MXPA96006108A - Monoclonal antibody active against cd44v6 - Google Patents

Abstract

The invention concerns an antibody active against an epitope coded by the exon v6 variant of the CD44 gene. The antibody concerned has characteristics superior to those of prior art antibodies and is suitable for use in therapy and diagnosis.

Description

MONOCLONAL ANTIBODIES AGAINST CD 44v6 DESCRIPTION PE THE INVENTION The invention concerns a monoclonal antibody against an epitope that is encoded by the v6 exon variant of the CD44 gene, as well as antibody molecules derived therefrom. Recently, it was demonstrated that the expression of variants of the surface glycoprotein CD44 is necessary and sufficient to trigger the so-called metastatic spontaneous behavior, both in a pancreatic adenocarcinoma cell line not targeting rats as well as in a fibrosarcora cell line no rat attentive goals (Günthert et al., 1991). While the smaller CD44 isoform, the standard form CD44s (or CD44std), is uitously expressed in a number of different tissues, including epithelial cells, certain splicing variants of CD44 (CD44v, CD44var) are only expressed in a subset of the epithelial cells. The CD44 variants are created by alternative splicing, so that the sequences of 10 exons (vl-vlO) in CD44s are completely separated by cutting, but can occur in the major variants in different combinations (Screaton et al., 1992; Tolg et al., 1993; Hof ann et al., 1991). The variants differ in that in a certain place of the extracellular portion of the protein REF: 23602 Different amino acid sequences are inserted. Variants of this type could be detected in different human tumor cells and in human tumor tissue. Thus, recently, the expression of CD44 variants was investigated in the course of colorectal carcinogenesis (Heider et al., 1993a). The expression of CD44 variants is lacking in the epithelium of the normal human colon, and only weak expression in the proliferating cells of the crypts can be detected. In later stages of tumor progression, for example in adenocarcinomas, all malignant degenerations express variants of CD44. The expression of tissue of variant CD44 at a high level could also be demonstrated in aggressive non-Hodgkin lymphomas (Koopman et al., 1993). A particular role, particularly in metastatic expansion, seems to be played by exon v6 (Rudy et al., 1993). In the animal model, antibodies against v6 specific epitopes could prevent the accumulation of metastatic cells and the development of metastases (Selter et al., 1993). In carcinomas of the colon, the expression of v6 correlates with tumor progression (Wielenga et al., 1993). In carcinomas of the stomach, the expression of v6 is an important diagnostic marker in the differentiation between intestinal tumors and those of diffuse type (Heider et al., 1993b). The expression of v6 was measured in the two works mentioned last with the help of antibodies against specific epitopes of ^ vß. Monoclonal antibodies against epitopes, which are encoded by exon v6, are known in the state of the art (Hofmann et al., 1991; Wielenga et al., 1993). Due to the high potential utility that this type of anticuexpos can have in diagnosis and therapy, there is a high demand for antibodies with improved properties. It was the object of the present invention to provide an antibody that exhibits significantly better properties with respect to the known v6-specific antibodies. This problem could be solved with the present invention. This concerns an antibody with the designation VFF-18. The invention also concerns a line of hybridoma cells secreted by this antibody and deposited, under the deposit number DSM ACC2174, in the DSM Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg Ib, D-38124 Braunsch eig , Germany. In the following, by the terms "antibodies" or "antibody molecules" are meant not only complete immunoglobulins, but also, in relation to specificity and binding affinity, equivalent substances such as those derived from antibodies and recombinant antibody molecules described. The antibody according to the invention was prepared with a method according to Example 1. The antibody can also be obtained from the deposited hybridoma cell line. It is in the knowledge of the person skilled in the art to prepare derivatives of the antibody according to the invention or, starting from an analysis of the sequence of this antibody and / or with the use of the hybridoma cell line that produces this antibody, to create recombinant antibodies with the same idiotype, that is to say antibody molecules that in the area of the binding site (complementarity determining regions, RDC) of the antigen have the same amino acid sequences as the antibody VFF-18. Therefore, derivatives and recombinant antibody molecules of this type are expressly included in the invention. From the complete immunoglobulin of the VFF-18 antibody, for example, Fab or F (ab ') 2 fragments or other fragments can be produced (Kreitman et al., 1993). For diagnostic procedures, VFF-18 antibody molecules, fragments thereof or recombinant antibody molecules with the same idiotype can be recombined, for example, with radioactive isotopes, such as 13lI, U1ln, "" Te, or radioactive compounds (Larson et al. al., 1991; Thomas et al., 1989; Srivastava, 1988), enzymes, such as peroxidase or alkaline phosphatase (Catty and Raykundalia, 1989), with fluorescent dyes (Johnson, 1989) or biotin molecules (Guesdon et al. , 1979). For therapeutic applications, VFF-18 or antibody molecules derived from VFF-18 can be linked to toxins (Vitetta et al., 1991; Vitetta and Thorpe, 1991; Kreitman et al., 1993; Theuer et al., 1993), cytostatics ( Schrappe et al., 1992), prodrugs (Wang et al., 1992; Senter et al., 1989) or radioactive substances. In addition, the antibody can be linked to a cytokine or other immunomodulatory polypeptide, for example with the tumor necrosis factor or interleukin-2. The person skilled in the art is, furthermore, in a position, after analysis of the amino acid sequence of the VFF-18 antibody and / or by using the hybridoma cell line which produces this antibody, in particular the genetic information contained therein, of prepare recombinant antibody molecules with the same idiotype as VFF-18. Corresponding procedures are known state of the art. Molecules of recombinant antibodies of this type can be, for example, humanized antibodies (Shin et al., 1989; GUssow and Seemann, 1991), biospecific antibodies (Weiner et al., 1993; Goodwin, 1989), single chain antibodies (scFv, Johnson and Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992; Barbas et al., 1992), antibodies created by chain drag (Winter et al., 1994). Humanized antibodies can be prepared for example by RDC grafting (EP 0239400). Frame regions can also be modified (EP 0519596). For the humanization of antibodies methods such as PCR (see for example EP 0368684, EP 0438310, WO 9207075) or computer modeling (see for example WO 9222653) can be used today. Fusion proteins can also be prepared, for example single chain / toxin antibody fusion proteins (Chaudhary et al., 1990; Friedman et al., 1993). Therefore, antibody molecules of this type are also included in the invention. Likewise, it is in the field of knowledge of the expert in the art to determine the exact epitope of VFF-18 and to prepare, with its knowledge, equivalent antibodies with the same binding specificity. This can happen for example by peptide binding studies as in Example 2, for example by variation of the Hui peptide. Therefore, antibodies of this type are also included in the invention. Another aspect of the invention is the use of VFF-18 or antibody molecules derived from VFF-18 or equivalents in diagnosis and therapy. Diagnostic procedures can be known methods with the use of the antibody molecules according to the invention, for example coupled immunoassays to enzymes (ELISA, Catty and Raykundalia, 1989), radioimmunoassays (Catty and Murphy, 1989), immunohistochemical procedures (Heider et al., 1993b) or Western blots. Procedures of this type can be conveniently carried out with tissue samples taken from the body or with body fluids, for example from biopsies. Investigations can be carried out qualitatively, semiquantitatively or quantitatively. In this case, the antibody or the antibody molecules can be used, as described for example in the state of the art for other v6-specific antibodies (WO 9500851), meaning the advantageous properties of the antibody according to the invention or of the antibody molecules according to the invention a significant improvement of such procedures. In addition to the in vitro diagnosis, the antibody molecules according to the invention are also suitable for in vivo diagnosis, especially of tumors. If the antibody molecule carries a detectable label, a detection of the label can be carried out for diagnostic purposes, for example for visualization of the tumor in vivo (imaging) or, for example, for surgery assisted by radio (radioguided surgery ). For the use of antibodies conjugated with radioactive isotopes for immunological scintigraphy (imaging) there exists for example a series of protocols on the basis of which the person skilled in the art can carry out the invention (Siccardi et al., 1989; Keenan et al., 1987; Perkins and Pimm, 1992; Colcher et al., 1987; Thompson et al., 1984). A therapeutic application can be carried out, for example, analogously to the use of the antibody ASML1.1 (Seiter et al., 1993). In this case, the application can be carried out systemically or topically, for example by intravenous injection / infusion (in the form of bolus or permanent infusion), intraperitoneal, intramuscular, subcutaneous or other. Individual organs or members can also be perfused. Protocols for the administration of conjugated or unconjugated antibodies (either in the form of complete immunoglobulins, fragments, recombinant chimera molecules or the like) are state of the art (Mulshine et al., 1991; Larson et al., 1991; Thorpe, 1991, Vitetta et al., 1991, Breitz et al., 1992, Press et al., 1989, Weiner et al., 1989, Chatal et al., 1989, Sears et al., 1982). The superior properties of VFF-18 with respect to other anti-DC44v6 antibodies are shown in Examples 2 to 4.

Drawings Figure 1: Schematic representation of the fusion protein GST-CD44 (v3-vlO). GST - glutathione-S-transferase from Schistosoma japonicum. v3-vl0 »variant insertion of CD44 keratinocytes. The arrow marks a place of dissociation of thrombin. Figure 2: Comparison of the exon v6 sequence of the CD44 gene between man and rat. The sequences of the Ral and Huí peptides, to which the antibodies 1.1ASML (anti-rat CD44v6) and VFF-18 (anti-human CD44v6) bind, are highlighted with bold. Identical amino acids are characterized by an asterisk. Figure 3: Binding of CD44v6-specific antibodies to synthetic peptides. The binding of the antibodies to the peptides was determined in an ELISA in which the peptides were immobilized and then incubated with antibody solutions. After the corresponding washing steps, the bound antibody was detected with anti-mouse IgG antibodies conjugated with peroxidase. (A): In the first experiment, the binding of the l.l.ASML antibody, specific for rat CD44v6, to the Ral peptide (KWFEN EWQGK NPPT) was determined. (B): In a second assay a peptide homologous to Ral was synthesized from the sequence of human CD44v6. Different supernatants of anti-human CD44v6 hybridoma were fixed to this Huí peptide (QWFGN RWHEG YRQT). In this case, it is shown that VFF-18 binds significantly better to the peptide than the other antibodies used. (C): For a quantitative evaluation, this The experiment was repeated with different concentrations of purified antibodies. Here again, it is demonstrated that VFF-18 has a higher binding affinity than the other 5-fold antibodies. Figure 4: Union of radioactively labeled antibodies to tumor cells. The three antibodies VFF-7 (anti-v6), VFF-18 (anti-v5) and VFF-18 (anti-v6) were radiolabelled with N-succinimyl [2, 3-3H] propionate and used to 1S "binding assays to different tumor cell lines." In this case, the following cell lines were used: CH0-CD44var: recombinant line of hamster cells (Chinese hamster ovary), which expresses human variant CD44 surface ( exons v3-vl0), HCT-116, CX-1, HT-29, CaCo, COLÓ i5 206: lines of human colon carcinoma, A431: line of carcinoma of the human flat epithelium.The specific binding of the antibodies to the different cell lines, while the binding of the v6 specific antibodies VFF-7 and VFF-18 to the recombinant cell line CHO-CD44var is located in the same order of magnitude, the antibodies show a very different binding behavior in the lines of tumor cells. In some cases, only a union of VFF-18 and, to a lesser extent, a union of VFF-8 (HT-29, CaCo, COLÓ 205) and in other lines of cells, VFF-18 is essentially better fixed than VFF-7. Figure 5: Detection of soluble CD44 variants, which contain exon v6, in normal human serum. In a sandwich ELISA, the three v6 specific antibodies VFF-4, VFF-7 or VFF-18 were used as coating antibodies. In all three cases, a specific antibody CD44std conjugated with peroxidase (BU-52, std-standard) was used as detection antibody. In these tests (((A) and (B)) the signal of two different human normal sera is shown at different dilutions, In both cases, with VFF-18, an essentially more intense signal is observed than with the other two antibodies. 6: Serum values of CD44 variants with v6 content With two different ELISAs, the content of soluble CD44var was determined in sera from 6 healthy donors.In this assay, it is used as coating antibody VFF-7 and in another, VFF -18, both antibodies recognize exon v6 As a comparative preparation, in both cases a recombinant soluble CD44 variant was prepared in CHO cells (exons v3-vl0) The ELISA with VFF-18 shows, on average, 3 values. , 5 times higher than the ELISA with VFF-7 This means that the soluble CD44var that occurs in the serum is better recognized by VFF-18 than by VFF-7.

Examples Example 1: Preparation of monoclonal antibody VFF-18 Cloning of pGEX fusion proteins The entire variant region of the HPKII type of CD44v (Hofmann et al., 1991) was amplified from human keratinocyte cDNA by polymerase chain reaction (PCR). The two 5 'PCR primers -CAGGCTGGGAGCCAAATGAAGAAAATG-3 •, positions 25-52, and 5' -TGATAAGGAACGATTGACATTAGAGTTGGA-3 ', positions 1013-984 of the variant region of LCLC97, as described by Hofmann et al, contained a of recognition Ir * 'of EcoRI that was used to clone the PCR product directly into the vector pGEX-2T (Smith et al., 1988). The resulting construct (pGEX CD44v HPKII, v3-vl0) encodes a fusion protein of -70 kD, consisting of glutathione-S-transferase from Schistosoma japonicum and exons v3-vl0 of human CD44 (figure 1, Heider et al., 1993a). The fusion protein was expressed in E. coli and then purified by affinity through glutathione-agarose (Smith et al., 1988).

Immunization and evaluation 0 Female Balb / c mice were immunized intraperitoneally with the affinity purified fusion protein according to the following scheme: immunization: 90 μg of fusion protein in complete Freund's adjuvant 5 2a and 3a immunizations: 50 μg protein fusion in incomplete Freund's adjuvant.

Immunizations were performed at intervals of in each case 4 weeks. 14 days after the last immunization, the animals were still immunized on three consecutive days in each case with 10 μg of fusion protein in PBS. The next day, cells from the spleen of an animal with a high titre of antibodies were fused with mouse myeloma cells P3.X63-Ag8.653 with the help of polyethylene glycol 4000. The hybridoma cells were then screened on? < * microtitre in HAT medium (Kóhler and Milstein, 1975; Kearney et al., 1979). The determination of the antibody titer in the serum or the evaluation of the hybridoma supernatants was carried out with the help of an ELISA. In this test, microtiter plates with fusion protein (GST-CD44v3-10) or only with glutathione-S-transferase were first coated. Then, it was incubated with serial dilutions of serum samples or hybridoma supernatants and the specific antibodies were detected with 0 antibodies against mouse immunoglobulin conjugated with peroxidase. Hybridomas, which only reacted with glutathione-S-transferase, were discarded. The remaining antibodies were first characterized in an ELISA with specific domain fusion proteins (exon v3, exon v5 5 + v6, exon v6 + v7, exon vβ-vlO) (Koopman et al., 1993). His immunohistochemical reactivity was tested in sections of the human skin. The VFF-18 antibody was then identified through binding to the synthetic peptide Hui (QWFGN RWHEG YRQT). The sequence of Hui is a fragment of exon v6 of human CD44.

Example 2: Fixation of CD44v6-specific antibodies to synthetic peptides The binding of CD44v6-specific antibodies to synthetic peptides was determined in an ELISA.

Solutions: Coating buffer: 0.05M sodium carbonate, pH 9.6 Assay buffer: PBS (phosphate buffered saline) 0.5% BSA (bovine serum albumin) 0.05% Tween 20 Substrate solution: trade name Kierkegaard & Perry Laboratories, Gaithersburg MD, USA; TMB peroxidase substrate: peroxidase B (H202) 1: 1 solution The peptides (50 μg / ml in coating buffer) were immobilized overnight at 4 ° C in NUNC immunoplates.

Maxisorp (1.1ASML) or Acti-A plates of the Bio trade name Products (VFF antibodies). In the case of Acti-A plates, the peptide is covalently bound to the plate. It was then washed with PBS / 0.05% Tween, the free adsorption sites were blocked on the surface of the plate with assay buffer (1 hour at room temperature) and again washed once with PBS / Tweßn. 20 to 0.05%. Acti-A plates were reduced after blocking with 10 mM sodium borohydride in 20 mM sodium hydrogencarbonate, pH 9.0 (shaking for 1 hour at room temperature) and then washed three times with PBS / 0 Tween 20, 05%. Hybridoma supernatants or antibody solutions in assay buffer in concentrations between 0.02 and 10.0 μg / ml were then added to the wells and incubated for 2 hours at room temperature in a plate shaker. Next, three washing steps were carried out with PBS / 0.05% Tween 20. Then, 100 μl / well of anti-mouse IgG antibodies conjugated with horseradish peroxidase were added at a suitable dilution in the assay buffer. After incubation for two hours at room temperature in the plate shaker, it was washed three times with PBS / 0.05% Tween 20 and stained with substrate solution. After development for 10-15 min, it was stopped with 2M sulfuric acid and the absorption was measured at 450 nm (reference 690 mm) in the photometer. In a first experiment, the binding of the rat CD44v6 specific antibody 1.1ASML to the peptide was determined Ral (KWFEN EWQGK NPPT) (Table 1, Figure 3 (A)).

Table 1 In another assay, a Ral homolog peptide of the human CD44v6 sequence (Hui, QWFGN RWHEG YRQT) was synthesized. Different anti-human CD44v6 antibodies were bound to Huí. In this case it is demonstrated that VFF-18 has a binding affinity surprisingly higher than all the other antibodies used (Table 2, Figure 3, (B)).

Table 2 For the quantitative evaluation, purified anti-human CD44v6 antibodies in different concentrations were fixed to the Hui peptide. Here, too, clearly improved binding affinity is demonstrated in comparison with the other antibodies (Table 3, Figure 3 (C)).

Table 3 Example 3: Fixation of radiolabelled CD44v6-specific antibodies to tumor cell lines Radioactive labeling of the N-succinimidyl- [2, 3-3H] -propionate antibodies 1 mCi ([3 H] -NSP, Amersham 1 mCi / ml) were concentrated by evaporation to near dryness in a sample tube siliconized to OQC in the vacuum of the water jet. 15 μg of antibody (1 mg / ml in PBS, pH 7.4) was added and incubated for 48 hours at 4 ° C. Then, excess [3H] -NSP was collected by reaction with 30 μl of 1 M Glycine in PBS (20 minutes at room temperature). The separation of the [3H] -glycine labeled antibody was carried out with a Sephadex-V-25- column (15ml column volume), where PBS / 0.5% BSA was used as buffer. The labeled antibody [5H] - appears in the volume of justification the amount of antibodies in an ELISA was determined to detect mouse immunoglobulin and the specific activity was calculated. Linkage of the labeled antibodies in tumor cells. The three antibodies VFF-7 (anti v6), VFF-8 (anti v5) and VFF-18 (anti v6) were labeled with radioactive N succinimidyl- [2,3- 5H] propionate and used in different tumor cells for testing of link. There, the following CHO-CD44var cell lines, a line of recombinant hamster cells (Chinese hamster ovary) expressing on the surface the human v44 CD44 (Exon v3-v10); HCT-116, CX-1, HT-29, CaCo, COLÓ 205; Human colon carcinoma lines; A431; carcinoma line of human flat epithelium. Cells were placed in 12-hole tissue culture plates, incubated overnight at 37 ° C in an incubator, then washed once with PBS and fixed with ethanol (1 min at room temperature). Then, it was washed once with culture medium (RPMI 1640/10% fetal calf serum) and the radioactive antibody was bound (250,000 dpm / well in the culture medium). After an incubation of 25 hours at room temperature in the plate shaker, it was washed three times with PBS / 0.5% BSA, the cells were solubilized with 0.1 M NaOH / 1% Triton X- -100 and the radioactivity was measured in the scintillation counter. The non-specific binding was determined in the presence of a 100-fold excess of unlabeled antibody. The binding was referred to the unit cell number (400,000 cells). After determination of the specific activity of the antibodies, the bound amount of antibodies can be expressed in fmol. Table 4 and Figure 4 show the specific binding of the antibodies to the different cell lines. While the binding of v6-specific VFF-7 and VFF-18 antibodies to the recombinant CH0-CD44var cell line is almost 0, the antibodies in. the lines of tumor cells show a very different binding behavior. In some cases, only one binding of VFF-18 and, to a lesser extent, of VFF-8 (HT-29, CaCo, COLÓ 205) is observed and in the other cell lines, VFF-18 binds substantially better than 5 VFF-7 Table 4 Example 4: ELISA for the determination in serum of soluble CD44v6 Solutions: Coating buffer: 0.05 M sodium carbonate, pH 9.6 Test buffer: PBS (phosphate buffered saline) 0.5% BSA (bovine serum albumin) 0.05% Tween 20 Sample diluent: trade name Bender MedSystems, Vienna, Austria Substrate solution:: Kierkegaard & Perry Laboratories, Gaithersburg MD, USA; TMB peroxidase substrate: B solution of peroxidase (H202) 1: 1 Microtiter plates (Nunc-Immunoplate MaxiSorp F96) were coated with 5 μg / ml of one of the CD44v6 specific antibodies (incubation at 4QC overnight). Then, was washed with PBS / 0.05% Tween, free adsorption sites were blocked on the surface of the plates with assay buffer (for 1 hour at room temperature) and again washed once with PBS / Tween 20 0.05% The serum samples were then previously diluted at least in the ratio 1: 5 in the sample diluent and were further diluted in serial dilutions of 1: 2 on the plate in the sample diluent. Then, 50 μl / well with horseradish peroxidase conjugated anti-CD44std antibodies (clone BU-52, The Binding Site, Birmingham) in a suitable dilution (1: 3,000 -1: 10,000) in the assay buffer was added. . After incubation for three hours at room temperature in the plate shaker, it was washed three times with PBS / 0.05% Tween 20 and typed with substrate solution. After development for 10-15 min, it was stopped with 2M sulfuric acid and the absorption was measured in the photometer at 450 nm (reference 690 nm). For the quantification, a serial dilution was arranged in the assay buffer of a standard preparation of CD44 soluble in parallel to the serum samples. This preparation was purified from a supernatant material of recombinant hamster cells (CHO), which express soluble CD44v3-vlO. In the case of CD44v3-vlO it is a variant of human CD44 containing the peptide sequences encoded by exons v3 to vlO. Table 5 and Figure 5 show the detection of soluble CD44 variants, which contain exon v6, in human normal serum. In a sandwich ELISA, the three v6-specific VFF-4, VFF-7 or VFF-18 antibodies were used as coating antibodies. In all three cases, a specific antibody of CD44std conjugated with peroxidase (BU-52, std-standard) was used as detection antibody. In these tests the signal of two different human normal sera is shown at different dilutions. In both cases ((A) and (B)), with VFF-18 an essentially more intense signal is observed than with the other two antibodies.

Table 5 A Serum 1 Table 5 B Serum 2 Table 6 and Figure 6 show serum values of CD44 variants with v6 content. With two different ELISAs, the content of soluble CD44var was determined in sera from 6 healthy donors. In this test VFF-7 was used and in the other VFF-18 as coating antibodies; both antibodies recognize exon v6. A recombinant soluble CD44 variant prepared in CHO cells (exons v3-vl0) was used as a comparative preparation in both cases. The ELISA with VFF-18 shows on average values 3.5 times higher than the ELISA with VFF-7. This means that the soluble CD44var that occurs in the serum, compared to the recombinant protein, is better recognized by VFF-18 than by VFF-7.

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SEQUENCE PROTOCOL "''!] GENERAL DATA:, i) APPLICANT: .a NAME: Boehnnger Ingelheim International GmbH CB] STREET: Binger Str. F 1] CITY: Ingeiheim [E] PH IS: Germany CF] POSTAL CODE: 55216 CG] TELEPHONE: 06132-772770 CH] T? LEFAX: 06132-774377 [A] NAME: Dr. Günther Adolf CB] STREET: Stiftgasse 15-17 / 10 CC] CITY: Vienna CS] COUNTRY: Austria CF] POSTAL CODE: A-1Ü7Ü f QÍ CA] NAME: Dr. Erik Patzelt CB] STREET: Hans-Buchmueller-Gasse 8 CC] CITY: Punkersdorg CE] COUNTRY: Austria CF] POSTAL CODE: A-3002 i? I) NAME OF THE INVENTION: Monoclonal antibodies against CD44v6 i iii) SEQUENCE NUMBER: 6 5 (iv) COMPUTER EDITION: CA] DTAOS CARRIER: Floppy disk CB] COMPUTER: compatible IBM PC Í C] OPERATING SYSTEM: PC-DOS / MS / DOS CD ] SOFTWARE: PatentIn Relay tfl.O, Version # 1.30 (EPA) í í] DATA ON SEQ ID No. 1: ii) CHARACTERISTICS OF THE SEQUENCE: C] LENGTH: 27 base pairs CB] TYPE: Nucleotides 0 CC] SHAPE FORM: Single strip CD] TOPOLOGY: linear iii) TYPE OF MOLECULE: other nucleic acids CA] DESCRIPTION: / desc = "Primary PCR".? I) DESCRIPTION OF THE SEQUENCE: SEQ. ID. NO: 1: CAJGCTGGGA GCCAAATGAA GAAAATG DATA ON SEC ID no. 2: (i) CHARACTERISTICS OF THE SEQUENCE: 5 CA] LENGTH: 30 base pairs CB] TYPE: Nucleotides iC] SHAPE FORM: Single strip CD] TOPOLOGY: linear vil) TYPE OF MOLECULE: other nucleic acids CA] DESCRIPTION : / desc = "Primary PCR" txu DESCRIPTION OF THE SEQUENCE: SEQ. ID. NO: 2: TGATAAGGA CGATTGACAT TAGAGTTGGA i 2) DATA ON SEC ID ID no. 3: r 'i i) CHARACTERISTICS OF THE. SEQUENCE: Í-Aj LENGTH: 14 amino acids [?] TYPE: amino acids C SHAPE FORM: Simple strip [D] TOPOLOGY: linear ?? i) TYPE D? MOLECULE: peptide (xi) DESCRON OF THE SEQUENCE: SEQ. ID. NO: 3: Lys Trp Phe Glu Asn Glu Trp Gln Gly Lys Asn Pro Pro Thr 1 5 10 .2) DATA ON SEC ID no. 4: (i) CHARACTERISTICS OF THE SEQUENCE: CA] LENGTH: 14 amino acids CB] TYPE: amino acids CG] FORM OF THE STRIP: CD single strip] TOPOLOGY: linear vil »TYPE OF MOLECULE: peptide (Xi) DESCRON OF THE SEQUENCE: I KNOW THAT. ID. NO: 4: Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr 1 5 10 .2) DATA ON SEQ ID no. 5: (i) CHARACTERISTICS OF THE SEQUENCE: CA] LENGTH: 42 amino acids CB] TYPE: amino acids I] FORM OF THE STRIP: Single strip [D] TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide ixi) DESCRON OF THE SEQUENCE : I KNOW THAT. ID. NO: 5: Frp rtla Asp Pro Asn Ser Thr Thr Glu Glu Ala Wing Thr Gln 1 5 10 Lys Glu Lys Trp Phe Glu Trp Gln Gly Lys Asn Pro Pro Thr 20 25 Pro Thr Pro Ser Glu Asp Ser His Val Thr Glu Gly Thr Thr U 35 '40 i, DATA ON SEC ID no. 6: (i) CHARACTERISTICS OF THE SEQUENCE: CH] LENGTH: 43 amino acids CB] TYPE: amino acids il] SHAPE FORM: Single strip CD1 TOPOLOGY: linear.? I, TYPE OF MOLECULE: peptide 5 (xi) DESCRON OF THE SEQUENCE: SEQ ID NO: 6: Gin Aia Thr Pro Ser Thr Thr Glu Glu Thr Thr Ala Gln 1 5 10 Lys Glu Glu Fen Gly Asn Arg Trp His Glu Gly Tyr Arg Gln 20 25 Thr Pro arg Glu Asp Ser His Ser Thr Thr Gly Thr Wing Ala It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects or products to which it refers. Having described the invention as above, property is claimed as contained in the following:

Claims (17)

1. CLAIMS 1. Antibodies against an epitope within the amino acid sequence that is encoded by the variable vd exon of the CD44 gene, characterized in that it is the VFF-18 antibody that is formed by the hybridoma cell line with the deposit number DSM ACC2174.
2. 2.- Hybridoma cell line with deposit number DSM ACC2174.
3. 3. Molecule of antibody, which can be obtained by chemical or enzymatic modification of an antibody according to claim 1.
4. 4.- Antibody molecule according to claim 3, characterized in that it is a fragment of an antibody according to claim 1.
5. 5. - Antibody molecule according to claim 4, characterized in that it is a Fab fragment or an F (ab ') 2 fragment.
6. 6. Molecule of antibody according to claim 3, characterized in that the antibody is linked to another molecule.
7. 7. The antibody molecule according to claim 6, characterized in that the other molecule is a polypeptide.
8. 8. Antibody molecule according to one of claims 3 to 5, characterized in that it is linked to a radioactive isotope.
9. 9. Recombinant antibody molecule with the idiotype of an antibody according to claim 1.
10. 10. Recombinant antibody molecule according to claim 9, characterized in that it is a chimeric antibody molecule, humanized, single chain or created by entrainment of the antibody. chain.
11. 11. Recombinant antibody molecule according to claim 9 or 10, characterized in that it is linked to another molecule or to a radioactive isotope.
12. 12. Molecule of antibody that recognizes the same epitope as the antibody according to claim 1.
13. 13. Use of an antibody or antibody molecule according to one of claims 1 or 3 to 12, in diagnostic procedures outside the human or animal body. .
14. 14. Use according to claim 13, characterized in that the method is an immunoassay coupled with enzymes or a radioimmunoassay.
15. 15. Use according to claim 13, characterized in that the method is an immunohistochemical procedure.
16. 16. Antibody or antibody molecule according to one of claims 1 or 3 to 12 for its pharmaceutical use.
17. 17. Use of an antibody or an antibody molecule according to one of claims 1 or 3 to 11 for the preparation of an agent 'for in vivo diagnosis.