Production of lymphotoxin (LTα) and a soluble dimeric form of its receptor using the baculovirus expression system

JOURNALOF IMMUNOLOGICAL METHODS ELSEVIER

Journal of ImmunologicalMethods 168 (1994) 79-89

Production of lymphotoxin (LTa) and a soluble dimeric form of its receptor using the baculovirus expression system P a u l D . C r o w e , T o d d L. V a n A r s d a l e , B a r b a r a C a r l F. W a r e

N. Walter, Kimberly M. Dahms,

Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, USA

(Received 9 July 1993, revised 15 September 1993, accepted 16 September 1993)

Abstract

Human LTa and a fusion protein (p60 : Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/1. Purified p60 : Fc binds human TNF and LTa with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LTa produced in mammalian or insect cells and naturally occurring LTa produced in T cells. The levels of human LTo~ produced in baculovirus-infected insect cells is estimated to be ~ 20 mg/1. Insect cell-derived human LTa is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60 : Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LTc~ and p60 : Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors. Key words: Baculovirus expression; Lymphotoxin; Tumor necrosis factor; Soluble receptor

I. Introduction

T u m o r necrosis factor (TNF, also known as T N F a ) and lymphotoxin ( L T a , previously known as TNF]3) are pleiotropic cytokines which exhibit similar, but non-overlapping spectra of biological activities (Aggarwal, 1990). TNF, a product of * Corresponding author. Tel.: (909) 787-5030; Fax: (909) 7872177. Aboreviations: TNF, tumor necrosis factor; LT, lymphotoxin; MOI, multiplicity of infection; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PMSF, phenylmethylsulfonylfluoride; PMA, phorbol 12-myristate 13-acetate.

activated monocytes and other ceils, plays a central role in inflammation and host defense against infection and malignant disease (Vilcek and Lee, 1991). Dysregulated production of T N F may contribute to the pathology of several disease states including septic shock, cachexia (chronic wasting) and autoimmunity (Beutler and Cerami, 1988; Jacob, 1992). L T a is produced specifically by activated T and B lymphocytes and although it shares inflammatory activities with T N F it is often less potent than T N F (Browning and Ribolini, 1989). In some cases L T a behaves as a partial agonist, suggesting a distinct role in immune function (Andrews et al., 1990). Mem-

0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(93)E0245-D

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Paul D. Croweet al. / Journal of Immunological Methods 168 (1994) 79-89

brane-anchored forms of both TNF and LTa have been observed; however, they utilize distinct mechanisms of attachment to the cell surface. TNF retains a long leader sequence which serves as a membrane anchor (Kriegler et al., 1988). LTa, on the other hand, is found in a heteromeric complex with the structurally related transmembrane protein, LT/3 (Browning et al., 1993). Human LTa has been produced in bacteria (Gray et al., 1984) and mammalian (Browning and Ribolini, 1989) expression systems but incomplete glycosylation or low levels of production limit the usefulness of these systems. Two distinct receptors of ~ 55-60 kDa (TNFR60) and ~ 75-80 kDa (TNFRs0) which bind the secreted forms of TNF and LTa with high affinity are present in different relative levels on a wide variety of cell types (Smith et al., 1990; Schall et al., 1990; Loetscher et al., 1990). The TNFR60 and TNFRs0 receptors show a high degree of amino acid sequence homology in the extracellular domain, whereas the intracellular domains are completely dissimilar. Naturally occurring, soluble TNF binding proteins derived by proteolytic processing of TNFR60 and TNFR80 may function to modulate the biological activities of TNF and LT in vivo by either competitively blocking ligand binding (Gray et al., 1990; Kohno et al., 1990; Lantz et al., 1990; Pennica et al., 1993) or by stabilizing ligand in solution (Aderka et al., 1992). Similarly, recombinant soluble dimeric TNF receptor fusion proteins have been created which bind soluble TNF and LTa with high-affinity and neutralize the biologic activity of these cytokines in vitro and in vivo (Ashkenazi et al., 1991; Lesslauer et al., 1991; Howard et al., 1993). In recent years, a large number of proteins have been produced in insect cells infected with recombinant baculoviruses exploiting high level expression of heterologous genes under control of the baculovirus polyhedrin promoter (O'Reilly et al., 1992). In contrast to protein expression in bacterial systems, a major advantage of baculovirus system is that insect cells appear to perform many of the posttranslational modifications seen in mammalian cells. To facilitate studies of secreted and membrane-anchored forms of TNF

and LT, we have constructed a soluble dimeric TNF receptor fusion protein comprised of the extracellular ligand-binding domain of the 60 kDa TNF receptor fused to the Fc portion of human IgG1 heavy chain (p60:Fc). We show here that p60:Fc, expressed to high levels utilizing a baculovirus expression system and purified by affinity chromatography, recognizes both recombinant and naturally occurring LTa. We also show that human LTa overexpressed using the baculovirus system is biologically active and antigenically similar to naturally occurring LTa. This production system provides sufficient tools for studying this complex cytokine system. 2. Materials and methods 2.1. Cell lines and reagents

The human T cell hybridoma, II-23.D7 (Ware et al., 1986) and the murine fibrosarcoma cell line, L929, were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics (penicillin and streptomycin, 100 /zg/ml) as described (Ware et al., 1991). COS-7 cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine and antibiotics. The insect cell lines BTI Tn 5B1-4 and Sf9, kindly provided by JRH Biosciences, were cultured in ExCell 401 serum-free medium (JRH Biosciences, Lenexa, KS) containing 10 /zg/ml gentamycin. Purified, recombinant TNF from E. coli and LTa from CHO cells were gifts from Dr. J. Browning, Biogen (Browning and Ribolini, 1989). The MAb 9B9 (mouse IgG1, made using human LTa produced by E. coli as an immunogen) was purchased from BoehringerMannheim. PMA (LC Services, Woburn, MA) was used at a concentration of 25 n g / m l to activate II-23.D7 cells to produce LTa. 2.2. Construction o f p60: Fc and production of recombinant baculoviruses

The p60:Fc fusion protein was made from a 640 bp NotI-BanI fragment encoding the entire extracellular domain of the 60 kDa TNF receptor (TNFR60) and a 710 bp BglII-NotI fragment encoding the CH2 and CH3 domains and part of

Paul D. Croweet aL /Journal of ImmunologicalMethods 168 (1994) 79-89 the hinge region (amino acid residues 231-447) of human IgG1. These restriction fragments were joined at the BanI and BgllI sites using the synthetic oligonucleotides: 5'-GCACCACAGAGCCCA-3' and 5'-GATCTGGGCTCTGTG3' which preserve the coding of TNFR60 up to the transmembrane region. The chimeric p60:Fc coding fragment was ligated into the mammalian shuttle vector pDC302 (Mosley et al., 1989) at the NotI site and used for transient transfection of COS-7 cells. The p60 :Fc-encoding cDNA was excised from pDC302 as a 1.4 kb NotI-XbaI restriction fragment and ligated into the baculovirus transfer vector pVL1932 (Invitrogen, San Diego, CA) adjacent to the polyhedrin promoter for transfection of insect cells as described below. A 1.4 kb Notl-BamHI restriction fragment encoding human LTo~ (Asn-26 version isolated from a RPMI 1788 cDNA library (Browning and Ribolini, 1989)) was excised from pCDM8-pLT1 (Biogen) and ligated into pVL1393 (Invitrogen). Monolayer cultures of Tn 5B1-4 cells were co-transfected with pVL1392-p60:Fc or pVL 1393-LTa and linearized, modified Autographa californica nuclear polyhedrosis virus (AcNPV) genome (BaculoGold, Pharmingen, San Diego, CA) using cationic liposomes (LipofectACE, Gibco). Briefly, 1 /zg pVL1392-p60 : Fc or pVL1393-LTa and 250 ng BaculoGold DNA were mixed in 3 ml ExCell 401 with 10 ~1 LipofectACE for 15 min at room temperature to allow liposome-DNA complexes to form. The mixture was added to a monolayer of 1.25 × 106 ceils in T25 tissue culture flasks and incubated for 7 days at 27°C. The resulting viral supernatants were amplified by two successive rounds of infection at an MOI of 0.1.

2.3. Plaque purification of recombinant uirus Amplified supernatants were plaque-purified to identify clonal recombinant viruses and titered by end-point dilution. Briefly, ten-fold dilutions of viral supernatants were used to infect 5 X 10 5 Tn 5B1-4 ceils in 60 mm dishes for 1 h at room temperature. The viral supernatant was removed and replaced with 4 ml of ExCell 401 containing 2% FBS and 1.5% low-melt agarose (SeaPlaque FMC, Rockland, ME). Once the agarose hard-

81

ened, the cells were incubated in a humidified chamber for 7 days. Individual plaques removed with a sterile pasteur pipet were eluted from the agarose plug by incubation overnight with gentle rocking and used to reinfect Tn 5B1-4 cells as described above. After two successive amplifications, the viral supernatants were titered by incubating 5000 cells per well in 96-well microtiter plates (Nunc) with dilutions of virus supernatant for 7 days at 27°C. Infected wells were scored by visual examination for cytopathic effects (cell fusion and enlargement) using a light microscope and virus titer calculated by the method of Reed and Muench (Reed and Muench, 1938).

2. 4. Transfection and biosynthetic labeling of COS7 cells COS-7 ceils (2 x 105) seeded in 60 mm dishes and cultured overnight were rinsed with PBS and incubated with 2 ml of serum-free DMEM containing 1 /zg pDC302-p60:Fc and 10/M LipofectACE. After 5 h at 37°C, 2 ml of DMEM containing 20% FBS was added. The following day, the medium was replaced with 4 ml fresh complete DMEM and 2 days later, the cells were pulselabeled with 35S-Cys/Met (Express label, NEN DuPont, Wilmington, DE) at a concentration of 0.2 m C i / m l in Cys/Met-deficient medium containing 10% dialyzed FBS. After 3 h at 37°C, an aliquot of the culture medium was removed for precipitation using protein G-Sepharose (GammaBind G, Pharmacia).

2.5. Biosynthetic labeling of infected insect cells Recombinant virus was used to infect Tn 5B1-4 or Sf9 cells (105 cells/cm 2) for 1 h at a multiplicity of infection (MOI) of 10. 24 h later cells were rinsed with PBS and incubated in ExCell 401 lacking methionine for 1 h prior to the addition of 35S-met/cys labeling mixture at a concentration of 0.2 mCi/ml. After 12-15 h, supernatants were collected and the cells lysed in PBS containing Ca 2+ and Mg 2+, 1 mM EDTA, 1% NP-40, 0.6% deoxycholate and 0.1% SDS. Supernatants and lysates were clarified by centrifugation for 10 min 12,000 x g. Immunoprecipitation of antigens and SDS-PAGE analysis were performed as previously described (Ware et al., 1991).

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Paul D. Croweet aL/Journal of lmmunological Methods 168 (1994) 79-89

Z 6. Affinity purification of p60: Fc Culture medium from Tn 5B1-4 cells infected with p60 : Fc virus was collected 5 days postinfection (p.i.) for purification of the fusion protein. The conditioned medium, adjusted to pH 6.4 with Tris-HCl was centrifuged and filtered to remove dead cells and the protease inhibitors PMSF (100 /xg/ml) and leupeptin (0.5 /xg/ml) were added. The protein was purified using a protein G-Sepharose affinity column (HiTrap, Pharmacia) equilibrated with PBS containing 20 mM Tris-HC1 pH 6.4 (TPBS). The column was washed with TPBS containing 1% NP-40 and bound p60:Fc eluted in a buffer containing 20 mM glycine, pH 3 and 150 mM NaC1 which was immediately brought to neutral pH with 1 M Tris pH 8.8. The protein concentration in each column fraction was determined by measuring the absorbance at 280 nm. Peak fractions were pooled and the protein concentration determined using a Coomassie dye protein assay reagent (Pierce Chemicals, Rockford, IL). 2. 7. TNF binding assays Production of p60 : Fc was quantitated using a ~25I-TNF binding assay. Dilutions of supernatants from infected cells were added in duplicate to plastic snap-wells (Immulon 2, Dynatech, Chantilly, VA) previously coated with goat anti-human Ig (500 ng/well) and allowed to bind for 1 h at room temperature. TNF, radioiodinated to a specific activity of 16.8 mCi//zg by the IodoGen method (Markwell and Fox, 1978) was used at a concentration of 5 nM and incubated for 1 h with the p60:Fc bound to plastic. Unbound ~25I-TNF was removed by washing four times with PBS0.02% Tween 20. Bound 125I-TNF in individual wells was detected using a gamma counter. Competition binding assays were performed using 1 ng/well p60:Fc. Graded amounts of CHO-LTa or TNF were incubated in the presence of ~zsITNF (0.5 nM) for 1 h. Wells were washed and counted as above. Non-specific binding was determined in the absence of p60:Fc. Non-linear least-squares regression analysis for curve fitting was performed using the Marquardt algorithm in

GraphPAD InPlot v. 3.0 (GraphPAD Software, San Diego, CA) 2.8. Cytotoxicity assay The L T a neutralizing activity of p60:Fc and the cytolytic activity of L T a were determined using a colorimetric assay for cell viability as described (Green et al., 1984). Briefly, L929 cells (2 × 104/well in 96-well flat-bottom microtiter plates) were incubated overnight at 37°C in complete medium containing 0.5 p~g/ml mitomycin C. Serial dilutions of CHO-LTa were added to the cells in duplicate and incubated 16-20 h at 37°C. A 20 /zl aliquot of 3-(4,5-dimethyl-thiazol2-yl)-2,5-diphenyltetrazolium bromide (MTI') dye (5 m g / m l in PBS) was added to each well and the cells incubated for 4 h at 37°C. The medium was removed by aspiration and the MTT formazan crystals solubilized in acidified isopropanol. The OD570 of each well was measured with a microplate spectrophotometer (Bio-Rad). The percent cytotoxicity was calculated using the following equation: % cytotoxicity = (1

-ODtreated/ODcontrol) )< 100

3. Results

3.1. Biosynthesis of p60:Fc in mammalian and insect cells The cDNA encoding the human TNF receptor-Ig fusion protein (p60:Fc) in pVL1392 was co-transfected with baculovirus DNA into cultured Tn 5B1-4 cells to obtain recombinant virus which was subsequently used to infect Tn 5B1-4 cells for protein production. Under non-reducing conditions, a major polypeptide band of ~ 110 kDa was precipitated from culture supernatants of p60: Fc baculovirus-infected Tn 5B1-4 cells biosynthetically labeled with 35S-Cys/Met using immobilized protein G (Fig. 1, lane 3). Under reducing conditions (lane 4), a single polypeptide band of ~ 55 kDa corresponding to the predicted molecular mass of p60:Fc monomer was seen demonstrating that Tn 5B1-4 secrete p60 : Fc as a disulfide-linked homodimer. In the cell-associated extracts, polypeptide bands of ~ 100-120 kDa and ~ 50-65 kDa were observed under

Paul D. Crowe et aL /Journal of lmmunological Methods 168 (1994) 79-89

non-reducing (lane 1) and reducing (lane 2) conditions respectively. The greater size heterogeneity of the cell-associated protein bands most likely represents glycosylation intermediates of the mature protein secreted by these cells. The minor bands below the 46 kDa marker and at ~ 200 kDa in cell extracts are nonspecific. In comparison, polypeptide bands of identical molecular weight were observed in cell extracts of biosynthetically labeled COS-7 cells transiently transfected with p60: Fc eDNA (Fig. 1, lanes 5 and 6) demonstrating that processing of p 6 0 : F c fusion protein is similar in mammalian and insect cells. Of practical note, a difference in the spe-

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Fig. 2. Kinetics of p60 : Fc production by Tn 5B1-4 insect cells. Cells were infected with p60: Fc virus at an MOI of 10. At the indicated time points, an aliquot of the culture supernatant was removed and p60:Fc protein production quantitated by solid phase lzsI-TNF binding assay as described in the materials and methods section. cific activity of biosynthetically labeled protein produced by COS-7 and Tn 5B1-4 cells necessitated a longer starvation period and labeling time for the insect cells and was most likely due to a large methionine pool in the insect cells and not to lower protein production (see below).

3.Z Kinetics of expression and purification of p60 : Fc

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Fig. 1. Expression of p60:Fc in insect and mammalian cells. Left panel: Tn 5B1-4 insect cells (2.5X106) infected with p60:Fc baculovirus were labeled with 35S-Cys/Met for 15 h. Right panel: COS-7 cells (5 X 105) transiently transfected with 1/xg pDC302-p60 : Fc were labeled with 35S-Cys/Met for 2 h. Protein G-Sepharose precipitates from whole cell extracts (lanes 1, 2, 5 and 6) or culture supernatants (lanes 3 and 4) were subjected to SDS-PAGE analysis under non-reducing (lanes 1, 3 and 5) or reducing (lanes 2, 4 and 6) conditions. Labeled proteins were visualized by autoradiography (24 h exposure) of the dried gels using intensifying screens.

An examination of the time course of p 6 0 : F c production by baculovirus-infected Tn 5B1-4 cells (MOI = 10) revealed that secreted protein levels peaked at a concentration of ~ 2 / z g / m l between 4 and 5 days postinfection (Fig. 2) as determined by the ~zSI-TNF binding assay. Similar kinetics and levels of protein production were seen with Sf9 insect cells (data not shown). In comparison, p60: Fc production in COS-7 cells never exceeded 50-100 n g / m l (data not shown). Using protein G affinity chromatography, p60 : Fc was purified in a single step from serumfree culture supernatants of recombinant baculovirus-infected insect cells. From a typical infection yielding 2-3 mg/1 p60:Fc, > 97% recovery was routinely achieved by this method. The product analyzed by SDS-PAGE was estimated to be > 95% pure by Coomassie brilliant blue R staining (Fig. 3). As predicted from biosynthetic labeling analysis, polypeptide bands of ~ 110 and

Paul D. Croweet aL /Journal of Immunological Methods 168 (1994) 79-89

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46

12SI-TNF (0.5 nM) binding to p60:Fc (1 ng/well bound to goat anti-human Ig). Data presented are percent of maximum specifically bound IaSI-TNF at each concentration of competing ligand. Non-specific binding was < 10% of the total cpm bound.

30

b i o a s s a y d e s c r i b e d in the m a t e r i a l s a n d m e t h o d s section ( G r e e n et al., 1984). T h e c o n c e n t r a t i o n of p 6 0 : F c which inhibits 50% of t h e m a x i m u m cytolytic activity (ICso) of 2 n M T N F was ~ 0.5 n M (Fig. 5). A t T N F c o n c e n t r a t i o n s of 0.2 n M a n d 0.02 n M t h e p 6 0 : F c IC50 was ~ 150 p M a n d 15 p M , respectively. W h e n t h e cytotoxicity of 0.2 a n d 2 n M T N F is p l o t t e d vs. t h e m o l a r ratio o f p60 : F c to T N F (Fig. 5, inset) a r a t i o of ~ 1.5 : 1 is o b t a i n e d at the p o i n t of c o m p l e t e n e u t r a l i z a -

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Fig. 3. SDS-PAGE analysis of purified p60:Fc. 2 tzg of affinity purified p60:Fc were subjected to electrophoresis in SDS under non-reducing (lane 1) or reducing (lane 2) conditions. The gel (8-12% linear gradient) was stained for protein with Coomassie brilliant blue dye. ~ 55 k D a c o r r e s p o n d i n g to p 6 0 : F c d i m e r a n d m o n o m e r w e r e s e e n u n d e r n o n - r e d u c i n g (lane 1) a n d r e d u c i n g ( l a n e 2) conditions.

3.3. Ligand binding and specific activity of purified p60 : Fc T h e affinity o f p 6 0 : F c for L T a a n d T N F was i n v e s t i g a t e d in a c o m p e t i t i o n b i n d i n g assay m e a suring d i s p l a c e m e n t o f 12SI-TNF (500 p M ) b o u n d to i m m o b i l i z e d p 6 0 : F c (Fig. 4). U n l a b e l e d T N F at a c o n c e n t r a t i o n o f ~ 150 p M i n h i b i t e d 50% of t h e m a x i m u m 125I-TNF b i n d i n g ( K i) to p 6 0 : F c . A slightly h i g h e r c o n c e n t r a t i o n o f L T a was req u i r e d to inhibit 125I-TNF b i n d i n g to p 6 0 : F c ( K i ~ 650 p M ) . T h e ability o f p60 : F c to a n t a g o n i z e T N F cytotoxicity in vitro was assessed using t h e L929

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iO2 iO3 ~.04 p60: Fc (pM) Fig. 5. Neutralization of TNF cytotoxic activity by purified p60:Fc. Mitomycin C-treated L929 cells were incubated with purified recombinant TNF at 2 nM (circles), 0.2 nM (squares) or 0.02 nM (triangles) in the presence of graded amounts of p60:Fc for 20 h at 37°C. The percentage cytotoxicty was determined using the MTT dye assay. Inset: % cytotoxicity data plotted vs. the molar ratio of p60 : Fc/TNF.

Paul D. Crowe et aL /Journal of lrnmunological Methods 168 (1994) 79-89

tion (0% cytotoxicity) demonstrating that p60:Fc is fully biologically active at stoichiometric concentration. Complete neutralization of TNF at concentrations significantly below the K d for p60:Fc ( ~ 150 pM) required higher molar ratios of p60:Fc. This appeared to be due to a larger fraction of free ligand available to initiate a biological response in L929 cells which occurs at low fractional receptor occupancy (Coffman et al., 1988).

ing that insect cell-derived LTa is: 1) antigenically similar to LTa produced in E. coil and 2) specifically binds the TNF receptor construct, The apparent molecular weight of LTa produced by insect ceils (~ 21 kDa) is comparable to CHO cell-derived LTa (lanes 2 and 4) indicating that insect cells and CHO cells process the protein similarly. In contrast, LTa was immunoprecipitated from supernatants of the PMA-activated human T cell line II-23.D7 using 9B9 (lane 8) or p60:Fc (lane 10) and migrates as a major polypeptide band of ~ 25 kDa and a minor band of ~ 23 kDa. The polypeptide band seen at ~ 31 kDa is a nonspecific protein incompletely cleared by murine IgG or human Ig. These results show that insect cells, like CHO cells, do not glycosylate LTa to the same extent as T cells. However, both insect cell- and CHO cell-derived recombinant LTa, as well as naturally occurring human LTo4 specifically bind the TNF receptor-Ig fusion protein.

3.4. Expression of L Ta in baculovirus-infected cells The cDNA encoding human LTa in pVL1393 was transferred into the baculovirus genome to produce recombinant virus for infection of insect cells as described for p60:Fc. In LTa baculovirus-infected Sf9 cells metabolically labeled with 35S-Cys/Met, a major polypeptide band of ~ 21 kDa was immunoprecipitated from culture supernatants using the anti-LTa MAb, 9B9 (Fig. 6, lane 4) or purified p60 : Fc (lane 6) demonstratCHO-LT

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Fig. 6. Expression of human LTa in Sf9, CHO-LT and activated II-23.D7 T cells. Sf9 insect cells (2.5 X 106) infected with recombinant LTa virus for 24 h were labeled with 35S-Cys/Met for 16 h at 27°C. CHO-LT (5 X 106) and II-23.D7 cells (5 x 106, activated for 4 h with 25 n g / m l PMA) were labeled with 35S-Cys/Met for 1 h at 37°C. Culture supernatants were subjected to immunoprecipitation with anti-LTa MAb 9B9 (lanes 2, 4 and 8) following a preclear with normal murine IgG1 (lanes 1, 3 and 7) or p60:Fc (lanes 6 and 10) following a preclear with normal human Ig (lanes 5 and 9). Left panel: 10% gel, 48 h exposure. Right panel: 8-12% gradient gel, 72 h exposure.

86

Paul D. Crowe et al. /Journal of lmrnunological Methods 168 (1994) 79-89

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tions greater than 1/107. No eytolytic activity was observed in culture supernatants from mock-infected cells at any dilution tested. Thus, using the biologic activity of C H O cell-derived recombinant L T a as a standard, we estimate the insect cellproduced L T a concentration to be ~ 20 m g / l .

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iO 3 ~04 105 ~06 :107 108 SupePnatant d i ] u t i o n -~ Fig. 7. Cytolytic activity of LTa produced in insect cells. Mitomycin C-treated L929 cells were incubated with dilutions of LTa baculovirus-infected (circles) or mock-infected (triangles) Tn 5B1-4 culture supernatants (circles) for 20 h at 37°C and percent cytotoxicity determined by the MTT dye assay as described. ~02

3.5. Biologic actiuity of LTe~ produced in insect cells To examine whether insect cell-derived L T a is biologically active, culture supernatants from cells infected with L T a baculovirus were tested for their ability to kill murine L929 cells in a 24 h cytotoxicity assay. As seen in Fig. 7, 50% maximal killing of L929 cells was seen with a 1 / 4 × 105 dilution of culture supernatant from L T a baculovirus-infected Tn 5B1-4 cells. Significant cytolytic activity was seen with supernatant dilu-

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Fig. 8. Neutralization of CHO LTa and baculovirus ETa bioactivity by p60:Fc. Mitomycin C-treated L929 cells were incubated with 100 pM CHO-LTa (circles) or a 1/4500 dilution of bacf)lovirus LTo~ supernatant (triangles) in the presence of graded amounts of affinity-purifiedp60 : Fc for 20 h at 37°C in 96-well microtiter plates. Cell viabilitywas determined using the MTT dye assay.

The ability of p60 : Fc to inhibit L T a - m e d i a t e d cytotoxicity of L929 target cells was investigated using the M T T dye reduction assay. As shown in Fig. 8, a p60"Fc concentration of ~ 400 pM inhibited 50% of the cell killing elicited by C H O L T a (100 pM) or baculovirus L T a supernatant (1/4500 dilution). These results show that the cytolytic activity of baculovirus L T a , when adjusted to comparable units of killing activity based on C H O - L T a , is also efficiently neutralized by p 6 0 : F c produced in insect cells. 4. Discussion The baculovirus expression system has proven useful for overexpressing a number of cytokines and receptors (Wojchowski et al., 1987; Chiou and Wu, 1990; M a e d a et al., 1985; Smith et al., 1983, 1985; Ingley et al., 1991; Matsuura et al., 1991; Loetscher et al., 1990). The studies reported here demonstrate that this system is ideally suited for producing human L T a and the soluble dimeric T N F receptor fusion protein, p60:Fc. Posttranslational processing and assembly of these proteins appears to be similar in mammalian and insect cells. However, the levels of protein production seen in insect cells far exceed that observed for mammalian cell systems. For example, for p60:Fc, transiently transfected COS-7 cells accumulate ~ 0.05-0.1 mg/1 over 7 days compared to ~ 2 - 3 mg/1 over 5 days for the insect cell line Tn 5B1-4. In addition, the insect cells described in this study are cultured in serum-free conditions, simplifying affinity purification of p60 : Fc. The p 6 0 : F c fusion protein produced in insect cells, like cellular T N F receptors, binds T N F and L T a with high affinity. T N F receptors bind trimeric T N F and L T a at the interface between

Paul D. Crowe et al. / Journal of Immunological Methods 168 (1994) 79-89

ligand subunits (Banner et al., 1993). Hence, a single T N F or L T a molecule is capable of binding up to three receptors simultaneously; a configuration which promotes the receptor clustering on the cell surface thought to be the initiating event in T N F signal transduction (Engelmann et al., 1990). Thus, complete neutralization of T N F bioactivity by p 6 0 : F c would be predicted at a molar ratio of 1:1 since the bivalent fusion protein could theoretically occupy two receptor binding sites on one T N F trimer, thereby preventing high avidity T N F binding and subsequent clustering of receptors on target cells. Accordingly, the present study demonstrates that p60 : Fc quantitatively neutralizes the bioactivity of a saturating T N F concentration at approximately equimolar stoichiometry confirming that the purified fusion protein is fully biologically active. The data presented in this p a p e r also establish the utility of p 6 0 : F c as specific ligand-precipitating reagent for recombinant and naturally occurring secreted L T a . Clearly, soluble receptor-mediated ligand precipitation (RMLP) will make it feasible to investigate TNFR60 interaction with the recently described m e m b r a n e - a n c h o r e d ligands formed between L T a and LT/3 and perhaps other members of the T N F family (Browning et al., 1993; Armitage et al., 1992; Hollenbaugh et al., 1992; Goodwin et al., 1993). In addition, the receptor fusion protein may be useful as an affinity reagent for purification of L T a or heteromeric complexes of L T a and LT/L We have also shown that the baculovirus system is suitable for expressing biologically active h u m a n L T a to levels approaching 20 m g / l . L T a produced in baculovirus-infected insect cells is antigenically similar to recombinant and naturally Occurring L T a secreted by T cells. Interestingly, glycosylation of L T a by insect cells and C H O ceils is not identical to T cells. Nevertheless, we have shown that incompletely glycosylated L T a secreted by insect cells is biologically active in a standard T N F / L T cytotoxicity assay. In conclusion, the baculovirus expression system was found to be ideal for high-level expression of biologically active h u m a n L T a and a soluble T N F receptor fusion protein for purification. Moreover, as an expression system, coinfec-

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tion of insect cells with different recombinant baculoviruses may prove to be an expedient method for studying heteromeric cytokine complex assembly.

Acknowledgements The authors thank Dr. Jeff Browning (Biogen) for the C H O and COS-7 cells and for providing purified recombinant L T a and TNF. We are also grateful to Dr. Ray Goodwin (Immunex) for the pDC302 plasmid and the TNFR60 and human IgG-Fc cDNAs. We thank Dr. Dave Johnson for helpful discussions. This research was supported by a grant from the American Cancer Society (IM663) and by funds from the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco-Related Diseases Research Program of the University of California, grant number RT0261.

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