CA2148274A1 - Protein p53 fragments and their uses in detecting and following-up pathological conditions - Google Patents

Abstract

A p53 protein fragment peptide specifically reacting with antibodies to p53 in the body fluids of cancer patients or precarcinomatous individuals. The peptide is preferably included in amino acid sequence 1-112 or 350-393 of protein p53, and may be used for detecting or monitoring diseased conditions, particularly carcinous conditions.

Description

WO 94/10306 ~ 1 4 8 2 ~ 4 PCI` / FR93 / 01082 FRAGMENTS OF PROI'EINE p53 AND THEIR USES
IN THE DETECTION AND MONITORING OF PATHOLOGICAL CONDITIONS
The present application relates to fragments ; of the protein p ~ 3 corresponding to epitopes immunodominant human p53 protein.
It is also related to their uses in state detection and monitoring pathological and in particular of cancerous states and . pre-cancerous.
- 10 Mutations in the p53 gene are alterations genes most often encountered in cancers humans. The frequency of mutations in the p53 gene is high for colon (1), breast (2) and breast cancer lung (3) as in leukemia (4), osteosarcomas (5) ~ ovarian cancer (6), stomach (7), and brain (8). Mutations hereditaries have recently been highlighted as support for Li-Fraumeni family cancer syndrome (9.10). Reported to the 10 most common cancers worldwide, alterations in the p53 gene are found in 40 to 45% of cancer patients. The most frequent alterations are mutations in 4 of the 5 regions - phylogenetically conserved (HCD) protein p53 (11.12).
In 1982, Benchimol et al. have shown by a radioimmunoassay that the p53 protein is specifically overexpressed in cells transformed and undetectable in normal cells (13). Many studies have since confirmed these results and shown that protein accumulation p53 is due to its stabilization. We now know that this stabilization is due in almost all cases to a point mutation that changes the conformation 35 - and the stability of the protein. These observations encourage systematic study by immunohistology :., ::, -,.
~ v '~;' ~.
......

WO9 ~ / 10306 ~ PCT / FR93 / 01082 D

of expression of the p53 protein over a wide range . tumors because there seems to be a good correlation ... between the mutation of the p53 gene and the accumulation of the protein (14, 15).
.t, 5 In 1982, Crawford et al. have detected i anti-pS3 antibody in patients with breast cancer (16). Caron de Fromentel et al. have then shown in 1987 that such antibodies were . present in the serum of children with very diverse cancers (17). The average frequency is 12% with a specific frequency of 20% in the Burkitt's lymphoma (17). More recently, Davidoff and al. (18) have shown that the presence of anti-p53 is associated with specific mutations of exons 5 and 6 of the gene. These mutated pxoteins are known to associate with the hsp70 protein and are even more oncogenic in vitro and in vivo.
The importance of these serum antibodies has been recently raised by the discovery that their presence is generally correlated with a poor prognosis for the patient.
The part of the human p53 protein exhibiting the most mutations being located approximately in the middle of the sequence of the p53 protein, we could have supposed that antibodies from cancer patients and producing such a mutee protein recognize . specifically this central part of the protein - preserltant mutations .. Indeed, this region of the human p53 protein being different in cancer patients it seemed logical to conceevoi.r ~ ue the body's immune response would preferentially be directed against the region modified.
; ' ~. The applicant has shown surprisingly that the regions recognized by the antibodies of .;,., ,. ..
... ..
..,.;

~ 1 - WO94 / 10306 ~ 1 4 ~ 2 7 4 PCT / FR93 / 01082 .

patients do not correspond to p53 regions '3 altered in cancers but has other regions of `human p53 protein which are located in amino and carboxy-terminal ends of p53. This original result could not be predicted by any previous study.
It should be noted that in the years 1980-1985, ¦ many murine monoclonal antibodies have been produced 3 either against murine p53 or against human p53.
! lo Two studies focused on the characterization of these monoclonal antibodies. Banks et al. (22) described the production of directed murine monoclonal antibodies against human p53. One of these antibodies (PAbl801) is widely used in various laboratories per hour . . . ~.
15 current. Using truncated p53 proteins, these authors have shown that the epitope recognized by pAbl801 or Pabl803 is between the amino acids 32 and 79.
In another article, WADE-EVANS and JENKINS
20 (30) mapped the epitopes of 4 antibodies monoclonals directed against mouse p53. For this study they used protein fragments ~~ - murine p53. The results are as follows:
the PAb242 antibody recognizes an epitope located between 25 amino acids 9 and 25, antibody PAb246 recognizes an epitope located between amino acids 88 and 109, the antibody PA ~ 248 recognizes an epitope located between amino acids 157 and 192 and the antibody PAb421 recognizes an epitope located between acids 30 amino 370 and 378.
All of these studies show that the epitopes recognized by these monoclonal antibodies are distributed all along the protein.
-:! The present invention relates to peptides ~ 35 or protein fragments, excluding the `,"

, , WO94 / 10306 PCTtFR93 / 01082 '~ 14827 ~ 4 p53 protein, exhibiting a specific reaction vis-against anti-p53 antibodies present in fluids ~ ::
biology of cancer patients or patients with cancer regardless of its origin. : ~
Advantageously, such a peptide is understood in the amino acid sequence of the amino region ..: ~
terminal (residues 1 to 112) or in the carboxy region terminal (residues 350 to 393) of the p53 protein. The; ~
sequence of the wild-type human p53 protein is `:. ~ - ~
described by SOUSSI et al (26). :
It advantageously partly comprises QU in one of the following sequences ~
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro ~ -Leu (SEQ ID N0: 1).: ~
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile ..
Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID
NO: 2). .
Preferably, such a peptide comprises in ~ `"
part or all of one of the following sequences. :;
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu (SEQ ID N0: 3) Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu ~
Leu Pro Glu Asn Asn (SEQ ID NO: 4) ~ `.
Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn ~.
Val Leu Ser Pro Leu (SEQ ID N0: 5) `
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile ~
Glu Gln Trp Phe Thr (SEQ ID NO 6) ~. .
Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro (SEQ ID N0: 7).
If such a pepti.de is included in the sequence amino acids 350 to 393 of the protein p53 it then preferably includes in part or in one of the following sequences:
Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala WO94 / 10306. ~ 1 4 8 2 7 4 PCT / FR93 / 01082 - ~ ly Lys Glu Pro Gly ~ SEQ ID NO: 8) ~:
Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly - ~
Gly Ser Arg Ala His (SEQ ID NO: 9) :::
Gly Lys & lu Pro Gly Gly Ser Arg Ala His Ser Ser His Leu Lys (SEQ ID NO: 10) -.
Gly Ser Arg Ala His Ser Ser His Leu Lys ~:
Ser Lys Lys Gly Gln (SEQ ID NO: 11) ~: -Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His (SEQ ID NO: 12) Ser Lys Lys Gly Gln Ser Thr Ser Ar ~ His - ~
Lys Lys Leu Met Phe (SEQ ID NO: 13) ~.
Ser Thr Ser Arg His Lys Lys Leu Met Phe:
Lys Thr Glu Gly Pro (SEQ ID NO: 14) Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp (SEQ ID NO: 15).
Determination of protein fragments -: ~
p53 with a specific reaction to antibodies from cancer patients can get to do by the means made available to man.
of the profession and in particular by test immunoenzymatic, radio-immunological, by immuno- ~
precipitation or immunoblotting. .
Such tests are well known to those of skill in the art.
trade and are described in general manuals (28.29). -The present invention also relates to ~:
a nucleotide, ribonucleotide (RNA) sequence or deoxyribonucleotide ~ DNA), coding for synthesis of one of the peptides object of the present request.
Such peptides can be used for the detection or monitoring of disease states, and especially for detecting or monitoring states ~ cancerous and precancerous.
Another object of the present invention is a WO94 / 10306 21 ~ ~ 2 ~! ~ PCT / FR93 / 01082; ~

method for determining presence of antibodies or for their determination in a tissue or a fluid characterized in that ~
- the tissue or the fluid is brought together and One of the peptides ob ~ and of the invention previously described, and.
- the presence of either the antibody is determined, either peptide or antibody-peptide pair and we performs the corresponding dosage.
The assay of antibody-peptide complexes can be performed either by assaying the antibody bond peptide, either by peptide or antibody analysis captured and entering the antibody-peptide complex.
This assay can be performed by methods known to those skilled in the art and in particular by physical or immunological methods such as immunoenzymological, radioimmunological or chemoimmunological or by more which are based on new techniques molecular biology such as immuno-PCR. According to this technique, the antigen-antibody complex is detected by a PCR reaction that involves a oligonucleotide linked to the revealing antibody. The complex is then visualized by electrophoresis.
Advantageously, the antibodies which are determines the presence or the dose are antibodies representative of pathological states and particular of cancerous or pre-cancerous states.
In order to implement the method, the peptide can be fixed on a solid phase.
The complexes formed can be revealed at using an antibody that specifically reacts with the peptide or the antibodies to be assayed or detected.
- This antibody allowing the revelation of complexes is advantageously combined with a darter in order to WO94 / 10306 ~ ~ 3 ~ 7 Ll PCT / FR93 / 01082 highlight the antibody-peptide complex.
Such a marker can be an enzyme, a chemiluminescent molecule, bioluminescent, fluorescent or radioactive.
Such a procedure can be advantageously implemented works with a box for dosing and determination of antibodies comprising at least ~
- a preparation of one or more of peptides as previously described and - a means allowing the revelation of the reaction or lack of reaction between any of these peptides and the antibodies to be assayed.
The antibody peptide complex formed during reaction is advantageously revealed using a lS antibody conjugated to a marker as defined above on it or using any molecules capable of detect the presence of such a complex.
The peptides which are the subject of the present invention are preferably obtained by synthesis from amino acids by any means known to those skilled in the art job .
They can also be obtained by genius genetic or by enzymatic or chemical breakdown of the p53 protein.
Antibodies allowing the revelation of the complex formed between the peptide and the antibodies are advantageously directed against determinants species specific antigenics of which is originating the fluid or fabric. So in the assay framework for antibodies of human origin, antibodies to reveal the complex will anti-human antibodies.
The present invention is illustrated without be limited by the following examples in which :

WO94 / 10306 ~ t ~ 27 4 PCT / FR93 / 01082 ~; ~

, ~

Figure 1 shows the profiles of interaction between the fragments of the p53 protein and antibodies present in lq serum of different cancer patients. Antibodies used for the hybridizations of Figures 1A to 1F are respectively a CMl rabbit serum immunized with the protein p53, a mouse serum immunized with protein p53, three sera from patients he had been determines that they had anti ~ antibodies p53 and a witness constituted by an anti-alkaline phosphatase.
Figures 2 to 7 show the reactivity of 77 peptides corresponding to the p53 protein, opposite of sera from three cancer patients lungs numbered 84, 37, 109, and three patients with breast cancer numbered 57, 27 and 2 ~
The antibody / peptide complex is revealed by colorimetry and optical density is measured by a microplate reader.
Figure 8 illustrates the frequency of recognition ~ on the ordinate) of 77 peptides (in abscissa) by different sera from patients with cancer.
The following abbreviations are used in the examples :
ER, estrogen receptor HCD, Domaine Hautement Conserv0 (highly conserved domain) of the p53 protein hsp70, 70 kilodalton heat shock protein MAB, monoclonal antibody PBS, ~ ampon phosphate PBST, PBS with 0.1% Tween 20 PBSTM, PBS with 0.1% Tween 20 and 3 ~ milk powder ~ skim PCR, polymerase chain reaction (polymerase WO94 / 10306 ~ 2 7 ~ pcT / FRs3 / o1o ~ 2 ~. '~ "''.
.
- chain reaction).
PhoA, E.coli alkaline phosphatase - PR, progestexxone receptor SDS, sodium dodecyl sulfate 5 EXAMPLE 1 - ' Highlighting and Sianification of Antibodies anti-~ 53 present in serum qe Datients affected cancer and localization of Drotein 'regions PS3 recognized ~ ar ces_antibodies.
1) MATERIALS AND METHODS
1.1 SERUMS.
The sera of 80 patients with invasive breast carcinoma and 20 patients with metastases were brought together after diagnosis histopathological and stored at -20'C.
1.2 Monoclonal antibodies BALB / c mice of haplotype H-2d are immunized intraperitoneally with 100 ~ g of normal human pS3. A booster injection is performed intravenously with 80 ~ g of protein ~ e same origin. Four days after the xappel, the splenocytes are removed and fused with NS-1 myeloma cells in the presence of polyethylene glycol (PEG 1500 'Boehringer). After selection in medium supplemented with 1% Hypoxanthine Aminopterin Thymine ~ HAT) (Gibco), the secretion of antibodies by hybridomas was sought by an ELISA test in using human p53 as an antigen. The hybridomas secreting antibodies capable of recognize the p53 were cloned by the technique of limit dilution.
For localization of epitopes on p53 human we used a series of peptides overlapping (length of 15 amino acids (aa), overlap of 10 aa) covering the entire 2l! ~ 8 ~ o protein p53. We have a series of 77 peptides biotinylated corresponding to all of p53. The First 76 peptides from the N- terminus terminal have a length of 15 amino acids each and the 77th peptide contains only 13 amino acids.
The H111 antibody recognizes an understood epitope between amino acids 291 and 300, the antibody HR231 recognizes an epitope between amino acids 371 and 380 and HT 21 ~ recognizes an epitope understood between amino acids 1 to 64.
1.3 EXPRESSION OF FRAGMENT OF ~
The vector pLip4 was used for the production of hybrid proteins. This plasmid is derived from pLipl (21) in which two Sac restriction I and Sal I are introduced between codons +6 and +7 of the E.coli PhOA gene. After cloning, the PhoA fusion protein is exported to the bacterial periplasm and can be extracted by shock cold osmotic (21).
~ 0 ~ the p53 protein was cut into 6 fragments well defined. Fragments 2 (residues 108 to 162), 3 (residues 158 to 219), 4 (residues 215 to 267) and 5 (residues 263 to 310) respectively contain the HCD regions II to V and correspond to the sites preferred for mutations (HOT-SPOT regions) in human cancers (11,12).
Fragments l (residues l to 112) and 6 (residues 306 to 393) correspond to the amino- and carboxy-terminal of the protein and are generally away from mutations.
Precise ~ fragments of human p53 cDNA
(clone H8, Varda Rotter, Weissman Institute, Israel) were amplified by PCR using Vent- ~ NA
~ polymerase to reduce incorporation errors then subcloned into a pLIP4 vector. The clones W0 ~ 4/10306, 7.1 4 ~ ~ 7 ~ PCT / FR93tO1082 - ~

1 1. -positive are checked for phosphatase activity: ~
alkaline and directly sequenced.
-The six fragments fused to the PhoA gene can be expressed in E, coli as proteins:
soluble. ~ e protein expression yield of fusion with fragment 6 is lower (2 to 5 times) than for the other fragments.
The antigenicity of the expressed protein is established by its reactivity with different antibodies monoclonals. (see table 2). PAbl801 (22) and HT 216 are specific for fragment 1; the PAb240 epitope is located on the fragm ~ nt 3 (23). Hlll is on the fragment 5; HR 231 and PAbi22 (24) are specific for:
the carboxy-terminal region of p53 (fragment 6).
All these monoclonal antibodies react in -immunoblot and immunoprecipitation with the protein of ~:
pLIP4 merger in accordance with the location of their epitope (see table 2) ~:
1. 4 METHOD OF ANALYSIS OF P5 3 PAR FRAGMENTS

2 0 I MMUNOBLOT. ~:
The Immunoblot was performed as described by.
Towbin et al. (20). Proteins are separated by polyacrylamide gel electrophoresis (10%
acrylamide) in Tris-Glycine buffer containing 0.1% of SDS, pH 8.3. Proteins are transferred to a nitrocellulose membrane (2 hours, 40 voltsj; la;:
membrane is immersed in PBSTM buffer to saturate the non-specific attachment sites then washed 5 times with PBST.
30 For Western blot of patient serum and E. coli extract, several pre-incubations are necessary to reduce non-specific bonds to the nitrocellulose membrane and remove the ~ anti-phosphatase antibodies present in some 35 sera. The serums are first incubated with the extract W094 / 10306 PCT / FRg3 / 01082 .,. :. ::
~ 4 12 bacteria expressing alkaline phosphatase E.coli for one hour at ~ 4'C then on a membrane of nitrocellulose containing a bacterial extract (without fusion protein) 12 hours at +4 C.
The nitrocellulose membrane with the hybrid protein is incubated with the sera prepared and diluted from 1/100 to 1/500 for 1 hour in PBSTM buffer at room temperature. After 5 washes in PBSTM, the membranes are incubated for 1 hour with a rabbit antiCQrps anti-mouse conjugate peroxidase (diluted 1/4000 in PBSTM buffer), washed 5 both in PBS buffer and revealed by Luminol (AmershamR).
After reading and interpretation, the filters are checked by immunological reaction (Immunoblot) for the amount of fusion protein actually deposited by incubating the membrane with an antibody anti-alkaline phosphatase.
Immunoprecipitation and electrophoresis in polyacrylamide-SDS gel (SDS-PAGE) were made according to the technique described by Soussi et al. (19).
2) RESULTS
2.1 Anticor ~ s anti- ~ S3 and clinical results.
The sera of 100 cancer patients 25 are tested for the presence of antibodies by immunoprecipitation and Western blot (Table 1). The circulating antibodies are detected in 14% of patients in all clinical stages.
The frequency is slightly higher than 30 that reported by Crawford et al. (10 ~) (16), in probably due to the use of purified protein recombinant p53 in place of cell lysate.
There is no correlation between the presence _ antibodies and the clinical stage of the disease. Any 35 frequency difference depending on the size of the WO94 / 10306 ~ 1 4 8 2 7 4 PCT / FR93 / 01082 tumor or lymph node spread has been shown. There is no correlation with others clinical parameters such as age, status hormonal. However, a correlation has been established with the histological stage of the tumor ~ SBR stage 3), an independent prognostic marker in cancers primitive breast. This is confirmed by the association between the presence of antibodies and the absence of hormonal receptors ~ ER-PR).
2.2 Analysis of the regions of ~ rotein ~ 53 im ~ linked in the immune response.
To determine if the immune response is raised against mutant or wild-type pS3, all positive sera and several negative sera controlled by immunoprecipitation against different p53 mutants (Hisl75, Val 143 and ProlS6). The recognition of wild and mutant types for sera will be identical.
In a second series of experiments, the serum CMl t25) has been checked. CM1 is an antibody polyclonal rabbit obtained by immunization with highly purified and frequently used human p53 in immunocytochemistry (24). Western techniques blot have shown that the CMl serum contains antibodies that mainly recognize fragments 1 and 6 and to a lesser degree fragment 5.
Similar results are obtained by immunoprecipitation. Control experience with an anti-alkaline phosphatase antibody allows to establish that identical quantities of the different fusion proteins are present on the gel (figure 1). To widen o ~ services we have controls the serum of a mouse immunized with immunopurified p53. The recognition profile is 3S almost identical to that of CMl serum. Of . .
~;

'' wo g4 / l0306 2'1 ~ 4 ~ 2 ~ 4 pcr / FR93 / o1o8?

identical results were obtained with three other mouse.
All of these results establish that the amino and carboxy terminal parts of the p53 protein are highly immunogenic in animals immune and correspond to preferred responses while the fragment 5 has a more low . Radiographic exposure times extended were unable to highlight a recognition birth of fragments 2 to 4 both by immunopre-cipitation only by immunohybridization timmunoblot).
We tested the recognition profile of patient sera containing anti-p53 antibodies (Figure 1. and Table 3). Immuno hybridizations have clearly shown that the answer immune system in patients is directed mainly against epitopes localized in fragments 1 and 6 of p53. The serum of some patients only recognize fragment 1 but none recognizes only fragment 6 which suggests that the primary response is directed mainly related to fragment 1. Results similar are obtained by immunoprecipitation. Of even as in animals, there are some variations in the immune response and some patients have antibodies directed against fragments 4 and 5.
No antibody directed against fragments 2 and 3 has could be detected. This phenomenon is not specific to breast cancer. Identical results have been obtained with sera from patients with ovarian or lung carcinomas (Figure 1 and example 2 ~.
These results clearly establish that the amino terminal part of p53 contains one or

3 ~ several dominant epitopes involved in the response WO94 ~ l0306 ~ 14 ~ 2 7 4 PCTtFR93 / 01082 B cell immune system in patients with various cancers. The terminal carboxy region contains also one or more immunodominant epitopes but their importance is less compared to the region amino terminal.
EXAMPLE 2:
Highlighting of PitoPes recognized by antic ~ anti-P53 orps resents in the serum of patients suffering from various types of cancer: analysis with synthesis peptides.
1) MATERIALS AND METHODS
The microtiter plates used are polyvinyl chloride sheets with pl, at, bottom 96 wells. Ref: M129B, Dynatech company.
Streptav ~ dine powder, reference (S4762), Sigma company.
Biotinilized peptides synthesized by the Company CAT (England) A series of overlapping peptides (length of 15 amino acids (aa), overlap of 10 aa) covering the entire p53 protein. We dispose currently from a series of 77 biotinylated peptides corresponding to all of p53. The first 76 peptides from the N-terminus have a length of 15 amino acids each and the 77th peptide contains only 13 amino acids (SEQ ID
NO: 15).
2 control peptides not corresponding to the p53 protein are used as a negative control.
ABTS 50 mg tablet; ref 1112422, Boehringer ABTS buffer ref 1112597, Boehringer Anti human coupled with IgG-GAH peroxidase;
P ~ SOF, Silenus Powdered milk WO 94tlO306 PCT / FR93 / 01082 2i4827 4 16 , ~:
; ``
PREPARATION OF MICROTITRATION PLAUSES.
100 yl of streptavidin (concentration 5yg / ml in distilled water) are deposited in the wells of the plaque.
The plates are then incubated at 37 ° C. in a dry oven for 48 hours. After this step dehydration, they can be packed individually and stored for several months at

4 C. -WASHING ~
The dehydrated plates are washed 5 times -with PBS (phosphate buffered saline) containing 0.05%
Tween 20. This PBS buffer with Tween (PBS-T) will used throughout the experiment as a solution washing. The 5 washes are done in the usual way in using 200 ~ l PBS-T buffer per well.
After the last wash, the plate is dried on absorbent paper. All washes are done this way.
SATURATION
100 μl of saturation solution are added in each well.
saturation solution: PBS buffer 0.2% Tween 20

5% Milk The plates are then incubated for 1 hour at 37C with slight agitation then are then washed as previously indicated.
ADDITION OF BIOTYNILIC PEPTIDES
50 μl of peptide solution (125 nanograms peptide diluted in PBS buffer containing 0.1%
Bovine Serum Albumin and 0.02% sodium azide) are added to each well.
Each well receives a different peptide corresponding to a specific region of p53.

.
WO94 / 1030

6 ~ 1 4 82 7 4 PCT / FR93 ~ 01082 ; ' The plates are incubated for 1 hour at 20 ° C. with slight agitation and are then washed as indicated previously.
ADDITION OF PATIENT SERUMS
The sera to be tested are dilated at lJ50 in a PBS solution containing 5% milk.
50 ~ l of serum thus diluted are added to each well of the plate.
The plates are incubated for one hour at 20 ° C. with slight agitation and are then washed as - - ~
indicated previously. ~ `
INCUBATION WITH SECONDARY ANTIBODY.
100 t ~ l of anti-monoclonal antibody human immunoglobulin are added to each well -(marketed by Silenus, antibody diluted 1/2500 in PBS 5 ~ 1ait).
Plates are incubated for 30 minutes at 37 ° C
with slight agitation and are then washed as indicated earlier. ~
REVELATION. -100 ~ 1 of substrate (ABTS 1 mM, markets by Boerhinger) are added to each well.
The plates are incubated at 20-C with a -slight agitation. `~
The result is read in a reading device ELISA (405 nm) 15 minutes, 30 minutes and 60 minutes; ~
after addition of the substrate.
2) RESULTS OBTAINED
In example l two phenomena were put in evidence.
i ~ The presence of anti-p53 antibodies is found in patients with an aggressive form breast cancer (poor prognosis).
ii) Anti-p53 antibodies recognize mainly a region of 112 amino acids located .

WO94 / 10306 ~ 1 4 ~ 2 7 4 PCT / FR93 / 01082 in the amino terminal part of p53 as well as a carboxy terminal region located between residues 306-393. The study could not provide details more precisely the region recognized by the antibodies anti-p53.
The study described in this example presents a very detailed study of these regions.
Analysis of more than 1,000 patient sera with various types of cancer has been made. 47 positive sera were chosen for a very detailed in order to determine the exact nature of the epitopes recognized by serum antibodies.
These 47 sera containing anti-p53 antibodies have been tested on a series of 77 peptides covering all of the wild-type human p53 protein. The method used is ELISA detection.
Examples of results obtained are described in Figures 2 to 7.
All the results are summarized in the table 5.
In this table, the peptides giving a response between 50 and 10% of the value ELISA maximum were shown in black. The peptides giving a response of between 20 and 50%
of the maximum ELISA value have been indicated in Grey.
The initials of the cancers studied are following:
LC: lung cancer CU: bladder cancer PA: pancreatic cancer BC: breast cancer BL: Burkitt's lymphoma H: liver cancer OV: ovarian cancer WO94 / 10306 2 ~ 4 8 2 7 4 PCT / FR93 / 01082 1 9:

L: leukemia TY: ~ anchor of the thyroid PR: prostate cancer.
Figure 8 illustrates the frequencies obtained for each of these peptides.
This histogram shows the frequency of recognition of each peptide by the 47 sera studied (see table 5). It shows, for example, that ~ -40% of the sera recognize at least peptide 3 and that 63% recognize at least peptide 4. ~ 8% of .
sera recognize at least one of the 5 peptides of the amino terminal region (N-ter) while only; ~
47% recognize one of the region's peptides carboxy terminal (C-ter). Of course, 100% of the sera recognize one of the 13 peptides described in this ~
board. -All of these results show that 98 ~ of patient sera recognize at least one of epitopes present in the 5 peptides of the region amino terminal. 47% of serums recognize at least one of the peptides of the terminal carboxy region. `~
It therefore appears that the use of these ~
peptides, amino or carboxy terminals, are of a -major importance ~ in the possibility of upgrading;
point an ELISA test to measure the presence of these antibodies in patients' biological fluids suffering from cancers or precancerous states. -:

, ~ ...

WO9 ~ / 10306 21 ~ 8 2 7 ~ PCT / FR93 / 01082 TABLE 1 ~

Correlation between the level of anti-p53 ~ antibodies:
in serum and clinical parameters and history of breast cancer patients Clinical parameters. N- P
_ _ Patients with 3/20 met ~ stasis or having undergone relapse primary breast cancer 17 ~ 80 Tumor size T -Tl 2/21 T3 2/19: ~.: -T4 1 ~ 7 State of the lymphatic nodules:
No 4/32 NS

Histological category categories 1 and 2 5/59 cO.05 category 3 6/17 Receiver status hormonal ER + PR +
ER- PR + 2/34 c0.05 ER + PR-_ NS = not significant.

......

~ 1 4 ~
WO 94/10306 21 PCr / FR93 / OtO82 a ~

; '~ ;; ~

; h ~ ~;

hl I ~ Ir.D ~

h; ~ ~ O

o ~ C ~
". ~ (O ~ 1 _ ~ r ~ _ ~ ( ~ I 1 ~

219 ~ 2 7 4 PCT / FIR93 / 01082 WO 94/1 03 ~ 6 - -r , ~

VS

I c ~ ~ ~ ~ ~

21 4 t ~ 2 7 4; PCr / FR93 / 01082 2 ~
TABLEA IJ 4 ~;

. BC 1 BC I BC BC BC BC I BC jBC BC 18C ¦BC I BCTC ~ -= 15 1 27 1 28 2 977 57 1 ~ 0 ~ 120 103 148 157 185 1 174 ~:
~ = - I == === = === ~
i _ _ _ _ .'x ''. ' ~ ................, ..; .. -. ~ ~
_ ~., ..................,., .--. _ _ ........... .. ~.
_ = = = - =. ~ ..., == = = ~
_ __ _ _:
__ _ _. _ = ~. ~. ~ ~ ~ ~ ~. _ _ ~. .................. .. ..
11 _ _,. , _ ___ _ ~ ~. ~ ~ `~ 'v ~ - ~ ~ ~
13. _ _ _ _. . ........................ _. .
~ _ == = = = = = ,. ~, ~ `.,:, === = ~ '.' ~ ._ l, _ _. _: ~.
~ == = = = = = ~ = = === ~ ". ' 19 _ ~ _ __ _:
~ 2 ~ 3! ~ ~
_. . .......
- ~ _ _ ..
=. == ~ = = = ~ =. _.
27. ~ `:
~ _ _ _ _. . ':.
== == = == == = == = '''' _ _ == == = == == = = = = ~ `,:` ':
~ - ------ ----: ~ ~
~ = = = = ~ ~ = = ==== =
1 ~ 7 l I. ll ~

2 7 4 PCI` / FR93 / 010 ~ 2 -`
WO 94/10306 2 1 4 ~

TABLE ~ ~ continued) ¦ BLT BL ¦ BL ¦ BL ¦ HI ovl LL TY ¦ TY ¦ TY rPR
1 1 2 1 5 1 6 1 155 1 80 1 48 ~ [23 1 24 1 1 ~ 8 151 . , - 3 ~
_ _ ::;, * ::, ~: _ _ _ _ _ _, ~., .. _: ~, _ __--, -,:,:. ~ _ = = ==

__ = _,. ~. :::: _ _ == _ = _ '' .: ".-- ~ ~ _ ~ .. ~ ~ = ~ = ~

~ t === == = = === =: ~
~ _ _ _ _ ~ _ ~
~ r ==== ~ = = ~ = = = = .......
~ _ ___ == == - __ _, ~,.,.,. ~.
~ t = = = ~ === = = = =; ~
2 ~ ~ _ ~ _ _ ~ - __ ___ _ _ _ _ _ _, ',''``.
___ __ == == ==, '"`''' ~ ':
_ _ _ _ _ _ = = = \,,.:
~ 1 __ __ _ ___ __ _ "''''';`,' ~ `", `' ~ r _ _ ___ = ===== =. .
.

'' ~ ~
"' .., ~.

/
. .

/ 0306 ~ 1 4 8 27 4 PCr / FR93 / 01082 W0 9 ~ 1 25 , TAEILEAU 4 (sulte) -~ ~ ~ ~ L ~; ~ L ~ 1 ~ ~ ~ - L ~ 1 = 8 ~ 37 182 147 149 1 ~ 3 ~ 2 148 18 9 ~ 13 !;

- ~ Cc = '_C = - =,,: = ~ = =, J. ~ .C _ _. ~ ~ C ~. ~ .CS. ~:.: Cc _ __ _ ~ _ _ _ _.,:. C5cs: ~,:, ..,; ~ =
/ = = = = = = =: ~. ,, ~ = = =:,.
8 _ _.
- _ _ ~., c,, c, c, c ~ .... ji ~, _ ~ ..:, ..,.,. ~ _, .- ~
= ~ s ~ 55 _ _, j ,. ~ CC ~ ', ~ =',: `', .......... ..' j ~: cC,: ~ i:
= = ~ = = = = = = = =. - ~
= - ~ = = ~ s ~ S = = = = = ~. , ','. ',', "'''', ~ 7_ = = = = = ~ = = = = ~
=== == = == ~ = ~:: ':,:: ..:. ~ `
~ 0- _ _, _ _ _.
2r _ ~ 5 ~ E ~ ~ _ _ _ __ _ ~ _ ~ __ _ _ - '~
, ~ _ ~ _ __ __ _ _, '' ~ ''"'.
== = = _ == _ - - '''' ~ `; '"
~ r __ _ _ _ _ ___ _ ~ _ _ ~
~ _ _ _ __ _ _ ___ - ~
- ~ = _. = _ - .. = _. ';''' -.` ': ~

PCr / FR ~ 3/01 082 WO94 / 10306 21 ~ Q2 ~ ~ 26 I

TA9LEAU 4 (continued) _ _ _ _ _ _ _ _ _ LCCC UC ~ ~ ~ PA ~ PA PA ~ PA
19 2037 ~ 1 lOl 10 ~ ~; ~ 0 82 1 11 7 12 1 _. . .
_ _ _ ..... _ ~ _ r ~ _ _ ~ _ _ ~ ... ~ "~ i _ ~
== _ _ ~ _ _ ''"
/. _ = = __ 8 _.
__ _ _ _ ~ _ _ _ _ _ _ 11 - ~ ~ ~ --_, ...

14 = = = == = ~ = = = =

1 ~ _ _ ........................................... ~ '" ., ~) '=== = - ~ - -~ == ~ ---------- & ~ i, ~ `~ ----; ~ ----- - - ~ - ~ ------: ~
~ --------- ---- --------; ~; --- ~ -------- --------: `:
~ r = = = == = === = =, ~
~ g__ _ _ _ ~
~ r _ _ .............................................. ..............::.
~ = = = = = = == = = -: ~

.
. . ~ .. ~.

~, ~ 214 ~ 2 7 4 PCTJFR93 / 01082 TABL ~ AIJ 4 tsuite) I BC I BC IE ~ C BC ¦ BC ¦ BC ¦ BC BC ¦ BC ¦ BC ¦ E3C ¦ ~
5 17 1 ~ 8 29 77 r57 1 109 20 1103 148 57 85 174 ~. ~ I = _ __, ,,. ~
~ = = - _ __ _- _ _ ~ _ E ~:

= = = = = - = ~ ~ = = = = =
= = = = = === = = === ..`. ~ ..: -., 1 ~ ~ ~ ~
~ _ _ _ _ _ ~ _ _ -. ~ ~ - _ ~

~. = = = = = = = = = _ = ~
~ - _. _ _ __ _ _- .- ~ I `~
~ - ~ EE _. ~ T ___--. ~, - ~ _ _ = = _ _ = = _ _ _., ~ ~ -. I -------- ~

_ - ~ ............. _ _ _ I ......
_ _ _ _ _.
"=== - = _ = = t = _ = = i ~ ~

2 ~ PCr / FR93 / 01082 WO 94/10306 2 1 4 8 2 7 ~
TABLEA ll 4 ~ ~ U ite) I BL BL I BL BL IH OV LL TY I TY I TY IPR I

_ _.
_ _ _ _ - ~ .. _ ~ r = _ = = = ~ = = = = _ =,::
~ = = = = ~ _ _ .........
_ _ __ ~ = ==== ==
= = = = = = = = _ _------ `.
,. ~ _ = = = = = = = = = = -. ';' ::
~ __ _ ~ ~ = == ___ _ ~ 1 ~ _ ~
: ~ _ _ _ _ _ _ _ _ _ _ _ . ~ ---- = = = = = = = = ,,,. ,,,,.,. ".
~; 7 = = = _ _ _ _---- = = =
~ = - ~ ~ ~ i _ _ _ = ~ = = _ : ~ ~ _ _ _ ~, - ...
~ = = = = = = = = = _,. . .
: ~ = = = ~ = = = = = = ~; = ''`'""~'','.`'. ~.
~ _ ___ __ _ ___ ~
: ~ :: ~ ~ ~

7 ~ ~ ~ _ ~ _ ~
/ ~ -; = # =. I
~ /. - _ == =. ,,., ~ = I = = '~''".. ~ .'`
. . . . . ......

: ~:
,. .

-` 2148274 WO 94/10306 PCr / FR93 / 01082 TABLEAIJ 4 (continued ~.
.

I LC i ~ 1 ~ 1 ~ ~ ~ ~ ~ ~ L ~ ~ ~. ~.

8 ~ 37 132 147 149 193 ~ 1 ~ 8 18 ~ 136 ~:
~ = F t ~ F = I-- ~ = F _ ; - ~
j47- _ l. _ _ ::
~ _ I _ _: ~. _ _ ':''' ~ ''' _ _ _ __ _ _ _ F--= ~ = - ~ - = = ~ i ~ E ~ - _ _ _ _ ~ _ _ t ~ ~:

214 8 2 ~ 4 Pcr / FRg3 / 01082 TABLEAll 4 ~ continued) L ~ I uC I ~ IU ~ - ~ U ~ I PA IF ~ AI PA I PA -PA
19 2 ~ 3 ~, ~ 101 10 ~ ~) E2 ¦ 111 1/123 = _ = = = = ~ = =, - ~,.
_. _ __ ~. = = '.

_ = = _ = = = =
= - == === ~ = = ~
~ F == == = = = = = = =
== = = = = = = _ _ _ ~ ,,,,., ","., .. ".
49 _ _ ~ -. .: ~
~ _ _ ~ ~ = = _ =: `. ~ ': ~'. '~' .- `' = ==== = = = == =. '~.'".
== = = = = = = = = = =
~ '''''' -` ~ ''''.
._. _ _ _ _ ::,: '-'; -.
.. _ _ _-- `: .. '~
_. ~ _ =, "~ = =...
~ = = = = ~ = = ~ _ _, '~
6i _ -: `~
~ F == _ = = == = _--`::` `` `
... I _ _ _ _ ~
_ ~.
~ j ~ _ _. _ ---- = ~. .
_. _ ..... `.i .. _` `
.... _ _;
_ I
7 ~ - _ t 7 ~ = = = - = _ = = I =
':
~ ,, r = == == ...............,., .. _ = I =
, = .--. ,,.,., = - = = --................... l =. , `.:
~ = = = .. l = = = l =

WO94 / 10306 ~ 1 4 3 2 7 4 PCT / FR93 / 01082 BIBLIOGRAPHY
1. Baker SJ Preisinger AC Jessup JM
Paraskeva C., Markowitz S., Wilson JKV, Hamilton S.
and Vogelstein B. P53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis. Cancer Res., SO: 7717 7722.1990.
2. Prosser J. Thompson AL, Cranston G. and 1 ~ Evans HJ Evidence that p53 behaves as a tumor suppressor gene in sporadic breast tumors.
Oncogene, 5: 1573-1579, 1990.
3. Takahashi T., Nau MM, Chiba I., Birrer MJ, Rosenberg RR Vinocour M., Levitt M., Pass H., Gazdar AF and Minna JD P53 a frequent target for genetic abnormalities in lung cancer. Science, 246:
491-494.1989.
4. Gaidano G. Ballerini P., Gong JZ, Inghirami G., Neri A., Newcomb EW Magrath IT, Knowles DM and Dallafavera R. P53 mutations in human lymphoid malignancies - association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc. Natl.
Acad. Sci. USA, 88: 5413-5417, 1991.
S. Miller CW Aslo A. Tsay C., Slamon D., Ishizaki K, Toguchida J., Yamamuro T., Lampkin B. and Koeffler HP Frequency and structure of p53 rearrangements in human osteosarcoma. Cancer Res, 50:
7950-7954, 1990.
6. Mazars R., Pujol P., Maudelonde T., Jeanteur P. and Theillet C. p53 mutations in ovarian cancer -A
late event. Oncogene, 6: 1685-1690, 1991.
7. Kim JH, Takahashi T., Chiba I., Park JG, Birrer MJ, Roh JK, Lee HD, Xim JP, Minna JD and Gazdar AF Occurrence o ~ pS3-gene ~ abnormalities in gastric carcinoma tumoxs and cell lines. J. Nat. Cancer Inst., 83: 938-943, 1991.

W ~ 94/10306 214 ~ ~ 7 4 PCT ~ FR93 / 01082 8. Mashiyama S., Murakami Y., Yoshimoto T., Sekiya T. and Hayashi X. Detection of p53 gene mutations in human brain tumors by sin ~ le-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene, 6: 1313-1318, 1991.

9. Malkin D., Li FP, Strong LC, Fraumeni JF, Nelson CE, Rim DH Kassel J., Gryka MA, Bischoff FZ Tainsky MA and Friend SH Germ line p53 mutations ~ na familial syndrome of breast cancer, sarcomas, and other neoplasms, Science, 250: 1233-1238, 1990.

10. Srivastava S., Zou ZQ, Pirollo X., Blattner W. and Chang EH Germ-line transmission of a mutated p53 gene in a cancer-prone family with li ~
fraumeni syndrome. Nature, 348: 747-749, 1990.

11. Caron de Fromentel C. and Soussi T. P53 ~ umor suppressor gene: a model for investigating human mutagenesis. Genes Chrom Cancer, 4: 1-15, 1992.

12. Hollstein M., Sidransky D., Vogelstein B.
and Harris CC P53 mutations in human cancers.
Science, 253: 49-53, l991.

13. Benchimol S., Pim D. and Crawford L.
Radioimmunoassay of the cellular protein p53 in mouse and human cell lines. EMBO J., 1: 1055-1062, 1982.

14.Hall PA, Ray A., Lemoine NR Midgley CA, Krausz T. and Lane DP P53 immunostaining as a marker of malignant disease in diagnostic cytopathology. Lancet, 338: 513, 1991.

15. Cattoretti G., Rilke F., Andrealo S., D'amato L. and Delia D. P53 expression in breast Cancer. Int. J. Cancer, 41: 178-183, 19BB.

16. Crawford LV, Pim DC and Bulbrook RD
Detection of antibodies against the cellular protein ~ p53 in sera from patients with breast cancer. Int. J.
Cancer, 30: 403-408, 1982.

W094 / 10306 21 ~ 8 2 7 4 PCT / FR93 / 01082 `i ,; ~. . ~.

17. Caron de Fromentel C., M ~ y-Levin F., Mouriesse H., Lemerle J., Chandrasekaran K. and May P.
Presence of circulating antibodies against cellular protein p53 in a notable proportion of children wlth B-cell lymphoma. Int. J. Cancer, 39: 185-189, 1987.

18. Da ~ idoff AM, Iglehart JD and Marks JR
Immune response to p53 is dependent upon p53 / HSP70 complexes in ~ breast cancers. Proc Natl. Acad. Sci USA, 89: 3439-3442, 1992.
~ 19. Soussi T., Caron de Fromentel C., StYrzbecher HW, Ullrich S., Jenkins J. and May P.
Evolutionary conservation of the biochemical ~ properties of p53- specific interaction of xenopus-laevisjp53 with simian virus 40 large ~ -antigen and mammalian heat shock proteins- 70. J. Virol., 63 3894-3901, 1989.
20. Towbin H., Staehelin R. and Gordon J.
Electrophoretic ~ transfer of proteins from polyacrylamide gels to nitrocellulose sheets:
20 ~ procedure and ~ somè applications. Proc Natl Acad. Sci.
USA,; ~ 76: 4350-4354, 1979.
21. Gillet D., Ducancel F., Pradel E., Léonetti M., Ménez A. and Boulain JC Insertion of a disulfide containing neurotoxin into E. coli alkaline 2 ~ 5; phosphatase: the hybrid protein retains both biologlcal actlvities. Protein Engng, 5: 273-278, 1992.
; 22. Banks L., Matlashewski G. and Crawford L.
Isolation of human-pS3 specific monoclonal antibodies - 30 and their use in the studies of human p53 expression.
Eur. J. Biochem., 159: -529-534, 1986.
23. Stephen CW and Lane DP Mutant conformation of p53- precise epitope mapping using a filamentous phage epitope library. J. Mol. Biol., ~ 25: 577-583, 1992.
, ., 2 1 4 8 ~ 7 4 34 24. Gurney EG, Harrison RO and Fenno J.
Monoclonal antibodies against simian vlrus 40 T
antigens; evidence for distinct subclasses of large T
antigen and for similarities among nonviral T
antigens. J. Virol., 34: 752-763, 1980.
25. Midgley CA, Fisher CJ, Bartek J.
Vojtesek B., Lane D. and Barnes D. Analysis of p53 expression in human tumors: an antibody raised against human p53 expressed in E. coli. J. Cell Science, 101:
183-189, l9g2.
26. Soussi T., Caron de Fromentel C. and May P.
Structural aspects of the p53 protein in relation to gene evolution. Oncogene, 5: 945-952, 1990.
27. Harlow E., Williamson NM, Ralston R., Helfman DM and Adams TE Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53. Mol. Cell. Biol. 5: 1601-1610, 1985.
28. Harlow E. and Lane D. (1988) Antibodies, A
laboratory manual. Cold Spring Harbor Laboratory Press, New York.
29. Maniatis T., Fr ~ tsch EF and Sambrook, J.
(1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New York.
30. Wade-Evans A. and Jenkins JR Precise epitope mapping of the murine transformation -associated protein, p53. The EMBO Journal 4, N'3, 699-706, 1985.

214827 ~ ~
WO94 / 10306. PCl ~ / FR93 / 01082 ~: ~
-;

LIST OF SEQUENCES

(1) GENERATE INFORMATION ~ LE ~
(i) DEPOSITOR ~
(A) NAME: EUROBIO LABORATORIES ~.
(B) STREET: 7, Avenue de Scandinavie --- ~
(C) CITY: LES ULIS CEDEX ~ ~.
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 91953:
(G) TELEPHONE: 69 07 94 77 (H) FAX: 69 07 95 34 (I) TELEX: 681 425 F
(ii) TITLE OF IN ~ ENTION: FRAGMENTS OF THE

AND TRACKING PATHOLOGICAL CONDITIONS
~ iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER-READABLE FORM: -(A) TYPE OF SUPPORT: Floppy disk (B) COMPUTER- IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release ~ 1.0, Version ~ 1.25 (EPO) (vi) DATA FROM THE PREVIOUS APPLICATION:
(A) DEPOSIT NUMBER: FR 9213110 (B) DEPOSIT DATE: 02-NOV-1992 (2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear:
(ii) TYPE OF MOLECULE: peptide;
(vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens I -(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser Pro Leu WO94 / 1 ~ 306 21 ~ 8 2 7 4 PCT / FR93 / 01082 ~;

(2) INFORMATION FOR SEQ ID NO: 2 ~
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid -(C) NUMBER OF STRANDS: simple.
(D3 CONFIGURATION: linear tii) TYPE OF MOLECULE: peptide ~ vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:;
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln Trp Phe ~ Thr Glu Asp Pro Gly Pro.

(2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:.
(A) LENGTH: 15 amino acids.
(B) TYPE: amino acid:.
(C) NUMBER OF STRANDS: simple ~ -.
(D) CONFIGURATION: linear: .. `
(ii) TYPE OF MOLECULE: peptide (vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE :: SEQ ID NO .: 3:
Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu 1 ~ -(2) INFORMATION FOR SEQ ID NO: 4: ~.
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide WO44 / 10306 21 4 ~ 2 7 4 PCT / FR93 / 01082. ~
37 ~:

:.
~ vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn (2) INFORMATION FOR SEQ ID NO: 5:
ti) CHARACTERISTICS OF THE SEQUENCE: - ~
(A) LENGTH: 15 amino acids ~ B) TYPE: amino acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear. ~.
(ii) TYPE OF MOLECULE: peptide . - .- ~
tvi) ORIGIN:
(A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu ~.

Ser Pro Leu. ~
~:
-:.
(2) INFORMATION FOR SEQ ID NO: 6:
..-. . ~.
(i) CHARACTERISTICS OF THE SEQUENCE:: ::
(A) LENGTH: 15 amino acids (B) TYPE: amino acid. :.
(C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Asp Asp Leu Met Leu Ser Pro Asp Asp Ile Glu Gln 5 10., `
Trp Phe Thr 2148 274 38 ~

(2) INEORMATION FOR SEQ ID NO: 7 ~
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid - (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear ~:
(ii) TYPE OF MOLECULE: peptide: ~
(vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:. ~: ~
Ser Pro Asp Asp Ile Glu Gln Trp Phe Thr Glu Asp .:

Pro Gly Pro .:
(2) INFORMATION FOR SEQ ID NO: 8::
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids.
(B) TYPE: amino acid (C) NUMBER OF STRANDS: simple :: ~
(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide; :: -(vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens '' - (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly -`

(2) INFORMATION FOR SEQ ID NO: 9:: ~
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid ~ C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGIN:

'' (A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser 1 5 ~ 10 Arg Ala His (2) INFORMATION FOR SEQ ID NO: 10::: ~
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LONGUEVR: 15 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS ~ .simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide (vi) ORIGIN:. :
(A) ORGANIZATION: Homo sapiens ::

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: `; ~
Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser l 5 10 His Leu Lys (2) INFORMATION FOR SEQ ID NO: 11:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) NUMBER OF STRANDS: single (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide ~ vi) ORIGIN:
(A) ORGANISM: Homo sapien ~

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
Gly Ser Arg Ala His Ser Ser His Leu Lys Ser Lys l 5 10 Lys Gly Gln _ 15 WO94 / 10306 214 8 2 7 ~ PCT ~ FR93 / 01082 ~: S

(2) INFORMATION FOR SEQ ID NO: 12:
(i) CHARACTERISTICS OF THE SEQUENCE:.
(A) LENGTH: 15 amino acids (B) TYPE: amino acid ::
(C) NUMBER OF STRANDS: single (D ~ CONFIGURATION: linear,.
(ii ~ TYPE OF MOLECULE: peptide (vi) ORIGIN::
(A) ORGANIZATION: Homo sapiens (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12 ~
Ser Ser His Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His ~ -t2) INFORMATION FOR SEQ ID NO: 13:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids: ~ E `:
~ B) TYPE: amino acid (C) NUMBER OF STRANDS: simple:
(D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide ~;
(vi) ORIGIN:; :
(A) ORGANISM: Homo sapiens ~. :

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:
Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys 1 5 10 '' Leu Met Phe 1 ~,.:
: ~:
(2) INFORMATION FOR SEQ ID NO: 14:: ~
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids:
(B) TYPE: amino acid (C) NUMBER OF STRANDS: single (OF CONFIGURATION: linear (ii) TYPE OF MOLECULE: peptide WO94 / 10306 21 4 8 2 7 4 PCT ~ FR93 / 01082 (xi ~ SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Ser Thr Ser Arg His Lys Lys Leu Met Phe Lys Thr Glu Gly Pro (2) INFORMATION FOR SEQ ID NO: 15:
(i) CHARACTERISTICS OF THE SEQUENCE:: ::
(A) LENGTH: 13 amino acids:
(B) TYPE: amino acid (C) NUMBER OF STRANDS: single. ~:
(D) CONFIGURATION: linear ~: ~
(ii) TYPE OF MOLECULE: peptide ~:

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15:
Lys Lys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser: ~

Asp ::

Claims (19)

42 1. Peptide, excluding p53 protein, showing a specific reaction to of anti-p53 antibodies present in the fluids biologics of patients suffering from cancer or precancerous. 2. Peptide according to claim 1, characterized in that the antibodies are those of patients with cancer regardless of its origin. 3. Peptide according to one of claims 1 and 2, characterized in that it is included in the sequence of amino acids 1 to 112 of the p53 protein. 4. Peptide according to one of claims 1 to 3, characterized in that it comprises in part or in one of the following sequences:
(SEQ ID NO:1) (SEQ ID NO:2). 5. Peptide according to one of claims 1 to 4, characterized in that it comprises in part or in one of the following sequences:
(SEQ ID NO:3 ) (SEQ ID NO:4) (SEQ ID NO: 5) (SEQ NO:6) (SEQ ID NO:7). 6. Peptide according to one of claims 1 and 2, characterized in that it is included in the sequence of amino acids 350 to 393 of the protein p53. 7. Peptide according to one of claims 1, 2 and 6, characterized in that it comprises in part or in one of the following sequences:
(SEQ ID NO:8) (SEQ ID NO:9) (SEQ ID NO:10) (SEQ ID NO:11) (SEQ ID NO:12) (SEQ ID NO:13) (SEQ ID NO:14) (SEQ ID NO:15). 8. Nucleotide sequence coding for the synthesis of a peptide according to one of claims 1 at 7. 9. Use of a peptide according to one of claims 1-7 for detection or tracking pathological states. 10. Use according to claim 9 for detecting or monitoring the evolution of states cancerous and pre-cancerous. 11. Method for determining the presence or for the determination of antibodies in a fluid or fabric characterized in that:

- the fluid or tissue is brought together with one peptides according to one of claims 1 to 7, and - the presence of either the antibody is determined either of the peptide or of the antibody-peptide couple and one performs the corresponding dosage. 12. Method according to claim 11, characterized in that the antibodies to be assayed or whose determines the presence are antibodies representative of pathological states. 13. Method according to one of claims 11 and 12, characterized in that said peptide is attached on a solid phase. 14. Method according to one of claims 11 to 13, characterized in that the complexes formed are revealed using an antibody reacting specifically with the antibodies to be assayed or whose determines presence. 15. Process according to claim 14, characterized in that the antibody allowing the revelation of the complexes is conjugated to a label. 16. Process according to claim 15, characterized in that the marker is an enzyme, a chemiluminescent molecule, bioluminescent, fluorescent or radioactive. 17. Box for dosing and determination antibody, characterized in that it comprises at less :
- a preparation of one or more peptides according to one of claims 1 to 7, and - a means allowing the revelation of the reaction between one of these peptides and the antibodies to dose . 18. Box according to claim 17, characterized in that the peptide-antibody complex formed during the reaction is revealed by an antibody conjugated to a label. 19. Box according to claim 18, characterized in that the marker is an enzyme, a chemiluminescent molecule, bioluminescent, fluorescent or radioactive.