MONOCLONAL ANTIBODIES TO HUMAN TUMOR NECROSIS FACTOR a: IN VITRO AND IN VIVO APPLICATION Achim Miiller,‘*
Franz Emling,2 Dietmar Blohm,3 Erich Schlick,’ Klaus Schollmeier’
Three stable murine hyhridoma cell lines, which secrete monoclonal antibodies (mAh) to human tumor necrosis factor (Y (TNFa), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon y (IFNy). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNFa. This antibody was further used during in vivo studies to neutralize human TNFa in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNFrv than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNFa! of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNFa, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7a has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7a in serum samples, with a limit of detection of 5 to 10 pg/mL.
o 1990 by W.B. Saunders Company.
Tumor necrosis factor 01 (TNFa) was originally described as a serum factor produced in mice after an infection with Bacillus Calmette-GuCrin and subsequently challenged with endotoxin.’ It causes necrosis of Meth A sarcoma in mice. Subsequent studies showed the cytotoxic or cytostatic features of TNFol for a number of cell lines in vitrozV3and antitumoral effects in several in vivo murine tumor models4 Recent evidence shows that TNFa is a regulatory polypeptide and mediates inflammation and cellular immune responses.5’6 It forms a complex network of interactive signals with other lymphokines as well as regulating production, growth, and differentiation of cells involved in inflammation, immunity, and hemopoiesis.’ Aggarwal, et al* described the purification of this cytotoxic factor from the human promyelocytic leukemia cell line HL60. Human TNFa is secreted as an unglycosylated 157-amino-acid polypeptide of relative
‘BASF Bioresearch Corp., Cambridge, MA, *Knoll AG, Department of Oncology, Ludwigshafen, FRG 3BASF AG, Hauptlaboratorium, Ludwigshafen, FRG *To whom reprint requests should be addressed. 0 1990 by W.B. Saunders Company. 1043-4666/90/0203-0002$05.00/O KEY WORDS: Monoclonal ELISA/In vivo use
162
antibodies/Tumor
necrosis
factor
a/
molecular mass of 17350 Dalton. Lipopolysaccaride (LPS) is a potent trigger for TNFa production.g’lO,l’ It is mainly secreted by macrophages but also produced by T lymphocytes and natural killer cells after stimulation. 12,13,14 TNFo( was cloned in Escherichia coli and a biologically active protein was expressed.15 Using recombinant DNA techniques, we were able to produce large quantities of TNFa for crystallization experiments. Jones et al.“j solved the structure of this lymphokine to a resolution of 2.9 A. TNFa has been linked to various toxic manifestations of infectious, neoplastic, or autoimmune diseases. For example, it has been implicated in the profound weight loss (cachexia) associated with chronic parasitic or bacterial diseases as well as with cancer.5,17,18S1g The circulatory collapse and shock associated with acute bacterial infection,20,2’ the death of animals with cerebral malaria,22 and the development of acute graft-versushost disease and graft rejection23’24 are other examples of the major involvement of TNFol in disease. Because TNFa plays a role in many life threatening diseases, the development of a molecule neutralizing the biological effects of TNFa would be of clinical value. In this report, we describe the isolation of neutralizing and non-neutralizing monoclonal anti-TNFa antibodies. They bind to TNFa with higher affinity than any of
CYTOKINE,
Vol. 2, No. 3 (May),
1990: pp 162-169
Murine monoclonal anti-human TNFoi antibodies / 163
the previously described monoclonal anti-TNFa antibodies. Furthermore, the mAb 195 shows a highly restricted species specificity not described before. This antibody has been used at equimolar concentrations to neutralize the biological effects of human TNFo( under in vitro and in vivo conditions. A combination of the antibodies mAb 199 and mAb 195 was used to develop a highly sensitive sandwich immunoassay. RESULTS Production
of Monoclonal
Antibodies
Spleen cells from mice showing high titers of antibody in their sera were fused with SP2/0-Ag14 cells. Two weeks after fusion, culture supernatants were harvested from each well and assayed for the secretion of anti-TNFa antibodies using an enzyme immunoassay and the inhibition of cytolytic activity as described in the Materials and Methods section. Twelve positive cultures were identified and after two subcloning steps performed by limiting dilution, three monoclonal anti-TNFor antibodies were further investigated. We designated these monoclonal antibodies mAb 114, mAb 195 and mAb 199. The general characteristics of these monoclonal antibodies are shown in Table 1. Epitope Mapping Antibodies
d10 1.6
B
1.4
of TNFcx Using Monoclonal 0
The number of distinct TNFol antigenic epitopes, defined by the panel of monoclonal antibodies, was determined by an enzyme immunoassay that used an antibody competition method. Three epitopes were identified, two of which reacted with the neutralizing antibodies mAb 114 and mAb 195. Fig. 1 depicts the inhibition experiments showing the competition between biotinylated mAb 114(a), mAb 195(b) or mAb 197(c) and the unlabeled mAbs. In all cases tested, the binding of the labeled mAb was completely inhibited by the corresponding unlabeled mAb. The data shown in Fig. 1 suggest that the epitopes recognized by mAb 114 and mAb 199 are in close proximity. mAb 195 did not compete with the other mAbs tested, suggesting that the epitope recognized by
1.4
C
0.1
10
1
Competitor
100
(q/ml)
Figure 1. Epitope mapping of TNFcY. TABLE 1. General characteristics of the anti-human TNFcv specific monoclonal antibodies mAb 114, mAb 195 and mAb 199
Isotype Affinity Constant x lo9 L/mole
mAb 114
mAb 195
G~,K 1.4
G3, K 3.5
mAb
Binding of biotin-labeled monoclonal antibodies (A-C) to recombinant human TNFa was competed by unlabeled monoclonal antibodies, using as competitors unlabeled mAb 114 (closed square, H), mAb 195 (closed triangle, A) and mAb 199 (open hourglass, X).
199
G~,K 2.0
The isotypes of the various mAbs were determined by specific reagents from Cappel. The determination of the affinity constants was performed with a non-radioactive immunoassay using nonlinear-least-square-fit models.
mAb 195 is located at a further distance than the other mAbs.. mAb 114 has an influence on the mAb 195 binding site but not vice versa. As TNFa can be detected on Western blots by the three different mAbs, this leads
164 / Miiller
CYTOKINE,
et al.
to the speculation that all three epitopes consist of a sequential stretch of amino acids on the TNFa molecule.
Characterization of Monoclonal Antibodies
1.6,
TABLE 2. Neutralization of the cytotoxic from different animal species
Mouse
The mAbs were tested for binding to TNFa and related cytokines by enzyme immunoassay, and for neutralization of cytolytic activity. Both natural and recombinant TNFa were recognized equally well, while the monoclonal antibodies did not crossreact with Lymphotoxin (TNFP), IL2, or IFNy (data not shown). The studies for neutralization of TNFa cytolytic activity were done in the L929 assay and are shown in Fig. 2. The best neutralizing antibody, mAb 195, had a neutralization titer of 1,000 U TNFol neutralized/pg of purified IgG. Under the assumption that the TNFa molecule forms a trimeric complex in solution,25 the activity of one TNFa molecule can be completely neutralized by three mAbs. The TNFa neutralizing activity of mAb 114 is reduced by a factor of 20 when compared to the activity of mAb 195. mAb 199, on the other hand, is able to neutralize the activity of TNFa! only at extremely high concentrations (neutralization titer of 1.6 U TNFa neutralized/pg of purified IgG), defining mAb 199 as non-neutralizing. Human Lymphotoxin (TNFP) shows the same cytotoxic effects on L929 cells in vitro as TNFcu. Neither of the TNFcv specific monoclonal antibodies mAb 114 and mAb 195 neutralized TNFP, even when tested at concentrations greater than two hundred times the amount required to neutralize TNFa.
1
Vol. 2, No. 3 (May 1990: 162-169) activity of TNFcv
mAb 114
mAb 195
-
-
Rat
-
-
Rabbit Dog Pig
+ +
-
Cynomolgus Rhesus
+ +
-
Baboon Chimpanzee Human
+ + +
+ +
The TNF~Y activities in the supernatants of peripheral mononuclear cells of the different animal species were determined by the L929 cytotoxic assay. For the neutralization studies a dilution was chosen, which gave more than 80% cytotoxicity. Two-fold serial dilutions of monoclonal antibodies were prepared and incubated with the different supernatants for 2 hr at room temperature before the assay.
Cross-Reactivity of Monoclonal Antibodies with TNFa from Diferent Animal Species The cross-reactivities of the monoclonal antibodies were tested by their abilities to neutralize TNFa in different species (Table 2). mAb 195 is highly specific. This is shown by its neutralization of only human and chimpanzee TNFa. mAb 114 neutralizes TNFa from other animal species, but does not react with mouse, rat, and rabbit TNFa.
Characterization of Monoclonal Antibodies by Immunoblot The monoclonal antibodies were examined for their binding to sodium dodecyl sulfate (SDS) denatured TNFa in the presence of human serum. This was done using the immunoblot technique.26 A representative blot is shown for mAb 195 in Fig. 3. The antibody bound the monomeric form of TNFa and did not show any cross-reactivity with human proteins. The same results were found for mAbs 114 and 199 (data not shown). These results indicate that the three monoclonal antibodies probably bind to sequential epitopes, which are not disrupted by treatment with SDS.
Quantification of TNFa Using an ELISA Method
0.1
1
Antibody
10
100
(ugiml)
Figure 2. Neutralization of the cytotoxic activity of human TNFa! by mAbs 114,195 and 199. A confluent monolayer of L929 cells is completely lysed by 12.5 ng/mL monclonal triangle, antigen sufficient
TNFol after 48 hr. In the neutralization experiments, the antibodies mAb 114 (closed square, n ), mAb 195 (closed A) and mAb 199 (open hourglass, X) were incubated with for 2 hr at room temperature prior to the assay. This time was to completely neutralize TNFol cytolytic activity.
A specific sandwich ELISA was developed as a potential diagnostic assay in order to monitor serum levels of TNFol in patients. Purified mAb 199 was adsorbed onto plastic micro-ELISA wells and used to specifically bind recombinant or natural TNFa. Biotinylated mAb 195, followed by the streptavidin-peroxidase complex, was then used to detect bound antigen. After incubation with TMB solution, the absorbance of each well was measured with an automated SLT Easy reader. A typical standard curve is shown in Fig. 4. The same curve can be seen when TNFa is titrated in 25% human
Murine monoclonal anti-human TNFa antibodies / 165
67 -
column fractions from a typical purification, using both types of assays. The ratio of bioactive to immunoreactive TNFs (i.e., the specific activity) of all of the TNFa fractions was relatively constant (data not shown). This result demonstrates that there was no significant difference in the levels of TNFcr that were measured by ELISA or by bioassay.
43 -
Neutralization of Human TNFa in the Mouse
M, x 10”
94 -
To investigate the potential in vivo use of the monoclonal antibodies, Balb/c mice were treated with a lethal dose of TNFol. The toxicity could be completely neutralized by mAb 195, as shown in Fig. 5. Tested at a 0.3 M ratio to TNFa, the mAb 195 demonstrated only a minor effect on the survival of the mice; while given at a 1:1.5 M ratio, mAb 195 completely blocked the toxic effects of human TNFa in the mouse. The control antibody, mAb 199, had no effect on human TNFa activity in the mice and neither monoclonal antibody mAb 19 5 nor monoclonal antibody mAb 199 alone had a toxic effect on the animals.
30 -
20.1 -
14.4 -
Figure
3.
TNFa
immunoblot
probed
DISCUSSION
with mAb 195.
Recombinant TNFo (0.2 pg) was mixed with human serum and chromatographed on a 15% Laemmli gel. The gel was blotted and tested with mAb 195. The position of the TNF~Y is shown by the arrow, designated A, which indicates the estimated molecular weight in Daltons x 10’.
serum (data not shown). The assay has a limit of detection of 5 to 10 pg/mL (2 times the standard deviation) and a linear range up to 300 pg/mL. The specificity was demonstrated by the lack of any signal generated by 10 pg/mL TNFP, IFNy and IL2. In order to compare the results obtained from ELISA with the bioassay, we measured TNFa levels in
O0
50
100
150 Concentration
200 (pg/ml)
250
300
In the present study, we have described the generation of three high affinity murine monoclonal antibodies to human TNFol, which we have designated mAb 114, 195, and 199. Although monoclonal antibodies against TNFa have previously been described,20,27,28,2p,30,31,32 the mAb 199 and, in particular, mAb 195 that are obtained here, are quite different and useful for evaluating the functions of TNFa under in vitro and in vivo conditions. We isolated two types of neutralizing monoclonal anti-TNFoc antibodies that bind to distinct antigenic epitopes. The epitope recognized by mAb 195 is re-
i 35(
Figure Figure
4.
Standard
curve for recombinant
and natural
TNFa!
5.
Neutralization
of human
TNFa!
in the mouse.
ELISA.
The open squares represent recombinant and the open circles natural TNFol. Up to 10 &mL TNF& IFNy and IL 2 gave no signal.
Survival of Balb/c mice was determined (1) 24 hr after the injection of TNFo (2); TNFa + mAb 195, ratio 1:0.3 (3); TNFa + mAb 195, ratio 1:1.5, and (4) TNFa + mAb 199.
166 / Mijller et al.
stricted to only human and chimpanzee TNFa. This antibody equally recognizes recombinant and native TNFcv and does not show any cross-reactivity with such lymphokines as lymphotoxin, IL 2, and IFNy. The high binding constant and the above-mentioned specificity enabled us to use this antibody efficiently in neutralization studies. Under in vivo and in vitro conditions, about 3 molecules of antibody per molecule of TNFcr are sufficient for blocking the lethal or cytotoxic effects. Furthermore, in our murine system, we could apply the monoclonal antibody, mAb 195, even after the injection of TNFol and still find complete survival of the mice. Fendly et a1.3’ postulated that mAbs neutralize the cytotoxicity of TNFcv by blocking the binding of TNFol to its receptor. By fluorescent activated cell sorter (FACS) analysis it was shown that the monoclonal antibody mAb 195 can still bind when TNFa occupies the receptor. This suggests an additional mechanism for neutralization of the biological activity of TNFol (Heilig B, MGller A, and Diirken B, manuscript submitted for publication). The TNFL~ molecule can then be divided into a receptor binding and a neutralization area. The second neutralizing antibody, mAb 114, can bind simultaneously with mAb 195 to TNFa. The epitope recognized by this monoclonal antibody is related spatially to mAb 199, the non-neutralizing antibody. These results support the above model that the biological activity of TNFcr is not restricted to one unique site. The monoclonal antibodies mAb 195 and mAb 199 were used to develop a sensitive sandwich ELISA assay. The ELISA was as sensitive as the bioassay and could discriminate between TNFa and TNFP, which the bioassay is unable to do. Furthermore, it is faster and easier to perform than the bioassay. The monoclonal antibodies, described here, should prove to be useful in the study of this cytokine’s mechanisms of actions. The sensitive ELISA assay can be used to identify and quantitate TNFa in human diseases and for the monitoring of TNFcr levels in patients undergoing TNFcr treatment. The neutralizing antibodies can selectively neutralize TNFa cytolytic activity in an in vitro system, thereby separating the effects of TNFa, from other cytokines in complex biological systems. Neutralizing anti-TNFa monoclonal antibodies may be able to block the side effects of TNFa in vivo, as shown by Tracey et al. 1987,33 for septic shock during lethal bacteremia in baboons. Studies with polyclonal anti-TNFa antibodies in mice have also shown that this treatment almost completely prevented the cutaneous and intestinal lesions of the acute-phase of Graft-versus-Host Disease and markedly reduced overall mortality.23 In the studies of Grau et al. ** the injection of a rabbit antibody to TNFa fully protects mice from cerebral malaria. TNFa-specific monoclonal antibodies may therefore provide a specific therapy for TNFa-mediated disorders.
CYTOKINE,
Vol. 2, No. 3 (May
1990: 162-169)
MATERIALS AND METHODS Recombinant Lymphokines Recombinant human and murine TNFa were produced in E. coli and purified to 8 x lo6 and 13 x lo6 laboratory U/mg,
respectively.34 The materials appeared homogeneous when analyzed by SDS-PAGE, stained with CoomassieBlue. Natural human TNFo( was purified from HL-60 cell line supernatants8 Recombinant human IFNy and human lymphotoxin (TNFP) were produced in E. coli and purified to homogeneity.
TNFcxfrom DiRevent Animal Species Blood was drawn from the following animal species: mouse, rat, rabbit, dog, pig, cynomolgus, rhesus, baboon and chimpanzee, and centrifuged through a ficoll gradient. Peripheral mononuclear cells (2 x IO6cells/ml) were suspended in RPM1 1640 supplemented with penicillin (50 U/mL), streptomycin (50 bg/mL), glutamine (2 mM) and 5% fetal calf serum (FCS) (Boehringer). 10 mL of this suspension was transferred to a T25 Falcon flask. TNFol was then induced by adding 10 Mg/mL LPS (Sigma, No. L400.5). The supernatants were collected after 16 hr and the TNFa! levels were either immediately estimated or stored at -80°C until the TNFol test was performed.
Biological Assayfor TNFol Activity The biological activity of TNFa was determined by the murine L929 fibroblast cytotoxic assay, as described by Aggarwal et al.* Briefly, L929 cells were seededat a density of 10,000 cells per well in 96-well microtiter plates (Nunc) using 0.1 mL Eagle’s Minimal Essential Medium that contained 10% fetal bovine serum (FBS). An equal volume of medium containing the desired dilutions of TNFol, with or without anti-TNFcu antibodies, was added and incubated at 37OCand 5% CO, for 48 hr in a humidified incubator. The cells were then stained with 0.5% crystal violet. After an incubation of 15 min, the plates were rinsed with tap water and then dried. Crystal violet was dissolved by the addition of 0.1 mL/well of 0.1% acetic acid in 50% ethanol. The absorbance at 580 nm was read with a SLT Easy Reader. The TNFol U/mL represent the reciprocal of the TNFcv dilution that causes50% cytotoxicity under the conditions of the assay.
Immunization, Fusion and Cloning Purified recombinant human TNFa, in an emulsion formed by equal volumes of TNFcv solution and Freund’s complete adjuvant, was injected into BALB/c mice i.p. (5 fig/mouse). Two, 4 and 6 weeks later, the mice received the same amount of antigen in Freund’s incomplete adjuvant. Three days after the last immunization spleen cells from mice showing high titers of antibody in their sera were fused with SP2/0-Ag14 cells at a ratio of 5:1, according to previously described techniques.35Two weeks after fusion, culture supernatants were assayed for secretion of anti-TNFa antibodies using the methods described. Positive hybridomas were subcloned twice by limiting dilution in 96-well plates, using BALB/c thymocytes ( lo6 cells/well) as feeder cells.
Murine monoclonal anti-human TNFa antibodies / 167
Selection TNFQ
of Monoclonal
Antibodies
to Human
Recombinant human TNFa was diluted in PBS to a concentration of 0.01 mg/mL. A 0.1 mL aliquot of this suspension was added to each well of a 96-well microtiter plate (Flow). The plate was incubated overnight at 4OC and the fluid was removed. Residual protein-binding sites were blocked by PBS containing 0.1% Tween 20 (PBS-T). Supernatants (0.1 mL/well), which contained monoclonal antibodies, were added and incubated for 2 hr at room temperature. After a washing step, 0.1 mL of a 1 ,OOO-fold diluted solution of goat anti-mouse IgG antibody labeled with horseradish peroxidase (Boehringer) was added to each well. After incubation for 1 hr at room temperature and washing with PBS-T, 0.1 mL of 0.42 mM 3,3’,5,5’-Tetramethylbenzidine (TMB, Boehringer Mannheim) in 0.1 M acetate-citrate buffer, pH 4.9 containing 0.004% H,O, was added. The reaction was stopped after 10 min by the addition of 2.0 N sulfuric acid and the absorbance at 450 nm of each well was determined using a SLT Easy Reader.
Monoclonal
Antibody
Isotyping
A 96-well microtiter plate (Flow) was coated with human rTNFa (0.01 mg/mL in PBS) overnight at 4°C and then blocked with PBS containing 1% bovine serum albumin (BSA). 0.1 mL of culture supernatants were added to wells and incubated for 1 hr at room temperature. Plates were washed with PBS-T and 0.05 mL of each isotype-specific rabbit anti-mouse Ig antibody (Cappel) was added. After 1 hr incubation at room temperature, the wells were washed 3 times and 0.05 mL of horseradish peroxidase labeled goat antirabbit IgG (Boehringer Mannheim) was added to each well. Incubation was continued for another hour at room temperature. Plates were washed 5 times with PBS-T and 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) was used as substrate as described previously.
0.05 M potassium phosphate, 0.5 M sodium chloride saccharose at pH 7.8.
Puvijication
of Monoclonal
and 5%
Antibodies
Hybridoma cells were cultivated in Iscove Medium containing the following supplements: 1.2 mg/mL BSA, 0.024 mg/mL soybean lipids, 0.034 mg/mL transferrin and 5 x 10m5 M @-mercaptoethanol. Cells were grown in spinner flasks, the medium was harvested by filtration through a 0.3 pm filter (Pall) and concentrated lo-fold by ultrafiltration using a PM10 membrane (Amicon). The concentrate was dialyzed against 1.5 M glycine and 3 M NaCl, pH 8.9 and the IgG purified by Protein A Sepharose chromatography (Pharmacia), according to the manufacturer’s recommendation.
Monoclonal
Antibodies
AJinity
Determination
96-well microtiter plates (Nunc) were coated for 16 hr at 4°C with 0.01 mg/mL TNFa (0.1 mL/well) in PBS. After that the wells were washed with PBS-T and post-coating was done with 1% BSA in PBS for 0.5 hr at 37OC. mAbs were serially diluted with 0.1% BSA in PBS and incubation was done for 4 hr at 37OC. The wells were washed again 5 times with PBS containing 0.1% Tween 20 (PBS-T) and incubated for 2 hr at 37OC with horseradish peroxidase conjugated rabbit anti-mouse IgG (0.1 mL/well, diluted 1:3,000 in 0.1% BSA in PBS). The wells were then washed 5 times with PBS-T and TMB was used as substrate, as described previously. Calculations of the affinity of the monoclonal antibodies were done by non-linear least square fits. These were based on the model of one monoclonal antibody binding site per TNFa subunit.
Enzyme-Linked
Immunosovbent
Assay (ELISA)
A 96-well microtiter plate (Flow) was coated with 0.001 mg/mL recombinant human TNFcv (0.1 mL/well) in PBS overnight at 4OC. Nonspecific binding sites were then blocked with a solution of 1% BSA in PBS for 0.5 hr at 37°C. A constant volume (0.05 mL) of an appropriate dilution of biotinylated monoclonal antibody was added to equal volumes of double dilutions of respective unlabeled monoclonal antibodies. The mixture was then added to the wells and incubated for 2 hr at 37OC. The washing and further processing were done as described for “ELISA.”
A 96-well microtiter plate (Nunc) was coated overnight with 0.1 mL/well of anti-TNFa mAb 199 (0.005 mg/mL) in PBS. Post coating was done with 1% BSA in PBS for 0.5 hr at 37OC. The plate was washed 5 times with PBS containing 0.1% Tween 20 (PBS-T). For a standard curve, TNFa was diluted in 20 mM Tris-HCl, 150 mM NaCl and 1% BSA, pH 7.4. TNFU-containing medium or serum (0.1 mL/well) was reacted for 3 hr at room temperature or overnight at 4°C. After washing, 0.1 mL/well of biotinylated mAb 195 (0.001 mg/ mL) was added and incubated for 2 hr at room temperature. After 3 washes with PBS-T, 0.1 mL/well of lO,OOO-fold diluted streptavidin-peroxidase complex (Boehringer Mannheim) was added and incubated again for 0.5 hr at room temperature. TMB was used as a substrate for developing the color as d.escribed.
Biotinylation
Gel Electrophovesis
Determination
of Epitope Specificities
of Monoclonal
Antibodies
The mAbs were biotinylated with D-biotinyle-e-amidocaproic-acid-N-hydroxysuccinimide-ester (Boehringer Mannheim). Ten mg of this reagent was dissolved in 1 mL DMSO. The monoclonal antibodies were adjusted to 5 mg/mL in PBS. A 35 molar excess of biotin was used to label the antibodies for 2 hr at room temperature. The unbound biotin was removed from modified monoclonal antibodies by Sepharose G25 column chromatography (1 x 15 cm). The column was run with
and Immunoblots
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed, as described by Laemmli et a1.36 Recombinant TNFa (0.2 Hg) and human serum were subjected to electrophoresis on a 15% acrylamide gel. Gels were stained with 0.15% Coomassie Blue R250 (wt/vol) in 25% isopropanol (vol/vol), 10% acetic acid (vol/vol), and destained with 10% methanol (vol/vol), 10% acetic acid (vol/vol). The binding of antibodies to TNFa, which are
168 / Miiller
CYTOKINE,
et al.
separated by SDS-PAGE, was assessed by the immunoblot technique. SDS-PAGE gels were transferred to nitrocellulose as previously described by Towbin et al.*‘j The non-specific reactive sites on the nitrocellulose were blocked by incubating for 30 min :j:ith 3% gelatin (wt/vol) in TBS. Test monoclonal antibodies v’-re diluted to 0.01 mg/mL in 1% gelatin (wt/vol) in TBS, and incubated with the nitrocellulose strips for 16 hr at 22OC. The strips were then washed for 15 min, each with three changes of PBS-T. Bound monoclonal antibodies were detected after incubating for 4 hr with horseradish peroxidase conjugated anti-murine IgG (Cappel) that was diluted 1:lOOO in 1% gelatin. The strips were washed as above and incubated 15 min with 4-chloro-1-naphthol (0.6 mg/mL in 20% methanol, 80% TBS containing 0.018% H202), rinsed with water and air dried.
In Vivo Neutralization of Human TNFa Four-6 week old BALB/c mice were randomized and divided into groups of 5. The substances were given i.v. into the lateral tail vein; the volume of injection did not exceed 10 mL/kg. TNFo( (2 mg/kg body weight) was applied first, followed 15 to 30 min later by the monoclonal antibodies (doses: 2 mg/kg and 10 mg/kg). The rate of survival was determined 24 hr later.
Protein Determination Protein determinations were performed by the dye binding method of Bradford,37 which uses bovine IgG as a standard. The protein concentration was also determined by absorbance using the extinction coefficients for pure recombinant TNFa, e2s01 mg/mL = 1.6.
Acknowledgments The authors thank M. Pan for help in writing the manuscript, J. Delzer and H. Hillen for fermentation and purification of the cytokines. We also gratefully acknowledge the expert technical assistance of M. Seitz, H. Hofmann, E. Klimm and R. Merx.
REFERENCES 1. Carswell EA, Old LJ, Kassel RL, Greene S, Fiore N, Williamson B (1975) An endotoxin-induced serum factor that causes necrosis of tumors. Proc Nat Acad Sci USA 72:3666-3670 2. Haranaka K, Satomi N (1981) Cytotoxic activity of tumor necrosis factor on human cancer cells in vitro. Jpn J Exp Med 51:191-194 3. Sugarman BJ, Aggarwal BB, Hass PE, Figari IS, Palladino MA, Jr, Shepard HM (1985) Recombinant human tumor necrosis factor-a: Effects of proliferation of normal and transformed cells in vitro. Science 230:943-949 4. Haranaka K, Satomi N, Sakurai A (1984) Antitumor activity of murine tumor necrosis factor against transplanted murine tumors and heterotransplanted human tumors in nude mice. Int J Cancer 341263-267 5. Beutler B, Cerami A (1987) Cachectin: More than a tumor necrosis factor. N Engl J Med 316:379-383 6. Kunkel SL, Remick DG, Strieter RM, Larrick JW (1989) Mechanisms that regulate the production and effects of tumor necrosis factor-a. Critical Rev Immunol9:93-117
Vol. 2, No. 3 (May 1990: 162-169)
7. Old LJ (1987) Polypeptide mediator network. Nature 326:330331 8. Aggarwal BB, Kohr WJ, Hass PE, Moffat B, Spencer SA, Henzel WJ, Bringman TS, Nedwin GE, Goeddel DV, Harkins RN (1985) Human tumor necrosis factor, production, purification characterization. J Biol Chem 260:2345-2354 9. Beutler B, M&ark IW, Cerami AC (1985) Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effects of endotoxin. Science 229:869-871 10. Chen AR, McKinnon KP, Koren HS (1985) Lipopolysaccharide (LPS) stimulates fresh human monocytes to lyse actinomycin D-treated WEHItarget cells via increased secretion of a monokine similar to tumor necrosis factor. J Immunol 135:3978-3984 11. Michie HR, Manogue KR, Spriggs DR, Revhaug A, O’Dwyer S, Dinarello CA, Cerami A, Wolff SM, Wilmore DW (1988) Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 318:1481-1486 12. Cuturi MC, Murphy M, Costa-Giomi MP, Weinmann R, Perussia B, Trinchieri G (1987) Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral bloodlymphocytes. JExpMed 165:1581-1585 13. Feinman R, Henriksen-DeStefano D, Tsujimoto M, Vilcek J (1987) Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes. J Immunol 138:635-641 14. Debets JMH, Van der Linden CJ, Spronken IEM, Buurman WA (1988) T-cell mediated production of tumor necrosis factor a by monocytes. Stand J Immunol27:601-608 15. Pennica D, Nedwin GE, Hayflick JS, Seeburg PH, Derynck R, Palladino MA, Kohr WJ, Aggarwal BB, Goeddel DV (1984) Human tumour necrosis factor: Precursor structure, expression and homology to lymphotoxin. Nature 312:724-729 16. Jones EY, Stuart DI, Walker NPC (1989) Structure of tumor necrosis factor. Nature 338:225-228 17. Cerami A, Ikeda Y, Le Trang N, Hotez PJ, Beutler B (1985) Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: The role of cachectin (tumor necrosis factor) Immunol Lett 11:173-177 18. Scuderi P, Sterling KE, Lam KS, Finley PR, Ryan KJ, Ray CG, Petersen E, Slymen DJ (1986) Raised serum levels of tumour necrosis factor in parasitic infections. Lancet 2: 1364- 1365 19. Shimomura K, Manda T, Mukumato S, Kobayashi K, Nakano K, Mori J (1988) Recombinant human tumor necrosis factor cy:Thrombus formation as a cause of anti-tumor activity. Int J Cancer 41~243-247 20. Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey TJ III, Zentella A, Albert JD, Shires GT, Cerami A (1986) Shock and tissue injury induced by recombinant human cachectin. Science 234:470-474 21. Waage A, Halstensen A, Espevik T (1987) Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet 1:355-357 22. Grau GE, Fajardo LF, Piguet P-F, Allet B, Lambert P-H, Vassalli P (1987) Tumor necrosis factor (Cachectin) as an essential mediator in murine cerebral malaria. Science 237:1210-1212 23. Piguet P-F, Grau GE, Allet B, Vassalli P (1987) Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs-host disease. J Exp Med 166:1280-1289 24. Maury CPJ, Teppo AM (1987) Raised serum levels of cachectin/tumor necrosis factor a in renal allograft rejection. J Exp Med 166:1132-1137 25. Smith RA, Baglioni C (1987) The active form of tumor necrosis factor is a trimer. J Biol Chem 262:6951-6955 26. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat1 Acad Sci USA 76:43504354 27. Bringman T, Aggarwal BB (1987) Monoclonal antibodies to human tumor necrosis factors alpha and beta: Application for affinity
Murine monoclonal anti-human TNFo( antibodies / 169 purification, immunoassays, and as structural probes. Hybridoma 6:489-507 28. Hirai M, Okamura N, Terano Y, Tsujimoto M, Nakazato H (1987) Production and characterization of monoclonal antibodies to human tumor necrosis factor. J Immunol Methods 96:57-62 29. Meager A, Parti S, Leung H, Peil E, Mahon B (1987) Preparation and characterization of monoclonal antibodies directed against antigenic determinants of recombinant human tumour necrosis factor (rTNF). Hybridoma 6:305-3 11 30. Liang CM, Liang SM, Jost T, Sand A, Dougas I, Allet B (1986) Production and characterization of monoclonal antibodies against recombinant human tumor necrosis factor/cachectin. Biothem Biophys Res Comm 137:847-854 31. Fendly BM, Toy KJ, Creasey AA, Vitt CR, Larrick JW, Yamamoto R, Lin LS (1987) Murine monoclonal antibodies defining neutralizing epitopes on tumor necrosis factor. Hybridoma 6:359-369 32. Shimamoto Y, Chen RL, Bollon A, Chang A, Khan A (1988) Monoclonal antibodies against human recombinant tumor
necrosis factor: Prevention of endotoxic shock. Immunol Lett 17:31 l318 33. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A (1987) Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330:662-665 34. Ziegler-Heitbrock HW, Moller A, Linke RP, Haas JG, Rieber EP and Riethmtiller (1986) Tumor necrosis factor as effector molecule in monocyte mediated cytotoxicity. Cancer Res 46:59475952 35. Oi V, Herzenberg L (1980) Immunoglobulin-producing hybrid cell lines. In Mishell B, Shiigi S (eds). Selected Methods in Cellular Immunology. Freeman Co., San Francisco, pp 351-372. 36. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 37. Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254
Franz Emling,2 Dietmar Blohm,3 Erich Schlick,’ Klaus Schollmeier’
Three stable murine hyhridoma cell lines, which secrete monoclonal antibodies (mAh) to human tumor necrosis factor (Y (TNFa), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon y (IFNy). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNFa. This antibody was further used during in vivo studies to neutralize human TNFa in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNFrv than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNFa! of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNFa, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7a has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7a in serum samples, with a limit of detection of 5 to 10 pg/mL.
o 1990 by W.B. Saunders Company.
Tumor necrosis factor 01 (TNFa) was originally described as a serum factor produced in mice after an infection with Bacillus Calmette-GuCrin and subsequently challenged with endotoxin.’ It causes necrosis of Meth A sarcoma in mice. Subsequent studies showed the cytotoxic or cytostatic features of TNFol for a number of cell lines in vitrozV3and antitumoral effects in several in vivo murine tumor models4 Recent evidence shows that TNFa is a regulatory polypeptide and mediates inflammation and cellular immune responses.5’6 It forms a complex network of interactive signals with other lymphokines as well as regulating production, growth, and differentiation of cells involved in inflammation, immunity, and hemopoiesis.’ Aggarwal, et al* described the purification of this cytotoxic factor from the human promyelocytic leukemia cell line HL60. Human TNFa is secreted as an unglycosylated 157-amino-acid polypeptide of relative
‘BASF Bioresearch Corp., Cambridge, MA, *Knoll AG, Department of Oncology, Ludwigshafen, FRG 3BASF AG, Hauptlaboratorium, Ludwigshafen, FRG *To whom reprint requests should be addressed. 0 1990 by W.B. Saunders Company. 1043-4666/90/0203-0002$05.00/O KEY WORDS: Monoclonal ELISA/In vivo use
162
antibodies/Tumor
necrosis
factor
a/
molecular mass of 17350 Dalton. Lipopolysaccaride (LPS) is a potent trigger for TNFa production.g’lO,l’ It is mainly secreted by macrophages but also produced by T lymphocytes and natural killer cells after stimulation. 12,13,14 TNFo( was cloned in Escherichia coli and a biologically active protein was expressed.15 Using recombinant DNA techniques, we were able to produce large quantities of TNFa for crystallization experiments. Jones et al.“j solved the structure of this lymphokine to a resolution of 2.9 A. TNFa has been linked to various toxic manifestations of infectious, neoplastic, or autoimmune diseases. For example, it has been implicated in the profound weight loss (cachexia) associated with chronic parasitic or bacterial diseases as well as with cancer.5,17,18S1g The circulatory collapse and shock associated with acute bacterial infection,20,2’ the death of animals with cerebral malaria,22 and the development of acute graft-versushost disease and graft rejection23’24 are other examples of the major involvement of TNFol in disease. Because TNFa plays a role in many life threatening diseases, the development of a molecule neutralizing the biological effects of TNFa would be of clinical value. In this report, we describe the isolation of neutralizing and non-neutralizing monoclonal anti-TNFa antibodies. They bind to TNFa with higher affinity than any of
CYTOKINE,
Vol. 2, No. 3 (May),
1990: pp 162-169
Murine monoclonal anti-human TNFoi antibodies / 163
the previously described monoclonal anti-TNFa antibodies. Furthermore, the mAb 195 shows a highly restricted species specificity not described before. This antibody has been used at equimolar concentrations to neutralize the biological effects of human TNFo( under in vitro and in vivo conditions. A combination of the antibodies mAb 199 and mAb 195 was used to develop a highly sensitive sandwich immunoassay. RESULTS Production
of Monoclonal
Antibodies
Spleen cells from mice showing high titers of antibody in their sera were fused with SP2/0-Ag14 cells. Two weeks after fusion, culture supernatants were harvested from each well and assayed for the secretion of anti-TNFa antibodies using an enzyme immunoassay and the inhibition of cytolytic activity as described in the Materials and Methods section. Twelve positive cultures were identified and after two subcloning steps performed by limiting dilution, three monoclonal anti-TNFor antibodies were further investigated. We designated these monoclonal antibodies mAb 114, mAb 195 and mAb 199. The general characteristics of these monoclonal antibodies are shown in Table 1. Epitope Mapping Antibodies
d10 1.6
B
1.4
of TNFcx Using Monoclonal 0
The number of distinct TNFol antigenic epitopes, defined by the panel of monoclonal antibodies, was determined by an enzyme immunoassay that used an antibody competition method. Three epitopes were identified, two of which reacted with the neutralizing antibodies mAb 114 and mAb 195. Fig. 1 depicts the inhibition experiments showing the competition between biotinylated mAb 114(a), mAb 195(b) or mAb 197(c) and the unlabeled mAbs. In all cases tested, the binding of the labeled mAb was completely inhibited by the corresponding unlabeled mAb. The data shown in Fig. 1 suggest that the epitopes recognized by mAb 114 and mAb 199 are in close proximity. mAb 195 did not compete with the other mAbs tested, suggesting that the epitope recognized by
1.4
C
0.1
10
1
Competitor
100
(q/ml)
Figure 1. Epitope mapping of TNFcY. TABLE 1. General characteristics of the anti-human TNFcv specific monoclonal antibodies mAb 114, mAb 195 and mAb 199
Isotype Affinity Constant x lo9 L/mole
mAb 114
mAb 195
G~,K 1.4
G3, K 3.5
mAb
Binding of biotin-labeled monoclonal antibodies (A-C) to recombinant human TNFa was competed by unlabeled monoclonal antibodies, using as competitors unlabeled mAb 114 (closed square, H), mAb 195 (closed triangle, A) and mAb 199 (open hourglass, X).
199
G~,K 2.0
The isotypes of the various mAbs were determined by specific reagents from Cappel. The determination of the affinity constants was performed with a non-radioactive immunoassay using nonlinear-least-square-fit models.
mAb 195 is located at a further distance than the other mAbs.. mAb 114 has an influence on the mAb 195 binding site but not vice versa. As TNFa can be detected on Western blots by the three different mAbs, this leads
164 / Miiller
CYTOKINE,
et al.
to the speculation that all three epitopes consist of a sequential stretch of amino acids on the TNFa molecule.
Characterization of Monoclonal Antibodies
1.6,
TABLE 2. Neutralization of the cytotoxic from different animal species
Mouse
The mAbs were tested for binding to TNFa and related cytokines by enzyme immunoassay, and for neutralization of cytolytic activity. Both natural and recombinant TNFa were recognized equally well, while the monoclonal antibodies did not crossreact with Lymphotoxin (TNFP), IL2, or IFNy (data not shown). The studies for neutralization of TNFa cytolytic activity were done in the L929 assay and are shown in Fig. 2. The best neutralizing antibody, mAb 195, had a neutralization titer of 1,000 U TNFol neutralized/pg of purified IgG. Under the assumption that the TNFa molecule forms a trimeric complex in solution,25 the activity of one TNFa molecule can be completely neutralized by three mAbs. The TNFa neutralizing activity of mAb 114 is reduced by a factor of 20 when compared to the activity of mAb 195. mAb 199, on the other hand, is able to neutralize the activity of TNFa! only at extremely high concentrations (neutralization titer of 1.6 U TNFa neutralized/pg of purified IgG), defining mAb 199 as non-neutralizing. Human Lymphotoxin (TNFP) shows the same cytotoxic effects on L929 cells in vitro as TNFcu. Neither of the TNFcv specific monoclonal antibodies mAb 114 and mAb 195 neutralized TNFP, even when tested at concentrations greater than two hundred times the amount required to neutralize TNFa.
1
Vol. 2, No. 3 (May 1990: 162-169) activity of TNFcv
mAb 114
mAb 195
-
-
Rat
-
-
Rabbit Dog Pig
+ +
-
Cynomolgus Rhesus
+ +
-
Baboon Chimpanzee Human
+ + +
+ +
The TNF~Y activities in the supernatants of peripheral mononuclear cells of the different animal species were determined by the L929 cytotoxic assay. For the neutralization studies a dilution was chosen, which gave more than 80% cytotoxicity. Two-fold serial dilutions of monoclonal antibodies were prepared and incubated with the different supernatants for 2 hr at room temperature before the assay.
Cross-Reactivity of Monoclonal Antibodies with TNFa from Diferent Animal Species The cross-reactivities of the monoclonal antibodies were tested by their abilities to neutralize TNFa in different species (Table 2). mAb 195 is highly specific. This is shown by its neutralization of only human and chimpanzee TNFa. mAb 114 neutralizes TNFa from other animal species, but does not react with mouse, rat, and rabbit TNFa.
Characterization of Monoclonal Antibodies by Immunoblot The monoclonal antibodies were examined for their binding to sodium dodecyl sulfate (SDS) denatured TNFa in the presence of human serum. This was done using the immunoblot technique.26 A representative blot is shown for mAb 195 in Fig. 3. The antibody bound the monomeric form of TNFa and did not show any cross-reactivity with human proteins. The same results were found for mAbs 114 and 199 (data not shown). These results indicate that the three monoclonal antibodies probably bind to sequential epitopes, which are not disrupted by treatment with SDS.
Quantification of TNFa Using an ELISA Method
0.1
1
Antibody
10
100
(ugiml)
Figure 2. Neutralization of the cytotoxic activity of human TNFa! by mAbs 114,195 and 199. A confluent monolayer of L929 cells is completely lysed by 12.5 ng/mL monclonal triangle, antigen sufficient
TNFol after 48 hr. In the neutralization experiments, the antibodies mAb 114 (closed square, n ), mAb 195 (closed A) and mAb 199 (open hourglass, X) were incubated with for 2 hr at room temperature prior to the assay. This time was to completely neutralize TNFol cytolytic activity.
A specific sandwich ELISA was developed as a potential diagnostic assay in order to monitor serum levels of TNFol in patients. Purified mAb 199 was adsorbed onto plastic micro-ELISA wells and used to specifically bind recombinant or natural TNFa. Biotinylated mAb 195, followed by the streptavidin-peroxidase complex, was then used to detect bound antigen. After incubation with TMB solution, the absorbance of each well was measured with an automated SLT Easy reader. A typical standard curve is shown in Fig. 4. The same curve can be seen when TNFa is titrated in 25% human
Murine monoclonal anti-human TNFa antibodies / 165
67 -
column fractions from a typical purification, using both types of assays. The ratio of bioactive to immunoreactive TNFs (i.e., the specific activity) of all of the TNFa fractions was relatively constant (data not shown). This result demonstrates that there was no significant difference in the levels of TNFcr that were measured by ELISA or by bioassay.
43 -
Neutralization of Human TNFa in the Mouse
M, x 10”
94 -
To investigate the potential in vivo use of the monoclonal antibodies, Balb/c mice were treated with a lethal dose of TNFol. The toxicity could be completely neutralized by mAb 195, as shown in Fig. 5. Tested at a 0.3 M ratio to TNFa, the mAb 195 demonstrated only a minor effect on the survival of the mice; while given at a 1:1.5 M ratio, mAb 195 completely blocked the toxic effects of human TNFa in the mouse. The control antibody, mAb 199, had no effect on human TNFa activity in the mice and neither monoclonal antibody mAb 19 5 nor monoclonal antibody mAb 199 alone had a toxic effect on the animals.
30 -
20.1 -
14.4 -
Figure
3.
TNFa
immunoblot
probed
DISCUSSION
with mAb 195.
Recombinant TNFo (0.2 pg) was mixed with human serum and chromatographed on a 15% Laemmli gel. The gel was blotted and tested with mAb 195. The position of the TNF~Y is shown by the arrow, designated A, which indicates the estimated molecular weight in Daltons x 10’.
serum (data not shown). The assay has a limit of detection of 5 to 10 pg/mL (2 times the standard deviation) and a linear range up to 300 pg/mL. The specificity was demonstrated by the lack of any signal generated by 10 pg/mL TNFP, IFNy and IL2. In order to compare the results obtained from ELISA with the bioassay, we measured TNFa levels in
O0
50
100
150 Concentration
200 (pg/ml)
250
300
In the present study, we have described the generation of three high affinity murine monoclonal antibodies to human TNFol, which we have designated mAb 114, 195, and 199. Although monoclonal antibodies against TNFa have previously been described,20,27,28,2p,30,31,32 the mAb 199 and, in particular, mAb 195 that are obtained here, are quite different and useful for evaluating the functions of TNFa under in vitro and in vivo conditions. We isolated two types of neutralizing monoclonal anti-TNFoc antibodies that bind to distinct antigenic epitopes. The epitope recognized by mAb 195 is re-
i 35(
Figure Figure
4.
Standard
curve for recombinant
and natural
TNFa!
5.
Neutralization
of human
TNFa!
in the mouse.
ELISA.
The open squares represent recombinant and the open circles natural TNFol. Up to 10 &mL TNF& IFNy and IL 2 gave no signal.
Survival of Balb/c mice was determined (1) 24 hr after the injection of TNFo (2); TNFa + mAb 195, ratio 1:0.3 (3); TNFa + mAb 195, ratio 1:1.5, and (4) TNFa + mAb 199.
166 / Mijller et al.
stricted to only human and chimpanzee TNFa. This antibody equally recognizes recombinant and native TNFcv and does not show any cross-reactivity with such lymphokines as lymphotoxin, IL 2, and IFNy. The high binding constant and the above-mentioned specificity enabled us to use this antibody efficiently in neutralization studies. Under in vivo and in vitro conditions, about 3 molecules of antibody per molecule of TNFcr are sufficient for blocking the lethal or cytotoxic effects. Furthermore, in our murine system, we could apply the monoclonal antibody, mAb 195, even after the injection of TNFol and still find complete survival of the mice. Fendly et a1.3’ postulated that mAbs neutralize the cytotoxicity of TNFcv by blocking the binding of TNFol to its receptor. By fluorescent activated cell sorter (FACS) analysis it was shown that the monoclonal antibody mAb 195 can still bind when TNFa occupies the receptor. This suggests an additional mechanism for neutralization of the biological activity of TNFol (Heilig B, MGller A, and Diirken B, manuscript submitted for publication). The TNFL~ molecule can then be divided into a receptor binding and a neutralization area. The second neutralizing antibody, mAb 114, can bind simultaneously with mAb 195 to TNFa. The epitope recognized by this monoclonal antibody is related spatially to mAb 199, the non-neutralizing antibody. These results support the above model that the biological activity of TNFcr is not restricted to one unique site. The monoclonal antibodies mAb 195 and mAb 199 were used to develop a sensitive sandwich ELISA assay. The ELISA was as sensitive as the bioassay and could discriminate between TNFa and TNFP, which the bioassay is unable to do. Furthermore, it is faster and easier to perform than the bioassay. The monoclonal antibodies, described here, should prove to be useful in the study of this cytokine’s mechanisms of actions. The sensitive ELISA assay can be used to identify and quantitate TNFa in human diseases and for the monitoring of TNFcr levels in patients undergoing TNFcr treatment. The neutralizing antibodies can selectively neutralize TNFa cytolytic activity in an in vitro system, thereby separating the effects of TNFa, from other cytokines in complex biological systems. Neutralizing anti-TNFa monoclonal antibodies may be able to block the side effects of TNFa in vivo, as shown by Tracey et al. 1987,33 for septic shock during lethal bacteremia in baboons. Studies with polyclonal anti-TNFa antibodies in mice have also shown that this treatment almost completely prevented the cutaneous and intestinal lesions of the acute-phase of Graft-versus-Host Disease and markedly reduced overall mortality.23 In the studies of Grau et al. ** the injection of a rabbit antibody to TNFa fully protects mice from cerebral malaria. TNFa-specific monoclonal antibodies may therefore provide a specific therapy for TNFa-mediated disorders.
CYTOKINE,
Vol. 2, No. 3 (May
1990: 162-169)
MATERIALS AND METHODS Recombinant Lymphokines Recombinant human and murine TNFa were produced in E. coli and purified to 8 x lo6 and 13 x lo6 laboratory U/mg,
respectively.34 The materials appeared homogeneous when analyzed by SDS-PAGE, stained with CoomassieBlue. Natural human TNFo( was purified from HL-60 cell line supernatants8 Recombinant human IFNy and human lymphotoxin (TNFP) were produced in E. coli and purified to homogeneity.
TNFcxfrom DiRevent Animal Species Blood was drawn from the following animal species: mouse, rat, rabbit, dog, pig, cynomolgus, rhesus, baboon and chimpanzee, and centrifuged through a ficoll gradient. Peripheral mononuclear cells (2 x IO6cells/ml) were suspended in RPM1 1640 supplemented with penicillin (50 U/mL), streptomycin (50 bg/mL), glutamine (2 mM) and 5% fetal calf serum (FCS) (Boehringer). 10 mL of this suspension was transferred to a T25 Falcon flask. TNFol was then induced by adding 10 Mg/mL LPS (Sigma, No. L400.5). The supernatants were collected after 16 hr and the TNFa! levels were either immediately estimated or stored at -80°C until the TNFol test was performed.
Biological Assayfor TNFol Activity The biological activity of TNFa was determined by the murine L929 fibroblast cytotoxic assay, as described by Aggarwal et al.* Briefly, L929 cells were seededat a density of 10,000 cells per well in 96-well microtiter plates (Nunc) using 0.1 mL Eagle’s Minimal Essential Medium that contained 10% fetal bovine serum (FBS). An equal volume of medium containing the desired dilutions of TNFol, with or without anti-TNFcu antibodies, was added and incubated at 37OCand 5% CO, for 48 hr in a humidified incubator. The cells were then stained with 0.5% crystal violet. After an incubation of 15 min, the plates were rinsed with tap water and then dried. Crystal violet was dissolved by the addition of 0.1 mL/well of 0.1% acetic acid in 50% ethanol. The absorbance at 580 nm was read with a SLT Easy Reader. The TNFol U/mL represent the reciprocal of the TNFcv dilution that causes50% cytotoxicity under the conditions of the assay.
Immunization, Fusion and Cloning Purified recombinant human TNFa, in an emulsion formed by equal volumes of TNFcv solution and Freund’s complete adjuvant, was injected into BALB/c mice i.p. (5 fig/mouse). Two, 4 and 6 weeks later, the mice received the same amount of antigen in Freund’s incomplete adjuvant. Three days after the last immunization spleen cells from mice showing high titers of antibody in their sera were fused with SP2/0-Ag14 cells at a ratio of 5:1, according to previously described techniques.35Two weeks after fusion, culture supernatants were assayed for secretion of anti-TNFa antibodies using the methods described. Positive hybridomas were subcloned twice by limiting dilution in 96-well plates, using BALB/c thymocytes ( lo6 cells/well) as feeder cells.
Murine monoclonal anti-human TNFa antibodies / 167
Selection TNFQ
of Monoclonal
Antibodies
to Human
Recombinant human TNFa was diluted in PBS to a concentration of 0.01 mg/mL. A 0.1 mL aliquot of this suspension was added to each well of a 96-well microtiter plate (Flow). The plate was incubated overnight at 4OC and the fluid was removed. Residual protein-binding sites were blocked by PBS containing 0.1% Tween 20 (PBS-T). Supernatants (0.1 mL/well), which contained monoclonal antibodies, were added and incubated for 2 hr at room temperature. After a washing step, 0.1 mL of a 1 ,OOO-fold diluted solution of goat anti-mouse IgG antibody labeled with horseradish peroxidase (Boehringer) was added to each well. After incubation for 1 hr at room temperature and washing with PBS-T, 0.1 mL of 0.42 mM 3,3’,5,5’-Tetramethylbenzidine (TMB, Boehringer Mannheim) in 0.1 M acetate-citrate buffer, pH 4.9 containing 0.004% H,O, was added. The reaction was stopped after 10 min by the addition of 2.0 N sulfuric acid and the absorbance at 450 nm of each well was determined using a SLT Easy Reader.
Monoclonal
Antibody
Isotyping
A 96-well microtiter plate (Flow) was coated with human rTNFa (0.01 mg/mL in PBS) overnight at 4°C and then blocked with PBS containing 1% bovine serum albumin (BSA). 0.1 mL of culture supernatants were added to wells and incubated for 1 hr at room temperature. Plates were washed with PBS-T and 0.05 mL of each isotype-specific rabbit anti-mouse Ig antibody (Cappel) was added. After 1 hr incubation at room temperature, the wells were washed 3 times and 0.05 mL of horseradish peroxidase labeled goat antirabbit IgG (Boehringer Mannheim) was added to each well. Incubation was continued for another hour at room temperature. Plates were washed 5 times with PBS-T and 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) was used as substrate as described previously.
0.05 M potassium phosphate, 0.5 M sodium chloride saccharose at pH 7.8.
Puvijication
of Monoclonal
and 5%
Antibodies
Hybridoma cells were cultivated in Iscove Medium containing the following supplements: 1.2 mg/mL BSA, 0.024 mg/mL soybean lipids, 0.034 mg/mL transferrin and 5 x 10m5 M @-mercaptoethanol. Cells were grown in spinner flasks, the medium was harvested by filtration through a 0.3 pm filter (Pall) and concentrated lo-fold by ultrafiltration using a PM10 membrane (Amicon). The concentrate was dialyzed against 1.5 M glycine and 3 M NaCl, pH 8.9 and the IgG purified by Protein A Sepharose chromatography (Pharmacia), according to the manufacturer’s recommendation.
Monoclonal
Antibodies
AJinity
Determination
96-well microtiter plates (Nunc) were coated for 16 hr at 4°C with 0.01 mg/mL TNFa (0.1 mL/well) in PBS. After that the wells were washed with PBS-T and post-coating was done with 1% BSA in PBS for 0.5 hr at 37OC. mAbs were serially diluted with 0.1% BSA in PBS and incubation was done for 4 hr at 37OC. The wells were washed again 5 times with PBS containing 0.1% Tween 20 (PBS-T) and incubated for 2 hr at 37OC with horseradish peroxidase conjugated rabbit anti-mouse IgG (0.1 mL/well, diluted 1:3,000 in 0.1% BSA in PBS). The wells were then washed 5 times with PBS-T and TMB was used as substrate, as described previously. Calculations of the affinity of the monoclonal antibodies were done by non-linear least square fits. These were based on the model of one monoclonal antibody binding site per TNFa subunit.
Enzyme-Linked
Immunosovbent
Assay (ELISA)
A 96-well microtiter plate (Flow) was coated with 0.001 mg/mL recombinant human TNFcv (0.1 mL/well) in PBS overnight at 4OC. Nonspecific binding sites were then blocked with a solution of 1% BSA in PBS for 0.5 hr at 37°C. A constant volume (0.05 mL) of an appropriate dilution of biotinylated monoclonal antibody was added to equal volumes of double dilutions of respective unlabeled monoclonal antibodies. The mixture was then added to the wells and incubated for 2 hr at 37OC. The washing and further processing were done as described for “ELISA.”
A 96-well microtiter plate (Nunc) was coated overnight with 0.1 mL/well of anti-TNFa mAb 199 (0.005 mg/mL) in PBS. Post coating was done with 1% BSA in PBS for 0.5 hr at 37OC. The plate was washed 5 times with PBS containing 0.1% Tween 20 (PBS-T). For a standard curve, TNFa was diluted in 20 mM Tris-HCl, 150 mM NaCl and 1% BSA, pH 7.4. TNFU-containing medium or serum (0.1 mL/well) was reacted for 3 hr at room temperature or overnight at 4°C. After washing, 0.1 mL/well of biotinylated mAb 195 (0.001 mg/ mL) was added and incubated for 2 hr at room temperature. After 3 washes with PBS-T, 0.1 mL/well of lO,OOO-fold diluted streptavidin-peroxidase complex (Boehringer Mannheim) was added and incubated again for 0.5 hr at room temperature. TMB was used as a substrate for developing the color as d.escribed.
Biotinylation
Gel Electrophovesis
Determination
of Epitope Specificities
of Monoclonal
Antibodies
The mAbs were biotinylated with D-biotinyle-e-amidocaproic-acid-N-hydroxysuccinimide-ester (Boehringer Mannheim). Ten mg of this reagent was dissolved in 1 mL DMSO. The monoclonal antibodies were adjusted to 5 mg/mL in PBS. A 35 molar excess of biotin was used to label the antibodies for 2 hr at room temperature. The unbound biotin was removed from modified monoclonal antibodies by Sepharose G25 column chromatography (1 x 15 cm). The column was run with
and Immunoblots
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed, as described by Laemmli et a1.36 Recombinant TNFa (0.2 Hg) and human serum were subjected to electrophoresis on a 15% acrylamide gel. Gels were stained with 0.15% Coomassie Blue R250 (wt/vol) in 25% isopropanol (vol/vol), 10% acetic acid (vol/vol), and destained with 10% methanol (vol/vol), 10% acetic acid (vol/vol). The binding of antibodies to TNFa, which are
168 / Miiller
CYTOKINE,
et al.
separated by SDS-PAGE, was assessed by the immunoblot technique. SDS-PAGE gels were transferred to nitrocellulose as previously described by Towbin et al.*‘j The non-specific reactive sites on the nitrocellulose were blocked by incubating for 30 min :j:ith 3% gelatin (wt/vol) in TBS. Test monoclonal antibodies v’-re diluted to 0.01 mg/mL in 1% gelatin (wt/vol) in TBS, and incubated with the nitrocellulose strips for 16 hr at 22OC. The strips were then washed for 15 min, each with three changes of PBS-T. Bound monoclonal antibodies were detected after incubating for 4 hr with horseradish peroxidase conjugated anti-murine IgG (Cappel) that was diluted 1:lOOO in 1% gelatin. The strips were washed as above and incubated 15 min with 4-chloro-1-naphthol (0.6 mg/mL in 20% methanol, 80% TBS containing 0.018% H202), rinsed with water and air dried.
In Vivo Neutralization of Human TNFa Four-6 week old BALB/c mice were randomized and divided into groups of 5. The substances were given i.v. into the lateral tail vein; the volume of injection did not exceed 10 mL/kg. TNFo( (2 mg/kg body weight) was applied first, followed 15 to 30 min later by the monoclonal antibodies (doses: 2 mg/kg and 10 mg/kg). The rate of survival was determined 24 hr later.
Protein Determination Protein determinations were performed by the dye binding method of Bradford,37 which uses bovine IgG as a standard. The protein concentration was also determined by absorbance using the extinction coefficients for pure recombinant TNFa, e2s01 mg/mL = 1.6.
Acknowledgments The authors thank M. Pan for help in writing the manuscript, J. Delzer and H. Hillen for fermentation and purification of the cytokines. We also gratefully acknowledge the expert technical assistance of M. Seitz, H. Hofmann, E. Klimm and R. Merx.
REFERENCES 1. Carswell EA, Old LJ, Kassel RL, Greene S, Fiore N, Williamson B (1975) An endotoxin-induced serum factor that causes necrosis of tumors. Proc Nat Acad Sci USA 72:3666-3670 2. Haranaka K, Satomi N (1981) Cytotoxic activity of tumor necrosis factor on human cancer cells in vitro. Jpn J Exp Med 51:191-194 3. Sugarman BJ, Aggarwal BB, Hass PE, Figari IS, Palladino MA, Jr, Shepard HM (1985) Recombinant human tumor necrosis factor-a: Effects of proliferation of normal and transformed cells in vitro. Science 230:943-949 4. Haranaka K, Satomi N, Sakurai A (1984) Antitumor activity of murine tumor necrosis factor against transplanted murine tumors and heterotransplanted human tumors in nude mice. Int J Cancer 341263-267 5. Beutler B, Cerami A (1987) Cachectin: More than a tumor necrosis factor. N Engl J Med 316:379-383 6. Kunkel SL, Remick DG, Strieter RM, Larrick JW (1989) Mechanisms that regulate the production and effects of tumor necrosis factor-a. Critical Rev Immunol9:93-117
Vol. 2, No. 3 (May 1990: 162-169)
7. Old LJ (1987) Polypeptide mediator network. Nature 326:330331 8. Aggarwal BB, Kohr WJ, Hass PE, Moffat B, Spencer SA, Henzel WJ, Bringman TS, Nedwin GE, Goeddel DV, Harkins RN (1985) Human tumor necrosis factor, production, purification characterization. J Biol Chem 260:2345-2354 9. Beutler B, M&ark IW, Cerami AC (1985) Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effects of endotoxin. Science 229:869-871 10. Chen AR, McKinnon KP, Koren HS (1985) Lipopolysaccharide (LPS) stimulates fresh human monocytes to lyse actinomycin D-treated WEHItarget cells via increased secretion of a monokine similar to tumor necrosis factor. J Immunol 135:3978-3984 11. Michie HR, Manogue KR, Spriggs DR, Revhaug A, O’Dwyer S, Dinarello CA, Cerami A, Wolff SM, Wilmore DW (1988) Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 318:1481-1486 12. Cuturi MC, Murphy M, Costa-Giomi MP, Weinmann R, Perussia B, Trinchieri G (1987) Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral bloodlymphocytes. JExpMed 165:1581-1585 13. Feinman R, Henriksen-DeStefano D, Tsujimoto M, Vilcek J (1987) Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes. J Immunol 138:635-641 14. Debets JMH, Van der Linden CJ, Spronken IEM, Buurman WA (1988) T-cell mediated production of tumor necrosis factor a by monocytes. Stand J Immunol27:601-608 15. Pennica D, Nedwin GE, Hayflick JS, Seeburg PH, Derynck R, Palladino MA, Kohr WJ, Aggarwal BB, Goeddel DV (1984) Human tumour necrosis factor: Precursor structure, expression and homology to lymphotoxin. Nature 312:724-729 16. Jones EY, Stuart DI, Walker NPC (1989) Structure of tumor necrosis factor. Nature 338:225-228 17. Cerami A, Ikeda Y, Le Trang N, Hotez PJ, Beutler B (1985) Weight loss associated with an endotoxin-induced mediator from peritoneal macrophages: The role of cachectin (tumor necrosis factor) Immunol Lett 11:173-177 18. Scuderi P, Sterling KE, Lam KS, Finley PR, Ryan KJ, Ray CG, Petersen E, Slymen DJ (1986) Raised serum levels of tumour necrosis factor in parasitic infections. Lancet 2: 1364- 1365 19. Shimomura K, Manda T, Mukumato S, Kobayashi K, Nakano K, Mori J (1988) Recombinant human tumor necrosis factor cy:Thrombus formation as a cause of anti-tumor activity. Int J Cancer 41~243-247 20. Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey TJ III, Zentella A, Albert JD, Shires GT, Cerami A (1986) Shock and tissue injury induced by recombinant human cachectin. Science 234:470-474 21. Waage A, Halstensen A, Espevik T (1987) Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet 1:355-357 22. Grau GE, Fajardo LF, Piguet P-F, Allet B, Lambert P-H, Vassalli P (1987) Tumor necrosis factor (Cachectin) as an essential mediator in murine cerebral malaria. Science 237:1210-1212 23. Piguet P-F, Grau GE, Allet B, Vassalli P (1987) Tumor necrosis factor/cachectin is an effector of skin and gut lesions of the acute phase of graft-vs-host disease. J Exp Med 166:1280-1289 24. Maury CPJ, Teppo AM (1987) Raised serum levels of cachectin/tumor necrosis factor a in renal allograft rejection. J Exp Med 166:1132-1137 25. Smith RA, Baglioni C (1987) The active form of tumor necrosis factor is a trimer. J Biol Chem 262:6951-6955 26. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat1 Acad Sci USA 76:43504354 27. Bringman T, Aggarwal BB (1987) Monoclonal antibodies to human tumor necrosis factors alpha and beta: Application for affinity
Murine monoclonal anti-human TNFo( antibodies / 169 purification, immunoassays, and as structural probes. Hybridoma 6:489-507 28. Hirai M, Okamura N, Terano Y, Tsujimoto M, Nakazato H (1987) Production and characterization of monoclonal antibodies to human tumor necrosis factor. J Immunol Methods 96:57-62 29. Meager A, Parti S, Leung H, Peil E, Mahon B (1987) Preparation and characterization of monoclonal antibodies directed against antigenic determinants of recombinant human tumour necrosis factor (rTNF). Hybridoma 6:305-3 11 30. Liang CM, Liang SM, Jost T, Sand A, Dougas I, Allet B (1986) Production and characterization of monoclonal antibodies against recombinant human tumor necrosis factor/cachectin. Biothem Biophys Res Comm 137:847-854 31. Fendly BM, Toy KJ, Creasey AA, Vitt CR, Larrick JW, Yamamoto R, Lin LS (1987) Murine monoclonal antibodies defining neutralizing epitopes on tumor necrosis factor. Hybridoma 6:359-369 32. Shimamoto Y, Chen RL, Bollon A, Chang A, Khan A (1988) Monoclonal antibodies against human recombinant tumor
necrosis factor: Prevention of endotoxic shock. Immunol Lett 17:31 l318 33. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A (1987) Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330:662-665 34. Ziegler-Heitbrock HW, Moller A, Linke RP, Haas JG, Rieber EP and Riethmtiller (1986) Tumor necrosis factor as effector molecule in monocyte mediated cytotoxicity. Cancer Res 46:59475952 35. Oi V, Herzenberg L (1980) Immunoglobulin-producing hybrid cell lines. In Mishell B, Shiigi S (eds). Selected Methods in Cellular Immunology. Freeman Co., San Francisco, pp 351-372. 36. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 37. Bradford, MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254
