WO1996018906A1

WO1996018906A1 - Detection of anti-p53 antibodies in body fluids

Info

Publication number: WO1996018906A1
Application number: PCT/EP1995/004904
Authority: WO
Inventors: Mathias Montenarh
Original assignee: Orga-Med
Priority date: 1994-12-16
Filing date: 1995-12-12
Publication date: 1996-06-20
Also published as: DE4444969C1; AU4261496A

Abstract

The invention concerns a test system for detecting anti-wild-type or mutant p-53 antibodies in body fluids. The invention further concerns a process for diagnosing tumours by detecting wild-type or mutant p-53 antibodies in body fluids.

Description

Detection of antibodies against p53 in body fluids

The invention relates to systems for the detection of antibodies against wild-type and mutant p53 proteins in body fluids, in particular in human serum. Furthermore, it relates to methods for the detection of wild-type and mutant p53 protein.

p53 is the most common mutated cellular gene in human tumors. The p53 protein encoded by this gene, as a growth suppressor protein, plays a regulatory role in cell proliferation processes. p53 is involved in the control of the cell cycle, DNA repair and synthesis, cellular differentiation and programmed cell death. Genetic changes in this growth-regulating gene can have a serious influence on the expression of a malignant phenotype of the cell. Apparently, these mutations in the p53 gene cause the loss of the growth control function, and the cell gets into an uncontrolled proliferation as a result of this loss of function.

Expression of a non-mutated wild-type p53, on the other hand, leads to a significant inhibition of cell growth. p53 suppresses the transcription of numerous promoters, this suppression taking place independently of the sequence and possibly via the interaction of p53 with cellular transcription factors. p53 has a specific DNA binding activity and is able to stimulate the expression of different genes. The genes that are transactivated by p53 include the GADD45, MDM2 and the WAF1 gene. p53 binds to a large number of viral and cellular proteins. The interaction with the E6 proteins of the human papillomaviruses HPV16 and HPV18 is closely related to the occurrence of cervical carcinomas. The complex formation of p53 with the hepatitis B antigen plays a role in the formation of hepatocellular carcinomas. The association of p53 with viral proteins leads to inactivation of the growth suppressor property of p53. The association of p53 with the cellular mdm2 protein also inactivates the growth suppressor properties of p53. In the case of other cellular proteins such as, for example, the protein kinase CK2 or the p34cdc-2 kinase, the complex formation with p53 may have an influence on the growth-regulating activities of the p53 protein. p53 therefore carries out its growth-regulating functions via various interlocking mechanisms in which, in addition to p53, other cellular factors are involved.

Cells that are exposed to DNA-damaging influences react with a p53 accumulation in the cell nucleus. At the same time, a stop of the cell cycle can be observed in the GI phase of the cell cycle. Cells without endogenous p53 do not show such Gl arrest. Artificial overexpression of wild-type p53 induces either a growth stop of the cells in the Gl phase or the programmed cell death. The p53 increase and accumulation in the nucleus that can be observed after exposure to DNA-damaging agents and the simultaneous cell cycle arrest in the Gl phase speak for a role of p53 in DNA repair mechanisms.

On the other hand, p53 also appears to be directly and directly involved in the mediation of programmed cell death from apoptosis. Cells with only one copy of the p53 gene are much more resistant to radiation-induced apoptosis than cells with two intact copies of the p53 gene. Such a gene dose effect has serious consequences for patients with germline mutations in the p53 gene, such as Li Fraumeni syndrome patients. Cells in which a p53 allele is mutated or deleted have a survival advantage in the presence of genotoxic agents compared to cells with intact p53 gene copies. Both the growth arrest and thus the possibility of DNA repair before replication is carried out, as well as the triggering of apoptosis prevent the multiplication of a tumor cell and thus the manifestation of a malignant tumor.

The p53 protein is expressed in large amounts in many tumor cells. In many, but not all cases, the presence of increased amounts of p53 correlates with the presence of mutations in the p53 gene. In normal cells, on the other hand, the p53 protein is present in hardly detectable amounts. The p53 protein can be detected immunohistochemically in the tumor tissue using a number of different monoclonal antibodies.

DeLeo et al. (Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse. Proc. Natl. Acad. Sei. USA 76: 2420-2424, 1979) was able to show in 1979 that the humoral immune response in mice genes induced by methylcholanthrene is directed against p53. Crawford et al. (Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer. Int. J. Cancer 30: 403-408, 1982) described antibodies against p53 in the serum of breast cancer patients in 1982. Caron de Mentel et al. (Presence of circulating antibodies against cellular protein p53 in a notable proportion of children with B-cell ly phoma. Int. J. Cancer 39: 185-189, 1987) later found that p53 antibodies were also found in human human sera with a wide variety of different tumors. The frequency of an immune response against p53 was about 12% in these children, with about 20% in the case of Burkitt lymphoma Tumor patients had antibodies against p53.

Davidoff et al. (Immune response to p53 is dependent upon p53 / HSP70 complexes in breast cancers. Proc. Natl. Acad. Sci. USA 89: 3439-3442, 1992) showed in 1992 that the appearance of p53 autoantibodies in patients with breast cancer with a spe ¬ specific subclass of p53 correlated, which binds the 70kDa heat shock protein very efficiently. Patients with these autoantibodies also had p53 mutations in the tumor material.

Winter et al. (Development of antibodies against p53 in lung cancer patients appears to be dependent on the type of p53 mutation. Cancer Res. 52: 4168-4174, 1992) were able to show in 1992 that the development of autoantibodies against p53 in lung cancer patients from Type of p53 mutations in the tumor depended. Antibodies against p53 were detected both with the help of the unprecipitation and in the immunoblot. In primary breast cancer patients, approximately 15% of all patients had antibodies against p53. A correlation was also found between the occurrence of such antibodies and a poor prognosis, which could essentially be correlated with the histological degree and the absence of the hormone receptor.

Other studies have shown that the antibodies against p53 from human patient sera were able to recognize both wild-type and mutant p53. The B-cell response is essentially directed against the p53 protein with two immunodominant regions in the carboxy-terminal and in the amino-terminal region of the polypeptide chains of p53. In most cases, the presence of p53 autoantibodies correlates with p53 accumulation or with p53 mutations. There are a number of different experimental options for performing such serological analyzes.

WO94 / 10575 describes a method for the detection of cancer is known, wherein human serum samples are brought into contact with test compositions which each comprise a single mutant p53 protein or several different mutant p53 proteins.

In this way, specific p53 antibodies are detected in the serum through interaction with mutant p53 proteins. The mutant p53 protein used (m-p53) comes from bacteria, for example E. coli, or from viral vectors, for example vaccinia vectors. Accordingly, the various m-p53 proteins are isolated from different tumors and then cloned and expressed using known cloning methods. The expressed m-p53 proteins are isolated and used for the detection of antibodies.

Post-translational modifications of the polypeptide chain of p53 are obviously of great importance for the regulation of functions of p53. Such post-translational changes are experienced by p53 as a function of the cellular environment. Thus, wild-type p53 can adopt a different conformation in a tumor cell than in a normal, non-transformed environment. Mutant p53 behaves differently in a normal cell than in a tumor cell. p53, which is expressed in bacteria, undergoes a different post-translational modification than a p53 protein which is expressed in a mammalian cell. The different post-translational modifications of p53 have a decisive influence on the conformation of the protein and thus to a very large extent on the recognizability by p53-specific antibodies.

However, the method disclosed in WO94 / 10575 does not take into account such post-translational modifications of the polypeptide chain of p53. Proteins cloned and expressed in prokaryotes or Fre d eukaryotes often differ from the native conformation of the protein, as described in normal cell environment. The informative value of detection systems that use such recombinant p53 proteins is therefore limited.

WO 94/08241 discloses a method for the detection of p53-specific antibodies in body fluids, p53 bound to a carrier material and / or binding regions for p53-specific antibodies containing fragments thereof being incubated with body fluids and the specific p53 and / or the fragments bound antibodies (a) react with marked antibodies (b) directed against the antibodies (a) or with unlabeled antibodies (b) and the latter react with marked antibodies (c) directed against the antibodies (b) leaves. The label should be non-radioactive. It is pointed out that the use of cell extracts often leads to non-specific antibody binding.

WO 92/00311 discloses various classes of mutations in the human p53 gene which produce precarcinogenic and carcinogenic cells with different phenotypes. These different phenotypes can be correlated with different forms of progression and different prognosis of carcinomas. Probes are disclosed which can recognize these mutations or groups of mutations and thus the different phenotypes.

It is an object of the present invention to provide a system for the detection of p53-specific antibodies which avoids the above-mentioned disadvantages of the prior art.

This object is achieved according to the invention by the features characterized in more detail in claim 1.

It is another object of the present invention To provide a method for the detection of p53-specific antibodies which avoids the disadvantages known from the prior art.

This object is achieved by the method characterized in more detail in claim 6.

The method and detection system according to the invention have the advantage that the detection of antibodies in sera containing p53 is made possible in a tumor cell environment. According to the invention, cell extracts from various tumor cells are used, each containing an mutated p53 and / or a wild-type p53. As a control and to rule out non-specific binding to tumor cell proteins, tumor cells which do not express a p53 protein are used according to the invention.

Positive sera are characterized in that they deliver a significantly higher signal with p53-containing tumor cell extracts than with extracts that do not contain p53. The use of cell extracts with wild type and / or with mutant p53 allows a characterization of the sera in relation to the determination whether the serum recognizes wild type or mutant p53 or whether the antibodies are directed against both forms of p53.

According to the invention, antibodies to both forms of p53 are detected in animal organisms, preferably in humans.

Positive sera against p53 are detected, for example, via a secondary antibody linked to horseradish peroxidase. The evaluation can be done qualitatively visually or quantitatively with a laser plate reader.

The test system according to the invention is preferably an ELISA system, a support surface being coated with a cell extract containing the p53 protein (wild type and / or mutated). is.

For this, tumor cells such as the tumor cell line HT29 (HT29: ATCC accession number HTB 38), which expresses a p53 with a mutation in amino acid 273, is digested with extraction buffer 100mM Tris, 100mM NaCl, 0.5% NP40, pH9.0. Alternatively, MCF-7 (MCF-7: ATCC accession number HTB 22) cells which express wild type p53 are digested with the extraction buffer. Other tumor cells which express either mutant or wild-type p53 can be used by the same method. As a control, cell extracts from tumor cells (e.g. SA0S2: ATCC accession number HTB 85) are used which do not express p53. The cell extract is freed from particulate matter by centrifugation. The cell extract thus clarified is incubated for 3 h in cavities with the plastic surface of plates. The plates are preferably coated with anti-p53 monoclonal antibodies prior to binding of the p53 proteins in order to increase the specificity of the binding of p53. Subsequently, unbound components of the cell extract are removed and the cavities are washed 3 times with washing buffer.

The body fluids to be analyzed, e.g. Serum or urine are incubated with p53 from cell extracts immobilized in the cavities. As a control, the body fluids to be examined are also incubated with cavities which contain p53-free tumor cell extracts. A further control is a human serum against p53, which gives a positive signal.

Antibodies against p53 are detected with a second antibody directed against human immunoglobulins, which is conjugated, for example, with horseradish peroxidase.

Binding of the second antibody against p53 antibody will visualized via a chromogen reaction with, for example, tetramethylbenzidine. The evaluation is carried out automatically with an Elisa reader or photometrically at a wavelength specific to the color reaction (eg 450 nm).

To carry out the test, the test arrangement containing the wild-type and / or mutant p53 protein is brought into contact with the sample to be examined, and the binding of the antibodies in the sample to be examined to the p53 proteins of the Test arrangement is determined. The p53 protein in the test arrangement can be present in suspended form in an aqueous medium or bound to a solid support. Examples of solid supports which can be used according to the invention are spheres, microspheres, gel particles, the surfaces of microtiter plates, etc.

A large number of methods can be used to determine the binding of the antibodies in the sample to be examined to the p53 protein in the test arrangement. Examples include immunoprecipitation, radio-immunoassay (RIA), fluorescence immunoassay (FIA), enzyme-linked immunosorbent assay (ELISA) etc. The exact composition of the test arrangement, ie whether the p53 protein is suspended or attached to a solid support is bound, will depend on the type of test method selected.

The test arrangement can comprise a single p53 protein or several different p53 proteins that come from different cell systems. Known wild-type and / or mutant p53-expressing cells which are either accessible to the person skilled in the art or which are still to be established can be used as cell systems.

The various p53 proteins can either be contained in one cavity or in several cavities separately from one another. The simultaneous detection of several different allows different antibodies that are directed against both wild-type and mutant p53 protein.

The invention is described in more detail below with reference to figures and an exemplary embodiment. However, the invention is not restricted to this specific example. The person skilled in the art can recognize that changes to the extent claimed are possible without inventive step. Such changes can include, for example: the tumor cell line used, the buffers, and all other solution systems, the method of binding the tumor cells to the solid support surface, the monoclonal antibody with which the p53 protein is bound to the solid support surface, the method to detect the binding of the autoantibodies to the p53 protein (s), the amounts of the reagents used, the composition and the type of the reagents used etc. These variants are within the scope of the specialist knowledge and skill of a person skilled in the art who is experienced in this field. All of these modifications are embraced by the present invention.

The attached figures show:

FIG. 1 shows a schematic representation of the “end product” of an embodiment of the method according to the invention (ELISA);

FIG. 2 shows an example of a microtiter plate division; L, reagent blank; K, positive control serum; x marked microtiter plate strips; empty fields are reserved for testing patient samples.

Figure 3 shows the graphic evaluation for a patient.

Measured absorbances for 1, reagent blank control in marked cavities; 2, reagents Blank value control in unmarked cavities; 3, positive control serum, incubated in labeled microtiter plate cavities (p53 antibody contained in the sample); 4, positive control serum incubated in unlabelled microtiter plate cavity (serum control); 5, normal serum sample incubated in labeled microtiter wells (no p53 antibodies contained in the sample); 6, normal serum sample, incubated in unlabelled microtiter plate cavity.

Test principle

The detection method of p53 autoantibodies according to the invention is preferably constructed as an ELISA method and particularly preferably as a sandwich ELISA method. According to the invention, cell extracts from tumor cells are used which contain either mutant p53 antigen or wild-type p53 antigen. This makes it possible to differentiate whether the patient sera contain antibodies against mutant p53 or against wild-type p53. Tumor cells without p53 are used as controls. As an alternative to this, the test systems can contain mutant or wild-type p53 antigens in a plate in several cavities or in one cavity.

In the p53 autoantibody ELISA, the sample (serum or plasma) is incubated in a first step with the p53 antigen, which is fixed to the microtiter plate with the aid of a monoclonal p53 antibody. The production of monoclonal antibodies against p53 lies within the specialist knowledge of a specialist (Goding, Monoclonal Antibodies, Principles and Practice, New York, Academic Press, 1983). Furthermore, such monoclonal antibodies have already been commercialized by various companies. commercially available (for example anti-p53 protein pan (clone PAb 122) from Boehringer, Mannheim). After a washing step, the p53 autoantibodies present are marked by a second incubation with anti-human-IgG-peroxidase conjugate. After a further washing step, substrate is added. In the presence of p53 autoantibodies, peroxidase is bound, which in turn converts the substrate enzymatically; the resulting color can be measured photometrically. As a confirmation test, the samples are simultaneously incubated in cavities in which the monoclonal p53 antibodies are present, but the p53 protein is missing.

Commercially available microtiter plates can be used as the microtiter plate. The type of plate used depends on the type of determination method. It can easily be selected by a person skilled in the art. Both the beschich¬ ended microtiter plates and the test reagents are preferably 4 ^β - stored 8 ° C in order to ensure a consistently high quality. The immunologist is known per se to carry out the detection method. The selection of the detection method is also within the range of specialist knowledge and skill of the specialist.

The method according to the invention is illustrated below using an exemplary embodiment.

ELISA for the detection of autoantibodies against p53

The following solutions and antibodies are used:

Solutions, antibodies

1. Carbonate buffer: 15mmol / l carbonate / bicarbonate buffer, pH 9.6 (1, 59g Na ₂ CO ₃ , 2, 94g NaHCO ₃ , 0, 2g NaN ₃ ad 11 aqua dest.)

2. PAb421:

Mouse antibody, monoclonal, antibody against p53, concentration lmg / ml.

3. ELISA buffer A:

PBS buffer pH 7.1 + 18 g / 1 Tween 20 - wash buffer lx

4. ELISA buffer B:

PBS buffer pH 7.1 + 1% BSA

5. cells:

HT29: p53-positive cell line SAOS2: p53-negative cell line

6. Extraction buffer: 100mM Tris, 100mM NaCl, 0.5% NP40, ad 300 ml Aqua bidest: pH 9.0.

7. Anti-human-IgG-POD conjugate: Company: Sigma, Product No. A-0170 Predilution: 1: 500 in ELISA buffer B Final dilution: 1: 50000 in ELISA buffer B POD = peroxidase

8. Acetate buffer:

25mmol / l sodium acetate pH 4.6

9. Substrate solution A:

TMB = tetramethylbenzidine; Company: Sigma; Product No. T-

8768

Concentration: 2.5 mg / ml 6 ml substrate solution A for a microtiter plate: 15 mg TMB dissolved in 3 ml DMSO + 3 ml acetate buffer

10. Substrate solution B:

0.02% H ₂ 0 ₂ in acetate buffer

11. Stop solution: 0.5 NH ₂ S0 ₄

Coating of the microtiter plates

1. Antibody coating:

Antibody: PAb421, mouse monoclonal antibody against p53 concentration: 1 mg / ml

Concentration for the coating: Dilute lμg / ml antibody in carbonate buffer to lμg / ml

Coating the microtiter plates with 100 μl antibody dilution per cavity; Incubation at 4 ° C overnight.

2. Post-coating with BSA:

Wash microtiter plates 3 times with wash buffer (= ELISA buffer A)

2% BSA in carbonate buffer; 200μl per cavity; Incubation at 4 ° C overnight.

3. Cell extract coating: Production of cell extracts:

The cell lawn of each dish (10 ⁶ cells) is taken up in 360 μl extraction buffer + 40 μl trasylol and incubated on ice for 1 h; Centrifugation: 10 min at 4 ° C and 1000 rpm

30 min at 4 ^β C and 13000 rpm

Discard pellet; Supernatant is used for coating

Coating: washing of the microtiter plates is not necessary since the sample buffer also contains BSA; Aspirate the BSA solution

Dilute cell extracts 1: 5 in sample buffer (= ELISA buffer B); 100μl per cavity; Incubation at 37 ^β C for 3 h, wash 3 times with wash buffer (»ELISA buffer A).

Execution of the sandwich ELISA

Washing: 3x with washing buffer (= ELISA buffer A);

Antiserum: All samples are diluted 1:20 in sample buffer (= ELISA buffer B), including the positive control. 100 μl of each serum dilution are pipetted into a SA0S2-coated and into an HT29-coated cavity. Negative control: Pipette 100 μl sample buffer into an SA0S2-coated and an HT29-coated cavity; 1 h incubation at 37 ° C; 3. Wash: 3x with wash buffer (»ELISA buffer A);

4. Conjugate: dilute anti-human-IgG-POD conjugate 1: 100 in sample buffer (»ELISA buffer B);

Pipette 0.1 ml into each well; 1 h incubation at 37 ^β C;

5. Wash: 3x with wash buffer (»ELISA buffer A)

6. Substrate: Pipette 50 μl substrate solution A and then (!) 50 μl substrate solution B into each cavity; 10 - 30 min. Incubation at room temperature;

7. Stop solution: Stop the substrate reaction by adding 100 μl stop solution;

8. Evaluation: Determine absorbance at 450 nm with an ELISA reader.

Due to the difficulties in the standardization of enzyme immunoassays, every clinical-chemical laboratory should use its own internal laboratory values.

3 shows a graphic evaluation for a patient.

When evaluating the data of a patient sample, it is important that there is a clear difference between the extinction value obtained in the marked cavity for this sample and the extinction value in the unlabelled cavity for the same sample was measured, is found.

The following can be used as an evaluation aid for a positive result Relationship can be used:

E - E. - E ₂

E »extinction difference obtained (E. should always be greater than [E ₂ + 20%]) E. - Absorbance of the sample from the marked cavity; E ₂ »Absorbance of the sample from unlabelled cavity.

Claims

- 18 -patent claims

1. Test arrangement, characterized in that it comprises one or more wild-type and / or mutant p53 proteins from one or more tumor cell extracts.

2. Test arrangement according to claim 1, characterized in that it comprises one or more human p53 proteins of the wild type and / or mutant type.

3. Test arrangement according to claim 1 or 2, characterized in that the p53 protein (s) are present bound on a solid support.

4. Test arrangement according to claim 1, 2 or 3, characterized in that the p53 protein (s) are bound to the support via a monoclonal or polyclonal antibody directed against wild type and / or mutant p53.

5. Test arrangement according to one or more of claims 1 to 4, characterized in that it further comprises one or more reagents for the qualitative and / or quantitative detection and / or for determining the binding of antibodies to the p53 proteins in the Test setup includes.

6. A method for the detection of antibodies against one or more wild-type or mutant p53 proteins in a test sample, characterized by

a) bringing a test sample into contact with a test arrangement which comprises one or more wild-type and / or mutant p53 proteins from one or more tumor cell extracts; and

b) quantitative and / or qualitative determination of the anti-wild-type or mutant p53 immune reaction in an individual by detection of the antibody binding to the wild-type or mutant p53 protein.

7. The method according to claim 6, characterized in that the amount of the antibodies directed against the wild-type and / or mutant p53 protein is determined in the test sample.

8. The method according to claim 6 or 7, characterized in that the test sample is a serum sample, plasma sample, urine or another body fluid.

9. The method according to one or more of claims 6 to 8, characterized in that a human test sample is used.

10. The method according to one or more of the preceding claims, characterized in that the mutant and / or wild-type p53 proteins are suspended in aqueous medium.

11. The method according to one or more of the preceding claims, characterized in that the mutant and / or wild-type p53 proteins are bound to a solid support.

12. The method according to one or more of the preceding claims, characterized in that the tumor cell extracts from HT29 and / or MCF-7-

Tumor cell lines are produced.

13. The method according to one or more of the preceding claims, characterized in that the detection of the antibody binding is carried out by a peroxidase-coupled antibody directed against human igG.

14. The method according to one or more of the preceding claims, characterized in that the detection of antibody binding for immunoprecipitation, radio-immunoassay (RIA), fluorescence immunoassay (FIA) or enzyme-linked immunosorbent assay (ELISA) is performed.

15. Use of the test arrangement and the method according to one or more of the preceding claims for the detection or prophylaxis of tumors.