JP2001519169A - Methods for detecting, confirming, obtaining or removing human or animal cells with impaired cell circulation control or human or animal cells having infinite proliferative or tumorigenic potential - Google Patents

Abstract

  (57) [Summary] To detect, confirm, obtain or remove human or animal cells with impaired cell circulation control, or human or animal cells that have infinite proliferative or tumorigenic potential, The presence of an association comprising a cdc37 protein having an extrachromosomal nucleic acid is detected. This can be, for example, using a detectable substance that has the ability to specifically bind, using a nucleic acid or oligonucleotide that hybridizes with the nucleic acid of the association, or by binding to a solid carrier. In this case, if the last method is used, such cells may be obtained or removed.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a cdc37 protein-nucleic acid-heteromer, a cdc37 protein, a nucleic acid and an antibody, which are suitable for the detection of a cell having an infinite proliferative ability or a tumor-forming ability, and such a heteromer, protein, nucleic acid or antibody. The present invention relates to a method for identifying or removing human or animal cells having an infinite proliferative ability or a tumor-forming ability.

[0002] All normally differentiated cells have a limited capacity for cell proliferation (Proliferation) before it undergoes cell senescence (aging phenomenon) and cell death after a defined number of cell divisions. The cellular biological course of cell division and senescence is based on intricately regulated genetic controls, the impairment of which can lead to cell death on the one hand or indefinite growth or tumor formation on the other hand .

Cell proliferation requires cell division, which takes place in a cascade of processes in temporal and spatial order (cell cycle). Control of this course is performed at two time points, the start of DNA-synthesis (S-phase of the cell cycle) and the start of mitosis (M-phase), and many of this course is involved in phosphorylation. Is achieved by a small group of protein kinases that regulate the activity of proteins that act. These cell cycle-controllable protein kinases are heterodimeric proteins with regulatory subunits (cyclins) whose content and association with other proteins characteristically change over the course of the cell cycle. The catalytic subunit of this protein kinase is called a cyclyn-dependent kinase (cdk) because it becomes active as a kinase only after association with cyclin. This cyclin-dependent kinase was first discovered in yeast and named for the gene that encodes it, for example, Schizosaccharomyce
spcombe) or cdc28 from Saccaromyces cerevisiae. Shortly thereafter, many proteins involved in cell cycle control were found to be highly conserved and exist as homologous genes with similar or identical functions in eukaryotic cells.

[0004] The course of the cell cycle in mammalian cells is controlled by a number of cyclin-dependent kinases in combination with various cyclin-molecules (CJSherr, Mammalia
n G1 Cyclins, Cell 73: 1059-1065, 1993). For example, Cdk2, Cdk4 and Cdk5 associate with the subunits cyclins D1, D2, D3, respectively,
It is involved in the initiation of the G1 phase of the cell cycle. In this case, the cyclin-dependent kinase can associate with different cyclins and control different points in the cell cycle. For example, Cdk2 associates with cyclin E to participate in the control of G1-S translocation, and Cdc2 associates with cyclins A and B to associate G2-M
Control the transition. The level of knowledge of this cell cycle control is very far from a single model, and additional Cdk- and cyclin molecules are being developed that still control the defined location of the cell cycle and consequent cell growth.

[0005] Although the cell cycle of normally proliferating cells is controlled in a coordinated manner by the mechanisms described above and by other previously unexplained processes, neoplasticly transformed cells are It is presumed to have one or more defects in one or more control mechanisms of the cycle. This assumption is made clear in the following example. Cyclin-dependent protein kinases can promote or inhibit the transition from one phase of the cell cycle to the next. However, damage to DNA by irradiation or chemical agents has a stabilizing effect on the tumor suppressor protein p53, thereby stimulating the synthesis of cyclin-dependent kinase inhibitors in particular. The kinase-inhibitor binds on its side to the G1-specific Cdk / cyclin complex, on which the cells remain until the DNA-wound is repaired and the p53 concentration during the G1-phase is reduced. This complicated adjustment is made up exclusively by D
NA—Effects the growth of cells that have completely repaired the wound. If this repair is not successful, the cell will undergo programmed cell death, thereby causing the existing DNA
-Prevention of cell deterioration until uncontrolled growth in wounds. However, p53
Mutation of the encoded gene did not result in a delay in the G1-S translocation induced by p53, which resulted in replication of the damaged DNA and damage to the cell without leading to programmed cell death. Cell proliferation occurs.

[0006] These and other deficits in this control of the cell cycle are presumed to contribute to permanent proliferation and ultimately to tumoral changes of the cells. Since almost all major control proteins and their modulators have not yet been identified, the current expert opinion on the lack of cell cycle-control in tumor degenerated cells is very flawed. Furthermore, the phenotype of neoplastic transformed cells can be very diverse, with many genetic phenotypes-morphological and physiological changes (which themselves are many other parameters, such as degree of differentiation, cell matrix, Surrounding cells, local growth factors
(Depending on density or immunological defenses) and the difficulty of molecularly defining common true properties. This problem is illustrated using the cell growth of normal and neoplastic transformed cells.

[0007] Normal human and animal cells have a limited capacity for cell growth, but neoplasticly transformed cells often show infinite cell division. This forms a constantly growing population of cells, which is responsible for the formation of tumors and ultimately other properties (eg reduced need for growth factors, continuous growth and contact-inhibition, induction of neovascularization, etc.). Together with the deformed manifestation of, leads to tumor disease. According to the current understanding of tumor development,
A cell has been transformed with great certainty into neoplasia (neoplastic) when (1) the cell grows in vivo indefinitely and / or (2) its cell death is stopped. This neoplastic transformation is a multi-step method, at the end of which the tumor cells are characterized (1) and / or
Or (2) appears with many other properties.

An early phenomenon in this process is endless proliferation (immortalization), characterized by the ability of cells to survive the cell cycle and undergo continuous cell division (Freund et al.). al., Oncogene 7: 1979-1992). However, due to this immortalization, the cells still do not acquire the capacity for tumorigenesis in one tissue population (Medina und Kttrell, Carcinogenesis 14: 25-28. 1993; Strauss und Griffi
n, Cabcer Cells 2: 360-365. 1990). This requires a number of additional "transformation phenomena" (not yet individually characterized).

On the other hand, induction of a neoplastic transformed phenotype does not require such immortalization (
O'Brien et al., Proc. Natl. Acad. Sci. USA 83: 8659-8663, 1986). This example can no longer be split, but its programmed cell death (Apopto
se) are chromosomal lymphocytic leukemia cells that have been stopped (Buschle et. al.,
J. Exp. Med. 177, 213-218, 1993).

[0010] The constant differentiation of progenitor cells always leads to the formation of new cells, which therefore inhibits their cell death. This constantly increases the number of cells, which leads to tumor formation.

[0011] Tumor cells are present at various stages of neoplastic transformation and can acquire various "tumor-associated" properties or have lost the properties of differentiated cells. . With various mechanisms leading to phenotypic heterogeneity and neoplastic transformation, it is extremely difficult to identify cells with infinite proliferative and tumorigenic potential around normal cells. However, for diagnosis and treatment, and ultimately for patient survival, it is a decision to find cells that no longer have normal growth control and cell cycle-regulation and have acquired infinite proliferative and tumorigenic potential. Is important.

[0012] In this case, one always distinguishes the growth of neoplastic transformed cells from the physiological growth of normal cells in the area of tissue regeneration or differentiation, for example during hematopoiesis or wound healing in the bone marrow. That is particularly difficult. Physiological growth occurs in response to a physiological stimulus, either for a short time or permanently, wherein the extent of biologically renewable growth can far exceed that of tumor cells. This is particularly important during the growth and differentiation of blood forming cells. Therefore, it is not possible to use the degree of proliferation of one cell (cell division per unit time) or the progress in the cell cycle and the degree of activation of the cell cycle-control protein (phosphorylation) for the identification of tumor cells. It is.

[0013] Known methods for detecting tumor cells are expressed on clinical, morphological-histological, cytological or physiological parameters or enhanced by proliferating (normal or tumor-) cells. Antibodies that recognize antigens (eg, Ki-67 antigen or PCN-antigen) are further based on irrelevant indirect parameters responsible for neoplastic transformation of cells. It is readily understood that in many possible processes that can result in neoplastic transformation of cells, mutations in advantageous genes are found in the presence of few tumors. Examples are mutations in the membrane location of intracellular signaling or in intracellular receptors (eg, fms, erb-B / A, src, yes), protein kinases (eg, mos, raf, ras) or nuclear transcription factors ( For example, mutations in fos, jun, myc) or cell cycle-associated control-proteins (eg, p53, rb).

It is therefore an object of the present invention to detect cells with injured cell cycle-controls or acquired infinite proliferative or tumorigenic potential in situ or in a cell suspension in a tissue community. Removing or inhibiting growth thereof, wherein the neoplastic transformed cells are distinguished from normal cells having transient or physiological proliferation.

This problem is solved by a method for the detection, identification, acquisition or removal of human or animal cells having injured cell controls or having infinite proliferative or tumorigenic potential, the method comprising the steps of: detecting the presence of an association from the cdc37 protein and the extrachromosomal nucleic acid. The polypeptide-portion of the polypeptide-nucleic acid association refers to the human or animal homologous protein to the yeast cdc37 protein and its subunits. This nucleic acid-portion contains the chromosomal DNa of each tumor cell.
-Consists of extrachromosomal DNA- or RNA-copy of the sequence (broadly cdc3
7-designated nucleic acid-heteromer). This cdc37-nucleic acid-heteromer is suitable for the detection of human or animal cells having infinite proliferative or tumorigenic potential. Therefore, another object of the present invention is to isolate from soluble fractions from human or animal cells that can be permanently divided, isolation by precipitation in the presence of 1.5 M NaCl and extraction with phenol and ethanol. Alternatively, a cdc37-nucleic acid-heteromer is obtained by precipitation using ammonium acetate.

The present invention takes advantage of the following unexpected properties of permanently proliferating or tumorigenic cells: (i) tumors, but not normal cell cells with physiological transient or permanent proliferation; In cells that have the ability to form or to grow indefinitely, the molecules of the cdc37 homologous protein associate together with the nucleic acid in the heteromer; (ii) the cdc37-nucleic acid heteromer is used to differentiate Suitable for the detection of cells having a tumorigenic or infinite proliferative potential, irrespective of the stage or organ from which the cells are derived; (iii) a molecule that binds to cdc37-nucleic acid heteromer, It is suitable for removing or increasing the concentration of cells having tumorigenic or infinite proliferative capacity and for suppressing their growth or initiating cell death of these cells.

From Proc. Natl. Acad. Sci. USA 90: 6519-6922 (1993) and PCT WO 95/02701, tumor cells or infinitely proliferating cells have extrachromosomal DNA associated with the protein. Is known. The described DNA-protein complex is enriched in a CsCl density gradient during equilibrium centrifugation in a fraction with a density of 1.82-1.86 g / cm ³ , and the DNA is SDS- and salt-stable. Associates with the protein by binding, and the proteins are 52 kD, 62 kD and 64 kD
Having a molecular weight of However, the cdc37-nucleic acid heteromers according to the invention are distinguished from the DNA-protein complex by the following properties:-The cdc37-protein has a molecular weight of about 40 kD. 52kDp, 62kD
Or a polypeptide having a molecular weight of 64 kD is not detectable in the cdc37-nucleic acid heteromers according to the invention.-The binding of the nucleic acid to the cdc37 protein is carried out in the presence of detergents at elevated temperatures, e.g. Can be released at 90 ° C., but does not bind the DNA-protein complex. The cdc37 protein-associated DNA sequence differs from that of the DNA-protein complex from PCT WO 95/02701.

To elaborate the present invention, an important level of knowledge on the biochemistry of the cdc37 protein is summarized below.

[0019] The cdc37 protein is known and was first isolated from yeast based on its properties that result in a cell cycle that has been impaired by mutation of this protein (Ferguson et al.
., 1986). Only very late has the isolation from Drosophila been successful by interfering with the mutations of cdc37 and hsp83 using the Signalweg, especially with the "sevenless" receptor tyrosine kinase. (Cutforth et al., Cell 77: 1027, 1994). Over the last three years, the mammalian homologous cdc37 protein has been transformed into a multiprotein complex containing a highly conserved amino acid sequence relative to the yeast cdc37 sequence and the cyclin-dependent kinase-4 (cdk4) and heat-shock protein hsp90. It was cloned based on some characteristics (Dai et al., J. Biol. Chem. 271).
: 22030, 1996; Lamphre et al., Oncogene 14: 1999, 1997). Furthermore, this cd
The c37 protein comprises many other proteins, such as the retinocytoma-protein RB and the virally encoded protein with transformed activity, such as SV40 large T, adenovirus E1A, papillon-virus E7, pp60V-src or raf.
(Ozaki und Sakiyama, DNA Cel)
l Biol. 15: 975, 1996; Perdew et al., Biochem. 36: 3600, 1997). This cdc37 protein further binds to hyaluronic acid (Grammatikakis et al., J. Bo.
l. Chem. 270: 16198, 1995). The diversity of this interaction between cdc37 and other molecules of the cell has not been previously known. hsp90 binds to the protein kinase cdk4 and stabilizes the cdc37 / cdk4 complex. Cdk4 in this form
Is activated by cyclin D1. After the transition of the cell through the G1 phase to the S-phase of the cell cycle, the complex breaks down and is then reconstituted only when the cell re-establishes the G1 transition position of the cycle (Stepanova et al.). al.
, GEnes Develop. 10: 1491, 1996). The cdc37 / hsp90 complex can be destabilized by the antibiotic geldanamycin, which results in the disintegration of this complex. The cells can no longer enter the S-phase of the cell cycle (Stepanova et al., GEnes Develop. 10: 1491, 1996). This level of knowledge derives from the fact that cdc37 is involved in controlling the G1-S transition of cells in the cell cycle and is therefore always expressed when the cells reach this point in the cell cycle ( Ozaki. Et al., DNA Cell biol. 14: 1017, 1995). This indicates that the gene expression of cdc37 protein in proliferating cells is exclusively G1
Is detectable during the transition of cells from the S-phase to the S-phase, consistent with observations that are stopped immediately after the transition into the S-phase. Thus, this cdc37 expression is about 24-3
It lasts up to 9 hours with a cycle time of 6 hours of proliferating cells and is undetectable during the other phases of this cell cycle.

According to Ozaki and Sakiyama (FEBS Lett. 375: 155, 1995), it is known that the cdc37 protein can bind DNA in vivo. This indicates that the authors incubated these with the genomic DNA from the Latte cells and incubated them under in vitro conditions with cdc37.
This is shown by cloning the DNA bound to. The author has in this case obtained one DNA clone of genomic DNA which binds to the cdc37 protein. The authors speculate that "cdc37 may play an important role in controlling cell cycle progression via a sequence-specific DNA binding mechanism." Contrary to this in vitro condition study and the author's assumption, even if the cdc37 protein was able to bind to chromosomal DNA,
DNA-association of the dc37 protein has not been previously demonstrated. Conversely, cdc37 protein in physiologically growing normal cells was not verifiably bound to DNA.

[0021] cdc3 in neoplastic transformed cells with infinite proliferative or tumorigenic potential
It was even more surprising that DNA bound to the 7 protein was found. However, this is extrachromosomal DNA, contrary to the assumptions made by Ozaki and Sekiyama. Therefore, the DNA sequence contained in this heteromer according to the invention is not homologous to the genomic DNA sequence isolated by Ozaki and Sekiyama. Therefore, heteromers from the cdc37 protein and extrachromosomal nucleic acids are novel and are objects of the present invention.

All known observations indicate that the cdc37 protein is exclusively responsible for the G1-S-cell cycle.
Injured cell cycle-control or cdc37-nucleic acid in cells with infinite growth or tumorigenic potential, as it is expressed during phase transition but suggests that it is not essentially during the whole cell cycle. It was surprising that the heteromer is expressed essentially independently of the cell cycle-phase. In contrast, cdc37-nucleic acid-heteromers are not expressed in normal cells, regardless of whether the cells are growing, quiescent or senescent. Thus, the detection method according to the present invention is independent of which cell cycle-phase the cell with this property is present.

Surprisingly, the cdc37-nucleic acid heteromers according to the invention are capable of transforming cells at different stages of neoplastic transformation, in particular at very early stages, by what mechanism they have been transformed into neoplastic. Can be used to detect independently. In particular, the cdc37-nucleic acid-heteromers according to the invention can be used to detect cells with impaired control of the cell cycle or cells with infinite, tumorigenic growth potential, but which have not yet formed tumors. . Suitable test substances are tissue biopsies or body fluids or cell mixtures from suitable cell- or cytoplasmic fractions, which detect the constitution of nucleic acids, proteins or their special heteromers or substrates or antibodies or other binding molecules. Detection of reactivity with
Used in specific reactions for the detection of dc37-nucleic acid-heteromers. Therefore,
Another object of the present invention is to provide a method for treating tumor cells and tumor cells using tissue biopsies, tissue debris, body fluids or appropriate cell lysates, whether or not a clinically demonstrable tumor has already formed. Detection of cells with infinite proliferative capacity. An advantageous use of this method of the invention is for precautionary purposes in tumor diagnostics or health care.

Starting materials for obtaining the cdc37-nucleic acid-heteromers according to the invention include:
In general, any human and animal, injured cell cycle-cells with controls or that demonstrate permanent growth or tumorigenic potential, permanent growth cells of various tissues or degrees of differentiation or species, and other tumors well known to those skilled in the art. Tumors or tumor-biopsies of cell-lineage or target tissue, degree of differentiation or species can be used. Surprisingly in this case,
It is important which process (eg, ionizing radiation), transformed gene (eg, viral oncogen), chemical carcinogen (eg, nitroso-urea) or other mechanism has transformed the corresponding cell into a neoplastic cell. is not. The cdc37 of the present invention determines which degree of differentiation the cells (eg, epithelium, lymphocytes, myeloid cells, fibroblasts, melanocytes) have, or from which species (eg, human, mouse, latte, Drosophila) the cells are derived. -Does not affect the isolatability of the nucleic acid-heteromer.

The repeated freezing and thawing of the cells from cytoplasmic fractions from permanently differentiable human or animal cells, for example using a surfactant such as NPO4 or SDS, or preferably in a solution containing Mg ^++. To obtain the cdc37-nucleic acid-heteromers of the invention. From this cytoplasmic fraction, mitochondria are then preferably separated using a sucrose gradient in a manner known to the person skilled in the art. c
Cytoplasmic fractions with dc37-nucleic acid heteromers can also be obtained by obtaining protein-containing cell fractions that do not contain nuclear DNA. Such a fraction is preferably used for cell lysis (Lyse) in the presence of NaCl and SDS.
And subsequent centrifugation (Hirt-Extraktion, J. Mol. Biol. 26: 3655-369, 1967)
Is obtained by From the supernatants of Hirt-Extraktion supernatants or mitochondria-free cell lysates, selective precipitation or electrophoretic or chromatographic methods, preferably by precipitation with 1.5 M NaCl and by precipitation with ethanol or ammonium sulphate, the cdc37- according to the invention. Nucleic acid heteromers can be obtained.

This cdc37-nucleic acid-heteromer shows unexpectedly poor solubility in both organic and aqueous solvents. Thus, c from the cytoplasmic lysate (Lysate) according to the invention
According to another method of isolating the dc37-nucleic acid heteromer, the lysate is extracted in an organic solvent, preferably phenol and chloroform. In this case, the cdc37-nucleic acid-heteromers according to the invention collect in the organic-aqueous mesophase of the extraction and can be separated from other molecules of the lysate by precipitation, preferably with ethanol.

Furthermore, the cdc37-nucleic acid-heteromers according to the invention have a surprisingly high stability towards proteinases and nucleases, but cytoplasmic tissue cells and membranes or mitochondrial or viral DNA or RNA are rapidly cleared by nucleases. Decomposed. Based on this property, the cdc37-nucleic acid-heteromers according to the invention are, according to another object of the invention, incubated for 1 hour at 60 ° C. in the presence of proteolytic enzymes, in particular proteinase K (100 μg / ml), and subsequently It can also be obtained by precipitation in the presence of ethanol or ammonium sulphate or, preferably, by chromatographic purification using HPL.

Although the protein-portion of this heteromer always contains the cdc37 protein, it has been surprisingly found that the nucleic acid sequence of the cdc37-nucleic acid-heteromer of the invention differs in cells of different degrees of differentiation and species. did. For example, the sequence of the associated DNA of cdc37-nucleic acid-heteromer according to the invention from melanoma cells is different from that from breast cancer or from colon cancer cells. Most cdc37-associated DNA-molecules are:
It has a length of about 30 bp to 1000 bp but is larger, for example 3000 bp
Molecules of up to length are also present in cells of acute myeloid leukemia.

From the cdc37-nucleic acid-heteromers according to the invention, the nucleic acids (RNA / DNA) can be prepared in a manner known to the person skilled in the art, preferably using proteolytic enzymes, detergents, high temperatures, enzymatic growth or molecular cloning. The sequence can be isolated; This cdc3
The nucleic acid sequence of the 7-nucleic acid-heteromer is preferably converted to the P with the use of reverse transcriptase or Klenov polymerase or the use of a thermostable DNA polymerase
When using the CR-method, it can also be grown enzymatically. Furthermore, the DNA sequence can be obtained by hybridization of a genomic or cDNA bank with a cdc37-nucleic acid heteromer according to the invention. Another advantageous method is to use affinity binding to the cdc37 protein, preferably for extrachromosomal DNA or for cDN.
Cdc37 formed under the use of S and by precipitation or use of an antigen-antibody reaction
The acquisition of this nucleic acid using a subsequent enrichment of the nucleic acid heteromer.

The obtained DNA sequence can then be used for the detection of human or animal cells with infinite proliferative or tumorigenic potential. For this, DNA- or RNA-hybridization methods known to those skilled in the art, sequence-specific oligonucleotides, which hybridize in situ to cell or tissue biopsies)
Enzymatic growth using the use of or binding of nucleic acid to cdc37-protein in a lysate or of protein to nucleic acid is advantageous.

Another subject of the present invention is the use of a cdc37 protein from a cdc37-nucleic acid heteromer to detect human or animal cells with infinite proliferative or tumorigenic potential. It is evident that these cells express the cdc37 protein and its mRNA with a larger number of molecules than normal cells having physiological proliferation. Comparison of the expression of cdc37 protein molecules can be determined by Western blot detection, especially cdc37.
Immunofluorescence or FACS analysis ("fluorescenc") using 37 protein specific antibodies.
e activated cell sorter ") (eg, Dai et al., J. Biol. Chem. 217).
: 22030-22034, 1996) or by antigen-antibody-recognition reaction using a binding molecule or cdc37-binding nucleic acid equivalent binding reaction (South-Western Blot) in a manner known to those skilled in the art (Harlow et al. , Antibodies, Cold Spring Harbor Laboratory Pre
ss, Cold Spring Harbor). Comparison of mRNA expression can be performed using a suitable hybridizing probe, such as the human cdc37 cDNA sequence (eg, db
EST database, is advantageously carried out using Northern blot hybridization Chillon under use of GenBenk ^(R) ID ^{cloneR87892).} Using these assays, for example, 5 fold higher cdc37 mRNA expression ((Northarn Blot Analyse) and 8-10 fold cdc37 protein expression in MCF-7 breast cancer cells compared to those in normal breast epithelial cells, for example. (Western Blot Analyse) and a 7-fold higher expression of cdc37 mRNA in L540 Hodgkin lymphoma cells is apparent as compared to that in normal lymphocytes.

When using the South-Western method or other methods of coupling nucleic acids to the cdc37 protein, DNA or RNA or single- or double-stranded desoxy- or oxy-oligonucleotides or labeled (Eg with digoxigenin or biotin)
Alternatively, it is advantageous to use a modified nucleic acid, for example, PNA (peptide nucleic acid). In vitro binding of nucleic acids to cdc37 protein in vitro is facilitated by the presence of Zn ²⁺ ions (preferably 8 mM) and monovalent ions such as Na ⁺ (preferably 8 mM) and ATP or GTP (preferably 2 mM). . The nucleic acid cdc37 interaction is a cdc
Closes at the N-terminus of the 37 polypeptide sequence.

The cdc37 protein can be obtained from the cdc37-nucleic acid heteromer according to the invention by treatment with DNase I, advantageously matrix-bound DNase I or by proteolysis or with detergents or at elevated temperatures, preferably 90 ° C. In the presence of
And the isolated cdc37 protein or a fragment thereof is isolated by chromatography or electrophoresis.

Another object of the present invention is an antibody or antiserum or recombinant molecule having specificity for the cdc37 protein-nucleic acid heteromer and antibody similarity binding specificity according to the present invention. These are suitable for the detection of human or animal cells having infinite proliferative or tumorigenic potential. These antibodies or antisera can be prepared in a manner known per se in polyclonal form by immunocompetent animals, such as mice, rabbits or sheep cd according to the invention.
It can be obtained by immunization with a nucleic acid of a c37-nucleic acid heteromer or a cdc37 protein or a heteromer. Monoclonal antibody-producing hybridoma
Clones can likewise be obtained by known methods by fusing antibody-binding lymphocytes with suitable myeloma-cells, for example Ag8.653 cells. Along with whole antibodies, antibody fragments obtained by known protein chemistry and molecular genetic methods can also be used. Immune competitive donor - V _H from lymphocytes - and / or V _L - using a cDNA sequence of an immunoglobulin chain, either alone or in combination can bind hybrid over molecules having antibody similarity specificity, preferably the Recombinant "single-chain" antibodies capable of binding the cdc37-nucleic acid heteromers according to the invention can be isolated. The isolation of recombinant antibodies from the "phage display library" is also advantageous, in which the appropriate recombinant phage is enriched by specific binding (panning) to the cdc37-nucleic acid heteromer. The antibody-like fusion protein on the surface of the phage or in a soluble form can then be used in a suitable detection method as well as the antibody. The use of combinatorial libraries is likewise possible for the isolation of recombinant antibodies having specificity for cdc37-nucleic acid heteromers and is known to the person skilled in the art (Orlandi et al., Proc.
Natl. Acad. Sci. USA 86: 3833-3837, 1989; Chiang et al., Biotechniques.
7: 360-366, 1989; Winter und Milstein, Nature 349: 293-299, 1991).

Surprisingly, antibodies obtained by immunization with the cdc37-nucleic acid heteromers according to the invention bind specifically to cells with infinite proliferative, injured cell cycle-control or tumorigenic potential, It can also be used in situ for labeling cells in a tissue community. In this case, the labeling of the target cells is advantageously performed in the cytoplasm, but normal cells that have physiological parameters and are transiently growing are not labeled. This suggests that normally growing cells are G-S-
It is even more surprising since it expresses the cdc37 protein during the transition to the phasic phase. Antibodies according to the present invention can be used to express cdc37-nucleic acid heteromers in tumor cells and cdc3
7 proteins.

Accordingly, a further object of the present invention are antibodies and antisera capable of binding to cdc37-nucleic acid heteromers. An advantageous use is the cdc37-nucleic acid heteromer of this cell for detection reactions in situ in biopsies or in tissue pieces, or in reaction with cell suspensions or body fluids or in lysates or body fluids For the detection reaction of
Or the use of antibodies or antisera or recombinant antibody analogs for removal or growth inhibition in tissues or in cell mixtures. The antibody or fragment or recombinant binding molecule thereof can be conjugated to a carrier or labeling molecule, such as a protein, sugar, sepharose, enzyme, dye, hapten or radioactive ion, and used for a suitable detection method. For this detection reaction, there are many known antigen-antibody recognition reactions, such as ELISA, RIA, fluorescence-immunoassay or Fluorescent Cell Sorter (FCAS).

One embodiment of the present invention is preferably a mitochondrial-free cytoplasmic fraction of the cells to be tested from a biopsy, a tissue debris or a cell suspension or from a soluble fraction of a body fluid. Measurement of nucleic acid heteromer, cdc37 protein-portion or nucleic acid content of this heteromer. Normal cells with physiological proliferative capacity and a regulated cell cycle do not contain such heteromers in the fraction, while cells with injured cell cycle-controls have a higher copy regardless of their cell cycle phase. It has this heteromer in number. For the determination of the content of cdc37-nucleic acid heteromers according to the invention, antigen-antibody reactions or nucleic acid hybridizations are advantageous. However, for those skilled in the art, various detection methods, such as ELISA, RIA, fluorescence-immunoassay, FA
Nucleic acid-hybridization on Southern blot or Southern-Western blots, PCR, etc. are provided.

The detection of the heteromer can also be effected by an enzymatic reaction of the heteromer with a substrate, preferably under cleavage of energetic phosphate bonds, or by binding to hyaluronic acid. The cdc37-protein-nucleic acids according to the invention have kinase activity and / or have a (self) kinase action, based on which the detection method is based.

Of particular importance for the present invention is that the cdc37-nucleic acid heteromer according to the invention can be found on the cell surface of injured cell cycle-control, cells with infinite proliferative or tumorigenic potential, but not physiologically. It was surprisingly confirmed that it was not found on the surface of normal cells having proliferative ability. Accordingly, another embodiment of the present invention is directed to a wounded cell cycle-control, infinite growth capacity using a reagent capable of identifying or binding a cdc37-nucleic acid heteromer according to the present invention on the surface of a cell. Alternatively, the detection of cells having tumorigenic ability. In this case, it is advantageous to use labeled antibodies, antisera or recombinant polypeptides having binding specificity for the cdc37-nucleic acid heteromers according to the invention. In a known manner for the immunostaining of biopsy or tissue preparations using a cdc37-nucleic acid heteromer binding antibody according to the invention or a derivative thereof coupled to a dye or enzyme or a fluorescent- or magnet-activated cell sorter It is particularly advantageous to detect cells in situ or in a mixture of cells under the use of cell enrichment according to.

The antibodies, antisera or recombinant antibody analogues according to the invention against the cdc37-nucleic acid heteromer on the surface of the cell membrane can also be used in vivo for the detection of cells with infinite proliferative or tumorigenic potential. . In this case, the antibody or recombinant antibody-like molecule or derivatives thereof of the present invention, for example, after an appropriate labeling in 99mTc or ¹²¹ I, it is advantageous to use in immune scintigraphy or other imaging methods. This antibody is increased in the organ where injured cell cycle-control, cells with infinite proliferative or tumorigenic potential are present. Organs with normal quiescent or proliferating cells (eg, brain or bone marrow) and cells that regenerate physiologically, eg, during wound healing, are not labeled in the methods of the invention.

Furthermore, the antibodies, nucleic acids or derivatives thereof according to the invention can be used to detect other detectable or reactive molecules or atoms, such as proteins, sugars, enzymes, biologically effective mediators, metal complexes, radioisotopes. Alternatively, it can be conjugated to a toxin and used for detection and / or destruction of damaged cell cycle-controls, cells with infinite proliferative or tumorigenic potential. The use of a fusion protein from a cdc37 protein-nucleic acid heteromer specific antibody and a cytotoxin, such as a Pseudomonas exotoxin, is advantageous, whereby a so-called immunotoxin is generated, which is specifically infinitely proliferative or tumor. Binds to and kills cells that have a formative potential. In addition, binding of the antibody to an immunologically acting protein, such as a cytokine or chemokine, is also possible, whereby a fusion protein that binds to the tumor cells and activates the immune cells there so as to lyse the tumor cells is obtained. Generated.

Known immuno-toxins or immuno-cytokines react via their antibody-parts almost exclusively to antigens expressed on tumor cells of a particular organ or degree of differentiation.
On the contrary, a fusion molecule according to the invention having specificity for the cdc37 protein-nucleic acid heteromer is independent of which organ the cell is derived from or which tumor cell has the degree of differentiation. In addition, it is reactive with many different tumor cells. Because most tumor cells lose many antigens on their surface with increasing malignancy and metastasis, known immuno-conjugates have found their binding to highly-malignant and metastatic cells. Lose. In contrast, fusion molecules according to the invention having specificity for the cdc37 protein-nucleic acid heteromer also bind to high-malignant and metastatic cells. This is because the fusion molecule binds to the molecule that the cell needs for each further cell division. This results in greater efficacy than that of known immuno-conjugates.

Even more surprisingly, an Fc-containing antibody according to the invention or a derivative thereof capable of binding a cdc37-nucleic acid heteromer of the invention can be used in cells with injured cell cycle control, infinite proliferation or tumorigenic potential. It has been shown that cell lysis can be initiated. This cell lysis is performed using complement activation by the Fc-portion of the antibody molecule.

The present invention therefore uses the molecule capable of binding a cdc37-nucleic acid heteromer according to the invention for the initiation of lysis of injured cell cycle-controls, cells with infinite proliferation or tumorigenic potential. In this connection too, use is made of the binding molecule itself or the fusion product of this molecule with other pharmaceutically active substances.

Furthermore, the nucleic acid of the cdc37 protein-nucleic acid heteromer or the derivative oligonucleotide of this nucleic acid can be used to suppress the proliferation of cells having infinite proliferation ability. To this end, a partial sequence (oligonucleotide) of this nucleic acid which is homologous to the binding region between the cdc37 protein and the nucleic acid is advantageous. The oligonucleotide interferes with the nucleic acid cdc37 protein, thereby destabilizing the heteromer. Surprisingly, in this case, the tumor cell ceases its infinite growth, leaves the cell cycle and leads to its death. Accordingly, one particular embodiment of the present invention provides for the binding of the cdc37 protein-nucleic acid heteromer cdc37-protein to the binding region of the nucleic acid and destabilizing oligonucleotides and nucleic acid derivatives to inhibit tumor cell growth and / or
Or to initiate cell death.

Other substances that alter the stability of the cdc37 protein-nucleic acid heteromer can also be used for this purpose. Surprisingly, such substances induce injured cell cycle-control, growth inhibition and / or cell death of cells with infinite proliferation and tumorigenesis. To this end, substances which interfere with heteromer formation by binding of nucleic acids or of cdc37 protein are advantageous. For this purpose, it is more advantageous to use a peptide sequence derived from the nucleic acid binding region of the cdc37 protein, in particular from amino acids 62 to 120 of the cdc37 protein sequence. In particular,

[0047]

Embedded image

Preference is given to the peptide of This peptide inhibits the growth of tumor cells, but does not inhibit physiologically growing normal cells. Suitable peptides cyclized via the N- and C-terminal cysteine-acid groups and / or whose basic amino acids have been exchanged for neutral amino acids may also be used for that purpose.

Furthermore, substances that block the function of the cdc37 protein-nucleic acid heteromer in controlling the G1 / S transition of the cell cycle, such as antibiotics, fungicides or other physiological or synthetic substances, are advantageous.

Thus, according to the present invention, a cdc37 protein-nucleic acid blocking or destabilizing substance is used for inhibiting the proliferation of cells having infinite growth and tumorigenicity and for introducing cell death of these cells. Used for

According to the present invention, a cell capable of binding to the cdc37-nucleic acid-heteromer according to the present invention can also be used to enrich or reduce cells having infinite proliferative capacity and tumorigenic capacity from a cell mixture. . For this purpose, antibodies, antisera or recombinant antibody analogs to the cdc37-nucleic acid heteromers according to the invention can be used. For this purpose, a method with a fluorescence-activated sorter (FACS) or a magnet-activated sorter (MACS) is advantageous. The antibodies or derivatives thereof reactive to the cdc37-nucleic acid heteromers according to the invention are also suitable for removing cells with infinite proliferative and tumorigenic potential from cell suspensions or organs, in particular from transplanted tissues, for example autologous. It can be used for purging bone marrow transplants. Advantageously, the antibody or its derivative with the binding domain for the cdc37-nucleic acid heteromer according to the invention is bound to a matrix, the cell mixture with this matrix is incubated and the unbound cells are eluted by chromatography. Using this method, cells having infinite proliferative ability or tumorigenic ability,
Regardless of which organ the tumor cells came from, and which neoplastic transformed cells in each cell mixture have any degree of differentiation, growth or malignancy, the cell mixture of normal cells can It was found that it can be effectively removed. As a cell mixture, peripheral blood, bone marrow or other body fluids are used. This method is suitable for removing cells with infinite proliferative or tumorigenic potential from blood, bone marrow or other body fluids.

It was further surprisingly revealed that the cdc37-nucleic acid heteromers according to the invention are secreted from cells having infinite proliferative or tumorigenic potential. Therefore, this c
The dc37-nucleic acid heteromer is used, for example, in the culture supernatant of tumor cells or in blood, bone marrow,
It can also be detected in serum, plasma, fluid, urine or other bodily fluids. Thus, injured cell cycle-control, human or animal cells with infinite proliferative or tumorigenic potential can be obtained by converting cdc37- according to the invention in body fluid or cell-free supernatant or filtrate.
It can be detected by detecting a nucleic acid heteromer.

The cdc37-nucleic acid heteromer is advantageously detected using an antibody that binds this heteromer, wherein the cdc37-nucleic acid heteromer isolated according to the invention can be introduced together as a standard. However, a combination of this method is also possible, advantageously enrichment with an antibody having binding specificity for a cdc37-nucleic acid heteromer according to the invention and enrichment with a second antibody or with a cdc37 molecule or cdc3.
Detection using nucleic acids capable of binding the nucleic acid of the 7-nucleic acid heteromer or using an enzymatic reaction starting from the cdc37-nucleic acid heteromer according to the invention itself, such as cleavage of the energetic phosphate bond of the substrate, is also possible.

The sensitivity of this method is to detect the associated nucleic acid of the cdc37-nucleic acid heteromer by hybridization with an appropriate nucleic acid-sonde, or enzymatically, advantageously, to sequence-specific or random-sequences as primers. Polymerase-chain-reaction using oligonucleotides (Mullis und Fallona, Meth. Enzymol. 155: 335-350,
1987; Saiki et al., Science 239: 487-491, 1988). Thus, in one particular embodiment of the method of the invention, the cdc37 protein-nucleic acid heteromer from a biopsy lysate or body fluid or cell supernatant is enriched by the use of a specific antibody and the associated nucleic acid is subsequently enzymatically To be increased.

The described embodiments of the method of the invention are suitable for the detection of malignant or benign tumors or their precursors or cells with injured cell cycle-controls, wherein biopsies, organ sections Or cells or their lysates or secreted complexes from human or animal body fluids are used as samples. The described method is likewise suitable for the discovery or localization of such cells in vitro or in vivo. Furthermore, these methods can be used, in suitable variants, for the isolation of cells from a cell mixture or for their enrichment or reduction and removal from a cell mixture. Finally, antibodies or recombinant molecules having binding specificity for the cdc37-nucleic acid heteromers according to the invention can be used for the removal of cells in vivo or in vitro, wherein the antibodies or recombinant molecules are used alone or Used as a fusion product with other bioactive molecules.

The present invention is explained in detail by the following examples.

Example 1 Isolation of cdc37-nucleic acid heteromers from MCF-7 breast cancer cells.

[0058] MCF-7 breast cancer cells (ATTC HTB22) were treated with 50 mM Tris-HCl,
The cells were thawed by several freeze-thaw cycles in the presence of pH 7.2, 10 mM EDTA, 1.5 mM MgCl ₂ , and cell nuclei were subsequently precipitated by centrifugation at 6000 × g for 30 minutes. The mitochondria were separated from the supernatant by a sucrose density gradient (J.
Biol. Chem. 249: 76991-1995, 1974). Mitochondrial-free fractions were collected from ribonuclease A (20 μg / ml) and ribonuclease T1.
(1000 U / ml) at 37 ° C for 30 minutes and then at 60 ° C for 2 minutes.
It was kept at a constant temperature together with proteinase K (100 μg / ml) for 0 minutes. To this batch amount was added SDS (ad 0.4% SDS w / v) and NaCl (ad 4M NaCl), cooled on ice for 60 minutes and then centrifuged at 6000 × g for 20 minutes. The supernatant was extracted in phenol (neutralized with Tris-HCl, pH 7.2) and subsequently in chloroform / isoamyl alcohol (24: 1 v / v). In this case, the cdc37 protein-nucleic acid-heteromer is enriched in a phase intermediate between the organic and aqueous phases. The intermediate phase is removed and the cdc37-nucleic acid heteromer is precipitated by the addition of 2 volumes of ethanol, 0.1 mM EDTA, 1 mM Tris-HCl.
M, pH 7.2.

The cdc37-nucleic acid heteromer is separated in native agarose (0.7%)-gel or polyacrylamide (4%)-gel, where the heteromer consisting of MCF-7 is a 100-500 bp protein It shows the moving speed of the DNS without containing. In a denaturing SDS-polyacrylamide-gel, the cdc37-nucleic acid heteromer migrates at approximately 37 kD, 48 kD, 82 kD, 90 kD.

Example 2 Molecular cloning of cdc37 protein-nucleic acid heteromer nucleic acid from tumor cells.

The cdc37 protein-nucleic acid heteromer (from Example 1) was converted to proteinase K (500
(g / ml) and 0.1% of SDS at 60 ° C for 10 hours. The batch amount was extracted again in phenol and subsequently in chloroform / isoamyl alcohol. In this case, the DNA released from the cdc37 protein-nucleic acid heteromer is collected in the aqueous phase of the extract. DNA was precipitated with ethanol and dissolved in 10 mM Tris-HCl, pH 7.2, 1 mM EDTA.

For cloning, the DNA of the cdc37 protein-nucleic acid heteromer (approximately 20
ng) in the presence of 10 mM Tris-HCl, pH 7.6, 6 mM β-mercaptoethanol, 50 mM NaCl, 10 mM MgCl ₂ and 0.1 ml each of TTP, dATP, dCTP, dGTP in Klenow polymerase (1
0U). Subsequently, the S-fragmented pUC19 vector was cloned into DNS in the presence of 10 U T4 DNA ligase. The obtained recombinant plasmid was used for E. coli. E. coli K12 bacteria. Four independent plasmid-clones were obtained (pMCF-7).
This plasmid-clone contains the inserted DNA sequence, which represents the nucleic acid content of the cdc37 protein-nucleic acid heteromer, and is suitable for detecting neoplastic transformed cells. Similarly to this method, the nucleic acid sequence of the cdc37 protein-nucleic acid heteromer was obtained from colon cancer cells, Hodgkin-lymphoma cells, melanoma cells, and acute myeloid leukemia cells. The DNA sequence is SEQ ID
This is shown in NO1.

Example 3 Obtaining antibodies against cdc37-nucleic acid heteromers.

Mouse Ehrlich-Aszites tumor cells (ATCC CCL77) and human MCF-7 (ATCC HTB22) breast cancer cells
It has a cdc37-nucleic acid heteromer in the cytoplasm, whereas this heteromer could not be detected in normal fetal mouse fibroblasts. Antibodies that specifically bind mouse-cdc37-nucleic acid heteromer as well as human-cdc37-nucleic acid heteromer from tumor cells, but do not react with normal cells were sought.

[0065] The cdc37-nucleic acid heteromer was transformed into mouse Ehrlich-Azuchites tumor cells (
ATCC CCL77) by the method described in Example 1. Associated DNA
A cdc37-nucleic acid heteromer having a content of about 100 ng (dissolved in 50 μl of PBS and supplemented with Freund) is injected subcutaneously into Balb / c mice. A second injection with the same amount of antigen without auxiliary substances ("boost injection")
) Was performed on days 32 and 54 after the first injection. On day 62, B for obtaining antiserum for post-analysis ("anti-cdc37-nucleic acid heteromer antiserum")
About 500 μl of ut was taken out.

On day 62 after injection, spleens of immunized mice were obtained and the myeloma line X3-A
To obtain hybridomas with g8.653 cells, they were fused using polyethylene glycol (Koehler G. and Milstein C. Nature 256: 495-49).
7, 1975; Koehler, G. (eds.) Hybridoma techniques. Cold Spring Harbor Labor
atory Press, Cold Spring Harbor, 1980). Selection of the resulting hybridoma cells was performed using HAT-selection medium (10 ^-4 M hypoxanthine in RPM1640 medium).
, Aminopterin 4 × 10 ⁻⁷ M, thymidine 1.6 × 10 ⁻⁵ M). Five days after fusion, cells were plated in "limit dilution" cultures at about 50-10 per well.
Seeded in microtiter plates with 0 cells. Day 28 and 35 after fusion
On day, approximately 400 culture supernatants were thoroughly examined for antibodies reactive with the cdc37-nucleic acid heteromer in tumor cells.

To do so, the following is performed: mouse Ehrlich-Azuchites cells,
Mouse fetal fibroblasts (as a negative control) and human MCF-7 breast cancer cells (approximately 100 cells per recess in a “Terasaki” microtiter plate) were each time supernatant of the hybridoma culture. Solution (approximately 10 μl per well, supernatant diluted 1: 100 in PBS) and incubated at 37 ° C. for 1 hour,
Subsequently, it is washed twice with PBS and subsequently incubated at 37 ° C. with a peroxidase-conjugated antibody (anti-mouse-lg antibody) against mouse-immunoglobulin. After another wash and reaction of the peroxidase with the substrate AEC,
In six out of about 400 supernatants, antibodies were found that reacted with the cytoplasm of mouse Ehrlich-Aztites tumor cells and human MCF-7 cancer cells, but did not react with normal fetal fibroblasts. Was.

The hybridoma cells of the culture were subcloned by “limit dilution” and final seeding of single cells. In this case, a total of about 300 cultures were made, and the supernatants of the cultures were thoroughly examined on day 24 again for reactive antibodies by the method described above. Hybridoma cells with reactive supernatant were subcloned again. In this case, mouse-cdc37-
Stable hybridoma clones ("3D6"), which show monoclonal antibodies with specificity for nucleic acid heteromers as well as human-cdc37-nucleic acid heteromers, but do not react with normal cells, were obtained.

Example 4 Cell-Use of the antibody 3D6, which is more specific for the cdc37 protein-nucleic acid heteromer, to detect tumor cells in the lysate.

The tumor cells are detected in a mixture with normal cells. Tissue preservation is often inadequate for histological analysis, thus demonstrating the detection of tumor cells in cell lysates in this example.

The human breast cancer cell line MCF-7 (ATCC HTB22) and human normal dermal fibroblasts were purified from Tris-HCl 50 mM, pH 7.2, EDTA 10 m.
Cell fragments, cell nuclei and cell organelles lysing in the presence of M, MgCl ₂ 3 mM, NP40 0.2% are separated by centrifugation at 8000 × g for 2 minutes. The supernatant (corresponding to approximately 10 ⁴ cells) as well as the isolated cdc37 protein-nucleic acid heteromer (as a positive control) were denatured in the presence of SDS and 12.5% SDS
The polyacrylamide gel is separated electrophoretically in the gel and electrophoretically transferred onto a nitrocellulose membrane. The membrane was washed with TBS (Tris 20 mM, NaCl 137 mM, p
H7.6) Incubate in 3% Tween 203%, 5% (w / v) dry milk powder for 1 hour, and subsequently antibody 3D6 (1: 100 hybridoma) specific for anti-cdc37 protein-nucleic acid heteromer (Supernatant), and the solution is kept at room temperature for 1 hour. The membrane is washed in TBS, Tween 203% and incubated for 1 hour with anti-mouse-lg antibody conjugated to peroxidase.
Subsequently, the membrane is again washed in TBS, Tween 203%, kept at a constant temperature together with the detection reagent ECL (Amerham), and subjected to autoradiography.

Results: Monoclonal antibody 3D6 showed a band of cdc37-nucleic acid heteromer (approximately 37 kD, 48 kD, 82 kD, 90 kD in human tumor cells) in cell lysates of MCF-7 breast cancer cells by Western blotting. Although specifically shown, however, the isolated cdc37-nucleic acid heteromer (positive control) does not react with cytoplasmic protein lysates from normal human fibroblasts.

Example 5 Monoclonal Antibody 3D6 with Specificity for cdc37-Nucleic Acid Heteromers
Marking Human Tumor Cells in Tissue Fragments Using Tissues There is the problem of presenting malignant melanoma cancer cells in biopsy tissue sections. To this end, the cryostat section of the tissue biopsy is placed in front and fixed with acetone / methanol. Endogenous peroxidase in methanol / PBS
Block by incubating with 1% (v / v) H ₂ O ₂ in 60% for 15 minutes, then wash this section and Triton X-100 in PBS.
Incubate with 2% for 10 minutes. After washing again, this section is kept at a constant temperature for 1 hour together with bovine serum albumin (3% w / v in PBS) and washed again. Continued,
Antibody 3D6 (1: 1 in PBS) specific for anti-cdc37-nucleic acid heteromer
(Hybridoma-supernatant diluted at 0) was kept at room temperature for 2 hours. As a control, use the buffer without antibody as the "apparent isothermal (Schein-Ink
ubationen) "and incubation with an anti-TAG72 antibody (B72-3) with specificity for the TGA72 antigen which is expressed on cells of the gastrointestinal tract but not on melamine or epidermal cells. Was used.
The section was washed four times with PBS and peroxidase-bound goat anti-mouse.
It was kept at room temperature for 1 hour together with an IgG (H + L) antibody (Dianova, Hamburg) (1:50 in PBS). After four washes, the reaction of peroxidase with the substrate AEC was performed for 20 minutes. Cell nuclei were stained with Haemalun.

Results: Malignant melanoma cells react in the cytoplasm with the antibody 3D6, which is specific for the cdc37-nucleic acid heteromer, whereas melanoma cell nuclei do not react with the 3D6 antibody. Normal cells surrounding melanoma as well as normal epidermal cells do not show coloration with the 3D6 antibody. Invasive tumor cells in normal tissues are confirmed by staining with the 3D6 antibody. Control markings without the 3D6 antibody show no specific staining and similarly no reaction with the anti-TAG72 antibody.

Example 6 Detection of cdc37-nucleic acid heteromer on the cell surface of tumor cells There is a problem of detecting viable cells of cdc37 protein-nucleic acid heteromer on the cell surface. The intact cell membrane of the surviving cells blocks entry of the antibody into the cells, whereas dead cells do so. Dead cells are confirmed by staining with propidium iodide and delimited by a subsequent assay.

[0076] Human MCF-7 breast cancer cells (A) and normal human fibroblasts (B) were
Wash in S, split into 4 batches each, and incubate at room temperature for 30 minutes with the following reagents: (1) Antibody 3D6 specific for anti-cdc37-nucleic acid heteromer (hybridoma in PBS) (Supernatant 1: 100) (2) Anti-TAG72 specific antibody B72-3 (hybridoma supernatant 1: 100 in PBS) (3) PBS without antibody (4) PBS without antibody Wash cells in PBS , Then the batch quantities (1), (2) and (3)
Anti-mouse-IgG antibody conjugated to ITC (Dianova, Hamburg) (1: 150
Together with 0) at room temperature for 30 minutes. Cells were washed again in PBS and analyzed using fluorescence activated cell sorting (FACS) (FACscan, Fa. Becton-Dickinson,
Mountain View, Cal.). In this case, by placing an appropriate measurement window ("gate"), dead cells are excluded from the analysis, and thus the signal obtained is exclusively from living cells with intact surface membranes.

Results: Batch amount (1) of MCF-7 breast cancer cells showed a FITC fluorescence signal. Cells in batch amounts (2) and (3) show a background fluorescence that is 5-10 fold weaker, this background fluorescence has the same intensity as the control batch amount (4) without secondary antibody and Shows the "intrinsic fluorescence". The marking of the batch amount (1) indicates that MCF-7 cells
It was marked by the D6 antibody, but not by the control antibody (B72-3), which reacts with an antigen that does not express MCF-7 cells.

Normal human fibroblasts differ from MCF-7 cells in batch volume (1)
Only the background fluorescence was shown, and the same was shown for the batch amounts (2), (3) and (4). This indicates that the 3D6 antibody converts reactive cdc37-nucleic acid heteromers to M
It can be inferred that it is expressed on the surface of CF-7 breast cancer cells, but not on the surface of normal fibroblasts.

Example 7 Detection of cdc37-Nucleic Acid Heteromer in Serum of Tumor Patients Serum was obtained from two patients each with breast carcinoma and melanoma and two healthy blood donors, and 2 μl of each serum was DNase I (10U, 1 hour, 37
℃) or proteinase K (10 μg, 1 hour, 60 ℃). Untreated serum and serum treated with DNase I or Proteinase K were incubated at 90 ° C. for 2 minutes in the presence of SDS (0.1 w / v%), and the SDS-
The gel was separated by electrophoresis in polyacrylamide, followed by electrophoresis with 50 mM Tris, 380 mM glycine, 0.1 SDS (w / v), 20 mM methanol.
% On a nitrocellulose membrane. Non-specific binding of the membrane to TBS (
Blocked for 1 hour at room temperature by incubating with 5% (w / v) dry milk powder in 20% Tris, 137 mM NaCl, pH 7.6) Tween 203% (v / v). . Subsequently, the membrane was treated with the antibody 3D6 (hybridoma supernatant 100 in blocking buffer) specific for the cdc37 protein-nucleic acid heteromer.
1) at room temperature for 1 hour, washed 3 times, and incubated with peroxidase-conjugated sheep anti-mouse Ig (Amersham) (1: 200) for 1 hour. After four washes, bound antibody was detected on X-ray film by reaction of peroxidase with ECL detection reagent (Amersham) and subsequent autoradiography.

Results: In the serum of patients with breast carcinoma and melanoma, the relative Mr about 37 kD,
Bands of the 3D6 antibody with 48 kD, 82 kD, 90 kD are detected, but show no reaction with serum from healthy subjects. The Mr detected corresponds to the relative migration rate of the isolated cdc37 protein-nucleic acid heteromer. The 3D6 antibody-reactive band in the serum of tumor patients can be degraded by pre-isothermal preservation of the serum, as well as with DNase I or with proteinase K. This indicates that the detected band contains a substance consisting of protein and DNS.

Example 8 Expansion of tumor cells from a mixture of normal cells using antibody 3D6 with specificity for the cdc37 protein-nucleic acid heteromer. To demonstrate the increase of tumor cells from a mixture with normal cells, the SK-Mel5 cells carcinoma added in the next mixing ratio lymphocyte ¹⁰⁶ normal human from peripheral blood (PBL): Sk-Mel5: PBL 1: 10,1: 100,1
: 1000, 1: 10000, 1: 100000. Sk-Mel5 melanoma-cells express HMW-MAA ("high molecular weight melanoma-associated antigen") on the surface and produce anti-H
It can be detected using a MW-MAA antibody. This allows the detection of melanoma cells in a population of normal cells, independent of the method used. Normal human lymphocytes do not express this antigen.

The cell mixture (approximately 10 ⁶ cells in 1 ml of PBS) was incubated with antibody 3D6 (200 μg of purified antibody) at room temperature for 1 hour, washed 4 times with PBS and subsequently marked with paramagnetism. Anti-mouse Ig antibody (about 50 μg) (Fa. Milt
(enyi, Bergisch-Gladbach) again for 1 hour. The cell suspension is washed four times, subsequently placed on a 20 ml separation column (Miltenyi) and exposed to a high magnetic field. The column is washed with 10 volumes of PBS to remove unmarked cells (flow-through fraction). Next, the column is removed from the magnetic field and the marked cells are eluted. The number of HMW-MAA + tumor cells in each fraction was
It is measured using the HMW-MAA antibody 763.74. The results of the FACS analysis are described in Table 1.

[0083]

[Table 1]

The measurement limit of the method lies in a counter-aptitude (Zaehlereignissen) of 5000 per measurement for about 10 cells.

The mean ± standard deviation of the 6 test runs is described.

Example 9 Removal of tumor cells from a cell mixture using the antibody 3D6 bound to a solid phase with specificity for the cdc37 protein-nucleic acid heteromer.

This example was carried out as in Example 8, but the separation was carried out with the antibody 3D6 bound to the solid phase.

To demonstrate the clearing of tumor cells from a cell mixture, the melanoma SK-Me
l5 cells is added in the next mixing ratio lymphocyte ¹⁰⁶ normal human from peripheral blood (PBL): Sk-Mel5: PBL 1: 10,1: 100,1: 1000,
1: 10000, 1: 100000. Sk-Mel5 melanoma-cells are HMW-
MAA ("high molecular weight melanoma-associated antigen") is expressed on the surface and anti-HMW-MAA
It can be detected using an antibody. Normal human lymphocytes do not express this antigen.

Antibody 3D6 (2 mg of purified protein) was bound to CNBr-activated Sepharose (0.5 ml) (Pharmacia), then Sepharose was added to PBS, dry milk powder 0.2% (w / v) Wash in. Cell mixture (PBS 1m
About 10 ⁶ cells / l) were placed on a column with Sepharose (0.5 ml) bound by the 3D6 antibody and incubated at room temperature for 1 hour. Continue to add 4 columns
The cells were washed with 10 times the volume of PBS to remove unbound cells (flow-through fraction). Bound cells were eluted in the presence of 2M MgCl2, 10 mM NaPO4, pH 7.2. The number of HMW-MAA + tumor cells in each fraction was determined by anti-H
Purification efficiency (deterioration factor) measured using MW-MAA antibody 763.74
Was measured. The results of the FACS analysis are described in Table 2.

[0090]

[Table 2]

The measurement limit of the method lies in a counter-fit (Zaehlereignissen) of 5000 per measurement for about 10 cells.

The mean ± standard deviation of the four test runs is reported.

Example 10 Detection of cdc37 Protein-Nucleic Acid Heteromers Using Nucleic Acid In Situ Hybridization Cells of Human Breast Carcinoma Line MCF7 (ATCC HTB22) (A) and Primary Skin Fibroblasts The cells (passage number 12) were incubated on a glass slide. Over 90% of the cells in the two cultures proliferate, in which case MCF-7 cells have infinite proliferative potential, but early human dermal fibroblasts have about 15-20 cells. After passage, growth occurs in vitro, aging and dies. In addition, subcutaneous injection of MCF-7 cells in Balb / c mice generated a coagulated tumor, which completed the freezing process of the tumor and was transferred to a glass slide (C).

The slides were briefly washed twice with PBS, coated with 200 μl of paraformaldehyde solution (5 v / v%) for fixation for 5 minutes and subsequently washed twice in 70% ethanol. For detection of cells with infinite proliferative potential, SEQ ID N
The DNA sequence designated O1 was used. This DNA sequence is the nucleic acid content of the cdc37 protein-nucleic acid heteromer from MCF-7 cells and was obtained by molecular cloning as described in Example 1a. The DNA sequence corresponds to the S of vector pUC19.
It has been cloned at the mal fragment position. The MCF-7 / sml DNA fragment of size 221 bp was released from the vector by restriction splitting of the DNA with Smal. This fragment was electrophoresed in an agarose gel electrophoretically with pUC19 vector D
Separated from NA (2.7 kb), eluted and precipitated with ethanol. MCF-
7 / sm1 DNA (1 μg of DNA) was denatured, and Klenow polymerase (1 U
) Using Tris-HCl 10 mM, pH 7.6, β-mercaptoethanol 6 mM, NaCl 50 mM, MgCl 2 10 mM and digoxigenin-1
1-dUTP 0.2 mM (Dig-dUTP) (Boehringer Mannheim), 2 hours at 37 ° C. in the presence of 0.1 mM each of dATP, dCTP, dGTP and hexanucleotide (Boehringer Mannheim) of random sequence as primer. Marking was performed by maintaining the temperature. Unincorporated nucleotides were separated by filtration through a Sepharose G50 column (Pharmacia).

For in situ hybridization, slides are coated with the cells to be fixed or tissue fragments are coated with 200 μl of hybridization solution (50% deionized formamide, 4 × SSC, 10% dextran sulfate, (1 × Denhardts solution, 0.25 mg / ml denatured tRNA, 0.5 mg / ml denatured herring sperm DNA). The solution on the slide glass was covered with a cover glass and kept at a constant temperature in a room humidified at 42 ° C. for 1 hour. Subsequently, the solution was removed and cells were labeled with Dig-dUTP of labeled MCF-7 / sm1 DNA.
(200 ng of DNA in 40 μl of hybridization solution) and incubated at 42 ° C. overnight. Control experiments ("apparent hybridization" (Scheinhybridisie)
rung) "), hybridization was performed without the digoxigen-labeled DNA sample. Finally, the slides were washed twice at 42 ° C. in 2 × SSC at 20 ° C.
Washed for minutes. For detection of the hybridized DNA sample, the cells were FI
It was incubated at room temperature for 20 minutes with anti-digoxigen-antibody conjugated with TC (Boehringer Mannheim). Unbound antibody was removed by two washes in PBS. The preparations were evaluated using fluorescence microscopy at an excitation wavelength of 470 nm.

Results: (A) MCF-7 tumor cells show a strong and well-defined fluorescent signal in the cytoplasm, while
The nucleus shows no fluorescence. Control experiments ("apparent hybridization" (Scheinhybr
idisierung) "), no fluorescent signal was detectable.

(B) No cell labeling was obtained after hybridizing early human skin fibroblasts.

(C) After hybridization of tumor tissue fragments induced by MCF-7 cells, tumor cells show exclusively fluorescence, while surrounding normal connective tissue, subcutaneous tissue and epidermis show no signal. . The hybridization signal of the tumor cells is likewise exclusively localized in the cytoplasm. Control hybridization ("apparent hybridization (Sc
heinhybridisierung) "and pUC19 DNA labeled with digoxigen
), No fluorescent signal was obtained.

Thus, a cytosolic cdc37 protein-nucleic acid heteromer can be detected by hybridization of an appropriate DNA sample with nucleic acid specifically associated with the heteromer in tumor cells. Performing in situ hybridization allows the identification of individual neoplastic transformed cells in tissue fragments or in biopsies.

Example 11 Detection of cdc37 Protein-Nucleic Acid Heteromer Using Antibodies and Polymerase Chain Reaction of Associated Nucleic Acid Acute myeloid leukemia (AML) cells contain a cytoplasmic cdc37 protein-nucleic acid heteromer, Can be increased from cell lysates using an antibody more specific for the cdc37 protein-nucleic acid heteromer, and the nucleic acid of the nucleic acid heteromer can be amplified by oligonucleotides specific for the polymerase chain reaction and sequence. .

AML (A) cells 104 and normal peripheral blood-lymphocytes (B) or 10 ^Four Mixture of 10 AML cells in 10 blood lymphocytes (C) or 10^FourIndividual blood
A mixture of 100 normal human fibroblasts in lymphocytes (D) was obtained. Erasure
Normal lymphocytes in blood must show no proliferation without antigen stimulation
Normal fibroblasts grow until the cells age after about 50-60 passages.
Proliferate logically. In contrast, acute myeloid leukemia cells have infinite proliferation and
And suppression of reaching physiological cell death. Live only when cells are restricted in vitro
Even when extended, it induces malignant tumors in vivo and shows infinite growth.

The cells in batches AD were sedimented and 100 μl of 10 mM Tris-HCl,
Resuspended in 1.5 mM MgCl _{2 at} pH 7.2, immediately frozen at −20 ° C. and thawed again. The sample was sedimented by centrifugation at 2000 × g for 10 minutes and the supernatant was placed in a recess of a microtiter plate whose surface was coated with the antibody 3D6 specific for cdc37 protein-nucleic acid heteromer. After 1 hour of incubation at room temperature, the samples were washed four times with PBS, 2% (w / v) bovine serum albumin. Now, on microtiter plates, cdc37 protein-nucleic acid heteromers from cells have increased. This increase ensured that in the next step of DNA amplification, only the DNA of the cdc37 protein-nucleic acid heteromer could be used as a matrix for DNA polymerases, but not for nuclear DNA or other contaminating DNA.
NA cannot be used.

[0103] Detection of the cdc37 protein-nucleic acid heteromer is performed by specific amplification of the associated DNA. A particular advantage is that this process can also be performed on microtiter plates following the increase.

The recess with the increased cdc37 protein-nucleic acid heteromer was incubated with proteinase K (100 μg / ml) at 55 ° C. for 1 hour and subsequently at 95 ° C. for 1 hour.
Heated for 0 minutes. Subsequently, the polymerase chain reaction (PCR) was performed by known methods (Mullis and Fallona, Methods Enzymol. 155: 335-350, 1987; Saiki et al., Science 239: 487-491, 1988). In this case, the next batch volume was selected.

3 μl of 10 × Taq buffer (Tris-HCl 200 mM, pH 8.4, KCl
250 mM, Tween 20 0.5%, 1 mg / ml BSA); 100 mM MgCl 2 0.5 μl; dNTP solution 1 μg (dATP 2 mM, dCTP 2 mM, dGTP 2 mM, dT
Taq-DNA polymerase 1 U (Boehringer Mannheim); DNA of cdc37 protein-nucleic acid heteromer from AML cells (SEQ-ID)
Primers for NO1)-100 n each of oligonucleotides A and B
g.

[0106]

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[0107]

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35 working cycles with the following temperature profile were performed: 1.1 × [95 ° C. for 2 minutes] 2.35 × [55 ° C. for 30 seconds; 72 ° C. for 90 seconds; 95 ° C. for 60 seconds] 1 × [72 ° C. for 15 minutes] The amplified DNA was separated by electrophoresis in a 1% agarose gel and visualized in UV light after incubation with ethidium bromide.

Results: When cells from acute leukemia (A) were analyzed, DNA of about 221 bp in length was amplified, but when normal human lymphocytes (B) were used, Not amplified. When a mixture of AML cells and normal lymphocytes (C) was used, DNA amplification was similarly obtained, but when normal lymphocytes were mixed with normal fibroblasts (D). Did not obtain DNA amplification. The mixing test of tumor cells and normal cells is performed by using the test batch amount described above.
It is shown that contamination of tumor cells with infinite growth can be detected in normal cells. The test batch volume is not compromised by contamination with physiologically growing cells, such as normal fibroblasts.

Furthermore, by performing the above tests, the contamination of the malignant tumor with the cells is such that (i) the cells in the sample to be analyzed are no longer intact, lysates of the cells are present, and (ii) Shows an ability to form tumors and grow indefinitely in vivo, but can also be detected when not shown in vitro.

Example 12 Inhibition of tumor cell growth using a synthetic peptide that binds to the nucleic acid of a cdc37 protein-nucleic acid heteromer.

Cells of human lymphoma lineage L540 (Sample A) as well as normal human lymphocytes from peripheral blood (Sample B) were isolated from RPMI 1640 medium, 10% (v / v), respectively.
3.) Seed at a concentration of 10 ⁴ cells per 100 μl of fetal bovine serum in ⁴ wells of a microtiter plate with 96 wells each. In addition, normal lymphocytes were stimulated in the presence of 10 μg of mitogen per μl of American pokeweed mitogen (PWM) (Sample C). The sample was kept at 37 ° C. for 12 hours. Peptides 1, 2 and 3 were combined with 2 μl of DOTAP “Transfection Agent” (Boehringer Mannheim, M
annheim, Cat.-No. 1811177), and put the samples A to C at a concentration of 40 μM of the peptide in the first to third recesses, respectively, and buffer (PBS) in the fourth recess.
And 2 μl of DOTAP “Transfection Agent”.

Peptides 1 and 2 are derived from the N-terminal sequence of the cdc37 protein and contain the nucleic acid binding region of the protein. Peptide 3 is a random sequence of peptides of the same length. The peptides are cyclized via N-terminal and C-terminal Cys groups, respectively.

[0114]

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The cultures were incubated at 37 ° C. for 3 days and the viability of the cells was subsequently determined by the XTT test (Boehri
nger Mannheim, Mannheim, Cat-No. 1465015). In this case, the tetrazolium salt XTT is added to the cells, and the XTT is degraded using the succinate-tetrazolium reductase system of the mitochondrial respiratory chain and converted to formazan. Subsequently, metabolites can be measured photometrically. The amount of metabolites is proportional to the number of viable cells and is used for assessing vitality and expanding cell populations.

Results: L540 lymphocytes (Sample A) had peptide 1 (recess 1) and peptide 2 (recess 1).
Only 15-20% metabolism of XTT compared to cultures in the presence of control peptide 3 (recess 3) in the presence of recess 2) and "blank" cultures without peptide (recess 4) Is shown. Human lymphocytes produced from terminal blood show the same XTT metabolism in the presence of the peptides (recesses 1 to 3) and in the "empty sample", and cultures with lymphocytes after mitogen stimulation (recess 1) The same applies to (4) to (4).

The growth of L540 lymphocytes was clearly regulated in the presence of Peptide 1 and Peptide 2, while the control peptide (Peptide 3) did not have the ability to trigger said growth inhibition. However, the proliferation of normal lymphocytes after stimulation with mitogens is not affected by this peptide. Sequence Listing (1) General Information: (i) Applicant: (A) Name: Univ. -Prof. Dr. med. Hinrich Abken (B) Town: Rhoenstrasse 1a (C) City: Meudt (E) Country: DE (F) Postal code: 56414 (ii) Title of the invention: Human cells or animal cells whose cell circulation control is impaired, Or a method for detecting, confirming, obtaining or removing human or animal cells having infinite proliferative ability or tumor-forming ability (iii) Number of sequences: 121 (iv) Computer-readable form: (A) Medium Type: Floppy disk (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS-DOS (D) Software: Patent-in release # 1.0, version # 1. (Vi) Priority Date: (A) Application Number: DE1974435.4 (B) Application Date: October 7, 1997 (vi) Priority Date: (A) Application Number: DE19749118.9 (B) Application Date: November 6, 1997 (2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 221 base pairs (B) Sequence type: nucleotide (C) Number of chains : Single-stranded (D) topology: linear (ii) sequence type: genomic DNA (vii) direct origin: (B) clone (E): MCF-7 / sm1 (xi) sequence: SEQ ID NO: 1:

[0118]

[Outside 1]

(2) Information on SEQ ID NO: 2: (i) Sequence characteristics: (A) Sequence length: 120 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB3 (xi) Sequence: SEQ ID NO: 2:

[0120]

[Outside 2]

(2) Information on SEQ ID NO: 3: (i) Sequence features: (A) Sequence length: 110 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB2 (xi) Sequence: SEQ ID NO: 3:

[0122]

[Outside 3]

(2) Information on SEQ ID NO: 4: (i) Sequence features: (A) Sequence length: 218 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB6 (xi) Sequence: SEQ ID NO: 4:

[0124]

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(2) Information on SEQ ID NO: 5: (i) Sequence features: (A) Sequence length: 184 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB1 (xi) Sequence: SEQ ID NO: 5:

[0126]

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(2) Information on SEQ ID NO: 6: (i) Sequence characteristics: (A) Sequence length: 131 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB5 (xi) Sequence: SEQ ID NO: 6:

[0128]

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(2) Information on SEQ ID NO: 7: (i) Sequence characteristics: (A) Sequence length: 323 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB7 (xi) Sequence: SEQ ID NO: 7:

[0130]

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[0131]

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(2) Information on SEQ ID NO: 8: (i) Sequence characteristics: (A) Sequence length: 30 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB9 (xi) Sequence: SEQ ID NO: 8:

[0133]

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(2) Information on SEQ ID NO: 9: (i) Sequence characteristics: (A) Sequence length: 219 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB11 (xi) Sequence: SEQ ID NO: 9:

[0135]

[Outside 10]

(2) Information on SEQ ID NO: 10: (i) Sequence features: (A) Sequence length: 179 base pairs (B) Sequence type: Nucleotide (C) Number of strands: Single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB12 (xi) Sequence: SEQ ID NO: 10:

[0137]

[Outside 11]

[0138]

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(2) Information on SEQ ID NO: 11: (i) Sequence characteristics: (A) Sequence length: 234 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB14 (xi) Sequence: SEQ ID NO: 11:

[0140]

[Outside 13]

(2) Information on SEQ ID NO: 12: (i) Sequence characteristics: (A) Sequence length: 269 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB16 (xi) Sequence: SEQ ID NO: 12:

[0142]

[Outside 14]

(2) Information on SEQ ID NO: 13: (i) Sequence features: (A) Sequence length: 256 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): 17 (xi) Sequence: SEQ ID NO: 13:

[0144]

[Outside 15]

(2) Information on SEQ ID NO: 14: (i) Sequence features: (A) Sequence length: 295 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB18 (xi) Sequence: SEQ ID NO: 14:

[0146]

[Outside 16]

(2) Information on SEQ ID NO: 15: (i) Sequence features: (A) Sequence length: 166 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB19 (xi) Sequence: SEQ ID NO: 15:

[0148]

[Outside 17]

(2) Information on SEQ ID NO: 16: (i) Sequence characteristics: (A) Sequence length: 244 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB20 (xi) Sequence: SEQ ID NO: 16:

[0150]

[Outside 18]

(2) Information on SEQ ID NO: 17: (i) Sequence characteristics: (A) Sequence length: 180 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB22 (xi) Sequence: SEQ ID NO: 17:

[0152]

[Outside 19]

(2) Information on SEQ ID NO: 18: (i) Sequence characteristics: (A) Sequence length: 176 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB24 (xi) Sequence: SEQ ID NO: 18:

[0154]

[Outside 20]

(2) Information on SEQ ID NO: 19: (i) Sequence characteristics: (A) Sequence length: 220 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB25 (xi) Sequence: SEQ ID NO: 19:

[0156]

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(2) Information on SEQ ID NO: 20: (i) Sequence features: (A) Sequence length: 192 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB27 (xi) Sequence: SEQ ID NO: 20:

[0158]

[Outside 22]

(2) Information on SEQ ID NO: 21: (i) Sequence characteristics: (A) Sequence length: 215 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB32 (xi) Sequence: SEQ ID NO: 21:

[0160]

[Outside 23]

(2) Information on SEQ ID NO: 22: (i) Sequence characteristics: (A) Sequence length: 222 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB34 (xi) Sequence: SEQ ID NO: 22:

[0162]

[Outside 24]

(2) Information on SEQ ID NO: 23: (i) Sequence characteristics: (A) Sequence length: 103 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB36 (xi) Sequence: SEQ ID NO: 23:

[0164]

[Outside 25]

(2) Information on SEQ ID NO: 24: (i) Sequence characteristics: (A) Sequence length: 253 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB37 (xi) Sequence: SEQ ID NO: 24:

[0166]

[Outside 26]

(2) Information on SEQ ID NO: 25: (i) Sequence features: (A) Sequence length: 245 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB39 (xi) Sequence: SEQ ID NO: 25:

[0168]

[Outside 27]

(2) Information on SEQ ID NO: 26: (i) Sequence characteristics: (A) Sequence length: 98 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB40 (xi) Sequence: SEQ ID NO: 26:

[0170]

[Outside 28]

(2) Information on SEQ ID NO: 27: (i) Sequence characteristics: (A) Sequence length: 243 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB41 (xi) Sequence: SEQ ID NO: 27:

[0172]

[Outside 29]

(2) Information on SEQ ID NO: 28: (i) Sequence characteristics: (A) Sequence length: 306 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB43 (xi) Sequence: SEQ ID NO: 28:

[0174]

[Outside 30]

(2) Information on SEQ ID NO: 29: (i) Sequence features: (A) Sequence length: 237 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB44 (xi) Sequence: SEQ ID NO: 29:

[0176]

[Outside 31]

(2) Information on SEQ ID NO: 30: (i) Sequence characteristics: (A) Sequence length: 212 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB45 (xi) Sequence: SEQ ID NO: 30:

[0178]

[Outside 32]

(2) Information on SEQ ID NO: 31: (i) Sequence characteristics: (A) Sequence length: 503 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML02 (xi) Sequence: SEQ ID NO: 31:

[0180]

[Outside 33]

[0181]

[Outside 34]

(2) Information on SEQ ID NO: 32: (i) Sequence characteristics: (A) Sequence length: 365 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML12 (xi) Sequence: SEQ ID NO: 32:

[0183]

[Outside 35]

(2) Information on SEQ ID NO: 33: (i) Sequence features: (A) Sequence length: 355 base pairs (B) Sequence type: nucleotide (C) Number of chains: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML18 (xi) Sequence: SEQ ID NO: 33:

[0185]

[Outside 36]

[0186]

[Outside 37]

(2) Information on SEQ ID NO: 34: (i) Sequence characteristics: (A) Sequence length: 385 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML19 (xi) Sequence: SEQ ID NO: 34:

[0188]

[Outside 38]

(2) Information on SEQ ID NO: 35: (i) Sequence characteristics: (A) Sequence length: 406 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML20 (xi) Sequence: SEQ ID NO: 35:

[0190]

[Outside 39]

(2) Information on SEQ ID NO: 36: (i) Sequence characteristics: (A) Sequence length: 167 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML28 (xi) Sequence: SEQ ID NO: 36:

[0192]

[Outside 40]

(2) Information on SEQ ID NO: 37: (i) Sequence characteristics: (A) Sequence length: 372 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML29 (xi) Sequence: SEQ ID NO: 37:

[0194]

[Outside 41]

(2) Information on SEQ ID NO: 38: (i) Sequence features: (A) Sequence length: 566 base pairs (B) Sequence type: Nucleotide (C) Number of strands: Single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML31 (xi) Sequence: SEQ ID NO: 38:

[0196]

[Outside 42]

(2) Information on SEQ ID NO: 39: (i) Sequence characteristics: (A) Sequence length: 680 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML35 (xi) Sequence: SEQ ID NO: 39:

[0198]

[Outside 43]

(2) Information on SEQ ID NO: 40: (i) Sequence characteristics: (A) Sequence length: 543 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML38 (xi) Sequence: SEQ ID NO: 40:

[0200]

[Outside 44]

(2) Information on SEQ ID NO: 41: (i) Sequence characteristics: (A) Sequence length: 580 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML40 (xi) Sequence: SEQ ID NO: 41:

[0202]

[Outside 45]

[0203]

[Outside 46]

(2) Information on SEQ ID NO: 42: (i) Sequence features: (A) Sequence length: 557 base pairs (B) Sequence type: Nucleotide (C) Number of strands: Single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML41 (xi) Sequence: SEQ ID NO: 42:

[0205]

[Outside 47]

(2) Information on SEQ ID NO: 43: (i) Sequence characteristics: (A) Sequence length: 593 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): AML47 (xi) Sequence: SEQ ID NO: 43:

[0207]

[Outside 48]

[0208]

[Outside 49]

(2) Information on SEQ ID NO: 44: (i) Sequence features: (A) Sequence length: 196 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-1 (xi) Sequence: SEQ ID NO: 44:

[0210]

[Outside 50]

(2) Information on SEQ ID NO: 45: (i) Sequence features: (A) Sequence length: 78 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-101 (xi) Sequence: SEQ ID NO: 45:

[0212]

[Outside 51]

(2) Information on SEQ ID NO: 46: (i) Sequence characteristics: (A) Sequence length: 122 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-103 (xi) Sequence: SEQ ID NO: 46:

[0214]

[Outside 52]

(2) Information on SEQ ID NO: 47: (i) Sequence characteristics: (A) Sequence length: 80 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-104 (xi) Sequence: SEQ ID NO: 47:

[0216]

[Outside 53]

(2) Information on SEQ ID NO: 48: (i) Sequence characteristics: (A) Sequence length: 63 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-105 (xi) Sequence: SEQ ID NO: 48:

[0218]

[Outside 54]

(2) Information on SEQ ID NO: 49: (i) Sequence characteristics: (A) Sequence length: 107 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-106 (xi) Sequence: SEQ ID NO: 49:

[0220]

[Outside 55]

(2) Information on SEQ ID NO: 50: (i) Sequence characteristics: (A) Sequence length: 36 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-107 (xi) Sequence: SEQ ID NO: 50:

[0222]

[Outside 56]

(2) Information on SEQ ID NO: 51: (i) Sequence features: (A) Sequence length: 231 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-11 (xi) Sequence: SEQ ID NO: 51:

[0224]

[Outside 57]

(2) Information on SEQ ID NO: 52: (i) Sequence characteristics: (A) Sequence length: 191 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-12 (xi) Sequence: SEQ ID NO: 52:

[0226]

[Outside 58]

(2) Information on SEQ ID NO: 53: (i) Sequence characteristics: (A) Sequence length: 246 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-14 (xi) Sequence: SEQ ID NO: 53:

[0228]

[Outside 59]

(2) Information on SEQ ID NO: 54: (i) Sequence features: (A) Sequence length: 281 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-16 (xi) Sequence: SEQ ID NO: 54:

[0230]

[Outside 60]

(2) Information on SEQ ID NO: 55: (i) Sequence characteristics: (A) Sequence length: 268 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-17 (xi) Sequence: SEQ ID NO: 55:

[0232]

[Outside 61]

(2) Information on SEQ ID NO: 56: (i) Sequence features: (A) Sequence length: 307 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-18 (xi) Sequence: SEQ ID NO: 56:

[0234]

[Outside 62]

(2) Information on SEQ ID NO: 57: (i) Sequence features: (A) Sequence length: 245 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-2 (xi) Sequence: SEQ ID NO: 57:

[0236]

[Outside 63]

(2) Information on SEQ ID NO: 58: (i) Sequence characteristics: (A) Sequence length: 256 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-20 (xi) Sequence: SEQ ID NO: 58:

[0238]

[Outside 64]

(2) Information on SEQ ID NO: 59: (i) Sequence characteristics: (A) Sequence length: 122 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-202 (xi) Sequence: SEQ ID NO: 59:

[0240]

[Outside 65]

(2) Information on SEQ ID NO: 60: (i) Sequence characteristics: (A) Sequence length: 132 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-203 (xi) Sequence: SEQ ID NO: 60:

[0242]

[Outside 66]

(2) Information on SEQ ID NO: 61: (i) Sequence characteristics: (A) Sequence length: 230 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-206 (xi) Sequence: SEQ ID NO: 61:

[0244]

[Outside 67]

(2) Information on SEQ ID NO: 62: (i) Sequence features: (A) Sequence length: 335 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-207 (xi) Sequence: SEQ ID NO: 62:

[0246]

[Outside 68]

[0247]

[Outside 69]

(2) Information on SEQ ID NO: 63: (i) Sequence characteristics: (A) Sequence length: 192 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-22 (xi) Sequence: SEQ ID NO: 63:

[0249]

[Outside 70]

(2) Information on SEQ ID NO: 64: (i) Sequence features: (A) Sequence length: 188 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-24 (xi) Sequence: SEQ ID NO: 64:

[0251]

[Outside 71]

(2) Information on SEQ ID NO: 65: (i) Sequence characteristics: (A) Sequence length: 232 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-24 (xi) Sequence: SEQ ID NO: 65:

[0253]

[Outside 72]

(2) Information on SEQ ID NO: 66: (i) Sequence characteristics: (A) Sequence length: 204 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-27 (xi) Sequence: SEQ ID NO: 66:

[0255]

[Outside 73]

(2) Information on SEQ ID NO: 67: (i) Sequence characteristics: (A) Sequence length: 192 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Type of sequence: genomic DNA (vii) Direct source: (B) Clone (E): HT29-3 (xi) Sequence: SEQ ID NO: 67:

[0257]

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(2) Information on SEQ ID NO: 68: (i) Sequence characteristics: (A) Sequence length: 227 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-32 (xi) Sequence: SEQ ID NO: 68:

[0259]

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(2) Information on SEQ ID NO: 69: (i) Sequence features: (A) Sequence length: 234 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-34 (xi) Sequence: SEQ ID NO: 69:

[0261]

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(2) Information on SEQ ID NO: 70: (i) Sequence characteristics: (A) Sequence length: 115 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-36 (xi) Sequence: SEQ ID NO: 70:

[0263]

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[0264]

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(2) Information on SEQ ID NO: 71: (i) Sequence characteristics: (A) Sequence length: 265 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-37 (xi) Sequence: SEQ ID NO: 71:

[0266]

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(2) Information on SEQ ID NO: 72: (i) Sequence characteristics: (A) Sequence length: 256 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-39 (xi) Sequence: SEQ ID NO: 72:

[0268]

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(2) Information on SEQ ID NO: 73: (i) Sequence characteristics: (A) Sequence length: 110 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-40 (xi) Sequence: SEQ ID NO: 73:

[0270]

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(2) Information on SEQ ID NO: 74: (i) Sequence features: (A) Sequence length: 255 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-41 (xi) Sequence: SEQ ID NO: 74:

[0272]

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(2) Information on SEQ ID NO: 75: (i) Sequence characteristics: (A) Sequence length: 318 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-43 (xi) Sequence: SEQ ID NO: 75:

[0274]

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(2) Information on SEQ ID NO: 76: (i) Sequence characteristics: (A) Sequence length: 249 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-44 (xi) Sequence: SEQ ID NO: 76:

[0276]

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(2) Information on SEQ ID NO: 77: (i) Sequence characteristics: (A) Sequence length: 224 base pairs (B) Sequence type: nucleotide (C) Number of chains: single stranded ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-45 (xi) Sequence: SEQ ID NO: 77:

[0278]

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(2) Information on SEQ ID NO: 78: (i) Sequence features: (A) Sequence length: 143 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-5 (xi) Sequence: SEQ ID NO: 78:

[0280]

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(2) Information on SEQ ID NO: 79: (i) Sequence features: (A) Sequence length: 76 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-6 (xi) Sequence: SEQ ID NO: 79:

[0282]

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(2) Information on SEQ ID NO: 80: (i) Sequence features: (A) Sequence length: 106 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-7 (xi) Sequence: SEQ ID NO: 80:

[0284]

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(2) Information on SEQ ID NO: 81: (i) Sequence characteristics: (A) Sequence length: 38 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HT29-9 (xi) Sequence: SEQ ID NO: 81:

[0286]

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(2) Information on SEQ ID NO: 82: (i) Sequence characteristics: (A) Sequence length: 78 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): BH1 (xi) Sequence: SEQ ID NO: 82:

[0288]

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(2) Information on SEQ ID NO: 83: (i) Sequence features: (A) Sequence length: 122 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): BH3 (xi) Sequence: SEQ ID NO: 83:

[0290]

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(2) Information on SEQ ID NO: 84: (i) Sequence characteristics: (A) Sequence length: 80 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): BH4 (xi) Sequence: SEQ ID NO: 84:

[0292]

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(2) Information on SEQ ID NO: 85: (i) Sequence characteristics: (A) Sequence length: 63 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): BH5 (xi) Sequence: SEQ ID NO: 85:

[0294]

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(2) Information on SEQ ID NO: 86: (i) Sequence characteristics: (A) Sequence length: 107 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): BH6 (xi) Sequence: SEQ ID NO: 86:

[0296]

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(2) Information on SEQ ID NO: 87: (i) Sequence characteristics: (A) Sequence length: 36 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): BH7 (xi) Sequence: SEQ ID NO: 87:

[0298]

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(2) Information on SEQ ID NO: 88: (i) Sequence features: (A) Sequence length: 184 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB1 (xi) Sequence: SEQ ID NO: 88:

[0300]

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(2) Information on SEQ ID NO: 89: (i) Sequence features: (A) Sequence length: 219 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB11 (xi) Sequence: SEQ ID NO: 89:

[0302]

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(2) Information on SEQ ID NO: 90: (i) Sequence characteristics: (A) Sequence length: 179 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB12 (xi) Sequence: SEQ ID NO: 90:

[0304]

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[0305]

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(2) Information on SEQ ID NO: 91: (i) Sequence characteristics: (A) Sequence length: 234 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB14 (xi) Sequence: SEQ ID NO: 91:

[0307]

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(2) Information on SEQ ID NO: 92: (i) Sequence characteristics: (A) Sequence length: 269 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB16 (xi) Sequence: SEQ ID NO: 92:

[0309]

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(2) Information on SEQ ID NO: 93: (i) Sequence characteristics: (A) Sequence length: 256 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB17 (xi) Sequence: SEQ ID NO: 93:

[0311]

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(2) Information on SEQ ID NO: 94: (i) Sequence characteristics: (A) Sequence length: 295 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB18 (xi) Sequence: SEQ ID NO: 94:

[0313]

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(2) Information on SEQ ID NO: 95: (i) Sequence characteristics: (A) Sequence length: 166 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB19 (xi) Sequence: SEQ ID NO: 95:

[0315]

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(2) Information on SEQ ID NO: 96: (i) Sequence characteristics: (A) Sequence length: 110 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB2 (xi) Sequence: SEQ ID NO: 96:

[0317]

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(2) Information on SEQ ID NO: 97: (i) Sequence characteristics: (A) Sequence length: 244 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB20 (xi) Sequence: SEQ ID NO: 97:

[0319]

[Outer 106]

(2) Information on SEQ ID NO: 98: (i) Sequence characteristics: (A) Sequence length: 180 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB22 (xi) Sequence: SEQ ID NO: 98:

[0321]

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[0322]

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(2) Information on SEQ ID NO: 99: (i) Sequence characteristics: (A) Sequence length: 176 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB24 (xi) Sequence: SEQ ID NO: 99:

[0324]

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(2) Information on SEQ ID NO: 100: (i) Sequence characteristics: (A) Sequence length: 220 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB25 (xi) Sequence: SEQ ID NO: 100:

[0326]

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(2) Information on SEQ ID NO: 101: (i) Sequence characteristics: (A) Sequence length: 192 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB27 (xi) Sequence: SEQ ID NO: 101:

[0328]

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(2) Information on SEQ ID NO: 102: (i) Sequence features: (A) Sequence length: 120 base pairs (B) Sequence type: nucleotides (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB3 (xi) Sequence: SEQ ID NO: 102:

[0330]

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(2) Information on SEQ ID NO: 103: (i) Sequence characteristics: (A) Sequence length: 215 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB32 (xi) Sequence: SEQ ID NO: 103:

[0332]

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(2) Information on SEQ ID NO: 104: (i) Sequence characteristics: (A) Sequence length: 222 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB34 (xi) Sequence: SEQ ID NO: 104:

[0334]

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(2) Information on SEQ ID NO: 105: (i) Sequence characteristics: (A) Sequence length: 103 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB36 (xi) Sequence: SEQ ID NO: 105:

[0336]

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(2) Information on SEQ ID NO: 106: (i) Sequence characteristics: (A) Sequence length: 253 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB37 (xi) Sequence: SEQ ID NO: 106:

[0338]

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(2) Information on SEQ ID NO: 107: (i) Sequence characteristics: (A) Sequence length: 245 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB39 (xi) Sequence: SEQ ID NO: 107:

[0340]

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(2) Information on SEQ ID NO: 108: (i) Sequence characteristics: (A) Sequence length: 98 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB40 (xi) Sequence: SEQ ID NO: 108:

[0342]

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(2) Information on SEQ ID NO: 109: (i) Sequence features: (A) Sequence length: 243 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB41 (xi) Sequence: SEQ ID NO: 109:

[0344]

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[0345]

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(2) Information on SEQ ID NO: 110: (i) Sequence characteristics: (A) Sequence length: 306 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB43 (xi) Sequence: SEQ ID NO: 110:

[0347]

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(2) Information on SEQ ID NO: 111: (i) Sequence features: (A) Sequence length: 237 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB44 (xi) Sequence: SEQ ID NO: 111:

[0349]

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(2) Information on SEQ ID NO: 112: (i) Sequence features: (A) Sequence length: 212 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Type of sequence: genomic DNA (vii) Direct origin: (B) Clone (E): HB45 (xi) Sequence: SEQ ID NO: 112:

[0351]

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(2) Information on SEQ ID NO: 113: (i) Sequence characteristics: (A) Sequence length: 131 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB5 (xi) Sequence: SEQ ID NO: 113:

[0353]

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(2) Information on SEQ ID NO: 114: (i) Sequence characteristics: (A) Sequence length: 218 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB6 (xi) Sequence: SEQ ID NO: 114:

[0355]

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(2) Information on SEQ ID NO: 115: (i) Sequence characteristics: (A) Sequence length: 323 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB7 (xi) Sequence: SEQ ID NO: 115:

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(2) Information on SEQ ID NO: 116: (i) Sequence characteristics: (A) Sequence length: 26 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (vii) Direct origin: (B) Clone (E): HB9 (xi) Sequence: SEQ ID NO: 116:

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(2) Information on SEQ ID NO: 117: (i) Sequence characteristics: (A) Sequence length: 26 amino acids (B) Sequence type: amino acids (C) Number of chains: single chain (D ) Topology: Both forms (ii) Sequence type: Peptide (xi) Sequence: SEQ ID NO: 117:

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(2) Information on SEQ ID NO: 118: (i) Sequence features: (A) Sequence length: 26 amino acids (B) Sequence type: amino acids (C) Number of chains: single chain (D ) Topology: Both forms (ii) Sequence type: Peptide (xi) Sequence: SEQ ID NO: 118:

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(2) Information on SEQ ID NO: 119: (i) Sequence characteristics: (A) Sequence length: 26 amino acids (B) Sequence type: amino acids (C) Number of chains: single chain (D ) Topology: Both forms (ii) Sequence type: Peptide (xi) Sequence: SEQ ID NO: 119:

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(2) Information on SEQ ID NO: 120: (i) Sequence characteristics: (A) Sequence length: 18 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: Linear (ii) Sequence type: Genomic DNA (xi) Sequence: SEQ ID NO: 120:

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(2) Information on SEQ ID NO: 121: (i) Sequence features: (A) Sequence length: 21 base pairs (B) Sequence type: nucleotide (C) Number of strands: single strand ( D) Topology: linear (ii) Sequence type: genomic DNA (xi) Sequence: SEQ ID NO: 121:

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[Sequence list]

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission Date] April 7, 2000 (200.4.7)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 43/00 111 43/00 111 C07K 14/47 C07K 14/47 16/18 16/18 16/30 16/30 G01N 33/53 D G01N 33/53 A61K 37/02 (31) Priority number 198 21 506.1 (32) Priority date May 13, 1998 (May 13, 1998) (33) Priority claim country Germany (DE) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL , PT, SE), JP, US F-term (reference) 4B063 QA01 QQ03 QQ08 QQ42 QQ79 QR16 QR31 QR32 QR48 QR51 QS25 QS34 QX02 4C084 AA02 AA06 BA44 CA17 CA25 DC50 MA01 NA14 ZB261 4C085 AA13 AA14 BB86 A01A A01 MA04 NA14 ZB26 4H045 AA11 BA18 EA51

Claims (31)

[Claims] (1) a human cell or an animal cell whose cell circulation control is impaired;
Or a method for detecting, confirming, obtaining or removing human cells or animal cells having an infinite proliferative ability or a tumor-forming ability, comprising the steps of: Detection, confirmation of human cells or animal cells having impaired cell circulation control, or human cells or animal cells having infinite proliferative capacity or tumorigenicity, characterized by detecting the presence thereof.
Methods for acquisition or removal. 2. The method according to claim 1, wherein the association is detected using a detectable substance capable of specifically binding to the association. 3. The method according to claim 2, wherein an antibody capable of binding to the association is used as the substance capable of specifically binding. 4. The method according to claim 3, wherein a polyclonal antibody, a monoclonal antibody, a recombinant antibody, or a fragment capable of binding these antibodies is used as the antibody. 5. The method according to claim 4, wherein an antibody directed to the cdc37 protein is used. 6. Use of an antibody directed against an extrachromosomal nucleic acid.
The described method. 7. The method according to claim 3, wherein a labeled antibody is used. 8. The method according to claim 7, wherein an antibody conjugated with an enzyme, a radionuclide, a dye or a toxicologically active substance is used. 9. The method according to claim 2, wherein a nucleic acid or an oligonucleotide that hybridizes with the nucleic acid of the association is used as the substance having a binding ability. 10. The method according to any one of claims 1 to 9, wherein the detection of the association is performed in a tissue biopsy, tissue collection, a body fluid, or a cell lysate. 11. The method according to claim 10, wherein the association is detected in a mitochondria-free cytoplasmic fraction or a body fluid. 12. The method according to claim 10, wherein the association is detected on the surface of the cell. 13. The detection of aggregates on the cell surface is carried out using a substance which is present bound to the solid carrier and which has the ability to separate cells bound thereby from the rest of the cells. 12. The method according to 12. 14. Use of the method according to claim 13 for removing cells having impaired cell circulation control or cells having infinite proliferative or tumorigenic potential from blood, bone marrow or another body fluid. . 15. The method according to any one of claims 3 to 8, wherein an Fc-supporting carrier is used, and the cell is held at a constant temperature together with a cell-containing substrate until the aggregate-supporting cells are in a lysed state.
A method according to any one of claims 10 to 14. 16. A cdc37 protein as an association having an extrachromosomal nucleic acid. 17. The method of claim 17, wherein the extrachromosomal nucleic acid is DNA of a tumor cell or such DN.
17. The association of claim 16, which is an RNA copy of A. 18. The method for obtaining an association according to claim 16 or 17, wherein a cytoplasmic fraction of human cells or animal cells having a permanently dividing ability is added to a surfactant or / and the cells are frozen and frozen. The method according to claim 1, wherein the mitochondria are separated from the treated cytoplasmic fraction by repeating the release, and the aggregate is obtained by selective preparation, electrophoresis or chromatography.
18. A method for obtaining the association according to 6 or 17. 19. The mitochondria-free cytoplasmic fraction is reduced to 1.5 M Na
19. Acquired by addition of Cl, removal of the formed precipitate and precipitation of the aggregate from the resulting supernatant with ethanol or ammonium sulfate.
The described method. 20. The method according to claim 1, wherein the cytoplasmic fraction containing the cell organelle and / or the membrane is kept at a constant temperature together with the protease, and the associated product is subsequently obtained from the lysate.
8. The method according to 8. 21. The method according to claim 20, wherein the aggregate is precipitated from the lysate using ethanol or ammonium sulfate and purified by chromatography. 22. The method according to claim 20, wherein proteinase K as a protease is kept at 50 to 70 ° C. for 0.5 to 3 hours. 23. The method according to any one of claims 20 to 22, wherein the cytoplasmic lysate is extracted using an organic solvent or a solvent mixture, and the associated product is obtained from an organic aqueous mesophase. 24. The method of claim 23, wherein the mesophase is extracted with phenol and chloroform. 25. The method according to claim 24, wherein the aggregate is precipitated from the organic aqueous interphase cells using ethanol. 26. cdc37 in labeled or unlabeled form.
An antibody having specificity for a protein-nucleic acid-heteromer. 27. The antibody of claim 26, which is conjugated to an enzyme, a radionuclide, a dye, or a toxicologically active substance. 28. The antibody of claim 26 conjugated to an enzyme, radionuclide, dye or toxin. 29. A pharmaceutical preparation comprising the antibody according to claim 26. 30. The preparation according to claim 29, comprising the antibody according to claim 27. A pharmaceutical preparation comprising a nucleic acid or a peptide that binds to the nucleic acid according to claim 17.