JP2003116549A - Method for detecting precancerous cells or cancer cells using P90 antibody or probe - Google Patents

Abstract

(57) Abstract: The present invention measures the expression level or gene amplification of p53 and dm2 and increases the level of either p53 or dm2 or both p53 and dm2 to cause cancer. And a method of diagnosing cancer. Also, the present invention measures the expression levels of p53 and dm2 or the gene amplification, and evaluates either p53 or dm2 or p53
The present invention provides a method for predicting the progression of cancer, which is judged to have a poor prognosis by increasing the levels of both dm2 and dm2. [Effect] The prognosis of cancer can be diagnosed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining the prognosis of cancer. This application is a PC filed on June 25, 1993
This is a continuation-in-part application of T-application PCT / US93 / 06063, which is a continuation-in-part of US patent application Ser. No. 08 / 018,649 filed Feb. 17, 1993. And the US patent application is June 2, 1992.
This is a continuation-in-part application of US patent application Ser. No. 07 / 904,766 filed on the 6th, and the US patent application Ser.
No. 6 is US Patent Application No. 07 filed on July 12, 1991.
This is a partial continuation application of / 703185. Further, its US application Ser. No. 07 / 703,185 was issued on June 27, 1990.
It is a continuation-in-part application of U.S. patent application Ser.

[0002]

BACKGROUND OF THE INVENTION It has been gradually recognized that mutations in proto-oncogenes in somatic cells have a great influence on the induction of human cancer. Examples of such oncogene mutations include neu, fes, fos, myc,
Examples include myb, fms, Ha-ras and Ki-ras. The mutation that converts the protooncogene into the oncogene is often a point mutation. Further studies are needed to understand how proto-oncogenes and their expression products transform normal cells into cancer cells. Oncogenes are generally considered to act dominantly. This is believed to mean that the conversion of protooncogenes to oncogenes results in a new function, namely enhanced conversion.

Another type of mutation associated with cancer occurs when a tumor suppressor gene is modified and its gene product loses tumor suppressor function. An example of a tumor suppressor gene is the retinoblastoma susceptibility gene Rb. Strictly speaking, tumor suppressor genes do not contribute to tumor formation, but tumor suppressor genes are sometimes referred to as recessive oncogenes. When both alleles are mutated, their phenotype is recessive because tumor formation is promoted by the absence of tumor suppressor genes.

The gene product characteristic of both dominant and recessive protooncogenes is the 53 kd phosphoprotein p53. It has been gradually established that mutations in the p53 gene are associated with many of various cancers. See, for example, Iggo et al., Lancet 335, page 675.
In 679 (1990), p53 is proposed to be the most common proto-oncogene mutated in lung cancer.

Much of what is known about p53 was obtained by examining the effects of transfecting murine fibroblasts with wild-type and mutant mouse p53. This study was carried out by Levine et al., "P53 Protooncogene And Its Products" in "General Mechanisms of Transformation by Small DNA Oncoviruses", L. et al. Villa
rreal edition, American Society f
or Microbiology Chapter 2 (1989)
); Chapter 7 by Hinds et al .; ibid, and BioEssays 12, page 60 by Levie.
~ 66 (1990) is being reviewed.

The p53 gene appears to be involved in transcriptional regulation (Fields, S and Jang, SK.
(1990) Science 249, page 104.
6-1049; Raycroft, L .; Wu, H .; And Lozano, G .; (1990) Science
249, pages 1049-1051; and Levi.
ne, A.N. J. Momand, J .; And Finla
y, C.I. A. (1991) Nature 351, pp. 453-456) and also acts as a regulatory checkpoint in the cell cycle and seems to capture cells in the G-1 phase (Martinez, J., Geo).
rgoff, I.D. And Levine, A .; J. (1
991) Genes Dev. 5, pages 151-1
59; Hupp, T .; R. , Meek, D .; W. , Mi
dgley, C.I. A. And Lane, D .; P. (19
1992) Cell 71, pages 875-886; and Yin, Y .; , Tainsky, M .; A. , Bisc
hoff, F.F. Z. Strong, C .; C. And W
ahl, E .; M. (1992) Cell 70, pages 937-948).

Genetic alterations of the p53 gene such as intragenic mutations, homozygous deletions and structural rearrangements are common in human cancers (Vogelstein, B. et al.
And Kinzler, K .; (1992) Cell 7
0, pages 523-526; Baker, S .; J. Et al. (1990) Cancer Res 50, page 7.
717-7722; Mori, N .; Et al (1989) Ca
ncer Res 49, pages 5130-5135; L
ee, J. H. Et al. (1990) Cancer Res
50, pages 2724-2728; Varley,
J. M. (1991) Oncogene 6, pages 413-421; Presti, J. et al. C. Et al (199
1) Cancer Res 51, page 5405;
And Dalbangi, G .; (1993) Dia
gnostic Molecular Patholog
y 2, pages 4-13). These modified forms of p53 reduce or block the activity of functional homotetramer units (Stenger JE et al. (199).
2 years) Mol Carcinog 5, page 102-
106; Sturzbecher, H .; W. Et al (199
2 years) Oncogene 7, pages 1513-152
3). Mutant p53 protein has a long half-life and slow degeneration, resulting in inactive complexes and self-aggregating molecules in the nucleus of tumor cells (Sturzbecher, H. et al.
W. Et al. (1987) Oncogene 1, page 20.
1-211; Halevy, O .; Et al (1989) Mo
CellBiol 9, pages 3385-339.
2).

[0008] In humans, the embryonic mutation of the p53 gene is caused by Li-Fraumeni.
Found in families affected by the syndrome. This syndrome has a rare autosomal dominant feature that predisposes individuals to a variety of tumors, including soft tissue sarcomas (L
i, F. P. And Fraumeni, J .; F. (1969
Year) Ann Intern Med 71, page 74.
7-752; Malkin, D .; (1990) N. et al.
Engl. J. Med. 250, pages 1233-12
38). More recently, p53 germline mutations have also been detected in cancer patients who are clearly not cancer families (Toguchida, J. et al. (1992) N. Eng.
l. J. Med. 326, pages 1301-130
8) and has also been detected in a small population of patients presenting with a second primary neoplasm (Malkin D. et al. (19).
1992) N. Engl. J. Med. 326, pages 1309-1315). Furthermore, somatic mutations of the p53 gene have been reported to occur in soft tissue sarcomas (Stratton, MR, et al. (1990).
Oncogene 5, pages 1297-1301; M
Ulligan, L .; M. Et al (1990) Proc
Natl Acad Sci USA 87, page 5
863-5867; Toguchida, J .; Et al (19
1992) Cancer Res 52, page 6194.
~ 6199; Drobnjak, M .; Et al. (1993)
Submitted; Latres, E .; (Submitted in 1993)).

Another gene recognized as a potential protooncogene is mouse double minutes-2.
(Mdm2) (Fakharzadeh et al. (19
1991), The EMBO Journal 10
(6), pages 1565-1569). The mouse and human mdm2 gene and protein sequences are known.
(Fakharzadeh et al., The EMBO Jo
internal10 (6), pages 1565-1569 (1
991); Oliner, J .; D. Et al. (1992)
Nature 358, pages 80-83; and Ca.
Hilly-Snyder et al., Somatic Cel
l and Molecular Genetics,
13 (3), pages 235-244 (1987)).
The sequence is evolutionarily constant among species containing mouse, rat, hamster and human genes (Ca
Hilly-Snyder et al., Somatic Cel
l and Molecular Genetics,
13 (3), pages 235-244 (1987)).

In the literature, the mdm2 gene is identified in MD
M2, 1MDM20, hdm2 (human species), and 1m
Called dm20 (mouse species). The 90 kD phosphoprotein mdm2 gene product is p90 in the literature.
, Which refers to both mouse and human species, and to the human species MDM2. The p90 protein is described in the applicant's relevant publication, Levine et al., 199.
International application PCT / US91 / 04 filed June 27, 1
608. Although dm2 is used throughout this application, it is meant to include the various terms used in the literature for the mdm2 gene and protein, including cognate species among all species.

1M in both osteosarcoma and soft tissue sarcoma
It has been demonstrated that there is proliferation of DM20 and overexpression of MDM2 (gene product) (Oliner, JD et al. (1
992) Nature 358, pages 80-83;
Ladanyi, M .; Et al. (1993) Cancer
Res 53, pages 16-18; Leach, F .;
S. (1993) Cancer Res 53, pages 2231-2234).

A human species of the hdm2 gene or the mdm2 gene called 1MDM20 or MDM2 was cloned and mapped to the long arm of chromosome 12 (12
q13-14) (Oliner et al., 1992, "Proliferation of genes encoding p53-related proteins in human sarcomas", Nature 358, pages 80-8).
3). In this region, two genes, SAS, previously found to grow in osteosarcoma and soft tissue sarcoma
And GLI. The SAS gene encodes a protein with an unidentified function. This gene was isolated from malignant fibrous histiocytoma (MFH) and
It has been shown to grow in FH and liposarcoma (Turc-Carel, C. et al. (1986) Can.
cer Genet Cytogenet 23, pages 291-299; Meltzer, P .; S. Et al (19
91) Cell Growth Diff 2, pages 495-501). The GLI gene encodes a DNA-binding zinc finger protein. This gene was first isolated from glioblastoma but has been reported to grow in rhabdomyosarcoma and osteosarcoma (Kinzl).
er, K.E. Et al. (1984) Science 236,
Pages 70-73).

[0013]

Prior to the present invention, mutated p53 was thought to be associated with cancer. Moreover, it was known prior to the present invention that overexpression of mdm2 is associated with tumors. However, prior to the present invention, the relationship between the altered p53 and dm2 genes, various modes of expression in cells, and their relationship to determine the clinical relevance or prognosis of cancer patients, as well as to make the diagnosis. It was not disclosed about how to use. An object of the present invention is to diagnose cancer and determine the prognosis of cancer patients by utilizing the relationship between the overexpressed or propagated p53 and dm2 genes.

[0014]

The present invention measures the levels of p53 and dm2 in a biological sample to determine p53 and d3.
It is intended to provide a method for diagnosing cancer, which shows the occurrence of cancer when the level of any one of m2 or both p53 and dm2 is increased.

Another object of the present invention is to measure the levels of p53 and dm2 in a biological sample,
It is achieved by predicting cancer progression as having a poor prognosis when either one of m2 or both p53 and dm2 levels are elevated.

The present invention also provides a method of classifying biological samples into three groups. This method uses p5
3 includes measuring whether the level of 3 or dm2 is abnormally elevated, and the first group includes p53 and dm.
The levels of 2 include the case where neither is abnormally increased,
In the second group, the level of p53 is abnormally elevated but the level of dm2 is not abnormally elevated, or the level of dm2 is abnormally elevated but the level of p53 is not abnormally elevated. Including, the third group is p53 and dm
Including the case where both levels of 2 are abnormally elevated.

Furthermore, the present invention provides a method for measuring cancer cells, which are biological samples, or cells at risk of becoming cancerous or precancerous, which cells contain at least one normal p53 allele. provide. This method comprises determining whether the level of dm2 in a biological sample is abnormally elevated, wherein the level of dm2 in the biological sample is higher than that of a normal biological sample. If elevated, it is indicated to be a cancer cell, or a cell at risk of becoming cancerous or precancerous.

The present invention also provides an isolated p53 / dm2 protein complex and an antibody against the dm2 protein.

[0019]

DETAILED DESCRIPTION OF THE INVENTION Definitions : p53 : As used herein, the term "wild-type" p53 refers to Matlashewski et al., EMBO J .; 13,
Pages 3257-3262 (1984); Zakut
-Houri et al., EMBO J. 4, pages 1251
1255 (1985); and Lamb and Crawf.
ord, Mol. Cell. Biol. 5, page 13
It means the nucleotide or amino acid sequence reported in 79-1385 (1986). This sequence is G
It is available from enBank. Wild-type p53 contains the proline / arginine polymorphism at amino acid 72 and the corresponding nucleotide polymorphism.

Mutations in the wild-type p53 gene or protein present precancerous or cancerous conditions. What are precancerous cells?
Usually a cell with one normal p53 allele and one mutated p53 allele. For example, the mutation is a point mutation. In cancer cells, usually both p53 alleles are mutated. For example, one mutation may be a point mutation and the other mutation may be due to a deletion of all or most of the p53 gene.

Dm2 : In the present invention, dm2 is 9
Family of proteins including 0 kD phosphoprotein (p90), protein p85 (85 kD), protein p76
(76 kD), protein p74 (74 kD) and protein p58-57 (58 kD and 58 kD) (p57
Is a mouse species equivalent to rat p58)
Represents a fragment or product of D phosphoprotein, and their cognate or similar species. p53 protein is dm2
Co-immunoprecipitates with the protein. For dm2 protein,
Arrayed Mouse Double Minutes-2 (mdm
2) 90 kD phosphoprotein is included. Also, dm2
The term refers to the gene encoding the above dm2 protein. The sequence of mdm2 gene and protein is Fak
harzadeh et al., The EMBO Journal
10 (6), pages 1565-1569 (1991).
); And Cahillly-Snyder et al., Som.
aticCell and Molecular Ge
netics, 13 (3), pages 235-244 (1
987). Sequences homologous to p90 dm2 are present in the genomes of several species such as human "hdm2". The rat, mouse and hamster as well as human genes have been sequenced and have a molecular weight of approximately 90 kD (Oliner, JD et al. (199).
2) Nature 358, pages 80-83). In a preferred embodiment, the dm2 gene and protein are human species.

The dm2 protein of approximately 90 kD clearly has a high probability that the p58 protein binds to p53.
3T3DM cells obtained from Balb / c3T3 responded to replication of the expanded mdm2 gene with 5 mdm2
Is generated excessively.

Assessment of dm2 and / or p53 levels
(Dm2 + and / or p53 +): as used herein
Increased levels of dm2 and / or p53 in cells
Shown is proliferation of the dm2 or p53 gene, or overexpression or accumulation of the dm2 or p53 protein product in a biological sample such as a cell. The level of dm2 or p53 protein is a total measurement of dm2 or p53 protein in a biological sample, preferably a cell,
It does not matter whether it is a free protein or a complex. In some cases, elevated levels of dm2 protein in the absence of dm2 proliferation indicate that dm2
It shows that a heterodimer / heterotetramer was formed between 2 and the mutated p53 product. The mutant p53 protein has a long half-life and is slow to degenerate and thus accumulates in the cell nucleus. p53 missense mutations account for the majority of at least about 85% of immunochemically detectable p53 mutations. However, in some cases elevated levels of wild-type p53 gene and protein in tumor tissue are detected.

Cancer cells, or cells at risk of becoming cancerous or precancerous, when the level of dm2 and / or p53 in the biological sample is elevated compared to a normal biological sample. Indicates. Biological samples include, but are not limited to, tissue extracts, samples of cells, or biological fluids such as lymph, blood or urine.

The present invention provides a method of classifying biological samples into three groups. The method involves determining whether the levels of either p53 or dm2, or both p53 and dm2, in a sample are abnormally elevated. Group A, which is the first group, has neither abnormal levels of p53 nor dm2 (p53
-/ Dm2-) is included. Group B, the second group
Indicates that the level of dm2 is abnormally elevated but the level of p53 is not abnormally elevated (p53− / dm2 +)
And the case where the level of p53 is abnormally elevated but the level of dm2 is not abnormally elevated (p53 + / dm2-), the third group, Group C, has both p53 and dm2 levels. If it is rising abnormally (p5
3 + / dm2 +).

The present invention classifies the various aspects of dm2 and p53 expression is of great clinical importance for diagnosing and predicting the clinical course of patients suffering from various cancers. Indicates that there is. Examples of cancer include sarcoma, carcinoma,
You have leukemia or lymphoma. In particular, sarcoma includes soft tissue sarcoma, osteosarcoma and the like. In other embodiments, the cancer comprises Bladder Cancer. In other aspects, colorectal cancer, lung cancer,
Ovarian cancer, cervical cancer, adrenocortical cancer, bone cancer, breast cancer, brain cancer, and chronic myelogenous leukemia are targeted, but are not limited thereto. Accordingly, the present invention provides a method of assessing a subject's prognosis by obtaining a biological sample from a subject's pre-cancerous tissue or tumor and determining which of the three biological sample groups it belongs to. provide. Of these categories, the third group shows the worst prognosis,
The group shows the best prognosis.

Group C (p53 + / dm2 +) : The present invention relates to cancer cells, or cells at risk of becoming cancerous or precancerous, by measuring whether or not the level of dm2 is elevated, Methods are provided for detecting cells containing at least one mutant p53 allele.
Unexpectedly, elevated levels of dm2 protein were found in p53
As the level rises, that is, p53 + / dm2 +
Clinicopathological variables that interact with elevated levels of p53 protein containing mutations as well as overexpressed wild-type p53 detected as a group (see Figure 4) and exhibit a poor prognosis such as survival or tumor progression Becomes Of these groups, Group C shows the worst prognosis. Example 2 and FIG. 3 show a very high survival when comparing Group A (no elevated levels of p53 or dm2) with Group B (elevated levels of only one of these proteins). The abnormal level rise of dm2 and p53 in the same tumor part which arose in a group with a low rate shows the state detected immunologically.

This is unexpected. because,
This is because, as discovered by the present inventor, the dm2 protein inactivates the transcriptional action of p53, so that there is no increase in the level of dm2 in cells in which the level of p53 protein is increased.

Group B (p53- / dm2 + and p
53 + / dm2-) : Group B is two subsets,
That is, when the level of p53 is normal but the level of dm2 is increased (p53− / dm2 +), and when the level of p53 is increased but the level of dm2 is normal (p53).
+ / Dm2-) is included. Group B out of 3 groups
Shows a worse prognosis than Group A but a better prognosis than Group C.

Subset p53 + / dm2 of group B
- : A precancerous or cancerous state when it is detected that the level of p53 protein such as wild-type p53 gene and protein is increased due to mutation or overexpression, that is, when the p53 + / dm2- group is detected Indicates.
Mutants of p53 inactivate the function of wild-type p53, and cells containing mutants of the p53 gene and proteins are likely tumorigenic. Also wild type p5
Increased levels of 3 protein contribute to worse prognosis. DNA damage or binding of viral protooncogene products in cells stabilizes wild-type p53 protein and increases its concentration.

Group B subset p53- / dm2
+ : In the present invention, BALB / c3T3 cells (3T3 DM cells) were unexpectedly increased even when the level of dm2 was increased.
Alternatively, it has been shown to give the same characteristics to cells such as pre-cancerous or cancerous cells when the level of p53 is increased. In either case, animals have elevated levels of dm2 or p53 in their cells, which increases the likelihood of tumorigenesis.

With regard to p53- / dm2 +, even if the level of the wild-type p53 allele in the cells is normal, if the level of dm2 is elevated, the possibility of tumorigenesis of the cells increases.

The present invention relates to cancer cells, or cells at risk of becoming cancerous or precancerous, when the cells contain at least two normal p53 alleles, ie wild type p53 or normal levels of p53. To detect whether the level of dm2 in the biological sample is abnormally elevated, the elevated level being normal biological levels. It means an increase in the level when compared to the level of dm2 in the sample (see Figure 4).

Group A (p53- / dm2-) : Group A includes both p53 and dm2 at normal levels. Pre-cancerous and cancerous cells in this group are likely due to causes other than elevated levels of p52 and / or dm2. Out of 3 groups
Group A has the best prognosis.

Detection of elevated levels of DM2 or P53 : Amplification : Amplification of the dm2 or p53 gene could be detected using known methods such as the nucleic acid probe method.
Higher copy numbers of DNA could be detected, for example, by Southern blotting, and increased amounts of RNA could be detected by Northern blotting (George, DL and Powers, VE, Cell 24 : 117-.
123 (1981); George, D .; L. EM
BOJ. , 4: 1199-1203 (1985); Si
ngh, L.N. And Jones, K .; W. , Nucle
ic Acids Res. , 12: 5627-563.
8 (1984)).

Overexpression : dm2 and p in biological samples
The 53 protein level is detected by a known method such as immunohistochemical staining using anti-dm2 and anti-p53 antibodies. By immunohistochemistry-based positive nuclear staining, p53,
Elevated levels of dm2 or p53 / dm2 complex are shown. Overexpressed p53 protein is associated with mutations in the p53 gene. In one embodiment of the present invention, nuclear overexpression of dm2 and p53 in various tumors is classified into one of the following three groups based on the staining ratio (%) in tumor cell nuclear staining: (a) negative ( <2
0%), (b) isomerism (20-70%), (c) homology (>
70%).

In another embodiment of the invention, dm2
2-1 of the level of a biological sample (such as a cell) in which the precancerous state or the cancerous state is normal for detecting the increase in the level
It is shown to be 00 times. In a preferred embodiment, the elevated level is 5-50 times that of a normal biological sample (such as cells).

Polyclonal and monoclonal antibodies will be prepared by known methods. Antibodies of the invention include those of Huse et al., Science 246: 1275.
-1281 (1989), recombinant polyclonal or monoclonal Fab fragments. See also: Burdon et al., "Laboratory Techniques in Biochemistry and Molecular Biology," Vol. 13, Elsevier.
Campbell, published in Science Publishers, Amsterdam (1985).
"Monoclonal antibody technology, production and characterization of rodent and human hybridomas". Methods for preparing polyclonal and monoclonal antibodies that express specificity for a single amino acid difference between oncogenes are described in McCor.
U.S. Pat. No. 4,798,787 to Mick et al.

Briefly, a polyclonal antibody is obtained by injecting a p53 protein capable of producing an antibody which distinguishes mutant p53 from wild-type p53 or a fragment thereof into a mammalian host such as rabbit, mouse, rat or goat. .. The injected peptide or peptide fragment will contain wild-type or mutant sequences. Serum from mammals is subjected to an extraction / screening process to obtain a polyclonal antibody specific for the peptide or peptide fragment.
Similar methods could be applied to the dm2 protein.

To produce a monoclonal antibody, a mammalian host is inoculated with the above peptide or peptide fragment,
This is propagated. A few days after the final growth, the spleen is excised from the inoculated mammal. The cell suspension obtained from the spleen was analyzed by Natler by Kohler and Milstein.
Fuse with tumor cells by the general method described in ure 256, pages 495-497 (1975). To be useful as a peptide fragment, the peptide fragment must contain sufficient amino acid residues to define the P53 or dm2 molecule epitope to be detected.

If the fragment is too short to be antigenic, it may be bound to a molecular carrier. Suitable carrier molecules include keyhole limpet hemocyanin, calf serum albumin. Coupling can be done by methods known in the art. As one of such methods,
There is a method of linking the cysteine residue of the fragment and the cysteine residue of the carrier molecule.

Peptide fragments can be synthesized by methods known in the art. Some of the preferred methods are Stu
"Solid Phase Peptide Synthesis" by Art and Young (2nd
Edition, Pierce Chemical Co. , 198
4 years).

Antibodies suitable for the co-immunoprecipitation method of p53 and dm2 include PAb421 and Ab2. PA
b421 recognizes the carboxy terminus of p53 obtained from various hosts including human, mouse, rat, etc., and has Ha
rlow et al. "Journal of Viro
log "39, 861-869 (1981). Ab2 is specific for the amino terminus of human p53 and is available from Oncogen Science, Inc., Manhasset, NY. RE that does not express p53.
When F cells were similarly treated with the same antibody, dm2
The protein does not immunoprecipitate.

The immunoprecipitate can be collected by centrifugation. Following centrifugation and elution from the column, dm2 is p53 / dm
It is separated from the two complexes by the SDS-PAGE method.
The single band at 90 KD is cut and sequenced. The present invention also provides an isolated dm2 / p53 complex, which contains dm2 and p5 from various transformed cells.
Obtained by co-immunoprecipitation of 3.

Another method for purifying dm2 is Aeber.
Sold et al., Proc. Natl. Acad. S
ci. USA 84 , 6970-6974 (1987).
The method described in 1.

The present invention provides a hybridoma expressing a monoclonal antibody against the dm2 (p90) gene product. Some of these hybridomas are Amer
ican Type Culture Collect
deposited at Ion (ATCC): 3G5 ATC
C deposit number HB11182), 4B11 (ATCC deposit number HB11183), 2A10 (ATCC deposit number H)
B11184), 2A9 (ATCC deposit number HB111)
85), and 4B2 (ATCC accession number HB1118)
6). Hybridoma 3G5, 4B11, 2A10, 2
A9 and 4B2 have been assigned ATCC deposit no.
2, HB11183, HB11184, HB11185
And HB11186 as Maryland 2 respectively
0852, Rockville, Park Lawn Drive 12
301 American Type Cultu
deposited with the Re Collection (ATCC).

Measure and regulate the level of Dm2 in cells
Assay for : dm2 and / or p53 in cells
Is capable of recognizing amplified or overexpressed dm2 and / or p53 genes or proteins,
It is determined by an assay method known in the art.

A variety of assays are available for detecting proteins using labeled antibodies. In a one-step assay, the target molecule, if present, is immobilized with the labeled antibody and incubated. The labeled antibody binds to the immobilized target molecule. After washing away unbound molecules, the assay checks the sample for the presence of label.

In a two-step assay, immobilized target molecule is incubated with unlabeled antibody. The target molecule-unlabeled antibody complex, if present, then binds to a second labeled antibody specific for the unlabeled antibody. The sample is washed and assayed for the presence of the label as described above.

The labeled anti-dm2 antibody can be used to detect dm2 by an imaging method. One method of imaging is to contact the tumor cells to be imaged with an anti-dm2 antibody labeled with a detectable marker. In this method, labeled antibody is dm2
Is performed under conditions that bind to. By detecting the antibody bound to dm2, dm2 is imaged and detected.

The marker used to label the antibody is chosen according to its application. Those skilled in the art can easily select and determine the marker. The labeled antibody can be used in immunoassays and histological applications to detect the presence of tumors. The labeled antibody may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody, and examples thereof include the above-mentioned antibodies deposited with the ATCC.

In a preferred embodiment of the invention the label is a radioactive atom, an enzyme or a chromophore. Examples of radioactive atoms include P ³² , I ¹²⁵ , H ³ and C ¹⁴ . Examples of enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose-6-phosphate dehydrogenase. Examples of chromophores include fluorescein and rhodamine. The binding between the antibody and these labels may be carried out by a known method. For example, enzymes and chromophores are linked to antibodies by coupling agents such as dialdehydes, carbodiimides, dimaleimides. Alternatively, the binding can be via a ligand-receptor pair. Examples of suitable ligand-receptor pairs include biotin-avidin, or-streptavidin, and antibody-antigen.

Antibodies to the dm2 protein can be used to detect elevated levels of the dm2 protein in cells and tissues. In one embodiment, antibodies directed against an epitope of dm2 can be used in situ by immunohistopathological techniques. These methods can be used for diagnosis in cases where there is a high risk of the tissue becoming cancerous, such as in polyps, atypical breast tissue, or in tumor cells where metastasis or recurrence is more likely.

Hybridoma 1F5, 6C10, 1D
6, 4B2, 2E12, 3F3, 3G5, 3F8, 6H
7, 2A9, 3G9, 1D11, 2A10, 1G2, 4
B11, and 5B10 produce monoclonal antibodies against the epitope of the dm2 protein (see Figure 1).
Some of these hybridomas have been deposited with the ATCC (see above). Monoclonal antibodies react with epitopes of both murine and human proteins, as well as those of other species, so long as the homologous dm2 sequences are conserved between host species.

In another embodiment of the invention, the cancer cells may release dm2, which increases the amount of dm2 in the blood or lymph of the patient. Therefore, an assay such as an immunoassay can detect a normal dm2 value in a cell or body fluid or a dm2 level exceeding the normal value.

Block dm2 binding to wild-type p53
Screening for therapeutic agents : d against wild-type p53
Screening for therapeutic agents to block m2 binding can be performed by various assays. The present invention discloses methods for selecting therapeutic agents that form a complex with dm2 having sufficient affinity to prevent deleterious binding of dm2 to wild-type p53. The method involves a variety of assays, including, for example, immobilizing dm2 on a support and contacting both wild-type p53 and a labeled therapeutic agent simultaneously or sequentially, which therapeutic agent binds to wild-type p53 of dm2. To determine whether it effectively competes with wild-type p53 so as to sufficiently prevent the binding of E. coli. In another aspect, wild-type p53 is labeled and the therapeutic agent is unlabeled.
In yet another embodiment, both p53 immobilized on a support and labeled dm2 and a therapeutic agent (or unlabeled dm2).
(Therapeutic agent labeled 2), and whether the amount of dm2 bound to p53 is decreased as compared to the case where no therapeutic agent is added is examined. dm2 is the anti-dm2 of the present invention
It can be labeled with an antibody.

In one embodiment, the method of the invention comprises the steps of: a) contacting a solid support with a predetermined amount of dm2 under conditions such that dm2 is attached to the surface of the support (eg, anti-dm2 antibody). Dm2 is contacted with a solid support using a), b) the dm2 that is not bound to the support is removed, and c) the solid support to which dm2 is bound, the wild type p5 is bound to the bound dm2.
3 binds to wild-type p53 under conditions such that 3 binds and forms a complex with it, d) removing unbound p53, and e) adding to the dm2 / p53 complex thus formed. Contacting a predetermined amount of sample labeled with a detectable marker under conditions such that the labeled potential therapeutic agent present in the sample binds to the binding dm2 in competition with wild-type p53, f ) Quantitatively determining the concentration of labeled potential therapeutic agent that is unbound to the solid support, g) dm
The concentration in the sample of potential therapeutic agent that specifically binds 2 and blocks dm2 from binding to wild-type p53 is determined.

The solid support can be easily selected and determined by those skilled in the art. In one preferred method, the solid support is a bead of an inert polymer, and in another, the solid support is a microwell. Those skilled in the art can arbitrarily select the marker used in the above method. Detectable markers are preferably enzymes, paramagnetic ions, biotin, fluorophores, chromophores, heavy metals, or radioisotopes. A more preferable marker is an enzyme, and most preferable is horseradish peroxidase or alkaline phosphatase.

According to a further aspect of the method of the invention, the potential therapeutic agent is enzymatically labeled and step f).
Removes the labeled potential therapeutic agent that was not bound to the solid support, which was then treated with a specific substrate for the enzyme under conditions such that the enzyme would react with the substrate to form a detectable product. Contact.

The present invention further provides a method of treating cancer in mammals, including mice, rats, hamsters, humans, wherein the deleterious binding of dm2 to wild-type p53 is blocked. According to one aspect of this method, blocking the binding of dm2 to wild-type p53 is accomplished by d
This is done by contacting m2 with a sufficient amount of anti-dm2 antibody. In another aspect, dm2 to wild-type p53
Block the binding of dm2 to an excess of anti-idiotype p
53 antibody. In another aspect, a dm2 anti-idiotype antibody that does not block the transcriptional promoter of p53 may be administered. In another method for treating tumors, the dm2 gene amplified by gene therapy is replaced with a normal number of dm2 genes to normalize elevated p53 levels. In yet another method of treating tumors, the dm2 gene amplified by antisense gene therapy is replaced with the antisense dm2 gene.

The method for determining relative binding affinity may be any method known in the art. For example, p5
Methods for determining whether the 3 protein is bound to hsc70 are described in Finley et al., Mol. and Cel
l Biol. 8, 531-539 (1988) and Hinds et al., Mol. and Cell Biol.
7, 2863-2869 (1987). In the method described in these papers, co-immunoprecipitation using anti-p53 and anti-hsc70 antibodies is performed.

[0062]

Example 1 Isolation of cDNA clones : A λgt11 cDNA library prepared from HeLa cells was screened under low stringency using mouse mdm2 cDNA as a probe. The cDNA insert was isolated from positive phage and subcloned into Bluescript vector for further characterization. A full length cDNA containing the entire coding region was reconstructed from two overlapping clones and completely sequenced by the method of Sanger (Sanger et al., 1977, "DNA sequence").
ncing with chain-terminat
ing inhibitors "Proc. Natl.
Acad. Sci. , USA 14 : 5463-546.
7).

Production of monoclonal antibodies against hdm2
Adult : c obtained by screening library
The DNA clone contained the N-terminal coding region of the hdm2 coding region and was truncated at the first methionine start codon. This cDNA is full-length hdm2
Recombinant with the cDNA gave the leader region and the first methionine-free coding region. This coding sequence is p
Insertion into the QE11 vector (Quiagen) resulted in a complete open reading frame with 6 histidine residues fused to the N-terminus of hdm2. The expression plasmid was transformed into E. It was introduced into E. coli and the histidine-hdm2 fusion protein was transferred to Ni-2 ⁺ -NTA-agarose (Quiage).
n) Purified by column chromatography. The mobilities of the major protein species in the purified product were comparable to the translated hdm2 in vitro.

Balb / C mice were transformed into E. coli strains producing the hdm2 protein. immunized with E. coli. The hybridoma was prepared by the usual procedure. Then, screening was carried out by an enzyme-linked immunosorbent assay and an immunoprecipitation method of the converted hdm2 protein in vitro. A stable clone was established by three times of cloning.

Isolation of human dm2 cDNA : A λgt11 library constructed from HeLa cells was screened with mouse mdm2 cDNA under low stringency. A total of 14 positive clones were isolated and the cDNA insert was subcloned into Bluescript vector for further analysis. By preliminary restriction mapping and partial sequencing, they were identified as human dm2 cDNA.
It was found to be part of the A clone (Fakha
rzadeh, S .; S. Et al., 1991, "Tumogenic Potential with Enhanced Expression of Amplified Genes in Mouse Tumor Cell Lines", EMBO J. et al. 10 : 1565-1569).
The full length coding region was constructed from two overlapping cDNA clones and sequenced. This c called hdm2
The DNA sequence of the DNA clone is hdm2 described in the literature.
Similar to the sequence (Oliner, JD et al.,
1992, "Amplification of the gene encoding the p53-related protein in human sarcoma", Nature 35.
8 : 80-83), which is completely the same in the code area and slightly different in the non-code area. These two cDNA clones were derived from two widely different cell sources (He
The fact that they have equivalent coding sequences, even though obtained from La cells and colon carcinomas), either represent the wild type hdm2 coding sequence or are systematic in different cancer cells. Suggests that there is a mutation.

Example 2 A cohort of 211 adult soft-tissue sarcomas whose clinical and pathological features were well understood was analyzed. 73
Tumors and normal specimens were available in a subgroup of 1 patient. Discussions from a molecular and biological perspective were based on this group of 73 patients.
However, in correlating with clinical pathology, immunohistochemical methods are used, and clinical and disease data that can be obtained by patients at Memorial Sloan-Kettering Cancer Center and is frequently updated. Information on 211 soft tissue sarcoma patients obtained from a physical information database was used.

Tissues In the cohort of 211 adult patients with soft tissue sarcoma (STS) used in this example, tumor lesion analysis results were 71 liposarcoma, 53 leiomyosarcoma,
There were 22 cases of malignant fibrous histiocytoma, 15 cases of fibrosarcoma, 15 cases of peripheral nerve sheath tumor (PNST), 13 cases of synovial sarcoma, 4 cases of rhabdomyosarcoma, and 18 cases of undifferentiated sarcoma. Most of the analyzed STSs were primary tumors (129 cases) and the remaining lesions were recurrent (39 cases) or metastatic (41 cases). The birth position of the two cases is unknown. In the 211 STS analyzed, 169 cases were classified as advanced sarcoma and 39 tumors were classified as less advanced lesions. The progress of the 3 cases is unknown. The median and mean follow-up time for a conhort of these patients was 29 and 34 months, respectively. Complete follow-up data could be obtained from 209 of the 211 patients. Tumor samples were embedded in cryopreservation solution (OCT compound, Miles Laboratories, Elkhart, IN) and snap frozen in isopentane-7.
Stored at 0 ° C. A representative part stained with hematoxylin-eosin is taken from each block and observed under a microscope to confirm the presence of tumors and to evaluate the percentage (%) of tumor cells present in these lesions and the extent of tumor necrosis. did. Tumor and normal tissue samples in the vicinity were collected and molecular genetic assays performed on 73 of 211 cases (see below). These tissue samples were surgically removed, immediately frozen, and stored at -70 ° C, and then the DNA was taken out.

Monoclonal Antibodies and Immunohistochemistry This study was conducted using a panel of mouse monoclonal antibodies against the gene product encoded by the p90 gene. Antibody 4B2 detects an epitope located in the amino terminal region. Antibodies 2A9 and 2A10 identify two distinct epitopes in the middle of p90. Antibody 4B11 recognizes a sequence located in the carboxy-terminal region of p90. p53
Three mouse monoclonal antibodies that detect different epitopes on the protein were used in this study. Anti-p53 antibody PAb1801 (Ab-2, "Oncogene Sc
ience ", Manhasset, NY) recognize an epitope located between amino acids (abbreviated as aa) 32-79 of both wild-type and mutant human p53 proteins (Banks, L. et al., 1986, Eu).
r. J. Biochem 159, 529-534).
Antibody PAb240 (Ab-3, "Oncogene S
science ") recognizes a conformational epitope located between aa156-335, which is characteristic of some of the mutant p53 products (Gan).
non, J. V. Et al., 1990, EMBO. J. 9,
1595-1602). Antibody PAb1620 (AB-
5, Oncogene Science) is a wild-type p
Reacts specifically with 53 (Ball, RK et al., 19
EMBO. J. 3, 1485-1492). MIgS-KpI, a mouse monoclonal antibody of the same subclass as anti-p90 and anti-p53 antibodies, was used as a negative control at the same dilution.

Avidin-containing 5 μm thick frozen tissue pieces fixed with cold methanol-acetone (1: 1 dilution).
The biotin peroxidase method was performed. Simply put,
The tissue pieces were cut for 15 minutes with 10% normal horse serum (Organo).
n Tecknika Corp. , West Chester, PA) and then appropriately diluted with primary antibodies (2A9, 4B2 and 4B11).
With a dilution ratio of 1: 100 and 2A10 with a dilution ratio of 1: 1000
(Used in) (Ab-2 is 200 ng / ml, Ab-3 is 2)
50 ng / ml and Ab-5 3 μg / ml) were incubated for 2 hours. After thorough washing, remove the tissue piece with 20
Biotinylated horseradish anti-mouse I diluted 0 times
gG antibody (Vector Laboratories,
Avidin-Biotin Peroxidase Complex (Vector) at Burlingame, CA for 30 minutes
Laboratories, diluted 1:25) for 30 minutes. Diaminobenzidine (0.0
6% DAB) was used as final chromogen and hemotoxylin was used for counterstaining of nuclei.

Immunohistochemical evaluations were performed by at least two independent investigators to calculate the approximate percentage of tumor cells that showed nuclear staining. The nuclear immunoreactivity of both p90 and p53 was classified into three categories: negative (<2
0% of tumor cells show nuclear staining), opposite (20-79%
Tumor cells show nuclear reactivity), and homology (> / =
80% of tumor cells show strong nuclear staining).

Southern Blotting Method and RELP Analysis The gene amplification was evaluated by Southern blotting method using pHDM (EcoRI) which is a 1.6 kb human dm2 cDNA fragment probe. The b-actin probe (EcoRI) was used as a control. Two probes were used for analysis of the allelic deletion of the short arm of chromosome 17: PYNZ22 (17p13.3, D17).
S5, TaqI) and php53B (17p13.
1, p53, BglII). The Southern blotting analysis was performed according to the description in the literature (Prestri, J. et al.
C. (1991), Cancer Res 51,
5405; Dalbagni, G et al., (1993), D.
iagnostic Molecular Patho
logy 2, 4-13). Simply put, DNA
Non-organically extracted from paired normal and tumor samples by the method developed by Ncor (Oncor, Gaithersburg, Md.), digested with appropriate restriction enzymes and in a 0.7% agarose gel. Perform electrophoresis,
Blotting onto a nylon membrane. Membranes were prehybridized with Hybrisol I (Oncor) for 1 hour at 42 ° C and overnight with [P32] dCTP labeled probe for high specific activity. The membrane was washed and radiographed at -70 ° C using an intensifying screen. Ultrascan XL Laser D
ensitometer (Pharmacia LKB Biotechnology, Piscataway, NJ)
And Betascope 630 Blot Analysis
The results were confirmed by measuring the concentration with a zer (Bettergen, Waltham, Mass.). It was considered that there was dm2 amplification when there were at least 5 copy genes / cell. Loss of atypia (LOH) was defined as loss of atypia when the signal intensity of alleles in tumor samples decreased by more than 40% (Prestr).
i, J. C. Et al., (1991), Cancer Res.
51, 5405; Olumi, A .; F. , (199
0), Cancer Res 50, 7081-708.
3).

Single Strand Conformational Polymorphism (SSCP) Analysis and
DNA Sequencing For these, Orita et al. (Orita, M. et al. (1989) Genomics 5, 874-87.
The method reported by 9) was carried out with a slight modification. Amplification is 100 ng of genomic DNA extracted from the above sample
Was performed using. The primers used were obtained from flanking exons 5 to 9 of the intron sequence of the human p53 gene, and this sequence has already been published (Moll, UM et al., (1992)).
Proc Natl Acad Sci USA 8
9, 7262-7266). DNA is Thermal
Cycler (Perkin Elmer Cetu
PCR (30 seconds at 94 ° C, exons 8 and 9)
For 30 seconds at 58 ° C, 30 seconds at 63 ° C for exons 5, 6, 7 and finally 60 seconds at 72 ° C for all samples). Amplified samples were denatured and placed on a non-denaturing acrylamide gel containing 10% glycerol and stored at room temperature for 12-16
Treated for 10-12 watts. The gel was dried under vacuum at 80 ° C and exposed to X-ray film at -70 ° C for 4-16 hours.

Amplification of the genomic DNA in the sequencing assay was independent of that used in the SSCP analysis, 35 cycles (60 seconds at 94 ° C., 60 seconds at 58 ° C. and 63 ° C. under the above conditions, and a further 72 seconds). Amplification was performed at 90 ° C. for 90 seconds. The DNA fragment was isolated from a 2% low melting point agarose gel and subjected to the dideoxy method (Sanger, F. et al.,
(1977), Proc Natl Acad Sc
i USA 74, 5463-5467). Both strands were sequenced for each DNA analyzed and genomic DNA from a control sample containing wild-type p53 was also sequenced in parallel to confirm the mutation.

Amplification of DM2 and overproduction of DM2 protein The amplification of the dm2 gene was examined in 11 cases of adult soft tissue sarcoma (STS) of 73 cases, and it was amplified 5 to 35 times. Amplification of dm2 was detected more frequently in cases of high disease progression (7 cases) than in cases of low disease progression (4 cases). Primary STS (4 out of 48 cases) in metastatic STS (3 out of 11 cases, 27%)
Amplification was more commonly observed than in (e.g., 8%).

The immunostaining pattern of the anti-dm2 antibody was first evaluated using 3T3-Balbc and 3T3-DM cells. Strong nuclear staining was observed in DM cells and amplified dm
It was reported to carry two genes and overexpress the dm2 protein, whereas Balb-c cells did not react (FIG. 4) with low dm2 protein levels. Six out of eleven amplified cases showed greater than 20% of tumor cells showing nuclear immunoreactivity with anti-dm2 antibody. However, no reaction was observed in the remaining 5 cases. Of the 62 cases, 17 cases with the dm2 gene apparently unamplified showed elevated levels of dm2 protein as detected by the dm2 antibody using tissue debris (dm2-positive phenotype).

P53 deletion, point mutations, and p53 nuclear immunity
73 pairs of reactive somatic and tumor DNA were transferred to chromosome 17
Was investigated using two different probes for the short arm of the. Deletion of the short arm of chromosome 17 was found in 27 out of 51 (53%) of the information notification cases investigated. Loss of chromosomal 17p dysmorphism (LOH) was observed in both aggressive and less aggressive sarcomas. Chromosome 17
LOH of p was found more frequently in metastatic tumors (6 of 8, 75%) than in primary tumors (13 of 33, 41%).

Since p53 appears to be associated with intracellular overexpression, 73 STSs were treated with SSCP to better characterize specific intragenic mutations of p53.
(Exons 5 to 9), and those that were positive in this assay were DNA sequenced. Existence of mutation is 1
It was confirmed in 4 cases. Of these 14 STSs, 11 showed p53 nuclear immunoreactivity with the antibody PAb1801. Sequencing characterized the point mutations in 7 of 11 sarcomas, and 5 cases of AT to G
Regarding C mutation, 2 cases showed mutation from GC to AT. Four
Changes in mobility were detected by SSCP in the examples, but were not sequenced to identify mutations. Three out of 14 mutations showed negative immunostaining results for PAb1801. One of the mutations produced a stop codon and was identified in codon 165. The other case had a C deletion at codon 278 and produced a stop codon at position 344. Another mutation occurred in exon 5, which affects the splice donor position. With the exception of one, short arm deletions associated with all informative mutants for the status of chromosome 17p were observed. In addition, 13 cases showed positive nuclear staining without evidence of point mutations to the exons under study.

Finally, 56 of the 211 STS analyzed showed a positive nuclear immunostaining pattern for PAb1801 (Table 1). There was a large difference between p53-positive phenotype and tumor progression. Moreover, patients suffering from STS showed p53 nuclear immunostaining with> 20% tumor cells and markedly poor survival.

[0079]

[Table 1]

Changes in genotype and phenotype of DM2 and P53
In relation to clinical pathology , only one case in a subgroup of 73 cases is amplified dm2
And a mutant of the p53 gene, which was a metastatic fibrosarcoma. However, 22 out of all 211 cohorts showed positive nuclear immunoreactivity in consecutive tissue sections against both dm2 and p53 proteins (Table 1).
The staining pattern of these molecules was generally heterogeneous when they were co-expressed. Combination of phenotypes (group A: dm2- / p53-; group B: dm2-
/ P53- and dm2- / p53 +; Group C: d
When m2 + / p53 +) was compared with clinicopathological parameters, there was a correlation between positive phenotype and poor prognosis. The data showed that Group A had the best prognosis and Group C had the poorest prognosis (see Figure 3).

Example 3 p90 can be used to prepare antibodies for immunoprecipitation of p90 or co-immunoprecipitation of p90 and p53. p90 is isolated from the sediment and purified. High p90
The antibody may be polyclonal or monoclonal. p
The method for preparing a polyclonal antibody against 90 is as follows.

Procedure for producing p90 antibody in rabbits : The p90 protein fragment is used at a concentration of 0.7 mg / ml. Immunization is performed as follows: Day 0: 200 μl PBS and 200 μl RIBI.
25 μg in (adjuvant), day 7: 200 μl PBS and 200 μl RIBI
50 μg in (adjuvant), day 14: 200 μl PBS and 200 μl RIB
50 μg in I (adjuvant), day 21: rest day 28: 200 μl PBS and 200 μl RIB
50 μg in I Day 39: Blood collection-Day 52 of assay: 200 μl PBS and 200 μl RIB.
50 μg added to I and administered to animals, Day 59: Blood collection-Assay 62 days 7 days after final infusion Day 62: Bleed dead

The assay was performed by applying the ELISA method and coating the wells (200 ng / well) at 4 ° C. overnight. Block with 2% BSA for 1 hour at 37 ° C. 1
Serum is added to% BSA, diluted and incubated at 37 ° C. for 2 hours. Secondary antibody dilution prepared with 1% BSA (TAGO goat anti-rabbit peroxidase-cat # 6
In 430), it is incubated at 37 ° C. for 1 hour. Serum antibody titer was 1: 25,000 in the first assay (cutoff absorbance: 0.400 at 450 nm), >> in the final assay.
50,000. Kirkegard & Perry
Develop with TMB peroxidase substrate solution, 450nm
Read the absorbance at.

Example 4 Hybridoma 1F deposited with ATCC (supra)
5, 6C10, 1D6, 4B2, 2E12, 3F3, 3
G5, 3F8, 6H7, 2A9, 3G9, 1D11, 2
A10, 1G2, 4B11, and 5B10 produce monoclonal antibodies against the epitope of p90 protein (see Figure 1). The protocol described below uses these monoclonal antibodies and other anti-p90 antibodies as transient antibodies to detect the DM2 gene-encoded product p90 protein in tissues, preferably human tissues, by immunohistochemical methods. It is a thing. The avidin-biotin immunoperoxidase method is a suitable method because of its high sensitivity. Human normal and tumor tissue sections are cut with a cryostat and mounted on microslides. These sections are incubated with blocking serum, followed by hydrogen peroxide and avidin-biotin blocking agent. The first antibody, anti-p90 antibody, is used at the appropriate concentration. This appropriate concentration is empirically determined by performing a titration for each antibody. The sections are then incubated with biotinylated secondary horse anti-mouse antibody followed by avidin-biotin peroxidase complex. The final reaction is performed with diaminobenzidine. Sections are counterstained with hematoxylin and mounted on a stage for final analysis.

In the immunohistochemistry method described above, typically only the p90 protein is detected in the tissues with elevated values deviating from the normal level because the monoclonal antibodies were not detected in the tissues tested in the above examples. This is because only an increase in p90 in cells can be detected.

Immunohistochemical avidin-biotin-peroxidase method Paraffin embedded section 1) A 5 μm tissue section is placed on a slide coated with poly-L-lysine. 2) Put the slices in an oven at 60 ° C for 30 minutes to melt the paraffin. 3) Cool slides to room temperature and treat for deparaffinization and rehydration. Xylene-3 times (5 minutes each time) 100% ethanol-3 times (3 minutes each time) 95% ethanol-3 times (3 minutes each time) 4) Wash in distilled water and transfer to PBS. 5) Cool with 1% H ₂ O ₂ for 15 minutes to remove endogenous peroxidase activity. 6) Wash in PBS (3 times). 7) To remove the endogenous peroxidase activity,
The avidin-biotin blocking kit (Vector) is applied to cells containing biotin such as liver, pancreas, and brain. Solutions should be used sequentially (avidin followed by biotin) and slides should be incubated with each solution for 15 minutes. 8) Wash in PBS.

9) Enzymatic degradation-enzyme selection is done empirically to be optimal for each antibody. Common Enzymes: Pepsin (porcine gastric mucosa, Sigma): Add 0.25 grams of pepsin to HCL solution (250 ml distilled water plus 200 μl HCl) and incubate for 30 minutes. Trypsin (bovine pancreas type I, Sigma): 25
Add 0.0625 grams trypsin to 0 ml TRIS and incubate for 5 minutes. Wash in distilled water,
Trypsin inhibitor (Sigma) (250 ml PBS)
And 0.025 grams of trypsin inhibitor) for 15 minutes. Pronase (Calbiochem Behring): 250 ml
To TRIS of 0.0055 grams of pronase,
Incubate for 4 minutes. Ficin (suspension, Sigma): Ready to use.
Add dropwise and incubate for 45 minutes. Saponin (detergent, Sigma): Add 0.125 grams of saponin to a mixture of 250 ml distilled water and incubate for 30 minutes.

10) Wash slides in distilled water, PB
Move to S. 11) Blocking serum-10% normal serum (specific species-same as secondary antibody) added and incubated for 20-30 minutes. 12) Aspirate off blocking serum with vacuum, add appropriately diluted primary antibody and incubate overnight at 4 ° C in a humid chamber. The first antibody is 1F5, 6C10, 1D
6, 4B2, 2E12, 3F3, 3G5, 3F8, 6H
7, 2A9, 3G9, 1D11, 2A10, 1G2, 4
Selected from anti-p90 monoclonal antibodies produced by the B11 and 5B10 hybridomas. First
The antibody is appropriately diluted empirically so as to have an optimum concentration. 13) Carefully wash with PBS. Change the PBS 3 times (5 minutes each time) 14) Add appropriately diluted biotinylated secondary antibody, and add 3
Incubate for 0 minutes. 15) Wash with PBS (3 times 5 minutes each). 16) Incubate the avidin-biotin complex (Vector) diluted 1:25 (equal ratio of A: B) for 30 minutes. 17) Wash with PBS (3 times 5 minutes each). 18) Chromogen substrate solution: perosidase-diaminobenzadine (0.06% DAB) (5 mg / DAB / 1
100 μl 0.3% H ₂ O _{2 in} 00 ml PBS). Incubate until the desired color strength (about 5
Minutes). 19) Counterstain with hematoxylin. Immunohistochemical Avidin-Biotin-Peroxidase Method Frozen Tissue Section 1) A 5 μm frozen tissue section is placed at room temperature for 30 minutes to thaw the section and dry. 2) Add appropriate fixative for 10 minutes (select the best fixative for each antibody). 3) Cool with 15% H ₂ O ₂ for 15 minutes to remove endogenous peroxidase activity. 4) Wash in PBS (3 times). 5) In order to eliminate the endogenous peroxidase activity,
Add avidin-biotin blocking kit into cells rich in endogenous biotin. Incubate for 15 minutes with avidin,
Wash with PBS and incubate with biotin for 15 minutes. 6) Wash in PBS (3 times). 7) Blocking serum-10% normal serum (specific species-same as secondary antibody) is added and incubated for 10-30 minutes in a humid chamber. 8) Vacuum up the blocking serum, remove, add appropriate diluted primary antibody and incubate for 1-2 hours. First
The antibodies are 1F5, 6C10, 1D6, 4B2, 2E1
2, 3F3, 3G5, 3F8, 6H7, 2A9, 3G
9, 1D11, 2A10, 1G2, 4B11, and 5
Selected from anti-p90 monoclonal antibodies produced by B10 hybridomas. The first antibody is appropriately diluted empirically so as to have an optimum concentration, but the volume ratio of the supernatant of the hybridoma to phosphate buffered serum (PBS) is typically 1 to 1000. 9) Carefully wash with PBS. 10) Add biotinylated secondary antibody diluted appropriately and
Incubate for 0 minutes. 11) Wash with PBS (3 times). 12) Incubate a 1:25 dilution (equal proportions of A: B) of the avidin-biotin complex (Vector) for 30 minutes. 13) Wash with PBS and PBS / Triton (3
Times). 14) Chromogenic substrate solution: perosidase-diaminobenzadine (0.06% DAB) for about 5 minutes. 15) Counterstain with hematoxylin.

[Brief description of drawings]

FIG. 1 shows the epitope of p90 protein that reacts with the anti-p90 single coulomb antibody of the present invention. Hybridoma 1F5, 6C10, 1D6, 4B
2, 2E12, 3F3, 3G5, 3F8, 6H7, 2A
9, 3G9, 1D11, 2A10, 1G2, 4B11,
The anti-p90 single coulombic antibodies produced by and 5B10 are shown below the p90 amino acid epitope map at the positions indicating the epitopes to which these antibodies react.

Figure 2 This graph shows 90 of 211 soft tissue sarcoma patients.
Shows overall survival over months.

FIG. 3 This graph shows elevated levels of p53 and mdm2 and survival in a group of 211 soft tissue sarcoma patients (see FIG. 2). The types of phenotypes shown in the graph are as follows. Group A: dm2- / p53- (d
There is no level increase in either m2 or p53). Group B: dm2 + / p53- and dm2- / p53 +. Group C: dm2 + / p53 + (both dm2 and p53 have elevated levels).

FIG. 4 shows an immunostaining pattern using an anti-dm2 antibody and an anti-p53 antibody. The upper two slides represent the control group. The upper left slide shows the staining pattern for the dm2-, 3T3-Balb / c cell line. The upper right slide shows the staining pattern for the dm2 + 3T3-DM cell line. The middle two slides show the staining pattern of human tumor tissue sections taken from the same patient. The center left slide shows a p53-staining pattern, and the center right slide shows a dm2 + staining pattern (group B subset p53- / d described below).
m2 + subset). The lower two slides show the staining pattern of human tumor tissue sections taken from another patient. The lower left slide shows the p53 + staining pattern and the lower right slide shows the dm2 + staining pattern (corresponding to group C described below).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07K 19/00 C12Q 1/02 4H045 C12N 5/10 1/68 A C12Q 1/02 G01N 33/566 1 / 68 33/574 A G01N 33/566 Z 33/574 33/577 B C12P 21/08 33/577 C12N 15/00 A // C12P 21/08 5/00 B (71) Applicant 399026731 Sloan-Kettering Institute The For Four Cancer Research, New York, USA 10021, York Avenue, York 1275, 1275 (72) Inventor Levin Arnold, J. USA 08540 Fritz Randolph Road, Princeton, NJ 138 (72) Inventor Finley, Kathy, A. USA United States 19067 Bayberry Lane, Yardley, Pennsylvania 660 (72) Inventor Cordon-Card, Carlos United States 10021 New York, New York, East 63 Street 504, APT 24 Earl F-term (reference) 4B024 AA11 BA36 BA41 CA03 CA04 CA05 CA07 CA09 DA02 DA06 EA04 GA01 GA11 GA18 GA19 HA03 HA14 4B063 QA01 QA12 QA18 QA19 QQ02 QQ42 QQ52 QQ58 QR32 QR38 QR41 QR55 QR66 QR82 QS12 QS25 QS34 QS36 QS39 QX01 QX07 4B064 AG27 CA10 CA20 CC24 CE12 DA14 4B065 AA91X AA93X AA93Y AB01 AB04 AC14 BA01 BA21 CA25 CA46 4C085 AA14 CC02 CC03 CC04 CC05 DD23 DD32 DD61 4H045 AA10 AA11 AA30 BA10 BA41 CA40 DA76 DA86 EA51 FA72 FA74

Claims (33)

[Claims] 1. A monoclonal antibody produced by hybridoma 3G5 deposited under ATCC Deposit No. HB11182. 2. A monoclonal antibody produced by hybridoma 4B11 deposited under ATCC Deposit No. HB11183. 3. A monoclonal antibody produced by hybridoma 2A10 deposited under ATCC Deposit No. HB11184. 4. A monoclonal antibody produced by hybridoma 2A9 deposited under ATCC Deposit No. HB11185. 5. A monoclonal antibody produced by hybridoma 4B2 deposited under ATCC Deposit No. HB11186. 6. The monoclonal antibody according to claim 1, which is bound to a toxin. 7. The monoclonal antibody according to claim 1, which is labeled with a detectable marker. 8. Hybridoma 3G5 and ATCC deposit number H deposited under ATCC deposit number HB11182
Hybridoma 4B1 deposited as B11183
Hybridoma 2A10 deposited under ATCC Deposit No. HB11184, ATCC Deposit No. HB111
Hybridoma 2A9 and A deposited as 85
A monoclonal antibody that recognizes the same epitope recognized by the monoclonal antibody produced by the hybridoma selected from the group comprising hybridoma 4B2 deposited under TCC Deposit No. HB11186. 9. Hybridoma 3G5 and ATCC deposit number H deposited under ATCC deposit number HB11182
Hybridoma 4B1 deposited as B11183
Hybridoma 2A10 deposited under ATCC Deposit No. HB11184, ATCC Deposit No. HB111
Hybridoma 2A9 and A deposited as 85
A monoclonal antibody that competes with the same epitope recognized by the monoclonal antibody produced by the hybridoma selected from the group comprising hybridoma 4B2 deposited under TCC Deposit No. HB11186. 10. Hybridoma 3G5 deposited under ATCC deposit number HB11182, hybridoma 4B1 deposited under ATCC deposit number HB11183.
Hybridoma 2A10 deposited under ATCC Deposit No. HB11184, ATCC Deposit No. HB111
Hybridoma 2A9 and A deposited as 85
The monoclonal antibody according to claim 9, which blocks in the range of 50 to 100% the same epitope as that recognized by the monoclonal antibody produced by the hybridoma selected from the group containing the hybridoma 4B2 deposited under TCC deposit number HB11186. 11. Hybridoma 3G5 deposited under ATCC Deposit No. HB11182, hybridoma 4B1 deposited under ATCC Deposit No. HB11183.
Hybridoma 2A10 deposited under ATCC Deposit No. HB11184, ATCC Deposit No. HB111
Hybridoma 2A9 and A deposited as 85
A hybridoma selected from the group comprising hybridoma 4B2 deposited under TCC Deposit No. HB11186. 12. An epitope recognized by a monoclonal antibody selected from the group of monoclonal antibodies according to claims 1-5. 13. An epitope recognized by the monoclonal antibody according to claim 3. 14. A diagnostic kit comprising (a) a container containing an anti-p53 antibody that recognizes a p53 protein, and (b) a container containing an anti-dm2 antibody that recognizes a dm2 protein. 15. The antibody according to claim 1, which is labeled.
4. The diagnostic kit according to 4. 16. The diagnostic kit according to claim 14, further comprising (a) a labeled antibody that recognizes an anti-p53 antibody, and (b) a labeled antibody that recognizes an anti-dm2 antibody. 17. A method for determining whether a cancer cell, or a cell at risk of becoming cancerous or precancerous, comprising at least one normal p53 allele is present in a biological sample. A method of detecting, comprising detecting whether the level of dm2 in a biological sample is abnormally elevated compared to the level of dm2 expression, wherein the level of dm2 in the biological sample is abnormal. A method of determining that the cancer cell is a cancer cell, or a cell that is at risk of becoming cancerous or precancerous due to the increase in the. 18. The method of claim 17, wherein the elevated levels are determined by the immunohistochemical staining pattern of the biological sample. 19. The method according to claim 17, wherein the level of proliferation or expression of the dm2 gene is determined using a probe. 20. The method of claim 19, wherein the probe is a nucleic acid probe. 21. The method of claim 19, wherein the probe is an antibody. 22. The method of claim 21, wherein the antibody is a monoclonal antibody. 23. The hybridoma 3 wherein the monoclonal antibody is deposited under ATCC Deposit No. HB11182.
23. The method of claim 22, which is produced by G5. 24. The hybridoma 4 wherein the monoclonal antibody is deposited under ATCC Deposit No. HB11183.
23. The method of claim 22, which is produced by B11. 25. The hybridoma 2 wherein the monoclonal antibody is deposited under ATCC Deposit No. HB11184.
23. The method of claim 22, which is produced by A10. 26. The hybridoma 2 wherein the monoclonal antibody is deposited under ATCC Deposit No. HB11185.
23. The method of claim 22, which is produced by A9. 27. The monoclonal antibody is hybridoma 4 deposited under ATCC Deposit No. HB11186.
23. The method of claim 22, which is produced by B2. 28. The hybridoma 3 wherein the monoclonal antibody is deposited under ATCC Deposit No. HB11182.
G5, hybridoma 4B11 deposited under ATCC deposit number HB11183, ATCC deposit number HB11
Hybridoma 2A10, AT deposited as 184
Hybridoma 2A9 deposited as CC Deposit No. HB11185, and ATCC Deposit No. HB11186
23. The method according to claim 22, which recognizes the same epitope as that recognized by the monoclonal antibody produced by the hybridoma selected from the group containing hybridoma 4B2 deposited as above. 29. The monoclonal antibody is hybridoma 3 deposited under ATCC Deposit No. HB11182.
G5, hybridoma 4B11 deposited under ATCC deposit number HB11183, ATCC deposit number HB11
Hybridoma 2A10, AT deposited as 184
Hybridoma 2A9 deposited as CC Deposit No. HB11185, and ATCC Deposit No. HB11186
23. The method according to claim 22, which competes with the same epitope recognized by the monoclonal antibody produced by the hybridoma selected from the group comprising hybridoma 4B2 deposited as above. 30. The competitive monoclonal antibody is ATCC.
Hybridoma 3G5 deposited under deposit number HB11182, Hybridoma 4B11 deposited under deposit number HB11183, ATCC deposit number HB
Hybridoma 2A10 deposited as 11184,
Hybridoma 2A9 deposited as ATCC Deposit No. HB11185, and ATCC Deposit No. HB111
30. The method of claim 29, which blocks 50-100% of the same epitopes recognized by the monoclonal antibody produced by the hybridoma selected from the group comprising hybridoma 4B2 deposited as 86. 31. The p53 / dm2 complex. 32. A method for treating a patient having a tumor, wherein the antibody is bound to the tumor by bringing the antibody of claim 6 into contact with the tumor. 33. A tumor image in which the tumor is imaged by contacting the anti-dm2 antibody according to claim 7 with the tumor under conditions such that the antibody binds to the tumor to be imaged and detecting the bound antibody. Method.