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Four epitopes on tumor necrosis factor-alpha defined by murine anti-tumor necrosis factor-alpha monoclonal antibodies
Immunology Letters, 41 (1994) 139-145 Elsevier Science B.V.
IMLET 2165
Four epitopes on tumor necrosis factor-alpha defined by murine anti-tumor necrosis factor-alpha monoclonal antibodies Bahija Karim, Roland B61iard, Jean-Jacques Huart and Dominique Bourel * Centre Rdgional de Transfusion Sanguine, rue Camille Gudrin, 59000 Lille, France
(Received 5 February 1994; revision received 7 April 1993; accepted 8 April 1994) Key words: Tumor necrosis factor; Monoclonal antibody; Anti-tumor necrosis factor-alpha; ELISA; BIAcore
I. Summary Eight murine anti-TNF,~ monoclonal antibodies (mAb) were produced after immunization of B A L B / c mice with rhTNF,~. Six of these mAbs were able to neutralize cytotoxic activity of TNF,, against L929 cells. Two other mAbs had no neutralizing effect. Epitope mapping studies were performed by ELISA and by using a BIAcore system (Pharmacia). The described mAbs were allowed to define 4 different epitopes on TNF,,. Three of them were involved in the binding of TNF,~ with its receptor (cytotoxic neutralization of TNF,~). Another epitope was defined by non-neutralizing mAbs.
2. Introduction Several studies have already shown that tumor necrosis factor (TNF) is a cytokine secreted by activated lymphocytes and monocytes [1-3]. Two forms of TNF were identified. TNF,~ is produced by monocytes/macrophages as an unglycosylated protein of 157 amino acids; its active form is a trimeric molecule [4]. TNFo, also called lymphotoxin, is produced by activated lymphocytes as a glycoprotein of 171 amino acids [5-7]. A comparison of TNF,~ and TNF0 reveals a high degree of homology in their amino acid sequences [5,8-10]. * Corresponding author: Dominique Bourel, Laboratoire d'Ing6nierie Cellulaire et Mol6culaire, CRTS, rue Camille Gu6rin, 59000 Lille, France. Tel: (33) 20-49-43-43; Fax: (33) 20-49-45-08. Abbreviations: ELISA, enzyme-linked immunosorbent assay; OPD, orthophenylenediamine; MTT, 3-(4,5 dimethyl thiazol-2yl)2-5 diphenyl tetrazolium bromide; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; NI-IS, N-hydroxysuccinimide; EDC, N-ethyl-N2 (dimethylaminopropyl-carbodiimide). SSDIO165-2478(94)OOO80-B
TNF~, in combination with gamma-interferon (INFv), is regarded as a major effector cytokine involved in tumor regression [11]. This anti-tumor effect was assessed in vitro against different murine and human cell lines. In addition, proliferation of different cell lines grafted into animals could be inhibited by injections of TNF~ [121. When TNF,~ is produced in excess, on account of a release of endotoxins, it induces inflammatory tissue destruction as observed in septic shock [13-15]. To prevent such toxic effects of TNF,~, anti-TNF~ monoclonal antibodies (mAbs) could be injected. The production of several murine anti-recombinant human TNF,~ (rhTNF,~) mAbs has already been described [16-19]. The aim of this work was to study the characteristics of 8 murine anti-TNF,~ (mAbs), some of which were neutralizing while others were non-neutralizing.
3. Materials and Methods
3.1. Preparation of monoclonal antibodies 3.1.1. Immunization protocol B A L B / c mice were immunized intraperitoneally (i.p.) and subcutaneously (s.c.) with 1.7/xg of rhTNF,~ (Genzyme), emulsified in incomplete adjuvant (Lutrol, BASF). Then, 3 other injections (i.p. and s.c.) of TNF,, (10 /xg) in incomplete adjuvant were done at 1-week intervals. After the fourth injection, the serum was collected and tested for its ability to prevent rhTNF,~ cytotoxic activity against L929 cells. Two weeks later, the mice were boosted with rhTNF,~: 5 /zg intravenously (i.v.) and 60 /zg s.c. 3.1.2. Production of monoclonal antibodies Splenocytes of immunized mice were fused with P3X63Ag8 653 mouse myeloma cells according to the 139
protocol described by K6hler and Milstein [20]. Supernatants were screened by ELISA and by a neutralization assay of TNF,~ cytotoxicity. Secreting hybridomas were cloned by limiting dilution at least 2 times to assure monoclonality. The production was performed by culture supernatants and by ascitic fluids. Classes and subclasses of the anti-TNF,~ mAbs were determined by ELISA. The antibodies were precipitated with an ammonium sulfate solution at 50% saturation and purified by affinity chromatography on protein A Sepharose (Pharmacia). Bound antibodies were eluted successively with 0.05 M sodium acetate buffer, pH 6 and pH 3. The pH was rapidly adjusted to 7 with Tris buffer and the antibodies were dialysed against phosphate-buffered saline (PBS). The concentration of the mAbs was measured by absorbance at 280 nm. Purity of the antibodies was controlled by SDS-PAGE according to the protocol described by Towbin et al. [21]. In addition, isoelectrofocusing analysis was performed with each mAb to assess their isoelectric point.
3.2. Selection of the anti-TNF~ monoclonal antibodies 3.2.1. Screening assay Micro-ELISA plates (Nunc) were coated with rhTNF,~ (170 ng/well) in 0.05 M carbonate buffer, pH 9.6, and incubated at 4°C overnight. The wells were saturated with 3% bovine serum albumin (BSA) in PBS ( w / v ) for 1 h at 22°C, and after each incubation, the plates were washed 3 times with PBS/Tween-20 (0.05%). Culture supernatants were added and incubated for 2 h at 22°C. Anti-TNF,~ mAbs were detected by incubating the plates for 1 h at 22°C with horseradish peroxidaseconjugated goat anti-mouse IgG (Diagnostic Pasteur). Then the plates were incubated with OPD (0.5 m g / m l ) in a 0.1 M phosphate citrate buffer, pH 5, and 0.04% H20 2. Optical densities (OD) were measured at 492 nm.
3.2.2. Cytotoxic activity of rhTNF~ Cytotoxicity of rhTNF,~ was tested with the L929 cell line. L929 cells (4 × 104) were cultured into 96-well microtiter plates (Nunc) in 50 /.d of Dulbecco's Modified Eagle's Medium (DMEM) overnight at 37°C. Actinomycin D (1 /zg/ml) was added with further incubation at 37°C for 4 h. Different concentrations of rhTNF~ were incubated overnight at 37°C. To detect viable cells, MTT (Sigma) [23-25] or Crystal Violet (Sigma) were used. MTF at 5 m g / m l in PBS was added for 4 h at 37°C. To dissolve the dye formation, 100 /zl of isopropanol in HC1 (0.04 mol/l) was added and the reagents were mixed thoroughly. The plates were read at 550 nm. Before coloration with Crystal Violet (0.5 g in 100 ml of 20% methanol), the cells were fixed with 140
methanol at 100%. After a last 10-min incubation at room temperature, the plates were washed several times and dried. The colored cells were solubilized with acetic acid at 33% and the plates were read at 550 nm. Cytotoxicity of TNF, was calculated as follows [26]: % cytotoxicity OD(control - ) - OD(test) OD(control - ) - OD(control + )
× 100
where control - : 100% viable cells (test without TNF~), and control + : 0% viable cells (test without TNF,~, plus Tween-20).
3.2.3. Neutralization of rhTNF~ cytotoxicity Activity of the mAbs was assessed by their efficacy to neutralize rhTNF,~ cytotoxicity [27]. L929 cells (4 X 104) were cultured as described previously. A preincubation of 6 n g / m l of TNF,~, with various dilutions of anti-TNF,~ mAbs ( v / v ) was performed for 1 h at 37°C. One hundred microliters were added to the cells and incubated overnight at 37°C. The amount of viable cells was measured with MTT.
3.3. Characterisation of the antibodies 3.3.1. Relative affinity of anti-TNF~ monoclonal antibodies Relative affinity of the anti-TNF,, mAbs was tested as already described [28]. Briefly, microtiter plates were coated with rhTNF~ at 1.7/xg/ml in 0.1 M bicarbonate buffer, pH 9.6, overnight at 4°C. The wells were saturated with PBS containing 3% BSA for l h at 22°C. Antibodies, at the concentration corresponding approximately to one-half the maximal absorbance (determined previously by indirect ELISA) were mixed with various concentrations of rhTNF~ and incubated in plates for 2 h at room temperature. Further processing was performed as described in the screening assay. The concentration or the molarity of rhTNF~ capable to inhibit 50% of the binding of the anti-TNF,~ mAbs on TNF,~, allowed determination of their relative affinity (TNF,~ MW: 52.8 kDa).
3.3.2. Epitope mapping Epitopic reactivity of the anti-TNF~ mAbs was studied by ELISA and with Biospecific Interaction Analysis (BIAcore, Pharmacia).
3.4. ELISA Ninety-six-well plates were coated with the first mAbs in 0.05 M bicarbonate buffer, pH 9.6. After an overnight incubation at 4°C, the plates were washed
with PBS containing 0.05% Tween-20. PBS with 3% BSA and 0.05% Tween-20 was added for 1 h at 22°C to prevent non-specific binding. After washings, rhTNF,~ was added at 400 p g / m l for 2 h at 22°C. The plates were washed again and a second biotinylated mAb was added for 1 h at 22°C. The plates were washed and streptavidin peroxidase was incubated for 45 mn at 22°C. Further processing was done as described above for E L I S A (screening assay).
a 120
loo =m ==
80
' =, L
60
23G5 41. 25G8 "--'11"-- 24A6
40
20
3.5. BIAcore analysis
i
0 The sensor surface was prepared by immobilization of rabbit anti-mouse IgG1 onto dextran as described. Briefly, the glass support was washed with one buffered solution (10 mM HEPES, pH 7.4, with 150 mM NaCI and 3.4 mM E D T A and 0.05% of a BIAcore surfactant P20 from Pharmacia) [29-31]. The sensor chip was then activated with a v / v mixture of 100 mM NHS in H 2 0 and 400 mM EDC in H 2 0 . The reactive surface was coated with a polyclonal rabbit anti-mouse IgG1 (RAM G1) at 10 / x g / m l and free reactive groups were saturated with ethanolamine. The coated surface was used to capture the first mAb present in culture supernatant. After injection of rhTNF,~ (Genzyme), 4 /zl of the first anti-TNF= was again added to eliminate falsepositive reactions and to saturate all potentially free sites. The binding of other mAbs was then analyzed. The sensor chip was regenerated with HCI (100 mM).
i
I
I
•
i
100 200 300 400 500 600
Mo Abs concentration n g / m l
120 :m
I O0
::m
80
'5
eo
L
40
~
20
..~
tl-
22E5 31H8 27F10
0
0
100 200 300 400 500 600
Mo Abs Concentration n g / m l Fig. 1. Neutralisation activity of rhTNF,~ by mAbs. The percentage of
surviving L929 cells was measured after preincubation of various concentrations of anti-TNF,~ mAbs with a same concentration of rhTNF,~ (6 ng/ml) at 37°C for 1 h.
4. Results
4.1. Production of anti-TNF~ monoclonal antibodies The hybridomas obtained after the fusion of splenocytes from immunized mice with myeloma cells were tested for antibody production by ELISA with rhTNF,~. A m o n g 800 tests, 40 culture supernatants were positive. Eight hybridomas were selected by E L I S A for their strong reactivity against rhTNF~. All these anti-TNF~ mAbs were of IgG1 subclass except Ab 31H8 which was of the IgG2 subclass.
4.2. Neutralization of rhTNF, cytotoxicity When the cytotoxic activity of rhTNF,~ was tested against L929 cells, maximal toxicity was obtained from 6 n g / m l of rhTNF,~. To determine the capacity of anti-TNF,~ mAbs to inhibit toxicity of rhTNF,,, various concentrations of purified antibodies were incubated with 6 n g / m l of rhTNF,~, and the percentage of surviving cells was determined (Fig. 1). The abilities of the 8 murine mAbs to neutralize rhTNF,~ were different, mAbs
TABLE 1 RELATIVEAFFINITIESOF anti-TNF,~ mAbs Various concentrations of rhTNF,~were mixed with antibodies at the concentration corresponding approximately to one-half the maximal absorbance determined previously. The activity was assessed by ELISA. Results corresponded to an average of 3 experiments (each point in duplicate). Anti-TNF,~ mAbs
mAbs * (/~g/ml)
TNF,~ ICs0 ** (ng/ml)
Relative affinities (10 8 M - 1)
22 E5 23 G5 24 A6 25 G8 27 F10 31 H8 31 F7 35 D6
0.80 0.07 1.25 0.12 0.30 0.20 1.25 0.12
0.40 0.30 0.40 0.25 0.40 0.40 0.60 0.25
1.35 1.40 1.30 2.00 1.20 1.20 0.85 2.00
* Mean concentrations of mAbs corresponding to 50% of maximal binding on rhTNF,,. ** Inhibiting concentrations of TNF,~ able to block 50% of the fixation of mAbs (IC50). 141
TABLE 2 EPITOPE MAPPING OF anti-TNF,~ mAbs TESTED BY ELISA According to their reactivity patterns the 8 mAbs tested identify 4 different epitopes. Average of at least 2 experiments (each point in duplicate). Second antibodies
First antibodies 24A6
25G8
22E5
23G5
27F10
31H8
31F7
35D6
24A6 25G8 22E5 23G5 27F10 31H8 31F7 35D6
+ + + + + + +
+ + + + +
+ + + + + +
+ + + . . . +
+ + +
+ + +
+ + +
-
+
+ + + + + +
.
.
.
.
.
. .
.
. +
+
. -
-
+, Groups of mAbs able to bind simultaneously to TNF,~: (OD > 1). - , Groups of mAbs interfering in binding to TNF,~: (OD < 0.2).
o f the c o n c e n t r a t i o n s tested, t h e s e t w o m A b s w e r e
23G5 and 2 5 G 8 p r e s e n t e d the s t r o n g e s t activity. A c o n c e n t r a t i o n o f 125 n g / m l o f t h e s e m A b s w a s sufficient to c o m p l e t e l y neutralize 6 n g / m l
o f rhTNF,~. O n
the o t h e r h a n d , it w a s n e c e s s a r y to add 250 n g / m l a n t i b o d i e s 2 4 A 6 or 22E5
non-neutralizing.
and 500 n g / m l
4.3. Affinity of anti-TNF~ monoclonal antibodies
of
or 1000
o f a n t i b o d i e s 3 1 H 8 a n d 27F10, r e s p e c t i v e l y , to
R e l a t i v e affinities o f anti-TNF,~ m A b s w e r e deter-
obtain the s a m e result. A n t i b o d i e s 31F7 and 3 5 D 6 w e r e
m i n e d b y E L I S A . T h e a n t i b o d y c o n c e n t r a t i o n s corre-
u n a b l e to b l o c k the c y t o t o x i c i t y o f rhTNF,~. R e g a r d l e s s
s p o n d i n g to 5 0 % o f m a x i m a l b i n d i n g o n T N F , w e r e
ng/ml
4000026000
35000-
B
25000
30000
24000
25000
23000 R
20000
R U
U 15000
22000
I0000
21000
5000
20000
0
19000
0
200
400
600
800 Time
1000 1200 1400 1600
"
0
, ,~ , , 200
400
600
800
i000
,
,
1200
1400
1600
1800
800 1000 1200 200
1400
1600
1800
Time
Is]
,t
[s]
27000
26800 26000 25800 25000 24800
R U
24000
23800
23000
22800
22000
21800
21000 0
200
400
600
800
Time
i000 [s]
1200
1400
1600
1800
200
400
600
Time Is]
Fig. 2. Epitope mapping study of anti-TNF,~ mAbs with a BIAcore (Pharmacia). a: sensorgrams obtained after immobilization of RAM G1 on sensor chip CM5; numbers indicate injections as follows: (1) NHS/EDC (surface activation); (2) RAM G1 (coating); (3) ethanolamine (free reactive group saturation); and (4) HCI (non-covalently bound material removal). B-D: examples of sensorgrams obtained with 2 anti-TNF,~ mAbs. (A) baseline; (A-B) first anti-TNF,~ mAb; (B-C) blocking antibody; (C-D) first anti-TNF, mAb, second injection; B and C: mAbl and mAb2 recognized different epitopes. D: mAbl and mAb2 recognized the same epitope. 142
variable. These values were used to measure the concentration of TNF,~ (IC50) able to inhibit 50% of the fixation of the Abs on the same TNF~-coated plates (Table 1). Relative affinities were calculated as described previously. Some antibodies were used at 0.3 /xg/ml (27 F10) and others at 1.25 /xg/ml (24A6 and 31F7). From these data, the affinities of anti-TNF~ mAbs for rhTNF~ were similar (0.85-2.108 M-l), 25G8 and 35D6 mAbs having the highest affinities.
TABLE3 EPITOPE MAPPING STUDIES WITH A BIAcore System (Pharmacia)
4.4. Epitope mapping
Results are presented as relative values of fixation of a second anti-TNF,~ mAb (resonance units). For example: 343(+)= specific fixation of a second mAb; 44(-) = negative fixation of a second mAb.
The number of epitopes recognized by the selected murine anti-TNF~ mAbs on the rhTNF~ was determined by ELISA and with BIAcore, using an antibody competition method.
4.4.1. ELISA Competition tests performed as described allowed to classify anti-TNF,, mAbs in several groups recognizing at least four different epitopes expressed by TNF~ molecules, 3 of which reacted with neutralizing antibodies (Table 2). Epitope 1 was defined only by mAb 24A6, and epitope 2 was recognized exclusively by mAb 22E5. Antibody 25G8 cross-reacted with both epitopes 1 and 2. Three other antibodies (23G5, 27F10 and 31H8) allowed definition of a third epitope. Epitope 4 was determined by mAb 35D6. In addition, mAb 31F7 was able to block the fixation of antibodies belonging to epitopes 3 and 4. 4.4.2. BIAcore Immobilization of a polyclonal rabbit anti-mouse IgG1 onto the dextran (Fig. 2a) was performed before injection of the first anti-TNF~ mAb. Picks EF were recorded after injection of a second anti-TNF~ mAb: 27F10 (Fig. 2b), 25G8 (Fig. 2c) and 31F7 (Fig. 2d). With 27F10 and 25G8 mAbs resonance signals were observed: picks EF being higher than picks DE. These data showed that antibodies 2 4 A 6 / 2 7 F 1 0 and 31F7/25G8 can bind simultaneously onto the rhTNF~. Conversely, antibodies 27F10/31F7 did not bind together onto TNF,~ (Fig. 2d). The results obtained with all the possible combinations of 6 anti-TNF~ mAbs are presented in Table 3 (numbers correspond to relative values: resonance units). In comparison with ELISA, no discrepancy was observed. The study of these mAbs with a BIAcore also allowed definition of 4 epitopes expressed by TNF~.
5. Discussion
In this report, the production of 8 murine anti-TNF~ mAbs was described after immunization of B A L B / c
Second First monoclonalantibody antibody 24A6 25G8 27F10
31F7
35D6
24A6 25G8 27F10 31F7 35D6
417(+) 561(+) 125(+) 36(-) 49(-)
262(+) 420(+) 220(+) 23(-) 17(-)
44(-) 113 (+) 602(+) 272(+) 469(+)
30(-) 70(-) 620(+) 251(+) 544(+)
343(+) 496(+) 55(-) 14(-) 132(+)
mice with rhTNF,~. The neutralizing activity of these mAbs was studied using MTI" assay with the L929 cell line. A pretreatment of L929 cells with actinomycin D increased their sensitivity for TNF~ [32]. Another test using Crystal Violet was also performed, but it only gave information about the percentage of dead cells. Although weaker values were obtained with MTI', this test remained more sensitive. MTT was enzymatically cleaved by mitochondrias present in viable cells to produce formazan crystals, and the reaction was proportional to the intensity of cellular metabolism. Numerous reports have already demonstrated that TNF,, interacts with cellular receptors [33,34] and elicits cytotoxic or growth regulatory responses. Among the 8 anti-TNF~ mAbs tested, 6 were able to prevent TNF,~ cytotoxicity. mAbs 23G5 and 25G8 were the more potent reagents, whereas mAbs 24A6 and 22E5 were weaker and mAbs 31H8 and 27F10 were less effective. These anti-TNF~ mAbs may inhibit binding of hTNF,~ to its receptors (TNF,,R) by masking some amino acid residues involved in the TNF~-TNF~R interactions. Two mAbs (31F7 and 35D6) were unable to block TNF, cytotoxicity; they recognized a part of TNF not involved in the binding to TNF receptors. For epitope mapping studies, comparable results were obtained with ELISA and with BIAcore studies. For ELISA it was necessary to use purified and biotinylated mAbs. BIAcore investigations allowed quick and sensitive analysis between several antibodies without any preparation of the mAbs. It was necessary to implement competition tests in reverse order because, depending on the first Ab used, different results could be obtained. This could be due to modifications of the microenvironment after the fixation of the first mAb. Anti-TNF mAbs could be classified into 6 different groups taking into account their reactivity (Table 4). Four groups of antibodies were clearly definable on the basis of their different reactivities: group I defined by mAb 22E5, group II defined by mAb 24A6, group III defined by 3 143
TABLE 4 CLASSIFICATIONOF THE 8 anti-TNF ANTIBODIES Group of antibodies
Antibodies
Epitopes
I II III IV V VI
24A6 22E5 23G5, 31H8, 27F10 35D6 a 25G8 31F7 a
1 2 3 4 1+ 2 3+ 4
a Non-neutralizing antibodies.
m A b s (23G5, 27F10, 31H8) and group IV by m A b 35D6. Two other antibodies corresponded to cross-reacting reagents: m A b 25G8 recognized an epitope expressed by both epitope 1 (mAb 24A6) and epitope 2 (mAb 22E5) and m A b 31F7 cross-reacted with both epitopes 3 and 4. Epitopes 1, 2 and 3 were well defined by antibody groups I, II and III, respectively. These 3 epitopes, characterized by neutralizing antibodies, could be involved in the binding region of TNF to its receptors. Epitope 4 could be located on an inactive part of the T N F molecule or a structural portion not implicated in the function of the molecule. Bringman and Aggarwal [35] have already defined 5 different epitopes on TNF,,, 3 of which reacted with neutralizing antibodies and 2 epitopes with non-neutralizing antibodies. Immunogenic regions of rhTNF,, have been mapped by Corti et al. [36]; "they studied interactions between various mouse anti-hTNF,, and synthetic hTNF,, peptides. They identified 3 immunogenic regions with residues 1 - 2 3 , 9 5 - 1 1 6 and 1 3 7 - 1 5 7 of rhTNF,~ and also 2 other less immunogenic regions with residues 3 7 - 5 5 and 117-136. They showed that the binding of m A b s to the antigenic sites 9 5 - 1 1 6 and 1 3 7 - 1 5 7 did not affect cytolytic activity. However, the neutralizing antibodies described in these 2 publications failed to recognize synthetic hTNF,~ peptides and it was concluded that they probably bound to conformationdependent or discontinuous epitopes. Our results are in accordance with the definition of at least 4 different epitopes expressed by T N F molecules, but it was impossible to define more precisely the reactivity of these mAbs. However, at the opposite of previously published results, all the described antibodies were able to recognize rhTNF, denatured during Western blot studies. A better knowledge of the precise reactivity of m A b s is necessary to select non-competitive reagents to prepare kits for the dosage of cytokines. For this purpose the use of a BIAcore system is very useful and time saving. Such a study could also be suitable for selection of antibodies with 144
complementary effects for the neutralisation of the TNF toxicity during therapeutical applications.
Acknowledgements W e wish to thank P. Seguin and L. Bellanger (CIS Biointernational, France) for BIAcore studies and also S. Martel for her assistance in the preparation of this manuscript.
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145
IMLET 2165
Four epitopes on tumor necrosis factor-alpha defined by murine anti-tumor necrosis factor-alpha monoclonal antibodies Bahija Karim, Roland B61iard, Jean-Jacques Huart and Dominique Bourel * Centre Rdgional de Transfusion Sanguine, rue Camille Gudrin, 59000 Lille, France
(Received 5 February 1994; revision received 7 April 1993; accepted 8 April 1994) Key words: Tumor necrosis factor; Monoclonal antibody; Anti-tumor necrosis factor-alpha; ELISA; BIAcore
I. Summary Eight murine anti-TNF,~ monoclonal antibodies (mAb) were produced after immunization of B A L B / c mice with rhTNF,~. Six of these mAbs were able to neutralize cytotoxic activity of TNF,, against L929 cells. Two other mAbs had no neutralizing effect. Epitope mapping studies were performed by ELISA and by using a BIAcore system (Pharmacia). The described mAbs were allowed to define 4 different epitopes on TNF,,. Three of them were involved in the binding of TNF,~ with its receptor (cytotoxic neutralization of TNF,~). Another epitope was defined by non-neutralizing mAbs.
2. Introduction Several studies have already shown that tumor necrosis factor (TNF) is a cytokine secreted by activated lymphocytes and monocytes [1-3]. Two forms of TNF were identified. TNF,~ is produced by monocytes/macrophages as an unglycosylated protein of 157 amino acids; its active form is a trimeric molecule [4]. TNFo, also called lymphotoxin, is produced by activated lymphocytes as a glycoprotein of 171 amino acids [5-7]. A comparison of TNF,~ and TNF0 reveals a high degree of homology in their amino acid sequences [5,8-10]. * Corresponding author: Dominique Bourel, Laboratoire d'Ing6nierie Cellulaire et Mol6culaire, CRTS, rue Camille Gu6rin, 59000 Lille, France. Tel: (33) 20-49-43-43; Fax: (33) 20-49-45-08. Abbreviations: ELISA, enzyme-linked immunosorbent assay; OPD, orthophenylenediamine; MTT, 3-(4,5 dimethyl thiazol-2yl)2-5 diphenyl tetrazolium bromide; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; NI-IS, N-hydroxysuccinimide; EDC, N-ethyl-N2 (dimethylaminopropyl-carbodiimide). SSDIO165-2478(94)OOO80-B
TNF~, in combination with gamma-interferon (INFv), is regarded as a major effector cytokine involved in tumor regression [11]. This anti-tumor effect was assessed in vitro against different murine and human cell lines. In addition, proliferation of different cell lines grafted into animals could be inhibited by injections of TNF~ [121. When TNF,~ is produced in excess, on account of a release of endotoxins, it induces inflammatory tissue destruction as observed in septic shock [13-15]. To prevent such toxic effects of TNF,~, anti-TNF~ monoclonal antibodies (mAbs) could be injected. The production of several murine anti-recombinant human TNF,~ (rhTNF,~) mAbs has already been described [16-19]. The aim of this work was to study the characteristics of 8 murine anti-TNF,~ (mAbs), some of which were neutralizing while others were non-neutralizing.
3. Materials and Methods
3.1. Preparation of monoclonal antibodies 3.1.1. Immunization protocol B A L B / c mice were immunized intraperitoneally (i.p.) and subcutaneously (s.c.) with 1.7/xg of rhTNF,~ (Genzyme), emulsified in incomplete adjuvant (Lutrol, BASF). Then, 3 other injections (i.p. and s.c.) of TNF,, (10 /xg) in incomplete adjuvant were done at 1-week intervals. After the fourth injection, the serum was collected and tested for its ability to prevent rhTNF,~ cytotoxic activity against L929 cells. Two weeks later, the mice were boosted with rhTNF,~: 5 /zg intravenously (i.v.) and 60 /zg s.c. 3.1.2. Production of monoclonal antibodies Splenocytes of immunized mice were fused with P3X63Ag8 653 mouse myeloma cells according to the 139
protocol described by K6hler and Milstein [20]. Supernatants were screened by ELISA and by a neutralization assay of TNF,~ cytotoxicity. Secreting hybridomas were cloned by limiting dilution at least 2 times to assure monoclonality. The production was performed by culture supernatants and by ascitic fluids. Classes and subclasses of the anti-TNF,~ mAbs were determined by ELISA. The antibodies were precipitated with an ammonium sulfate solution at 50% saturation and purified by affinity chromatography on protein A Sepharose (Pharmacia). Bound antibodies were eluted successively with 0.05 M sodium acetate buffer, pH 6 and pH 3. The pH was rapidly adjusted to 7 with Tris buffer and the antibodies were dialysed against phosphate-buffered saline (PBS). The concentration of the mAbs was measured by absorbance at 280 nm. Purity of the antibodies was controlled by SDS-PAGE according to the protocol described by Towbin et al. [21]. In addition, isoelectrofocusing analysis was performed with each mAb to assess their isoelectric point.
3.2. Selection of the anti-TNF~ monoclonal antibodies 3.2.1. Screening assay Micro-ELISA plates (Nunc) were coated with rhTNF,~ (170 ng/well) in 0.05 M carbonate buffer, pH 9.6, and incubated at 4°C overnight. The wells were saturated with 3% bovine serum albumin (BSA) in PBS ( w / v ) for 1 h at 22°C, and after each incubation, the plates were washed 3 times with PBS/Tween-20 (0.05%). Culture supernatants were added and incubated for 2 h at 22°C. Anti-TNF,~ mAbs were detected by incubating the plates for 1 h at 22°C with horseradish peroxidaseconjugated goat anti-mouse IgG (Diagnostic Pasteur). Then the plates were incubated with OPD (0.5 m g / m l ) in a 0.1 M phosphate citrate buffer, pH 5, and 0.04% H20 2. Optical densities (OD) were measured at 492 nm.
3.2.2. Cytotoxic activity of rhTNF~ Cytotoxicity of rhTNF,~ was tested with the L929 cell line. L929 cells (4 × 104) were cultured into 96-well microtiter plates (Nunc) in 50 /.d of Dulbecco's Modified Eagle's Medium (DMEM) overnight at 37°C. Actinomycin D (1 /zg/ml) was added with further incubation at 37°C for 4 h. Different concentrations of rhTNF~ were incubated overnight at 37°C. To detect viable cells, MTT (Sigma) [23-25] or Crystal Violet (Sigma) were used. MTF at 5 m g / m l in PBS was added for 4 h at 37°C. To dissolve the dye formation, 100 /zl of isopropanol in HC1 (0.04 mol/l) was added and the reagents were mixed thoroughly. The plates were read at 550 nm. Before coloration with Crystal Violet (0.5 g in 100 ml of 20% methanol), the cells were fixed with 140
methanol at 100%. After a last 10-min incubation at room temperature, the plates were washed several times and dried. The colored cells were solubilized with acetic acid at 33% and the plates were read at 550 nm. Cytotoxicity of TNF, was calculated as follows [26]: % cytotoxicity OD(control - ) - OD(test) OD(control - ) - OD(control + )
× 100
where control - : 100% viable cells (test without TNF~), and control + : 0% viable cells (test without TNF,~, plus Tween-20).
3.2.3. Neutralization of rhTNF~ cytotoxicity Activity of the mAbs was assessed by their efficacy to neutralize rhTNF,~ cytotoxicity [27]. L929 cells (4 X 104) were cultured as described previously. A preincubation of 6 n g / m l of TNF,~, with various dilutions of anti-TNF,~ mAbs ( v / v ) was performed for 1 h at 37°C. One hundred microliters were added to the cells and incubated overnight at 37°C. The amount of viable cells was measured with MTT.
3.3. Characterisation of the antibodies 3.3.1. Relative affinity of anti-TNF~ monoclonal antibodies Relative affinity of the anti-TNF,, mAbs was tested as already described [28]. Briefly, microtiter plates were coated with rhTNF~ at 1.7/xg/ml in 0.1 M bicarbonate buffer, pH 9.6, overnight at 4°C. The wells were saturated with PBS containing 3% BSA for l h at 22°C. Antibodies, at the concentration corresponding approximately to one-half the maximal absorbance (determined previously by indirect ELISA) were mixed with various concentrations of rhTNF~ and incubated in plates for 2 h at room temperature. Further processing was performed as described in the screening assay. The concentration or the molarity of rhTNF~ capable to inhibit 50% of the binding of the anti-TNF,~ mAbs on TNF,~, allowed determination of their relative affinity (TNF,~ MW: 52.8 kDa).
3.3.2. Epitope mapping Epitopic reactivity of the anti-TNF~ mAbs was studied by ELISA and with Biospecific Interaction Analysis (BIAcore, Pharmacia).
3.4. ELISA Ninety-six-well plates were coated with the first mAbs in 0.05 M bicarbonate buffer, pH 9.6. After an overnight incubation at 4°C, the plates were washed
with PBS containing 0.05% Tween-20. PBS with 3% BSA and 0.05% Tween-20 was added for 1 h at 22°C to prevent non-specific binding. After washings, rhTNF,~ was added at 400 p g / m l for 2 h at 22°C. The plates were washed again and a second biotinylated mAb was added for 1 h at 22°C. The plates were washed and streptavidin peroxidase was incubated for 45 mn at 22°C. Further processing was done as described above for E L I S A (screening assay).
a 120
loo =m ==
80
' =, L
60
23G5 41. 25G8 "--'11"-- 24A6
40
20
3.5. BIAcore analysis
i
0 The sensor surface was prepared by immobilization of rabbit anti-mouse IgG1 onto dextran as described. Briefly, the glass support was washed with one buffered solution (10 mM HEPES, pH 7.4, with 150 mM NaCI and 3.4 mM E D T A and 0.05% of a BIAcore surfactant P20 from Pharmacia) [29-31]. The sensor chip was then activated with a v / v mixture of 100 mM NHS in H 2 0 and 400 mM EDC in H 2 0 . The reactive surface was coated with a polyclonal rabbit anti-mouse IgG1 (RAM G1) at 10 / x g / m l and free reactive groups were saturated with ethanolamine. The coated surface was used to capture the first mAb present in culture supernatant. After injection of rhTNF,~ (Genzyme), 4 /zl of the first anti-TNF= was again added to eliminate falsepositive reactions and to saturate all potentially free sites. The binding of other mAbs was then analyzed. The sensor chip was regenerated with HCI (100 mM).
i
I
I
•
i
100 200 300 400 500 600
Mo Abs concentration n g / m l
120 :m
I O0
::m
80
'5
eo
L
40
~
20
..~
tl-
22E5 31H8 27F10
0
0
100 200 300 400 500 600
Mo Abs Concentration n g / m l Fig. 1. Neutralisation activity of rhTNF,~ by mAbs. The percentage of
surviving L929 cells was measured after preincubation of various concentrations of anti-TNF,~ mAbs with a same concentration of rhTNF,~ (6 ng/ml) at 37°C for 1 h.
4. Results
4.1. Production of anti-TNF~ monoclonal antibodies The hybridomas obtained after the fusion of splenocytes from immunized mice with myeloma cells were tested for antibody production by ELISA with rhTNF,~. A m o n g 800 tests, 40 culture supernatants were positive. Eight hybridomas were selected by E L I S A for their strong reactivity against rhTNF~. All these anti-TNF~ mAbs were of IgG1 subclass except Ab 31H8 which was of the IgG2 subclass.
4.2. Neutralization of rhTNF, cytotoxicity When the cytotoxic activity of rhTNF,~ was tested against L929 cells, maximal toxicity was obtained from 6 n g / m l of rhTNF,~. To determine the capacity of anti-TNF,~ mAbs to inhibit toxicity of rhTNF,,, various concentrations of purified antibodies were incubated with 6 n g / m l of rhTNF,~, and the percentage of surviving cells was determined (Fig. 1). The abilities of the 8 murine mAbs to neutralize rhTNF,~ were different, mAbs
TABLE 1 RELATIVEAFFINITIESOF anti-TNF,~ mAbs Various concentrations of rhTNF,~were mixed with antibodies at the concentration corresponding approximately to one-half the maximal absorbance determined previously. The activity was assessed by ELISA. Results corresponded to an average of 3 experiments (each point in duplicate). Anti-TNF,~ mAbs
mAbs * (/~g/ml)
TNF,~ ICs0 ** (ng/ml)
Relative affinities (10 8 M - 1)
22 E5 23 G5 24 A6 25 G8 27 F10 31 H8 31 F7 35 D6
0.80 0.07 1.25 0.12 0.30 0.20 1.25 0.12
0.40 0.30 0.40 0.25 0.40 0.40 0.60 0.25
1.35 1.40 1.30 2.00 1.20 1.20 0.85 2.00
* Mean concentrations of mAbs corresponding to 50% of maximal binding on rhTNF,,. ** Inhibiting concentrations of TNF,~ able to block 50% of the fixation of mAbs (IC50). 141
TABLE 2 EPITOPE MAPPING OF anti-TNF,~ mAbs TESTED BY ELISA According to their reactivity patterns the 8 mAbs tested identify 4 different epitopes. Average of at least 2 experiments (each point in duplicate). Second antibodies
First antibodies 24A6
25G8
22E5
23G5
27F10
31H8
31F7
35D6
24A6 25G8 22E5 23G5 27F10 31H8 31F7 35D6
+ + + + + + +
+ + + + +
+ + + + + +
+ + + . . . +
+ + +
+ + +
+ + +
-
+
+ + + + + +
.
.
.
.
.
. .
.
. +
+
. -
-
+, Groups of mAbs able to bind simultaneously to TNF,~: (OD > 1). - , Groups of mAbs interfering in binding to TNF,~: (OD < 0.2).
o f the c o n c e n t r a t i o n s tested, t h e s e t w o m A b s w e r e
23G5 and 2 5 G 8 p r e s e n t e d the s t r o n g e s t activity. A c o n c e n t r a t i o n o f 125 n g / m l o f t h e s e m A b s w a s sufficient to c o m p l e t e l y neutralize 6 n g / m l
o f rhTNF,~. O n
the o t h e r h a n d , it w a s n e c e s s a r y to add 250 n g / m l a n t i b o d i e s 2 4 A 6 or 22E5
non-neutralizing.
and 500 n g / m l
4.3. Affinity of anti-TNF~ monoclonal antibodies
of
or 1000
o f a n t i b o d i e s 3 1 H 8 a n d 27F10, r e s p e c t i v e l y , to
R e l a t i v e affinities o f anti-TNF,~ m A b s w e r e deter-
obtain the s a m e result. A n t i b o d i e s 31F7 and 3 5 D 6 w e r e
m i n e d b y E L I S A . T h e a n t i b o d y c o n c e n t r a t i o n s corre-
u n a b l e to b l o c k the c y t o t o x i c i t y o f rhTNF,~. R e g a r d l e s s
s p o n d i n g to 5 0 % o f m a x i m a l b i n d i n g o n T N F , w e r e
ng/ml
4000026000
35000-
B
25000
30000
24000
25000
23000 R
20000
R U
U 15000
22000
I0000
21000
5000
20000
0
19000
0
200
400
600
800 Time
1000 1200 1400 1600
"
0
, ,~ , , 200
400
600
800
i000
,
,
1200
1400
1600
1800
800 1000 1200 200
1400
1600
1800
Time
Is]
,t
[s]
27000
26800 26000 25800 25000 24800
R U
24000
23800
23000
22800
22000
21800
21000 0
200
400
600
800
Time
i000 [s]
1200
1400
1600
1800
200
400
600
Time Is]
Fig. 2. Epitope mapping study of anti-TNF,~ mAbs with a BIAcore (Pharmacia). a: sensorgrams obtained after immobilization of RAM G1 on sensor chip CM5; numbers indicate injections as follows: (1) NHS/EDC (surface activation); (2) RAM G1 (coating); (3) ethanolamine (free reactive group saturation); and (4) HCI (non-covalently bound material removal). B-D: examples of sensorgrams obtained with 2 anti-TNF,~ mAbs. (A) baseline; (A-B) first anti-TNF,~ mAb; (B-C) blocking antibody; (C-D) first anti-TNF, mAb, second injection; B and C: mAbl and mAb2 recognized different epitopes. D: mAbl and mAb2 recognized the same epitope. 142
variable. These values were used to measure the concentration of TNF,~ (IC50) able to inhibit 50% of the fixation of the Abs on the same TNF~-coated plates (Table 1). Relative affinities were calculated as described previously. Some antibodies were used at 0.3 /xg/ml (27 F10) and others at 1.25 /xg/ml (24A6 and 31F7). From these data, the affinities of anti-TNF~ mAbs for rhTNF~ were similar (0.85-2.108 M-l), 25G8 and 35D6 mAbs having the highest affinities.
TABLE3 EPITOPE MAPPING STUDIES WITH A BIAcore System (Pharmacia)
4.4. Epitope mapping
Results are presented as relative values of fixation of a second anti-TNF,~ mAb (resonance units). For example: 343(+)= specific fixation of a second mAb; 44(-) = negative fixation of a second mAb.
The number of epitopes recognized by the selected murine anti-TNF~ mAbs on the rhTNF~ was determined by ELISA and with BIAcore, using an antibody competition method.
4.4.1. ELISA Competition tests performed as described allowed to classify anti-TNF,, mAbs in several groups recognizing at least four different epitopes expressed by TNF~ molecules, 3 of which reacted with neutralizing antibodies (Table 2). Epitope 1 was defined only by mAb 24A6, and epitope 2 was recognized exclusively by mAb 22E5. Antibody 25G8 cross-reacted with both epitopes 1 and 2. Three other antibodies (23G5, 27F10 and 31H8) allowed definition of a third epitope. Epitope 4 was determined by mAb 35D6. In addition, mAb 31F7 was able to block the fixation of antibodies belonging to epitopes 3 and 4. 4.4.2. BIAcore Immobilization of a polyclonal rabbit anti-mouse IgG1 onto the dextran (Fig. 2a) was performed before injection of the first anti-TNF~ mAb. Picks EF were recorded after injection of a second anti-TNF~ mAb: 27F10 (Fig. 2b), 25G8 (Fig. 2c) and 31F7 (Fig. 2d). With 27F10 and 25G8 mAbs resonance signals were observed: picks EF being higher than picks DE. These data showed that antibodies 2 4 A 6 / 2 7 F 1 0 and 31F7/25G8 can bind simultaneously onto the rhTNF~. Conversely, antibodies 27F10/31F7 did not bind together onto TNF,~ (Fig. 2d). The results obtained with all the possible combinations of 6 anti-TNF~ mAbs are presented in Table 3 (numbers correspond to relative values: resonance units). In comparison with ELISA, no discrepancy was observed. The study of these mAbs with a BIAcore also allowed definition of 4 epitopes expressed by TNF~.
5. Discussion
In this report, the production of 8 murine anti-TNF~ mAbs was described after immunization of B A L B / c
Second First monoclonalantibody antibody 24A6 25G8 27F10
31F7
35D6
24A6 25G8 27F10 31F7 35D6
417(+) 561(+) 125(+) 36(-) 49(-)
262(+) 420(+) 220(+) 23(-) 17(-)
44(-) 113 (+) 602(+) 272(+) 469(+)
30(-) 70(-) 620(+) 251(+) 544(+)
343(+) 496(+) 55(-) 14(-) 132(+)
mice with rhTNF,~. The neutralizing activity of these mAbs was studied using MTI" assay with the L929 cell line. A pretreatment of L929 cells with actinomycin D increased their sensitivity for TNF~ [32]. Another test using Crystal Violet was also performed, but it only gave information about the percentage of dead cells. Although weaker values were obtained with MTI', this test remained more sensitive. MTT was enzymatically cleaved by mitochondrias present in viable cells to produce formazan crystals, and the reaction was proportional to the intensity of cellular metabolism. Numerous reports have already demonstrated that TNF,, interacts with cellular receptors [33,34] and elicits cytotoxic or growth regulatory responses. Among the 8 anti-TNF~ mAbs tested, 6 were able to prevent TNF,~ cytotoxicity. mAbs 23G5 and 25G8 were the more potent reagents, whereas mAbs 24A6 and 22E5 were weaker and mAbs 31H8 and 27F10 were less effective. These anti-TNF~ mAbs may inhibit binding of hTNF,~ to its receptors (TNF,,R) by masking some amino acid residues involved in the TNF~-TNF~R interactions. Two mAbs (31F7 and 35D6) were unable to block TNF, cytotoxicity; they recognized a part of TNF not involved in the binding to TNF receptors. For epitope mapping studies, comparable results were obtained with ELISA and with BIAcore studies. For ELISA it was necessary to use purified and biotinylated mAbs. BIAcore investigations allowed quick and sensitive analysis between several antibodies without any preparation of the mAbs. It was necessary to implement competition tests in reverse order because, depending on the first Ab used, different results could be obtained. This could be due to modifications of the microenvironment after the fixation of the first mAb. Anti-TNF mAbs could be classified into 6 different groups taking into account their reactivity (Table 4). Four groups of antibodies were clearly definable on the basis of their different reactivities: group I defined by mAb 22E5, group II defined by mAb 24A6, group III defined by 3 143
TABLE 4 CLASSIFICATIONOF THE 8 anti-TNF ANTIBODIES Group of antibodies
Antibodies
Epitopes
I II III IV V VI
24A6 22E5 23G5, 31H8, 27F10 35D6 a 25G8 31F7 a
1 2 3 4 1+ 2 3+ 4
a Non-neutralizing antibodies.
m A b s (23G5, 27F10, 31H8) and group IV by m A b 35D6. Two other antibodies corresponded to cross-reacting reagents: m A b 25G8 recognized an epitope expressed by both epitope 1 (mAb 24A6) and epitope 2 (mAb 22E5) and m A b 31F7 cross-reacted with both epitopes 3 and 4. Epitopes 1, 2 and 3 were well defined by antibody groups I, II and III, respectively. These 3 epitopes, characterized by neutralizing antibodies, could be involved in the binding region of TNF to its receptors. Epitope 4 could be located on an inactive part of the T N F molecule or a structural portion not implicated in the function of the molecule. Bringman and Aggarwal [35] have already defined 5 different epitopes on TNF,,, 3 of which reacted with neutralizing antibodies and 2 epitopes with non-neutralizing antibodies. Immunogenic regions of rhTNF,, have been mapped by Corti et al. [36]; "they studied interactions between various mouse anti-hTNF,, and synthetic hTNF,, peptides. They identified 3 immunogenic regions with residues 1 - 2 3 , 9 5 - 1 1 6 and 1 3 7 - 1 5 7 of rhTNF,~ and also 2 other less immunogenic regions with residues 3 7 - 5 5 and 117-136. They showed that the binding of m A b s to the antigenic sites 9 5 - 1 1 6 and 1 3 7 - 1 5 7 did not affect cytolytic activity. However, the neutralizing antibodies described in these 2 publications failed to recognize synthetic hTNF,~ peptides and it was concluded that they probably bound to conformationdependent or discontinuous epitopes. Our results are in accordance with the definition of at least 4 different epitopes expressed by T N F molecules, but it was impossible to define more precisely the reactivity of these mAbs. However, at the opposite of previously published results, all the described antibodies were able to recognize rhTNF, denatured during Western blot studies. A better knowledge of the precise reactivity of m A b s is necessary to select non-competitive reagents to prepare kits for the dosage of cytokines. For this purpose the use of a BIAcore system is very useful and time saving. Such a study could also be suitable for selection of antibodies with 144
complementary effects for the neutralisation of the TNF toxicity during therapeutical applications.
Acknowledgements W e wish to thank P. Seguin and L. Bellanger (CIS Biointernational, France) for BIAcore studies and also S. Martel for her assistance in the preparation of this manuscript.
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145