©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Epitopic Regions for Antibodies against Tumor Necrosis Factor
ANALYSIS BY SYNTHETIC PEPTIDE MAPPING (*)

(Received for publication, February 3, 1995; and in revised form, May 29, 1995 )

Kenji Yone (§) Sandrine Bajard (¶) Noriyuki Tsunekawa Jun Suzuki

From the Biotechnology Research Laboratories, Teijin Limited, 4-3-2 Asahigaoka, Hino, Tokyo 191, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The location of biologically relevant epitopes on human tumor necrosis factor alpha (hTNF-alpha) was evaluated by testing the immunoreactivity of anti-TNF-alpha antibodies against 149 sequential, overlapping octamer peptides. A goat polyclonal antibody raised against recombinant hTNF-alpha, which neutralizes hTNF-alpha biological activities, reacted with oligopeptides corresponding to hTNF-alpha residues 7-11, 17-23, 30-39, 42-49, 106-112, and 135-142. A possible assembled epitopic region (residues 25, 27, and 144) for neutralizing murine monoclonal antibodies designated 11D7G4 and 9C4G5 was deduced from the fact that they bound to tripeptides, mimicking a discontinuous epitope. These antigenic regions were found to include or adjoin poorly conserved amino acids and they were located in the turns between beta-sheets on the surface of the molecule. Three of the sequential epitopic regions and an assembled region were closely related to the receptor binding sites proposed in several other studies. These antibodies appear to neutralize TNF-alpha activities by directly masking receptor binding sites.


INTRODUCTION

Tumor necrosis factor alpha (TNF-alpha) (^1)with a molecular weight of 17,000 was originally characterized by its ability to cause hemorrhagic necrosis in certain transplanted tumors (1) . This factor, produced mainly by activated macrophages, has been shown to have a wide variety of biological activities and is considered to be an acute phase inflammatory cytokine(2) . The biological functions of the molecule are mediated by high affinity binding to receptors expressed on many cell types(3, 4, 5) . The three-dimensional structure of human TNF-alpha (hTNF-alpha) has been determined(6, 7) , and such studies suggest that the trimeric quaternary structure corresponds to the most stable state of the molecule and that the TNF monomer folds to form a sandwich of two beta-pleated sheets.

Several attempts have been made to assign functions to particular regions of the TNF-alpha molecule. By mutational analysis(8, 9) , several sites crucial for hTNF-alpha biological activity have been localized in loops at the base of the molecule. Furthermore, residues 32, 36, 84, and 86(9) , 31-35, 84-87, and 143-148(10) , 31, 35, 87, 95, 133, and 147(11) , 11-13, 37-42, 49-57, and 155-157 (7) are putative receptor binding sites. Single amino acid substitutions at positions 29 and 32 reduce binding activities with the p75 receptor, although they still interact with the p55 receptor(12) . Banner et al.(13) reported the complex structure of the extra-cellular domain of human 55-kDa TNF receptor and TNF-beta. Their x-ray crystallographic study revealed that two beta-turns in the TNF-beta monomer form three grooves between each monomer on the trimer. These grooves were demonstrated to be the interaction sites of the p55 receptor(13) .

Monoclonal antibodies (mAbs) against hTNF-alpha have been reported previously(14, 15, 16, 17) . mAbs bind discrete epitopes on the molecule and affect its binding to the receptors. Epitope analysis for mAbs against whole TNF molecules could be useful for elucidating the receptor binding sites. The screening of short, sequential overlapping peptides for antibody reactivity as described by Geisen et al.(18, 19) permits the simultaneous screening of entire molecules for linear immunoreactive epitopes. This approach has been effective in mapping epitopes of the outer protein VP1 of foot-and-mouth disease virus (18, 20) and human beta-interferon(21) .

Using Geisen's methods, possible epitopic regions for two types of mAbs (neutralizing and non-neutralizing) and a goat polyclonal antibody were investigated to determine their relationship with the receptor binding sites on the three-dimensional structure of hTNF-alpha.


EXPERIMENTAL PROCEDURES

Tumor Necrosis Factors

Recombinant hTNF-alpha (rhTNF-alpha) was produced in Escherichia coli (E. coli) using a chemically synthesized gene (22) and purified to 2 10^6 units/mg according to the method of Shirai et al.(23) . The TNF-alpha units were determined by the biological assay as described later under ``Experimental Procedures.'' rhTNF-alpha was further purified to 5 10^7 units/mg by anti-TNF mAb 10B7E11 affinity column chromatography. For affinity purification, mAb 10B7E11 was coupled to CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions.

Natural hTNF-alpha was secreted from a human myelomonocytic cell THP-1 (24) , kindly provided by Dr. T. Watanabe (Kyushu University, Fukuoka, Japan). The cells were incubated at a density of 1 10^6 cells/ml for 24 h using serum-free RPMI 1640 that contained 100 ng/ml phorbol 12-myristate 13-acetate (Sigma). The culture supernatant was concentrated using a YM-10 ultrafiltration membrane (Amicon, Danvers, MA), then stored at -80 °C until use.

Murine tumor necrosis serum (mTNS) was prepared as described by Green et al.(25) . BALB/c mice (Japan SLC, Hamamatsu, Japan) were primed with 1 mg/animal of Corynebacterium porvum (Ribi Immunochem Research, Hamilton, MT). Then, 2 weeks later, 10 µg/animal of lipopolysaccharide from E. coli O55B5 (Difco) was injected into the primed mice. Two hours later, sera were collected and stored at -80 °C. RhTNF-beta was purchased from Bender Medsystems (Vienna, Austria).

Preparation of Goat Anti-rhTNF Antiserum

Anti-rhTNF goat antiserum was prepared by JIMRO (Maebashi, Japan) using affinity-purified rhTNF-alpha. The antiserum was prepared by inoculating a Shiba goat intradermally eight times at 2-week intervals with 0.5 mg of rhTNF-alpha initially in Freund's complete adjuvant (Difco) and subsequently in Freund's incomplete adjuvant (Difco). The antiserum titer was tested regularly using the Ouchterloney immunodiffusion method. Blood specimens were taken after the last immunization, when the antibody titer had reached a plateau.

Monoclonal Antibody Production

mAbs against rhTNF-alpha were produced according to the method of Köler and Milstein(26) . BALB/c mice (Japan SLC) were immunized subcutaneously with 5 µg of rhTNF-alpha in Freund's complete adjuvant. RhTNF-alpha was administered three times at 2-week intervals. Four days after the last immunization, spleen cells from mice were fused with murine myeloma P3-x63-Ag8U1 (kindly provided by Dr. T. Watanabe) using 50% polyethylene glycol 1540. Two weeks after fusion, culture supernatants were assayed for the secretion of anti-TNF-alpha antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive hybridomas were subcloned by limiting dilution on a feeder layer of syngenic splenocytes. mAbs were purified from the supernatant of hybridoma culture by affinity chromatography using protein A-Sepharose CL4B (Pharmacia). Antibody subclasses were determined by an immunodiffusion system (The Binding Site, Birmingham, United Kingdom). Six mAbs were chosen for further investigation (Table 1).



ELISA for Selection of Antibodies to TNF-alpha

RhTNF-alpha was suspended in phosphate-buffered saline (PBS) at a concentration of 10 µg/ml, and a 0.1-ml aliquot of the suspension was added to each well of a 96-well microtiter plate (Titertek, Flow Lab, The Netherlands). The plate was incubated overnight at 4 °C and the fluid removed. Nonspecific protein-binding sites were blocked by incubation with PBS containing 0.5% bovine serum albumin at 37 °C for 1 h. After washing with PBS containing 0.5% bovine serum albumin and 0.1% Tween 20, 0.1 ml of the hybridoma culture supernatant was added and incubated for 1 h at 37 °C. The plate was then washed three times and 0.1 ml of a 1/1000 diluted solution of alkaline phosphatase-conjugated goat anti-mouse IgG(H+L) antibody (Tago, Burlingame, CA) was added to each well. After incubation for 1 h at room temperature and washing, 0.1 ml of enzyme substrate (1 mg/ml of p-nitrophenyl phosphate in 9.6% diethanolamine, pH 9.8, containing 0.24 mM MgCl(2)) was added, and the plates were incubated for 20 min at room temperature. The reaction was stopped by adding 0.05 ml of 3 N NaOH, and absorbance at 405 nm was determined using an ETY-96 ELISA analyzer (Toyo-Sokki, Tokyo, Japan).

Gel Electrophoresis and Immunoblots

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli(27) . RhTNF-alpha (0.2 µg) was subjected to electrophoresis on a 4-20% gradient gel (SDS-PAGE plate, Daiichi Kagaku, Tokyo, Japan). Gels were stained with 0.15% Coomassie Brilliant Blue R-250 in 10% methanol, 10% acetic acid, and destained with 10% methanol and 10% acetic acid.

The binding of antibodies to rhTNF-alpha was performed using the immunoblot technique(28) . After separation by SDS-PAGE, the proteins were electrophoretically (50 V for 1.5 h) transferred to nitrocellulose membranes (Bio-Rad). After transferring, the sheets were incubated in Tris buffer (20 mM Tris, 0.5 M NaCl, pH 7.5) containing 3% gelatin at 25 °C for 2 h, washed twice with Tris buffer containing 0.05% Tween 20 (TBS-T), and incubated in TBS-T containing 1% gelatin and 1/3000 diluted test antiserum or 2.0 µg/ml of test mAbs at 25 °C for 16 h. After washing, the sheets were incubated with horseradish peroxidase-conjugated anti-goat IgG rabbit antibody (Bio-Rad) for a test serum or anti-mouse IgG rabbit antibody (Bio-Rad) for test mAbs dissolved in TBS-T containing 1% gelatin at 25 °C for 2 h, washed twice with TBS-T, and subjected to color development using 4-chloro-1-naphthol (Bio-Rad).

Biological Assay for TNF-alpha and Anti-TNF-alpha Antibodies

The murine L929 fibroblast cytolytic assay was performed as described by Ruff et al.(29) . Briefly, L929 cells (kindly provided by Dr. A. Uchida Kyoto University, Japan) were seeded at a density of 4 10^4 cells/well in 96-well microtiter plates (Costar, Cambridge, MA) using 0.1 ml of RPMI 1640 that contained 10% fetal bovine serum. An equal volume of PBS containing the desired dilutions of TNF-alpha was added and incubated in the presence of 1 mg/ml of actinomycin D at 37 °C and 5% CO(2) for 17 h in a humidified incubator. The cells were then stained with 0.1% crystal violet, 1% methanol in PBS. After incubation for 15 min, the plates were washed with tap water and allowed to dry. The crystal violet was dissolved by adding of 0.1 ml of 0.5% SDS to each well. The absorbance at 595 nm was read with an ELISA analyzer. The TNF-alpha units/ml represents the reciprocal of the maximum TNF-alpha dilution that causes 50% cytotoxicity under the conditions of the assay.

In the neutralization experiments, antibodies were incubated with antigen for 1 h at 37 °C prior to the assay. The incubated mixture was then added to the L929 cell culture.

Monoclonal Antibodies Affinity Determination

The binding affinities of mAbs were calculated by Scatchard plot analysis. Protein A-purified mAbs were iodized using immobilized lactoperoxidase glucose oxidase (Enzymobeads, Bio-Rad). I-Labeled mAb was allowed to bind to hTNF-alpha coated on a well of a microtiter plate (Falcon, Becton Dickinson, Oxnard, CA) in the presence of various concentrations of similar but non-labeled mAb. The well was washed five times with PBS containing 0.05% Tween 20, and the radioactivity bound to each well was measured with a counter (Aloka, Tokyo, Japan).

Peptide Synthesis

Duplicate sets of 149 octamer peptides, representing the entire sequence of hTNF-alpha in a sequential and overlapping manner, were synthesized on prederivatized polyethylene pins (Cambridge Research Biochemicals, Northwich, UK) according to the manufacturer's instructions. All dipeptide and extended tripeptide combinations were synthesized in the same way.

Peptides corresponding to selected sequences of hTNF-alpha were synthesized using a peptide synthesizer (model 430A, Applied Biosystems, Foster City, CA) and purified to homogeneity by reverse-phase high-performance liquid chromatography. The amino acid sequences of the peptides were confirmed using an amino acid sequencer (model 471A, Applied Biosystems).

ELISA for mAbs Reactivity toward Peptides

Antibody reactivities toward solid-phase peptides were determined by ELISA. The peptide pins were incubated in PBS containing 1% bovine serum albumin, 1% ovalbumin, and 0.1% Tween 20 for 1 h. The pins were then incubated with an antibody under investigation at 4 °C overnight. After washing three times with PBS containing 0.1% Tween 20, the pins were incubated with an appropriate antibody-horseradish peroxidase conjugate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) for 1 h, washed five times with PBS containing 0.1% Tween 20 and once with PBS, and then subjected to color development using 2,2`-azino-di-[3-ethyl-benzthiazolinesulfonic acid] (Boehringer Mannheim, Mannheim, Germany). The absorbance at 405 nm was determined using an ELISA analyzer.


RESULTS

Production of Monoclonal Antibodies

The six mAbs chosen for investigation were divided into two groups according to their effects on the cytolytic activity of TNF-alpha. One group of mAbs, including 11D7G4, 9C4G5, 10B7E11, and 1G7D3, bound to TNF-alpha and neutralized its cytolytic activity to L929 fibroblast cells. The other group of mAbs, including 1F12A7 and 1A10D4, bound to TNF-alpha but did not neutralize its cytolytic activity. The general characteristics of these mAbs are shown in Table 1.

Specificity of the Neutralizing Activities of Antibodies

Fig. 1a shows the neutralizing activity of goat anti-TNF-alpha antiserum against the L929 fibroblast cytolytic effect of rhTNF-alpha. By adding 1/2000 diluted antiserum, the cytolytic activity of this cytokine was reduced from 2.6 10^5 units to 1.7 10^3 units. From these, the neutralizing activity was calculated at 3.0 10^5 neutralizing units/ml. The neutralizing activities of mAbs were calculated in the same way and shown in Table 1.


Figure 1: Effects of anti-rhTNF-alpha antiserum on L929 fibroblast cytolytic activities of TNFs. L929 cells were exposed to the indicated dilution series of rhTNF-alpha (3 µg/ml) (a), 40 times concentrated THP-1 cell culture supernatant (b), rhTNF-beta (10 µg/ml) (c), or mTNS (d) in RPMI 1640 containing 5% fetal bovine serum and 1 µg/ml of actinomycin D at 37 °C for 17 h. Each antigen was incubated with (closed circles) or without (open circles) 1/100 (a, c) or 1/1000 (b, d) diluted goat anti-rhTNF-alpha antiserum for 1 h at 37 °C prior to the assay. Viabilities were determined by crystal violet staining of viable cells compared with control cultures.



The antiserum also inhibited the cytolytic activity of natural hTNF-alpha secreted from a human myelomonocytic cell THP-1 stimulated by phorbol 12-myristate 13-acetate (Fig. 1b). The neutralization of natural hTNF-alpha was also seen by adding neutralizing mAbs. (^2)This indicates that the topography of the site related to the cytolytic activity on rhTNF-alpha in relation to the epitopes recognized by these antibodies is almost the same for natural hTNF-alpha. Therefore, it is likely that the tertiary structure of rhTNF-alpha is similar to that of natural hTNF-alpha, at least for the region concerning the function of cytolytic activity. However, the antiserum cross-reacted with neither mTNS nor hTNF-beta (Fig. 1, c and d). Similar specificity was observed using neutralizing mAbs. (^3)Therefore, the antibodies showed species-specific reactivity to TNF-alpha, and they also distinguished hTNF-alpha from hTNF-beta.

Characterization of the Antibodies by Immunoblot

The antiserum was examined for its binding to SDS-denatured rhTNF-alpha in the presence of human serum and E. coli lysate. The results obtained using the immunoblot technique are shown in Fig. 2a. The polyclonal antibody in the antiserum bound to the monomeric form of TNF-alpha and did not show any cross-reactivity with human or E. coli proteins.


Figure 2: RhTNF-alpha immunoblot probed with the antiserum and monoclonal antibodies. a, RhTNF-alpha (0.15 mg) was mixed with 50 µl of 1/50 diluted human serum (lane A) or 100 µl of E. coli lysate (6.4 mg/ml) (lane B). Samples were subjected to SDS-PAGE on a 4-20% gradient gel, and the proteins were transferred to nitrocellulose. Blots were probed with goat anti-rhTNF-alpha antiserum. b, RhTNF-alpha in E. coli lysate immunoblot probed with 11D7G4 (lane C) and with 1F12A7 (lane D) in the same procedure as a. The position of the TNF-alpha is shown by the arrow, which indicates the estimated molecular mass in kilodaltons.



Fig. 2b shows the mAbs 1F12A7 and 11D7G4 bound to SDS-denatured rhTNF-alpha in the presence of E. coli lysates. The same specificities of these two mAbs were shown by the other mAbs. (^4)

Screening of Antibody Reactivity to Octamer Peptides

Overlapping, sequential octamer peptides corresponding to the entire hTNF-alpha amino acid sequence were treated with selected mAbs and the antiserum to identify linear epitopes (Fig. 3). The goat polyclonal antibody in the antiserum was revealed to have six major areas of binding with four peaks of reactivity near the amino terminus and two other peaks near the carboxyl terminus (Fig. 3a). In each area, common residues among peptides which were recognized strongly by this antibody had residue numbers 7-11, 17-23, 30-39, 42-49, 106-112, and 135-142.


Figure 3: Antibody reactivity to hTNF-alpha octamer peptides. Bindings to octapeptides of (a) the goat polyclonal antibody in the antiserum (1/1000 dilution), (b) 1F12A7 (2.3 µg/ml) were detected in an ELISA. The absorbance is represented as the height of the vertical bar for each octapeptide whose amino-terminal residue is designated by the peptide number. The numerical assignment of the peptide is such that peptide number 1 represents residues 1-8, number 2 represents residues 2-9, etc., to the last peptide number 150 that represents residues 150-157. Results are the mean values from duplicate samples.



A murine non-neutralizing mAb, 1F12A7, recognized one octapeptide with initial residue 104, one of the six major areas of binding by the polyclonal antibody (Fig. 3b). In cases of overlapping sequential hexamer peptides, this mAb demonstrated exactly the same reactivity. The only hexapeptide this mAb recognized had initial residue 106. (^5)

On the other hand, neutralizing mAbs, 11D7G4, 9C4G5, and 1G7D3, did not bind to any sequential octamer peptide, even when the concentration of mAb was 10-fold higher.

Inhibition of mAb Binding by Peptides

A peptide composed of residues 98-127, including a 1F12A7 binding region (residues 106-111), was synthesized. The inhibition of the binding of 1F12A7 to rhTNF-alpha was measured over a wide range of peptide concentrations using an antigen-binding ELISA system described in the figure legend. The peptide inhibited the binding of 1F12A7 in a dose-dependent fashion (Fig. 4).


Figure 4: Inhibition of binding of mAb 1F12A7 to rhTNF-alpha by a synthetic peptide composed of hTNF-alpha residues 98-127. mAb 1F12A7 was preincubated for 1 h at 37 °C with various amounts of a peptide composed of residues 98-127 and tested by the same ELISA procedure for selection of antibodies. Ratios of the mAb 1F12A7 bound to hTNF-alpha were calculated from changes in absorbance at 405 nm per minute.



Identification of Dipeptides Contributing to Neutralizing mAb Binding

Neutralizing mAbs failed to react with any of the sequential octamer peptides, suggesting that these mAbs might be directed toward assembled or discontinuous epitopes. A search for binding peptides was performed according to the mimotope strategy by Geisen et al.(19) .

One set of 380 dipeptides made from the L- and D-optical isomers of common amino acids was synthesized. An amino acid derivatives set for synthesis provided by the manufacturer included L- and D-isomer of the common amino acids except for D-alpha-cysteine. All combinations of dipeptides and following extended tripeptides, except for peptides containing D-alpha-cysteine, were synthesized and treated with neutralizing mAbs. Table 2summarizes the major reactions of 11D7G4, 9C4G5 and the polyclonal antibody. The dipeptide L-Phe-D-Gln (F-q) was found to give the greatest binding activity to both neutralizing mAbs. The defined pair F-q was chosen for further extension.



Extending the Reactive Dipeptide

A total of 76 tripeptides based on reacting dipeptide F-q were synthesized, and their reactivities to neutralizing mAbs were tested. Fig. 5shows that the two mAbs (11D7G4 and 9C4G5) and the polyclonal antibody bound to some tripeptides. Among these 76 tripeptides, F-q-q, F-q-G, and several other tripeptides showed high reactivities to 11D7G4 (Fig. 5a). Compared with 11D7G4, mAb 9C4G5 showed similar but slightly different reactivities to these tripeptides (Fig. 5b). F-q-q, F-q-r, and F-q-h were the highly reactive peptides. The polyclonal antibody showed more diverse reactivities to these tripeptides (Fig. 5c).


Figure 5: Antibody reactivity to F-q-based extended trimer peptides. Based on the reacting pair F-q, two sets of tripeptides were synthesized with the general formulae acetyl-@-F-q-solid support and acetyl-F-q-@-solid support. The symbol ``@'' represents all L- and D-alpha-amino acids. Each set was composed of 38 tripeptides. The antibody binding activities were determined by reacting each of the 76 tripeptides in an ELISA with the mAbs 11D7G4 (a), 9C4G5 (b), and the goat polyclonal antibody (c). The vertical bars are proportional to the ELISA absorbance. Every group of 76 bars corresponds to a 38-trimer set for @-F-q and another 38-trimer set for F-q-@. Within each group of 38 bars, the left-hand bar corresponds to L-alpha-alanine (A)-containing tripeptide, and the successive 19 bars are then in alphabetic order according to the single letter code for the amino acids. The following 18 bars correspond to D-alpha-amino acids in the same order as L-alpha-amino acids. Several highly reactive peptides are indicated in the figure.




DISCUSSION

The present study was conducted to characterize the antibody binding to hTNF-alpha. By scanning the entire sequence of hTNF-alpha for immunoreactivity of sequential, overlapping octapeptides, we have identified six major sequential epitopic areas that were recognized by the goat polyclonal antibody against rhTNF-alpha. One of them was also recognized by non-neutralizing mAb 1F12A7.

As sequential epitopes, non-conservative regions in amino acid sequences of TNF-alpha between human and other species were to be recognized. The high degree of amino acid conservation among the various TNF-alpha sequences has been studied(30) . Thus, the non-conservative regions are highly limited. All of the epitopic regions were found to either contain or adjoin to poorly conserved sequences by comparing amino acid sequences of murine(31) , goat(30) , and human (32) TNF-alpha. Therefore, it is likely that species-specific regions are recognized as sequential epitopes.

Those sequential epitopes were thought to be positioned on the surface area of the TNF-alpha molecule through examination of the three-dimensional structure of rhTNF-alpha (Fig. 6). Eck and Sprang (7) determined the three-dimensional structure of hTNF-alpha at 2.6 Å resolution by x-ray crystallography, and the atomic coordinates have been deposited in the Brookhaven Protein Data Bank by the same authors (identification code 1TNF). The locations of all the sequential epitopes on the tertiary structure of the TNF monomer were examined using the BIOGRAF software (Molecular Simulation Inc., Pasadena, CA). They were all positioned on the surface area of the TNF-alpha monomer (Fig. 6). These results support the work of Sprang and Eck(30) , describing that all but 12 of the 45 poorly conserved residues correspond to solvent-accessible amino acids in the three-dimensional structure of the hTNF trimer. The resulting six epitopic regions were revealed to contain or adjoin those solvent-accessible poorly conserved amino acids. In addition, five of six epitopic regions are positioned on short polypeptide turn structures between antiparallel beta-strands. The rest (residue numbers 30-39) include a turn and a short beta-strand. Similar results were obtained from studies of antibody recognition sites of gp120, the external envelope protein of human immunodeficiency virus type 1. The sequence Gly-Pro-Gly, a candidate for a polypeptide beta-turn in the RP135 disulfide loop within gp120, has been proposed as an epitope of neutralizing antibodies(33) . Therefore, it is likely that those sequential epitopes which are located in beta-turns correspond to antibody recognition sites.


Figure 6: HTNF-alpha trimer showing the deduced epitopic regions recognized by goat polyclonal antibody. A, a view of the hTNF-alpha trimer. The positions of C-alpha carbon atoms of three monomers of the TNF trimer are traced in yellow, aqua, and purple lines. All atoms of the epitopic regions on the yellow subunit are shown in orange (residues 7-11), magenta (residues 17-23), light blue (residues 30-39), red (residues 42-49), cyan (residues 106-112), and green (residues 135-142). All the epitopic regions are positioned on turn structures between beta-sheets. B, a Corey-Pauling-Koltun model of the hTNF-alpha monomer. All atoms of the epitopic regions are shown in the same color as Fig. 7A. All the epitopic regions include solvent-accessible amino acids on the surface area of an hTNF-alpha monomer molecule.




Figure 7: A possible assembled epitopic region for neutralizing mAbs 11D7G4 and 9C4G5. a, a view of hTNF-alpha showing Phe (cyan) and Gln, Gln, and Gln (magenta) on a Corey-Pauling-Koltun model of a monomer. The C-alpha carbon atoms of the other two monomers are traced in aqua and purple lines. b, a stick model showing Phe (cyan) positioned onto a beta-turn of the g-h loop apart from Gln, Gln, and Gln (magenta) onto the other beta-turn of the a-a` loop. c, superposition of F-q-q tripeptide (orange) on Phe, Gln, and Gln shows that it may mimic an assembled epitope. The tripeptide was built, superposed mainly on the side chains of Phe, Gln, and Gln, and its energy was minimized to find a conformation that corresponded to the nearest local minimum in the potential energy surface using BIOGRAF.



A non-neutralizing mAb 1F12A7 recognized a linear epitope spanning residues 106-111 (Fig. 3b). This was supported by the fact that a peptide composed of residues 98-127 inhibited the binding of 1F12A7 to rhTNF-alpha in a dose-dependent fashion (Fig. 4). mAb 1F12A7 did not inhibit hTNF-alpha binding to its receptor. These results suggest that residues 106-111 are not involved in the TNF-receptor interaction. The peptide composed of residues 98-127 had neither rhTNF-alpha agonistic nor antagonistic activity at concentrations up to 0.15 mM. (^6)

The goat polyclonal antibody neutralizes the biological activity of hTNF-alpha. Thus, the activity was inhibited by bound antibodies. Therefore, some of the epitopic regions might be closely related to the functional domains of this molecule. In these epitopes, except for the one common to mAb 1F12A7, there might be sequential epitopes for neutralizing antibodies. As for the receptor binding site of hTNF-alpha, several common areas have been proposed. Residues 32 and 36(9) , 31-35 (10) , 31 and 35(11) , and 37-42 (7) are candidates for the receptor binding site, which are consistent with the two epitopic regions for the polyclonal antibody, residues 31-39 and 42-49. Owing to the direct binding to these possible receptor binding sites, this antibody might neutralize TNF-alpha activities by interfering with the interaction between TNF-alpha and its receptor.

Neutralizing mAbs 11D7G4, 9C4G5, and 1G7D3 failed to react with any peptide of 149 sequential, overlapping octamer peptides. This indicates that these mAbs might bind assembled or discontinuous epitopes that are dependent on the tertiary folding. Testing combinations of dipeptides and the extended tripeptides based on the dipeptide F-q, mAbs 11D7G4 and 9C4G5 have been revealed to react with tripeptides, including phenylalanine and glutamine residues. The possibility that these tripeptides could mimic an assembled epitope has been shown by the close positions of Phe and Gln, Gln, and Gln on the tertiary structure of the hTNF-alpha monomer (Fig. 7, a and b). The monomer contains four Phe residues and ten Gln residues. Among these, Phe has been revealed to be the closest to Gln, Gln, and Gln. Moreover, all these are positioned onto two beta-turns of two independent loops and on the solvent accessible surface of the trimer molecule. The simulation of the F-q-q tripeptide superposing on Phe, Gln, and Gln (Fig. 7c) shows that it may mimic a certain portion of the TNF molecule. Gln of hTNF-alpha, which aligns with Glu of murine TNF-alpha, is not conserved between the two species. Several species specificities are also observed on residues 19-27. These show that the region including these residues possibly forms an assembled epitope for neutralizing mAbs.

Replacements at positions 29 and 146 clearly reduced cytotoxicity only when poorly conserved alterations were induced(9) . From the results of a number of experiments using site-directed mutagenesis, Jones et al.(10) reported the two hot spot regions situated on separate sides of the TNF monomer, consisting of residues 31-35, 84-87, and 143-148. Banner et al.(13) reported that the receptor fragment binds in the groove between two adjacent TNF-beta subunits of a trimer molecule. This groove formed between the d-e loop (^7)(residues 105-110 which align with residues 84-89 in hTNF-alpha^8) of one subunit and the a-a" region (residues 36-52 which align with residues 19-35 in hTNF-alpha) of the next. Additionally, there is the upper contact region flanked by the end of strand g (His and Asp which align with Gln and Asn in hTNF-alpha) of one subunit and the a-a` loop (residues 37-40 which align with residues 20-23 in hTNF-alpha) of the next. Three-dimensional structures of TNF-alpha and -beta (34) are strikingly similar and they bind to the same receptors. It is, therefore, reasonable to deduce the regions in TNF-alpha from the alignment of amino acids with TNF-beta. The receptor binding site and contact regions of TNF-beta are in agreement with the two hot spot regions reported by Jones et al.(10) , the assembled epitopic region for neutralizing mAbs, and three sequential epitopic regions for the polyclonal antibody (residues 17-23, 30-39, and 135-142). Therefore, recognizing species-specific regions that are in close proximity to the receptor binding sites, neutralizing antibodies tested appear to neutralize TNF activities by directly masking the receptor binding sites.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

Present address: Laboratoire de Biometrie, URA-CNRS 243, Universite Claude Bernard, Lyon 1, 43 boulevard du 11 novembre 1918, 69622 Villeurbanne, France.

(^1)
The abbreviations used are: TNF-alpha, tumor necrosis factor alpha; hTNF-alpha, human TNF-alpha; mAbs, monoclonal antibodies; rhTNF-alpha, recombinant hTNF-alpha; mTNS, murine tumor necrosis serum; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
K. Yone, unpublished observation.

(^3)
K. Yone, unpublished observation.

(^4)
K. Yone and J. Suzuki, unpublished observation.

(^5)
K. Yone, unpublished observation.

(^6)
K. Yone and N. Tsunekawa, unpublished observation.

(^7)
The beta strands for TNF-beta monomer are labeled in the notation of Eck et al. (34).

(^8)
The alignment of amino acid sequences of TNF-alpha and TNF-beta are in accordance with Banner et al. (13).


ACKNOWLEDGEMENTS

We thank Yasunobu Takano for operating the computer systems, Kazue Shirakura for her technical assistance, and Tomoko Morita for preparing the manuscript.


REFERENCES

  1. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson, B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72,3666-3670 [Abstract]
  2. Beutler, B. M., and Cerami, A. (1992) in Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine (Beutler, B., ed) pp. 1-10, Raven Press, New York
  3. Aggarwal, B. B., Eessalu, T. E., and Has, P. E. (1985) Nature 318,665-667 [Medline] [Order article via Infotrieve]
  4. Loetscher, H., Pan, Y. C. E., Lahm, H. W., Gentz, R., Brockhaus, M., Tabuchi, H., and Lesslauer, W. (1990) Cell 61,351-359 [Medline] [Order article via Infotrieve]
  5. Smith, C. A., Davis, T., Anderson, D., Solam, L., Beckmann, M. P., Jerzy, R., Dower, S. K., Cosman, D., and Goodwin, R. G. (1990) Science 248,1019-1023 [Medline] [Order article via Infotrieve]
  6. Jones, E. Y., Stuart, D. I., and Walker, N. P. C. (1989) Nature 338,225-228 [CrossRef][Medline] [Order article via Infotrieve]
  7. Eck, M. J., and Sprang, S. R. (1989) J. Biol. Chem. 264,17595-17605 [Abstract/Free Full Text]
  8. Yamagishi, J., Kawashima, H., Matsuo, N., Ohue, M., Yamayoshi, M., Fukui, T., Kotani, H., Furuta, R., Nakano, K., and Yamada, M. (1990) Protein Eng. 3,713-719 [Abstract]
  9. Van Ostade, X., Tavernier, J., Prange, T., and Fiers, W. (1991) EMBO J. 10,827-836 [Abstract]
  10. Jones, E. Y., Stuart, D. I., and Walker, N. P. C. (1992) in Tumor Necrosis Factors: Structure, Function, and Mechanism of Action (Aggarwal, B. B., and Vilcek, J., eds) pp. 93-127, Marcel Dekker, Inc., New York
  11. Zhang, X.-M., Weber, I., and Chen, M.-J. (1992) J. Biol. Chem. 267,24069-24075 [Abstract/Free Full Text]
  12. Van Ostade, X., Vandenabeele, P., Everaerdt, B., Loetscher, H., Gentz, R., Brockhaus, M., Lesslauer, W., Tavernier, J., Brouckaert, P., and Fiers, W. (1993) Nature 361,266-269 [CrossRef][Medline] [Order article via Infotrieve]
  13. Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.-J., Broger, C., Loetscher, H., and Lesslauer, W. (1993) Cell 73,431-435 [Medline] [Order article via Infotrieve]
  14. Bringman, T. S., and Aggarwal, B. B. (1987) Hybridoma 6,489-507 [Medline] [Order article via Infotrieve]
  15. Fendly, B. M., Toy, K. J., Creasey, A. A., Vitt, C. R., Larrick, J. W., Yamamoto, R., and Lin, L. S. (1987) Hybridoma 6,359-370 [Medline] [Order article via Infotrieve]
  16. Möller, A., Emling, F., Blohm, D., Schlick, D., and Schollmeier, K. (1990) Cytokine 2,162-169 [Medline] [Order article via Infotrieve]
  17. Rathjen, D. A., Cowan, K., Furphy, L. J., and Aston, R. (1991) Mol. Immunol. 28,79-86 [Medline] [Order article via Infotrieve]
  18. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U. S. A., 81,3998-4002 [Abstract]
  19. Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G., and Schoofs, P. G. (1987) J. Immunol. Methods 102,259-274 [CrossRef][Medline] [Order article via Infotrieve]
  20. Geysen, H. M., Barteling, S. J., and Meloen, R. H. (1985) Proc. Natl. Acad. Sci. U. S. A.. 82,178-182 [Abstract]
  21. Redlich, P. N., Hoeprich, P. D., Jr., Colby, C. B., and Grossberg, S. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,4040-4044 [Abstract]
  22. Nakamura, S., Masegi, T., Kitai, K., Ichikawa, Y., Kudo, T., Aono, R., and Horikoshi, K. (1990) Agric. Biol. Chem. 54,3241-3250 [Medline] [Order article via Infotrieve]
  23. Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W., and Wallace, R. B. (1985) Nature 313,803-806 [Medline] [Order article via Infotrieve]
  24. Williamson, B. D., Carswell, E. A. Rubin, B. Y., Prendergast, J. S., and Old, L. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,5397-5401 [Abstract]
  25. Green, S., Dobrjansky, A., Chiasson, M. A., Carswell, E., Schwartz, M. K., and Old, L. J. (1977) J. Natl. Cancer Inst. 59,1519-1522 [Medline] [Order article via Infotrieve]
  26. Köhler, G., and Milstein, C. (1976) Eur. J. Immunol. 6,511-519 [Medline] [Order article via Infotrieve]
  27. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  28. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 [Abstract]
  29. Ruff, M. R., and Gifford, G. E. (1980) J. Immunol. 125,1671-1877 [Abstract/Free Full Text]
  30. Sprang, S. R., and Eck, M. J.(1992) in Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine (Beutler, B., ed) pp. 11-32, Raven Press, New York
  31. Pennica, D., Hayflick, J. S., Bringman, T. S., Palladino, M. A., and Goeddel, D. V. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,6060-6064 [Abstract]
  32. Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B., and Goeddel, D. V. (1984) Nature 312,724-729 [Medline] [Order article via Infotrieve]
  33. Javaherian, K., Langlois, A. J., McDanal, C., Ross, K. L., Eckler, L. I., Jellis, C. L., Profy, A. T., Rusche, J. R., Bolognesi, D. P., Putney, S. D., and Matthews, T. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86,6768-6772 [Abstract]
  34. Eck, M. J., Ultsch, M., Rinderknecht, E., de Vos, A. M., and Sprang, S. R. (1992) J. Biol. Chem. 267,2119-2122 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.






This Article
Abstract
Full Text (PDF)
Purchase Article
View Shopping Cart
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Copyright Permissions
Google Scholar
Articles by Yone, K.
Articles by Suzuki, J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Yone, K.
Articles by Suzuki, J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   Biochemistry and Molecular Biology Education 
Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.