EP1109832A1

EP1109832A1 - Ozf protein-specific monoclonal antibodies and their diagnostic therapeutic uses

Info

Publication number: EP1109832A1
Application number: EP99941716A
Authority: EP
Inventors: Gérard GOUBIN; Didier Ferbus; Martine Muleris; Marie-Thérèse PROSPERI
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut Curie
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut Curie
Priority date: 1998-09-08
Filing date: 1999-09-08
Publication date: 2001-06-27
Also published as: FR2782998A1; CA2342963A1; AU5522699A; WO2000014118A1; FR2782998B1

Abstract

The invention concerns an OZF protein-specific monoclonal antibody and a pharmaceutical composition comprising said antibody for preventing, treating or diagnosing pathology related with abnormal OZF protein expression such as cancer. The invention further concerns methods for detecting OZF protein and methods for selecting compounds capable of interacting with the OZF protein.

Description

MONOCLONAL ANTI-PROTEIN OZF ANTIBODIES AND THEIR APPLICATIONS IN THE DIAGNOSTIC AND THERAPEUTIC FIELD.

The present invention relates to a monoclonal antibody specific for the OZF protein as well as a pharmaceutical composition comprising said antibody intended for the prevention, treatment or diagnosis of pathology linked to the abnormal expression of OZF protein such as cancer. The invention further includes methods for detecting the OZF protein as well as methods for selecting compounds capable of interacting with the OZF protein. Most proteins with zinc finger motifs are characterized by their ability to bind to RNA or DNA. This zinc finger motif has been found in genes controlling development, transcription factor genes or genes linked to the formation of tumors, thus attesting to the importance of this zinc finger structure in the regulation of the expression of Genoa. Among the genes coding for these proteins with zinc finger motifs, a gene coding for a protein consisting solely of zinc finger motifs, called the OZF gene (OZF for “Only Zinc Fingers”) has been isolated (Le Chalony et al., 1994 ) and the corresponding cDNA has been cloned and sequenced. The amino acid sequence of the putative protein encoded by the OZF gene contains 292 amino acid residues, including 10 finger zinc patterns of 28 amino acid residues, for an estimated molecular weight of 33 kDa. Comparative analysis of the OZF protein has shown that the domain containing the finger to zinc structure has great homologies with other proteins with finger to zinc motifs.

On the other hand, Ferbus et al. (Ferbus et al., 1996) were able to highlight certain characteristics of the OZF protein from a recombinant OZF protein produced in E. coli. In particular, these authors have shown that the recombinant OZF protein is capable of binding zinc ions, DNA and heparin. In addition, these authors were also able to show, using a polyclonal antibody obtained from a recombinant OZF protein fused with a maltose-fixing protein (MBP), that the OZF protein was expressed in human mammary cells of the epithelial type, preferably in the nucleus, while it was very weakly expressed in the myoepithelial and stromatic cells of this breast tissue. These authors were also able to demonstrate that the OZF protein was expressed in a tumor cell line established from a pancreatic tumor. The inventors have surprisingly demonstrated, using polyclonal antibodies to the OZF protein, that the OZF protein is overexpressed in primary tumors which have an amplification of the OZF gene but also in primary tumors which do not exhibit amplification of the gene. On the other hand, the inventors have also demonstrated that the overexpression of the OZF protein in these primary tumors is restricted to tumor cells.

These results thus show that the OZF protein appears as a marker for tumor cells. Obtaining antibodies capable of specifically recognizing the OZF protein would thus make it possible to detect tumor cells characterized by an abnormal expression of the OZF protein.

Polyclonal anti-OZF protein antibodies have already been described (Ferbus et al., 1996). These antibodies were prepared by immunizing rabbits against a recombinant protein obtained by fusion of the OZF protein and of the MBP protein, then purified on an immunoabsorbent consisting of the fused recombinant OZF-MBP protein coupled on a 4B sepharose column. These polyclonal antibodies can thus recognize several epitopes of the OZF protein and in particular certain epitopes common to human or mammalian proteins with zinc finger patterns, taking into account the great sequence homology observed between the OZF protein and other proteins with zinc finger patterns.

The inventors were thus able to demonstrate non-specific reactions obtained with certain proteins with zinc finger motifs for these polyclonal antibodies. If these polyclonal antibodies can prove to be sufficient specificity for particular applications such as the detection of OZF protein in isolated homogeneous cells or tissues, their specificity and possibly their affinity could prove to be insufficient as recognized by the authors. of the publication describing these polyclonal antibodies. These authors indeed specify in their conclusion that it would be necessary to have anti-OZF antibodies making it possible to precisely identify the types of cells expressing the OZF protein. Obtaining such antibodies, directed against an epitope specific for the human OZF protein, would undoubtedly make it possible not only to identify the types of cells expressing normally or abnormally the OZF protein but also to search for target nucleic sequences of the OZF protein, in particular nuclear, and to identify the genes capable of being regulated by the OZF protein.

Thus, there is today a need for a monoclonal antibody directed specifically against the human OZF protein. Such an antibody could not only be useful for studying and thus better understanding the role played by the OZF protein in the regulation of genes but also would be useful with regard to the results presented by the inventors in the present invention, for other applications such as therapeutic applications, diagnostics, in particular in vivo or in vitro from a heterogeneous biological sample, or for the targeting of compounds capable of modulating the biological activity of the OZF protein.

The present invention is based on the discovery of the possibility of obtaining, by a particular preparation approach, a monoclonal antibody characterized by a specificity never previously obtained. It has thus been discovered that by immunizing a mouse with a recombinant OZF protein, it is possible to obtain an antibody recognizing an epitope specific for the OZF protein and which does not recognize other proteins with zinc finger motifs from mammals.

Thus, the subject of the present invention is a monoclonal antibody or one of its fragments capable of binding specifically to an epitope of the OZF protein, preferably human. The term “epitope” is understood to mean in the present description, any determinant of the protein responsible for the specific interaction with the antibody. Epitopic determinants usually consist of groups of molecules having chemically active surfaces such as amino acids or sugar side chains and having a specific three-dimensional structure and / or a characteristic specific charge.

The monoclonal antibody fragments according to the invention comprise any fragment of said monoclonal antibody capable of binding to the epitope of the OZF protein on which the monoclonal antibody from which said fragment is derived binds. Examples of such fragments include in particular single-chain monoclonal antibodies or monovalent fragments Fab or Fab 'and divalent fragments such as F (ab') 2, which have the same binding specificity as the monoclonal antibody from which they are derived . A fragment according to the invention may also be a single chain Fv fragment produced by methods known to those skilled in the art and as described for example by Skerra et al., 1988 and King et al., 1991. According to the present invention, fragments of monoclonal antibodies of the invention can be obtained from the monoclonal antibodies as described above by methods such as digestion with enzymes, such as pepsin or papain and / or by cleavage of disulfide bridges by chemical reduction. In another way, the fragments of monoclonal antibodies included in the present invention can be synthesized by automatic peptide synthesizers such as those supplied by the company Applied Biosystems, etc., or can be prepared manually using techniques known in the art. 'skilled in the art and as described for example by Geysen et al., 1978. In general, for the preparation of monoclonal antibodies or their fragments, reference may be made to the techniques which are in particular described in the manual “Antibodies ”(Harlow et al., 1988) or to the technique of preparation from hybridomas described by Kohler and Milstein in 1975.

The monoclonal antibodies according to the invention can be obtained for example from the cell of an animal immunized against the OZF protein, or one of its fragments, comprising the epitope recognized specifically by said monoclonal antibodies according to the invention. Said OZF protein, or one of its fragments, may in particular be produced, according to the usual procedures, by genetic recombination from a nucleic acid sequence contained in the cDNA sequence coding for the OZF protein or by synthesis peptide from an amino acid sequence included in the peptide sequence of the OZF protein.

The monoclonal antibodies according to the invention can for example be purified on an affinity column on which the OZF protein or one of its fragments comprising the epitope specifically recognized by said monoclonal antibodies according to the invention has previously been immobilized.

Preferably, the monoclonal antibody or a fragment thereof according to the present invention binds to the linear or conformational epitope of the N-terminal domain of the human OZF protein, the sequence of which has a low rate of homology compared with the sequences d other proteins with zinc finger patterns.

More preferably, the monoclonal antibody or one of its fragments according to the invention binds to an epitope located on the first fifteen amino acids, of the N-terminal domain of the OZF protein as described by Le Chalony et al., 1994, in Figures 1B and 1C page 400. Thus, the invention relates to a monoclonal antibody or one of its fragments according to the invention, characterized in that the epitope of the OZF protein is located on the N-terminal part.

Preferably, the invention relates to a monoclonal antibody or a fragment thereof according to the invention, characterized in that the epitope of the OZF protein is located on the N-terminal part comprising the tyrosine residue located in position 10 of the sequence of the human OZF protein as described by Le Chalony et al., 1994, in FIG. 1B on page 400, preferably said epitope is carried by the fragment of sequence aa7-aa17 of said human OZF sequence. Also preferably, the invention relates to a monoclonal antibody or a fragment thereof according to the invention, characterized in that it is capable of further recognizing the human OZF protein whose sequence has a lysine / arginine polymorphism in position 8 of the sequence of the human OZF protein as described by Le Chalony et al., 1994, in FIG. 1B on page 400. Among the monoclonal antibodies according to the invention, the monoclonal antibody or one of its fragments, in particular, is preferred. what it is produced by a cell as deposited at the National Center for Culture of Microorganism (CNCM) (Institut Pasteur, Paris, France) on September 6, 1999 under the number I-2308. The hybridoma as deposited at the CNCM on September 6, 1999 under the number I-2308, also forms part of the present invention.

The invention further comprises a monoclonal antibody or one of its fragments according to the invention, characterized in that it is chosen from humanized, chimeric or anti-idiotypic antibodies.

The humanized monoclonal antibodies according to the invention or their fragments can be prepared by techniques known to those skilled in the art (Carter et al.,

1992; Singer et al., 1992 and Mountain et al., 1992). Such humanized monoclonal antibodies according to the invention are preferred for their use in in vivo diagnostic and therapeutic methods.

The monoclonal antibodies or their chimeric type fragments according to the invention can be produced using the techniques of genetic recombination. For example, the chimeric monoclonal antibody can be produced by cloning a recombinant DNA comprising a promoter and a sequence coding for the variable region of a monoclonal antibody according to the invention and a sequence coding for the constant region of human antibody. A chimeric antibody of the invention encoded by such a recombinant gene will be for example a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from murine DNA and its isotype determined by the constant region derived from human DNA (Verhoeyn et al., 1988).

The monoclonal antibodies or their fragments according to the present invention also include anti-idiotype antibodies produced by methods known to those skilled in the art (Cozenza, et al., 1976 and Harlow et al., 1988).

Also included in the invention are the monoclonal antibodies or their fragments according to the present invention obtained by genetic recombination or by chemical synthesis. The invention also comprises a monoclonal antibody or one of its fragments according to the invention, characterized in that it is labeled.

The monoclonal antibodies according to the invention or their fragments can also, according to the invention, be in the form of labeled antibodies in order to obtain a detectable and / or quantifiable signal. The monoclonal antibodies labeled according to the invention or their fragments include, for example, antibodies called immunoconjugates which can be conjugated, for example, with enzymes such as peroxidase, alkaline phosphatase, β-D-gaiactosidase, glucose oxidase, glucose amylase , carbonic anhydrase, acetyl-cholinesterase, lysozyme, malate dehydrogenase or glucose-6 phosphate dehydrogenase or by a molecule such as biotin, digoxigenin or 5-bromo-deoxyuridine. Fluorescent markers can also be conjugated to the monoclonal antibodies or their fragments of the invention and include in particular fluorescein and its derivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for "Green Fluorescent Protein"), dansyl, umbelliferone, etc. In such conjugates, the monoclonal antibodies of the invention or their fragments can be prepared by methods known to those skilled in the art. They can be coupled to enzymes or fluorescent markers directly or through a spacer group or a linking group such as a polyaldehyde, such as glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or in the presence of coupling agents such as periodate, etc. The conjugates comprising markers of the fluorescein type can be prepared by reaction with an isothiocyanate.

Other conjugates can also include chemiluminescent markers such as luminol and dioxetanes or bioluminescent markers such as luciferase and luciferin. Among the markers which can be attached to the monoclonal antibody or one of its fragments according to the invention, radioactive markers such as

¹ C, ³⁶ CI, ⁵⁷ Co, ∞Co, ⁵¹ Cr, ¹⁵² Eu, ⁵⁹ Fe, ³ H, ¹²⁵ l, ¹³¹ l, ³² P, ^M S, ⁷⁵ SE and " ^m Tc which can be detected by known means such as the gamma or scintillation counter, by autoradiography, etc.

The present invention also comprises the labeled monoclonal antibodies or their fragments according to the invention in which the conjugate can be a detectable marker chosen from the markers which can be used in the in vivo imaging application. Examples of such markers according to the invention are ⁷² As, ⁶⁷ Cu, ⁶⁷ Ga, Ga, ¹²³ l, ¹²⁵ l, ¹³¹ l, ¹¹¹ ln, ⁹⁷ Ru, " ^m Tc, ²⁰¹ TI and ∞Zr.

The term “in vivo imaging” should be understood in the present description as any method allowing the detection of a labeled monoclonal antibody according to the present invention or a fragment thereof which specifically binds to the epitope of the OZF protein in the body. of the patient. The patient will preferably be a man likely to present tumor cells abnormally expressing the protein OZF.

The present invention also includes the labeled monoclonal antibodies or their fragments according to the invention in which the conjugate can be a marker chosen from paramagnetic markers which can be used in the in vivo imaging application. Such markers according to the invention are for example the paramagnetic isotopes particularly used in magnetic resonance imaging (MRI) and which notably include ^S2 Cr, ¹⁶² Dy, ∞Fe, ¹⁵⁷ Gd and ∞Mn.

As mentioned previously, for the preparation of monoclonal antibody conjugates, the isotopic or paramagnetic marker can be attached to the antibody according to the invention or one of its fragments either directly or indirectly using an intermediate functional group. According to the invention, the monoclonal antibody or one of its fragments can be labeled by any technique known to those skilled in the art such as, for example, those described by Wagner et al., 1979 and Saha et al., 1976. The radiolabelled monoclonal conjugates according to the present invention can, for example, be iodinated by contact with sodium or potassium iodide and with an oxidizing chemical agent such as sodium hypochlorite or an enzymatic oxidizing agent such as lactoperoxidase.

The type of detection of the instrument used will be a major factor in the selection of the marker. For example, radioactive or paramagnetic isotopes will be chosen, in particular for in vivo imaging, depending on the type of instrument used that will guide the selection of these markers. Preferably, the radioactive markers chosen must have a period which is detectable by the type of instrument chosen. These monoclonal antibodies can thus be used for example as an imaging agent, in vivo or ex vivo on biological samples in any conventional method making it possible to visualize by image a diagnosis of pathology linked to the abnormal expression of the OZF protein. These imaging agents or reagents for in vitro diagnosis, characterized in that they comprise a labeled monoclonal antibody according to the invention as well as their uses in methods of in vitro diagnosis or of diagnosis by in vivo imaging for the diagnosis of pathology linked to the abnormal expression of the OZF protein are of course part of the present invention.

The invention also comprises a monoclonal antibody or a fragment thereof according to the invention, characterized in that it is coupled to a cytotoxic compound. Cytotoxic agents which can be conjugated to the monoclonal antibodies according to the invention notably include, but are not limited to, alkylating compounds such as mechlorethamine, triethylene phosphoramide, triaziquone, camustine, semustine, methotrexate, mercaptopurine , cytarabine, fluorouracil, antibiotics such as actinomycin, hormones or hormone antagonists such as corticosteroids, such as prednisone or progestins. The monoclonal antibody conjugates according to the invention can be prepared by conjugating cytotoxic substances containing either the intact toxin or their chain A derived with the monoclonal antibody or one of its fragments according to techniques of a person skilled in the art (Chaudry et al., 1993; Sung et al., 1993 and Selvaggi et al., 1993).

The monoclonal antibody according to the invention can also be a heteroconjugate monoclonal antibody such as a hybrid molecule composed of two or more antibodies. A heteroconjugate includes, for example, a single chain monoclonal antibody according to the invention and a single chain monoclonal antibody which is specific for a cellular receptor (Kerr et al., 1990 and Hsieh-Ma et al., 1992).

In another aspect, the subject of the invention is a pharmaceutical composition for the treatment, prevention or for the in vivo diagnosis of pathology linked to the abnormal expression of the OZF protein, characterized in that it comprises: a) a monoclonal antibody or a fragment thereof, according to the invention; and b) a pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition according to the invention is characterized in that said pathology is chosen from cancers, in particular cancer of the pancreas, of the colon or of the breast.

A subject of the invention is also the use of a monoclonal antibody or one of its fragments according to the invention for the manufacture of a medicament intended for the prevention or treatment of cancer, in particular pancreatic cancer, cancer of the colon or breast cancer.

In general, the dose of labeled monoclonal antibodies or one of their fragments for the in vivo diagnosis of pathology linked to the abnormal expression of the OZF protein, may vary depending on parameters such as age, conditions, sex , the extent of the patient's disease, contraindications if there are any, concomitant therapies or other variables that a person skilled in the art will be able to adjust.

The administration of these compounds to the patient can be local or systemic and accomplished by the intravenous, intra-arterial route, by means of the spinal fluid, etc. The administration can also be carried out by intradermal route or by intracavitary route, oral or nasal, depending on the part of the body to be examined. After a sufficient time allowing the monoclonal antibody to bind to the OZF protein, for example from 30 minutes to 48 hours, the part of the body which must be examined by conventional imaging techniques is examined, or scintillation imaging. The exact protocol will necessarily depend on specific factors related to the patient, the part of the body to be examined, the method of administration and the type of markers used. The specific procedures may be determined by those skilled in the art. The distribution of the fixed radioactive isotopes and their decrease over time will then be recorded and monitored. By comparing the results obtained with those obtained for clinically normal individuals, the presence and location of tumor cells can be determined and controlled.

The amount of monoclonal antibodies included in the pharmaceutical compositions according to the present invention and necessary for an effective therapy will depend on various factors such as the mode of administration, the targeted part of the body, the physiological state of the patient, the administration other drugs, possible side effects, etc. The dosage for such preventions or therapeutic treatments should be carried out in such a way as to optimize its safety and effectiveness. In general, assays used in vitro can provide an indication for the amounts used for administration of monoclonal antibodies in situ and thus animal models can be used to determine the effective amounts monoclonal antibodies according to the invention for the treatment of particular pathology.

These methods are in particular discussed in works such as Gilman et al.

(1990) and Remington's Pharmaceutical Sciences (1990) for administration methods such as oral, intravenous, intraperitoneal, intramuscular or transdermal methods. The pharmaceutically acceptable excipients include in particular water, saline solutions, buffers or any other compound described for example in the Merck Index.

The invention further comprises the use of a monoclonal antibody or one of its fragments for the treatment, prevention or for the in vitro or in vivo diagnosis of pathology linked to the abnormal expression of the OZF protein, in particular for the treatment, prevention or diagnosis of cancer such as cancer of the pancreas, colon or breast, said use being included in the invention.

The invention also relates to a method for detecting and / or assaying the OZF protein in a biological sample, characterized in that it comprises the following steps: a) bringing a biological sample into contact with a monoclonal antibody according to the invention. invention; and b) detecting and / or assaying the binding of said antibody to the OZF protein contained in the biological sample. Furthermore, the monoclonal antibodies or their fragments according to the invention can also be used for the identification, the localization, in particular tissue or cell, and / or the assay of the OZF protein, said use being included in the invention.

The monoclonal antibodies or their fragments according to the invention also constitute a means of immunocytochemical or immunohistochemical analysis of the expression of the OZF protein on sections of specific tissues, for example by immunofluorescence, by enzymatic, radioactive or gold labeling. . They make it possible in particular to demonstrate and quantify the normal or abnormal specific presence of the OZF protein in tissues or biological samples, which makes them useful for the identification and localization of the expression of the OZF protein, for the diagnosis of pathologies linked to the abnormal presence of the OZF protein but also for monitoring the development of methods of prevention or treatment of pathology requiring said detection or said assay.

More generally, the monoclonal antibodies or their fragments according to the invention can be advantageously used in any situation where the expression of the OZF protein must be observed qualitatively and / or quantitatively.

Preferably, the biological sample consists of a biological fluid, such as serum, whole blood, cells, a tissue sample or biopsies of human origin.

Any conventional procedure or test can be used to carry out such a detection and / or assay. Said test can be a competition or sandwich test, or any test known to those skilled in the art dependent on the formation of an antibody-antigen immune complex. Depending on the applications according to the invention, the monoclonal antibody or one of its fragments can be immobilized or labeled. This immobilization can be carried out on numerous supports known to those skilled in the art. These supports may in particular include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, or natural or modified celluloses. These supports can be either soluble or insoluble. For example, a preferred method involves immunoenzymatic processes according to the ELISA technique, by immunofluorescence, or radioimmunological (RIA) or equivalent.

The invention also includes a kit for determining the presence of OZF protein in a biological sample comprising an antibody or one of its fragments according to the invention.

Also within the scope of the invention is a kit for the detection and / or assay of the OZF protein according to the invention in a biological sample, characterized in that it comprises the following elements: a) a monoclonal antibody or one of its fragments according to the invention; b) where appropriate, the reagents for constituting the medium suitable for the immunological reaction; c) reagents for the detection of antigen-antibody complexes produced by the immunological reaction.

In a last aspect, the subject of the invention is a method for evaluating the affinity of a compound to be tested for the OZF protein, characterized in that it comprises: a) bringing a sample containing said protein into contact OZF with i) a monoclonal antibody or a fragment thereof according to the invention; and ii) said compound to be tested; and b) measuring the quantity of said monoclonal antibody or one of its fragments, said quantity being inversely proportional to the quantity of test compounds attached to said OZF protein.

The monoclonal antibodies according to the invention or their fragments can also be used in in vitro methods for the selection of compounds capable of binding to the OZF protein with the desired affinity. Thus, the present invention also comprises methods of selection by competition of pharmaceutical compounds in which the monoclonal antibodies of the invention or their fragments enter into competition with the test compound capable of binding to the OZF protein. In this way, Panticoφs monoclonal or its fragments is used to detect the presence of any compound such as a polypeptide or any other organic compound which may have one or more recognition sites of the epitope of the OZF protein recognized by the anticoφs according to the invention. Thus these compounds, identified by the method according to the invention can be used to occupy these binding sites on the OZF protein and act as an agent modulating or antagonist of the biological activity of the OZF protein.

Other characteristics and advantages of the invention appear in the following description with the examples and the figures, the legends of which are shown below.

LEGENDS OF FIGURES

FIGURES 1A and 1B. Southern blot analysis of pancreatic cell lines and primary tumors.

Figure 1A: Southern blot hybridization of total genomic DNA from placenta and twelve cell lines of pancreatic adenocarcinoma digested with BglII. Three cell lines, AICPC-1, BxPC-3, Su.86.86 show co-amplification of OZF with RAC. PANC-1 presents an amplification of RAC only.

Figure 1B: Southern blot hybridization of the total genomic DNA of AICPC-1 (Figure

1A), placenta (P) and twelve primary tumors of pancreatic adenocarcinoma digested with Bglll. Comparison of the intensities of the OZF hybridization signals with those obtained after rehybridization with an N-myc probe shows an increase in the number of copies of the gene in the T6 and T9 tumor samples.

FIGURE 2. Expression of OZF mRNA in pancreatic cell lines.

The RNA, of the twelve pancreatic adenocarcinoma cell lines analyzed by Southern blot in FIGS. 1 A and 1 B, was analyzed by Northern blot with a probe. of cDNA containing the open OZF reading phase. AICPC-1, BxPC-3, Su.86.86 having amplified OZF, exhibit a high level of expression. High levels of OZF expression are observed in the absence of amplification in Capan-1, -2, MIA PaCa-2 and PANC-1. The absence or very weak expression was observed in a sample of normal adult pancreas (Clontech). The corresponding gel, stained before transfer to ethidium bromide, is shown below, with the ribosomal RNA bands 28S and 18S.

FIGURES 3A, 3B and 3C. Western blot analysis of pancreatic cell lines and primary tumors. Identical amounts of protein (10 μg), determined by coloring the extracts after electrophoresis on SDS gel (not shown) were transferred to nitrocellulose. The nitrocellulose was then blocked and hybridized with a polyclonal anti-OZF antibody.

Figure 3A: Pancreatic cell lines previously analyzed by Southern and Northern blot (Figure 1A and Figure 2). Figure 3B Immunoblot of OZF from samples of primary pancreatic adenocarcinoma tumors.

Figure 3C: Immunoblot of human normal pancreas OZF. Proteins were extracted under the same conditions for the four independent samples of normal pancreas (P1-P4), the MIA line PaCa-1 (MP) and for a pancreatic carcinoma (T6) expressing OZF. Nitrocellulose has been hybridized with anti-OZF and anti-pag polyclonal anticoφs in order to verify the integrity of proteins. Anti-pag antibodies are hen anticoφs purified under the same conditions as anti-OZF. FIGURES 4A, 4B and 4C. Immunohistochemistry detection of OZF in human pancreatic carcinomas. Peroxidase staining was obtained with an affinity purified anti-OZF antibody. Anti-OZF antibodies show nuclear granular staining in AICPC-1 cells grafted in athymic mice (Figure 4A), in an adenocarcinoma expressing OZF (Figure 4B), and the absence of staining in healthy pancreas not expressing OZF (Figure 4C) (magnification: 1000X). FIGURE 5. Western blot analysis of crude extracts of human 293 cells not transfected and transfected with an OZF expression vector. Detection of the endogenous OZF protein (left) and after transient detection by an expression vector of the OZF gene (right) in triplicate, with an anti-OZF monoclonal antibody and a secondary anti-mouse antibody coupled to peroxidase, by chemo-luminescence. FIGURE 6. Immunocytochemical labeling of the OZF protein in cells of the AICPC-1 line derived from a pancreatic adenocarcinoma overexpressing the OZF protein. The cells are cultured on a slide, fixed with 4% of paraformaldehyde in PBS, then permeabilized with 0.1% of triton X100. Non-specific sites are saturated with 2% BSA. The cells are incubated for 1 hour with the anti-OZF monoclonal antibody (1/200), washed and then brought into contact for 30 minutes with the second anti-mouse antibody coupled to a fluorochrome, washed and incubated for 4 minutes with DAPI (0.2 μg / ml) which specifically marks the nuclei (photos on the left). A specific OZF nuclear marking is observed (photos on the right). At low magnification at the top and at high magnification at the bottom.

FIGURE 7. Expression of the OZF gene in colon cancer. Western blot analysis.

Using monoclonal antibodies, we examined the expression of the protein

OZF in colon cancer. The study involved 16 colonic samples. Of the 16 samples, 10 are from a colonic tumor (T1 - T3 - T5 - T6 - T7 - T8 - T9 - T10 - T11 - T12), and 6 from healthy colonic mucosa (C2 - C4 - C5 - C6 - C8 - C12). The T5 and C5 samples come from the same patient, as well as T6 - C6, T8 - C8, and T12 - C12. We deposited in the electrophoresis wells equivalent amounts of protein for each colonic sample, the samples are separated by migration on polyacrylamide gel, then transferred to a PVDF membrane. The OZF protein is revealed by the anti-OZF monoclonal antibody and a mouse anti-antibody coupled to peroxidase.

In the four cases where we had a healthy colonic sample and a cancerous colonic sample taken from the same person, we found a greater accumulation of the OZF protein in the tumor tissue than in the normal tissue.

By comparing the amount of OZF protein accumulated, we find that the tumor tissues T1 - T3 - T5 - T7 - T8 - T9 - T10 - T12 have a higher amount of OZF protein compared to healthy tissues. FIGURE 8. Comparison of the N-terminal amino acid sequences of the OZF proteins according to their origin and their reactivity with the monoclonal antibodies. Comparison of the N-terminal region of two alleles of the human OZF protein (hu- OZF) which differ by a polymorphism at position 8 (K / R) with the murine OZF protein (mu-OZF) and bovine ( bo-OZF). The sequence of the synthetic peptide used in the competitive inhibition experiments is indicated above (PEPTIDE). The tyrosine residue in position 10 is underlined. The sequence of the MBP carrier protein is indicated in small capital letters for the MBP-OZF protein. The H / C consensus link ("H / C link") preceding the first zinc finger corresponds to position 11-17. * indicates perfectly preserved positions;

⁰ indicates K / R polymorphism.

The reactivity with the monoclonal antibody 5B8 according to the invention is indicated in the right column (+: positive; -: negative).

EXAMPLES

Materials and methods

Tumor samples and cell lines. Eleven pancreatic adenocarcinoma cell lines were obtained from the American Type Culture Collection (Rockville, USA) and cultured as prescribed by the supplier. An additional cell line (AICPC-1) has been established in the laboratory from the primary tumor of a sixty-year-old patient with adenocarcinoma of the pancreas. The cells were cultured in modified Dubelcco medium supplemented with 20% (vol / vol) of fetal calf serum. Tumor tissue was also obtained from patients with primary carcinoma of the exocrine pancreas (n = 13; 6 men and 7 women).

Analysis by Southern and by Northern blot. DNA extraction was carried out according to standard methods for cell lines and for frozen tumors (Sambrook et al., 1989). The fragments obtained after digestion of the genomic DNA (5 μg) with the Bgl1 endonuclease were separated by electrophoresis on 0.7% agarose gel, transferred to nitrocellulose (BA85, Schleicher and Schuell) and hybridized with probes marked with P32 (Le Chalony et al., 1996). The tumor cell lines were hybridized with an OZF probe obtained after amplification by PCR using internal primers to generate a 1 kb fragment containing the entire open reading phase (847-1840). Primary pancreatic carcinomas were hybridized with a cloned fragment Xbal / Scal (652-1852) (Le Chalony et al., 1994). The RAC probe (2.2 kb) was prepared from a pUC-RAC clone using universal primers located on either side of the multiple cloning site (Cheng et al., 1992). The quantitative evaluation of the hybridization signals was carried out with the phosphorimager (BioRad). Total RNAs were extracted using the Rneasy kit (Qiagen). For the Northern blot analyzes, 10 μg of RNA were deposited on 1% / 2.2 M agarose gel formaldehyde, transferred after migration onto a nitrocellulose membrane (BA85, Schleicher and Schuell) and hybrids with the Xbal / Scal probe.

Production of polyclonal antibodies. Antisera were developed in chickens using the purified OZF protein as an immunogen (Ferbus et al., 1996). Chicken egg yolk IgY immunoglobulins were prepared as described (Akita et al., 1992). The specific antibodies were obtained from anti-OZF IgY after affinity purification on a column of MBP-OZF coupled to Sepharose 4B (Pharmacia) (Ferbus et al., 1996). The specificity of anti-OZF antibodies was checked by immunoelectrophoresis. A single band of 33 kDa was observed in the nuclear extracts of the cells expressing OZF.

Method for preparing the monoclonal antibody. The recombinant fusion protein MBP-OZF produced in E. coli was purified on an amylose column by affinity for MBP (Ferbus et al., 1996). Mice were immunized with the purified MBP-OZF protein. The immunization of Balb / c mice is carried out by three subcutaneous injections two weeks apart of 50 μg of purified MBP-OZF protein. The anti-OZF immune response is monitored by ELISA and immunoblotting using the protein MBP-OZF. Three days before the fusion, 100 μg of MBP-OZF protein are injected intravenously into the mice having the highest antiserum titers. After the screening test in ELISA, we selected for the fusion step the mouse which presented the best response. The splenic leukocytes were fused with the cells of the myeloma line P3X63 / 8.653 in the presence of 50% polyethylene glycol. After "HAT" selection in the crude culture, the hybridomas are individually encapsulated in droplets of biotinylated agarose and labeled with the protein MBP-OZF conjugated to fluorescin isothiocyanate (+) and with the protein MBP-PAG conjugated with phycoerythrine (-). The droplet suspension is then analyzed by flow cytometry and only the hybridomas reacting with the MBP-OZF conjugate are isolated and cloned according to the "secretion capture report web" (SCRW) technique (Kenney et al., 1995).

The monoclonal antibodies are purified from the culture supernatant of the hybridomas by affinity chromatography on a protein G Sepharose column (Pharmacia).

The immunoglobulins produced are mainly IgG. The monoclonal antibody specific for the human OZF protein recognizes the latter in the native or denatured state (in ELISA, in Western, in immunohistochemistry, by immunoprecipitation). ELISA technique for the selection and characterization of monoclonal antibodies.

- Incubation of 96-well microplates for ELISA in the presence of 50 μl per well of a solution of MBP-OZF or MBP-PAG (control) at 10 μg / ml in 0.05 M carbonate-bircarbonate buffer, pH 9.6, overnight at 4 ° C.

- Saturation with a 2% serum bovine albumin solution in phosphate-buffered saline pH 7.3 (PBS).

- Addition of 25 μl of crude supernatant of hybridoma culture with 25 μl of a 0.1% Tween solution in PBS buffer and incubation for 2 hours at 37 ° C. - After washing, addition of 50 μl of a 1/10 000 solution of anti-mouse goat anticoφs conjugated with alkaline phosphatase (Sigma, St Louis, MO) and incubation for 1 hour at 37 ° C.

- Detection with a solution of p-nitrophenyl phosphate at 1 mg / ml in diethanolamine buffer, pH 9.8, MgCl _{2 2} 0.5 mM. - Measurement of absorbance at 405 nm with a microplate reader (Titertek Multiscan from Labsystem, Les Ulis, France).

The isotypes of the monoclonal antibodies are determined by ELISA using a kit for the subtyping of murine hybridoma (Boehringer-Mannheim, Germany).

The recognition epitope is located at its NH ₂ terminal end. It recognizes the truncated recombinant OZF protein, having only the first two zinc fingers, and does not recognize the homologous murine protein which differs from the human protein at the level of its first fifteen amino acids. Protein extraction and analysis by Western blot with polyclonal antibodies. The frozen tumor samples and the cell extracts were dissolved in SDS buffer in the presence of β-mercaptoethanol and heated to boiling for 5 minutes. Equal amounts of protein (10 μg) were loaded onto 12% polyacrylamide SDS gel and transferred after migration onto a PDVF membrane (Amersham). The membranes were hybridized with anti-OZF antibodies and then with rabbit anti-hens conjugated to alkaline phosphatase (Sigma), diluted respectively 200 and 15,000 times. Chemiluminescence was produced by incubation of the membrane with CDP star (Dupont) and the protein was revealed by autoradiography on an X-OMAT AR5 film.

Protein extraction and Western blot analysis with monoclonal antibodies. The cells are cultured at confluence, trypsinized, concentrated by centrifugation and lysed with buffer (65 mM Tris-HCl, pH 6.8, 0.01% blue bromophenol, 5% β-mercaptoethanol, 10% glycerol, 2% SDS) in the presence of protease inhibitors, heated to boiling and then sonicated. 50 μg of cell extract or 0.05 μg of recombinant protein are loaded into each well. The proteins are separated by electrophoresis on a 12% SDS polyacrylamide gel and transferred to a PDVF membrane (Immobilon-P, Millipore). The efficiency of the protein transfer is controlled by Ponceau S staining. Analysis by Western blot is carried out according to the technique described in Ferbus et al., 1996. The blots are hybridized for 2 hours with anti-OZF antibodies diluted to 1 / 500 then with goat anti-mouse IgG conjugated to alkaline phosphatase diluted to 1/10 000. The same conditions are used for the positive control with anti-chicken anti-OZF antibodies diluted to 1/200 and anti-chicken IgY antibodies of rabbit at 1: 10,000. Alkaline phosphatase is detected by chemiluminescence with the reagent CDP-star (NEN Live Science Products) and visualized by autoradiography (X-OMAT AR, Kodak, Rochester, NY). Immunofluorescence. The cells are cultured in Lab-Teck chamber slides (Nunc Inc., Naperville, IL) for 24 hours. The Lab-Teck slides are washed twice in PBS and fixed with a 4% solution of paraformaldehyde in PBS (w / v) for 15 minutes at room temperature. The cells are then rinsed in PBS and incubated twice for 30 minutes in a 0.1 M glycine solution, 0.2 M Tris-HCl pH 7.5 and then permeabilized in a 0.1% triton X100 solution (v / v) in PBS for 5 minutes. The purified anti-OZF antibodies are incubated for one hour at 37 ° C (1/100 dilution) in PBS buffer at 0.5% serum bovine albumin (w / v). After washing, the cells are incubated for 30 minutes at 37 ° C. with anti-goat anti-goat IgG antibodies (H + L) conjugated to FITC (fluorescein isothiocyanate) diluted 1/80 (Biosys SA, France). Slides are stained by contrast with DAPI (4'6-diamidino-2-phenylindole) for staining the chromatin DNA, fixed by a solution (Vectashield, Vector Laboratoires, Inc., Burlingame, CA) and observed under a fluorescence microscope. Immunohistochemical labeling of OZF. Staining was obtained by an anti-hen coupled to peroxidase after incubation with a purified hen anti-OZF antiserum, on sections of frozen tissue (5 μm) as described (Terris et al., 1995). Transfection of NIH3T3 cells. OZF overexpression is achieved by transfection of a NIH3T3 murine cell line with a plasmid vector expressing the OZF protein in eukaryotic cells. The culture medium is seeded with a cell density of 0.8 x 10 ⁶ cells in petri dishes 60 mm in diameter, then the cells are transfected 24 h later with 2 μg of plasmid using the calcium phosphate technique. 5 to 6 h after transfection the precipitates are eliminated and the cells are returned to the culture medium. The following day, the cells are transferred to Lab-Tek chamber slides and fixed before analysis by immunofluorescence.

EXAMPLE 1 Amplification of the OZF Gene in the Pancreatic Adenocarcinoma Lines

To evaluate the number of copies of the OZF gene in the cells of the tumor lines and to determine whether it belongs to the amplicon carrying the RAC gene, the total genomic DNAs, of 12 cell lines and of a placenta, were digested with l restriction enzyme Bglll, separated on agarose gel and hybridized by Southern blot with DNA probes OZF and RAC. As shown in FIG. 1A, three lines, AICPC-1, BxPC-3, SU.86.86 show an amplification signal in a BglII genomic fragment of 5.3 kb in size. Quantification of the amplification levels reveals that the OZF gene is approximately 60 copies, 6 copies and 30 copies in AICPC-1, BxPC-3, SU.86.86 respectively. Co-amplification with RAC is observed in these three lines, however PANC-1 shows an amplification of RAC alone. The copy number of the RAC and OZF genes is similar in BxPC-3. The relative intensity of the amplification signal is higher for the OZF gene than for RAC in AICPC-1 and lower in SU.86.86. These experiments show that the OZF gene is co-amplified with RAC in three of the four cell lines. Contrary to what had been reported, the amplification of RAC in the AsPC-1 line is not detected (Cheng et al., 1996; Miwa et al., 1996). Karyotypic analyzes of the cell lines confirmed the ploidy and the specificity of the chromosomal labeling of each line including AsPC-1 (Curtis et al., To be published). The amplification previously described in AsPC-1 may be due to a subclone or may have been lost during propagation in culture.

The state of amplification of OZF was examined in tumor samples. Twelve primary pancreatic adenocarcinomas, taken at random, were analyzed by Southern blot with an OZF probe (Figure 1B). Two tumors (T6 and T9) show an increase in the hybridization signal (from 3 to 5 times) compared to placental DNA, after normalization with the signal obtained after rehybridization with a single copy of the N-myc gene. This signal is considered significant for amplification given the high proportion of non-tumor cells in tumor tissue.

EXAMPLE 2 Expression of OZF in Pancreatic Lines and in Tumor Samples The expression of OZF was examined first in tumor lines by

Northern blot (Figure 2). Different levels of expression were detected in all of the lines. The AICPC-1 and SU.86.86 lines which have the highest levels of amplification express the highest levels of mRNA OZF. Moderate expression is observed in BxPC-3, Capan-1, Capan-2, MIAPaCa-2 and PANC-1, and lower expression is found in the remaining four lines.

The expression of OZF was also sought at the protein level by Western blot using a polyclonal antibody directed against the recombinant OZF protein expressed in E. coli (Ferbus et al., 1996). The OZF protein was detected in all cell lines (Figure 3A). The highest level of protein is found in cell lines which exhibit amplification of the OZF gene (AICPC-1, BxPC-3 and SU.86.86). As the pancreatic tissue has RNAs which are often degraded, the expression of OZF has been studied in tumors and the normal pancreas by determining the accumulation of its protein. In order to compare the level of the OZF protein in the normal adult pancreas with those found in tumors, proteins from four different pancreatic samples were extracted according to the standard protocol and analyzed by Western blot (Figure 3C). The integrity of the extracts was checked by the use of a polyclonal antibody directed against the ubiquitous protein PAG of 22 kDa which is expressed in the proliferating cells (Prospéri et al., 1993). Among the eight samples of primary pancreatic adenomas studied, three show a low level of the protein OZF as in the samples of healthy pancreas. Five tumors (T6-T9, T12) have the highest levels of OZF (Figures 3B and 3C). Consequently, the OZF gene is expressed at high levels in more than half of the adenocarcinomas analyzed. EXAMPLE 3 Restriction of OZF expression to tumor cells in pancreatic tumors

The possible use of anti-OZF antibodies in immunohistochemical studies has been determined. The expression of OZF was detected (FIG. 4A) in a tumor obtained after grafting of AICPC-1 cells in athymic mice. Immunohistochemistry was performed on sections of frozen pancreatic carcinomas to determine if other cell types in addition to tumor cells, contribute to the expression of OZF. An example of pancreatic carcinoma showing expression of OZF is shown in Figure 4B. Anti-OZF antibodies reveal by staining a nuclear granulation which predominates in tumor cells. Other cell types such as fibroblasts and endothelial cells show very little or no staining. Thus in pancreatic tumors the expression of OZF is mainly restricted to tumor cells and anti-OZF antibodies can be used to detect tumor cells in heterogeneous pancreatic tumors. EXAMPLE 4 Expression of the OZF Gene in Colon Cancers Using monoclonal antibodies, the expression of the OZF protein was examined in colon cancers. The study involved 16 colonic samples. Of the 16 samples, 10 are from a colonic tumor (T1 - T3 - T5 - T6 - T7 - T8 - T9 - T10 - T11 - T12), and 6 from healthy colonic mucosa (C2 - C4 - C5 - C6 - C8 - C12). The T5 and C5 samples come from the same patient, as well as T6 - C6, T8 - C8, and T12 - C12.

Western blot analysis:

Equivalent amounts of protein for each colonic sample are deposited in the electrophoresis wells. The samples are separated by migration on polyacrylamide gel, then transferred to a PVDF membrane. The OZF protein is revealed by the anti-OZF monoclonal antibody and a mouse anti-antibody coupled to peroxidase. The result is shown in Figure 7.

In the four cases where the healthy colonic sample and the cancerous colonic sample are taken from the same person, a greater accumulation of the OZF protein is observed in the tumor tissue than in the normal tissue. By comparing the amount of OZF protein accumulated, it is found that the tumor tissues T1 - T3 - T5 - T7 - T8 - T9 - T10 - T12 have a greater amount of OZF protein compared to healthy tissues.

According to the results, 50% of the cancer tissues analyzed exhibit a strong overexpression of OZF and 80% of the tumor samples analyzed have a level of expression of the OZF protein higher than that observed in normal tissues. These observations show that the OZF gene is overexpressed in colon cancer.

The results obtained in the previous examples show that the amplification of the OZF gene was observed in 3 of the 12 adenocarcinoma lines pancreas (AICPC-1, BxPC-3 and SU.86.86). The increase in the number of copies of the gene was also found in 2 of the 12 samples of primary tumors (T6 and T9), although at lower levels than those observed in the lines. Because primary pancreatic tumors generally contain a large excess of normal cells, Southern blot signal hybridization may have been attenuated in T6 and T9 tumors. In addition, samples containing few tumor cells, exhibiting a moderate amplification signal, escape detection. Xenografts on athymic mice should allow us to better estimate the amplification of the OZF gene in primary pancreatic tumors. In addition to these results, the amplification of OZF was identified by in situ fluorescence hybridization in the tumor used to generate the AICPC-1 line (Curtis et al., To be published). Thus, amplification of OZF takes place in primary pancreatic tumors and in cell lines.

All pancreatic adenocarcinoma lines express mRNA and OZF protein but at different levels. The highest levels of OZF mRNA were found in the lines that amplified the OZF gene (AICPC-1, BxPC-3 and SU.86.86). Although the estimation of the protein may be less precise, the level of the OZF protein was compared in the two primary tumors exhibiting an amplification of the copy number (T6 and T9), with the cell line MIAPaCa-2. Both tumors express high levels of OZF protein. Thus, the OZF gene, which is highly expressed in all pancreatic tumors with amplification, is not inactivated in the amplicon 19q13.1.

Gene amplification is an important mechanism for increasing the expression of genes involved in carcinogenesis, although overexpression is often observed in the absence of amplification. Expression studies of four independent samples of normal pancreas and three samples of primary tumors show very low or undetectable levels of OZF protein. On the contrary, all the lines express OZF and four of them show high levels of OZF mRNA in the absence of amplification. The most significant case is that of the PANC-1 line which has two copies of the OZF gene and expresses a high level of mRNA 2 to 4 times lower than that of the line SU.86.86 which has 30 copies of the OZF gene . High levels of the OZF protein are also seen in primary tumors without amplification of the OZF gene, and immunohistochemistry shows that the OZF protein is detected primarily in tumor cells. Apart from gene amplification status, OZF is overexpressed in 7 of the 12 tumor lines and in 5 of 8 primary tumors. The higher levels of OZF expression in a large sample of tumors compared to the normal pancreas suggest that this gene is involved in the oncogenic process. In addition, it could be used as a marker to detect rare tumor cells in the pancreas. Besides the OZF gene, the q13.1 region of chromosome 19 includes several candidate genes. The best characterized is the RAC gene, located near OZF, which codes for a serine-threonine kinase (The Human Gene Map, 1997). The two genes are co-amplified in cell lines, however only the RAC gene has been found amplified in PANC-1. However, in the PANC-1 line, in which the RAC gene is amplified, the mRNA levels are only slightly lower than those observed in cell lines which exhibit amplification of the OZF gene. This cell line also contains an abundant OZF protein. Thus, overexpression of OZF in PANC-1 may be due to a mechanism other than gene amplification as in the case of other known genes. For example in breast and ovarian cancers, the prevalence of HER2 / neu overexpression occurs more frequently than amplification and has been used to predict survival rate (Slamon et al., 1989).

EXAMPLE 5 Selection of Hybridomas and Characterization of Monoclonal Antibodies

A. Selection of hybridomas

After immunization then cell fusion, the anticoφs secreted by a single hybridoma are examined using the SCRW technique and selected one by one by flow cytometry. 22 supernatants reacting by FACS (automatic cell sorter by fluorescence flow cytometry) with the recombinant OZF protein and not reacting with the MBP carrier protein are tested for their activity with the recombinant MBP-OZF protein. Half of the cell lines are positive by ELISA and immunoblotting (cf. table 1). No reactivity is observed after ELISA on the protein MBP-PAG indicating that these supernatants are specific for the OZF polypeptide (Prospéri MT et al., 1993). Since certain monoclonal antibodies can react with epitopes resulting from the fusion with the MBP carrier protein, the reactivity of the supernatants has been examined with the OZF protein produced by eukaryotic cells. After immunoblot and immunofluorescence analysis in the presence of endogenous OZF proteins from human HBL breast cells 100, six monoclonal antibodies were selected on the basis of their high reactivity with the human OZF protein (cf. table 1 below).

TABLE 1: Screening and selection of anti-OZF monoclonal antibodies.

Legend of Table 1: The crude culture supernatants of 22 hybridoma clones selected by the SCRW technique were tested successively by ELISA against the recombinant proteins MBP-OZF and MBP-PAG, then then by immunoblot and immunofluorescence (IFA) MBP-OZF protein and endogenous OZF protein from human HBL 100 cells. The recombinant protein MBP-PAG was used as a negative control.

B. Characterization of the monoclonal antibodies After purification on a protein G Sepharose column, the immunoglobulin subtypes of the six selected monoclonal antibodies were determined. Five antibodies (5B8, 5C10, 5G10, 5H7, 4H10) belong to the lgG1 subtype and one antibody to the lgG3 (5D9) subtype. The monoclonal antibodies were also tested by immunoblotting and immunofluorescence with various extracts from mammalian cells. Western blot analysis on human extracts (breast, kidney, colon and pancreas cells) detect a unique band of 33 kDA characteristic of the OZF protein. Unexpectedly, the monoclonal antibodies do not recognize the bovine and murine OZF proteins despite their strong identity (95%) with the human OZF protein (Le Chalony, C. et al., 1996; Blottière L. et al., 1999). The lack of reactivity does not result from the translation process since the murine OZF proteins translated in vitro are also not detected. The same results are observed by immunofluorescence (cf. table 2 below), a strong nuclear expression of OZF is observed after transfection of murine NIH3T3 cells with the expression vector. No fluorescence is visible on murine NIH3T3 cells not transfected. Consequently, the six monoclonal antibodies thus produced and characterized according to the invention are highly specific for the human protein OZF.

EXAMPLE 6 Determination of the Epitope Since the monoclonal antibodies react only with the human OZF protein, the human, bovine and murine OZF sequences were compared in order to determine in a first step the localization of the epitope.

Most of the variable domains are located in the first three zinc fingers and in the 10 amino acid leader peptide (PL) sequence preceding the zinc finger domain (Le Chalony et al., 1996; Blottière et al., 1999) .

This first step suggests that the location of the epitope is plausibly located in the N-terminal part of the OZF protein. In view of the fact that the protein MBP-OZF for which the first five amino acids of the protein OZF are missing, reacts with the monoclonal antibodies according to the invention, it is very likely that the epitope against which these monoclonal antibodies are directed is located at the junction between the leader peptide and the first zinc finger (cf. FIG. 8).

In order to confirm this hypothesis, an ELISA technique by competitive inhibition is carried out in the presence of a peptide whose sequence corresponds to the aa4-aa17 fragment of the human OZF protein. This peptide appears to be a powerful competitive inhibitor vis-à-vis the monoclonal antibodies according to the invention (see Table 2 below).

TABLE 2: Characterization of the selected monoclonal antibodies.

Detection method Antigen Antibody

Y-OZF 5B8 5C10 5D9 5G10 5H7 4H10

Monoclonal anticoLISs ELISA IgY ig igi lg3 igi igi igi

MBP-OZF + + + + + + +

MBP-OZF

+ competition by peptide aa4 - aa17

Human OZF immunoblot + + + + + + + +

Bovine OZF + - - - - - - ^ ι

Murine OZF + - - - - - -

Murine OZF translated in vitro + - - nf nf - nf

Human OZF immunofluorescence + + + + + + + +

Murine NIH3T3 cells + - - - - - -

Murine NIH3T3 cells transfected with human OZF + + + + + + +

Legend of Table 2: Six monoclonal anti-OZF antibodies were tested by ELISA, immunoblot and immunofluorescence. The monoclonal antibodies and polyclonal anti-OZF chicken antibodies were diluted 1/500 and 1/100 respectively. The competition is carried out in the presence of 0.05 mg / ml of the synthetic peptide aa4-aa17 (cf. FIG. 8).

For the immunoblot, the following extracts were used:

- human origin: breast HBL100 cells, kidney 293 cells, pancreatic SU86-86 cells, colon RC8 and TC7 cells;

- murine origin: fibroblasts NIH3T3; - bovine origin: endothelium FBHE cells and LB9THY lymphocytes.

Immunofluorescence was carried out on HBL100 and NIH3T3 cell lines. The anti-OZF chicken polyclonal antibody was used as a positive control (Y-OZF). (+): detection of OZF; (-): no OZF detection; nf: not done.

These results demonstrate that the six monoclonal antibodies according to the invention tested recognize a common epitope of the human OZF protein located at the junction between the leader peptide and the first zinc finger. In this region, the comparison of the human and bovine sequences of the OZF protein highlights two substitutions: - a first conservative substitution in position 8 resulting from a lysine / arginine polymorphism in humans which does not modify the reactivity of the monoclonal antibodies ; and

- the substitution of a tyrosine residue by a leucine residue in position 10 in the bovine sequence. In the sequence of the murine OZF protein, a cysteine residue is found in position 10 and two substitutions in positions 13 and 14.

Consequently, these results show that the presence of a tyrosine residue at position 10 is critical for the recognition of monoclonal antibodies and probably influences the conformation of this epitopic region. Comparison of the sequence of this epitope with sequences of proteins from databases has not made it possible to identify other proteins carrying this epitope.

Thus, six monoclonal anticoφs effective for the detection of the recombinant and native OZF protein by ELISA, western blot or immunofluorescence were thus purified and characterized. Five antibodies are of type lgG1 and one of type lgG3. At on the contrary, polyclonal anticoφs which detect numerous zinc finger proteins other than the human OZF protein, these monoclonal anticoφs according to the invention recognize a unique epitope of the human OZF protein. The sequence of this unique epitope located at the junction between the first ten amino acids and the zinc finger domain is not present in other Kruppel proteins, including OZF proteins of murine and bovine origin. These monoclonal antibodies according to the invention are highly specific for the endogenous human OZF protein, both in its denatured and native form.

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Claims

1 / Anticoφs monoclonal or one of its fragments capable of binding specifically to an epitope of the OZF protein. 2 / Anticoφs monoclonal or one of its fragments according to claim 1 characterized in that the OZF protein is of human origin.

3 / Anticoφs monoclonal or one of its fragments according to claim 1 or 2, characterized in that the epitope of the OZF protein is located on the N-terminal part. 4 / monoclonal antibody or a fragment thereof according to claim 3, characterized in that the epitope of the OZF protein is located on the N-terminal part comprising the tyrosine residue located in position 10 of the sequence of the human OZF protein.

5 / Anticoφs monoclonal or one of its fragments according to one of claims 1 to 4, characterized in that it is produced by a cell as deposited at the CNCM on September 6, 1999 under the number I-2308.

6 / Anticoφs monoclonal or one of its fragments according to one of claims 1 to 4, characterized in that it is chosen from humanized, chimeric, anti-idiotypic antibodies. 7 / Anticoφs monoclonal or one of its fragments according to one of claims 1 to 6, characterized in that it is marked.

8 / Anticoφs monoclonal or one of its fragments according to one of claims 1 to 6, characterized in that it is coupled to a cytotoxic compound.

9 / Cell capable of producing a monoclonal anticoφs according to one of claims 1 to 5 as filed with the CNCM on September 6, 1999 under the number I-2308.

10 / Pharmaceutical composition for the treatment or prevention of pathology linked to the abnormal expression of the OZF protein, characterized in that it comprises: a) a monoclonal antibody or one of its fragments according to one of claims 1 to 6 and 8; and b) a pharmaceutically acceptable excipient.

11 / Pharmaceutical composition for the in vivo diagnosis of pathology linked to the abnormal expression of the OZF protein, characterized in that it comprises: a) a monoclonal anticoφs or one of its fragments according to one of claims 1 to 7; and b) a pharmaceutically acceptable excipient.

12 / Pharmaceutical composition according to claim 10 or 11, characterized in that said pathology is chosen from cancers, in particular cancer of the pancreas, colon or breast.

13 / Use of a monoclonal anticoφs or one of its fragments according to one of claims 1 to 6 and 8, for the manufacture of a medicament intended for the treatment or prevention of cancer, in particular pancreatic cancer, cancer colon or breast cancer.

14 / Use of a monoclonal anticoφs or one of its fragments according to one of claims 1 to 7, for the in vitro diagnosis of pathology linked to the abnormal expression of the protein OZF.

15 / Method for detecting and / or assaying OZF protein in a biological sample characterized in that it comprises the following steps: a) bringing a biological sample into contact with an anticoφs according to one of claims 1 to 7; and b) detecting and / or assaying the binding of said anticoφs to the OZF protein contained in the biological sample. 16 / Kit for determining the presence of OZF protein in a biological sample comprising an antibody or one of its fragments according to one of claims 1 to 7.

17 / Method for evaluating the affinity of a compound for the OZF protein, characterized in that it comprises: a) bringing a sample containing said OZF protein into contact with i) a monoclonal antibody or one of its fragments according to one of claims 1 to 7; and ii) said compound to be tested; and b) measuring the quantity of said monoclonal antibody or one of its fragments, said quantity being inversely proportional to the quantity of compounds to be tested attached to said OZF protein.