Tosko~, Vd. 30, No . 7, pp . 723-731, 1992. Printed in Gtaat Hrit.ln.
0041-0101/92 SS .00 + .00 © 1992 PaPmon Pnew Ltd
MONOCLONAL ANTIBODIES TO TOXIN II FROM THE SCORPION ANDROCTONUS AUSTRALIS HECTOR: FURTHER CHARACTERIZATION OF EPITOPE SPECIFICITIES AND NEUTRALIZING CAPACITIES NOUARA YAI~u, G~nersTiwlvE Dgvwux, PwscwL MANSUELLE, MARIA-L~Riw D~rrnnvl and CLAUDE GRANIER' CNRS URA 1455, Laboratoire de Biochimie, Faculté de Médecine Secteur Nord, Boulevard Pierre Dramard, 13326 Marseille Cedex 15, France (Received 8 November 1991 ; accepted 27 Jmluary 1992)
N. YAin, C. DEVAUX, P. MANSUELLE, M.-L. DSFENDIPiI and C. GRANIER. Monoclonal antibodies to toxin II from the scorpion Androctonus australis Hector : further characterization of epitope specificities and neutralizing capacities . Toxicon 30, 723-731, 1992 .-The epitope specificities of two previously prepared monoclonal antibodies (mAb) to the toxin II from Androctonus australis Hector were characterized. Neither mAb 4C 1 nor mAb 3C5 was able to recognize any of the 58 overlapping synthetic heptapeptides which cover the whole sequence of toxin II . Thus, both mAbs probably recognize conformation-dependent epitopes at the surface of the toxin. Experiments were designed to check whether or not the two mAbs, or their Fab fragments, were able to bind simultaneously to the toxin. The results indicated that the epitopes recognized by the two antibodies are probably close together at the surface of the toxin, thus preventing the simultaneous binding of both mAbs to a single toxin molecule. Given the proximity of the two epitopes and the fact that mAb 4C1 is known to be a neutralizing antibody, the capacity of mAb 3C5 to inhibit the toxic effects of the toxin was re-evaluated in C57BL/6 mice . A clear, but weak, neutralizing effect was found, consistent with the low affinity binding of the mAb in the proximity of a neutralizing site of the toxin. INTRODUCTION
ScoRP1oN toxins are the major components of venoms responsible for the toxic effects that occur in man after a scorpion sting. They act by binding with high allïnity to receptor sites of voltage dependent sodium channels (CATTHtALL, 1977). Despite their small mol. wt (about 7000) these toxins are immunogenic in horse, rabbit and mouse. Polyclonal antibodies to various toxins have been obtained which have been very useful for assessing the antigenic site number (EL A~ et al., 1983) or the location of the main antigenic 'Author to whom correspondence should be addressed . 723
724
N. YAHI et al.
regions of one toxin (GRANIER et al., 1989). More recently, two monoclonal antibodies, mAb 3C5 and mAb 4C1, to the toxin II of the North African species Androctom~ts australis Hector (AaH In have been obtained (BAHRAOUI et al., 1988). MAb 4C1 is very interesting in that it displays a high affinity for the antigen (Kd = 0.8 nM) and powerful in vitro and in vivo neutralizing properties . Some of the residues of the toxin that are probably involved in the epitope recognized by mAb 4C1 have been identified using chemically modified derivatives of AaH II, in a competitive radioimmunoassay (BAHRAOUI et al., 1988). The other monoclonal, mAb 3C5, was more difficult to characterize because it has a much lower affinity for the toxin than mAb 4C1 (Kd = 0.61tM). In the experimental conditions used to demonstrate the neutralizing capacity of mAb 4C1, mAb 3C5 had no neutralizing effect (BAHRAOUI et al., 1988). This could be explained ifmAb 3C5 recognized on the toxin an epitope located on a region opposite to the region interacting with the receptor . For example, the epitope could be the a-helix region, which is known to be antigenic (B.~hntwoul et al., 1986) but does not contact the receptor during binding (EL AYIm et al., 198 . A better definition of the epitope recognized by mAb 3C5, together with its spatial relationship with the mAb 4C 1 epitope, is thus of interest . The present study is an attempt to characterize the epitope recognized by mAb 3C5 on the Attdroctonus australis Hector toxin II and to re-evaluate its neutralizing capacity .
Furfficatiort ojrnAbs 3C5 ared 4Cl
MATERIALS AND METHODS
Clones 4C1 and 3C5, which have been described previously (B~newotn et al., 1988), were expanded in BALB/C mice primed with pristane (2,6,10,14 ~etn~~yl-pentadecane, Sigma, St . Louis, MO, U.S.A.). Monoclonal antibodies were purified from aecitic fluids by ammonium sulphate precipitation followed by DEAE~Cellulose chromatogisphy (DE52, whatman, Maidstone, U.K.). The homogeneity of the preparation was checked by SDS-PAGE electrophoresis. The antibody concentration was assessed by spectrophotometric measurement, at 280 nm, taking absorption coefficient e 1% (1 cm) = 13.5. The molar concentration of these yl IgGs was estimated assuming a mol. wt of 150,000. MAbs were stored at -20°C in small aliquots. Fab fragment preparation followed the general conditions described by Port~e (1958). Briefly, 5 mg of mAb were incubated for 60 min at 37°C with 1% (w : w) pepsin (Sigma) in the presence of 20 mM EDTA, and 15 pM ßmercaptoethanol . The n~ction was stopped by addition of 20 mM iodacetamide (Fluke). The Fab fragments were recovered in the void-volume fractions of a protein A-Sepharose column (Pharmacia, Uppsala, Sweden). Solid~phase radioinuraoroarsays
AaH II was labelled with Na'~I (Amereham), as described by Raccxer et al. (1977). Direct binding of ~I-Aohl II to plastic coated rnAbs . Fifty microlitrea of varying dilutions of mAbs 4C1 or 3C5 in phosphate buffered saline (PBS) were incubated in wells of Falcon microtest III flexible plates (2 hr, 37°C). The wells were saturated with 2Y° bovine serum albumin (BSA) in PBS (1 hr, 37°C), and washed with PBS. Then, 50 WI of a 10 -' M solution of '~I-AaH II in PBS were added to each well and the plates incubated for 3hr at 37°C, then overnight at 4°C. After five washes, each well was cut and its radioactivity measured (Packard, Crystal IIn. Each assay was in duplicate. In general, duplicates did not differ by more than 10%. Non-specific binding was measured with a mouse non-immune serum and subtracted from the total radioactivity in each well.
Assay for competttiwr between mAbs or Fab fragments
For studies on competition between mAbs, 50 pl of a 7.9 x 10 -' M solution of mAb 4C1 were adsorbed onto each well of a 96-well Falcon plate (2 hr, 37°C). After saturation and washing as above, a mixture of 25 pl of varying dilutions of mAb 4C1, or mAb 3C5 and of 25 Wl of a 2 x 10 -9 M solution of'~I-AaH II was added to each well and the plates were incubated at 37°C for 3 hr and then at 4°C overnight. The subsequent steps of the assay were as described above. The assay for oompe6tion between Fab fragments were as described above. Fab 4C1 was coated to the wells at a 10 -'M concentration. Fab 3C5 was used as the competitor in a concentration range from 5 x 10-' to 8 x 10 -' M.
Monoclonal Antibodies to Scorpion Toxins
72 5
11folecrdar weight dletermination of Fab-toxin canplxes The elution volumes of a series of flue mol. wt markets (12,580 to 240,000) were measured using a Sephadex G150 column (30 x 1 cm). A reference curve of elution volumes (ml) against mol. wt was then plotted. Three different preparations of Fab-'~I-AaH II complexes were each independently chromatographed on the column and the elution volumes of the main radioactive peaks noted. The mol. wt of the complex was determined from the calibration curve. Two independent experiments were performed, which gave results differing by a maximum of 10% from the mean value. For the complex with the 3C5 Fab we found two experimental values which were close but quite elevated (see results). The three preparations were as follows: (1)'vI-AaH II (5 x 10-9 M, 501il) + Fab 4CI (2 x 10'°M, 501il) + 50mM phosphate buffer pH7.4 (100 lil) ; (2) 'vI-AaH II (5 x 10-' M, 50 lil) + Fab 3C5 (2 x 10 -° M, 50 lil) + 50 ttilvi phosphate buffer pH 7.4 (1001il) ; and (3) '~I-AaH II (5 x 10_ 9 M, 501il) + Fab 4C1 (2 x 10- ° M, 50 lil) + Fab 3C5 (2 x 10 -° M, 50 lil) 50 tnM phosphate buffer pH 7.4 (50 lily. All three mixtures were incubated for 90 min at 37°C, then overnight at 4°C, and then used immediately for chromatography.
Binding ojmrtibodies to synthetic heptapeptidea The assay format was that described by GeYw et d. (1984, 1987). Every heptapeptide derived from the sequence of AaH II (Rocxer et al., 1972) wes synthesized following the instructions of the commercial epitope trapping kit (Cambridge Research Biachetnicals, Cambridge, U.K.) . Briefly, synthetic peptides were assembled, in duplicate, on polyethylene rods arranged in the format of a standard 96-well ELISA plate. After completion of the synthetic steps, rods were thoroughly washed in methanol, water and then PBS and ptecoated with PHS-T (1 % BSA, 0.05% Tween 20 in PBS) . Then, the antibody solution (monoclotuil or polyclonal) at the appropriate dilution was dispensed in a 96-well ELISA plate and incubated overnight at 4°C with rod-attached peptides . After extensive washing, the antibody binding to the peptide was measured by incubating (90 rain at room temperature) blacks of trods with a rabbit anti-mouse IgG coupled to alkaline phosphatase or a goat anti-rabbit IgG coupled to alkaline phosphatase, depending on the source of antibody leafed . Then, the patanitrophenyl phosphate substrate (Sigma) was added and, after the colour had developed (60-90 rain), the absorbance at 405 nm of each well was measured in a Titertek Mullisten MCC (Flow Laboratories, McLean, U.S .A.) . Results were expressed as the signal/noise ratio (S/I~. The `noise' was calculated in each experiment as the mean absorbance given by the 50% peptides showing the lowest absorbance values. Between each test, rod-bound antibodies were removed from the peptides by sonicating the blocks (30 min, 60°C) in 0.2 M sodium phosphate containing I% sodium dodecyl sulphate and 0.1% ß-mereaptoethanol, according to the manufacturer's instructions.
Imnuoioaffrnity pwijrcatian of toxin II jrom Androctonus australis Hector venom For preparation of the immuaoaffmity gel, 4CI ascitic fluid (12 ml) was incubated overnight with 1 .8 g of protein A-Sepharoae (Pharmacia) in 3 M NaCI, 1 .5 M Glycine, pH 8.9, then washed with the setae buffer. IgGs were eluted with 0.1 M sodium citrate, pH 6.0 . Then, 4C1 purified IgG (56tag) was coupled to 4 g of CNHr activated Sepharoee 4B, as described by the manufacturer (Pharmacia) . For purification of AaH II, the venom from .lndroctarua mrstralis Hector was extracted by water as previously described (Mtwu~tne et al., 1970; M~ternv and RocrteT, 1986) and incubated with 4C1 IgG-CNHr Sephatbse in 20 mM Tris-HCI, 150 tnM NaCI, pH 7.4, by rotating overnight at 4°C. The IgG-Sepharose was then extensively washed. Fractions were eluted with 350 mM formic acid, 150 mM NaCI, pH 2, immediately neutralized by 1 M Tris-HCI, 150 tnM NaCI, pH 8.8 and then checked on a 20% SDS-PAGE (Phast System, Pharmacie) with or without ß-tner~captoethanol . Silver staining showed two bands of apparent mol. wta about 7000 and 14,000. In an immunoblotting experiment, only the mol. wt 7000 band was itnmunoreacdve with anti-AaH II serum. The immunoabsorbed material was further purified by reverse phase HPLC . Buffer A was 0.1 % trifluoroaoetic acid (TFA), buffer B was 0 .1% TFA in acetonitrile. The column (RPB, Lichrosphsr, Merck, Darmstadt, F.R.G .) was equilibrated in l7% buffer B for 10 mn and eluted with a linear gradient of 175% buffer B over a period of 126 min. The elution pattern showed two peaks at 56 rain (peak I) and 97 min (peak 2) which each migrated as a single band in SDS-PAGE with apparent mol. wts of 7000 (peak 1) and 14,000 (peak 2). The material corresponding to peak 1 was homogeneous in SDS-PAGE . An aliquot was hydrolysed by 6N HCI (20 hr, 110°C) and analysed on a Beckman 6300 amino acid analyser . The amino acid composition showed good agreement with that of toxin II purified by conventional methods. The toxin was quantified according to
726
N. YAI-II et al.
6
11
16
21
26
31
36
41
46
Sl
56
PEPTIDES
FIk3 . 1. BINDINß Od+ ~lDNOCLONAL AND POLYCLONAL ANTl80DIE9 10 OYP1tLAPPINO i18PTAPEPT'lDF3'.OF AaH II s~uelvce . Every heptapeptide from the sequence of AaH II was synthesized in duplicate on polyethylene rods and tested in an ELISA assay for its capacity to bind monoclonal antibody 4C1 (A), 3C5 (B) or a polytonal anti-AaH II serum (C), according to the procedure of GsYSBx et ol. (1984, 1987). IgG from 4C1 or 3C5 mAbs were used at a concentration of 30 ~g/ml and the polyclonal anti-AaH II antiserum was diluted 400afold . Results are expressed as the signal/noise ratio (S/I~: the mean optical density of each duplicate peptide was divided by the background absorbante, as deSned in Materials and Methods.
the results of amino acid analysis. The toxicity of this preparation was found to be 1 .9 lIg/20 g mouse, by i.c.v injxtion into C57 BL/6 mak mice.
Neutralizattort auoy For neutralisation assays, 20 .1k1 aliquots of a solution of toxin at 41kg/ml (i.e. titrated to contain slightly over 2 kn~/pl) were incubated with ~ pl of variooa dilutions of mAb 3C5 (15 mg/ml; 8.7 mg/ml; 2.8 mg/ml) or mAb 4C 1 (3.4 mg/ml) or PBS as a control. After 90 min at 37°C and 4hr at 4°C, 2 pl of each solution was injected i.c.v to 20 g C57 BL/6 mice . Dead mice were counted 18 hr later.
Monoclonal Antibodies to Scorpion Toxins
727
h N
'+ mAb, log M
Fro. 2. Sor.nrrxe.~ anvnnaa oe'~I-AaH II To mAb 4Cl wn mAb 3C5. The two mAbs were coated on 96-wellflexible plastic plates at varying dilutions. The labelled toxin was added to each well and the radioactivity remaining in each well after washings was measured . The symbol / shows binding to mAb 4C1 ; the symbol p shows the binding to mAb 3C5. Each point is the mean of duplicate experiments. M is the molar concentration of the different mAbs. RESULTS
Binding ofmAb 4C1 and mAb 3C5 to overlapping heptapeptide segments of AaH II MAb 4C1 and mAb 3C5 were assayed for their capacity to. bind each of 58 synthetic
peptides representing every possible heptapeptide (i .e. 1-7, 2-8, ~9 . .. 584) in the amino acid sequence of AaH II, according to the epitope mapping method of GsxsErr et al. (1984, 198 . In this assay, peptides were synthesized on polyethylene rods and the immunoreactivity of the rod-coupled peptides tested directly in an ELISA format . Neither mAb bound significantly any of the heptapeptide (Fig. lA, B) . Even at high concentrations of purified IgG (301tg/ml), the signal/noise ratio of both mAbs in all cases was under the cutoff value of 2, used to distinguish positive peptides . These results suggest that the two mAbs recognize discontinuous amino acids on the surface of the toxin, which cannot be mimicked by sequential heptapeptides. In contrast, a polyclonal rabbit antibody to AaH II recognized different peptides derived from the amino acid sequence (Fig. 1 C), showing that a positive response can be clearly detected by this system . Moreover, the antigenic regions mapped in this short-peptide assay agree well with antigenic regions defined previously by conventional methods (C . DEVAUX, manuscript in preparation). Inhibition of the binding of ~~I-AaH II to mAb 4C1, by mAb 3C5 and its Fab fragments
The ability of the two mAbs to bind '2SI-AaH II in a solid phase assay was determined (Fig. 2). Much larger amounts of mAb 3C5 than of mAb 4C1 were required to bind the same amount of toxin. This is consistent with the previous observation that their ICds for binding to AaH II differ by about three orders of magnitude. However, mAb 3C5 was able to inhibit fully the binding of '~sI-AaH II to plastic~oated mAb 4C1 (Fig. 3A). Half-inhibition of '~I-AaH II binding to mAb 4C1 was obtained with a concentration of either 2 x 10 -' M mAb 3C5 or 6 x 10 - a M mAb 4C1 . The capacity of mAb 3C5 to inhibit the fixation of mAb 4C 1 to the toxin suggests that the epitopes recognized by the two mAbs could be close to each other. However, these results could also be due to steric hindrance between two antibodies recognizing distant parts of the toxin. Monovalent Fab
728
N. YAHI et al.
Fta. 3.
Fab 3C5, Log M
of mAba 4C1 ~uvo 3C5, ox 1~ Fab Fxeo~ns, m '~I-AaH-II. (A) Inhibition of the binding of'~I-AaH II to mAb 4C1, by mAb 4C1 or mAb 3C5. mAb 4C1 was coated to 9Crwell flexible plastic plates, then a mixture of'~I-AaH II and mAb 4C1 (/) or mAb 3C5 (p) in varying dilutions was added. After incubation, the radioactivity bound to each well after washings was measured. (H) Inhibition of the binding of'~I-AaH II to Fab 4C1, by Fab 3C5. Experimental conditions were the same as above, except that the 4C1 Fab fragment was coated on the plate and the 3C5 Fab used as a competitor . M is the molar concentration of the different mAbs or Fabs. H and Bo are, respectively, the radioactivity bound to the wells in the presence or the absence of the tested antibody . CO1(PErITION
FOR HIIdDINO
fragments of the two mAbs were prepared . Solid-phase inhibition experiments using Fab fragments were performed and are reported in Fig. 3B. The Fab fragments of mAb 3C5 inhibited the fixation of '~I-AaH II to 4C1 Fab, which is not consistent with the stark hindrance hypothesis . Molecular weiglet determination of Falrtoxin complexes Fab-toxin complexes were prepared in solution, as described in Materials and Methods. The mol. wt of performed '2sI-AaH II-Fab complexes was estimated by a chromatographic method (see Materials and Methods). The experimental mol. wt (61,300) of a mixture of the two different monovalent Fab fragments with labelled toxin was clearly different from the expected mol. wt (about 110,000) of a ternary complex, which would have formed if the two monoclonal antibodies had bound simultaneously to the toxin.
Monoclonal Antibodies to Scorpion Toxins
729
This experimental mol. wt also compares favourably with the mol. wt of either Fab 4C1toxin (65,500) or Fab 3C~toxin (84,700) . These results show that the two different Fab fragments do not bind simultaneously to AaH II when the toxin is freely accessible in solution. This inability was not due to a limited toxin surface accessibility since previous experiments (EI. AY>~ et al., 1983) using Fab fragments of polyclonal anti-AaH II antibodies have shown that the surface of the toxin can accommodate up to four Fabs . 4C1 and 3C5 The in vivo neutralizing capacity of mAb 4C1 has been established in conditions where 3C5 did not show measurable neutralizing capacity (BA>:ntwoul et al., 1988). That the epitope recognized by 3C5 is close in space to a neutralizing epitope is, however, consistent with mAb 3C5 possessing some neutralizing activity . To check for a possible neutralizing capacity of mAb 3C5, the antibody was used at the highest concentration possible, so that the antibody/toxin ratio would be as large as possible . AaH II was prepared by a new immunoafimity procedure and judged pure by several criteria (see Materials and Methods). A titrated solution of the toxin was prepared and incubated with different dilutions of atAb 3C5. Mice received 2 Etl of the mixture, by an i.c.v. route which contained slightly over 2 Ln~ of AaH II. Preincubation with 15 mg/ml mAb 3C5 partly neutralized the toxin (four surviving mice of eight injected), the apparent toxicity of the solution being lowered by a factor of about two. However, this effect was not observed at mAb concentrations of 8.7 mg/ml or lower. In the case of mAb 4C1, the previously described potent neutralizing effect (BAFntwoul et al., 1988) was observed at an antibody concentration of 3.4 mg/ml (two surviving mice of two injected). In control assays, AaH II in PBS was found to be fully potent (no surviving mice out of five injected). Neutralizing capacities of mAbs
DISCUSSION
The first conclusion drawn from this study is that the epitopes recognized by mAbs 4C1 and 3C5 on AaH II are discontinuous and close epitopes. Our previous results (BeItitAOUI et al., 1988) have shown that mAb 4C1 failed to recognize the reduced carboxymethylated form of AaH II, clearly indicating that the epitope recognized by 4C1 is dependent on the native conformation of the toxin. This result is confirmed here by the observation that mAb 4C1 failed to bind any of the possible linear heptapeptides derived from the AaH II sequence . Moreover, when peptides larger in size than heptapeptides were tested, they also showed no reactivity with mAb 4C1 (BAxitAOUI et al., 1988). MAb 3C5 was also unable to bind significantly to any of the synthetic heptapeptides of AaH II. Although one cannot formally exclude that peptides larger in size than heptapeptides would have shown some binding, it is likely that the binding of mAb 3C5 is also dependent on the conformation of the toxin. The vast majority of antibodies recognize discontinuous regions of the protein surface (BSRZOFSRY, 1985 ; LAVER et al., 1990) and, in this respect, mAbs 4C1 and 3C5 follow the general rule. However, some proteins of the size and general structural orgânization of a scorpion toxin elicited mAbs able to bind to rod-attached peptides (Hosrl:zcx et al., 1989) which indicates that tightly folded mini-proteins can contain linear epitopes . The control experiment (Fig. 1 C), shows that some polyclonal anti-toxin antibodies bound to the synthetic heptapeptides, indicating that peptide-specific antibodies
73 0
N. YAHI et al .
are generated in the polyclonal response to AaH II. These antigenic specificities, however, were not selected during the hybridoma screening procedure which led to mAbs 4C1 and 3C5. It was not possible to find which particular residues of AaH II participate in the epitope recognized by mAb 3C5 by using modified derivatives of the toxin in a competitive immunoassay, due to the low affinity of mAb 3C5 for its antigen (huge amounts of modified AaH II would have to be used to inhibit a binding reaction with an affinity constant around 10_e M). Thus, other experiments were designed to determine if the epitope recognized by mAb 3C5 is topographically distant from the epitope for mAb 4C1. The two antibodies or their Fab fragments could not bind simultaneously to the toxin, either in solid-phase (Fig. 3) or solution conditions (results of the mol. wt determination) . This was not anticipated since the toxin is able to accommodate four Fab fragments from a polyclonal anti-AaH II serum (EL AYE et al., 1983). Moreover, reports in the literature (JncxsoN et al., 1988; I~ATHJSN and UNn~woon, 1986) indicate that insulin-sized polypeptides can bind two mAbs of different specificity, simultaneously. From these considerations, we concluded that the epitopes of mAbs 3C5 and 4C1 are close enough on the toxin surface to preclude the simultaneous binding of the two Fab fragments . Since mAb 4C1 exhibits a strong neutralizing capacity, the evidence that the epitope recognized by mAb 3C5 could be close to the 4C1 epitope led us to reinvestigate the neutralizing capacity of 3C5. It was previously shown (B~xitnout et al., 1988) that the mAb 4C 1 acts by binding to a region of the toxin which is close to the site of binding to pharmacological receptor . The spatial proximity of the 4C1 and 3C5 epitopes makes it likely that the mAb 3C5 could neutralize the toxin effect by a similar mechanism and not, for example, by destabilizing the toxin-receptor complex by binding to a distal part of the toxin (BOULAIN and MErrBZ, 1982). In the experimental protocol used in this study, a clear neutralizing effect of 3C5 was observed, but only at a very high antibody concentration and was by no means comparable to the strong neutralizing capacity of mAb 4C1. In the in vivo assay used in our previous study (B~xi~,otn et al., 1988), we tested the neutralizing capacity of 3C5 at a mAb/toxin ratio of 4.3 without any discernible neutralization of the toxin. Here, a ratio of 175/1 was used and the antibody showed some capacity to diminish the AaH II toxicity . The poor neutralizing activity of 3C5 in the in vivo test could be explained by differences in the binding affinities displayed by AaH lI for the antibody molecule and for the receptor sites of mouse brain. AaH II binds to the rat brain sodium channel with high affinity (1~Cd around 0.2 nM; association constant 1 .5 x 10' M -' sec- ' ; dissociation constant 1 .6 x 10_s sec- ') (Jovmt et al., 1978); thus, to display any neutralizing capacity, a monoclonal antibody should have an affinity enabling it to bind a significant proportion of the toxin in the presence of the receptor . Otherwise, the toxin will dissociate rapidly from the antigen antibody complex and bind to the sodium channel receptor, leading the animal to death. This is what probably occurs with mAb 3C5 for which the affuûty of the toxin is in the micromolar range. In the case of mAb 331 against the snake toxin abungarotoxin, it was observed that the injection of mAb-toxin complex into mice slows down the onset of the poisoning symptoms (Knsa et al., 1989), perhaps reflecting a slow release of the toxin from its antibody complex. In conclusion, our study showed that the two mAbs 4C1 and 3C5 each probably recognize discontinuous groups of amino acids at the surface of AaH II and that these epitopes are sufficiently close to preclude the simultaneous binding of the two corresponding Fabs . The strong neutralizing capacity of mAb 4C1 was confirmed and a weak neutralizing effect was found for mAb 3C5, consistent with the low affinity binding of mAb 3C5 in the proximity of a main neutralizing site of the toxin .
Monoclonal Antibodies to Scorpion Toxins
73 1
address our wann thanks to Dr E. Betnewout and to Dr J. Ptcxox for their initial preparation of the hybridomas used in this study. The akilfiil assistance of M. ALVYrAE and T. Baertno and the support of Prof. H. Rocrrwr are appreciated. This work was supported by institutional funds of the Centre National de la Recherche Scientifique and the Institut National de la Santé et de la Recherche Médicale . Acknowkdgm~ents-We
REFERENCES B~uout, E., Et Area, M., Vety Rmrscllorex, J., Roctr~r, H. and Gruxme, C. (1986) Immunochemistry of scorpion a-toxins: study with synthetic peptides of the antigenicity of four regions of toxin II of Andrcetonees austrerlts Hector. Mokc. Irrrrem. 23, 357-366. Bwweeotn, E., Ptcx~ox, J., Mrn.~e, J. M., D~neox, H., Er . Ar®, M., Gam, C., MwrlVtrnr, J. and RocFUr, H. (1988) Monoclonal antibodies to scorpion toxins. Characterization and molecular mechanisms of neutralization. J. Imnrutt. 141, 214-220. Hertza+stlr, J. A. (1985) Intrinsic and extrinsic factors in protein antigenic structure. Science 229, 932940 . Hout.tnr, J. C. and Mr~z, A. (1982) Neurotoxin-spadfic immunoglobulins acceh:rate dissociation of the neurotoxin-acetylcholine receptor wmplex. Science 217, 732-733. Ce~r.r., W. A. (1977) Membrane potential dependant binding of scorpion toxins to the action potential Na+ ionophore. J. biol. Cherr. 252, 8660,8668 . Et. AYIT9, M ., M~nrtx, M. F., Dra.oat, P., Beaus, G. and Roctu+r, H. (1983) Immunochemistry of scorpion a-neurotoxins. Determination of the antigenic site number and isolation of a highly enriched antibody specific to a single antigenic site of toxin II of Mdroctorrus australis Hector. Mokc. Ieeerreun. 20, 697-708. E~ Ar®, M., B~uorn, E., G~x~e, C. and Rocrrwr, H. (1986) Use of antibodies to defined regions of scorpion a-toxin to study its interaction with its receptor site on the sodium channel. Blocherristry 2S, 6671678. Gersex, M. H., BARTSLING, S. J. and MEr.oex, R. H. (1984) Use of peptide synthesis to probe viral antigens for epitope to a resolution of single amino acid . Proc. natee. Aced. Sci. U.S.A . 82, 178-182. Gersex, M. H., RODDA, S. J., Mesox, T. J., TRtearctc, G. and Scr~oops, P. G. (1987) Strategies for epitope analysis using peptide synthesis. J. Imrrror . Methods 102, 259-274. Gw~rr~e, C., Novorxr, J., Foxreca.t .~-C~s, J. C., Foun~r, P., Er. Avee, M. and Bnrnuorn, E. (1989) The antigenic structure of a scorpion toxin. Mokc . Irrrreoe. lb, 503-513. Hoerxic~r, P. D., Jx, Lerrorox, B. C., Z,rr~uvo, J:W. and Tner, J. P. (1989) Identification of immunodominant regions of transforming growth factor a. Implications for structure and function. J. biol. Chem . 264, 19,086-19,091 . IACR90N, D. C., Porn®ountos, P, and Wrm~e, D. O. (1988) Simultaneous binding of two monoclonal antibodies to epitopes separated in sequence by only three residues. Molec. Irr»run. 25, 465-471 . Jovea, E., M~rertx-Mouror, N., Cor~rm, F. and Rocx~r, H. (1978) Scorpion toxin: specific binding to rat synaptosomes . Biochem. biophys. Res. Commeoe. 85, 377-382. K~se, R., Krrt c~we, H., I3~rwstu, K., Tt xoue, K. and Ix~aext, F. (1989) Neutralizing monoclonal antibody specific for a-bungarotoxin : preparation, characterization of the antibody and localization of antigenic rogion of a-bungarotoxin. FEBS Lctt. 254, 106-110. Ltvea, W. G., Atx, G. M., Weesx~e, R. G. and S~rx-Gu.r., S. J. (1990) Epitopes on proteins : misconceptions and ttalitiea. Cell 61, 553-556. M~rtmv, M:F. and Roctr~r, H. (1986) Large scale purification of toxins from We venom of the scorpion Atulroctoreus australis Hector. Toxicon 24, 1131-1139. MrRwrroe, F., Korer~uv, C., Rocxwr, H. and Lisstrzw, 3. (1970) Purification of animal neurotoxina: isolation and characterization of eleven neurotoxins from the venons of the scorpions Aredroctoeeees mvtralis Hector, Buthus occittmus tunetmrees and Leiurus quinquestriatus quietquestriatus . Eur. J. Blocherr. 16, 514-519. Portrete, R. R (1958) Separation and isolation of fractions of rabbit gamma globulin containing the antibody and antigenic sites. Nateoe 182, 67071. Rertrrex, D. A. and UxneRwooo, P. A. (1986) Identification of antigenic determinants on insulin molecule recognized by monoclonal antibodies. Mokc. Irnnaere . 23, 44150. Roc~rwr, H., Rocxer, C., Simmer, F. and M~xne, F. (1972) The amino acid sequence of neurotoxin II of Androctortus australis hector . Eur. J. Biocherr . 28, 381-388. ROCEUr, H., Tax, M., M~u~ne, F. and Lissrtzter, S. (1977) Radioiodination of scorpion and snake neurotoxins . Arurlyt. Biocherr . 85, 532-538.
,BTOX445P 00 000008559 0112 00008
0041-0101/92 SS .00 + .00 © 1992 PaPmon Pnew Ltd
MONOCLONAL ANTIBODIES TO TOXIN II FROM THE SCORPION ANDROCTONUS AUSTRALIS HECTOR: FURTHER CHARACTERIZATION OF EPITOPE SPECIFICITIES AND NEUTRALIZING CAPACITIES NOUARA YAI~u, G~nersTiwlvE Dgvwux, PwscwL MANSUELLE, MARIA-L~Riw D~rrnnvl and CLAUDE GRANIER' CNRS URA 1455, Laboratoire de Biochimie, Faculté de Médecine Secteur Nord, Boulevard Pierre Dramard, 13326 Marseille Cedex 15, France (Received 8 November 1991 ; accepted 27 Jmluary 1992)
N. YAin, C. DEVAUX, P. MANSUELLE, M.-L. DSFENDIPiI and C. GRANIER. Monoclonal antibodies to toxin II from the scorpion Androctonus australis Hector : further characterization of epitope specificities and neutralizing capacities . Toxicon 30, 723-731, 1992 .-The epitope specificities of two previously prepared monoclonal antibodies (mAb) to the toxin II from Androctonus australis Hector were characterized. Neither mAb 4C 1 nor mAb 3C5 was able to recognize any of the 58 overlapping synthetic heptapeptides which cover the whole sequence of toxin II . Thus, both mAbs probably recognize conformation-dependent epitopes at the surface of the toxin. Experiments were designed to check whether or not the two mAbs, or their Fab fragments, were able to bind simultaneously to the toxin. The results indicated that the epitopes recognized by the two antibodies are probably close together at the surface of the toxin, thus preventing the simultaneous binding of both mAbs to a single toxin molecule. Given the proximity of the two epitopes and the fact that mAb 4C1 is known to be a neutralizing antibody, the capacity of mAb 3C5 to inhibit the toxic effects of the toxin was re-evaluated in C57BL/6 mice . A clear, but weak, neutralizing effect was found, consistent with the low affinity binding of the mAb in the proximity of a neutralizing site of the toxin. INTRODUCTION
ScoRP1oN toxins are the major components of venoms responsible for the toxic effects that occur in man after a scorpion sting. They act by binding with high allïnity to receptor sites of voltage dependent sodium channels (CATTHtALL, 1977). Despite their small mol. wt (about 7000) these toxins are immunogenic in horse, rabbit and mouse. Polyclonal antibodies to various toxins have been obtained which have been very useful for assessing the antigenic site number (EL A~ et al., 1983) or the location of the main antigenic 'Author to whom correspondence should be addressed . 723
724
N. YAHI et al.
regions of one toxin (GRANIER et al., 1989). More recently, two monoclonal antibodies, mAb 3C5 and mAb 4C1, to the toxin II of the North African species Androctom~ts australis Hector (AaH In have been obtained (BAHRAOUI et al., 1988). MAb 4C1 is very interesting in that it displays a high affinity for the antigen (Kd = 0.8 nM) and powerful in vitro and in vivo neutralizing properties . Some of the residues of the toxin that are probably involved in the epitope recognized by mAb 4C1 have been identified using chemically modified derivatives of AaH II, in a competitive radioimmunoassay (BAHRAOUI et al., 1988). The other monoclonal, mAb 3C5, was more difficult to characterize because it has a much lower affinity for the toxin than mAb 4C1 (Kd = 0.61tM). In the experimental conditions used to demonstrate the neutralizing capacity of mAb 4C1, mAb 3C5 had no neutralizing effect (BAHRAOUI et al., 1988). This could be explained ifmAb 3C5 recognized on the toxin an epitope located on a region opposite to the region interacting with the receptor . For example, the epitope could be the a-helix region, which is known to be antigenic (B.~hntwoul et al., 1986) but does not contact the receptor during binding (EL AYIm et al., 198 . A better definition of the epitope recognized by mAb 3C5, together with its spatial relationship with the mAb 4C 1 epitope, is thus of interest . The present study is an attempt to characterize the epitope recognized by mAb 3C5 on the Attdroctonus australis Hector toxin II and to re-evaluate its neutralizing capacity .
Furfficatiort ojrnAbs 3C5 ared 4Cl
MATERIALS AND METHODS
Clones 4C1 and 3C5, which have been described previously (B~newotn et al., 1988), were expanded in BALB/C mice primed with pristane (2,6,10,14 ~etn~~yl-pentadecane, Sigma, St . Louis, MO, U.S.A.). Monoclonal antibodies were purified from aecitic fluids by ammonium sulphate precipitation followed by DEAE~Cellulose chromatogisphy (DE52, whatman, Maidstone, U.K.). The homogeneity of the preparation was checked by SDS-PAGE electrophoresis. The antibody concentration was assessed by spectrophotometric measurement, at 280 nm, taking absorption coefficient e 1% (1 cm) = 13.5. The molar concentration of these yl IgGs was estimated assuming a mol. wt of 150,000. MAbs were stored at -20°C in small aliquots. Fab fragment preparation followed the general conditions described by Port~e (1958). Briefly, 5 mg of mAb were incubated for 60 min at 37°C with 1% (w : w) pepsin (Sigma) in the presence of 20 mM EDTA, and 15 pM ßmercaptoethanol . The n~ction was stopped by addition of 20 mM iodacetamide (Fluke). The Fab fragments were recovered in the void-volume fractions of a protein A-Sepharose column (Pharmacia, Uppsala, Sweden). Solid~phase radioinuraoroarsays
AaH II was labelled with Na'~I (Amereham), as described by Raccxer et al. (1977). Direct binding of ~I-Aohl II to plastic coated rnAbs . Fifty microlitrea of varying dilutions of mAbs 4C1 or 3C5 in phosphate buffered saline (PBS) were incubated in wells of Falcon microtest III flexible plates (2 hr, 37°C). The wells were saturated with 2Y° bovine serum albumin (BSA) in PBS (1 hr, 37°C), and washed with PBS. Then, 50 WI of a 10 -' M solution of '~I-AaH II in PBS were added to each well and the plates incubated for 3hr at 37°C, then overnight at 4°C. After five washes, each well was cut and its radioactivity measured (Packard, Crystal IIn. Each assay was in duplicate. In general, duplicates did not differ by more than 10%. Non-specific binding was measured with a mouse non-immune serum and subtracted from the total radioactivity in each well.
Assay for competttiwr between mAbs or Fab fragments
For studies on competition between mAbs, 50 pl of a 7.9 x 10 -' M solution of mAb 4C1 were adsorbed onto each well of a 96-well Falcon plate (2 hr, 37°C). After saturation and washing as above, a mixture of 25 pl of varying dilutions of mAb 4C1, or mAb 3C5 and of 25 Wl of a 2 x 10 -9 M solution of'~I-AaH II was added to each well and the plates were incubated at 37°C for 3 hr and then at 4°C overnight. The subsequent steps of the assay were as described above. The assay for oompe6tion between Fab fragments were as described above. Fab 4C1 was coated to the wells at a 10 -'M concentration. Fab 3C5 was used as the competitor in a concentration range from 5 x 10-' to 8 x 10 -' M.
Monoclonal Antibodies to Scorpion Toxins
72 5
11folecrdar weight dletermination of Fab-toxin canplxes The elution volumes of a series of flue mol. wt markets (12,580 to 240,000) were measured using a Sephadex G150 column (30 x 1 cm). A reference curve of elution volumes (ml) against mol. wt was then plotted. Three different preparations of Fab-'~I-AaH II complexes were each independently chromatographed on the column and the elution volumes of the main radioactive peaks noted. The mol. wt of the complex was determined from the calibration curve. Two independent experiments were performed, which gave results differing by a maximum of 10% from the mean value. For the complex with the 3C5 Fab we found two experimental values which were close but quite elevated (see results). The three preparations were as follows: (1)'vI-AaH II (5 x 10-9 M, 501il) + Fab 4CI (2 x 10'°M, 501il) + 50mM phosphate buffer pH7.4 (100 lil) ; (2) 'vI-AaH II (5 x 10-' M, 50 lil) + Fab 3C5 (2 x 10 -° M, 50 lil) + 50 ttilvi phosphate buffer pH 7.4 (1001il) ; and (3) '~I-AaH II (5 x 10_ 9 M, 501il) + Fab 4C1 (2 x 10- ° M, 50 lil) + Fab 3C5 (2 x 10 -° M, 50 lil) 50 tnM phosphate buffer pH 7.4 (50 lily. All three mixtures were incubated for 90 min at 37°C, then overnight at 4°C, and then used immediately for chromatography.
Binding ojmrtibodies to synthetic heptapeptidea The assay format was that described by GeYw et d. (1984, 1987). Every heptapeptide derived from the sequence of AaH II (Rocxer et al., 1972) wes synthesized following the instructions of the commercial epitope trapping kit (Cambridge Research Biachetnicals, Cambridge, U.K.) . Briefly, synthetic peptides were assembled, in duplicate, on polyethylene rods arranged in the format of a standard 96-well ELISA plate. After completion of the synthetic steps, rods were thoroughly washed in methanol, water and then PBS and ptecoated with PHS-T (1 % BSA, 0.05% Tween 20 in PBS) . Then, the antibody solution (monoclotuil or polyclonal) at the appropriate dilution was dispensed in a 96-well ELISA plate and incubated overnight at 4°C with rod-attached peptides . After extensive washing, the antibody binding to the peptide was measured by incubating (90 rain at room temperature) blacks of trods with a rabbit anti-mouse IgG coupled to alkaline phosphatase or a goat anti-rabbit IgG coupled to alkaline phosphatase, depending on the source of antibody leafed . Then, the patanitrophenyl phosphate substrate (Sigma) was added and, after the colour had developed (60-90 rain), the absorbance at 405 nm of each well was measured in a Titertek Mullisten MCC (Flow Laboratories, McLean, U.S .A.) . Results were expressed as the signal/noise ratio (S/I~. The `noise' was calculated in each experiment as the mean absorbance given by the 50% peptides showing the lowest absorbance values. Between each test, rod-bound antibodies were removed from the peptides by sonicating the blocks (30 min, 60°C) in 0.2 M sodium phosphate containing I% sodium dodecyl sulphate and 0.1% ß-mereaptoethanol, according to the manufacturer's instructions.
Imnuoioaffrnity pwijrcatian of toxin II jrom Androctonus australis Hector venom For preparation of the immuaoaffmity gel, 4CI ascitic fluid (12 ml) was incubated overnight with 1 .8 g of protein A-Sepharoae (Pharmacia) in 3 M NaCI, 1 .5 M Glycine, pH 8.9, then washed with the setae buffer. IgGs were eluted with 0.1 M sodium citrate, pH 6.0 . Then, 4C1 purified IgG (56tag) was coupled to 4 g of CNHr activated Sepharoee 4B, as described by the manufacturer (Pharmacia) . For purification of AaH II, the venom from .lndroctarua mrstralis Hector was extracted by water as previously described (Mtwu~tne et al., 1970; M~ternv and RocrteT, 1986) and incubated with 4C1 IgG-CNHr Sephatbse in 20 mM Tris-HCI, 150 tnM NaCI, pH 7.4, by rotating overnight at 4°C. The IgG-Sepharose was then extensively washed. Fractions were eluted with 350 mM formic acid, 150 mM NaCI, pH 2, immediately neutralized by 1 M Tris-HCI, 150 tnM NaCI, pH 8.8 and then checked on a 20% SDS-PAGE (Phast System, Pharmacie) with or without ß-tner~captoethanol . Silver staining showed two bands of apparent mol. wta about 7000 and 14,000. In an immunoblotting experiment, only the mol. wt 7000 band was itnmunoreacdve with anti-AaH II serum. The immunoabsorbed material was further purified by reverse phase HPLC . Buffer A was 0.1 % trifluoroaoetic acid (TFA), buffer B was 0 .1% TFA in acetonitrile. The column (RPB, Lichrosphsr, Merck, Darmstadt, F.R.G .) was equilibrated in l7% buffer B for 10 mn and eluted with a linear gradient of 175% buffer B over a period of 126 min. The elution pattern showed two peaks at 56 rain (peak I) and 97 min (peak 2) which each migrated as a single band in SDS-PAGE with apparent mol. wts of 7000 (peak 1) and 14,000 (peak 2). The material corresponding to peak 1 was homogeneous in SDS-PAGE . An aliquot was hydrolysed by 6N HCI (20 hr, 110°C) and analysed on a Beckman 6300 amino acid analyser . The amino acid composition showed good agreement with that of toxin II purified by conventional methods. The toxin was quantified according to
726
N. YAI-II et al.
6
11
16
21
26
31
36
41
46
Sl
56
PEPTIDES
FIk3 . 1. BINDINß Od+ ~lDNOCLONAL AND POLYCLONAL ANTl80DIE9 10 OYP1tLAPPINO i18PTAPEPT'lDF3'.OF AaH II s~uelvce . Every heptapeptide from the sequence of AaH II was synthesized in duplicate on polyethylene rods and tested in an ELISA assay for its capacity to bind monoclonal antibody 4C1 (A), 3C5 (B) or a polytonal anti-AaH II serum (C), according to the procedure of GsYSBx et ol. (1984, 1987). IgG from 4C1 or 3C5 mAbs were used at a concentration of 30 ~g/ml and the polyclonal anti-AaH II antiserum was diluted 400afold . Results are expressed as the signal/noise ratio (S/I~: the mean optical density of each duplicate peptide was divided by the background absorbante, as deSned in Materials and Methods.
the results of amino acid analysis. The toxicity of this preparation was found to be 1 .9 lIg/20 g mouse, by i.c.v injxtion into C57 BL/6 mak mice.
Neutralizattort auoy For neutralisation assays, 20 .1k1 aliquots of a solution of toxin at 41kg/ml (i.e. titrated to contain slightly over 2 kn~/pl) were incubated with ~ pl of variooa dilutions of mAb 3C5 (15 mg/ml; 8.7 mg/ml; 2.8 mg/ml) or mAb 4C 1 (3.4 mg/ml) or PBS as a control. After 90 min at 37°C and 4hr at 4°C, 2 pl of each solution was injected i.c.v to 20 g C57 BL/6 mice . Dead mice were counted 18 hr later.
Monoclonal Antibodies to Scorpion Toxins
727
h N
'+ mAb, log M
Fro. 2. Sor.nrrxe.~ anvnnaa oe'~I-AaH II To mAb 4Cl wn mAb 3C5. The two mAbs were coated on 96-wellflexible plastic plates at varying dilutions. The labelled toxin was added to each well and the radioactivity remaining in each well after washings was measured . The symbol / shows binding to mAb 4C1 ; the symbol p shows the binding to mAb 3C5. Each point is the mean of duplicate experiments. M is the molar concentration of the different mAbs. RESULTS
Binding ofmAb 4C1 and mAb 3C5 to overlapping heptapeptide segments of AaH II MAb 4C1 and mAb 3C5 were assayed for their capacity to. bind each of 58 synthetic
peptides representing every possible heptapeptide (i .e. 1-7, 2-8, ~9 . .. 584) in the amino acid sequence of AaH II, according to the epitope mapping method of GsxsErr et al. (1984, 198 . In this assay, peptides were synthesized on polyethylene rods and the immunoreactivity of the rod-coupled peptides tested directly in an ELISA format . Neither mAb bound significantly any of the heptapeptide (Fig. lA, B) . Even at high concentrations of purified IgG (301tg/ml), the signal/noise ratio of both mAbs in all cases was under the cutoff value of 2, used to distinguish positive peptides . These results suggest that the two mAbs recognize discontinuous amino acids on the surface of the toxin, which cannot be mimicked by sequential heptapeptides. In contrast, a polyclonal rabbit antibody to AaH II recognized different peptides derived from the amino acid sequence (Fig. 1 C), showing that a positive response can be clearly detected by this system . Moreover, the antigenic regions mapped in this short-peptide assay agree well with antigenic regions defined previously by conventional methods (C . DEVAUX, manuscript in preparation). Inhibition of the binding of ~~I-AaH II to mAb 4C1, by mAb 3C5 and its Fab fragments
The ability of the two mAbs to bind '2SI-AaH II in a solid phase assay was determined (Fig. 2). Much larger amounts of mAb 3C5 than of mAb 4C1 were required to bind the same amount of toxin. This is consistent with the previous observation that their ICds for binding to AaH II differ by about three orders of magnitude. However, mAb 3C5 was able to inhibit fully the binding of '~sI-AaH II to plastic~oated mAb 4C1 (Fig. 3A). Half-inhibition of '~I-AaH II binding to mAb 4C1 was obtained with a concentration of either 2 x 10 -' M mAb 3C5 or 6 x 10 - a M mAb 4C1 . The capacity of mAb 3C5 to inhibit the fixation of mAb 4C 1 to the toxin suggests that the epitopes recognized by the two mAbs could be close to each other. However, these results could also be due to steric hindrance between two antibodies recognizing distant parts of the toxin. Monovalent Fab
728
N. YAHI et al.
Fta. 3.
Fab 3C5, Log M
of mAba 4C1 ~uvo 3C5, ox 1~ Fab Fxeo~ns, m '~I-AaH-II. (A) Inhibition of the binding of'~I-AaH II to mAb 4C1, by mAb 4C1 or mAb 3C5. mAb 4C1 was coated to 9Crwell flexible plastic plates, then a mixture of'~I-AaH II and mAb 4C1 (/) or mAb 3C5 (p) in varying dilutions was added. After incubation, the radioactivity bound to each well after washings was measured. (H) Inhibition of the binding of'~I-AaH II to Fab 4C1, by Fab 3C5. Experimental conditions were the same as above, except that the 4C1 Fab fragment was coated on the plate and the 3C5 Fab used as a competitor . M is the molar concentration of the different mAbs or Fabs. H and Bo are, respectively, the radioactivity bound to the wells in the presence or the absence of the tested antibody . CO1(PErITION
FOR HIIdDINO
fragments of the two mAbs were prepared . Solid-phase inhibition experiments using Fab fragments were performed and are reported in Fig. 3B. The Fab fragments of mAb 3C5 inhibited the fixation of '~I-AaH II to 4C1 Fab, which is not consistent with the stark hindrance hypothesis . Molecular weiglet determination of Falrtoxin complexes Fab-toxin complexes were prepared in solution, as described in Materials and Methods. The mol. wt of performed '2sI-AaH II-Fab complexes was estimated by a chromatographic method (see Materials and Methods). The experimental mol. wt (61,300) of a mixture of the two different monovalent Fab fragments with labelled toxin was clearly different from the expected mol. wt (about 110,000) of a ternary complex, which would have formed if the two monoclonal antibodies had bound simultaneously to the toxin.
Monoclonal Antibodies to Scorpion Toxins
729
This experimental mol. wt also compares favourably with the mol. wt of either Fab 4C1toxin (65,500) or Fab 3C~toxin (84,700) . These results show that the two different Fab fragments do not bind simultaneously to AaH II when the toxin is freely accessible in solution. This inability was not due to a limited toxin surface accessibility since previous experiments (EI. AY>~ et al., 1983) using Fab fragments of polyclonal anti-AaH II antibodies have shown that the surface of the toxin can accommodate up to four Fabs . 4C1 and 3C5 The in vivo neutralizing capacity of mAb 4C1 has been established in conditions where 3C5 did not show measurable neutralizing capacity (BA>:ntwoul et al., 1988). That the epitope recognized by 3C5 is close in space to a neutralizing epitope is, however, consistent with mAb 3C5 possessing some neutralizing activity . To check for a possible neutralizing capacity of mAb 3C5, the antibody was used at the highest concentration possible, so that the antibody/toxin ratio would be as large as possible . AaH II was prepared by a new immunoafimity procedure and judged pure by several criteria (see Materials and Methods). A titrated solution of the toxin was prepared and incubated with different dilutions of atAb 3C5. Mice received 2 Etl of the mixture, by an i.c.v. route which contained slightly over 2 Ln~ of AaH II. Preincubation with 15 mg/ml mAb 3C5 partly neutralized the toxin (four surviving mice of eight injected), the apparent toxicity of the solution being lowered by a factor of about two. However, this effect was not observed at mAb concentrations of 8.7 mg/ml or lower. In the case of mAb 4C1, the previously described potent neutralizing effect (BAFntwoul et al., 1988) was observed at an antibody concentration of 3.4 mg/ml (two surviving mice of two injected). In control assays, AaH II in PBS was found to be fully potent (no surviving mice out of five injected). Neutralizing capacities of mAbs
DISCUSSION
The first conclusion drawn from this study is that the epitopes recognized by mAbs 4C1 and 3C5 on AaH II are discontinuous and close epitopes. Our previous results (BeItitAOUI et al., 1988) have shown that mAb 4C1 failed to recognize the reduced carboxymethylated form of AaH II, clearly indicating that the epitope recognized by 4C1 is dependent on the native conformation of the toxin. This result is confirmed here by the observation that mAb 4C1 failed to bind any of the possible linear heptapeptides derived from the AaH II sequence . Moreover, when peptides larger in size than heptapeptides were tested, they also showed no reactivity with mAb 4C1 (BAxitAOUI et al., 1988). MAb 3C5 was also unable to bind significantly to any of the synthetic heptapeptides of AaH II. Although one cannot formally exclude that peptides larger in size than heptapeptides would have shown some binding, it is likely that the binding of mAb 3C5 is also dependent on the conformation of the toxin. The vast majority of antibodies recognize discontinuous regions of the protein surface (BSRZOFSRY, 1985 ; LAVER et al., 1990) and, in this respect, mAbs 4C1 and 3C5 follow the general rule. However, some proteins of the size and general structural orgânization of a scorpion toxin elicited mAbs able to bind to rod-attached peptides (Hosrl:zcx et al., 1989) which indicates that tightly folded mini-proteins can contain linear epitopes . The control experiment (Fig. 1 C), shows that some polyclonal anti-toxin antibodies bound to the synthetic heptapeptides, indicating that peptide-specific antibodies
73 0
N. YAHI et al .
are generated in the polyclonal response to AaH II. These antigenic specificities, however, were not selected during the hybridoma screening procedure which led to mAbs 4C1 and 3C5. It was not possible to find which particular residues of AaH II participate in the epitope recognized by mAb 3C5 by using modified derivatives of the toxin in a competitive immunoassay, due to the low affinity of mAb 3C5 for its antigen (huge amounts of modified AaH II would have to be used to inhibit a binding reaction with an affinity constant around 10_e M). Thus, other experiments were designed to determine if the epitope recognized by mAb 3C5 is topographically distant from the epitope for mAb 4C1. The two antibodies or their Fab fragments could not bind simultaneously to the toxin, either in solid-phase (Fig. 3) or solution conditions (results of the mol. wt determination) . This was not anticipated since the toxin is able to accommodate four Fab fragments from a polyclonal anti-AaH II serum (EL AYE et al., 1983). Moreover, reports in the literature (JncxsoN et al., 1988; I~ATHJSN and UNn~woon, 1986) indicate that insulin-sized polypeptides can bind two mAbs of different specificity, simultaneously. From these considerations, we concluded that the epitopes of mAbs 3C5 and 4C1 are close enough on the toxin surface to preclude the simultaneous binding of the two Fab fragments . Since mAb 4C1 exhibits a strong neutralizing capacity, the evidence that the epitope recognized by mAb 3C5 could be close to the 4C1 epitope led us to reinvestigate the neutralizing capacity of 3C5. It was previously shown (B~xitnout et al., 1988) that the mAb 4C 1 acts by binding to a region of the toxin which is close to the site of binding to pharmacological receptor . The spatial proximity of the 4C1 and 3C5 epitopes makes it likely that the mAb 3C5 could neutralize the toxin effect by a similar mechanism and not, for example, by destabilizing the toxin-receptor complex by binding to a distal part of the toxin (BOULAIN and MErrBZ, 1982). In the experimental protocol used in this study, a clear neutralizing effect of 3C5 was observed, but only at a very high antibody concentration and was by no means comparable to the strong neutralizing capacity of mAb 4C1. In the in vivo assay used in our previous study (B~xi~,otn et al., 1988), we tested the neutralizing capacity of 3C5 at a mAb/toxin ratio of 4.3 without any discernible neutralization of the toxin. Here, a ratio of 175/1 was used and the antibody showed some capacity to diminish the AaH II toxicity . The poor neutralizing activity of 3C5 in the in vivo test could be explained by differences in the binding affinities displayed by AaH lI for the antibody molecule and for the receptor sites of mouse brain. AaH II binds to the rat brain sodium channel with high affinity (1~Cd around 0.2 nM; association constant 1 .5 x 10' M -' sec- ' ; dissociation constant 1 .6 x 10_s sec- ') (Jovmt et al., 1978); thus, to display any neutralizing capacity, a monoclonal antibody should have an affinity enabling it to bind a significant proportion of the toxin in the presence of the receptor . Otherwise, the toxin will dissociate rapidly from the antigen antibody complex and bind to the sodium channel receptor, leading the animal to death. This is what probably occurs with mAb 3C5 for which the affuûty of the toxin is in the micromolar range. In the case of mAb 331 against the snake toxin abungarotoxin, it was observed that the injection of mAb-toxin complex into mice slows down the onset of the poisoning symptoms (Knsa et al., 1989), perhaps reflecting a slow release of the toxin from its antibody complex. In conclusion, our study showed that the two mAbs 4C1 and 3C5 each probably recognize discontinuous groups of amino acids at the surface of AaH II and that these epitopes are sufficiently close to preclude the simultaneous binding of the two corresponding Fabs . The strong neutralizing capacity of mAb 4C1 was confirmed and a weak neutralizing effect was found for mAb 3C5, consistent with the low affinity binding of mAb 3C5 in the proximity of a main neutralizing site of the toxin .
Monoclonal Antibodies to Scorpion Toxins
73 1
address our wann thanks to Dr E. Betnewout and to Dr J. Ptcxox for their initial preparation of the hybridomas used in this study. The akilfiil assistance of M. ALVYrAE and T. Baertno and the support of Prof. H. Rocrrwr are appreciated. This work was supported by institutional funds of the Centre National de la Recherche Scientifique and the Institut National de la Santé et de la Recherche Médicale . Acknowkdgm~ents-We
REFERENCES B~uout, E., Et Area, M., Vety Rmrscllorex, J., Roctr~r, H. and Gruxme, C. (1986) Immunochemistry of scorpion a-toxins: study with synthetic peptides of the antigenicity of four regions of toxin II of Andrcetonees austrerlts Hector. Mokc. Irrrrem. 23, 357-366. Bwweeotn, E., Ptcx~ox, J., Mrn.~e, J. M., D~neox, H., Er . Ar®, M., Gam, C., MwrlVtrnr, J. and RocFUr, H. (1988) Monoclonal antibodies to scorpion toxins. Characterization and molecular mechanisms of neutralization. J. Imnrutt. 141, 214-220. Hertza+stlr, J. A. (1985) Intrinsic and extrinsic factors in protein antigenic structure. Science 229, 932940 . Hout.tnr, J. C. and Mr~z, A. (1982) Neurotoxin-spadfic immunoglobulins acceh:rate dissociation of the neurotoxin-acetylcholine receptor wmplex. Science 217, 732-733. Ce~r.r., W. A. (1977) Membrane potential dependant binding of scorpion toxins to the action potential Na+ ionophore. J. biol. Cherr. 252, 8660,8668 . Et. AYIT9, M ., M~nrtx, M. F., Dra.oat, P., Beaus, G. and Roctu+r, H. (1983) Immunochemistry of scorpion a-neurotoxins. Determination of the antigenic site number and isolation of a highly enriched antibody specific to a single antigenic site of toxin II of Mdroctorrus australis Hector. Mokc. Ieeerreun. 20, 697-708. E~ Ar®, M., B~uorn, E., G~x~e, C. and Rocrrwr, H. (1986) Use of antibodies to defined regions of scorpion a-toxin to study its interaction with its receptor site on the sodium channel. Blocherristry 2S, 6671678. Gersex, M. H., BARTSLING, S. J. and MEr.oex, R. H. (1984) Use of peptide synthesis to probe viral antigens for epitope to a resolution of single amino acid . Proc. natee. Aced. Sci. U.S.A . 82, 178-182. Gersex, M. H., RODDA, S. J., Mesox, T. J., TRtearctc, G. and Scr~oops, P. G. (1987) Strategies for epitope analysis using peptide synthesis. J. Imrrror . Methods 102, 259-274. Gw~rr~e, C., Novorxr, J., Foxreca.t .~-C~s, J. C., Foun~r, P., Er. Avee, M. and Bnrnuorn, E. (1989) The antigenic structure of a scorpion toxin. Mokc . Irrrreoe. lb, 503-513. Hoerxic~r, P. D., Jx, Lerrorox, B. C., Z,rr~uvo, J:W. and Tner, J. P. (1989) Identification of immunodominant regions of transforming growth factor a. Implications for structure and function. J. biol. Chem . 264, 19,086-19,091 . IACR90N, D. C., Porn®ountos, P, and Wrm~e, D. O. (1988) Simultaneous binding of two monoclonal antibodies to epitopes separated in sequence by only three residues. Molec. Irr»run. 25, 465-471 . Jovea, E., M~rertx-Mouror, N., Cor~rm, F. and Rocx~r, H. (1978) Scorpion toxin: specific binding to rat synaptosomes . Biochem. biophys. Res. Commeoe. 85, 377-382. K~se, R., Krrt c~we, H., I3~rwstu, K., Tt xoue, K. and Ix~aext, F. (1989) Neutralizing monoclonal antibody specific for a-bungarotoxin : preparation, characterization of the antibody and localization of antigenic rogion of a-bungarotoxin. FEBS Lctt. 254, 106-110. Ltvea, W. G., Atx, G. M., Weesx~e, R. G. and S~rx-Gu.r., S. J. (1990) Epitopes on proteins : misconceptions and ttalitiea. Cell 61, 553-556. M~rtmv, M:F. and Roctr~r, H. (1986) Large scale purification of toxins from We venom of the scorpion Atulroctoreus australis Hector. Toxicon 24, 1131-1139. MrRwrroe, F., Korer~uv, C., Rocxwr, H. and Lisstrzw, 3. (1970) Purification of animal neurotoxina: isolation and characterization of eleven neurotoxins from the venons of the scorpions Aredroctoeeees mvtralis Hector, Buthus occittmus tunetmrees and Leiurus quinquestriatus quietquestriatus . Eur. J. Blocherr. 16, 514-519. Portrete, R. R (1958) Separation and isolation of fractions of rabbit gamma globulin containing the antibody and antigenic sites. Nateoe 182, 67071. Rertrrex, D. A. and UxneRwooo, P. A. (1986) Identification of antigenic determinants on insulin molecule recognized by monoclonal antibodies. Mokc. Irnnaere . 23, 44150. Roc~rwr, H., Rocxer, C., Simmer, F. and M~xne, F. (1972) The amino acid sequence of neurotoxin II of Androctortus australis hector . Eur. J. Biocherr . 28, 381-388. ROCEUr, H., Tax, M., M~u~ne, F. and Lissrtzter, S. (1977) Radioiodination of scorpion and snake neurotoxins . Arurlyt. Biocherr . 85, 532-538.
,BTOX445P 00 000008559 0112 00008
