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10 Journal of Immunological Methods 1 (2000) 000–0004 www.elsevier.nl / locate / jim

12 Critical residues of epitopes recognized by several anti-p5313 monoclonal antibodies correspond to key residues of p5314 involved in interactions with the mdm2 protein

a , a b*15 Jean-Michel Portefaix , Sabine Thebault , Florence Bourgain-Guglielmetti ,a a a a

16 Maguy Del Rio , Claude Granier , Jean-Claude Mani , Isabelle Navarro-Teulon ,a b a

17 Michel Nicolas , Thierry Soussi , Bernard Paua ˆ18 CNRS UMR9921, CRLC Val d’Aurelle /Bat Recherche, Rue de la Croix Verte, 34298 Montpellier Cedex 5, France

b19 CNRS UMR218, Institut Curie, Pavillon Trouillet-Rossignol, 26 Rue d’Ulm, 75231 Paris Cedex 5, France

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

21

22 Abstract

23 The aim of this work was to study the reactivity of antibodies directed against the N-terminus of p53 protein. First, we24 analysed the cross-reactivity of anti-p53 antibodies from human, mouse and rabbit sera with peptides derived from human,25 mouse and Xenopus p53. Next, we characterized more precisely a series of monoclonal antibodies directed against the26 N-terminal part of p53 and produced by immunizing mice with either full length human or Xenopus p53. For each of these27 mAbs we localized the epitope recognized on human p53 by the Spot method of multiple peptide synthesis, defined critical28 residues on p53 involved in the interaction by alanine scanning replacement experiments and determined kinetic parameters29 using real-time interaction analysis. These antibodies could be divided into two groups according to their epitopic and kinetic30 characteristics and their cross-reactivity with murine p53. Our results indicate that critical residues involved in the interaction31 of some of these mAbs with p53 correspond to key residues on p53 involved in its interaction with the mdm2 protein. These32 antibodies could, therefore, represent powerful tools for the study of p53 regulation. 2000 Elsevier Science B.V. All33 rights reserved.

34 Keywords: p53; mdm2; Monoclonal antibody; Spot; Surface plasmon resonance35

36 1. Introduction ptotic or growth arrest pathways in proliferating cells 39

(Hartwell and Kastan, 1994). Among the various 40

37 The p53 protein plays a crucial role in the cellular biochemical activities exerted by the p53 protein, 41

38 response to DNA damage by activating either apo- one main function seems to be its ability to activate 42

transcription of genes containing a p53 response 43

element. The transcription domain is localized in the 44

amino-terminal part of the protein (residues 1–42), 455 *Corresponding author. Tel.: 133-4-67-613745; fax: 133-4-whereas the DNA binding domain is localized in the 466 67-613041.

7 E-mail address: [email protected] (J.-M. Portefaix). central region (residues 90–290). The carboxy-termi- 47

1 0022-1759/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.2 PII : S0022-1759( 00 )00246-5

3 8660 DTD410 182JUL22000 17:04:39.46 v1.4.154

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144 2 J.-M. Portefaix et al. / Journal of Immunological Methods 1 (2000) 000 –000

48 nal part of p53 is involved in the negative regulation peptide libraries or recombinant fragments of p53 96

49 of its DNA binding activity. It also contains the protein or by Pepscan analysis (Legros et al., 1994a; 97

50 tetramerization domain of the protein (Soussi and Wade-Evans and Jenkins, 1985; Stephen and Lane, 98

51 May, 1996). 1992; Stephen et al., 1995). 99

52 Molecular alteration of the p53 gene is found in Anti-p53 antibodies are detected in the serum of 100

53 most tumor types. Missense mutations occur pre- patients with different types of cancer (Caron de 101

54 dominantly non-randomly within the central region Fromentel et al., 1987; Crawford et al., 1982; Soussi, 102

55 of the protein and inactivate its DNA binding 1996). They have been shown to be associated with 103

56 properties (Beroud and Soussi, 1997). Metabolism of the accumulation of mutant p53 in tumor cells. 104

57 wild-type p53 protein is characterized by a rapid Epitopes recognized by these human antibodies are 105

58 turnover, and the actual level present in the nuclei of very similar to those recognized by antibodies from 106

59 normal cells is below the sensitivity of immuno- immunized animals, i.e. they are located in the 107

60 histochemical detection methods. In tumor cells, amino- and carboxy-terminal regions (Schlichtholz et 108

61 there is an accumulation of nuclear, inactive, p53 al., 1992; Schlichtholz et al., 1994; Lubin et al., 109

62 protein. Several analyses have shown that there is a 1993). Two important conclusions may be drawn 110

63 close correlation between accumulation of the p53 from these findings: (i) the similarity of the immune 111

64 protein and mutation of the p53 gene (Lane, 1994). responses of both cancer patients and hyperimmun- 112

65 Recent studies suggest that such an accumulation is ized animals suggests that accumulation of the p53 113

66 due to a lack of degradation by the mdm2 protein, protein in tumor cells drives the patients’ immune 114

67 which is normally involved in the catabolism of p53 response; (ii) the amino-terminal region (residues 1 115

68 (Haupt et al., 1997; Kubbutat et al., 1997; Midgley to roughly 95) and the carboxy-terminal region 116

69 and Lane, 1997). (residues 300–393) of p53 are highly exposed and 117

70 Monoclonal antibodies (mAbs) directed against the accessible on the protein surface, whereas the central 118

71 p53 protein have been invaluable tools for both region seems to be buried in the interior of the 119

72 clinical and basic research. In clinical laboratories, molecule. 120

73 the use of various p53 mAbs has led to an extensive All these observations have been performed with 121

74 series of immunohistochemical analyses for the rapid human p53 (hp53). Recently, we have produced a 122

75 identification of p53 alteration (Dowell et al., 1994). new panel of mAbs directed against the Xenopus 123

76 In the area of basic research research, these mAbs laevis p53 (Xp53) (Bessard et al., 1998). Preliminary 124

77 have permitted detailed studies of the various con- mapping of the epitopes recognized by these mAbs 125

78 formations of the p53 protein (Milner, 1984; Gannon indicated that they also recognize determinants local- 126

79 et al., 1990; Legros et al., 1994b). The production of ized in the amino- and carboxy-terminus of the hp53 127

80 several panels of mAbs directed against human p53 protein. 128

81 has led to the various observations. Firstly, p53 is In the present report, we have performed a de- 129

82 clearly a highly immunogenic protein. Secondly, tailed study of the epitopes recognized by mAbs 130

83 more than 95% of the various mAbs recognize produced against either hp53 or Xp53. We used the 131

84 epitopes which are localized in the amino, or (to a recently described Spot method of multiple peptide 132

85 lesser extent) in the carboxy, terminus of the protein synthesis (Frank, 1992; Molina et al., 1996) to 133

86 (Legros et al., 1994a; Bartek et al., 1993). The only prepare immobilized peptides which can be easily 134

87 way to obtain mAbs towards the central region of the tested for reactivity with mAbs. Here, we demon- 135

88 p53 protein is through the immunization of mice strate that: (i) the immunodominant epitopes in the 136

89 with a truncated form of p53, lacking both ex- amino-terminus of p53 are conserved in various 137

90 tremities (Legros et al., 1994b; Vojtesek et al., 1995). species; (ii) all these epitopes are linear sequences 138

91 Thirdly, out of over 100 anti-p53 mAbs described in which are 4–6 residues long; and (iii) amino acid 139

92 the literature, only two (PAb1620 and PAb246) residues Phe-19 and Trp-23 which are key residues 140

93 recognize non-sequential epitopes, which have not in the binding of p53 to mdm2 (Kussie et al., 1996) 141

94 yet been mapped. The binding sites of all the other are also essential residues of the epitopes recognized 142

95 mAbs were identified by the use of phage-displayed by several mAbs. 143

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228 J.-M. Portefaix et al. / Journal of Immunological Methods 1 (2000) 000 –000 3

145 2. Materials and methods the alanine analogs of the latter sequences. The 187(P)phosphorylated peptide TFS DLWKLLP was syn- 188

146 2.1. Monoclonal antibodies thesized by incorporating phosphorylated Fmoc- 189

serine. Derivatized membranes were from Abimed 190

147 The production of monoclonal antibodies has been (Langenfeld, Germany). Fmoc-amino acids were 191

148 described previously: anti-Xenopus p53 mAbs: X18, from Novabiochem. 192

149 X44, X61, X73, X77, X87 and X91 (Bessard et al.,150 1998); anti-human p53 mAbs: H279, H447 and 2.4. Immunoassay on immobilized peptides 193151 H461 (Legros et al., 1993, B17 and C36 (Legros et152 al., 1994a), DO7 (Vojtesek et al., 1992), Pab 1801 This immunoassay has been described elsewhere 194153 (Banks et al., 1986). Xenopus p53 mAbs were used (Molina et al., 1996). The intensities of the blue 195154 as culture supernatants. All the other mAbs were precipitate was quantified after scanning the mem- 196155 purified antibodies. brane using NIH image software. 197

Membranes were then washed with dimethylform- 198156 2.2. Pepscan ELISA amide, 6 M urea and 10% (v/v) acetic acid in 199

ethanol in order to eliminate the precipitated sub- 200157 The Pepscan ELISA was performed as previously strate and bound antibodies. Membranes could then 201158 described (Legros et al., 1994a). Peptide 1 sequences be reused several times. 202159 correspond to the first 15 amino acids of either160 human, murine or Xenopus p53 sequences. Peptides

2.5. Synthesis of soluble peptides 203161 2–6 are 15-mer overlapping peptides frameshifted by162 five residues. All these peptides were biotinylated on

Peptides 96009 and 96011 were synthesized using 204163 their N-terminus. They were synthesized by Cam-the Abimed AMS 422 synthesizer and the Fmoc 205164 bridge Research Biochemicals (UK). These se-coupling strategy. Peptides were deprotected and 206165 quences are as follows: peptides 1cleaved from the resin by trifluoroacetic acid treat- 207166 MEEPQSDPSVEPPLS (hp53), MEESQSDISLELPLment. Peptides were lyophilized and purified to 208167 (mp53), MEPSSETGMDPPLS (Xp53); peptides 2greater than 90% homogeneity by HPLC. Peptide 209168 SDPSVEPPLSQETF (hp53), SDISLELPLSQETFS96009P was obtained from Neosystem (Strasbourg, 210169 (mp53), ETGMDPPLSQETFE (Xp53); peptides 3,France) and peptide A from Cambridge Research 211170 EPPLSQETFSDLWK (hp53), ELPLSQETFSGLWKBiochemicals (UK). A spacer sequence (GSGS) and 212171 (mp53), PPLSQETFEDLWSL (Xp53); peptides 4a biotin residue were added to the N-terminus of 213172 QETFSDLWKLLPEN (hp53), QETFSGLWKLLPPEeach peptide. 214173 (mp53), ETFEDLWSLLPDPL (Xp53); peptides 5

174 DLWKLLPENNVLSP (hp53), GLWKLLPPEDILPS2.6. Real-time analysis of peptide-antibody 215175 (mp53), LWSLLPDPLQTVTCR (Xp53); peptides 6

interaction by BIAcore 216176 LPENNVLSPLPSQA (hp53), LPPEDILPSPHCMD177 (mp53), PDPLQTVTCRLDNL (Xp53).

The BIAcore apparatus was from BIAcore (Upp- 217

sala, Sweden). The following N-terminally 218178 2.3. Peptide synthesis on cellulose membrane using21biotinylated peptides (1 mg ml in Hepes-buffered 219179 Spot technology

saline) were immobilized at a flow-rate of 5 22021

ml min on a streptavidin-coated sensor chip. Pep- 221180 All the following peptides were synthesized on atide A is an N-terminal biotinylated peptide whose 222181 cellulose membrane according to the protocol previ-sequence corresponds to the first 60 amino acids of 223182 ously described (Molina et al., 1996): 51 10-merhp53; peptide 96009, biot-(SGSG)- 224183 peptides frameshifted by one residue and covering15 29SQETFSDLWKLLPEN ; peptide 96009P, biot- 225184 the first 60 residues of the human p53 sequence, all

15 (p) 29 (p)185 the peptides from 3 to 10 residues long from the (SGSG)- SQETFS DLWKLLPEN with S rep- 226

186 sequence TFSDLWKLLP and PDDIEQWFT and all resenting an O-phosphorylated serine; peptide 227

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38 55229 96011, biot-(SGSG)- DDLMLSPDDIEQWFT . p53 as previously demonstrated (Legros et al., 273

230 Injection of mAbs (culture supernatant diluted 10- 1994a). It also bound to mp53- and Xp53-derived 27421

231 fold, or purified mAb at 50 mg ml in HBS buffer) peptides 3 and 4. Serum LC37 recognized peptides 4 275

232 on immobilized peptides was performed at 50 derived from both hp53 and Xp53 but exhibited no 27621

233 ml min and 258C. Dissociation was then monitored cross-reaction with any peptides from the mouse p53 27721

234 in running buffer at a flow-rate of 50 ml min . The sequence. Similarly, S11 recognized two peptides 278

235 dissociation rate k was determined from a plot of from Xp53 and from the human p53 protein but did 279off

236 ln(R /R) vs. time, where R is the surface plasmon not cross-react with any peptide from the mouse 2800

237 resonance signal at time t, using BIAevaluation 3.0 sequence. Finally, mouse serum S1 recognized main- 281

238 software. The half-life of the complex (t ), time ly peptides from the homologous sequence, and 2821 / 2

239 required to remove half of the bound mAb from the showed a slight reactivity with peptides from hp53 283

240 peptide, was calculated using the formula t 5 and no reactivity with Xenopus peptides. 2841 / 2

241 ln(2) /k .This value is independent of the initial The set of observations described above prompted 285off

242 concentration of the complex. us to undertake a fine epitope analysis using syn- 286

thetic peptides derived from the sequence of human, 287

mouse and Xenopus p53. 288

243 3. Results

244 3.1. Cross-reactivity of anti-p53 antibodies from 3.2. Fine epitope specificity analysis of anti-p53 289

245 human, mouse and rabbit sera with peptides monoclonal antibodies 290

246 derived from human, mouse and Xenopus p53All our previous studies involving the Pepscan 291

247 During the course of our work on anti-p53 mAbs, ELISA for mapping the epitopes of the various 292

248 we performed an extensive cross-reactivity study of anti-p53 mAbs used 15-mer peptides frameshifted by 293

249 the different mAbs raised against different p53 5 residues (Gannon et al., 1990). For more precise 294

250 proteins. We observed that a series of anti-hp53 mapping, we developed three approaches based on 295

251 mAbs cross-reacted with Xenopus p53 (Xp53) but the Spot technology: (i) synthesis of 10-mer peptides 296

252 not with mouse p53 (mp53). The converse situation frameshifted by one residue covering the first 60 297

253 was also true; namely, several anti-Xp53 mAbs residues of the p53 protein in order to identify the 298

254 cross-reacted with hp53 but not with mp53. All these common sequence in reactive peptides; (ii) synthesis 299

255 mAbs recognized epitopes localized at the amino- of a series of shortened peptides in order to define 300

256 terminus of p53. Such observations are in accordance the minimal sequence recognized by a given mAb; 301

257 with the recent epitope analysis of the mp53 tumor (iii) synthesis of alanine-substituted peptides of the 302

258 suppressor protein (Lane et al., 1996). No mAbs consensus binding site sequence to define the key 303

259 produced against murine p53 cross-reacted with the residues involved in the interaction. 304

260 amino terminus of either hp53 or Xp53, despite a261 significant sequence homology between these pro-262 teins. To verify that this was not due to a bias in 3.3. Localization of the epitopes on the human p53 305

263 mAb selection, we analyzed polyclonal sera: a rabbit sequence 306

264 serum raised against hp53 (CM1), a rabbit serum265 raised against Xp53 (S11), a serum from a mouse The amino-terminal region of the p53 protein that 307

266 bearing an SV40 induced tumor (S1) and also a had been shown previously to enclose continuous 308

267 serum from a patient with lung cancer and producing epitopes was further analyzed. Overlapping decapep- 309

268 autoantibodies to p53 (LC37). All these sera were tides frameshifted by one residue corresponding to 310

269 tested against a panel of overlapping peptides corre- the first 60 amino acids of hp53 were synthesized 311

270 sponding to the amino termini of human, mouse and and their immunological reactivity with 14 mono- 312

271 Xenopus p53 (Fig. 1). As expected, CM1 recognized clonal antibodies assessed [Fig. 2(a) and Table 1]. 313

272 all the peptides from the amino-terminus of human All tested mAbs reacted with one or more synthetic 314

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317

318 Fig. 1. Pepscan ELISA of various sera with p53 specific antibodies. The six peptides used in this experiment correspond to overlapping319 peptides from the amino-terminus of various p53 proteins (see Section 2): human, black square; murine, hatched square; Xenopus, empty320 square. CM1 is a rabbit serum raised against human p53. LC37 is the serum of a human patient with lung cancer with p53 specific321 antibodies. S11 is a rabbit serum raised against Xenopus p53. S1 was obtained from a mouse bearing a tumor originating from mouse SV40322 transformed cells.

323 peptides. As previously demonstrated (Legros et al., 3.4. Determination of the minimum reactive 343

324 1994a), two regions clearly appear to be immuno- peptide 344

325 dominant. The first one, encompassing residues 19–326 26 from hp53, was recognized by mAbs derived In an attempt to verify that the sequence of the 345

327 from mice immunized with either Xp53 (mAbs X18, epitopes deduced from the analysis of the reactivity 346

328 X44, X61, X73, X77, X87, X91) or hp53 (mAbs of overlapping peptides did correspond to reactive 347

329 B17, C36, H461, DO7). The second immuno- peptides, all the peptides from 3- to 10-residues long 348

330 dominant region was localized between residues 47– included in the sequence of the 10-mer reactive 349

331 54 of hp53; it was recognized by three of the peptide were synthesized. In all cases but three 350

332 monoclonal antibodies tested (mAbs H279, H447 (mAbs DO7, C36, Pab1801), we observed that the 351

333 and PAb 1801). This region was specific for hp53 as size of the minimum reactive peptide defined in this 352

334 none of these mAbs cross-reacted with either mp53 way corresponded to the epitope sequence that had 353

335 or Xp53. By identifying for each mAb the sequence been deduced from the previous analysis (Table 1). 354

336 shared by reactive peptides, it was possible to These results show that all the mAbs tested recog- 355

337 improve the information obtained from the previous nized linear peptide sequences of 5–8 residues. 356

338 analysis using 15-mer peptides frameshifted by 5339 residues (Legros et al., 1994a) 3.5. Determination of critical residues of epitopes 357

340 Table 1 summarises the results and shows that by alanine replacement analysis 358

341 there is a very good correlation between these results342 and those from the previous study. The contribution to antibody binding of each 359

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362

363 Fig. 2. Epitope analysis of H447 mAb. (a) Epitope localization using overlapping decapeptides; the epitope determined (DIEQWF) is the364 common sequence of immunoreactive peptides 15, 16, 17, 18 and 19. (b) Determination of residues contributing to the binding of H447365 mAb by alanine scanning. Each residue of the immunoreactive peptide PDDIEQWFTE (control) was successively replaced by alanine.366 Boxed letters correspond to critical residues based on the observation that their substitution by alanine induced a loss of mAb binding greater367 than 80%.

368 amino acid in the consensus binding sequence was raised against Xenopus p53 (mAbs X18, X44, X61, 377

369 assessed by preparing a series of alanine analogues X73, X77, X87, X91), the critical residues were 378

370 of each sequence. A residue was defined as critical Phe-19 and Trp-23. For some of these mAbs (X44, 379

371 for binding if its replacement by an alanine residue X61, X87, X91), a significant loss of signal (40– 380

372 induced a decrease in the signal greater than 80% of 60% of the signal obtained with control peptide) was 381

373 the value observed with the unmodified peptide also observed when Asp-21, Leu-22, Leu-26 and 382

374 epitope [Figs. 2(b) and 3]. Pro-27 were replaced by alanine, suggesting that 383

375 For the first antigenic region, replacements were these residues also contribute to antibody recogni- 384

376 performed on peptide TFSDLWKLLP. For mAbs tion. The same group of mAbs also exhibited lower 385

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387 Table 1388 Identification and characterization of epitopes recognized by a panel of anti-p53 mAbs (peptide sequences refer to the human p53 sequence389 unless otherwise stated)390

a391 mAb Pepscan analysis Spot analysis392

b393 Common sequence in Common sequence in Minimum reactive Critical residues in aa c394 reactive peptides reactive peptides peptide consensus peptide

39516 30 19 26396 H461 QETFSDLWKLLPENN FSDLWKLL FSDLWKLL TFSDLWKLLP21 25 21 24397 DO7 DLWKL DLWK FSDLWK TFSDLWKLLP16 30 21 26398 C36 QETFSDLWKLLPENN DLWKLL LWKLLP TFSDLWKLLP16 30399 ETFEDLWSLLPDPLQ (Xp53)16 30 19 26400 X44 QETFSDLWKLLPENN FSDLWKLL FSDLWKLL TFSDLWKLLP16 30401 ETFEDLWSLLPDPLQ (Xp53)16 30 19 26402 X61 QETFSDLWKLLPENN FSDLWKLL FSDLWKLL TFSDLWKLLP16 30403 ETFEDLWSLLPDPLQ (Xp53)16 30 19 26404 X91 QETFSDLWKLLPENN FSDLWKLL FSDLWKLL TFSDLWKLLP16 30405 ETFEDLWSLLPDPLQ (Xp53)16 30 19 26406 X87 QETFSDLWKLLPENN FSDLWKLL FSDLWKLL TFSDLWKLLP16 30407 ETFEDLWSLLPDPLQ (Xp53)16 25 19 23408 B17 QETFSDLWKL FSDLW FSDLW TFSDLWKLLP16 25409 QETFSGLWKL (mp53)16 26410 ETFEDLWSLL (Xp53)16 25 19 23411 X77 QETFSDLWKL FSDLW FSDLW TFSDLWKLLP16 30412 QETFSGLWKLLPPED (mp53)16 25413 ETFEDLWSLL (Xp53)16 25 19 25414 X18 QETFSDLWKL FSDLWKL FSDLW TFSDLWKLLP16 30415 QETFSGLWKLLPPED (mp53)16 25416 ETFEDLWSLL (Xp53)16 25 19 25417 X73 QETFSDLWKL FSDLWKL FSDLW TFSDLWKLLP16 30418 QETFSGLWKLLPPED (mp53)16 25419 ETFEDLWSLL (Xp53)46 55 48 53420 PAb1801 SPDDIEQWFT DDIEQW PDDIEQWF PDDIEQWFTE46 55 48 53421 H279 SPDDIEQWFT DIEQWF DIEQWF PDDIEQWFTE46 55 48 53422 H447 SPDDIEQWFT DIEQWF DIEQWF PDDIEQWFTE

423a424 From Legros et al. (1994a).b425 This work.c426 Critical residues in bold characters.

427 binding on the phosphorylated peptide the binding to C36 and that a larger set of residues 441(P)

428 TFS DLWKLLP. Therefore, on the basis of these would have been identified by substitution with other 442

429 results, Xp53 mAbs could be separated into two amino acids. In the case of mAb DO7, used as 443

430 groups, according to their reactivity with human p53 control, the most crucial residues for binding were 444

431 peptides. Asp-21 and Lys-24, as previously shown (Picksley et 445

432 In contrast, for each of the three anti-hp53 mAbs al., 1994). 446

433 tested, the epitopic specificity as well as the key For the second immunodominant region, the reac- 447

434 residues were different. mAb B17 required three tivity of alanine analogues of peptide PDDIEQWF 448

435 residues for its interaction with the p53 peptide, was investigated. The two mAbs H279 and H447 449

436 Phe-19, Leu-22, Trp-23; mAb H461 was dependent recognized the same epitope (DIEQWF) and re- 450

437 on five amino acids (Phe-19, Asp-21, Leu-22, Trp-23 quired the same four residues (Ile-50, Glu-51, Trp-53 451

438 and Leu-26) for its binding whereas mAb C36 and Phe-54) for their interaction with p53 (Table 1). 452

439 required only Leu-22 (Table 1 and Fig. 3). In this Pab 1801 recognized an epitope which is slightly 453

440 latter case it is possible that leucine alone determines shifted compared with the epitope of the two previ- 454

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457

458 Fig. 3. Identification by alanine scanning of key residues required for mAb binding. Each bar represents the color intensity of the substituted459 peptide as a percentage of the intensity obtained with a control peptide TFSDLWKLLP. The sequence of the phosphorylated peptide is

(p) (p)460 TFS DLWKLLPEN with S representing an O-phosphorylated serine.

461 ous mAbs and whose critical residues are Asp-48, limited set of amino acid residues of which several 464

462 Ile-50 and Trp-53. These results indicate that the are hydrophobic (Phe-49, Ile-50, Trp-53 and Phe-54) 465

463 anti-p53 mAbs interact on the p53 protein through a and two are negatively charged (Asp-48 and Glu-51). 466

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476 3.6. Assessment of the kinetic parameters of the peptide 96011, biot-(SGSG)- 48938 55

477 interaction between mAbs and peptides from the DDLMLSPDDIEQWFT . The values of the half- 490

478 N-terminus of hp53 using BIAcore technology lives of the antigen–antibody complexes were de- 491

termined and are shown in Fig. 4. It is interesting to 492

479 In order to quantify the interaction of Xenopus p53 note that all the Xenopus mAbs had longer binding 493

480 and human p53 mAbs with their cognate peptide half-lives with the phosphorylated peptide 96009P 494

481 epitopes, using BIAcore technology, we measured than with the original peptide 96009. Two classes of 495

482 the off-rate value (k ) of the binding reaction of anti-Xenopus p53 antibodies could also be defined 496off

483 these antibodies with Peptide A, an N-terminal according to their behavior with peptide A. One 497

484 biotinylated peptide whose sequence corresponds to group corresponded to mAbs showing a very high 498

485 the first 60 amino acids of hp53; peptide 96009, half-life (mAbs X18, X73, and X77) and a second 49915 29

486 biot-(SGSG)- SQETFSDLWKLLPEN ; peptide group (mAbs X44, X61, X87 and X91) having 4-fold 50015 (p) 29

487 96009P, biot-(SGSG)- SQETFS DLWKLLPEN lower half-lives. Interestingly, these two groups 501(p)

488 with S representing an O-phosphorylated serine; correspond to the two groups determined on the basis 502

469

470 Fig. 4. Determination of the half-life (t ) of the complex between various mAbs and biotinylated N-terminal peptides from hp53 as1 / 2

471 measured using BIAcore technology. The off-rate (k ) of p53 mAbs from N-terminus peptides was measured. The value of the complexoff

472 half-life is given by t 5 ln 2 /k . Peptide A is an N-terminal biotinylated peptide whose sequence corresponds to the first 60 amino acids1 / 2 off29473 of hp53; peptide 96009, biot-(SGSG)-15SQETFSDLWKLLPEN29; peptide 96009P, biot-(SGSG)-15SQETFS(p)DLWKLLPEN with S(p)

38 55474 representing an O-phosphorylated serine; peptide 96011, biot-(SGSG)- DDLMLSPDDIEQWFT . HR231, an antibody directed against the475 C-terminus of p53, was used as a negative control.

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504 of their differential recognition of alanine-substituted are very labile regions. However, a peptide corre- 547

505 peptides. sponding to residues 15–29 of hp53 was cocrystal- 548

lized with mdm2 by Kussie et al. (1996), who 549

506 3.7. Recognition of peptides derived from murine determined that this peptide adopts an amphipathic a 550

507 and Xenopus p53 helical conformation with all side chains of charged 551

residues on one side of the helix and all side chains 552

508 Since the antibodies tested recognized regions of hydrophobic residues clustered on the other side. 553

509 highly conserved during evolution, it was of interest Interestingly, we found that the critical residues for 554

510 to determine whether the mAbs were able to recog- mAb DO7 recognition are only charged residues 555

511 nize peptides derived from mouse and Xenopus p53 (Asp-21 and Lys-24), whereas the critical residues 556

512 sequences. Using either Pepscan analysis or Spot for recognition by Xenopus mAbs are only hydro- 557

513 technology, the antibodies could be divided into two phobic residues (Phe-19 and Trp-23), suggesting that 558

514 classes (Table 1). The first group is composed of the two faces of the a helix are antigenic. 559

515 mAbs that recognized peptides from murine, Despite the fact that the various immunodominant 560

516 Xenopus and human p53 (mAbs B17, X18, X73, epitopes are highly conserved through evolution and 561

517 X77). The second group is composed of mAbs C36, are similar in both species, we noted some differ- 562

518 X61, X87, X44 and X91 which recognized peptides ences. We defined two groups of Xp53 mAbs accord- 563

519 from the Xenopus and human p53 sequence but did ing to their recognition pattern of hp53, Xp53 and 564

520 not recognize peptides from the murine protein mp53. The first group of mAbs (X44, X61, X87, and 565

521 sequence even though no residues determined as X91) cross-reacted with hp53 and Xp53 but did not 566

522 crucial were substituted in the murine sequence. The recognize mp53. Substitution of the Asp-21 residue 567

523 common features of these antibodies are: (i) their of hp53 by a Gly in mp53 may lead to destabilization 568

524 lower binding half-lives; and (ii) their lower recogni- of the a helix both by partial loss of its amphipathic 569

525 tion of the phosphorylated peptide and peptides in nature and by elimination of the (Asp-21)–(Thr-18) 570

526 which Leu-26 and Pro-27 had been replaced by an hydrogen bond necessary for the initiation of the 571

527 alanine residue. helix (Kussie et al., 1996). We suggest that this 572

change in the conformation could also contribute to 573

the weak immunogenicity of mp53 described by 574

528 4. Discussion Lane et al., 1996. This group of mAbs was also 575

characterized by its lower complex half-life with 576

529 In this study, we characterized epitopes recognized peptide A and a significant loss of recognition when 577

530 by 14 mAbs directed against either the human or Asp-21, Leu-22, Leu-26 and Pro-27 were replaced 578

531 Xenopus p53 N-terminus. We confirmed that there by an alanine residue, indicating that these residues 579

532 are two immunodominant regions in this part of the also contribute to antibody recognition. The same 580

533 protein. All epitopes could in fact be shortened to 5- group of mAbs also exhibited a lower binding to the 58118 (p) 27

534 to 8-residue long linear sequences in which only a phosphorylated peptide TFS DLWKLLP in the 582

535 few residues are critical for antibody binding. In the Spot assay. 583

536 case of the anti-hp53 mAb B17, for example, we The second group recognized hp53, Xp53, mp53 58418 23

537 defined the epitope recognized as TFSDLW and is composed of mAbs X18, X73, X77. This 585

538 where F, L and W were determined as critical. These group is characterised by its shorter minimum reac- 586

539 results are in total agreement with those we obtained tive peptide size, since it is composed of FSDLW. We 587

540 using a 15-mer peptide phage-displayed library presume that in the case of this shorter epitope, the 588

541 (results not shown). Indeed, more than 50% of the disruption of the a helix is not sufficient to abolish 589

542 peptides sequenced after panning on mAb B17 the recognition of mp53. 590

543 possessed Phe, Leu and Trp (unpubl. data). These Strikingly, amino acids residues Phe-19, Trp-23, 591

544 results validate the method we describe here for the Trp-53, Phe-54, which are key residues for p53 592

545 dissection of linear epitopes. transactivation functions, are also essential residues 593

546 N- and C-terminal regions from the p53 protein of the epitopes of several anti-p53 mAbs. The N- 594

UNCORRECTED PROOF

JIM8660


596 terminal part of p53 contains the transactivation activity of the two N-terminal activation domains of 644

597 domain of the p53 and therefore must be accessible p53. 645

598 in order to interact with proteins involved in the599 transcription machinery such as TBP, TAF40 and600 TAF60 (Thut et al., 1995). Indeed, mutation at Acknowledgements 646

601 residues Leu-22 and Trp-23 leads to a total loss of602 the binding of numerous proteins which downregu- We are grateful to Dr. S.L. Salhi for critical 647

603 late p53 function such as E1b or mdm-2 (Lin et al., reading of the manuscript. This work was supported 648

604 1994). It should be stressed that mdm2 requires the by a grant from the Association pour la Recherche 649

605 same residues (Phe-19, Trp-23 and Leu-26) for its sur le Cancer. JMP was supported by a doctoral 650

606 interaction with p53 as do mAbs X44, X61, X87 and fellowship from the Association pour la Recherche 651

´ ´607 X91. Recently it has been reported that a new sur le Cancer and the Comite de l’Herault de la 652

608 activation domain maps between amino acids 40–83 Ligue Nationale Contre le Cancer. 653

609 (Candau et al., 1997). Residues Trp-53 and Phe-54610 were defined as critical for function both in yeast and611 mammalian cells. These same amino acids were also References 654

612 found to be critical for the binding of mAbs H279613 and H447. Banks, L., Matlashewski, G., Crawford, L., 1986. Isolation of 655

human-p53-specific monoclonal antibodies and their use in the 656614 This information indicates that some of our mAbsstudies of human p53 expression. Eur. J. Biochem. 159, 529. 657615 may interfere with the same residues on p53 that are

Bartek, J., Bartkova, J., Lukas, J., Staskova, Z.,Vojtesek, B., Lane, 658616 used by many transcription and regulation factors to D.P., 1993. Immunohistochemical analysis of the p53 onco- 659617 bind to p53. Given the similarities in the site of protein on paraffin sections using a series of novel monoclonal 660618 interaction of p53 with mdm2 and with the group of antibodies. J. Pathol. 169, 27. 661

Beroud, C., Soussi, T., 1997. p53 and APC gene mutations: 662619 mAbs X44, X61, X87 and X91, we hypothesize thatsoftware and databases. Nucleic Acids Res. 25, 138. 663620 the complementary determining regions of these

Bessard, A.C., Garay, E., Lacronique, V., Legros, Y., Demarquay, 664621 mAbs and the interacting region of mdm2 may also C., Houque, A., Portefaix, J.M., Granier, C., Soussi, T., 1998. 665622 present a certain degree of homology. Such mimicry Regulation of the specific DNA binding activity of Xenopus 666623 between an antibody and a protein has already been laevis p53: evidence for conserved regulation through the 667

carboxy-terminus of the protein. Oncogene 16, 883. 668624 described (Ducancel et al., 1996). The third com-Candau, R., Scolnick, D.M., Darpino, P., Ying, C.Y., Halazonetis, 669625 plementary determining region of the heavy chain of

T.D., Berger, S.L., 1997. Two tandem and independent sub- 670626 F12, a human monoclonal antibody derived from a activation domains in the amino terminus of p53 require the 671627 pemphigus patient and directed against desmosomal adaptor complex for activity. Oncogene 15, 807. 672628 and hemidesmosomal plaques, was shown to share a Caron de Fromentel, C., May-Levin, F., Mouriesse, H., Lemerle, 673

J., Chandrasekaran, K., May, P., 1987. Presence of circulating 674629 four-amino-acid sequence (GSSG) with the intracel-antibodies against cellular protein p53 in a notable proportion 675630 lular domains of desmoglein 1 and bullous pem-of children with B-cell lymphoma. Int. J. Cancer 39, 185. 676

631 phigoid antigen. Furthermore, a peptide from the Crawford, L.V., Pim, D.C., Bulbrook, R.D., 1982. Detection of 677632 VH-CDR3 containing the GSSG motif was able to antibodies against the cellular protein p53 in sera from patients 678633 inhibit the binding of mAb F12 to the target antigens with breast cancer. Int. J. Cancer 30, 403. 679

Dowell, S.P., Wilson, P.O., Derias, N.W., Lane, D.P., Hall, P.A., 680634 (Gilbert et al., 1997). Similarly, in our case, it would1994. Clinical utility of the immunocytochemical detection of 681635 be worth trying to localize a peptide sequencep53 protein in cytological specimens. Cancer Res. 54, 2914. 682

636 homologous to mdm2 in the variable regions of one Ducancel, F., Merienne, K., Fromen-Romano, C., Tremeau, O., 683637 of our anti-p53 mAbs and to verify its capacity to Pillet, L., Drevet, P., Zinn-Justin, S., Boulain, J.C., Menez, A., 684638 bind p53 as well as to inhibit the binding of mdm2. 1996. Mimicry between receptors and antibodies. Identifica- 685

tion of snake toxin determinants recognized by the acetyl- 686639 Consequently, we suggest that the anti-p53 mAbscholine receptor and an acetylcholine receptor-mimicking 687640 described here represent potentially powerful toolsmonoclonal antibody. J. Biol. Chem. 271, 31345. 688

641 for studying the function and regulation of the p53 Frank, R., 1992. Spot synthesis: an easy technique for positionally 689642 protein. It would be of great interest to use these addressable, parallel chemical synthesis on membrane support. 690643 antibodies to block or modify independently the Tetrahedron 46, 9217. 691

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JIM8660


693 Gannon, J.V., Greaves, R., Iggo, R., Lane, D.P., 1990. Activating cells is not determined by mutation but is dependent on Mdm2 742694 mutations in p53 produce a common conformational effect. A binding. Oncogene 15, 1179. 743695 monoclonal antibody specific for the mutant form. Embo. J. 9, Milner, J., 1984. Different forms of p53 detected by monoclonal 744696 1595. antibodies in non-dividing and dividing lymphocytes. Nature 745697 Gilbert, D., Courville, F., Brard, F., Joly, P., Petit, S., Bernardi, E., 310, 143. 746698 Schoofs, A.R., Lauret, P., Tron, F., 1997. A complementary- Molina, F., Laune, D., Gougat, C., Pau, B., Granier, C., 1996. 747699 determining region peptide of antidesmosome autoantibody Improved performances of spot multiple peptide synthesis. 748700 may interact with the desmosomal plaque through molecular Pept. Res. 9, 151. 749701 mimicry with a cytoplasmic desmoglein 1 sequence. Eur. J. Picksley, S.M., Vojtesek, B., Sparks, A., Lane, D.P., 1994. 750702 Immunol. 27, 1055. Immunochemical analysis of the interaction of p53 with 751703 Hartwell, L.H., Kastan, M.B., 1994. Cell cycle control and cancer. MDM2; fine mapping of the MDM2 binding site on p53 using 752704 Science 266, 1821. synthetic peptides. Oncogene 9, 2523. 753705 Haupt, Y., Maya, R., Kazaz, A., Oren, M., 1997. Mdm2 promotes Schlichtholz, B., Legros, Y., Gillet, D., Gaillard, C., Marty, M., 754706 the rapid degradation of p53. Nature 387, 296. Lane, D., Calvo, F., Soussi, T., 1992. The immune response to 755707 Kubbutat, M.H., Jones, S.N., Vousden, K.H., 1997. Regulation of p53 in breast cancer patients is directed against immuno- 756708 p53 stability by Mdm2. Nature 387, 299. dominant epitopes unrelated to the mutational hot spot. Cancer 757709 Kussie, P.H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Res. 52, 6380. 758710 Levine, A.J., Pavletich, N.P., 1996. Structure of the MDM2 Schlichtholz, B., Tredaniel, J., Lubin, R., Zalcman, G., Hirsch, A., 759711 oncoprotein bound to the p53 tumor suppressor transactivation Soussi, T., 1994. Analyses of p53 antibodies in sera of patients 760712 domain. Science 274, 948. with lung carcinoma define immunodominant regions in the 761713 Lane, D.P., 1994. On the expression of the p53 protein in human p53 protein. Br. J. Cancer 69, 809. 762714 cancer. Mol. Biol. Rep. 19, 23. Soussi, T., 1996. The humoral response to the tumor-suppressor 763715 Lane, D.P., Stephen, C.W., Midgley, C.A., Sparks, A., Hupp, T.R., gene-product p53 in human cancer: implications for diagnosis 764716 Daniels, D.A., Greaves, R., Reid, A., Vojtesek, B., Picksley, and therapy. Immunol. Today 17, 354. 765717 S.M., 1996. Epitope analysis of the murine p53 tumour Soussi, T., May, P., 1996. Structural aspects of the p53 protein in 766718 suppressor protein. Oncogene 12, 2461. relation to gene evolution: a second look. J. Mol. Biol. 260, 767719 Legros, Y., Lacabanne, V., d’Agay, M.F., Larsen, C.J., Pla, M., 623. 768720 Soussi, T., 1993. Production of human p53 specific mono- Stephen, C.W., Helminen, P., Lane, D.P., 1995. Characterisation of 769721 clonal antibodies and their use in immunohistochemical epitopes on human p53 using phage-displayed peptide li- 770722 studies of tumor cells. Bull. Cancer 80, 102. braries: insights into antibody-peptide interactions. J. Mol. 771723 Legros, Y., Lafon, C., Soussi, T., 1994a. Linear antigenic sites Biol. 248, 58. 772724 defined by the B-cell response to human p53 are localized Stephen, C.W., Lane, D.P., 1992. Mutant conformation of p53. 773725 predominantly in the amino and carboxy-termini of the Precise epitope mapping using a filamentous phage epitope 774726 protein. Oncogene 9, 2071. library. J. Mol. Biol. 225, 577. 775727 Legros, Y., Meyer, A., Ory, K., Soussi, T., 1994b. Mutations in Thut, C.J., Chen, J.L., Klemm, R., Tjian, R., 1995. p53 transcrip- 776728 p53 produce a common conformational effect that can be tional activation mediated by coactivators TAFII40 and 777729 detected with a panel of monoclonal antibodies directed TAFII60. Science 267, 100. 778730 toward the central part of the p53 protein. Oncogene 9, 3689. Vojtesek, B., Bartek, J., Midgley, C.A., Lane, D.P., 1992. An 779731 Lin, J., Chen, J., Elenbaas, B., Levine, A.J., 1994. Several immunochemical analysis of the human nuclear phosphopro- 780732 hydrophobic amino acids in the p53 amino-terminal domain tein p53. New monoclonal antibodies and epitope mapping 781733 are required for transcriptional activation, binding to mdm-2 using recombinant p53. J. Immunol. Methods 151, 237. 782734 and the adenovirus 5 E1B 55-kD protein. Genes Dev. 8, 1235. Vojtesek, B., Dolezalova, H., Lauerova, L., Svitakova, M., Havlis, 783735 Lubin, R., Schlichtholz, B., Bengoufa, D., Zalcman, G., Tredaniel, P., Kovarik, J., Midgley, C.A., Lane, D.P., 1995. Conforma- 784736 J., Hirsch, A., de Fromentel, C.C., Preudhomme, C., Fenaux, tional changes in p53 analysed using new antibodies to the 785737 P., Fournier, G. et al., 1993. Analysis of p53 antibodies in core DNA binding domain of the protein. Oncogene 10, 389. 786738 patients with various cancers define B-cell epitopes of human Wade-Evans, A., Jenkins, J.R., 1985. Precise epitope mapping of 787739 p53: distribution on primary structure and exposure on protein the murine transformation-associated protein, p53. Embo. J. 4, 788740 surface. Cancer Res. 53, 5872. 699. 789741 Midgley, C.A., Lane, D.P., 1997. p53 protein stability in tumour

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