in B S A / T f m e d i u m was higher than that in s e r u m - s u p p l e m e n t e d m e d i u m . S u p e r n a t a n t s from h y b r i d o m a s TABLE III HUMAN IMMUNOGLOBULIN PRODUCTION AND ANTI-TETANUS TOXOID ANTIBODY PRODUCTION BY HUMAN x HUMAN HYBRIDOMAS IN SERUM-FREE AND SERUM-SUPPLEMENTED MEDIA Clone a
Medium b Anti-TT antibody c
IgM c
IgG c
(ttg/ml)
(/tg/ml per 10 6 cells)
(/~g/ml) (/~g/ml per 10 6 cells)
(/tg/ml)
XY134-1 BSA/Tf 10% FBS
4.5 2.7
9.6 9.5
25.6 16.2
54.5 57.9
< 0.07 < 0.07
XY134-2 BSA/Tf 10% FBS
1.4 1.5
3.6 1.9
29.6 8.6
75.9 11.4
< 0.07 < 0.07
a XY134-1 and XY134-2 are subclones of a human x human hybridoma secreting anti-TT antibody. b The media compared are 0.5% BSA in RPMI 1640 with 10 #g/ml Tf (BSA/Tf) and RPMI 1640 supplemented with 10% FBS (10% FBS). c Hybridoma supernatants were collected 6 days after plating and tested in an ELISA for anti-TT antibody and IgM and IgG levels. Supernatants were diluted as follows: for anti-TT, 1 : 5 and 1 : 25; for IgM, 1 : 50 and 1 : 100; for IgG, 1 : 25. Values shown are the mean of duplicate determinations.
277
AIH and B5C were tested for reactivity with human lung tumor cell lines and the results are shown in Table II. No significant differences in serum-free and serumsupplemented cultures were observed.
Discussion
In the present study we have shown that human × human hybridomas may be successfully grown in serum-free medium. Furthermore, their capacity to synthesize human Ig is not impaired in such medium. Both growth and immunoglobulin synthesis in serum-free medium are comparable to that in serum-supplemented medium (Fig. 1, Tables II and III) although viability studies indicate that cells do not achieve as high a cell density in serum-free medium (Table I). Nevertheless, when Ig levels are expressed as #g/106 ceils, the production of Ig in both media are similar except in the case of one hybridoma (XY134-2) in which production was higher in BSA/Tf medium. Several investigators have reported growth of human lymphoid cells in serum-free medium. In 2 cases, the cells were cultured in Iscove's modified Dulbecco's medium (IMDM) (Uittenbogaart et al., 1983; Farrant et al., 1984). In a third instance, cells were cultured in a 1 : 1 mixture of Ham's F-12 medium and IMDM (Sharath et al., 1984). Farrant et al. (1984) found that human cells of the B cell lineage had an absolute requirement for exogenous lipids for proliferation, differentiation and antibody production while those of the T cell lineage did not. Sharath et al. (1984) have reported that human B cells are capable of producing Igs in response to polyclonal activation in the absence of exogenous lipids. However, as in our experiments, they did not use delipidated BSA and it may well be that the undelipidated BSA serves as a source of essential lipids. It has been shown in the murine system that hybridomas derived from various myeloma fusion partners differ as to the dependence of their growth on individual components of serum-free medium (Kovar and Franek, 1984). It is not yet known whether the BSA/Tf medium described in this study will support the growth of human hybridomas derived from human fusion partners other than KR-4. A number of laboratories have reported the production of murine monoclonal antibodies in serum-free culture. More recently, Cleveland et al. (1983) have reported murine monoclonal antibody production in protein-free culture medium. Their strategy was to replace serum components with mixtures of trace elements. It is possible that human × human hybrids could be successfully grown in similar protein-free medium. However, our human hybrids grow as suspension cultures and do not thrive well at densities greater than 1 × 106 cells/ml whereas success in the mouse system depended on the ability to grow hybridomas as attached monolayers of high cell density. Nevertheless, studies are currently in progress to examine the feasibility of growing human x human hybridomas in protein-free medium. In addition to facilitating purification of Igs and other secretory products, a protein-free system may be useful in studies aimed at defining the metabolic requirements of human hybridomas and their responses to immunomodulating substances.
278
Acknowledgements The expert technical assistance of I. Louwman is gratefully acknowledged. We thank Dr. A. Lagarde for helpful discussions and C. Jackson for her help in the preparation of the manuscript.
References Abrams, P.G., J.J. Ochs, S.L. Giardina, A.C. Morgan, S.B. Wilburn, A.R. Wilt, R.K. Oldham and K.A. Foon, 1984, J. Immunol. 132, 1611. Cleveland, W.L., I. Wood and B.F. Erlanger, 1983, J. Immunol. Methods, 56, 221. Cole, S.P.C., B.G. Campling, I.H. Louwman, D. Kozbor and J.C. Roder, 1984, Cancer Res. 44, 2750. Cole, S.P.C., D. Kozbor and J.C. Roder, 1985, in: Hybridomas in Biotechnology and Medicine, ed. T.A. Springer (Plenum Press, New York), in press. Farrant, J., C.A. Newton, M.E. North, C. Weyman and M.K. Brenner, 1984, J. Immunol. Methods 68, 25. Kovar, J. and F. Franek, 1984, Immunol. Lett. 7, 339. Kozbor, D. and J.C. Roder, 1981, J. Immunol. 127, 1275. Kozbor, D., A.E. Lagarde and J.C. Roder, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 6651. Roder, J.C., D. Kozbor, S.P.C. Cole, T. Atlaw, B.G. Campling and R. McGarry, 1985, in: Human Hybridomas and Monoclonal Antibodies, eds. E.G. Engelman, S. Foung, J. Larrick and A. Raubitschek (Plenum Press, New York), in press. Russell, W.C., C. Newman and D.H. Williamson, 1975, Nature (London) 253, 461. Sharath, M.D., S.B. Rinderknecht and J.M. Weiler, 1984, J. Lab. Clin. Med. 103, 739. Truitt, K.E., J.W. Larrick, A.A. Raubitschek, D.W. Buck and S.W. Jacobson, 1984, Hybridoma 3, 195. Uittenbogaart, C.H., Y. Cantor and J.L. Fahey, 1983, In Vitro 19, 67.
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Elsevier JIM 03455
Antibody Production by Human × Human Hybridomas in Serum-Free Medium S.P.C. Cole 1,2, E.H. Vreeken and J.C. Roder 3 Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6. Canada
(Received 12 November 1984, accepted 19 December 1984)
Four human x human hybridomas were adapted to growth in serum-free medium consisting of RPMI 1640 supplemented with bovine serum albumin and transferrin (BSA/Tf medium). Production of specific monoclonal antibodies was maintained for more than 2 months. Although the maximal cell density achieved was lower than that in serum-supplemented medium, immunoglobulin production was similar or higher when results were expressed on a per viable cell basis. Thus it is feasible to grow human × human hybridomas in serum-free culture and it is possible that this will become the method of choice for large scale production of human monoclonal antibodies. Key words: serum-free medium - human bybridomas - albumin - transferrin
Intrmiuction O n e of the p r o b l e m s r e m a i n i n g in h u m a n m o n o c l o n a l a n t i b o d y t e c h n o l o g y is that o f b u l k p r o d u c t i o n . M u r i n e h y b r i d o m a s m a y be r e a d i l y g r o w n as ascites t u m o r s in syngeneic mice, increasing the yield of a n t i b o d y u p to 1000-fold. W i t h h u m a n h y b r i d o m a s , the use of i m m u n o d e f i c i e n t mice is required to avoid x e n o g r a f t rejection. A b r a m s et al. (1984) have r e p o r t e d that m u r i n e × h u m a n h y b r i d o m a s secreting h u m a n i m m u n o g l o b u l i n grow r e a d i l y in d o u b l y p r i s t a n e - p r i m e d a t h y m i c (nude) mice. I n o u r hands, this a p p r o a c h has not b e e n successful with h u m a n × h u m a n h y b r i d o m a s (Cole et al., 1985; R o d e r et al., 1985). Instead, we a n d others (Truitt et al., 1984) have f o u n d it necessary to passage h u m a n h y b r i d o m a s in n u d e mice as s u b c u t a n e o u s solid tumors, followed b y culture in vitro, b e f o r e i n t r a p e r i t o n e a l i n o c u l a t i o n of the cells in a second n u d e m o u s e to grow as ascites. U n f o r t u n a t e l y , I To whom correspondence should be addressed. 2 Recipient of grants from the Queen's University Dean's Medical Research Council Fund, Ontario Respiratory Disease Foundation and the Ontario Cancer Foundation. 3 Recipient of grants from the Terry Fox Special Initiatives Program of the National Cancer Institute, the Natural Sciences and Engineering Research Council and the Medical Research Council. Abbreviations: FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; RIA, radioimmunoassay; Tf, transferrin; Ig, immunoglobulin; TT, tetanus toxoid; IMDM, Iscove's modified Dulbecco's medium. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
272
the volume of ascites fluid per mouse is usually small ( < 2 ml) and the fraction of mice developing ascites is only about one-third. Furthermore, this is rather an expensive and time-consuming procedure and the maintenance of an adequate animal care facility for immunodeficient mice is beyond the resources of many laboratories. Thus ascites production appears limited as a routine method for production of large quantities of human monoclonal antibodies. As an alternative to ascites production, hybridomas may be grown in large scale tissue culture systems. However, to be practical and economically feasible, a serumfree culture system is essential. Elimination of serum would also facilitate purification of human immunoglobulins from spent culture medium. In the present study we describe the adaptation of several human X human hybridomas to growth in serumfree culture. In our system human immunoglobulin production and specific antibody production were maintained for more than 2 months in culture. Thus it is feasible to grow human hybrids in serum-free culture and it is possible that this will be the method of choice for large scale production of human monoclonal antibodies.
Materials and Methods
Source of materials Cells were routinely cultured in the absence of antibiotics in RPMI 1640 medium in 25 cm 2 flasks in a 5% CO 2 atmosphere at 37°C. All cells were checked monthly for mycoplasma contamination using the DAPI (4',6-diamidino-2-phenylindole) DNA-binding assay (Russell et al., 1975) and found to be negative. Bovine serum albumin (RIA grade) (BSA) was obtained from Sigma Chemical Co. and iron-saturated transferrin (Tf) was obtained from Boehringer, Mannheim. Tetanus toxoid and standardized anti-tetanus toxoid antisera were obtained from Connaught Laboratories and prepared as described (Kozbor and Roder, 1981). Human X human hybridomas Human hybridomas B5C and A1H are 2 subclones of a hybridoma obtained from the fusion of Epstein-Barr virus (EBV)-transformed peripheral blood B lymphocytes of a patient with small cell lung cancer and KR-4, our thioguanine- and ouabain-resistant human B lymphoblastoid cell line (Kozbor et al., 1982; Cole et al., 1984). These clones had been in culture for more than a year and had maintained stable antibody production. XY134-1 and XY134-2 are 2 subclones of a human x human hybridoma secreting anti-tetanus toxoid (TT) antibody obtained as described elsewhere (Kozbor et al., 1982). The original anti-TT-producing hybrid was cloned at 1 cell per well and 5 clones producing 0.5 /xg/ml to 5 /xg/ml human anti-TT antibody were obtained. Two clones were selected for adaptation to serum-free culture. The 4 human hybrids were adapted to serum-free culture in a step-wise fashion over a period of 4 - 6 weeks. At the time of testing, the cells had been cultured in 0.5% BSA and 10 /~g/ml transferrin in RPMI 1640 medium for 6-12 weeks. The clones were compared to their counterparts grown in RPMI 1640 supplemented with
273
10% fetal bovine serum (FBS) for cell growth, human immunoglobulin production and specific antibody production. In the case of B5C and A1H, supernatants were tested for reactivity with lung tumor cells and, in the case of XY134-1 and XY134-2, for anti-TT antibody production. f I
Cell growth curves Cultures were seeded at 5 × 104 cells/well in 24-well Costar plates and were counted by trypan blue exclusion using a hemocytometer 3, 4, 6 and 7 days after plating. Triplicate wells were counted on each day. Human immunogiobulin assay For quantitation of human immunoglobulin, polyvinylchloride plates (Falcon 3912) were coated with F(ab')2 fragments of goat anti-human immunoglobulin (Cappel) (10 /~g/ml in 50 mM bicarbonate buffer, pH 9.6) for 1½ h at room temperature. After washing 4 times with phosphate-buffered saline (PBS), RPMI/10% FBS was added to the wells for 1 h to block non-specific binding. After washing, hybridoma supernatants and standards were added and antibodies that bound were detected with heavy chain-specific goat anti-human IgG or anti-IgM conjugated to alkaline phosphatase (1/1000) (Sigma). Color was developed with p-nitrophenylphosphate (Sigma) (1 mg/ml in 50 mM bicarbonate buffer, pH 9.6 and 1 mM MgC12). The enzyme reaction was stopped by the addition of 2 M NaOH. Amounts of immunoglobulin were calculated by interpolation of standard curves (IgG range: 70 ng/ml to 8.6 lag/ml; IgM range: 50 ng/ml to 1 /~g/ml) obtained with affinity-purified human IgG and IgM (Cappel). Tissue culture medium was used as a negative control. In addition, IgG standards were included in anti-/~ chain assays while IgM standards were included in anti-~ chain assays in each experiment to test for cross-reactivity. All sample supernatants were tested in duplicate and at several dilutions. All determinations were performed at least twice in separate assays. Assay for anti-tetanus toxoid monoclonal antibodies For quantitation of anti-TT antibody from hybrids XY134-1 and XY134-2, polystyrene microtiter plates (Dynatech) were coated with 1 t~g/well of "IT in 50 rrrM bicarbonate pH 9.6 overnight at room temperature. After washing the plates 4 times with PBS/0.05% Tween 20, RPMI/10% FBS was added to the wells for 2 h at room temperature to block non-specific binding. After washing, standard anti-TT antibodies (range: 10 ng/ml to 1 btg/ml) and XY134-1 and XY134-2 hybridoma supernatants were added to the wells and incubated overnight at 4°C. The following day the plates were washed and Tr-specific antibody bound was detected by addition of alkaline phosphatase-conjugated heavy chain-specific anti-human immunoglobulin antibody (Sigma) diluted 1/1000 in 0.5% BSA/PBS for 3 h at room temperature. Color was developed with p-nitrophenylphosphate as described above, Negative controls included wells with no antigen and wells with antigen and tissue culture medium. Amounts of anti-'IT antibody were calculated by interpolation of the standard curve and corrected by multiplication by 0.8 since only 80% of the standard antibody is specific for "IT (Kozbor and Roder, 1981).
274
Reactivity of hybridoma supernatants with tumor cells S u p e r n a t a n t s from h y b r i d o m a s B5C a n d A I H were screened using an enzymelinked i m m u n o s o r b e n t assay (ELISA) against glutaraldehyde-fixed l u n g t u m o r cells as described previously (Cole et al., 1984). Negative controls i n c l u d e d K R - 4 supern a t a n t (contains IgG) a n d purified IgM ( 1 0 0 / t g / m l ) .
Results Human hybridomas were initially cultured in RPMI 1640 medium containing 10% FBS. The cells were subsequently fed with medium containing decreasing amounts of FBS. The cells were allowed 1 - 2 weeks at a given percentage of FBS before decreasing the FBS once again. At 2½% FBS, the cells generally did very
XYI34-1
BSC
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60
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I 3
I 5
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i I
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I 5
I 7
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AIH
._J --J
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80
60
40
20
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Fig. 1. Growth curves of human ×human hybridomas in serum-free and serum-supplemented media. Cultures were set up in 24-well plates at 5 × 104 cells/ml in RPMI 1640 medium supplemented with (a) 0.5% BSA and 10 #g/ml transferrin (BSA/Tf) (O ©) or (b) 10% FBS (e -e). Viable cells were counted using a hemocytometer. Each point represents the mean of cell counts from 3 replicate wells.
275 TABLE I VIABILITY O F H U M A N × H U M A N MENTED MEDIA Days a
Medium b
H Y B R I D O M A S IN S E R U M - F R E E A N D S E R U M - S U P P L E -
% viability ~ ( + SD; n = 3) XY134-1
XY134-2
AIH
B5C
3
BSA/Tf 10% FBS
72 + 1 86+4
68 + 3 97_+2
71 + 2 94+3
86 5:2 95+1
4
BSA/Tf 10% FBS
75 + 1 88 + 2
73 + 5 91 5-1
76 + 4 94 5-1
76 + 2 92 + 3
6
BSA/Tf 10% FBS
58 5:5 84 5- 7
65 5- 3 73 5- 0
65 5-1 85 5:6
65 __3 91 + 3
7
BSA/Tf 10% FBS
41 + 6 795-5
56 + 3 69-t-3
48 5- 2 805-6
48 _+1 84+1
Cultures were set up in 24-well Costar plates at 5 × 10 4 cells/ml per well and tested on the specified days after seeding cultures. b The media compared were 0.5% BSA in RPMI 1640 with 10 ~tg/ml transferrin (BSA/Tf) and RPMI 1640 supplemented with 10% FBS (10% FBS). Viability of triplicate cultures was determined by trypan blue exclusion using a hemocytometer. a
T A B L E II H U M A N I M M U N O G L O B U L I N P R O D U C T I O N A N D REACTIVITY O F A N T I B O D I E S F R O M H U M A N × H U M A N H Y B R I D O M A S IN S E R U M - F R E E A N D S E R U M - S U P P L E M E N T E D M E D I A Clone a
Medium b
ELISA A 4 9 2 n m c Cell line tested d A549
KD-EBV
Immunoglobulin level e IgM (/t g / m l )
IgG (/t g / m l )
Cells/ml f ( × 10 - 6 )
IgM (pg/rrd per 106 cells)
AIH
BSA/Tf 10% FBS
0.359 0.286
0.578 0.431
7.4 7.8
< 0.07 < 0.07
0.52 0,83
14.2 9.4
B5C
BSA/Tf 10% FBS
0.655 0.598
0.861 0.643
14.1 16.5
< 0.07 < 0.07
0,65 1,00
21.7 16.5
a H u m a n hybridomas A I H and B5C are 2 subclones of a hybridoma obtained from the fusion of EBV-transformed B lymphocytes of a small cell lung cancer patient and KR-4. b The media compared are 0.5% BSA in RPMI 1640 with 10 ~tg/ml Tf (BSA/Tf) and R P M I 1640 supplemented with 10% FBS (10% FBS). c Hybridoma supernatants were collected 7 days after plating and tested in an ELISA on glutaraldehyde-fixed cells in 96-well plates. Negative controls (KR-4 supematant, myeloma IgM) gave absorbances of less than 0.030. Values shown are the m e a n of duplicate determinations. d A549 is a h u m a n lung adenocarcinoma cell line and KD-EBV is a cell line derived from peripheral blood lymphoeytes of a small cell lung cancer patient. e Hybridoma s u p e m a t a n t s were collected 7 days after plating, diluted 1 : 10 and 1 : 50 and tested in an ELISA for IgM and IgG content by comparison to affinity-purified standards. The values shown are the mean of duplicate determinations. f The density of viable cells was determined by trypan blue exclusion. The values shown are t h e mean of triplicate cultures.
276 p o o r l y . This effect was not transient since viability c o n t i n u e d to be very low even after 4 weeks in this m e d i u m . By contrast, when transferred to m e d i u m c o n t a i n i n g 1% BSA a n d 10 f f g / m l transferrin, the cells flourished. Even after r e d u c i n g the a m o u n t of BSA to 0.5%, g o o d cell g r o w t h was observed. A f t e r growing the h y b r i d s in B S A / T f m e d i u m for 6 - 1 2 weeks, g r o w t h curves were d e t e r m i n e d a n d the results are shown in Fig. 1. H y b r i d s grew to a m a x i m a l cell d e n s i t y of a b o u t 0.8-1.1 x 10 6 c e l l s / m l in R P M I / 1 0 % F B S while in B S A / T f , the m a x i m a l cell d e n s i t y achieved was generally lower a n d r a n g e d f r o m 4.6 x 105 c e l l s / m l to 6.5 x 10 5 c e l l s / m l . Cell viability was also d e t e r m i n e d a n d the d a t a are s u m m a r i z e d in T a b l e I. It can be seen that, in a d d i t i o n to cessation of growth, cell v i a b i l i t y b e g a n to d i m i n i s h once m a x i m a l cell d e n s i t y was reached. T h e decrease in cell viability was m o r e p r o n o u n c e d in serum-free cultures than in s e r u m - s u p p l e m e n t e d cultures. However, the differences b e t w e e n cell viability in 10% F B S a n d B S A / T f were n o t consistent a m o n g the 4 h y b r i d s tested. P r o d u c t i o n of h u m a n I g G a n d I g M of h y b r i d s in s e r u m - s u p p l e m e n t e d m e d i u m a n d B S A / T f m e d i u m was q u a n t i t a t e d b y E L I S A . T h e c o n c e n t r a t i o n s of h u m a n i m ~ u n o g l o b u l i n s (Igs) were d e t e r m i n e d in culture s u p e r n a t a n t s h a r v e s t e d 6 or 7 d a y s after i n i t i a t i n g fresh cultures. T h e s u p e r n a t a n t s were stored at - 2 0 ° C a n d then tested in an E L I S A . T h e results are p r e s e n t e d in T a b l e s II a n d III. As i n d i c a t e d , Ig p r o d u c t i o n of all 4 h y b r i d o m a s in serum-free m e d i u m was c o m p a r a b l e to o r in the case of XY134-2 higher t h a n that o b t a i n e d in s e r u m - s u p p l e m e n t e d m e d i u m . Specific a n t i b o d y p r o d u c t i o n was also tested in serum-free cultures. S u p e r n a t a n t s f r o m h y b r i d s XY134-1 a n d XY134-2 were tested for a n t i - T T activity a n d the results are shown in T a b l e III. N o significant decreases in anti-q-T a n t i b o d y p r o d u c t i o n in serum-free a n d s e r u m - s u p p l e m e n t e d cultures were found. In fact, w h e n expressed on a p e r viable cell basis, p r o d u c t i o n of a n t i - T T b y XY134-2 in B S A / T f m e d i u m was higher than that in s e r u m - s u p p l e m e n t e d m e d i u m . S u p e r n a t a n t s from h y b r i d o m a s TABLE III HUMAN IMMUNOGLOBULIN PRODUCTION AND ANTI-TETANUS TOXOID ANTIBODY PRODUCTION BY HUMAN x HUMAN HYBRIDOMAS IN SERUM-FREE AND SERUM-SUPPLEMENTED MEDIA Clone a
Medium b Anti-TT antibody c
IgM c
IgG c
(ttg/ml)
(/tg/ml per 10 6 cells)
(/~g/ml) (/~g/ml per 10 6 cells)
(/tg/ml)
XY134-1 BSA/Tf 10% FBS
4.5 2.7
9.6 9.5
25.6 16.2
54.5 57.9
< 0.07 < 0.07
XY134-2 BSA/Tf 10% FBS
1.4 1.5
3.6 1.9
29.6 8.6
75.9 11.4
< 0.07 < 0.07
a XY134-1 and XY134-2 are subclones of a human x human hybridoma secreting anti-TT antibody. b The media compared are 0.5% BSA in RPMI 1640 with 10 #g/ml Tf (BSA/Tf) and RPMI 1640 supplemented with 10% FBS (10% FBS). c Hybridoma supernatants were collected 6 days after plating and tested in an ELISA for anti-TT antibody and IgM and IgG levels. Supernatants were diluted as follows: for anti-TT, 1 : 5 and 1 : 25; for IgM, 1 : 50 and 1 : 100; for IgG, 1 : 25. Values shown are the mean of duplicate determinations.
277
AIH and B5C were tested for reactivity with human lung tumor cell lines and the results are shown in Table II. No significant differences in serum-free and serumsupplemented cultures were observed.
Discussion
In the present study we have shown that human × human hybridomas may be successfully grown in serum-free medium. Furthermore, their capacity to synthesize human Ig is not impaired in such medium. Both growth and immunoglobulin synthesis in serum-free medium are comparable to that in serum-supplemented medium (Fig. 1, Tables II and III) although viability studies indicate that cells do not achieve as high a cell density in serum-free medium (Table I). Nevertheless, when Ig levels are expressed as #g/106 ceils, the production of Ig in both media are similar except in the case of one hybridoma (XY134-2) in which production was higher in BSA/Tf medium. Several investigators have reported growth of human lymphoid cells in serum-free medium. In 2 cases, the cells were cultured in Iscove's modified Dulbecco's medium (IMDM) (Uittenbogaart et al., 1983; Farrant et al., 1984). In a third instance, cells were cultured in a 1 : 1 mixture of Ham's F-12 medium and IMDM (Sharath et al., 1984). Farrant et al. (1984) found that human cells of the B cell lineage had an absolute requirement for exogenous lipids for proliferation, differentiation and antibody production while those of the T cell lineage did not. Sharath et al. (1984) have reported that human B cells are capable of producing Igs in response to polyclonal activation in the absence of exogenous lipids. However, as in our experiments, they did not use delipidated BSA and it may well be that the undelipidated BSA serves as a source of essential lipids. It has been shown in the murine system that hybridomas derived from various myeloma fusion partners differ as to the dependence of their growth on individual components of serum-free medium (Kovar and Franek, 1984). It is not yet known whether the BSA/Tf medium described in this study will support the growth of human hybridomas derived from human fusion partners other than KR-4. A number of laboratories have reported the production of murine monoclonal antibodies in serum-free culture. More recently, Cleveland et al. (1983) have reported murine monoclonal antibody production in protein-free culture medium. Their strategy was to replace serum components with mixtures of trace elements. It is possible that human × human hybrids could be successfully grown in similar protein-free medium. However, our human hybrids grow as suspension cultures and do not thrive well at densities greater than 1 × 106 cells/ml whereas success in the mouse system depended on the ability to grow hybridomas as attached monolayers of high cell density. Nevertheless, studies are currently in progress to examine the feasibility of growing human x human hybridomas in protein-free medium. In addition to facilitating purification of Igs and other secretory products, a protein-free system may be useful in studies aimed at defining the metabolic requirements of human hybridomas and their responses to immunomodulating substances.
278
Acknowledgements The expert technical assistance of I. Louwman is gratefully acknowledged. We thank Dr. A. Lagarde for helpful discussions and C. Jackson for her help in the preparation of the manuscript.
References Abrams, P.G., J.J. Ochs, S.L. Giardina, A.C. Morgan, S.B. Wilburn, A.R. Wilt, R.K. Oldham and K.A. Foon, 1984, J. Immunol. 132, 1611. Cleveland, W.L., I. Wood and B.F. Erlanger, 1983, J. Immunol. Methods, 56, 221. Cole, S.P.C., B.G. Campling, I.H. Louwman, D. Kozbor and J.C. Roder, 1984, Cancer Res. 44, 2750. Cole, S.P.C., D. Kozbor and J.C. Roder, 1985, in: Hybridomas in Biotechnology and Medicine, ed. T.A. Springer (Plenum Press, New York), in press. Farrant, J., C.A. Newton, M.E. North, C. Weyman and M.K. Brenner, 1984, J. Immunol. Methods 68, 25. Kovar, J. and F. Franek, 1984, Immunol. Lett. 7, 339. Kozbor, D. and J.C. Roder, 1981, J. Immunol. 127, 1275. Kozbor, D., A.E. Lagarde and J.C. Roder, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 6651. Roder, J.C., D. Kozbor, S.P.C. Cole, T. Atlaw, B.G. Campling and R. McGarry, 1985, in: Human Hybridomas and Monoclonal Antibodies, eds. E.G. Engelman, S. Foung, J. Larrick and A. Raubitschek (Plenum Press, New York), in press. Russell, W.C., C. Newman and D.H. Williamson, 1975, Nature (London) 253, 461. Sharath, M.D., S.B. Rinderknecht and J.M. Weiler, 1984, J. Lab. Clin. Med. 103, 739. Truitt, K.E., J.W. Larrick, A.A. Raubitschek, D.W. Buck and S.W. Jacobson, 1984, Hybridoma 3, 195. Uittenbogaart, C.H., Y. Cantor and J.L. Fahey, 1983, In Vitro 19, 67.