Cytotechnology 10: 15-23, 1992. 9 1992KluwerAcademicPublishers. Printed in the Netherlands.

Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivity Fusao Makishima l, Satoshi Terada 2, Tadashi Mikami 2 and Eiji Suzuki 2 1Institute for Genetics, University of Cologne, Weyertal 121, W-5000 Cologne 41, Germany; and 2Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan Received 5 February 1992; accepted in revisedform 14 July 1992

Key words: antibody productivity, growth suppression, hybridoma, interleukin-6, specific productivity Abstract Monoclonal antibody production by hybridoma cells at moderately slowed growth states would be favorable for commercial scale production since cells can devote their resources to performing the differentiated function, immunoglobulin production. We found that a purified recombinant human interleukin-6, which had been reported to support or stimulate proliferation of B cell hybridoma/ plasmacytoma cells, suppressed growth of a hybridoma cell line in serum-free medium. In the presence of the interleukin, the growth-suppressed cells were viable for remarkably long periods in batch culture, and after removal of the interleukin from the culture medium, they started to proliferate at their normal growth rate. As the concentration of the interleukin increased in the culture, the growth rate decreased and the specific antibody productivity (antibody production rate per cell) increased to 5-fold of control at 10 U m1-1 (2 ng m1-1) of the interleukin.

Abbreviations: IL-1, 2, and 6: interleukin-1,2, and 6; rhIL-6: recombinant human interleukin-6; MCAb: monoclonal antibody; TNP: trinitrophenyl; unit (U) of interleukin-6 : A unit (U) is equivalent to the amounts of IL-6 which gives one-half maximal IgM secretion by SKW6-CL4 cells ( 1 U m1-1 = 200 pg m1-1).

Introduction For the commercial scale production of monoclonal antibody (MCAb) by hybridoma cells, many efforts have been expended to develop high cell density culture systems. Enhancement of production rate in unit number of viable cells has received scant attention, although it is desirable and important. Interleukin-6 (IL-6) is a pleiotropic cytokine, which induces: final maturation of B cells into

immunoglobulin-secreting cells (Hirano et al., 1985); IL-2 production, proliferation and differentiation of T cells (Takai et al., 1988; Okada et al., 1988; Garman et al., 1987; Lotz et al., 1988); proliferation and differentiation of hematopoietic precursors (Ikebuchi et al., 1987); and production of acute phase proteins by hepatocytes (Andus et al., 1987; Gauldie et al., 1987). IL-6, a glycoprotein, has also been shown to support proliferation of plasmacytomas, B cell hybridomas, myeloma cells, and Epstein Barr virus-transformed B lyre-

16 phoblastoid cells (Van Damme et al., 1987; Van Snick et al., 1988, Kawano et al., 1988; Van Snick et al., 1986; Van Snick et al., 1987; Nordan et al., 1986). Furthermore, Muraguchi et al. (1988) demonstrated that IL-6 is essential for immunoglobulin secretion of all isotypes by B ceils activated with Pokeweed mitogen. Hence, IL-6 would be a potent factor acting on hybridoma cells to increase their specific MCAb productivity. We investigated the effect of a recombinant human interleukin-6 (rhIL-6) on the growth and MCAb productivity of five mouse hybridoma cell lines which were independently obtained and able to grow in serum-free medium for at least one month. We found that four of the five hybridoma cell lines were growth-inhibited in the presence of the rhIL-6. This observation is noticeable, considering that IL-6 has so far been shown as a hybridoma/plasmacytoma growth factor and that growth suppression of hybridoma cells by a naturally-occurring cytokine is a rare phenomenon. Onozaki et al. (1989) reported the growth-suppressive effect of IL-6, whether alone or in synergy with IL-1, on several murine and human myeloid leukemia cell lines. In the presence of the IL-6, these cells were induced to differentiate into macrophages, although the effect varies depending on cell types (Onozaki et al., 1989; Miyaura et al., 1988). A dependence of MCAb production rate on growth rate was predicted and confirmed in certain cases (Dean et al., 1988; Miller et al., 1988). According to the model proposed by Suzuki and Ollis (1990), the MCAb production rate would increase if cell growth were moderately slowed. Therefore, growth-suppressed hybridoma cultures should be favorable for large-scale production of MCAb since the cells may devote their resources to performing the differentiated function, that is, MCAb production. In our hybridoma cultures which were growth-suppressed by the rhIL-6, we examined MCAb secreted into the culture supernatants and found that the specific MCAb productivity of the hybridoma cells increased 5-fold to the control. The hybridoma culture where the rhIL-6 suppresses cell growth would be a model

system for analyzing a growth-associated kinetics of MCAb production.

Materials and methods Reagents

Recombinant human interleukin-6 (5 x 106 units mg -1, purity over 98%) was purchased from Wako Junyaku (Tokyo). Cell line a n d culture conditions

Cell line 2E3 employed throughout this study is a P3U1 (P3X63 AG8U.1) derived mouse hybridoma that was obtained by electric fusion. The hybridoma is a high producer of an IgG1 MCAb specific to a trinitrophenyl(TNP)-hapten. P3U1 myeloma cell line was provided from Japanese Cancer Research Resources Bank (JCRB). The myeloma and hybridoma cells were cultured in a Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; heat inactivated), 0.2% NaHCO 3, 2 mM L-glutamine, 80 gg m1-1 kanamycin sulfate, and 20 mM N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid (HEPES, pH 7.3). 2E3 cells were also maintained in chemically defined serum-free media, ASF103 (Ajinomoto, Tokyo) or SF-O (Sanko Junyaku Co., Tokyo; bovine serum albumin free). Determination o f antibody concentration

The MCAb concentration was measured using ELISA following a procedure described by Engvail (1980). For each sample evaluated, 2-fold serial dilutions with 1% bovine serum albumin (BSA) in phosphate-buffer saline were carried out. The MCAb was bound to plates precoated with TNP-BSA or anti-mouse IgG antibody, and then detected by horseradish peroxidase activity. The plates were read at 5-15 minutes interval with a Sjeia auto reader Model ER-8000 (Sanko Junyaku Co., Tokyo) with a dual wavelength mode which measures peak absorbance at 492 nm

17 and reference absorbance at 630 nm. T h e amount o f M C A b was determined by c o m p a r i n g absorbance with that of k n o w n a m o u n t o f the purified M C A b secreted b y 2E3 ceils.

Cell proliferation assay Cell growth was m e a s u r e d by counting cell n u m bers with Coulter Counter Z M (Coulter Electronics Ltd, UK). Viability o f the cells was determ i n e d by the trypan blue dye exclusion method. Cell proliferation was also studied measuring [3HI thymidine incorporated into D N A with a liquid scintillation counter. With each of serial dilutions o f the rhlL-6, each culture of the 2 x 103 cells in a 0.2 ml well was incubated and then pulsed with 0.1 g C i of [3HI thymidine for 6 hours. All e x p e r i m e n t s were p e r f o r m e d in triplicate.

Neutralization of IL-6 bioactivity

suppressed b y the rhIL-6 (Terada and M a k i s h i m a , unpublished results). H e r e we report the observation obtained with only 2E3 cell line, one of the five lines we tested, because the cytostatic effect o f the rhIL-6 on the cell line is m o r e r e m a r k a b l e than the other cell lines, and because this cell line can be maintained in SF-O m e d i u m for m o r e than 10 months with reproducible results. The representative results in Fig. 1 demonstrate that the rhIL-6 inhibited growth of 2E3 cells in a dosedependent manner. E v e n in the presence of minute a m o u n t ( 2 . 5 - 5 U m1-1 o f rhIL-6, apparent growth inhibition was observed. F r o m the results, cell doubling time was calculated in a period between 39 and 83 hours after the rhIL-6 addition. T h e y were 35 hours and 102 hours w h e n the cells were 10 7 "7

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IL-6 is a growth-suppressive factor for a hybridoma cell line, 2E3 W e investigated the effect o f the rhIL-6 on the cell growth o f five h y b r i d o m a cell lines which were unrelated. The exponentially growing h y b r i d o m a ceils in the serum- and B S A - f r e e m e d i u m (SF-O) were washed, suspended in the m e d i u m , and cultured in 24-well plates with various concentrations purified rhIL-6. A m o n g the five h y b r i d o m a lines tested, we found rhIL-6 inhibited growth of four lines, although their sensitivity to the antiproliferative activity of the rhIL-6 varied f r o m line to line. On the other hand, another line did not respond to the rhIL-6 at all ( M a k i s h i m a and Terada, unpublished). It is noteworthy that one m o u s e m y e l o m a cell line, P3X63.Ag8.653, adapted to the serum-free m e d i u m was also growth-

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m i g h t secrete s o m e s o l u b l e factors w h i c h w o u l d d i m i n i s h the s u s c e p t i b i l i t y to the r h I L - 6 a n d acc o r d i n g l y the s u s c e p t i b i l i t y m i g h t b e l o w e r at h i g h e r cell d e n s i t y . T h e cells w e r e c u l t u r e d at v a r i o u s cell d e n s i t i e s f r o m 1 x 104 to 1 x 105 cells m1-1 w i t h 20 U m1-1 o f the r h I L - 6 . T h e cells at m o s t d o u b l e d in all the c u l t u r e e x a m i n e d , a n d s a t u r a t i o n o f g r o w t h w a s o b s e r v e d e v e n at the l o w cell d e n s i t y a n d m a i n t a i n e d for a l o n g p e r i o d (Fig. 4). T h e s e r e s u l t s i n d i c a t e that the g r o w t h s u p p r e s s i v e effect o f the r h l L - 6 was n o t i n f l u e n c e d b y the cell d e n s i t y . T h i s g r o w t h - s u p p r e s s i v e effect o f the r h I L - 6 on 2E3 was neutralized by anti-IL-6 antibody

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Fig. 3. Removal of rhlL-6 dependent growth suppression by eliminating the IL-6 from culture medium. After cultured in ASF103 medium supplemented with rhlL-6 at 15 U ml-I, 2E3 cells were further cultured in the medium free of IL-6 from day (0) 5, (A) 7, (ll) 9, or (O) 11 of the IL-6 supplemented cultures, respectively. (ZI): Control, cultured in the IL-6 free medium from the beginning.

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Fig. 4. Growth-suppression of 2E3 cells by rhIL-6 at various cell densities. The hybridoma 2E3 cells were cultured starting at various initial cell densities in ASF103 medium supplemented witfi the rhlL-6 at 20 U m1-1.

( T a b l e 1). I n a d d i t i o n , w e o b s e r v e d that a n o t h e r source o f I L - 6 , c u l t u r e s u p e m a t a n t o f a m y e l o m a t r a n s f e c t e d w i t h r h I L - 6 c D N A ( T o h y a m a et al., 1990) s u p p r e s s e d the g r o w t h o f 2E3. T h e s e results c o n f i r m e d that this g r o w t h s u p p r e s s i v e effect was d u e n o t to s o m e c o n t a m i n a n t s c o n t a i n e d i n o u r r h I L - 6 p r e p a r a t i o n , b u t to h u m a n I L - 6 itself.

Increase in specific M C A b productivity o f 2E3 cells W e d e t e r m i n e d the a m o u n t o f M C A b a c c u m u l a t e d i n the s u p e m a t a n t s o f the c u l t u r e s w h o s e g r o w t h data are s h o w n i n Fig. l a , a n d the results are

Table 1. Effect of anti-rhlL-6 antibody on growth-suppression of 2E3 due to rhlL-6 Anti-rhlL-6 Ab (lag m1-1)

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Cell concentration at 44 hours (104 cells m1-1)

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2E3 cells were cultured 1.64 • 104 cells m1-1 with or without rhlL-6 and with or without anti-rh!L-6 goat polyclonal antibody. The cell concentrations were measured at 44 and 106 hours of the cultures.

20 shown in Fig. lb. The cell cultures with low concentration of the rhIL-6 (0.3-10 U m1-1) produced more MCAb than the control culture; in particular, 2.5-5 U ml -I of rhlL-6 enhanced the total batch MCAb production 2-fold. On the other hand, the culture supplemented with 20 U m1-1 of rhIL-6 produced only 50% of MCAb in comparison with the control culture during the first 100 hours of culture. However, the cells could continuously produce the MCAb throughout the batch culture period even after the control culture terminated MCAb production due to cell death. As a result, MCAb accumulated in the culture three times more than the control. From the results shown in Figs. la and lb, we calculated the specific MCAb productivity (gg/ 10-6 cells day) during the period between 39 and 83 hours of the culture where cells in all cultures were growing exponentially. As shown in Fig. 5, the specific MCAb productivity was improved even in the culture with low concentrations of IL-6 (0.3-1.25 U ml-I). In the presence of 2.5-10 U m1-1, the productivity increased four to five times in comparison with the control.

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Fig. 5. Effect of the rhlL-6 on specific M C A b productivity of 2E3 cells. The specific M C A b productivity was calculated for 2E3 cells cultured at various concentrations o f the IL-6, using the cell density and the M C A b concentration data between 39 and 83 hours o f culture time shown in Fig. 1.

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Doubling time (hour) Fig. 6. Relationship between growth rate and M C A b productivity of 2E3 cells cultured with rhIL-6. Growth-rates were represented by doubling times of cell number. (O) Control, free of IL-6, (O) supplemented with IL-6.

Relationship between growth-rate and specific MCAb productivity With the results obtained throughout this work with rhIL-6, the relationship between the growthrate (represented by doubling time of the cell number) and the specific MCAb productivity is shown in Fig. 6. The calculated results fall into two groups. In the first, where doubling times were less than 40 hours, slight decrease in the growth rate by rhIL-6 always brought about a steep increase in the specific productivity. In the second group, where the doubling times were extended to over 40 hours due to the higher dose of the rhlL-6, further increases in the specific MCAb productivity were not achieved. It is notable that extending the doubling time (about 120 hours) did not cause a drop of the specific productivity in comparison with control. This fact supports the notion that rhIL-6 at the concentration examined here is not a strong cytotoxic factor, but a cytostatic factor, to 2E3 cells.

Possible mechanisms of enhanced specific MCAb productivity at slowed growth state The specific MCAb productivities measured be-

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Fig. 7. Dependence of specific MCAb productivity on specific growth rate. Specific MCAb productivities between 0 and 39 hours after addition of rhIL-6 were selected from the data in Fig. 5. Specific growth rates were calculated from the doubling times as In 2/doubling time.

tween 0 and 39 hours after the addition of IL-6 were selected from the data in Fig. 6, and plotted against the specific growth rate in Fig. 7. The plot indicated that the addition of rhIL-6 influenced both the growth rate and the MCAb productivity earlier than 39 hours, presumably in 20 hours. Therefore, there could be a mechanism that could bring the enhanced productivity in 20 hours after treating the cells with rhIL-6 for suppressing the growth. According to Suzuki and Ollis's model, the enhanced productivity at the slowed growth state could be brought by co-functioning of the following two mechanisms. The first is that messenger RNAs encoded for MCAb accumulate in growthsuppressed cells and accordingly MCAb synthesis is accelerated. The second is that cells in growth-suppressed culture accumulate at the late G1 phase of the cell cycle where they reportedly produce MCAb at a rate several fold larger than in the other phases of the cell cycle (Garatun-Tjeldstr et al., 1976). The first mechanism needs two to three days for accumulating twice the amount of mRNA in the cell (Suzuki et al., 1991). Therefore, it cannot fully explain the quick enhance-

ment shown in Fig. 6. The second mechanism can result in quick enhancement of MCAb productivity in a normal cell cycle period (less than 20 hours), although accumulation of the growthsuppressed cells and maximum MCAb synthesis rate at the phase of the cell cycle (possibly late G1) are yet to be experimentally confirmed with this cell line. However, there could be some other mechanisms responsible for this quick enhancement. As an example, we propose that the MCAb production could be enhanced by rhIL-6 independently from growth suppression, although this hypothesis needs to be experimentally proven. Another possible mechanism which is consistent with the quick enhancement of MCAb productivity accompanied by the growth suppression is that cellular resources required for protein synthesis such as ATP, GTP, and ribosomes could be diverted to MCAb synthesis from that of cellular components required for cell growth, resulting in enhanced MCAb synthesis when the growth is suppressed. Flickinger and his coworkers proposed such a mechanism and built a mathematical model simulating ATP allocation between MCAb synthesis and the other cellular reactions (Flickinger et al., 1991).

Conclusions

A hybridoma cell line, 2E3 which can be viably growth-suppressed by rhlL-6 was established. The cell line has reproducibly responded to IL-6 for more than 10 months since they were first established. Applied together with rhIL-6, 2E3 cell line can be a useful tool for elucidating mechanisms of the cell cycle control, especially when it is experimentally compared with two other existing cell lines, F12-28 (Tohyama et al., 1990) whose growth is enhanced by the rhIL-6 and VII H-8 (Reuveny et al., 1986) whose growth is independent of the rhlL-6 (data not shown). The supplementation of rhIL-6 to the cultures of 2E3 cells enhanced specific MCAb productivity 5-fold and their total batch production 3-fold

22 w h i l e it s u p p r e s s e d c e l l g r o w t h . S i n c e the enh a n c e m e n t of specific M C A b productivity and the s u p p r e s s i o n o f g r o w t h o c c u r r e d a l m o s t s i m u l taneously, we proposed two possible mechanisms: t h e first w a s that r h I L - 6 c o u l d h a v e a c t i v a t e d i n t e r c e l l u l a r p a t h w a y s o f a n t i b o d y p r o d u c t i o n inc l u d i n g e x p r e s s i o n o f i m m u n o g l o b u l i n g e n e s ind e p e n d e n t l y f r o m the g r o w t h s u p p r e s s i o n , a n d the s e c o n d w a s that d i v e r s i o n o f a v a i l a b l e c e l l u l a r r e s o u r c e s to s y n t h e s i s o f M C A b f r o m that o f c e l l u lar c o n s t i t u e n t s at the s l o w e d g r o w t h rate c o u l d h a v e h e l p e d the e n h a n c e m e n t . H o w e v e r , t h e s e h y p o t h e s e s n e e d to b e e x p e r i m e n t a l l y t e s t e d yet.

Acknowledgements W e w i s h to t h a n k P r o f H. N i s h i m u r a a n d D r K. K i k u c h i f o r t h e i r e n c o u r a g e m e n t a n d h e l p f u l disc u s s i o n t h r o u g h o u t this w o r k . W e are also g r a t e ful to D r N. T o h y a m a as she o f f e r e d us the c u l t u r e supematant of a myeloma transfected with rhIL-6 c D N A . This w o r k was partly supported by a grant for 'Biodesign Research Program' from RIKEN to F. M a k i s h i m a .

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Address for offprints: S. Terada, Department of Chemical Engineering, Faculty of Engineering, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan.