AU726704B2 - Method of diagnosing and treating epithelioma - Google Patents

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S016704J.200 Boehringer Ingelheim International GmbH Case 12/178,181 Forschungszentrum Karlsruhe GmbH Process for the diagnosis and treatment of squamous cell carcinomas The invention relates to processes for the diagnosis and therapy of squamous cell carcinomas which are based on the expression of the variable exon v6 of the CD44 gene, agents for these processes and the use thereof.

It has recently been shown that the expression of variants of the surface glycoprotein CD44 is necessary and sufficient to trigger so-called spontaneous metastatic behaviour both in a non-metastasising pancreas-adenocarcinoma cell line of the rat and also in a non-metastasising fibrosarcoma cell line of the rat (Gtinthert et al., 1991).

Whereas the smallest CD44-isoform, the standard form CD44s, are expressed ubiquitously in a series of different tissues, including epithelial cells, certain splice variants of CD44 (CD44v) are expressed only in a subgroup of epithelial cells. The CD44-isoforms are produced by alternative splicing in such a way that the sequences of 10 exons (vl -v 10) in CD44s are excised completely, but may occur in different combinations in the larger variants (Screaton et al., 1992; Heider et al., 1993; Hofmann et al., 1991). The variants differ in that different amino acid sequences are inserted at a specific site of the extracellular part of the protein. Such variants can be detected in different human tumour cells and in human tumour tissue. Thus, the expression of CD44-variants in the course of colorectal carcinogenesis has recently been investigated (Heider et al., 1993). There is no expression of CD44-variants in normal human colon epithelium and only slight expression can be detected in the proliferating cells of the cavities. At later stages of the tumour progression, e.g. in adenocarcinomas, all malignant degenerations express variants of CD44. Moreover, the expression of CD44-splice variants has recently been demonstrated in activated lymphocytes and in non-Hodgkin's lymphomas (Koopman et al., 1993).

Various approaches have been adopted for making use of the differential expression of variant exons of the CD44-gene in tumours and normal tissue for diagnostic and therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0531300).

SThe expression of variant CD44-molecules in squamous cell carcinomas has also

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2 been investigated. Salmi et al. (1993) found, with the v6-specific antibody Var3.1, that there was a reduction in the v6-expression in tumour cells compared with normal cells.

With the v6-specific antibody 11.9, Brooks et al. (1995) obtained a heterogeneous staining ofnasopharyngeal carcinomas. Strong staining was achieved in only 2/12 cases, whilst in the majority of cases only a slight focal v6-expression could be detected by immunohistology.

The aim of the present invention was to develop new methods for the diagnosis and therapy of squamous cell carcinomas and to provide agents for such methods.

This objective has been achieved by means of the present invention. It relates to processes for the diagnosis and therapy of squamous cell carcinomas which are based on the expression of the variant exon v6 of the CD44 gene as a molecular marker or target. In particular, the present invention relates to processes based on the powerful homogeneous expression of v6 in squamous cell carcinomas, which was surprisingly detected, in contradiction to the teaching known from the prior art. Antibody molecules of corresponding specificity are particularly suitable as vehicles for selectively reaching squamous cell carcinomas in vivo.

The preferred processes are those characterised in that an antibody molecule is used which recognises the amino acid sequence QWFGNRWHEGYRQT, more preferably the amino acid sequence WFGNRWHEGYR. The monoclonal antibody BIWA-1 (clone VFF-18) secreted by a hybridoma cell line which was deposited on 7.6.1994 under Accession No. DSM ACC2174 at the DSM-Deutsche Sammlung fhr Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany (WO 95/33771), and derivatives of this antibody, are particularly preferred.

Other aspects of the present invention are the use of antibody molecules of this kind in the processes according to the invention and agents for carrying out these processes.

The nucleic and amino acid sequence of the variant exon v6 of the CD44-gene is known (Screaton et al., 1992, Talg et al., 1993). The existence of degenerate or allelic variants is of no significance to the implementation of the invention; such variants are therefore expressly included.

The sequence of exon v6 of the human CD44-gene is: SI t 3 Q A T P S S T T E E T A T Q TC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAG K E Q W F G N R W H E G Y R Q AAG GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA T P R E D S H S T T G T A ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G.

The invention may be carried out with polyclonal or monoclonal antibodies which are specific to an epitope coded by exon v6, particularly an epitope within the amino acid sequence QWFGNRWHEGYRQT, most preferably within the amino acid sequence WFGNRWHEGYR. The preparation of antibodies against known amino acid sequences may be carried out according to methods knownper se (Catty, 1989). For example, a peptide of this sequence may be prepared synthetically and used as an antigen in an immunisation procedure. Another method is to prepare a fusion protein which contains the desired amino acid sequence, by integrating a nucleic acid (which may be prepared synthetically or, for example, by polymerase chain reaction (PCR) from a suitable probe) which codes for this sequence into an expression vector and expressing the fusion protein in a host organism. The fusion protein, optionally purified, can then be used as an antigen in an immunisation procedure and insert-specific antibodies or, in the case of monoclonal antibodies, hybridomas which express insert-specific antibodies can then be selected by suitable methods. Processes of this kind are known in the art. Heider et al. (1993, 1996a) and Koopman et al. (1993) describe the preparation of antibodies against variant epitopes of CD44.

However, for the process according to the invention, it is also possible to use other antibody molecules, which are derived from poly- or monoclonal antibodies, e.g. Fab- or F(ab') 2 -fragments of immunoglobulins, single-chain antibodies (scFv) prepared by the recombinant method, chimeric or humanised antibodies and other molecules which bind specifically to epitopes coded by exon v6. Fab- or F(ab')2-fragments or other fragments may be produced, for example, from the complete immunoglobulin of the antibody BIWA-1 (VFF-18) or other antibodies (Kreitman et al., 1993). The skilled person is also in a position to produce recombinant v6-specific antibody molecules. In particular, after analysing the amino acid sequence of the antibody BIWA-1 (VFF-18) and/or using the hybridoma cell line which produces this antibody, particularly the genetic information contained therein, he can produce recombinant antibody molecules having the same idiotype as BIWA-1 (VFF-18), i.e. antibody molecules which have the same amino acid sequence in the region of the antigen binding site (complementarity-determining regions, CDR) as the antibody BIWA- 1 (VFF-18). Such processes are known in the art.

Recombinant antibody molecules of this kind may be, for example, humanised antibodies (Shin et al., 1989; Giissow and Seemann, 1991), bispecific or bifunctional antibodies (Weiner et al., 1993; Goodwin, 1989, Featherstone, 1996), single-chain antibodies (scFv, Johnson and Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992; Barbas et al., 1992) or antibodies produced by chain shuffling (Winter et al., 1994). Humanised antibodies may be prepared, for example, by CDR-grafting (EP 0239400). Framework regions can also be modified (EP 0519596; WO 9007861). For humanising antibodies, methods such as PCR (see for example EP 0368684; EP 0438310; WO 9207075) or computer-modelling (see for example WO 9222653) may be used nowadays. It is also possible to prepare and use fusion proteins, e.g. single-chain antibody/toxin fusion proteins (Chaudhary et al., 1990; Friedman et al., 1993). The terms "antibodies" and "antibody molecules" are intended to cover not only polyclonal and monoclonal antibodies but all the compounds discussed in this paragraph which can be structurally derived from immunoglobulins and may be prepared by methods known per se.

It is also within the capabilities of the average skilled person with a knowledge of the epitope (cf. Fig. 1, Fig. 4) of BIWA-1 (VFF-18), to produce equivalent antibodies with the same binding specificity. Such antibodies are therefore also included in the invention.

For diagnostic purposes, antibody molecules, preferably BIWA-1 antibody molecules, fragments thereof or recombinant antibody molecules with the same idiotype, may be linked for example with radioactive isotopes such as 1251, 1311, 1 1 1In, 9 9 mTc or radioactive compounds (Larson etal., 1991; Thomas etal., 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 purposes v6-specific antibody molecules, preferably BIWA-1 (VFF-18)antibody molecules or VFF-18-derived antibody molecules, e.g. fragments thereof or recombinant antibody molecules with the same idiotype, may be linked to radioisotopes such as 9 0 y, 1311, 18 6 Re, 18 8 Re, 1 53 Sm, 6 7 Cu, 2 12 Bi, 2 13 Bi, 17 7 Lu (Quadri et al., 1993; Lenhard et al., 1985, Vriesendorp et al., 1991; Wilbur et al., 1989, Maraveyas et al., 1995a, Jurcic et Scheinberg, 1994), toxins (Vitetta et al., 1991; Vitetta et Thorpe, 1991; Kreitman et al., 1993; Theuer et al., 1993), cytostatics (Schrappe et al., 1992), prodrugs (Wang et al., 1992; Senter et al., 1989), photoactivatable substances (Hemming et al., 1993), an antibody molecule with a different specificity or radioactive compounds. The antibody molecule may also be linked to a cytokine or some other immunomodulatory ,.polypeptide, e.g. with tumour necrosis factor, lymphotoxin (Reisfeld et al., 1996) or interleukin-2 (Becker et al., 1996). The antibody molecules may also be modified for use in a pretargetting system, e.g. with streptavidin or biotin (Goodwin, 1995).

Advantageously, the diagnostic process according to the invention may be used to investigate samples from patients, e.g. from biopsies, where there is a suspicion of squamous cell carcinoma or the diagnosis has already been made but the tumour needs to be further characterised. The detection of variant CD44-molecules which contain an amino acid sequence coded by the variable exon v6 may be carried out at the protein level using antibodies or at the nucleic acid level using specific nucleic acid probes or primers for polymerase chain reaction (PCR). The invention consequently also relates to antibody molecules and nucleic acids which are suitable as probes or primers for such processes, and the use of these antibodies and nucleic acids for diagnosing and analysing squamous cell carcinomas. For example, tissue sections can be examined immunohistochemically using antibodies by methods knownper se. Extracts taken from tissue samples or body fluids may also be investigated by other immunological methods using antibodies, e.g. in Western blots, enzyme-linked immunosorbent assays (ELISA, Catty and Raykundalia, 1989), radioimmunoassays (RIA, Catty and Murphy, 1989) or related immunoassays. The investigations may be qualitative, semi-quantitative or quantitative.

Apart from in vitro diagnosis, antibody molecules with specificity according to the invention are also suitable for the in vivo diagnosis of squamous cell carcinomas. If the antibody molecule carries a detectable label, the label can be detected for diagnostic purposes, e.g. for imaging the tumour in vivo or for radioguided surgery, for example. For using antibodies conjugated with radioactive isotopes for immunoscintigraphy (imaging), for example, there are numerous procedures which can be used by the skilled person as the basis for performing the invention (Siccardi et al., 1989; Keenan et al., 1987; Perkins and Pimm, 1992; Colcher et al., 1987; Thompson et al., 1984).

Data obtained by the detection and/or quantifying of the expression of the variant CD44-epitope v6 can thus be used for diagnosis and prognosis. It may be advantageous to combine these data with other prognostic parameters, e.g. the grade of tumour.

Antibody molecules having the specificity according to the invention, optionally combined with a cytotoxic agent, may advantageously be used for the treatment of squamous cell carcinomas. They may be administered systemically or topically, e.g. by intravenous route (as a bolus or continuous infusion) or by intraperitoneal, intramuscular, subcutaneous or other injection/infusion. Procedures for administering conjugated or nonconjugated antibodies (be they in the form of complete immunoglobulins, fragments, recombinant humanised molecules or the like) are known in 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). They may be used therapeutically, for example, in the same way as the antibody 1.1ASML (Seiter et al., 1993). Unmodified monoclonal antibodies can be used directly for therapeutic purposes if they have the intrinsic effector function suitable for a cytotoxic effect, e.g. for complementinduced or antibody-induced cell cytotoxicity (Riethmiiller et al. 1994). Suitable monoclonal antibodies for this application are murine antibodies of isotype IgG2a or antibodies of the human IgG -type. Unmodified antibodies may also be administered in order to induce the patient's own antitumoral reaction through an anti-idiotypic mechanism (Baum et al., 1993; Khazaeli et al., 1994).

According to a preferred embodiment of a therapeutic application, a humanised v6specific immunoglobulin or a F(ab') 2 fragment thereof is linked with 9 0 Y (Quadri et al., 1993; Vriesendorp et al., 1995), 1311 (Maraveyas et al., 1995a, 1995b; Juweid et al., 1995; Press et al., 1995; Thomas et al., in: Catty 1985, p. 230-239) 1 8 6 Re (Breitz et al., 1992, 1995) or another suitable radioisotope and used for radioimmunotherapy of squamous cell carcinomas. For example, the antibody BIWA-1, a humanised version of BIWA-1 or a F(ab') 2 fragment of BIWA-1 or the humanised antibody may be linked to 9 0 Y using a chelating linker such as ITCB-DTPA (isothiocyanatobenzyldiethylenetriaminepentaacetate), achieving a specific activity of 5-20 mCi/mg, preferably mCi/mg. This agent can then be administered to a patient with an antigen-positive tumour in a dose of 0.1 to 1 mCi/kg of body weight, preferably 0.3 to 0.5 mCi/kg of body weight. If the antibody molecule is linked to 1311, a possible dosage plan might be, for example, 2 x 150 mCi at 6 week intervals for a specific activity of 2 mCi/mg. The skilled person can determine the maximum possible dosages using methods known per se (Maraveyas et al., 1995a, 1995b). When the total quantity of protein to be administered is 2 to 5 mg it may be given in the form of a fast intravenous bolus injection. In the case of larger amounts of protein an infusion might be a more appropriate method of administration. With monoclonal antibodies it may be necessary to mix the agent with an excess a tenfold molar excess) of the non-radioactive antibody before administration; in this case, it is better to administer the agent in the form of an intravenous infusion, e.g.

over 15 minutes. This can be repeated. The therapy may be combined with external radiation therapy. It may also be backed up by bone marrow transplantation; this is particularly necessary when a dosage of more than 1.6 Gy is reached in the bone marrow during therapy.

Antibody molecules according to the invention may also be used ex vivo to purify CD34-positive stem and precursor cell preparations (immunopurging). Radiation or chemotherapy of squamous cell carcinomas may also be backed up with autologous bone marrow transplantation. The preparation of haematopoietic stem and precursor cells thus administered must be free from tumour cells. This can be achieved by incubation with antibody molecules according to the invention, e.g. antibody-toxin conjugates (Myklebust et al., 1994; DE P 196 48 209.7).

Antibody molecules according to the invention may also be introduced into the T-cell receptor of T-lymphocytes in the form of recombinant constructs. Reprogrammed Tlymphocytes of this kind bind selectively to the antigen-expressing tumour cells and develop a cytotoxic activity, with the result that they can be used for the treatment of squamous cell carcinomas (PCT/EP9604631; Altenschmidt et al., 1996).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

a a a C a. Figures Fig. 1: Determining the epitope specificity ofBIWA-1 by binding to synthetic peptides derived from the human CD44v6 sequence. The corresponding peptide from rat CD44v6 was tested with the antibody 1.1ASML. Binding was determined in an ELISA in which the peptides were immobilised on microtitre plates (cf. Heider et al., 1996b, Fig. 2).

no binding, slight binding, strong binding.

Fig. 2: Immunohistochemical analysis of a squamous cell carcinoma of the larynx and a liver metastasis of a carcinoma of the oesophagus with the CD44v6-specific monoclonal antibody BIWA-1. In both cases the reactivity of the antibody with the membrane of the tumour cells can be observed. Original magnification counterstaining haematoxylin.

Fig. 3: Comparison of antigen binding of various CD44v6-specific mAbs. The binding of four different CD44v6-specific mAbs to human SCC A-431-cells was measured in a cell ELISA. MAb BIWA-1 shows a higher affinity for the tumour cells than the other mAbs.

Fig. 4: Refined epitope mapping of the mAb BIWA-1. The binding of BIWA-1 to various overlapping synthetic peptides which span the amino acids 18-32 of the CD44v6coded region was measured by competitive ELISA. The minimum binding sequence (peptide v6 (19-29)) is underlined.

Fig. 5: Biodistribution of' 2 5 I-BIWA-1 in A-431 xenotransplanted nude mice. The accumulation of the antibody is given as ID/g (mean SEM) at 4, 24, 48, 120 and 168 hours post injection.

Examples Example 1: Expression of CD44v6 in squamous cell carcinoma Tissue A total of 126 cases of paraffin-embedded tumour samples were analysed immunohistochemically with the mAb BIWA-1 (clone VFF-18) for expression of CD44v6.

The samples included 31 cases of primary squamous cell carcinomas (15 cases larynx, 16 cases skin), 91 cases of lymph node metastases (larynx, n=38; lung, n=27; oesophagus, n= 11; oral cavity, n= 11; tonsils, n=4) and 4 cases of liver metastases (oesophagus).

Antibodies The total 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 PCR primers 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3', positions 25-52, and 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3', positions 1013-984 of the LCLC97-variant region as described by Hofmann et al., contained an EcoRI recognition site which was used in order to clone the PCR product directly into the vector pGEX-2T (Smith et al., 1988). The resulting construct (pGEX CD44v HPKII, v3-vl0) codes for a fusion protein of -70 kD, consisting of glutathione-S-transferase from Schistosoma japonicum and the exons v3-vl0 of human CD44 (Fig. 1; Heider et al., 1993). The fusion protein was expressed in E. coli and then subjected to affinity purification over glutathioneagarose (Smith et al., 1988).

Female Balb/c mice were intraperitoneally immunised with the affinity-purified fusion protein according to the following plan: 1st immunisation: 90 pg of fusion protein in complete Freund's adjuvant 2nd and 3rd immunisations: 50 tg of fusion protein in incomplete Freund's adjuvant.

The immunisations were given at intervals of 4 weeks. 14 days after the last immunisation the animals were immunised on three successive days with 10 pg of fusion protein in PBS. On the following day, the spleen cells from an animal with a high antibody titre were fused with P3.X63-Ag8.653 mouse myeloma cells using polyethyleneglycol 4000. The hybridoma cells were then selected in microtitre plates in HAT-medium (Kihler and Milstein, 1975; Keamey et al., 1979).

Measurement of the antibody titre in the serum or screening of the hybridoma supernatants were carried out using an ELISA. In this test, first of all microtitre plates were covered with fusion protein (GST-CD44v3-10) or only with glutathione-Stransferase. They were then incubated with serial dilutions of serum samples or hybridoma supematants and the specific antibodies were detected using peroxidase-conjugated antibodies against mouse immunoglobulin. Any hybridomas which reacted only with glutathione-S-transferase were discarded. The remaining antibodies were first characterised in an ELISA with domain-specific fusion proteins (exon v3, exon v5 v6, exon v6 v7, exon v8 v10) (Koopman et al., 1993). Their immunohistochemical reactivity was tested on human skin sections.

BIWA-1 (VFF-18; for preparation and properties see also WO 95/33771) bound only to fusion proteins which contained a domain coded by the exon v6. In order to further restrict the epitope of the antibody, various synthetic peptides which represented parts of the v6 domain were used in ELISA binding assays (Fig. The 14 amino acid peptide v6D showed the strongest binding. Consequently, the epitope of BIWA-1 is wholly or partly within the sequence QWFGNRWHEGYRQT of the domain coded by exon v6. This sequence is homologous to the binding epitope of the antibody 1.1ASML, which was used in a therapeutic rat model and which is specific to rat CD44v6 (Fig. 1).

Immunohistochemistry Before incubation with the primary antibody, paraffin sections (4 ptm) in Rotihistol (Roth, Germany) were deparaffinated 3 times for 10 minutes each time and then rehydrated in a rising alcohol series. The sections were briefly washed with distilled water and then cooked in a microwave oven (Sharp Model R-6270) 3 times for 10 minutes each time at 600 Watts in 0.01 M Na-citrate buffer. After each microwave incubation the sections were cooled for 20 minutes. After the final cooling stage the carriers were washed in PBS and pre-incubated with normal goat serum (10% in PBS). After 3 washes in PBS the sections were incubated with primary antibody (BIWA-1: 5 pg/ml; murine IgG (isotypecorresponding negative control) 5 jig/ml in PBS/1% BSA) for 1 hour. The positive control used for the staining reaction consisted of normal human skin sections, as keratinocytes express a CD44-isoform which contains v3-v10. Endogenous peroxidases were blocked with 0.3% H 2 0 2 in PBS, and the sections were incubated for 30 minutes with the biotinylated secondary antibody (anti-mouse IgG-F(ab') 2 DAKO Corp.). In order to 11 develop the stain, the sections were incubated for 30 minutes with horseradish peroxidase which was coupled to biotin as a streptavidin-biotin-peroxidase complex (DAKO Corp.).

The sections were then incubated for 5-10 minutes in 3 3 -amino-9-ethyl-carbazole substrate (Sigma Immunochemicals), the reaction was stopped with H 2 0 and the sections were counterstained with haematoxylin. The stains were evaluated using a Zeiss Axioskop Light Microscope and the colour intensities were quantified as follows: strong expression; moderate expression; weak expression; unclear or no expression detected. Only tumour cells with a clear membrane stain were evaluated as positive. The percentage of positive tumour cells in each section was roughly estimated and two groups were formed: focally positive tumours (less than 10% of the tumour cells reacted with the antibody) and positive tumours (10 or more of the tumour cells positive). If fewer than of the tumour cells in the positive cells reacted with the antibody, the corresponding percentage was indicated.

126 cases of squamous cell carcinomas of various origins were analysed using the CD44v6-specific monoclonal antibody BIWA-1. The expression of isoforms containing CD44v6 was observed in all but one tumour sample. The majority of the samples exhibited expression of the antigen on 80-100% of the tumour cells and the staining was restricted to the membrane of the tumour cells. No reaction was observed with stroma tissue, lymphocytes, muscle cells or endothelium.

In order to quantify the expression of CD44v6-molecules on these tumour cells, sections of normal human skin were stained parallel to the tumour sections. Normal skin keratinocytes express high levels of CD44-isoforms and are among the strongest expressors of CD44v6 of the normal cells which have been described hitherto. Consequently, keratinocyte staining was used as the reference and classified as "strong" in our system of evaluation. In the majority of the tumour samples examined, the staining of the tumour cells was comparable with or even greater than the staining of the skin keratinocytes, with only a few cases showing weak tumour staining (3 cases of lymph node metastasis) or moderate tumour staining (2 primary carcinomas, 10 metastases). The staining reaction was very homogeneous within a given tumour section, with the majority of tumour cells in the section having the same stain intensity. No significant differences were observed in the CD44v6-expression pattern between the primary tumours and metastases. A detailed summary of the results is shown in Table 1 with the Examples shown in Fig. 2.

Table 1: Expression of CD44v6 in squamous cell carcinomas Sample Type of tumour BIWA-1 Reactivity 46937 86 Primary Larynx 4687 90 Primary Larynx 8372 90 Primary Larynx 17427 90 Primary Larynx 27298 90 Primary Larynx 46908 90 Primary Larynx 51334 90 Primary Larynx 51402 91 Primary Larynx 60414 91 Primary Larynx 61733 91 Primary Larynx 12280 92 Primary Larynx 23140 92 Primary Larynx 31792 92 Primary Larynx 32214 92 Primary Larynx 10209 95 Primary Larynx 2366 86 Primary Skin 2574 86 Primary Skin 9916 86 Primary Skin 2696 87 Primary Skin 8906 87 Primary Skin 8191 88 Primary Skin 8354 88 Primary Skin 11963 88 Primary Skin 5590 90 Primary Skin 530 92 Primary Skin 2583 94 Primary Skin 11337 94 Primary Skin 10901 95 Primary Skin 11557 95 Primary Skin 11744 95 Primary Skin 11917 95 Primary Skin 4688 90 I Lymph node metastasis Larynx 4688 90 II Lymph node metastasis Larynx 8374 90 Lymph node metastasis Larynx 17428 90 Lymph node metastasis Larynx 27300 90 Lymph node metastasis Larynx 36942 90 Lymph node metastasis Larynx 46909 90 Lymph node metastasis Larynx 51336 90 Lymph node metastasis Larynx 41108 91 Lymph node metastasis Larynx 51398 91 Lymph node metastasis Larynx 60416 91 Lymph node metastasis Larynx 61734 91 Lymph node metastasis Larynx 1318 92 I Lymph node metastasis Larynx 1318 92 II Lymph node metastasis Larynx 1318 92 III Lymph node metastasis Larynx 1318 92 IV Lymph node metastasis Larynx 2863 92 I Lymph node metastasis Larynx 2863 92 II Lymph node metastasis Larynx 5745 92 I Lymph node metastasis Larynx 5745 92 II Lymph node metastasis Larynx 8969 92 I Lymph node metastasis Larynx 8969 92 II /Lymph node metastasis Larynx 8969 92 III Lymph node metastasis Larynx 8969 92 IV Lymph node metastasis Larynx 8969 92 2/I Lymph node metastasis Larynx 8969 92 2/II Lymph node metastasis Larynx 8969 92 2/III Lymph node metastasis Larynx 8969 92 2/IV Lymph node metastasis Larynx 9366 92 Lymph node metastasis Larynx A 9509 92 9566 92 12283 92 14046 92 31787 92 49228 92 29228 93 29829 93 29804 95 15293 91 1667 92 2757 92 2757 92 2757 92 2757 92 4790 92 6168 92 6168 92 6168 92 6168 92 1 7206 92 7531 92 I 7531 92 I 7531 92 I 7531 92 I 10324 92 10519 92 I 10519 92 F

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Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Lymph node metastasis Larynx Larynx Larynx Larynx Larynx Larynx Larynx Larynx Larynx Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung Lung I f 13055 92 Lymph node metastasis Lung 13055 92 II Lymph node metastasis Lung focal 13055 92 III Lymph node metastasis Lung 15663 92 Lymph node metastasis Lung 16713 92 Lymph node metastasis Lung 14980 91 I Lymph node metastasis Oesophagus 14980 91 II Lymph node metastasis Oesophagus 16641 91 I Lymph node metastasis Oesophagus 16641 91 II Lymph node metastasis Oesophagus 16641 91 III Lymph node metastasis Oesophagus 1059 92 Lymph node metastasis Oesophagus 1710 92 I Lymph node metastasis Oesophagus 1710 92 II Lymph node metastasis Oesophagus 1710 92 III Lymph node metastasis Oesophagus 11502 92 I Lymph node metastasis Oesophagus 11502 92 II Lymph node metastasis Oesophagus 202 92 Lymph node metastasis Oral cavity 6030 92 Lymph node metastasis Oral cavity 7335 92 I Lymph node metastasis Oral cavity 7335 92 II Lymph node metastasis Oral cavity 15324 92 II Lymph node metastasis Oral cavity 16164 92 I Lymph node metastasis Oral cavity 16164 92 II Lymph node metastasis Oral cavity 16412 92 Lymph node metastasis Oral cavity 16836 92 I Lymph node metastasis Oral cavity 16836 92 II Lymph node metastasis Oral cavity 16836 92 III Lymph node metastasis Oral cavity 6228 92 I Lymph node metastasis Tonsil 6228 92 II Lymph node metastasis Tonsil 6618 92 Lymph node metastasis Tonsil 11840 92 Lymph node metastasis Tonsil 14172 91 4 Liver metastasis Oesophagus 14172 91 5 Liver metastasis Oesophagus 4131 94 1 Liver metastasis Oesophagus 8438 94 Liver metastasis Oesophagus focal 80-100% of the tumour cells reacted positively with BIWA-1. In cases where fewer tumour cells reacted with the antibody, the percentage obtained is given.

Example 2: Expression of CD44v6 in kidney cell carcinomas, prostate carcinomas and liver metastases of colon carcinomas Tissue 19 cases of kidney cell carcinomas (12 cases of clear cell, 5 cases of chromophilic, 1 case of chromophobic, 1 oncocytoma), 16 primary adenocarcinomas of the prostate and 19 cases of lymph node metastases of prostate carcinoma, and 30 cases of liver metastases of colon carcinomas were analysed.

Antibody BIWA-1 (see Example 1).

Immunohistochemistry For method see Example 1.

In contrast to the squamous cell carcinomas, no or only focal expression of CD44v6-isoforms could be detected in the majority of the kidney cell and prostate carcinomas investigated. In the case of a more than focal expression in the prostate carcinomas the staining was predominantly diffusely cytoplasmic and weak or heterogeneous, compared with the staining of normal prostate epithelium. In 50% of the liver metastases of colon carcinomas investigated, a more than focal expression of CD44v6 isoforms was detected. The staining in the majority of cases was faint to moderate, but generally fewer than 100% of the tumour cells in a sample showed any staining with BIWA-1. The results are summarised in Table 2.

Table 2: Expression of CD44v6 in prostate adenocarcinomas, kidney cell carcinomas and liver metastases of colorectal carcinomas Type of tumour Prostate Primary n 16 BIWA-1 Reactivity negative focal pos.

8 3 positive adenocarcinoma Prostate adenocarcinoma Kidney cell carcinoma Colorectal Lymph node 19 metastases Primary 19 Liver metastases 30 15 15 2 2 17 0 2 7 8 carcinoma Example 3: Characterisation ofCD44v6-specific antibodies Cell line The human SCC cell line A-431 (spontaneous epidermoid carcinoma of the vulva) was obtained from the American Type Culture Collection (Rockwell MD) and cultured in accordance with the manufacturer's instructions. The surface expression of CD44v6containing isoforms was determined by FACS analysis, using an FITC-linked mAb BIWA-1.

Analysis of the kinetic constants The affinity and kinetics of the monoclonal antibody CD 4 4v6-interaction was determined by Surface Plasmon Resonance (SPR), using a BIAcore 2000 system (Pharmacia Biocensor). A glutathione-S-transferase-CD44-fusion protein which contained the region coded by the exons v3-v10 (GST/CD44 v3-v10) was immobilised on a Sensor Chip, the amine coupling method being carried out in accordance with the manufacturer's instructions. Antibodies in various concentrations (8-132 nM) in HBS mM HEPES pH 7.4, 150 mM sodium chloride, 3.4 mM EDTA, 0.05% BIA core Ssurfactant P20) was injected over the antigen-specific surface at a flow rate of 5 pl/min.

The interaction was recorded as a change in the SPR signal. Dissociation of the antibody was observed for 5 minutes in the buffer flow (HBS). The surface of the chip was regenerated with a single pulse of 15 tl 30 mM HC1. The data analysis and calculation of the kinetic constants were carried out using the Pharmacia Biocensors BIA Evaluation Software, Version 2.1.

In this way, the antigen affinity of BIWA-1 was compared with other CD44v6specific mAbs (VFF4, VFF7, BBA-13 (IgGI, R&D Systems, Abingdon, Kinetic and affinity constants of the various antibodies were determined in two independent experiments. Table 3 shows the values of the association rates dissociation rates (kd) and dissociation constants (Kd) for the 4 mAbs. All the mAbs showed similar ka and kd, with the exception of BBA-13, which has a 3-times lower ka and VFF7, which has a significantly higher dissociation rate (factor 5) compared with the other mAbs. This results in a lower binding affinity for VFF7 and BBA-13 compared with VFF4 and BIWA-1.

BIWA-1 shows the lowest Kd of all the antibodies investigated.

Table 3: Kinetic and affinity constants of various CD44v6-specific mAbs Antibody ka (M-s- 1 kd(s- 1 Kd (M) VFF4 1.1 x 10 5 2.6 x 10 5 2.4 x 10-10 VFF7 1.1 x 10 5 1.2 x 10 4 1.1 x 10- 9 BIWA-1 1.3 x 10 5 2.2 x 10- 5 1.7 x 10-10 BBA-13 3.7 x 104 2.3 x 10- 5 6.2 x 1010 Analysis of the antibody-protein interaction using ELISA.

CD44v6 expressing A-431 cells were cultivated in 96-well plates (Falcon Microtest III, Becton Dickinson, Lincoln Park, NJ) in numbers of 5 x 104 per well in RPMI 1640 with 10% foetal calf serum overnight at 37°C. After washing with PBS/0.05% Tween 20 the cells were fixed for 1 minute with ice cold ethanol, followed by a washing step. Incubation with the primary antibodies (VFF4, VFF7, BIWA-1, BBA-13, 1 ng/ml to 600 ng/ml, in each case in assay buffer: PBS/0.5% BSA/0.05% Tween 20) was carried out for 1 hour at ambient temperature and followed by 3 washing steps. The secondary antibody used was a rabbit-antimouse-IgG horseradish peroxidase-conjugated antibody (DAKO Corporation, .t I1 19 Copenhagen, Denmark; dilution 1:6000 in assay buffer) (1 hour at room temperature).

After 3 washing steps the colour was developed using TMB solution (Kirkegaard and Perry, Gaithersburg, USA). The extinction was measured using a Hewlett-Packard

ELISA

Reader.

Figure 3 shows that the relative affinities of the antibodies as determined by BIAcore analysis are reflected in their interaction with the tumour cells, with BIWA-1 clearly showing the highest binding affinity.

The protein domain which is coded by the CD44-exon v6 consists of 45 amino acids (Figure In order to define more accurately the epitope which is recognised by BIWA-1, a series of synthetic peptides were used in ELISA assays. Preliminary experiments showed binding to a centrally located 14-mer (amino acid groups 18-31; Figure 4; cf. also Figure 1) but not to peptides outside this region. A second series of peptides were therefore synthesised and tested in competitive ELISAs (Figure The results show that the peptide 19-29 (WFGNRWHEGYR) represents the minimum structure required for high affinity binding. Elimination of the C-terminal arginine groups resulted in a more than 100 times weaker binding.

Example 4: Biodistribution of radio-iodinated CD44v6-antibodies in xenotransplantcarrying nude mice A-431-Xenotransplant model 8 week old female BALB/c nu/nu nude mice (B K Universal, Renton, WA) were subcutaneously injected in the left-hand median line with 5 x 106 cultivated A-431 cells (human epidermoid carcinoma of the vulva). Xenotransplanted animals carrying A-431 tumours were used for biodistribution experiments within two weeks (weight of tumours: 40-50 mg).

Radio-iodination of BIWA-1 Protein G-purified mAb BIWA-1 (murine IgG1) was coupled to streptavidin, using the heterobifunctional crosslinker succinimidyl 4 -(N-maleimido-methyl)cyclohexane-1carboxylate. Streptavidin-lysyl groups were linked to reduced antibody-cysteinyl groups produced by preliminary treatment of the antibody with dithiothreitol. The 1:1 conjugates obtained 90%) were further purified by ion exchange chromatography. For Sbiodistribution experiments, BIWA-1/SA was labelled via primary amines of lysine with 125I, using p-iodophenyl labelling reagent (PIP; NEN Dupont, Wilmington, DE), followed by the process of Willbur et al. (1989). Labelling BIWA-1 with SA or 125I did not affect the immunoreactivity or the pharmacokinetics of the antibody in mice.

Biodistribution experiments Nude mice which were xenotransplanted with human A-431 tumours were intravenously injected through the lateral caudal vein with 5-7 ptCi 125I on 50 pg ofmAb BIWA-1 (specific activity 0.1-0.14 mCi/mg). Time-biodistribution studies were carried out in groups of n=3 animals for each point of time at 4, 24, 48, 120 and 168 hours postinjection. At selected times, mice were weighed, blood was extracted from the retro-orbital plexus and they were killed by cervical dislocation. Nine organs and tissues were collected and weighed: blood, tail, lung, liver, spleen, stomach, kidney, intestine and tumour. The radioactivity in the tissues was counted in a gamma-scintillation counter (Packard Instrument Company, Meriden, CT) by comparison with standards of the injected antibody preparation, the energy window being set at 25-80 keV for 1 25 1. The percentage of injected dose per gram of tissue was calculated ID/g).

Preliminary experiments had shown that BIWA-1 did not cross-react with murine CD44v6-antigen. Table 4 and Figure 5 show the absorption of radioactivity in tumours and normal tissue. Iodinated BIWA-1 showed a rapid tumour absorption injected dose/g at 4 hours post-injection) which increased to more than 18% ID/g after 48 hours and then remained constant for up to 120 hours. Seven days post-injection (168 hours) the tumour still contained 15.3% ID/g of tissue. Tumour:tissue ratios were calculated for individual times and these are shown in Table 4. At 24 hours post-injection the tumour:blood ratio was 0.48 and increased to 3.16 on day 7. The uptake in normal tissue was low and most probably caused by blood-pool background in the tissue biopsies. Selective in vivo targeting of human SCC-xenotransplants in nude mice with 12 5 I-labelled BIWA-1 shows that this monoclonal antibody has a high potential as a targeting vehicle for diagnostic and therapeutic use in SCC patients.

Table 4: Tumour:tissue ratios of 25 I-BIWA-1 in various times post-injection A-431-tumour-carrying nude mice at Ratio of tumour to 24h 48h 120h 168h Blood 0.22a 0.48 1.31 2.60 3.16 Tail 1.18 2.62 7.70 12.28 13.06 Lung 0.40 1.03 2.65 7.04 4.82 Liver 0.94 1.18 2.28 3.57 3.24 Spleen 1.40 1.84 4.00 4.86 4.42 Stomach 3.89 7.37 19.40 25.56 33.96 Kidney 0.82 1.31 2.72 2.79 2.53 Intestine 3.54 6.24 11.94 19.24 27.78 a mean values SD are <7% Example 5: Different expression of CD44v6 in a large number of human tumours In a wider investigation, a total of 544 tumour samples were examined immunohistochemically with the monoclonal antibody BIWA 1 (clone VFF-18) for the expression of CD44v6. The samples were either embedded in paraffin or frozen in liquid nitrogen immediately after surgical removal and stored at -70 0 C until required. The following tumours were analysed: basalioma adenocarcinoma (AC) of the breast AC of the colon squamous cell carcinomas (SCC) of the head and neck (n=125), lung carcinomas (n=120), prostate AC kidney cell carcinomas (n=27), SCC of the skin (n=l 5) and AC of the stomach The tissues were obtained by routine surgery or biopsy and normal tissue was obtained to accompany the tumour samples. The immunohistochemical investigations were carried out as in Example 1.

Table 5 shows a summary of the immunohistochemical analysis of 397 different types of tumour with the mAb BIWA 1.

22 Table 5: Expression of CD44v6 in human tumours Type Total number Positive cases* n% Basalioma Primary tumour 16 10 62 Breast AC Primary tumour 17 15 88 Lymph node metastases 34 31 91 Liver metastases 4 4 100 Colon AC Lymph node metastases 51 21 41 Liver metastases 26 13 Brain metastases 6 6 100 Larynx SCC Lymph node metastases 18 18 100 Lung AC Primary tumour 35 15 43 Lung SCC Primary tumour 9 9 100 Oesphagus Primary tumour 20 20 100 C. Prostate AC

RCC

SCLC

Stomach AC Primary tumour Lymph node metastases Primary tumour Primary tumour Primary tumour Lymph node metastases Liver metastases Total number 16 18 27 31 22 43 4 397 5 0 5 7 15 16 4 31 0 18 23 68 37 100

I

2 10% of tumour cells positive AC: adenocarcinoma; RCC: renal cell carcinoma SCLC: small cell lung cancer; SCC: squamous cell carcinoma In small cell lung cancers, renal cell carcinomas and AC of the prostate, no or only little reactivity was observed. All the other types of tumour investigated expressed CD44v6-containing isoforms to varying degrees. The majority of the AC of the breast investigated showed reactivity with BIWA 1, and the SCC tested (larynx, lung and oesophagus) expressed CD44b6 in 100% of all cases.

185 additional cases of SCC of various types and classification were investigated for their reactivity with BIWA 1. These included 67 cases of primary SCC (larynx, oral cavity, n=16; oropharynx, n=3; skin, n=15), 77 samples of lymph node metastases (larynx, n=12; lung, n=27; oesphagus, n=l 1; oral cavity, n=6; oropharynx, n=7; hypopharynx, n=10; tonsil, and 3 samples of liver metastases (oesphagus). Table 6 summarises the immunohistochemical analysis of all the SCC samples investigated.

Table 6: Expression of CD44v6 in squamous cell carcinomas Type Total number Negative Focal pos. Positive n n n Hypopharynx LNM 10 0 0 0 0 10 100 Oropharynx PT 3 0 0 0 0 3 100 LNM 7 0 0 0 0 7 100 Larynx PT 15 0 0 0 0 15 100 LNM 30 1 3 0 0 29 97 Lung PT 18 2 11 0 0 16 89 LNM 27 0 0 1 4 26 96 Oesphagus PT 20 0 0 1 5 19 LNM 11 0 0 0 0 11 100 LM 3 0 0 0 0 3 100 Oral cavity PT 16 0 0 0 0 16 100 LM 6 0 0 0 0 6 100 Skin PT 15 0 0 0 0 15 100 Tonsil LNM 4 0 0 0 0 4 100 Total number 185 Focal pos.: <10% of tumour cells positive; LNM: lymph node metastases; PT: primary tumour; LM: liver metastases Expression of CD44v6 containing isoforms was found in all but three tumour samples (one case of larynx, 2 cases of lung). The majority of the samples showed expression of the antigen on 80 to 100% of the tumour cells within a single section, the staining being concentrated chiefly on the membrane of the tumour cells. The most homogeneous staining pattern was found in carcinomas of the larynx, oesphagus and hypopharynx, with the majority of tumour cells in the section having the same intensity of staining.

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I, 32 SEQUENCE LISTING GENERAL DATA:

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NAME: Boehringer Ingelheim International GmbH STREET: Rheinstrasse TOWN: Ingelheim COUNTRY: Germany POSTCODE: 55216 TELEPHONE: +49-(0)-6132-772770 FAX: +49-(0)-6132-774377 NAME: Forschungszentrum Karlsruhe GmbH STREET: Postfach 3640 TOWN: Karlsruhe COUNTRY: Germany POSTCODE: 76021 NAME: Heider, Karl-Heinz STREET: Hervicusgasse 4/3/21 TOWN: Vienna COUNTRY: Austria POSTCODE: 1120 NAME: Adolf, Guenther STREET: Stiftgasse 15-17/10 TOWN: Vienna COUNTRY: Austria POSTCODE: 1070 NAME: Ostermann, Elinborg STREET: Mauerbachstr. 56/6 TOWN: Vienna COUNTRY: Austria fT2 POSTCODE: 1140 NAME: Patzelt, Erik STREET: Hans-Buchmueller-Gasse 8 TOWN: Purkersdorf COUNTRY: Austria POSTCODE: 3002 NAME: Sproll, Marlies STREET: Schwenkgasse 3 TOWN: Vienna COUNTRY: Austria POSTCODE: 1120 (ii) TITLE OF THE INVENTION: Process for the diagnosis and treatment of squamous cell carcinomas (iii) NUMBER OF SEQUENCES: 16 (iv) COMPUTER-READABLE

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AUTHORS: Screaton, GR Bell, MV Jackson, DG Cornelis, FB Gerth, U Bell, JI TITLE: Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons JOURNAL: Proc. Natl. Acad. Sci. U.S.A.

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DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996

W

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAG AAG 47 Gin Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys 1 5 10 GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA ACA CCC Glu Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro 25 AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G 129 Arg Glu Asp Ser His Ser Thr Thr Gly Thr Ala DATA RELATING TO SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids NATURE: amino acid TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gln Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys Glu 1 5 10 Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro Arg 25 Glu Asp Ser His Ser Thr Thr Gly Thr Ala DATA RELATING TO SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr 1 5 DATA RELATING TO SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs NATURE: Nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: other nucleic acid DESCRIPTION: /desc "PCR primer" PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CAGGCTGGGA GCCAAATGAA GAAAATG 27 DATA RELATING TO SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs NATURE: Nucleotide STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: other nucleic acid DESCRIPTION: /desc "PCR primer" PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGATAAGGAA CGATTGACAT

TAGAGTTGGA

*I L 38 DATA RELATING TO SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg 1 5 DATA RELATING TO SEQ ID NO: 7: SEQUENCE

CHARACTERISTICS:

LENGTH: 43 amino acids NATURE: amino acid STRAND. FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Gin Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys Glu 1 5 10 Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro Arg 25 Glu Asp Ser His Ser Thr Thr Gly Thr Ala Ala DATA RELATING TO SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 L (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys 1 5 DATA RELATING TO SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Glu Glu Thr Ala Thr Gin Lys Glu Gin Trp 1 5 DATA RELATING TO SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 11 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Thr Ala Thr Gln Lys Glu Gln Trp Phe Gly Asn 1 5 DATA RELATING TO SEQ ID NO: 11: SEQUENCE

CHARACTERISTICS:

LENGTH: 14 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr 1 5 DATA RELATING TO SEQ ID NO: 12: SEQUENCE

CHARACTERISTICS:

LENGTH: 11 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro 1 5 DATA RELATING TO SEQ ID NO: 13: SEQUENCE

CHARACTERISTICS:

LENGTH: 11 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide i k 43 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Glu Gly Tyr Arg Gin Thr Pro Arg Glu Asp Ser 1 5 DATA RELATING TO SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Thr Pro Arg Glu Asp Ser His Ser Thr Gly DATA RELATING TO SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 42 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION

INFORMATION:

DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Trp Ala Asp Pro Asn Ser Thr Thr Glu Glu Ala Ala Thr Gln Lys Glu 1 5 10 Lys Trp Phe Glu Asn Glu Trp Gln Gly Lys Asn Pro Pro Thr Pro Ser 25 Glu Asp Ser His Val Thr Glu Gly Thr Thr DATA RELATING TO SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids NATURE: amino acid STRAND FORM: single strand TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 195 45 472.3 FILING DATE: 06-DEC-1995 PUBLICATION INFORMATION: DOCUMENT NUMBER: DE 196 15 074.4 FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Lys Trp Phe Glu Asn Glu Trp Gln Gly Lys Asn Pro Pro Thr 1 5

Claims (12)

1. A method for treating squamous cell carcinomas, characterised in that this method is based on the expression of the variable exon v6 of the gene CD44 as the molecular marker.

2. Method according to claim 1, characterised in that it is based on the binding of an antibody molecule to an epitope which is coded by the variable exon v6 of the gene CD44.

3. Method according to claim 2, characterised in that an antibody molecule is used which recognises the amino acid sequence WFGNRWHEGYR.

4. Method according to claim 3, characterised in that the monoclonal antibody BIWA-1 (VFF-18), which is formed by the hybridoma cell line with the Accession Number DSM ACC2174, or a derivative of this antibody is used. Method according to one of claims 1 to 4, characterised in that the antibody molecule is a monoclonal antibody, a Fab- or F(ab') 2 -fragment of an immunoglobulin, an antibody prepared by recombinant methods, a chimeric or humanised antibody prepared by recombinant methods, a bifunctional or a single-chain antibody (scFv). p

6. Use of an antibody molecule which is specific to an epitope which is coded by the variant exon v6 of the CD44-gene, in a method according to one of claims 1 to 4. S 7. Use according to claim 6, characterised in that the antibody molecule binds to the amino acid sequence WFGNRWHEGYR.

8. Use according to claim 6 or 7, characterised in that the antibody molecule is the monoclonal antibody BIWA-1 (VFF-18), which is formed by the hybridoma cell line with the Accession Number DSM ACC2174, or a derivative of this antibody. P:\OPER\JMS\l 1773-97CLM- 121W0 -47-

9. Use according to one of claims 6 to 8, characterised in that the antibody molecule is a monoclonal antibody, a Fab- or F(ab') 2 -fragment of an immunoglobulin, an antibody prepared by recombinant methods, a chimeric or humanised antibody prepared by recombinant methods, a bifunctional or a single-chain antibody (scFv). Use of an antibody molecule which is specific to an epitope within the amino acid sequence which is coded by the variable exon v6 of the CD44-gene, for the treatment of squamous cell carcinomas.

11. Use according to claim 10, characterised in that the antibody molecule binds to the amino acid sequence WFGNRWHEGYR.

12. Use according to claim 10 or 11, characterised in that the antibody molecule is the monoclonal antibody BIWA-1 (VFF-18), which is formed by the hybridoma cell line with the Accession Number DSM ACC2174, or a derivative of this antibody. S13. Use according to one of claims 10 to 12, characterised in that the antibody molecule is a monoclonal antibody, a Fab- or F(ab') 2 -fragment of an immunoglobulin, an antibody prepared by recombinant methods, a chimeric or humanised antibody prepared by recombinant methods, a bifunctional or a single-chain antibody (scFv).

14. Use according to one of claims 10 to 13, characterised in that the antibody molecule is linked to a radioactive isotope, a photoactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytostatic, a prodrug, an antibody molecule with a different specificity, a cytokine or another immunomodulatory polypeptide. P:\OPER\JMS\1 1773-97.CLM 12/9/00 -48- Use of an antibody molecule which is specific to an epitope within the amino acid sequence which is coded by the variable exon v6 of the CD44 gene, for the preparation of a pharmaceutical composition for the treatment of squamous cell carcinomas.

16. Method according to claim 1, substantially as hereinbefore described with reference to the drawings and/or Examples.

17. Use according to claim 6, claim 10 or claim 15, substantially as hereinbefore described with reference to the drawings and/or Examples. DATED this 12th day of September, 2000 Boehringer Ingelheim International GmbH AND Forschungszentrum Karlsruhe GmbH By their Patent Attorneys Davies Collison Cave •oooo