MXPA99005544A - Method for diagnosis and therapy of hodgkin lymphomas - Google Patents

Abstract

The invention relates to a method for the diagnosis and therapy of Hodgkin-lymphomas (lymphogranulomatosis), which is founded on the expression of the variant exon v10 of the CD44-gene as a molecular marker target. There is a significant correlation between v10-expression and stage as well as prognosis of the disease. In an advantageous embodiment, v10-specific antibody molecules are used to measure the expression of the exon in samples. In a further advantageous embodiment, radio-labelled v10-specific antibodies are used for therapy of Hodgkin lymphomas.

Description

ANTIBODY MOLECULE AND ITS EMPLOYMENT FOR THE DIAGNOSIS AND THERAPY OF LYMPHOMA OF HODGKIN Field of the Invention The present invention relates to methods for the diagnosis and therapy of Hodgkin's lymphomas (lymphogranulomatosis) which are based on the expression of the variant exon of the CD44 gene as a molecular target, to agents for these procedures ^ as well as to the use of these agents. The highly glycosylated cell surface protein CD44 participates in the interaction between cells and the extracellular matrix, such as the migration and activation of leukocytes in the case of inflammation and immunological surveillance, formation of precursors of leukocytic and myeloid cells in the bone marrow, as well as in the development of lymphoid organs and in the interaction of cells with the extracellular matrix (Lesley et al., 1993. Günthert 1993, País et al., 1993, Mackay et al., 1994). The human CD44 gene is composed of at least 19 exons, of which at least 12, which encode the extracellular region, are alternatively cut and spliced (Screaton et al., 1992). The CD44 gene is transcribed in a series of normal tissues and carcinomas (Fox et al., 1994). While REF. : 30411 that the standard CD44 molecule (CD44s) is ubiquitously expressed in epithelial and mesenchymal tissues, the different isoforms, which are created by alternative RNA splicing, are in a very limited distribution (Heider et al. , 1993). Some of the variant isoforms participate in the activation of lymphocytes and manifest themselves in association with the formation of metastases (Mackay et al., 1994, Günthert et al., 1991, Rudy et al., 1993, Koopman et al., 1993) . Although a direct biological role of variant CD44 expression in the formation of metastasises in rat pancreatic carcinoma was demonstrated (Günthert et al., 1991, Seiter et al., 1993), its role in human tumors is still unknown.

BACKGROUND OF THE INVENTION Different reports were published in which it was shown that certain forms of CD44 cut and spliced alternatively were expressed in human metastatic tumors (Heider et al., 1993 and 1996, Fox et al., 1994, Friedrichs et al. ., 1995, Kaufmann et al., 1995, Salles et al., 1993, Stauder_et al., 1995, Koopman et al., 1993, Tanabe et al., 1993). Studies of the expression of CD44 in non-Hodgkin's lymphomas (NHL) were concentrated in the analysis of the so-called lymphocyte receptor receptor CD44H or CD44s (Horst et al., 1990a, Horst et al., 1990b, Jalkanen et al., 1991, Moller et al., 1992). While some authors (Horst et al., 1990a, Jalkanen et al 1991, Picker et al., 1988, País et al., 1989, Fujiwara et al., 1993) found a correlation between an increased expression of CD44s and a prognosis. unfavorable, others (Terpe et al., 1994) could not confirm these findings. Recently, a high regulation of CD44v3 and CD44v6 isoforms was found in NHL with an unfavorable pathological state (Koopman et al., 1993, Terpe et al., 1994, Salles et al., 1993, Stauder et al., 1995), using CD44 monoclonal antibodies (acms) specific for variants (Mackay et al., 1994, Koopman et al., 1993, Fox et al., 1993).

Description of the invention Various attempts have been made to exploit the differential expression of variant exons of the CD44 gene in tumors and in normal tissues for diagnostic and therapeutic processes' (WO 94/02633, WO 94/12631, WO 95/00658, WO 95). / 00851, EP 0531300.

It was the object of the present invention to develop new methods for the diagnosis and therapy of Hodgkin lympholas (lymphogranulomatosis) as well as the provision of agents for such procedures. This problem could be solved with the present invention. This refers to procedures for the diagnosis and therapy of Hodgkin's lymphomas (lymphogranulomatosis) which are based on expression of the variant vlO exon of the CD44 gene as a marker or molecular target. Molecules of antibodies with a corresponding specificity are particularly suitable as vehicles in order to selectively access in vivo Hodgkin lymphomas. In this case, methods are preferred which are characterized in that an antibody molecule that specifically binds to the amino acid sequence SEQ ID NO. 2 (see the sequence protocol). Other aspects of the present invention are the use of antibody molecules of this type in the procedures according to the invention, as well as agents for carrying out these procedures.

The invention further relates to the use of an antibody molecule that is specific for an epitope within the amino acid sequence, which is encoded by the variable vlO exon of the CD44 gene, for the preparation of a pharmaceutical composition for diagnosis and / or therapy of tumor diseases. Preferably, in the case of the tumor disease it is a Hodgkin lymphoma (lymphogranulomatosis). The invention further relates to an antibody molecule that is specific for an epitope within the amino acid sequence that is encoded by the variable exon of the CD44 gene, for pharmaceutical use. Preferably, an antibody molecule of this type is characterized in that it binds to SEQ ID NO. 2 In this case, it can be in particular a monoclonal antibody, a Fab or F (ab ') 2 fragment of an immunoglobulin, a recombinantly produced antibody, a chimeric or humanized antibody produced recombinantly or a chain antibody simple (scFv). Preferably, such an antibody molecule is linked to a radioactive isotope, a radioactive compound, an enzyme, a toxin, a cytostatic agent, a prodrug, a cytokine or other immunomodulatory polypeptide.

The nucleic acid and amino acid sequence of the variant vlO exon of the CD44 gene is known (Screaton et al., 1992, Tdlg et al., 1993). These sequences are found in the sequence protocol (SEQ ID Nos. 1 and 2). The existence of degenerate or allelic variants is not of importance for the embodiment of the invention; therefore, variants of this type are expressly included. The invention can be carried out with polyclonal or monoclonal antibodies that are specific for an epitope that is encoded by the vlO exon. The preparation of antibodies against known amino acid sequences can be carried out according to methods known per se (Catty, 1989). For example, a peptide of this sequence can be produced synthetically and, in the form of an antigen, used in an immunization protocol. Another route is the preparation of a fusion protein containing the desired amino acid sequence, by integrating a nucleic acid (which can be produced synthetically or, for example, by polymerase chain reaction (PCR) from a sample suitable), which encodes this sequence, in an expression vector, and the fusion protein is expressed in a host organism. The optionally purified fusion protein can then be used as an antigen in an immunization protocol and selected, with suitable methods, specific antibodies to the insertion or, in the case of monoclonal antibodies, hybridomas that express specific antibodies to the insert. Procedures of this type are known state of the art. Heider et al. (1993, 1996) and Koopman et al. (1993) describe the preparation of antibodies against epitopes variant of CD44. However, other antibody molecules which are derived from polyclonal or monoclonal antibodies, for example Fab or F (ab ') 2 fragments of immunoglobulins, single chain antibodies (scFv) produced can also be used for the method according to the invention. recombinantly, chimeric or humanized antibodies, as well as other molecules that bind specifically to epitopes that are encoded by the vlO exon. From a complete immunoglobulin, for example, Fab or F (ab ') 2 fragments or other fragments can be created (Kreitman et al., 1993). The person skilled in the art is also able to produce vlO-specific recombinant antibody molecules. 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; Güssow and Seemann, 1991), bispecific antibodies (Weiner et al., 1993; Goodwin, 1989), single chain antibodies ( ScFv, Johnson and Bird, 1991), complete or fragmented immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992; Barbas et al., 1992) or antibodies created by chain rearrangement (Winter et al., 1994), Humanized antibodies can be produced, for example, by CDR 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 produced and used, for example single chain / toxin antibody fusion proteins (Chaudhary et al., 1990; Friedman et al., 1993). Under the general concepts "antibodies" and "antibody molecules" must be included, in addition to polyclonal and monoclonal antibodies, all the compounds discussed in this paragraph, as well as other compounds that can be derived structurally from immunoglobulins and that can be produced with methods known per se.

For diagnostic procedure, antibody molecules can be linked, for example with radioactive isotopes such as 131I, 111In, 99p? Tc or radioactive compounds (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1988), enzymes such such as peroxidase or alkaline phosphatase (Catty and Raykurtdalia, 1989), with fluorescent dyes (Johnson, 1989) or with biotin molecules (Guesdon et al., 1979). For therapeutic applications, vlO-specific antibody molecules can be linked to radioisotopes such as 90Y,? NIn, 131I, 186Re (Quadri et al., 1993; Lenhard et al., 1985; Vriesendorp et al., 1991; Wilbur et al., 1989), toxins (Vitetta et al., 1991, Vitetta and Thorpe, 1991, Kreitman et al., 1993, Theuer et al., 1993), cytostatic agents (Schrappe et al., 1992), prodrugs (Wang et al., 1992; et al., 1989) or radioactive compounds. The antibody can also be linked to a cytokine or other immunomodulatory polypeptide, for example with tumor necrosis factor or interleukin-2. Advantageously, samples from patients, for example from biopsies, in which there is a suspicion of a Hodgkin's lymphoma (lymphogranulomatosis), can be examined with the diagnosis method according to the invention, or the diagnosis is already available, but The tumor must be characterized with greater precision. The detection of variant CD44 molecules, which contain an amino acid sequence that is encoded by the variable vlO exon, can be carried out in the plane of the proteins by antibodies or in the plane of the nucleic acids by means of specific probes of nucleic acids or primers for the poly-erasa chain reaction (PCR). Accordingly, the invention also relates to antibody molecules and nucleic acids which are suitable as probes or primers for such procedures, and to the use of antibodies and nucleic acids of this type for the diagnosis and analysis of Hodgkin lymphomas. . For example, tissue sections can be examined immunohistochemically with antibodies with methods known per se. Extracts obtained from tissue or body fluid samples can also be examined with other immunological methods using antibodies, for example in Western blots, analysis with immunosorbent-linked enzyme (ELISA, Catty and Raykundalia, 1989), radioimmunoassays ( RÍA, Catty and Murphy, 1989) or related immunoassays. The investigations can be carried out qualitatively, semiquantitatively or quantitatively. The expression of the splicing variant of CD44 viO in Hodgkin's disease is associated with aggressive tumor behavior and a high risk of recurrence. This expression correlates with an advanced phrase and a poor prognosis of NSHD (nodular sclerosis Hodgkin's disease). In addition to the in vitro diagnosis, antibody molecules with a specificity according to the invention are also suitable for the in vivo diagnosis of Hodgkin lymphomas. If the antibody molecule carries a detectable marker, a marker detection can be performed for diagnostic purposes, for example visualization of the tumor as a live one (imaging) or, for example, for radioguided surgery. For the use of antibodies conjugated with radioactive isotopes for immunoscintillation (imaging) there are, for example, a series of protocols, based on which the person skilled in the art can execute the invention (Siccardi et al., 1989; Keenan et al. al., 1987, Perkins and Pimm, 1992, Colcher et al., 1987, Thompson et al., 1984). By detecting and / or quantifying the variant epitope expression of CD44 vlO, the collected data can thus be incorporated into the diagnosis and prognosis. In this case, the combination with other prognostic parameters, for example with the tumor grade, may be advantageous. Molecules of antibodies with the specificity according to the invention and, eventually, linked with a cytotoxic agent, can be used advantageously for the therapy of Hodgkin lymphomas (lymphogranulomatosis). 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 others. Protocols for the administration of conjugated or unconjugated antibodies (either as complete immunoglobulins, fragments, recombinant humanized molecules or the like) are known state of the art (Mulshine et al., 1991; Larson et al., 1991; Vitetta and Thorpe, 1991; Vitetta et al., 1991; Breitz et al., 1992, 1995; Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989; Sears et al., 1982). The antibody molecules can be formulated in a manner known per se. For example, they can be presented in an aqueous solution which is optionally buffered with a physiologically compatible buffer. A solution of this type can be characterized by the addition of suitable stabilizers and adjuvants. The antibody molecules can, however, also be in the form of a lyophilized preparation (freeze-dried) which is reconstituted before use with a suitable solvent, for example water. A preferred embodiment of a therapeutic application is to bind a humanized-specific immunoglobulin or a F (ab ') 2 fragment thereof with 90Y (Quadri et al., 1993; Vriesendorp et al., 1995), 131I ( Juweid et al., 1995; Press et al., 1995, Thomas et al., In: Catty 1985, pp. 230-239), 186Re (Breitz et al., 1992, 1995) or with another suitable radioisotope and use it for radioimmunotherapy for Hodgkin lymphomas. For example, an antibody molecule of this type can be linked to 90Y using a chelator-forming linker such as ITCB-DTPA (isothiocyanatobenzyl-diethylenetriamine pentaacetate), a specific activity of 5-20 mCi / mg, preferably 10-20 mCi / mg, should be achieved. mCi / mg. This agent can then be administered to a patient with an antigen-positive tumor in a dosage of 0.1 to 1 mCi / kg of body weight, preferably 0.3 to 0.5 mCi / kg of body weight, particularly preferably 0.4 mCi / kg. In the case of a total protein amount to be administered from 2 to 5 mg, this can be done in the form of a rapid intravenous bolus injection. In the case of monoclonal antibodies it may be necessary to mix the agent, prior to administration, with an excess (eg, a tenfold molar excess) of the non-radioactive antibody; in this case, administration is best effected in the form of an intravenous infusion, for example over 15 minutes. The application can be repeated. Therapy can be supported by a bone marrow transplant.

Description of the figures Fig. 1; A. Single cell HRS (Hodgkin and Reed-Sternberg) of a patient without recurrence that reacts with the acm VFF16 (CD44vlO). The arrowheads point to non-reactive HRS cells. B. > 50% of the HRS cells of patients with recurrence show reactivity (ABC, x400). Fig. 2; Expression of CD44v10 in HRS cells in different groups of patients. It should be noted that patients with a poor clinical course, ie recurrence or involvement of the bone marrow, show exclusively more than 10% of positive HRS cells, while patients without recurrence have less than 10% of positive HRS cells.

The difference between these two groups is very statistically significant. Fig. 3; RT-PCR analysis using specific primers of CD44v10 (half of the right): the main transcript of approximately 470 bp in all samples and a weaker transcript of 660 bp in samples 2, 3 and 4 confirm isoforms with content in CD44vl0 in all 5 cases. A dominant band of 440 bp in the case of the use of primers that are specific for the constant 5 'and 3' regions is indicated by the standard form of CD44 (half of the left). EXAMPLES Example 1: Preparation of vlO specific antibodies The entire variant region of the HPKII type of CD44v (Hofmann et al., 1991) was amplified from human keratinocyte cDNAs by polymerase chain reaction (PCR). PCR primers 5 '-CAGGCTGGGAGCCAAATGAAGAAAATG-3', positions 25-52, and 5 '-TGATAAGGAACGATTGACATTAGAGTTGGA-3', positions 13-984 of the variant region LCLC97, as described by Hofmann et al, contained a recognition site of EcoRI, which 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 the exons v3-vl0 of human CD44 (Heider et al., 1993). The fusion protein was expressed in E. Coli and then affinity purified on glutathione-agarose (Smith et al., 1988). Balb / c female mice were immunized intraperitoneally with the fusion protein purified by affinity according to the following scheme: immunization: 90 μg of fusion protein in complete Freund's adjuvant 2a and 3a immunizations: 50 μg of fusion protein in incomplete Freund's adjuvant. The immunizations were carried out at intervals of in each case 4 weeks. 14 days after the last immunization, the animals were still immunized for three consecutive days in each case with 10 μg of fusion protein in PBS. The next day, spleen cells from an animal with a high titer of antibodies were fused with mouse myeloma cells P3.X63-Ag8.653 with the help of polyethylene glycol 4000. The hybridoma cells were then selected in microtiter plates in medium HAT (Kóhler and Milstein, 1975; Kearney et al., 1979).

The determination of the antibody titer in the serum or the screening of the hybridoma supernatants was carried out with the aid of an ELISA. In this assay, microtiter plates were first coated with the fusion protein (GST-CD44v3-10) or only with glutathione-S-transferase. It was then incubated with serial dilutions of serum samples or hybridoma supernatants and specific antibodies were detected with peroxidase-conjugated antibodies against mouse immunoglobulin. Hybridomas that only reacted with glutathione-S-transferase were discarded. The remaining antibodies were first characterized in an ELISA with domain-specific fusion proteins (exon v3, exons v5 + v6 +, exons v6 + v7, exons v8-vlO, exon vlO) (Koopman et al., 1993). Their immunohistochemical reactivity was tested on human skin strips. Antibodies from the supernatants of the hybridoma clones VFF-14 and VFF-16 bind only to fusion proteins containing a domain that is encoded by the vlO exon.

Example 2: Immunohistochemical investigation of tissue samples Tissues and patients 37 paraffin-embedded lymph node samples from 29 patients with NSHD (nodular sclerosis Hodgkin's disease according to the Rye classification) were obtained from the Collection of the Department of Pathology, School University for Medicine, Graz, Austria, and was divided into three groups; group 1: 11 patients with NSHD previously treated before treatment (5 patients in phase I, 6 in phase II), were free of recurrence for more than 6 years; group 2: 9 patients with previously treated NSHD before treatment (4 in phase I, 5 in phase II) showed a recurrence of the disease in the space of one to three years. Of 7 of these 9 patients, two to three consecutive lymph node segments of NSHD relapses were included in this study; group 3: 9 patients with involvement of the bone marrow at the time of the original diagnosis (phase IV).

Immunohistochemistry The lymph node samples were stained with the following acms: standard CD44 (s) recognized by the SFF2 acm; CD44v5 detected by the VFF8 acm; CD44v6 detected by acms VFF7 and VFF18; CD44 lO detected by acms VFF14 and VFF16. The SFF2 mAb recognizes an epitope that is common for all isoforms of CD44. Acms VFF7 and VFF18 recognize different epitopes, but overlapping, which are encoded by exon v6. The VFF8 acm is specific for exon v6. The acms VFF14 and VFF16 react with an epitope that is encoded by the vlO exon. Immunohistochemistry was performed in microwave-treated sections (Gerdes et al., 1992), using the avidin-biotin complex (ABC) -peroxidase method (Guesdon et al., 1979). The paraffin sections were deparaffinized in xylene, rehydrated and the endogenous peroxidase blocked with H202 in methanol. The slides were placed on a glass support and moistened in 500 ml of 0.01 M citrate buffer (2.1 g of citric acid in 1 l of deionized water, pH adjusted to 6.0 with 2 N NaOH). The microwave treatment was carried out for 35 minutes at a maximum concentration (600 W) in a microwave (BioRad). After the microwave treatment for 9 minutes, the vaporized buffer was completed with deionized water.

After irradiation by microwaves, the solution was cooled for 20 minutes. Then, the slides were rinsed in phosphate buffered saline (PBS) and not immunostained by diaminobenzidine development (DAB) For comparative purposes, 10 samples of frozen lymph nodes (3 of patients from group 1, 2 from group 2 and 5 from cases only recently collected) were also incubated with acms VFF14 (vlO) and VFF16 (vlO), using the method of alkaline phosphatase-anti-alkaline phosphatase (APAAP) (Cordell et al., 1984). For control purposes, sections of normal human epidermis which is known to contain the respective antigens (positive controls) were tested. The replacement of the primary antibody by normal serum always provided negative results (negative controls). For other control purposes, immunohistochemical stains for the expression of CD44vl0 and CD44v6 were repeated twice in each case. In order to confirm the findings, the cases were further incubated in another laboratory, using the APAAP method (Cordell et al., The percentage of HRS cells (Hodgkin and Reed-Sternberg) that stained with the antibodies was classified as 0 %, less than 10%, 10-50% and greater than 50% Care was taken that the immunoreactive HRS cells were clearly tumor cells (for example, with the help of the presence of characteristic details of the nucleus), particularly in cases in which less than 10% of the HRS cells expressed the respective antigens All the cases were inspected separately by 2 of the inventors The distribution of the staining, for example in the membrane, in the cytoplasm or in both, was recorded as well as immunological reactivity in cells that were not HRS cells.

Statistical analysis CD44 expres models were analyzed, using the chi square calculation of Pearson and the Mantel-Haenszel test for linear association, using the SPSS program for Windows. The P values were equal to or less than 0.05, were considered significant.

Results of immunohistochemical stains Table 1 shows an overview of the results obtained with antibodies directed against CD44s, CD44v5, v6 and vlO in HRS cells. The main part of the antigenic reactivity of HRS cells was in all cases on the surface of the cell. A variable number of HRS cells provided a cytoplasmatic and / or similar perinuclear activity at spots with or without surface staining that likely reproduce the reactivity of CD44 molecules in the Golgi apparatus or in the endoplasmic reticulum. The expres of CD44vlO correlates with the advanced phase and a poor prognosis of NSHD. The expres of CD44s, CD44v5 (detected by acm VFF8) and CD44v6 (detected by acm VFF18) in HRS cells was found in the majority of the cases, notwithstanding a great variability in the number of stained cells (Table 1).

The expres of CD44v6 (detected by acm VFF7) was found only in a few cases and, when present, was then limited to a minority of HRS cells (Table 1) . In patients without recurrence, expres of CD44vlO (detected by acms VFF14 and VFF16) was found only in very few cases, and the proportion of reactive HRS cells was < 10% (Figs 1A and 2). In contrast, in all cases of patients with recurrence or initial involvement of the bone marrow, expres of CD44v10 was observed. In most of these cases, there was a clear suppres of CD44v10 with > 50% of reactive HRS cells (Figs IB and 2). This supra-expres of CD44v10 was also found in lymph node sections investigated for recurrence (Table 2). Lymph node sections - frozen lymphatics from 3 patients without recurrence and from 2 patients with recurrence gave identical results to paraffin sections. The two antibodies (VFF14 and VFF16) specific for the exon vlO showed identical results in the reaction on the suerficie and / or in the cytoplasm of the HRS cells. Using an exact Fisher's test, differences in the expres of CD44 isoforms between groups of patients with nonaggressive and aggressive NSHD provided the following p values: CD44s (p = 0.3625), CDD44v5 (p = 0.2415), CD44v6 (detected with acm VFF7) (p = 0.2093), CD44v6 (detected with acm VFF18) (p = 0.1836), CD44vlO (detected by acms VFF14 and VFF16) (p = 0.001). This shows that the different expres models of CD44vl0 (detected by acms VFF14 and VFF16) within these groups of NSHD were statistically very significant. The expres of the variant CD44 by splicing of vlO in Hodgkin's disease is associated with an aggressive behavior of the tumor and a high risk of recurrence. It correlates with an advanced phase and a poor prognosis of NSHD. In contrast to previous immunohistochemical studies of CD44 expres in relation to a prognostic relevance in neoplasms of different histogenetic origin, which were exclusively performed in frozen sections (Koopman et al., 1993, Terpe et al., 1994, Ristamáki et al. ., 1994, 1995, Stauder et al., 1994, Heider et al., 1993, Horst et al., 1990a, b, Heider et al., 1996, Kaufmann et al., 1995, Mulder et al., 1994), it could be demonstrated with the present invention that the CD44 acms can also be applied to paraffin-embedded material when a microwave treatment is used. This procedure requires constant fixation and microwave treatment in order to provide reproducible results. For the validation of the immune reactivity that was obtained with material embedded in paraffin, the immunohistochemical analysis was carried out in parallel in frozen samples, and the same results were obtained. Additionally, the results of CD44vlO expression were confirmed by RT-PCR. For the expression of CD44v6, a correlation with a poor prognosis in NHL could be demonstrated (Koopman et al., 1993, Salles et al., 1993, Terpe et al., 1994, Ristamaki et al., 1994, Stauder et al., 1994), breast cancer (Kaufmann et al., 1995) and colon carcinomas (Heider et al., 1993). However, in the cases of NSHD investigated by the authors of the invention, only a few cases were CD44v6-positive, and could not establish any correlation with the prognosis, using two different antibodies against CD44v6. Since these two acms used against v6 recognize different epitopes of the amino acid sequence encoded by exon v6, this lack of detectable CD44v6 can not be explained in most cases (see Table 1) by a modification or masking of epitopes. In contrast to the frequent expression of CD44v5 in gastric adenocarcinomas (Heider et al., 1993), the data concerning the expression of CD44v5 within the three NSHD groups were not statistically significant. The vlO exon is - in addition to exons v3 and v6 - a variable exon that is constitutively expressed in lymphocytes (Stauder et al., 1994). Until now, the expression of CD44v10 in NHLs has not been systematically analyzed, and this exon was rarely detected in carcinomas (Heider et al., 1996). The present invention demonstrates in the example of two different antibodies against CD44vlO (VFF14 and VCFF16) a statistically significant high regulation of the expression of CD44v10 in HRS cells of NSHD with a poor prognosis (groups 2 and 3). In this case, the two exo vlO-specific antibodies showed identical results both on the surface and also (and / or) in the cytoplasm of the HRS cells. The detection of CD44vlO expression with 2 different acms is important since, for example in the case of breast carcinoma, divergent data were obtained from different authors who used different acms of the same specificity towards the exon (Friedrichs et al., 1995, Kaufmann et al., 1995). For further confirmation of the surprising results of the authors of the invention, all cases were immunostained inde pendently in another laboratory (using another method of staining) with identical results. The results for the expression of CD44vlO in HRS cells of NSHD are the first data showing a correlation of the expression of CD44vlO with the phase and the prognosis of the disease. The methods according to the invention therefore provide the doctor with valuable diagnosis and prognosis information for the disease in Hodgkin's lymphoma. In addition, CD44vlO is a suitable molecular target for therapeutic interventions in this disease. Table 1: Reactivity of HRS cells Group 1 Table 2: Reactivity (%) of CD44v10 (VFF14 and VFF16) and CD44v6 (VFF18) in HRS cells of Group 2 patients (relapse) Example 3: Use of vlO specific RT-PCR for diagnostic purposes Polymerase chain reaction (PCR) with reverse transcription Five cases allowed the isolation of mRNA, and were further analyzed by polymerase chain reaction with reverse transcriptase (RT-PCR). 1 μg of total RNA was isolated and reverse transcribed, as described in the literature (Günthert et al., 1991). 5 μl of the first strand cDNA were amplified with Taq polymerase (Promega, Madison, USA) in a volume of 50 μl using the buffer conditions that were recommended by the manufacturer. The concentration of the primer was 0.2 mM. In order to test the quality and periodicity of cDNA synthesis, a GAPDH-PCR was carried out with oligonucleotides that were homologous to positions 8-29 and 362-339 of the published GAPDH-cDNA sequence (Alien et al., 1987). To a previous incubation * for 5 min. at 95 ° C followed 25 rounds of amplification (30 s at 95 ° C, 1.5 min at 62 ° C) and an extension cycle of 7 min at 72 ° C. Then, 10 μl of the reaction was analyzed on a 2% agarose gel and the amplification product was visualized under UV light, once the gel had been stained with ethidium bromide. For the amplification of CD44vlO-containing cDNAs, primers were used that were homologous to the 3 'end of the vlO exon (positions 986-1013, Hofmann et al., 1991) and to the 5' -constant region of CD44 (positions 513-540). , Stamenkovic et al., 1989). For the amplification of isoforms with standard CD44 content, instead of the specific primer to CD44v10, a 3'-constant CD44 primer (positions 934-958, Stamenkovic et al., 1989) was used. After 40 cycles of amplification (94 ° C for 30 s, 62 ° C for 1.5 min), 10 μl of the reaction mixture was analyzed as above. For control purposes, distilled water (negative control) or a plasmid with CD44v3-vlO content (positive control, Heider et al., 1996) was used instead of RNA.

In the 5 cases in which RNA isolation was possible, the RT-PCR analysis confirmed the expression of CD44 isoforms with C 44v10 content (since these are only recently obtained samples, the subsequent evolution of the disease of these patient is not yet known). The amplified fragments correspond to transcripts of CD44 that present the constant portion of CD44 in combination with the variant vlO exon (460 bp band) or of the variant vlO exon plus another variant exon (660 bp band) (Figure 3, half of the right) . For control purposes, parallel cDNAs were amplified with primers that were specific for the 5 'and 3' constant region of CD44 (figure 3, half of the left), obtaining a dominant band of 440 bp that indicates the standard form of CD44. Genetic, molecular results correlate with immunohistochemical findings, where in all 5 cases a high proportion of HRS cells (which make up less than 10% of the total number of cells in a sample) expressed CD44vl0, and most of the cells (HRS plus non-tumor cells) reacted with the anti-CD44 antibody.

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LIST OF SEQUENCES (1) GENERAL DATA: (i) APPLICANT: (A) NAME: Boehringer Ingelheim International GmbH (B) STREET: Rheinstrasse (C) LOCALITY: Ingelheim (D) COUNTRY. Germany - (E) POSTAL CODE: 55216 (F) TELEPHONE: 06132-77-2770 (G) TELEFAX: 06132-77-4377 (ii) TITLE OF THE INVENTION: Procedure for diagnosis and therapy of lymphogranulomatosis (iii) NUMBER SEQUENCES: 4 (iv) LEGIBLE VERSION PER COMPUTER: (A) DATA SUPPORT: soft disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) LOGICAL EQUIPMENT: Patent In Relay No. 1.0, Version No. 1.30 (EPA) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERIZATION OF THE SEQUENCE: (A) LENGTH: 204 base pairs (B) TYPE: nucleotide (C) FORM OF THE CHAIN: both (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: DNA of the genome (ix) FEATURE: (A) NAME / KEY: exon (B) POSITION: 1 204 (C) OTHER DETAILS: / product = "Exon vlO of human CD44" / note = "accession number to the library L05419 / appointment = ([1]) (ix) FEATURE: (A) NAME / KEY: CDS (B) POSITION: 3 203 ( x) PUBLICATION INFORMATION: (A) AUTHORS: Screaton, GR Be ll, M V Jackson, D G Cornelis, F B Gerth, U Bell, J I (B) TITLE: The genomic structure of DNA encoding the lymphocyte receptor receptor reveals at least 12 exons cut and spliced (C) PUBLICATION: Proc. Nati Acad. Sci. U.S.A. (D) VOLUME: 89 (E) PAGES: 12160-12164 (F) DATE: December 1992 (G) IMPORTANT REMAINS IN SEQ ID NO. 1: FROM 1 TO 204 (xi) DESCRITION OF THE SEQUENCE: SEQ I D NO: 1: AT AGG AAT GAT GTC ACA GGT GGA AGA AGA GAC CCA AAT CAT TCT GAA 47 Arg Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn Hi s Ser Glu 1 5 10 15 GGC TCA ACT TTA CTG GAA GGT TAT ACC TCT CAT TAC CCA CAC AGG 95 Gly Ser Thr Thu Leu Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr 20 25 30 AAG GAA AGC AGG ACC TTC ATC CCA GTG ACC TCA GCT AAG ACT GGG TCC 143 Lys Glu Ser Arg Thr Phe lie Pro Val Thr Ser Wing Lys Thr Gly Ser 35 40 45 TTT GGA GTT ACT GCA GTT ACT GTT GGA GAT TCC AAC TCT AAT GTC ATT 191 Phe Gly Val Thr Ala Val Thr Val Gly Asp Ser Asn Ser Asn Val Asn 50 55 60 CGT TCC TTA TCA G 204 Arg Ser Leu Ser 65 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERIZATION OF THE SEQUENCE: (A) LENGTH: 67 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: protein (ix) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Arg Asn Asp Val Thr Gly Gly Arg Arg Asp Pro Asn His Ser Glu Gly 1 5 10 15 Be Thr Thr Leu Leu Glu Gly Tyr Thr Ser His Tyr Pro His Thr Lys 20 25 30 Glu Ser Arg Thr Phe He Pro Val Thr Ser Wing Lys Thr Gly Ser Ph 35 - 40 45 Gly Val Thr Wing Val Thr Val Gly Asp Ser Asn As Asn Val Asn Arg 50 55 60 Ser Leu Ser 65 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERIZATION OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleotide (C) CHAIN FORM: single chain (D) TOPOLOGY: linear (ii) TYPE OF THE MOLECULE: any other nucleic acid (A) DESCRIPTION: / desc = "PCR primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CAGGCTGGGA GCCAAATGAA GAAAATG 27 \ 2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERIZATION OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleotide (C) CHAIN FORM: single chain (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: any other nucleic acid (A) DESCRIPTION: / desc = "PCR primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: TGATAAGGAA CGATTGACAT TAGAGTTGGA It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property:

Claims (16)

1. RE I VI ND I CAC I ONE S 1. Procedure for the diagnosis or therapy of Hodgkin's lymphomas (lymphogranulomatosis), characterized in that this procedure is based on the expression of the variant vlO exon of the CD44 gene as a molecular marker. The method according to claim 1, characterized in that it is based on the binding of an antibody molecule to an epitope that is encoded by the variable vlO exon of the CD44 gene. 3. Method according to claim 2, characterized in that in this case an antibody molecule is used that recognizes the amino acid sequence according to SEQ ID NO. The method according to one of claims 1 to 3, characterized in that the antibody molecule is a monoclonal antibody, a Fab or F (ab ') 2 fragment of an immunoglobulin, a recombinantly produced antibody, a humanized chimeric antibody produced recombinantly or a single chain antibody (scFv). 5. Use of an antibody molecule that is specific for an epitope that is encoded by the variant vlO exon of the CD44 gene, in a method according to one of claims 1 to 4. 6. Use of an antibody molecule that is specific for an epitope within the amino acid sequence that is encoded by the variable vlO exon of the CD44 gene for the therapy of Hodgkin's lymphomas (lymphogranulomatosis). 7. Use according to claim 6, characterized in that the antibody molecule is fixed to the amino acid sequence according to SEQ ID NO. 2. Use according to one of claims 6 or 7, characterized in that the antibody molecule is a monoclonal antibody, a Fab or F (ab ') 2 fragment of an immunoglobulin, a recombinantly produced antibody, a humanized chimeric antibody. produced recombinantly or a single chain antibody (scFv). Use according to one of claims 6 to 8, characterized in that the antibody molecule is linked to a radioactive isotope, a radioactive compound, an enzyme, a toxin, a cytostatic agent, a prodrug, a cytokine or another polypeptide in a unmodulator. 10. Use of an antibody molecule that is specific for an epitope within the amino acid sequence that is encoded by the variable vlO exon of the CD44 gene, for the preparation of a pharmaceutical composition for the diagnosis and / or therapy of tumor diseases. 11. Use according to claim 10, characterized in that in the case of the tumor disease it is a Hodgkin lymphoma (lymphogranulomatosis). 12. Use according to claim 10 or 11, characterized in that the antibody molecule is fixed to the amino acid sequence according to SEQ ID NO: 2. 13. Antibody molecule that is specific for an epitope within the amino acid sequence, characterized in that it is encoded by the variable vlO exon of the CD44 gene, for pharmaceutical use. 14. The antibody molecule according to claim 13, characterized in that it is fixed to the amino acid sequence according to SEQ ID NO.
2. 2. The antibody molecule according to claim 13 or 14, characterized in that it is a monoclonal antibody, a Fab or F (ab ') 2 fragment of an immunoglobulin, a recombinantly produced antibody, a chimeric humanized antibody produced recombinantly or a single chain antibody (scFv). 16. The antibody molecule according to one of claims 13 to 15, characterized in that it is linked to a radioactive isotope, a radioactive compound, an enzyme, a toxin, a cytostatic agent, a prodrug, a cytokine or another immunomodulatory polypeptide.