FR2698367A1 - Fragments of the p53 protein and their uses in the detection and monitoring of disease states. - Google Patents

Abstract

Peptide, fragment of the p53 protein, exhibiting a specific reaction to anti-p53 antibodies present in the biological fluids of patients suffering from cancer or pre-cancer. Preferably, this peptide is included in the sequence of amino acids 1 to 112 of the p53 protein. It can be used for the detection or monitoring of pathological conditions, in particular cancerous conditions.

Description

The present application relates to fragments of the p53 protein
It is also related to their uses in the detection and monitoring of pathological conditions and in particular cancer and pre-cancerous conditions.

 The p53 gene mutations are the most frequently encountered gene alterations in human cancers. The frequency of mutations of the p53 gene is high for colon (1), breast (2) and lung (3) cancers, as well as in leukemias (4), osteosarcomas (5), cancers of the ovary (6), stomach (7), and brain (8). Hereditary mutations have recently been shown to support the Li-Fraumeni family cancer syndrome (9,10).

When compared to the 10 most common cancers in the world, p53 alterations are found in 40 to 45% of cancer patients. The most frequent alterations are point mutations located in 4 of the 5 HCD regions of the p53 protein (11,12).

 Ten years ago, Benchimol et al. have shown by radioimmunoassay that the p53 protein is specifically overexpressed in transformed cells and undetectable in normal cells (13). Numerous studies have since confirmed these results and showed that the accumulation of the p53 protein is due to its stabilization.

It is now known that this stabilization is due in almost all cases to a point mutation which modifies the conformation and the stability of the protein. These observations encourage the systematic immunohistological study of p53 protein expression on a wide range of tumors as there appears to be a good correlation between p53 gene mutation and protein accumulation (14, 15).

 In 1984, Crawford et al. detected anti-p53 antibodies in patients with breast cancer (16).

Caron de Fromentel et al. then showed that such antibodies were present in the serum of children with very diverse cancers (17). The mean frequency is 12% with a particular frequency of 20% in
Burkitt (17). More recently, Davidoff et al (18) have shown that the presence of anti-p53 antibodies is associated with specific mutations of exons 5 and 6 of the gene. These mutated proteins are known to associate with the hsp70 protein and are even more oncogenic in vitro and in vivo.

 The Applicant has shown that there is a correlation between the clinical stage of breast cancer and the presence of anti-p53 antibodies in the serum.

He has also surprisingly shown that only certain parts of the p53 protein are involved in the reaction between the antibodies of patients with cancer and this protein.
The subject of the present invention is peptides having a specific reaction with respect to antip53 antibodies present in the biological fluids of precancerous or cancer patients, preferably breast cancer, lung cancer or liver cancer. Prostate, ovarian or leukemia
Advantageously, such a peptide is included in the amino acid sequence 1 to 112 of the p53 protein. The sequence of the p53 protein has been described by Harlow et al (31).

It advantageously comprises in part or in full one of the following sequences
EPPLSQETFSDLWKLLPENNVLSPL DDLMLS PDD I EQWFTEDPGP
Preferably, such a peptide comprises in part or in full one of the following sequences
EPPLSQETFSDLWKL
QETFSDLWKLLPENN
DLWKLLPENNVLSPL DDLMLS PDD I EQWFT
SPDDIEQWFTEDPGP
The determination of the fragments of the p53 protein exhibiting a specific reaction with respect to the antibodies of cancer patients may be carried out by the means made available to those skilled in the art and in particular by radioimmunoassay, radio immunologically, by immunoprecipitation or by Western-Blot.

 Such tests are well known to those skilled in the art and are described in general manuals (32, 33).

The present invention further relates to a nucleotide sequence, ribonucleotide (RNA) or deoxyribonucleotide (DNA), coding for the synthesis of one of the peptides subject of the present application
Such peptides may be used for the detection or monitoring of pathological conditions, and in particular for the detection or monitoring of cancerous and precancerous conditions.

Another subject of the present invention is a method for determining the presence of antibodies or for their determination in a tissue or a fluid, characterized in that
the tissue or the fluid is brought into contact with one of the peptides which are the subject of the invention described above, and
the presence of either the antibody, the peptide or the antibody-peptide pair is determined and the corresponding assay is carried out
The assay of the antibody-peptide complexes can be carried out either by assaying the antibody peptide binding or by assaying the peptide or the antibody captured and entering the antibody-peptide complex.

 This assay can be carried out by methods known to those skilled in the art and in particular by physical or immunological methods such as immunoenzymological, radioimmunological or chemo-immunological methods.

 Advantageously, the antibodies whose presence is determined or that are measured are antibodies representative of pathological conditions and in particular of cancerous or pre-cancerous states.

 In order to carry out the method, the peptide can be fixed on a solid phase.

The complexes formed can be revealed by means of an antibody reacting specifically with the peptide or antibodies to be assayed or detected. This antibody allowing the development of the complexes is advantageously conjugated to a marker in order to demonstrate the antibody-peptide complex.
Such a marker can be an enzyme, a chemiluminescent, bioluminescent, fluorescent or radioactive molecule.

Such a method can advantageously be implemented using a kit for assaying and determining antibodies comprising at least:
a preparation of one or more of the peptides as previously described and
a means allowing the revelation of the reaction or the absence of reaction between one of these peptides and the antibodies to be assayed
The peptide antibody complex formed during the reaction is advantageously revealed using an antibody conjugated to a label as defined above.

The subject peptides of the present invention are preferably obtained by synthesis from amino acids by any means known to those skilled in the art.
They can also be obtained by genetic engineering or by enzymatic or chemical cutting of the p53 protein.

 The antibodies allowing the development of the complex formed between the peptide and the antibodies are advantageously directed against antigenic determinants specific to the species from which the fluid or tissue originates. Thus, in the context of assaying antibodies of human origin, the antibodies allowing the development of the complex will be anti-human antibodies.

The present invention is illustrated without being limited by the following examples in which
Figure 1 shows the interaction profiles between the fragments of the p53 protein and antibodies present in the serum of different patients with cancer. The antibodies used for the hybridizations of FIGS. 1A to 1F are, respectively, a rabbit serum immunized with the p53 protein, a mouse serum immunized with the p53 protein, three sera from patients which had been determined to possess anti-inflammatory antibodies. -p53 and a control (Figure 1) constituted an anti-alkaline phosphatase antibody.

Figures 2 to 7 show the reactivity of seven peptides of the portion comprising amino acids 1 to 112 of the p53 protein, vis-à-vis sera of six patients numbered 84, 37, 109, 57, 27 and 29. The The reaction is evidenced by measuring the optical density of the reaction product of each peptide-serum pair.
The following abbreviations are used
ER, estrogen receptor
HCD, Highly conserved domain of p53 hsp70 protein, 70 kilodalton heat shock protein
MAB, monoclonal antibody
PBS, phosphate buffer
PBST, PBS with 0.1% Tween 20
PBSTM, PBS with 0.1% Tween 20 and 3% skim milk powder
PCR, polymerase chain reaction.

PhoA, E.coli alkaline phosphatase
PR, progesterone receptor
SDS, sodium dodecyl sulfate
EXAMPLE 1
Demonstration and significance of anti p53 anticorPs present in the serum of cancer patients and localization of the regions of the p53 protein recognized by these antibodies.

1) MATERIALS AND METHODS
SERA.

 Sera of 80 patients with invasive carcinoma of the breast and 20 patients with metastases were reunited after histopathological diagnosis and stored at 20C.

Analysis of the p53.

 Wild type human p53 cDNA (clone H8) was donated by Dr. Varda Rotter (Weizman Institute, Rehovot, Israel).

 The labeled p53 protein is obtained by transcription and translation in vitro of the corresponding cDNA, as described by SOUSSI et al (19).

The Western blot was performed as described by Towbin et al. (20). The proteins are separated by electrophoresis in polyacrylamide gel (10% acrylamide) in Tris-Glycine buffer containing 0.1% of SDS, pH 8.3. The proteins are transferred to a nitrocellulose membrane (2 hours, 40 volts); the membrane is immersed in PBSTM buffer to saturate the nonspecific binding sites and then washed 5 times with PBST. For the
Western blot of the patient's serum and E.coli extract, several pre-incubations are necessary to decrease non-specific binding to the nitrocellulose membrane and eliminate the anti-phosphatase antibodies present in some sera. first incubated with the bacterial extract expressing alkaline phosphatase E.coli for one hour at +4 C and then on a nitrocellulose membrane containing a bacterial extract (without fusion protein) for 12 hours at +4 C. The nitrocellulose membrane with the hybrid proteins is incubated with the sera prepared and diluted 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 peroxidase conjugated rabbit anti-mouse antibody (diluted 1/4000 in PBSTM buffer), washed 5 times in PBS buffer and revealed by Luminol (AmershamR).

After reading and interpretation, the filters are monitored by immunological hybridization (Immunoblot) for the amount of fusion protein actually deposited by incubating the membrane with an anti-alkaline phosphatase antibody.

 Immunoprecipitation and SDS polyacrylamide gel electrophoresis (SDS-PAGE) were performed according to the technique described by SOUSSI et al (19).

 Plasmid construction.

 The vector pLip4 was used for the production of hybrid proteins. This plasmid is derived from pLipl (21) in which two Sac I and Sal I restriction sites 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 cold osmotic shock (21).

The p53 protein has been decoupled into 6 well-defined fragments. Fragments 2 (residues 108 to 162), 3 (residues 158 to 219), 4 (residues 215 to 267) and 5 (residues 263 to 310) respectively contain regions HCD II to V and correspond to regions HOTSPOT for mutations in human cancers (11,22). Fragments 1 (residues 1 to 112) and 6 (residues 306 to 393) correspond to the amino and carboxy-terminal regions of the protein and are generally isolated from mutations. Precise cDNA fragments were amplified by PCR using Vent-DNA polymerase to reduce incorporation errors and then subcloned into a pLIP4 vector. Positive clones are monitored for alkaline phosphatase activity and sequenced directly. The six fragments fused to the PhoA gene can be expressed in E. coli as soluble proteins. The expression efficiency of fusion protein with fragment 6 is lower (2- to 5-fold) than for the other fragments
The antigenicity of the expressed protein is established by its reactivity with different monoclonal antibodies. (see Table 2).

 2) RESULTS.

Anti-p53 antibodies and clinical results.

Sera from 100 patients with breast cancer are tested for the presence of antibodies by immunoprecipitation and
Western blot (Table 1). Circulating antibodies are detected in 14% of patients at all clinical stages.

 The frequency is slightly higher than that reported by Crawford et al. (10%) (16), probably because of the use of recombinant purified p53 protein instead of cellular lysate.

 There is no correlation between the presence of antibodies and the clinical stage of the disease. No difference in frequency as a function of tumor size or lymph node dissemination has been shown. There is no correlation with other clinical parameters such as age, hormonal status. On the other hand, a correlation has been established with the histological stage of the tumor (SBR stage 3), an independent prognostic marker in primary breast cancers. This is confirmed by the association between the presence of antibodies and the absence of hormone receptors (ER-PR).

Analysis of the recursions of the p53 protein involved in the immune response.

 To determine whether the immune response is directed against mutant or wild-type p53, all positive sera and several negative sera are monitored by immunoprecipitation against different p53 mutants (His175, Val 143 and Pro156). The recognition of wild and mutant types by sera is identical.

 To determine the regions of the protein recognized by these antibodies, the human p53 fragments are expressed in the pLIP4 vector producing alkaline phosphatase soluble fusion proteins (21). In a control experiment, all fusion proteins are tested with monoclonal antibodies specific for known epitopes (Table 2). PAbl801 (23) and HT 216 (24) are specific for fragment 1; the PAb240 epitope is located on fragment 3 (25). Hlll is on fragment 5; HR 231 (24) and PAb122 (26) are specific for the carboxy-terminal region of p53 (fragment 6). All these monoclonal antibodies react in immunoblot and immunoprecipitation with the pLIP4 fusion protein in accordance with the localization of their epitope.

In a second series of experiments, serum CM1 (27) was monitored. CM1 is a rabbit polyclonal antibody obtained by immunization with highly purified human p53 and frequently used in immunocytochemistry (27). Western blotting techniques have shown that CM1 serum contains antibodies that primarily recognize fragments 1 and 6 and to a lesser extent fragment 5. Similar results are obtained by immunoprecipitation
A control experiment with an anti-alkaline phosphatase antibody makes it possible to establish that identical quantities of the different fusion proteins are indeed present on the gel (FIG. 1). To expand observations we monitored the serum of a mouse immunized with immunopurified p53.

The recognition profile is almost identical to that of serum CM1. Identical results were obtained with three other mice.

 All of these results make it possible to establish that the amino and carboxy terminal parts of the p53 protein are highly immunogenic in the immunized animal and correspond to preferential responses while the fragment 5 has a lower response. Elongated radiographic exposure times failed to demonstrate recognition of fragments 2 to 4 by both immunoprecipitation and immunohybridization (immunoblotting).

 We then tested the recognition profile of the sera of patients containing anti-p53 antibodies (Figure 1 and Table 3). The immuno hybridizations carried out clearly showed that the immune response in the patients is directed mainly against localized epitopes in the fragments. 1 and 6 of the p53. The serum of some patients recognizes only fragment 1, but none recognizes only fragment 6, suggesting that the primary response is mainly directed against fragment 1. Similar results are obtained by immunoprecipitation. As in animals, there are some variations in the immune response and some patients have antibodies against fragments 4 and 5. No antibodies directed against fragments 2 and 3 could be detected. This phenomenon is not specific to breast cancer. Similar results were obtained with sera from patients with carcinomas of the ovary or lung (Figure 1).

 These results clearly establish that the amino terminal part of p53 contains one or more dominant epitopes involved in the B cell immune response in patients with various cancers.

EXAMPLE 2
Demonstration of epitopes recognized by the anti-p53 antibodies present in the serum of patients suffering from various types of cancer: analysis with synthetic peptides
Equipment
The microtiter plates used are flat-bottomed, 96-well poly (vinyl chloride) plates.

Ref: M129B, Dynatech Company.

Streptavidin powder, reference (4762), Company
Sigma.

Biotinylated peptides synthesized by the CAT Company (England)
A series of overlapping peptides (length of 15 amino acids (aa), 5 aa overlap) covering the entire p53 protein. There is currently a series of 75 biotinylated peptides corresponding to the entire p53. 2 control peptides not corresponding to the p53 protein are used as a negative control.

 Peptides were synthesized by CAT (England).

 Amersham Enzyme Immunoassay Kit, N933.

METHODS OF THE ELISA TESTING
PREPARATION OF MICROTITRATION PLATES.

 100 μl of streptavidin (concentration 5 μg / ml in distilled water) are deposited in the wells of the plate.

 The plates are then incubated at 37 ° C. in a dry oven for 48 hours. After this dehydration step, they can be individually wrapped and stored for several months at 4 C.

WASHING
The dried plates are washed 6 times with PBS (phosphate buffer saline) containing 0.5% Tween 20. This buffer
PBS with Tween (PBS-T) will be used throughout the experiment as a wash solution. The first wash is always by immersion of the plate in the solution of
PBS-T (300 ml solution for 1 plate). The following 5 washes are done in the usual manner using 200 μl of PBS-T buffer per well.

After the last wash, the plate is dried on an absorbent paper
All washes are done this way.

SATURATION
100 μl of saturation solution is added to each well.

saturation solution: PBS phosphate buffer
0.2% Tween 20
10% goat serum (Ref.

J9023, Sigma)
The plates are then incubated for 1 hour at 37 ° C. with slight stirring and are then washed as indicated previously.

ADDITION OF BIOTYNILIC PEPTIDES
50 μl of peptide solution (125 mg of 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 the p53.

The plates are incubated for 1 hour at 20 ° C. with gentle stirring and are then washed as indicated previously.
ADDITION OF PATIENT SERUMS
The sera to be tested are diluted 1/50 in a solution of PBS containing 10% goat serum.

 50 μl of serum thus diluted are added to each well of the plate.

 The plates are incubated for 1 hour at 20 ° C. with gentle stirring and are then washed as indicated previously.

INCUBATION WITH SECONDARY ANTIBODY.

100 l of monoclonal anti-human immunoglobulin antibody are added to each well (source: kit
Amersham N 933, antibody diluted 1/1000 in PBS 10% goat serum).

 The plates are incubated for 30 minutes at 37 ° C. with gentle stirring and are then washed as indicated previously.

 A seventh wash with distilled water is performed.

REVELATION.

100 μl of substrate (ABTS 1 mM, from: Amersham kit
N 933) are added to each well.

 The plates are incubated at 20 ° C. with gentle stirring.

The result is read in an ELISA reader (405 nm) 15 minutes, 30 minutes and 60 minutes after addition of the substrate
RESULTS OBTAINED
In example 1 two phenomena were highlighted.

 i) The presence of anti-p53 antibodies is found in patients with an aggressive form of breast cancer (poor prognosis).

 ii) Anti-p53 antibodies mainly recognize a region of 112 amino acids located in the amino-terminal part of p53. The study could not allow to detail more precisely the region recognized by the anti-p53 antibodies.

 For a more detailed study, 15 residue synthetic peptides were used as a substrate to map the epitopes present in these sera.

 The serum of 12 patients with various types of cancers were tested by this ELISA-peptide to search for the exact nature of the p53 protein sequences recognized by the anti-p53 antibodies present in these sera.

patient 84 cancer: lung patient 132 cancer: lung patient 37 cancer: liver patient 109 cancer: breast patient 57 cancer: breast patient 103 cancer: breast patient 15 cancer: breast patient 27 cancer: breast patient 28 cancer: breast patient 29 cancer: breast patient 48 cancer: patient leukemia 51 cancer: prostate
Each ELISA was performed twice except for serum 84 where it was performed 5 times in order to verify the reproducibility of the results obtained.

 The results obtained for respectively the sera of patients 84, 37, 109, 57, 27 and 29 are shown in the profiles of FIGS. 2 to 7.

 They are summarized for the first 20 p53 peptides in Table 4.

 All the results show that the antibodies are directed against epitopes very localized on the p53 molecule and more particularly in the amino terminal region of the protein.

The sequences recognized mainly by the antibodies are the following: Peptide 3 EPPLSQETFSDLWKL peptide 4 QETFSDLWKLLPENN peptide 5 DLWKLLPENNVLSPL peptide 9 DDLMLSPDDIEQWFT peptide 10 SPDDIEQWFTEDPGP
All sera tested at present recognize at least three of these peptides.

TABLE 1
Correlation between anti-p53 antibody level
in serum and clinical parameters and
histological findings of patients with breast cancer
NP clinical parameters
Patients with 3/20 metastases or who have relapsed primary breast cancer 17/80
Tumor size T-T1 2/21
T2 7/33 NS
T3 2/19
T4 1/7
State of the lumpy nodules
No 4/32 NS
N- 7/43 Histological category categories 1 and 2 5/59 <0.05 category 3 6/17
State of the hormonal receptors
ER + PR +
ER- PR + 2/34 <0.05 ER + PR
ER- PR- 5/13
NS = not significant TABLE 2
Characterization of p53 fragments cloned into plasmid pLIP4
Fragments of p53
1 2 3 4 5 6
Antibody Ref. (1-112) (108-162) (158-219) (215-267) (263-310) (306-393) monoclonal antibodies
PAb1801 23 + - - - -
PAb122 26 - - - - - +
PAb240 25 - - + - -
HR231 24 - - - - - +
HT216 24 + - - - -
HI11 24 - - - - + - TABLE 3
Characterization of the antigenic determinants of p-53 by
serum antibodies of patients with carcinoma
Patients (1) p53 (2) Prot 1 Prot 2 Prot 3 Prot 4 Prot 5 Prot 6 PhoA
26 ++ ++ - - - + +
33 ++ ++ - - - - +
37 (3) ++ ++ - - - - -
57 ++ ++ - - + ++ -
60 ++ ++ - - - - -
63 ++ + - - - - -
80 (4) ++ ++ - - - - -
84 (3) ++ ++ - - - - +
90 ++ ++ - - - - -
94 ++ ++ - - - - ++
103 ++ + - - - - -
109 ++ ++ + - + - +
134 ++ ++ - - + - -
Prot = protein fragment.

(1) All patients have breast cancer unless otherwise indicated (2) p-53 intact human (3) lung carcinoma (4) ovarian carcinoma.

TABLE 4
Immunological reactivity of the first 20 peptides
straddling the p53 protein

<SEP> Peptide <SEP> 84 <SEP> 132 <SEP> 37 <SEP> 109 <SEP> 57 <SEP> 103 <SEP> 15 <SEP> 27 <SEP> 28 <SEP> 29
<tb> 1 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> ++ <SEP> - <SEP> - <SEP>
<tb><SEP> 3 <SEP> ++ <SEP> + <SEP> - <SEP> ++ <SEP> - <SEP> + <SEP> + <SEP> + <SEP> + <SEP> +
<tb><SEP> 4 <SEP> ++ <SEP> ++ <SEP> + <SEP> + <SEP> ++ <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + +
<tb><SEP> 5 <SEP> - <SEP> ++ <SEP> - <SEP> + <SEP> ++ <SEP> - <SEP> - <SEP> - <SEP> - <SEP> ++
<tb><SEP> 6 <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<tb> 7 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP>
<tb><SEP> 8 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> +
<tb><SEP> 9 <SEP> ++ <SEP> - <SEP> ++ <SEP> ++ <SEP> ++ <SEP> - <SEP> + <SEP> ++ <SEP> + <SEP > ++
<tb><SEP> 10 <SEP> ++ <SEP> - <SEP> + <SEP> + <SEP> ++ <SEP> + <SEP> + <SEP> ++ <SEP> + <SEP> +
<tb><SEP> 11 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> +
<tb><SEP> 12 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<SEP> 13 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<tb><SEP> 14 <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<tb> 15 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP>
<tb><SEP> 16 <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<tb><SEP> 17 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<tb> 18 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<tb> 19 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP>
<tb> 20 <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> + <SEP> + <SEP> +
<Tb>
BIBLIOGRAPHY
1 Baker SJ, Preisinger AC, Jessup JM, Paraskeva C., Markowitz S., Willson JKV, Hamilton
S.and Vogelstein B. P53 gene mutations occur in combination with allelic deletions and late events in colorectal tumorigenesis. Cancer Res., 50: 7717-7722, 1990.

 Prosser J., Thompson A., Cranston, G.and Evans, H. J. Evidence that p53 behaves as tumor suppressor gene in sporadic breast tumors. Oncogene, 5: 1573-1579, 1990.

3 Takahashi T., Nau MM, Chiba I., Birrer MJ, Rosenberg RK, Vinocour M., Levitt M., Pass H.,
Gazand AFand Minna JD P53 - a frequent target for cancer abnormalities in lung cancer. Science, 246: 491-494, 1989.

Gaidano G., Ballerini P., Gong JZ, Inghirami G., Neri A., Newcomb EW, Magrath IT, Knowles
DMand Dallafavera R. P53 mutations in human Iymphoid malignancies - association with Burkitt
Iymphoma and chronic Iymphocytic leukemia. Proc Natl Acad Sci USA, 88: 5413-5417, 1991.

Miler 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., Jeaneur P.and Theillet C. P53 mutations in ovarian cancer - A late event. Oncogene, 6: 1685-1690, 1991.

 Kim J. H., Takahashi T., Chiba I., Park J.G., Birrer M.J., Roh J.K., Lee H.D., Kim J.P., Minna J.

D.and Gazdar AF Occurrence of p53-gene abnormalities in gastric carcinoma tumors and cell lines. J Nat
Cancer Inst, 83: 938-943, 1991.

 8 Mashiyama S., Murakami Y., Yoshimoto T., Sekiya T.and Hayashi K. Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene, 6: 1313-1318, 1991.

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

 S. Srivastava, Zou Z. Q., Pirollo K., Blattner W. and Chang E. H. Germ-line transmission of a mutated p53 gene in a cancer-prone family with li-fraumeni syndrome. Nature, 348: 747-749, 1990.

 He Caron of Fromentel C.and Soussi T. TP53 Tumor suppressor gene: a model for investigating human mutagenesis. Genes Chrom Cancer, 4: 1-15, 1992.

 12 Hollstein M., Sidransky D., Vogelstein B.and Hardis C. C. P53 mutations in human cancers.

Science, 253: 49-53, 1991.

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

 14 Hall P.A., Ray A., Lemoine N.R., MidgleyC.A., Krausz T.and Lane D.P. P53 Immunostaining as a marker of malignant disease in cytopathology. Lancet, 338: 513, 1991.

 Cattoretti G., Rilke F., Andrealo S., D'amato L.and Delia D. P53 expression in breast cancer. Int.

J. Cancer, 41: 178-183, 1988.

 Crawford L.V., Pim D.C. and Bulbrook R.D. Detection of antibodies against the cellular protein p53 will be from patients with breast cancer. Int. J. Cancer, 30: 403-408, 1982.

 17 Fromentel Caron C., May-Levin F., Mouriesse H., Lemerle J., Chandrasekaran K.and May P.

Presence of circulating antibodies against cellular protein p53 in a significant proportion of children with B-cell
Iymphoma. Int. J. Cancer, 39: 85-189, 1987.

 18 Davidoff A.M., Iglehart J.D.and Marks J.R. Immune response to p53 Is dependent on p53 / HSP70 complexes in breast cancers. Proc Natal Acad Sci USA, 89: 3439-3442, 1992.

 19 Soussi T., Caron Fromentel C., Strzbecher H.W., Ullrich S., Jenkins J.and May P.

Evolutionary conservation of the biochemical properties of p53 - specific interaction of xenopus-laevis p53 with simian virus 40 broad T-antigen and mammalian heat shock proteins-70. J. Virol., 63: 3894-3901, 1989.

 Towbin H., Staehelin R. and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA, 76: 4350-4354, 1979.

 Gillet D., Ducancel F., Pradel E., Leonetti M., Ménez A.and Boulain J. C. Insertion of a disulfidecontaining neurotoxin in E. coli alkaline phosphatase: the hybrid protein retains both biological activities.

Protein Engng, 5: 273-278, 1992.

22 Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hostetter R., Cleary K., Bigner SH,
Davidson N., Baylin S., Devilee P., Glover T., FS Collins, Weston A., Modali R., Harris CCand
Vogelstein B. Mutations in the p53 gene occur in various human tumourtypes. Nature, 342: 705-708, 1989.

 23 Banks L., Matlashewski G. and Crawford L. Isolation of human-specific p53 monoclonal antibodies and their use in the studies of human p53 expression. Eur. J. Biochem., 159: 529-534. 1986.

24 Legros Y., Lacabanne V., Agay M., Larsen C., Pla M. and Soussi T. Isolation of human p53 specific monoclonal antibodies and their use in immunohistochemical studies of tumor cells. Bull. of
Cancer, in press: 1992.

 Stephen C. W.and Lane D. P. Mutant conformation of p53 - precise epitope mapping using a filamentous phage epitope library. Mol Mol, 225: 577-583, 1992.

 26 Gumey E.G., Harrison R. O.and Fenno J. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct subclasses of large T antigen and for similarities among nonviral T antigens.

J. Virol., 34: 752-763, 1980.

 Midgley C.A., Fisher C. J., Bartek J., Vojtesek B., Lane D.and Barnes D. Analysis of p53 expression in human tumors: an antibody raised agains human p53 expressed in E. coli. J. Cell Science, 101: 183-189, 1992.

 Davidoff A.M., Kerns B.J.M., Iglehart J.D.and Marks J.R. Maintenance of p53 alterations throughout breast cancer progression. Cancer Res, 51: 2605-2610, 1991.

 Hinds, P.W., Finlay, C.A., Quartin R.S., Baker S.J., Fearon E.R., Vogelstein, B.and, Levine, A.J.

Mutant p53 DNA clones from human colon carcinomas co-operate with ras in trasnforming primary rat cells: a comparison or kills "hot spot" mutant phenotypes. Cell Growth and Differentiation, 1: 571-580, 1990.

 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.

31 Harlow E., Williamson NM, Ralston R., Helfman DM, and Adams TE Molecular cloning and in vitro expression of a cDNA clone for human cellulotumorantigen p53. Mol. Cell. Biol. 5: 1601-16101985
32 Harlow E. and Lane D. (1988) Antibodies, A laboratory manual. Cold Spring Harbor Laboratory
Press, New York.

33 Maniatis T., Fritsch EF and Sambrook, J. (1982) Molecular cloning.A laboratory manual. Cold
Spring Harbor Laboratory Press, New York.

Claims (17)

 1. Peptide having a specific reaction against anti-p53 antibodies present in the biological fluids of cancer or precancerous patients.  2. Peptide according to claim 1, characterized in that the antibodies are those of patients suffering from cancer of the breast, liver, lungs, prostate, ovary or leukemia.  3. Peptide according to one of claims 1 and 2 characterized in that it is included in the amino acid sequence 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 full one of the following sequences  EPPLSQETFSDLWKLLPENNVLSPL  DDLMLSPDDIEQWFTEDPGP  5. Peptide according to one of claims 1 to 4, characterized in that it comprises in part or in full one of the following sequences  EPPLSQETFSDLWKL  QETFSDLWKLLPENN  DLWKLLPENNVLSPL DDLMLSPDD I EQWFT  SPDDIEQWFTEDPGP  6. A nucleotide sequence coding for the synthesis of a peptide according to one of claims 1 to 5.  7. Use of a peptide according to one of claims 1 to 5 for the detection or monitoring of pathological states  8. Use according to claim 7 for detecting or monitoring the evolution of cancerous states.  9. A method for determining the presence or for the determination of antibodies in a fluid or tissue characterized in that:  the fluid or tissue and one of the peptides according to one of claims 1 to 5 are brought into contact, and  the presence of either the antibody or the peptide or the antibody-peptide pair is determined and the corresponding assay is carried out  10. Process according to claim 9, characterized in that the antibodies to be assayed or whose presence is determined are antibodies representative of pathological states.  11. Method according to one of claims 9 and 10 characterized in that said peptide is fixed on a solid phase  12.Procédé according to one of claims 9 to 11, characterized in that the complexes formed are revealed with the aid of an antibody reacting specifically with the antibodies to be assayed or whose presence is determined.  13. The method of claim 11, characterized in that the antibody for revealing the complexes is conjugated to a marker  14.Procédé according to claim 13, characterized in that the marker is an enzyme, a chemiluminescent molecule, bioluminescent, fluorescent or radioactive  15. Box for the determination and determination of antibodies, characterized in that it comprises at least  - a preparation of one or more peptides according to one of claims 1 to 5, and  a means allowing the revelation of the reaction between one of these peptides and the antibodies to be assayed  16. The kit according to claim 15, characterized in that the peptide-antibody complex formed during the reaction is revealed by an antibody conjugated to a marker.  17. The kit according to claim 16, characterized in that the marker is an enzyme, a chemiluminescent molecule, bioluminescent, fluorescent or radioactive.