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Critical residues of epitopes recognized by several anti-p53 monoclonal antibodies correspond to key residues of p53 involved in interactions with the mdm2 protein

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Jean-Michel Portefaix a , *, Sabine Thebault a , Florence Bourgain-Guglielmetti b , Maguy Del Rio a , Claude Granier a , Jean-Claude Mani a , Isabelle Navarro-Teulon a , Michel Nicolas a , Thierry Soussi b , Bernard Pau a ˆ Recherche, Rue de la Croix Verte, 34298 Montpellier Cedex 5, France CNRS UMR9921, CRLC Val d’ Aurelle /Bat b CNRS UMR218, Institut Curie, Pavillon Trouillet-Rossignol, 26 Rue d’ Ulm, 75231 Paris Cedex 5, France

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Received 7 April 1999; received in revised form 16 September 1999; accepted 18 October 1999

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Abstract

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The aim of this work was to study the reactivity of antibodies directed against the N-terminus of p53 protein. First, we analysed the cross-reactivity of anti-p53 antibodies from human, mouse and rabbit sera with peptides derived from human, mouse and Xenopus p53. Next, we characterized more precisely a series of monoclonal antibodies directed against the N-terminal part of p53 and produced by immunizing mice with either full length human or Xenopus p53. For each of these mAbs we localized the epitope recognized on human p53 by the Spot method of multiple peptide synthesis, defined critical residues on p53 involved in the interaction by alanine scanning replacement experiments and determined kinetic parameters using real-time interaction analysis. These antibodies could be divided into two groups according to their epitopic and kinetic characteristics and their cross-reactivity with murine p53. Our results indicate that critical residues involved in the interaction of some of these mAbs with p53 correspond to key residues on p53 involved in its interaction with the mdm2 protein. These antibodies could, therefore, represent powerful tools for the study of p53 regulation.  2000 Elsevier Science B.V. All rights reserved.

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Keywords: p53; mdm2; Monoclonal antibody; Spot; Surface plasmon resonance

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1. Introduction

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The p53 protein plays a crucial role in the cellular response to DNA damage by activating either apo-

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*Corresponding author. Tel.: 133-4-67-613745; fax: 133-467-613041. E-mail address: [email protected] (J.-M. Portefaix).

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ptotic or growth arrest pathways in proliferating cells (Hartwell and Kastan, 1994). Among the various biochemical activities exerted by the p53 protein, one main function seems to be its ability to activate transcription of genes containing a p53 response element. The transcription domain is localized in the amino-terminal part of the protein (residues 1–42), whereas the DNA binding domain is localized in the central region (residues 90–290). The carboxy-termi-

0022-1759 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0022-1759( 00 )00246-5

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peptide libraries or recombinant fragments of p53 protein or by Pepscan analysis (Legros et al., 1994a; Wade-Evans and Jenkins, 1985; Stephen and Lane, 1992; Stephen et al., 1995). Anti-p53 antibodies are detected in the serum of patients with different types of cancer (Caron de Fromentel et al., 1987; Crawford et al., 1982; Soussi, 1996). They have been shown to be associated with the accumulation of mutant p53 in tumor cells. Epitopes recognized by these human antibodies are very similar to those recognized by antibodies from immunized animals, i.e. they are located in the amino- and carboxy-terminal regions (Schlichtholz et al., 1992; Schlichtholz et al., 1994; Lubin et al., 1993). Two important conclusions may be drawn from these findings: (i) the similarity of the immune responses of both cancer patients and hyperimmunized animals suggests that accumulation of the p53 protein in tumor cells drives the patients’ immune response; (ii) the amino-terminal region (residues 1 to roughly 95) and the carboxy-terminal region (residues 300–393) of p53 are highly exposed and accessible on the protein surface, whereas the central region seems to be buried in the interior of the molecule. All these observations have been performed with human p53 (hp53). Recently, we have produced a new panel of mAbs directed against the Xenopus laevis p53 (Xp53) (Bessard et al., 1998). Preliminary mapping of the epitopes recognized by these mAbs indicated that they also recognize determinants localized in the amino- and carboxy-terminus of the hp53 protein. In the present report, we have performed a detailed study of the epitopes recognized by mAbs produced against either hp53 or Xp53. We used the recently described Spot method of multiple peptide synthesis (Frank, 1992; Molina et al., 1996) to prepare immobilized peptides which can be easily tested for reactivity with mAbs. Here, we demonstrate that: (i) the immunodominant epitopes in the amino-terminus of p53 are conserved in various species; (ii) all these epitopes are linear sequences which are 4–6 residues long; and (iii) amino acid residues Phe-19 and Trp-23 which are key residues in the binding of p53 to mdm2 (Kussie et al., 1996) are also essential residues of the epitopes recognized by several mAbs.

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nal part of p53 is involved in the negative regulation of its DNA binding activity. It also contains the tetramerization domain of the protein (Soussi and May, 1996). Molecular alteration of the p53 gene is found in most tumor types. Missense mutations occur predominantly non-randomly within the central region of the protein and inactivate its DNA binding properties (Beroud and Soussi, 1997). Metabolism of wild-type p53 protein is characterized by a rapid turnover, and the actual level present in the nuclei of normal cells is below the sensitivity of immunohistochemical detection methods. In tumor cells, there is an accumulation of nuclear, inactive, p53 protein. Several analyses have shown that there is a close correlation between accumulation of the p53 protein and mutation of the p53 gene (Lane, 1994). Recent studies suggest that such an accumulation is due to a lack of degradation by the mdm2 protein, which is normally involved in the catabolism of p53 (Haupt et al., 1997; Kubbutat et al., 1997; Midgley and Lane, 1997). Monoclonal antibodies (mAbs) directed against the p53 protein have been invaluable tools for both clinical and basic research. In clinical laboratories, the use of various p53 mAbs has led to an extensive series of immunohistochemical analyses for the rapid identification of p53 alteration (Dowell et al., 1994). In the area of basic research research, these mAbs have permitted detailed studies of the various conformations of the p53 protein (Milner, 1984; Gannon et al., 1990; Legros et al., 1994b). The production of several panels of mAbs directed against human p53 has led to the various observations. Firstly, p53 is clearly a highly immunogenic protein. Secondly, more than 95% of the various mAbs recognize epitopes which are localized in the amino, or (to a lesser extent) in the carboxy, terminus of the protein (Legros et al., 1994a; Bartek et al., 1993). The only way to obtain mAbs towards the central region of the p53 protein is through the immunization of mice with a truncated form of p53, lacking both extremities (Legros et al., 1994b; Vojtesek et al., 1995). Thirdly, out of over 100 anti-p53 mAbs described in the literature, only two (PAb1620 and PAb246) recognize non-sequential epitopes, which have not yet been mapped. The binding sites of all the other mAbs were identified by the use of phage-displayed

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The production of monoclonal antibodies has been described previously: anti-Xenopus p53 mAbs: X18, X44, X61, X73, X77, X87 and X91 (Bessard et al., 1998); anti-human p53 mAbs: H279, H447 and H461 (Legros et al., 1993, B17 and C36 (Legros et al., 1994a), DO7 (Vojtesek et al., 1992), Pab 1801 (Banks et al., 1986). Xenopus p53 mAbs were used as culture supernatants. All the other mAbs were purified antibodies.

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2.2. Pepscan ELISA

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The Pepscan ELISA was performed as previously described (Legros et al., 1994a). Peptide 1 sequences correspond to the first 15 amino acids of either human, murine or Xenopus p53 sequences. Peptides 2–6 are 15-mer overlapping peptides frameshifted by five residues. All these peptides were biotinylated on their N-terminus. They were synthesized by Cambridge Research Biochemicals (UK). These sequences are as follows: peptides 1 MEEPQSDPSVEPPLS (hp53), MEESQSDISLELPL (mp53), MEPSSETGMDPPLS (Xp53); peptides 2 SDPSVEPPLSQETF (hp53), SDISLELPLSQETFS (mp53), ETGMDPPLSQETFE (Xp53); peptides 3, EPPLSQETFSDLWK (hp53), ELPLSQETFSGLWK (mp53), PPLSQETFEDLWSL (Xp53); peptides 4 QETFSDLWKLLPEN (hp53), QETFSGLWKLLPPE (mp53), ETFEDLWSLLPDPL (Xp53); peptides 5 DLWKLLPENNVLSP (hp53), GLWKLLPPEDILPS (mp53), LWSLLPDPLQTVTCR (Xp53); peptides 6 LPENNVLSPLPSQA (hp53), LPPEDILPSPHCMD (mp53), PDPLQTVTCRLDNL (Xp53).

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2.3. Peptide synthesis on cellulose membrane using Spot technology

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All the following peptides were synthesized on a cellulose membrane according to the protocol previously described (Molina et al., 1996): 51 10-mer peptides frameshifted by one residue and covering the first 60 residues of the human p53 sequence, all the peptides from 3 to 10 residues long from the sequence TFSDLWKLLP and PDDIEQWFT and all

2.4. Immunoassay on immobilized peptides

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This immunoassay has been described elsewhere (Molina et al., 1996). The intensities of the blue precipitate was quantified after scanning the membrane using NIH image software. Membranes were then washed with dimethylformamide, 6 M urea and 10% (v / v) acetic acid in ethanol in order to eliminate the precipitated substrate and bound antibodies. Membranes could then be reused several times.

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2.1. Monoclonal antibodies

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2.5. Synthesis of soluble peptides

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the alanine analogs of the latter sequences. The phosphorylated peptide TFS ( P ) DLWKLLP was synthesized by incorporating phosphorylated Fmocserine. Derivatized membranes were from Abimed (Langenfeld, Germany). Fmoc-amino acids were from Novabiochem.

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Peptides 96009 and 96011 were synthesized using the Abimed AMS 422 synthesizer and the Fmoc coupling strategy. Peptides were deprotected and cleaved from the resin by trifluoroacetic acid treatment. Peptides were lyophilized and purified to greater than 90% homogeneity by HPLC. Peptide 96009P was obtained from Neosystem (Strasbourg, France) and peptide A from Cambridge Research Biochemicals (UK). A spacer sequence (GSGS) and a biotin residue were added to the N-terminus of each peptide.

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2.6. Real-time analysis of peptide-antibody interaction by BIAcore 

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The BIAcore apparatus was from BIAcore (Uppsala, Sweden). The following N-terminally biotinylated peptides (1 mg ml 21 in Hepes-buffered saline) were immobilized at a flow-rate of 5 ml min 21 on a streptavidin-coated sensor chip. Peptide A is an N-terminal biotinylated peptide whose sequence corresponds to the first 60 amino acids of hp53; peptide 96009, biot-(SGSG)15 SQETFSDLWKLLPEN 29 ; peptide 96009P, biot(SGSG)- 15 SQETFS (p ) DLWKLLPEN 29 with S (p ) representing an O-phosphorylated serine; peptide

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2. Materials and methods

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3.1. Cross-reactivity of anti-p53 antibodies from human, mouse and rabbit sera with peptides derived from human, mouse and Xenopus p53

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During the course of our work on anti-p53 mAbs, we performed an extensive cross-reactivity study of the different mAbs raised against different p53 proteins. We observed that a series of anti-hp53 mAbs cross-reacted with Xenopus p53 (Xp53) but not with mouse p53 (mp53). The converse situation was also true; namely, several anti-Xp53 mAbs cross-reacted with hp53 but not with mp53. All these mAbs recognized epitopes localized at the aminoterminus of p53. Such observations are in accordance with the recent epitope analysis of the mp53 tumor suppressor protein (Lane et al., 1996). No mAbs produced against murine p53 cross-reacted with the amino terminus of either hp53 or Xp53, despite a significant sequence homology between these proteins. To verify that this was not due to a bias in mAb selection, we analyzed polyclonal sera: a rabbit serum raised against hp53 (CM1), a rabbit serum raised against Xp53 (S11), a serum from a mouse bearing an SV40 induced tumor (S1) and also a serum from a patient with lung cancer and producing autoantibodies to p53 (LC37). All these sera were tested against a panel of overlapping peptides corresponding to the amino termini of human, mouse and Xenopus p53 (Fig. 1). As expected, CM1 recognized all the peptides from the amino-terminus of human

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3. Results

3.2. Fine epitope specificity analysis of anti-p53 monoclonal antibodies

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p53 as previously demonstrated (Legros et al., 1994a). It also bound to mp53- and Xp53-derived peptides 3 and 4. Serum LC37 recognized peptides 4 derived from both hp53 and Xp53 but exhibited no cross-reaction with any peptides from the mouse p53 sequence. Similarly, S11 recognized two peptides from Xp53 and from the human p53 protein but did not cross-react with any peptide from the mouse sequence. Finally, mouse serum S1 recognized mainly peptides from the homologous sequence, and showed a slight reactivity with peptides from hp53 and no reactivity with Xenopus peptides. The set of observations described above prompted us to undertake a fine epitope analysis using synthetic peptides derived from the sequence of human, mouse and Xenopus p53.

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96011, biot-(SGSG)- 38 DDLMLSPDDIEQWFT 55 . Injection of mAbs (culture supernatant diluted 10fold, or purified mAb at 50 mg ml 21 in HBS buffer) on immobilized peptides was performed at 50 ml min 21 and 258C. Dissociation was then monitored in running buffer at a flow-rate of 50 ml min 21 . The dissociation rate k off was determined from a plot of ln(R 0 /R) vs. time, where R is the surface plasmon resonance signal at time t, using BIAevaluation 3.0 software. The half-life of the complex (t 1 / 2 ), time required to remove half of the bound mAb from the peptide, was calculated using the formula t 1 / 2 5 ln(2) /k off .This value is independent of the initial concentration of the complex.

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All our previous studies involving the Pepscan ELISA for mapping the epitopes of the various anti-p53 mAbs used 15-mer peptides frameshifted by 5 residues (Gannon et al., 1990). For more precise mapping, we developed three approaches based on the Spot technology: (i) synthesis of 10-mer peptides frameshifted by one residue covering the first 60 residues of the p53 protein in order to identify the common sequence in reactive peptides; (ii) synthesis of a series of shortened peptides in order to define the minimal sequence recognized by a given mAb; (iii) synthesis of alanine-substituted peptides of the consensus binding site sequence to define the key residues involved in the interaction.

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3.3. Localization of the epitopes on the human p53 sequence

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The amino-terminal region of the p53 protein that had been shown previously to enclose continuous epitopes was further analyzed. Overlapping decapeptides frameshifted by one residue corresponding to the first 60 amino acids of hp53 were synthesized and their immunological reactivity with 14 monoclonal antibodies assessed [Fig. 2(a) and Table 1]. All tested mAbs reacted with one or more synthetic

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Fig. 1. Pepscan ELISA of various sera with p53 specific antibodies. The six peptides used in this experiment correspond to overlapping peptides from the amino-terminus of various p53 proteins (see Section 2): human, black square; murine, hatched square; Xenopus, empty square. CM1 is a rabbit serum raised against human p53. LC37 is the serum of a human patient with lung cancer with p53 specific antibodies. S11 is a rabbit serum raised against Xenopus p53. S1 was obtained from a mouse bearing a tumor originating from mouse SV40 transformed cells.

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peptides. As previously demonstrated (Legros et al., 1994a), two regions clearly appear to be immunodominant. The first one, encompassing residues 19– 26 from hp53, was recognized by mAbs derived from mice immunized with either Xp53 (mAbs X18, X44, X61, X73, X77, X87, X91) or hp53 (mAbs B17, C36, H461, DO7). The second immunodominant region was localized between residues 47– 54 of hp53; it was recognized by three of the monoclonal antibodies tested (mAbs H279, H447 and PAb 1801). This region was specific for hp53 as none of these mAbs cross-reacted with either mp53 or Xp53. By identifying for each mAb the sequence shared by reactive peptides, it was possible to improve the information obtained from the previous analysis using 15-mer peptides frameshifted by 5 residues (Legros et al., 1994a) Table 1 summarises the results and shows that there is a very good correlation between these results and those from the previous study.

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3.4. Determination of the minimum reactive peptide

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In an attempt to verify that the sequence of the epitopes deduced from the analysis of the reactivity of overlapping peptides did correspond to reactive peptides, all the peptides from 3- to 10-residues long included in the sequence of the 10-mer reactive peptide were synthesized. In all cases but three (mAbs DO7, C36, Pab1801), we observed that the size of the minimum reactive peptide defined in this way corresponded to the epitope sequence that had been deduced from the previous analysis (Table 1). These results show that all the mAbs tested recognized linear peptide sequences of 5–8 residues.

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3.5. Determination of critical residues of epitopes by alanine replacement analysis

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The contribution to antibody binding of each

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Fig. 2. Epitope analysis of H447 mAb. (a) Epitope localization using overlapping decapeptides; the epitope determined (DIEQWF) is the common sequence of immunoreactive peptides 15, 16, 17, 18 and 19. (b) Determination of residues contributing to the binding of H447 mAb by alanine scanning. Each residue of the immunoreactive peptide PDDIEQWFTE (control) was successively replaced by alanine. Boxed letters correspond to critical residues based on the observation that their substitution by alanine induced a loss of mAb binding greater than 80%.

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amino acid in the consensus binding sequence was assessed by preparing a series of alanine analogues of each sequence. A residue was defined as critical for binding if its replacement by an alanine residue induced a decrease in the signal greater than 80% of the value observed with the unmodified peptide epitope [Figs. 2(b) and 3]. For the first antigenic region, replacements were performed on peptide TFSDLWKLLP. For mAbs

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raised against Xenopus p53 (mAbs X18, X44, X61, X73, X77, X87, X91), the critical residues were Phe-19 and Trp-23. For some of these mAbs (X44, X61, X87, X91), a significant loss of signal (40– 60% of the signal obtained with control peptide) was also observed when Asp-21, Leu-22, Leu-26 and Pro-27 were replaced by alanine, suggesting that these residues also contribute to antibody recognition. The same group of mAbs also exhibited lower

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Table 1 Identification and characterization of epitopes recognized by a panel of anti-p53 mAbs (peptide sequences refer to the human p53 sequence unless otherwise stated)

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binding on the phosphorylated peptide TFS ( P ) DLWKLLP. Therefore, on the basis of these results, Xp53 mAbs could be separated into two groups, according to their reactivity with human p53 peptides. In contrast, for each of the three anti-hp53 mAbs tested, the epitopic specificity as well as the key residues were different. mAb B17 required three residues for its interaction with the p53 peptide, Phe-19, Leu-22, Trp-23; mAb H461 was dependent on five amino acids (Phe-19, Asp-21, Leu-22, Trp-23 and Leu-26) for its binding whereas mAb C36 required only Leu-22 (Table 1 and Fig. 3). In this latter case it is possible that leucine alone determines

Pepscan analysis

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Minimum reactive peptide

Critical residues in a consensus peptide c

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FSDLWKLL 26 DLWK 24 21 DLWKLL 26

FSDLWKLL FSDLWK LWKLLP

TFSDLWKLLP TFSDLWKLLP TFSDLWKLLP

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FSDLWKLL 26

FSDLWKLL

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PAb1801 H279 H447

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DDIEQW 53 DIEQWF 53 48 DIEQWF 53 48

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TFSDLWKLLP

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From Legros et al. (1994a). This work. c Critical residues in bold characters.

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QETFSDLWKLLPENN 30 DLWKL 25 16 QETFSDLWKLLPENN 30 16 ETFEDLWSLLPDPLQ 30 (Xp53) 16 QETFSDLWKLLPENN 30 16 ETFEDLWSLLPDPLQ 30 (Xp53) 16 QETFSDLWKLLPENN 30 16 ETFEDLWSLLPDPLQ 30 (Xp53) 16 QETFSDLWKLLPENN 30 16 ETFEDLWSLLPDPLQ 30 (Xp53) 16 QETFSDLWKLLPENN 30 16 ETFEDLWSLLPDPLQ 30 (Xp53) 16 QETFSDLWKL 25 16 QETFSGLWKL 25 (mp53) 16 ETFEDLWSLL 26 (Xp53) 16 QETFSDLWKL 25 16 QETFSGLWKLLPPED 30 (mp53) 16 ETFEDLWSLL 25 (Xp53) 16 QETFSDLWKL 25 16 QETFSGLWKLLPPED 30 (mp53) 16 ETFEDLWSLL 25 (Xp53) 16 QETFSDLWKL 25 16 QETFSGLWKLLPPED 30 (mp53) 16 ETFEDLWSLL 25 (Xp53) 46 SPDDIEQWFT 55 46 SPDDIEQWFT 55 46 SPDDIEQWFT 55

H461 DO7 C36

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the binding to C36 and that a larger set of residues would have been identified by substitution with other amino acids. In the case of mAb DO7, used as control, the most crucial residues for binding were Asp-21 and Lys-24, as previously shown (Picksley et al., 1994). For the second immunodominant region, the reactivity of alanine analogues of peptide PDDIEQWF was investigated. The two mAbs H279 and H447 recognized the same epitope (DIEQWF) and required the same four residues (Ile-50, Glu-51, Trp-53 and Phe-54) for their interaction with p53 (Table 1). Pab 1801 recognized an epitope which is slightly shifted compared with the epitope of the two previ-

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Fig. 3. Identification by alanine scanning of key residues required for mAb binding. Each bar represents the color intensity of the substituted peptide as a percentage of the intensity obtained with a control peptide TFSDLWKLLP. The sequence of the phosphorylated peptide is TFS ( p ) DLWKLLPEN with S ( p ) representing an O-phosphorylated serine.

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ous mAbs and whose critical residues are Asp-48, Ile-50 and Trp-53. These results indicate that the anti-p53 mAbs interact on the p53 protein through a

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limited set of amino acid residues of which several are hydrophobic (Phe-49, Ile-50, Trp-53 and Phe-54) and two are negatively charged (Asp-48 and Glu-51).

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3.6. Assessment of the kinetic parameters of the interaction between mAbs and peptides from the N-terminus of hp53 using BIAcore technology

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In order to quantify the interaction of Xenopus p53 and human p53 mAbs with their cognate peptide epitopes, using BIAcore technology, we measured the off-rate value (k off ) of the binding reaction of these antibodies with Peptide A, an N-terminal biotinylated peptide whose sequence corresponds to the first 60 amino acids of hp53; peptide 96009, biot-(SGSG)- 15 SQETFSDLWKLLPEN 29 ; peptide 96009P, biot-(SGSG)- 15 SQETFS ( p) DLWKLLPEN 29 with S ( p ) representing an O-phosphorylated serine;

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peptide 96011, biot-(SGSG)38 DDLMLSPDDIEQWFT 55 . The values of the halflives of the antigen–antibody complexes were determined and are shown in Fig. 4. It is interesting to note that all the Xenopus mAbs had longer binding half-lives with the phosphorylated peptide 96009P than with the original peptide 96009. Two classes of anti-Xenopus p53 antibodies could also be defined according to their behavior with peptide A. One group corresponded to mAbs showing a very high half-life (mAbs X18, X73, and X77) and a second group (mAbs X44, X61, X87 and X91) having 4-fold lower half-lives. Interestingly, these two groups correspond to the two groups determined on the basis

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Fig. 4. Determination of the half-life (t 1 / 2 ) of the complex between various mAbs and biotinylated N-terminal peptides from hp53 as measured using BIAcore technology. The off-rate (k off ) of p53 mAbs from N-terminus peptides was measured. The value of the complex half-life is given by t 1 / 2 5 ln 2 /k off . Peptide A is an N-terminal biotinylated peptide whose sequence corresponds to the first 60 amino acids of hp53; peptide 96009, biot-(SGSG)-15SQETFSDLWKLLPEN29; peptide 96009P, biot-(SGSG)-15SQETFS(p)DLWKLLPEN 29 with S(p) representing an O-phosphorylated serine; peptide 96011, biot-(SGSG)- 38 DDLMLSPDDIEQWFT 55 . HR231, an antibody directed against the C-terminus of p53, was used as a negative control.

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3.7. Recognition of peptides derived from murine and Xenopus p53

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Since the antibodies tested recognized regions highly conserved during evolution, it was of interest to determine whether the mAbs were able to recognize peptides derived from mouse and Xenopus p53 sequences. Using either Pepscan analysis or Spot technology, the antibodies could be divided into two classes (Table 1). The first group is composed of mAbs that recognized peptides from murine, Xenopus and human p53 (mAbs B17, X18, X73, X77). The second group is composed of mAbs C36, X61, X87, X44 and X91 which recognized peptides from the Xenopus and human p53 sequence but did not recognize peptides from the murine protein sequence even though no residues determined as crucial were substituted in the murine sequence. The common features of these antibodies are: (i) their lower binding half-lives; and (ii) their lower recognition of the phosphorylated peptide and peptides in which Leu-26 and Pro-27 had been replaced by an alanine residue.

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In this study, we characterized epitopes recognized by 14 mAbs directed against either the human or Xenopus p53 N-terminus. We confirmed that there are two immunodominant regions in this part of the protein. All epitopes could in fact be shortened to 5to 8-residue long linear sequences in which only a few residues are critical for antibody binding. In the case of the anti-hp53 mAb B17, for example, we defined the epitope recognized as 18 TFSDLW 23 where F, L and W were determined as critical. These results are in total agreement with those we obtained using a 15-mer peptide phage-displayed library (results not shown). Indeed, more than 50% of the peptides sequenced after panning on mAb B17 possessed Phe, Leu and Trp (unpubl. data). These results validate the method we describe here for the dissection of linear epitopes. N- and C-terminal regions from the p53 protein

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are very labile regions. However, a peptide corresponding to residues 15–29 of hp53 was cocrystallized with mdm2 by Kussie et al. (1996), who determined that this peptide adopts an amphipathic a helical conformation with all side chains of charged residues on one side of the helix and all side chains of hydrophobic residues clustered on the other side. Interestingly, we found that the critical residues for mAb DO7 recognition are only charged residues (Asp-21 and Lys-24), whereas the critical residues for recognition by Xenopus mAbs are only hydrophobic residues (Phe-19 and Trp-23), suggesting that the two faces of the a helix are antigenic. Despite the fact that the various immunodominant epitopes are highly conserved through evolution and are similar in both species, we noted some differences. We defined two groups of Xp53 mAbs according to their recognition pattern of hp53, Xp53 and mp53. The first group of mAbs (X44, X61, X87, and X91) cross-reacted with hp53 and Xp53 but did not recognize mp53. Substitution of the Asp-21 residue of hp53 by a Gly in mp53 may lead to destabilization of the a helix both by partial loss of its amphipathic nature and by elimination of the (Asp-21)–(Thr-18) hydrogen bond necessary for the initiation of the helix (Kussie et al., 1996). We suggest that this change in the conformation could also contribute to the weak immunogenicity of mp53 described by Lane et al., 1996. This group of mAbs was also characterized by its lower complex half-life with peptide A and a significant loss of recognition when Asp-21, Leu-22, Leu-26 and Pro-27 were replaced by an alanine residue, indicating that these residues also contribute to antibody recognition. The same group of mAbs also exhibited a lower binding to the phosphorylated peptide 18 TFS ( p) DLWKLLP 27 in the Spot assay. The second group recognized hp53, Xp53, mp53 and is composed of mAbs X18, X73, X77. This group is characterised by its shorter minimum reactive peptide size, since it is composed of FSDLW. We presume that in the case of this shorter epitope, the disruption of the a helix is not sufficient to abolish the recognition of mp53. Strikingly, amino acids residues Phe-19, Trp-23, Trp-53, Phe-54, which are key residues for p53 transactivation functions, are also essential residues of the epitopes of several anti-p53 mAbs. The N-

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We are grateful to Dr. S.L. Salhi for critical reading of the manuscript. This work was supported by a grant from the Association pour la Recherche sur le Cancer. JMP was supported by a doctoral fellowship from the Association pour la Recherche ´ sur le Cancer and the Comite´ de l’Herault de la Ligue Nationale Contre le Cancer.

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References

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activity of the two N-terminal activation domains of p53.

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Banks, L., Matlashewski, G., Crawford, L., 1986. Isolation of human-p53-specific monoclonal antibodies and their use in the studies of human p53 expression. Eur. J. Biochem. 159, 529. Bartek, J., Bartkova, J., Lukas, J., Staskova, Z., Vojtesek, B., Lane, D.P., 1993. Immunohistochemical analysis of the p53 oncoprotein on paraffin sections using a series of novel monoclonal antibodies. J. Pathol. 169, 27. Beroud, C., Soussi, T., 1997. p53 and APC gene mutations: software and databases. Nucleic Acids Res. 25, 138. Bessard, A.C., Garay, E., Lacronique, V., Legros, Y., Demarquay, C., Houque, A., Portefaix, J.M., Granier, C., Soussi, T., 1998. Regulation of the specific DNA binding activity of Xenopus laevis p53: evidence for conserved regulation through the carboxy-terminus of the protein. Oncogene 16, 883. Candau, R., Scolnick, D.M., Darpino, P., Ying, C.Y., Halazonetis, T.D., Berger, S.L., 1997. Two tandem and independent subactivation domains in the amino terminus of p53 require the adaptor complex for activity. Oncogene 15, 807. Caron de Fromentel, C., May-Levin, F., Mouriesse, H., Lemerle, J., Chandrasekaran, K., May, P., 1987. Presence of circulating antibodies against cellular protein p53 in a notable proportion of children with B-cell lymphoma. Int. J. Cancer 39, 185. Crawford, L.V., Pim, D.C., Bulbrook, R.D., 1982. Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer. Int. J. Cancer 30, 403. Dowell, S.P., Wilson, P.O., Derias, N.W., Lane, D.P., Hall, P.A., 1994. Clinical utility of the immunocytochemical detection of p53 protein in cytological specimens. Cancer Res. 54, 2914. Ducancel, F., Merienne, K., Fromen-Romano, C., Tremeau, O., Pillet, L., Drevet, P., Zinn-Justin, S., Boulain, J.C., Menez, A., 1996. Mimicry between receptors and antibodies. Identification of snake toxin determinants recognized by the acetylcholine receptor and an acetylcholine receptor-mimicking monoclonal antibody. J. Biol. Chem. 271, 31345. Frank, R., 1992. Spot synthesis: an easy technique for positionally addressable, parallel chemical synthesis on membrane support. Tetrahedron 46, 9217.

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terminal part of p53 contains the transactivation domain of the p53 and therefore must be accessible in order to interact with proteins involved in the transcription machinery such as TBP, TAF40 and TAF60 (Thut et al., 1995). Indeed, mutation at residues Leu-22 and Trp-23 leads to a total loss of the binding of numerous proteins which downregulate p53 function such as E1b or mdm-2 (Lin et al., 1994). It should be stressed that mdm2 requires the same residues (Phe-19, Trp-23 and Leu-26) for its interaction with p53 as do mAbs X44, X61, X87 and X91. Recently it has been reported that a new activation domain maps between amino acids 40–83 (Candau et al., 1997). Residues Trp-53 and Phe-54 were defined as critical for function both in yeast and mammalian cells. These same amino acids were also found to be critical for the binding of mAbs H279 and H447. This information indicates that some of our mAbs may interfere with the same residues on p53 that are used by many transcription and regulation factors to bind to p53. Given the similarities in the site of interaction of p53 with mdm2 and with the group of mAbs X44, X61, X87 and X91, we hypothesize that the complementary determining regions of these mAbs and the interacting region of mdm2 may also present a certain degree of homology. Such mimicry between an antibody and a protein has already been described (Ducancel et al., 1996). The third complementary determining region of the heavy chain of F12, a human monoclonal antibody derived from a pemphigus patient and directed against desmosomal and hemidesmosomal plaques, was shown to share a four-amino-acid sequence (GSSG) with the intracellular domains of desmoglein 1 and bullous pemphigoid antigen. Furthermore, a peptide from the VH-CDR3 containing the GSSG motif was able to inhibit the binding of mAb F12 to the target antigens (Gilbert et al., 1997). Similarly, in our case, it would be worth trying to localize a peptide sequence homologous to mdm2 in the variable regions of one of our anti-p53 mAbs and to verify its capacity to bind p53 as well as to inhibit the binding of mdm2. Consequently, we suggest that the anti-p53 mAbs described here represent potentially powerful tools for studying the function and regulation of the p53 protein. It would be of great interest to use these antibodies to block or modify independently the

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cells is not determined by mutation but is dependent on Mdm2 binding. Oncogene 15, 1179. Milner, J., 1984. Different forms of p53 detected by monoclonal antibodies in non-dividing and dividing lymphocytes. Nature 310, 143. Molina, F., Laune, D., Gougat, C., Pau, B., Granier, C., 1996. Improved performances of spot multiple peptide synthesis. Pept. Res. 9, 151. Picksley, S.M., Vojtesek, B., Sparks, A., Lane, D.P., 1994. Immunochemical analysis of the interaction of p53 with MDM2; fine mapping of the MDM2 binding site on p53 using synthetic peptides. Oncogene 9, 2523. Schlichtholz, B., Legros, Y., Gillet, D., Gaillard, C., Marty, M., Lane, D., Calvo, F., Soussi, T., 1992. The immune response to p53 in breast cancer patients is directed against immunodominant epitopes unrelated to the mutational hot spot. Cancer Res. 52, 6380. Schlichtholz, B., Tredaniel, J., Lubin, R., Zalcman, G., Hirsch, A., Soussi, T., 1994. Analyses of p53 antibodies in sera of patients with lung carcinoma define immunodominant regions in the p53 protein. Br. J. Cancer 69, 809. Soussi, T., 1996. The humoral response to the tumor-suppressor gene-product p53 in human cancer: implications for diagnosis and therapy. Immunol. Today 17, 354. Soussi, T., May, P., 1996. Structural aspects of the p53 protein in relation to gene evolution: a second look. J. Mol. Biol. 260, 623. Stephen, C.W., Helminen, P., Lane, D.P., 1995. Characterisation of epitopes on human p53 using phage-displayed peptide libraries: insights into antibody-peptide interactions. J. Mol. Biol. 248, 58. Stephen, C.W., Lane, D.P., 1992. Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library. J. Mol. Biol. 225, 577. Thut, C.J., Chen, J.L., Klemm, R., Tjian, R., 1995. p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60. Science 267, 100. Vojtesek, B., Bartek, J., Midgley, C.A., Lane, D.P., 1992. An immunochemical analysis of the human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53. J. Immunol. Methods 151, 237. Vojtesek, B., Dolezalova, H., Lauerova, L., Svitakova, M., Havlis, P., Kovarik, J., Midgley, C.A., Lane, D.P., 1995. Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein. Oncogene 10, 389. Wade-Evans, A., Jenkins, J.R., 1985. Precise epitope mapping of the murine transformation-associated protein, p53. Embo. J. 4, 699.

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Gannon, J.V., Greaves, R., Iggo, R., Lane, D.P., 1990. Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. Embo. J. 9, 1595. Gilbert, D., Courville, F., Brard, F., Joly, P., Petit, S., Bernardi, E., Schoofs, A.R., Lauret, P., Tron, F., 1997. A complementarydetermining region peptide of antidesmosome autoantibody may interact with the desmosomal plaque through molecular mimicry with a cytoplasmic desmoglein 1 sequence. Eur. J. Immunol. 27, 1055. Hartwell, L.H., Kastan, M.B., 1994. Cell cycle control and cancer. Science 266, 1821. Haupt, Y., Maya, R., Kazaz, A., Oren, M., 1997. Mdm2 promotes the rapid degradation of p53. Nature 387, 296. Kubbutat, M.H., Jones, S.N., Vousden, K.H., 1997. Regulation of p53 stability by Mdm2. Nature 387, 299. Kussie, P.H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A.J., Pavletich, N.P., 1996. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 274, 948. Lane, D.P., 1994. On the expression of the p53 protein in human cancer. Mol. Biol. Rep. 19, 23. Lane, D.P., Stephen, C.W., Midgley, C.A., Sparks, A., Hupp, T.R., Daniels, D.A., Greaves, R., Reid, A., Vojtesek, B., Picksley, S.M., 1996. Epitope analysis of the murine p53 tumour suppressor protein. Oncogene 12, 2461. Legros, Y., Lacabanne, V., d’Agay, M.F., Larsen, C.J., Pla, M., Soussi, T., 1993. Production of human p53 specific monoclonal antibodies and their use in immunohistochemical studies of tumor cells. Bull. Cancer 80, 102. Legros, Y., Lafon, C., Soussi, T., 1994a. Linear antigenic sites defined by the B-cell response to human p53 are localized predominantly in the amino and carboxy-termini of the protein. Oncogene 9, 2071. Legros, Y., Meyer, A., Ory, K., Soussi, T., 1994b. Mutations in p53 produce a common conformational effect that can be detected with a panel of monoclonal antibodies directed toward the central part of the p53 protein. Oncogene 9, 3689. Lin, J., Chen, J., Elenbaas, B., Levine, A.J., 1994. Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein. Genes Dev. 8, 1235. Lubin, R., Schlichtholz, B., Bengoufa, D., Zalcman, G., Tredaniel, J., Hirsch, A., de Fromentel, C.C., Preudhomme, C., Fenaux, P., Fournier, G. et al., 1993. Analysis of p53 antibodies in patients with various cancers define B-cell epitopes of human p53: distribution on primary structure and exposure on protein surface. Cancer Res. 53, 5872. Midgley, C.A., Lane, D.P., 1997. p53 protein stability in tumour

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