NZ324314A

NZ324314A - Process for the diagnosis and treatment of squamous cell carcinomas based on the expression of the variable exon v6 of the CD44 gene

Info

Publication number: NZ324314A
Application number: NZ324314A
Authority: NZ
Inventors: Karl-Heinz Heider; Gunther Adolf; Elinborg Ostermann; Erik Patzelt; Marlies Sproll
Original assignee: Karlsruhe Forschzent; Boehringer Ingelheim Int
Priority date: 1995-12-06
Filing date: 1996-12-05
Publication date: 2000-02-28
Also published as: CO4520233A1; EE03783B1; MX9804459A; WO1997021104A1; NO319903B1; BG102513A; AR004360A1; AU1177397A; PL327066A1; KR100473824B1; ES2190484T3; AU726704B2; ATE235056T1; EP0865609A1; CN1151377C; EE9800164A; DK0865609T3; PT865609E; CN1207811A; DE59610248D1

Abstract

The invention concerns a method of diagnosing and treating squamous cell carcinomas, the method being based on the expression of the variant exon v6 of the CD44 gene as the molecular target. In a preferred embodiment, v6-specific antibody molecules, in particular the monoclonal antibody BIWA-1 (VFF-18), are used for this purpose. 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.

Description

<div class="application article clearfix" id="description"> 5 S016704J 200 324314 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 the use for the diagnosis and therapy of squamous cell 10 carcinomas which are based on the expression of the variable exon v6 of the CD44 gene Ji It has recerdy been shown that the expression of variants of the surface glycoprotein CD44 is necessary and sufficient to trigger so-called spontaneous metastatic 15 behaviour both in a non-metastasising pancreas-adenocarcmoma cell line of the rat and also in a non-metastasising fibrosarcoma cell line of the rat (Giinthert etal, 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 1 he CD44-isoforms are 20 produced by alternative splicing in such a way that the sequences of 10 exons (vl-vlO) m CD44s are excised completely, but may occur m different combinations in the larger variants (Screaton et al, 1992, Hcider et al, 19P3, Hofmann etal, 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 25 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 cavitics At later stages of the tumour progression, e g in adenocarcinomas, all malignant degenerations express variants of 30 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 35 therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0531300) The expression of variant CD44-molecules in squamous cell carcinomas has also 1 1-LLLli.vJn, , I > 2-Kw, I'Jj j> 2 2431 10 15 20 25 30 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 119, Brooks etal (1995) obtained a heterogeneous staining of nasopharyngeal 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 lmmunchistology 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 m squamous cell carcinomas, which was surprisingly detected, m contradiction to the teaching known from the pnor 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 ammo 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, Maschcroder Weg lb, D-38124 Braunschweig, Germany (WO 95/33771), and derivatives of this antibody, are particularly preferred One aspect of the present invention is the use of antibody molecules which bind to the amino acid sequence WFGNRWHEGYR, for the preparation of n pharmaceutical composition lor the treatment of squamous cell carcinomas The nucleic and amino acid sequence of the variant exon v6 of the CD44-gene is known (Screaton et al, 1992, T6lg 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 0 i LL . / V. I L 32431* Q A TC CAG GCA K E Q 5 AAG GAA CAG T P R ACA CCC AGA 1 o The invention may be earned 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 witliin the amino acid sequence WFGNRWHEGYR. The preparation of antibodies against known ammo acjd sequences may be earned out according to methods known per se (Catty, 1989) For example, a 15 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 20 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, m 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 Heiderefa/ (1993,1996a) and Koopman et al (1993) describe the preparation of antibodies against variant epitopes 25 of CD44 However, 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 30 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 ' 35 analysing the amino acid sequence of the antibody BIWA-1 (VFF-18) and/or using the hybndoma cell line which produces this antibody, particularly the genetic information contained therein, he can produce recombinant antibodj molecules having the same ldiotype as BIWA-1 (VFF-18), l e antibody molecules which have the same amino acid ' i, < l > I.' i i i f 1 Rfrprn/r-n Jj T P S S T T ACT CCT AGT AGT ACA ACG W F G N R W TGG TTT GGC AAC AGA TGG E D S H S T GAA GAC TCC CAT TCG ACA E E T A T Q GAA GAA ACA GCT ACC CAG H E G Y R Q CAT GAG GGA TAT CGC CAA T G T A ACA GGG ACA GCT G 4 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, humamsed antibodies (Shin et al, 1989, Gusscw and Seemann, 1991), bispecific or Afunctional antibodies 5 (Werner 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) Humamsed antibodies may be prepared, for example, by CDR-graftmg , (EP 0239400) Framework regions can also be modified (EP 0519596, WO 9007861) For lo humanising antibodies, methods such as PCR (see for example EP 0368684, EP 0438310, \V i 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 15 monoclonri 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 . 20 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 ldiotype, maybe linked for example with radioactive isotopes such as 125jt 131 1 lljn) 99m-fc or 25 radioactive compounds (Larson et al, 1991, Thomas et al, 1989, Snvastava, 1988), enzymes such as peroxidase or alkaline phosphatase (Catty and Raykundalia, 1989), with fluorescent dyes (Johnson, 1989) or btotin molecules (Guesdon etal, 1979) For therapeutic purposes v6-specific antibody molecules, preferably BIWA-1 (VFF-18)- / antibody molecules or VFF-18-denved antibody molecules, e g fragments thereof or 30 recombinant antibody molecules with the same ldiotype, may be linked to radioisotopes such as 90 Y, 131^ 186^ 188rc> 153gm> 67^U) 212b}, 213b,? 177lu (Quadn etal, 1993, Lenhard et al, 1985, Vnesendorp et al ,1991, Wilbur et al, 1989, Maraveyas et al, 1995a, Jurcic et Scheinberg, 1994), toxins (Vitettae/a/, 1991, Vitetta et Thorpe, 1991, Kxeitman et al, 1993, Theuer et al, 1993), cytostatics (Schrappe et al, 1992), prodrugs 35 (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 (Reisfeid et al, 1996) or mterleukin-2 (Becker et al, 1996) The antibody molecules may also be modified for use m a pretargetting system, e g with streptavidm or biotm (Goodwin, 1995) Advantageously, the diagnostic process according to the invention may be used to mvestigate 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 charactensed The detection of vanant CD44-molecules which contain an ammo acid sequence coded by the vanable exon v6 may be earned 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 mvention consequently also relates to antibody molecules and nucleic acids which are suitable as probes or pnmers 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 known per 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 Raykundaha, 1989), radioimmunoassays (RIA, Catty and Murphy, 1989) or related immunoassays The , investigations may be qualitative, semi-quantitative or quantitative Apart from in vitrj) diagnosis, antibody molecules with specificity according to the invention are also suitable for the m 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 m 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 r 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 systernically or topically, e g by intravenous route (as a bolus or continuous infusion) or by intraperitoneal, intramuscular, subcutaneous or other injection/infusion Procedures for administenng conjugated or non-conjugated antibodies (be they in the form of complete immunoglobulins, fragments, 6 recombinant humanised molecules or the like) are known m the art (Mulshine etal, 1991, Larson et al, 1991, Vitetta and Thorpe, 1991, Vitetta et al, 1991, Breitz et al, 1992, 1995, Press et al., 1989, Werner et al, 1989, Chatal et al, 1989, Sears et al, 1982) They may be used therapeutically, for example, m the same way as the antibody 1.1ASML (Seiter et al, 5 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 complement-induced or antibody-induced cell cytotoxicity (Riethmuller ef a/ 1994) Suitable monoclonal antibodies for this application are murine antibodies of isotype IgG2a or antibodies of the human IgGl-type Unmodified antibodies may also be administered in order to induce 10 the patient's own antitumoral reaction through an anti-idiotypic mechanism (Baum et al, 1993, Khazaeh etal, 1994) According to a preferred embodiment of a therapeutic application, a humamsed v6-specific immunoglobulin or a F(ab')2 fragment thereof is linked with (Quadri et al, 15 1993, Vnesendorp etal, 1995), (Maraveyas et al, 1995a, 1995b, Juweide/a/, 1995, Press et al, 1995, Thomas et al, in Catty 1985, p 230-239) 1 ^Re (Breitz et c', 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 humamsed antibody may be linked to ^®Y using a 20 chelating linker such as 1TCB-DTPA (lsothiocyanatobenzyl- diethylenetriammepentaacetate), achieving a specific activity of 5-20 mCi/mg, preferably 10 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 ^' I, a possible dosage plan might be, for 25 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 30 administration With monoclonal antibodies it may be necessary to mix the agent with an excess (e g 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 thirapy may be combined with external radiation therapy It may also be backed up by bone marrow transplantation, this is 35 particularly necessary when a dosage of more than 1 6 Gy is reached in the bone marrow during therapy ! , Antibody molecules accordmg to the invention may also be used ex vivo to purify CD34-positive stem and precursor cell preparations (lmmunopurging) 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 accordmg to the invention, e.g antibody-toxin conjugates (Myklebust et al, 1994, DE P 196 48 209 7) Antibody molecules accordmg to the mvention may also be introduced into the T-cell receptor of T-lymphocytes m the form of recombinant constructs Reprogrammed T-lymphocytes of this land bmd selectively to the antigen-expressing tumour cells and develop a cytotoxic activity; with the result that they can be used for the tieatment of squamous cell carcinomas (PCT/EP9604631, Altenschmidt et al, 1996) qp 8 Figures Fig. 1 Determining the epitope specificity of BIWA-1 by binding to synthetic 5 peptides derivedfrom the human CD44v6 sequence The corresponding peptide from rat CD44v6 was tested with the antibody 1 1ASML Binding was determined m an ELISA in which the peptides were immobilised on microtitre plates (cf Heider et al, 1996b, Fig 2) - no binding, +/- slight binding, + strong binding 10 Tig. 2 Immunohistochemical analysis of a squamous cell carcinoma of the larynx (a) and a liver metastasis of a carcinoma of the oesophagus (b) 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 40x, counterstaimng haematoxyhn 15 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 20 Fig. 4: Refined epitope mapping of the mAb BIJVA-1 The binding of BIWA-1 to various overlapping synthetic peptides which span the ammo acids 18-32 of the CD44v6-coded region was measured by competitive ELISA. The minimum binding sequence (peptide v6 (19-29)) is underlined 25 Fig. 5 Biodistribution of'25I-BIWA-l 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 xggp 9 Examples Example 1: Expression of CD44v6 m squamous cell carcinoma 5 Tissue j , " 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 10 cases skm), 91 cases of lymph node metastases (larynx, n=38, lung, n=27, oesophagus, n=l 1, oral cavity, n=l 1, tonsils, n=4) and 4 cases of liver metastases (oesophagus) Antibodies 15 The total variant region of the HPKII type of CD44v (Hofmann etal, 1991) was amplified from human keratmocyte-cDNA by polymerase chain reaction (PCR) The two PCR primers 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3', positions 25-52, and 5'-TGATAAGGAACGATTGACAlTAGAGTTGGA-3', positions 1013-984 of the LCLC97-variant region as described by Hofmann et al, contained an EcoRI recognition 20 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-vl 0) codes for a \ ( fusion protein of ~70 kD, consisting of glutathione-S-transferase from Schistosoma i japomcum and the exons v3-vl0 of human CD44 (Fig 1, Heider et al, 1993) The fusion protein was expressed in E coh and then subjected to affinity purification over glutathione-25 agarose (Smith et al, 1988) Female Balb/c mice were intraperitoneal^ immunised with the affinity-purified fusion protein according to the following plan 30 1st immunisation 90 |ig of fusion protein in complete Freund's adjuvant 2nd and 3rd immunisations 50 p.g of fusion protein in incomplete Freund's adjuvant 35 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 jag of fusion protein m PBS On the foliowmg day, the spleen cells from an animal with a high antibody titre were fused with P3 X63-Ag8 653 mouse myeloma cells using polyethyleneglycol 10 4000 The hybndoma cells were then selected in microtitre plates in HAT-medium (Kohler and Milstein, 1975, Kearney et al, 1979) Measurement of the antibody titre in the serum or screening of the hybridoma 5 supernatants were earned out using an ELISA In this test, first of all microtitre plates were covered with fusion protein (GST-CD44v3-10) or only with glutathione-S-transferase They were then incubated with serial dilutions of serum samples or hybndoma supernatants and the specific antibodies were detected usmg peroxidase-conjugated antibodies against mouse immunoglobulin Any hybridomas which reacted only with 10 glutathione-S-transferase were discarded The remaining antibodies were first charactensed in an ELISA with domain-specific fusion proteins (exon v3, exon v5 + v6, exon v6 + v7, exon v8 - vlO) (Koopman et al, 1993) Their immunohistochemical reactivity was tested on human skin sections 15 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 vb domain were used m ELISA binding assays (Fig 1)' The 14 amino acid peptide v6D showed the strongest binding Consequently, the epitope of BIWA-1 is wholly,or 20 partly within the sequence QWFGNRWHEGYRQT of the domain coded by exon v6 This sequence is homologous to the binding epitope of the antibody d 1ASML, which was used in a therapeutic rat model and which is specific to rat CD44v6 (Fig 1) Immunohistochemistry 25 Before incubation with the pnmary antibody, paraffin sections (4 |im) in Rotihistol (Roth, Germany) were deparaffinated 3 times for 10 minutes each time and then rehydrated in a rising alcohol senes 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 30 600 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 m PBS the sections were incubated with pnmary antibody (BIWA-1 5 pg/ml, munne IgG (lsotype-correspondmg negative control) 5 ng/ml in PBS/1% BSA) for 1 hour The positive control 35 used for the staining reaction consisted of normal human skin sections, as keratmocytes express a CD44-isoform which contains v3-v 10 Endogenous peroxidases were blocked with 0 3% H2O2 in PBS, and the sections were incubated for 30 mmutes with the biotmylated secondary antibody (anti-mouse IgG-F(ab')2, DAKO Corp) In order to , t 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 mcubated for 5-10 minutes in 3,3-aimno-9-ethyl-carbazole substrate (Sigma Immunochemicals), the reaction was stopped with H20 and the sections 5 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 10 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 80% of the tumour cells in the positive cells reacted with the antibody, the corresponding percentage was indicated 15 126 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 m 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 20 tissue, lymphocytes, musele cells or endothelium In order to quantify the expression of CD44v6-molecules on these tumour cells, sections of normal human skm were stained parallel to the tumour sections Normal skin keratinocytes express high levels of ,CD44-isoforms and are among the strongest expressors 25 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 r 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 30 metastasis) or moderate tumour staimng (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 m the CD44v6-expression pattern between the pnmary tumours and metastases A detailed summary of the results is shown in Table 1 with the Examples 35 shown m Fig 2 \ < s s- \\ ~ /I A 12 Table 1 Expression of CD44v6 in squamous cell carcinomas Sample Type of tumour BIWA-1 Reactivity Ami 86 Primary Larynx +++* 4687 90 Pnmary Larynx +++ 8372 90 Pnmary Larynx +++ 17427 90 Pnmary Larynx +-H- 27298 90 Pnmary Larynx +++ 46908 90 Pnmary Larynx +++ 51334 90 Pnmary Larynx +++ 51402 91 Pnmary Larynx -H-+ 60414 91 Pnmary Larynx +++ 61733 91 Primary Larynx +++ 12280 92 Primary Larynx +++ 23140 92 Primary Larynx -H-+ 31792 92 Pnmary Larynx +++ 32214 92 Primary Larynx +++ 10209 95 Primary Larynx +++ 2366 86 Primary Skin +++ 2574 86 Pnmary Skin' +++ 9916 86 Primary Skin HH-/+-H* 2696 87 Primary Skin +++ 8906 87 Primary Skin -H-f 8191 88 Primary Skin +++ 8354 88 Pnmary Skin ++50% 11963 88 Pnmary Skin '++ 5590 90 Pnmary Skin ++/+-H- 530 92 Primary Skin +++ 2583 94 Pnmary Skm -H-+ 11337 94 Pnmary Skm +++ 10901 95 Pnmary Skm +++ / 13 11557 95 Pnmary Skm +++ 11744 95 Pnmaiy Skin +++ 11917 95 Pnmary Skm +++ 4688 90 I Lymph node metastasis Larynx ++/+-H- 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 1 -1-1 61734 91 Lymph node metastasis Larynx +++ 1318 92 I Lymph node metastasis Larynx +-H- 1318 92 II Lymph node metastasis Larynx +++ 1318 92 III Lymph node metastasis Larynx 1 I- 1 1318 92 IV Lymph node metastasis Larynx 1 I'l1 2863 92 I Lymph node metastasis Larynx +++ 2863 92 II Lymph node metastasis Larynx +++ 5745 92 0 I Lymph node metastasis Larynx +++ 5745 92 i'n Lymph node metastasis Larynx +++ 8969 92 I Lymph node metastasis Larynx +++ 8969 92 II Lymph node metastasis Larynx +++ 8969 92 IU Lymph node metastasis Larynx *H- 8969 92 IV Lymph node metastasis Larynx +-H- 8969 92 2/1 Lymph node metastasis Larynx +++ 8969 92 2(11 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 +++ Y © 14 9509 92 Lymph node metastasis Larynx +++ 9566 92 Lymph node metastasis Larynx +++ 12283 92 Lymph node metastasis Larynx +++ 14046 92 Lymph node metastasis Larynx +++ 31787 92 Lymph node metastasis Larynx +++ 49228 92 Lymph node metastasis Larynx +++ 50% 29228 93 Lymph node metastasis Larynx +++ 29829 93 Lymph node metastasis Larynx ++ 29804 95 l Lymph node metastasis Larynx ++/+-H- 15293 91 Lymph node metastasis Lung + 25% 1667 92 Lymph node metastasis Lung + 20% 2757 92 I Lymph node metastasis Lung +++ 2757 92 II Lymph node metastasis Lung +++ 2757 92 III Lymph node metastasis Lung +-H- I 2757 92 IV Lymph node metastasis Lung +++ lj 4790 92 ( Lymph node metastasis Lung +++ j 6168 92 I Lymph node metastasis Lung ++ 50% | 6168 92 ' II Lymph node metastasis Lung \\ +++ \ 6168 92 III Lymph node metastasis Lung +++ \ 6168 92 IV Lymph node metastasis Lung +++ ^ 7206 92 Lymph node metastasis Lung +++ 7531 92 I Lymph node metastasis Lung ill" 7531 92 II Lymph node metastasis Lung +++ 7531 92 III Lymph node metastasis Lung ++/-H-+ 7531 92 IV Lymph node metastasis Lung +-H- fr' 10324 92 Lymph node metastasis Lung +++ 10519 92 II Lymph node metastasis Lung +++ 10519 92 RM II Lymph node metastasis Lung +-H- 10958 92 Lymph node metastasis Lung +++ 11425 92 I Lymph node metastasis Lung +++ 11425 92 n Lymph node metastasis Lung +++ 15 13055 92 Lympn node metastasis Lung ++/+++ 13055 92 n Lymph node metastasis Lung focal +++ 13055 92 in Lymph node metastasis Lung +-H- 15663 92 Lymph node metastasis Lung +-H- 16713 92 Lymph node metastasis Lung +++ 14980 91 I Lymph node metastasis Oesophagus -HH- 14980 91 II Lymph node metastasis Oesophagus +-H- 16641 91 I Lymph node metastasis Oesophagus +++ 16641 91 n Lymph node metastasis Oesophagus -H-+ 16641 91 ni Lymph node metastasis Oesophagus +++ 1059 92 Lymph node metastasis Oesophagus + 1710 92 i Lymph node metastasis Oesophagus 1 1 1 1710 92 ii Lymph node metastasis Oesophagus +++ 1710 92 in Lymph node metastasis Oesophagus +++ 11502 92 i Lymph node metastasis Oesophagus -1 -I 1 11502 92 n Lymph node metastasis Oesophagus ++ 202 92 Lymph node metastasis Oral cavity ++ 60% 6030 92 i Lymph node metastasis Oral cavity +/++/+++ 25% 7335 92 i Lymph node metastasis Oral cavity +-H- 7335 92 ii Lymph node metastasis Oral cavity +++ 15324 92 ii Lymph node metastasis Oral cavity +++ 70% 16164 92 i Lymph node metastasis Oral cavity +++ 16164 92 ii IS Lymph node metastasis Oral cavity +++ 50% 16412 92 Lymph node metastasis Oral cavity ++/+++ 16836 92 i Lymph node metastasis Oral cavity +++ 16836 92 n Lymph node metastasis Oral cavity +++ 16836 92 m Lymph node metastasis Oral cavity +-H- 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 ++ 1/ 16 14172 91 4 Liver metastasis Oesophagus +++ r 14172 91 5 Liver metastasis Oesophagus +++ 4131 94 1 Livei metastasis Oesophagus +/++ 8438 94 Liver metastasis Oesophagus focal -H-/-H-+ 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 5 Example 2: Expression of CD44v6 in kidney cell carcinomas, prostate carcinomas and liver metastases of colon carcinomas Tissue 10 19 cases of kidney cell carcinomas (12 cases of clear cell, 5 cases of chromophilic, 1 case of chromophobic, 1 oncocytoma), 16 pnmary 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 15 Antibody BIWA-1 (see Example 1) Immunohistochemistry 20 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 majonty of the kidney cell and prostate 25 carcinomas mvestigated In the case of a more than focal expression m 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 majonty of cases was faint to moderate, but 30 generally fewer than 100% of the tumour cells in a sample showed any staining with BIWA-1 The-results are summansed m Table 2 17 Table 2 Expression of CD44v6 in prostate adenocarcinomas, kidney cell carcinomas and liver metastases of colorectal carcinomas Type of tumour n BIWA-1 Reactivity negative focal pos positive Prostate adenocarcinoma Pnmary 16 8 3 5 Prostate adenocarcinoma Lymph node metastases < 19 15 2 2 Kidney cell carcinoma Pnmary 19 17 0 2 Colorectal carcinoma Liver metastases 30 7 8 15 Example 3* Characterisation of CD44v6-specific antibodies Cell line The human SCC cell lme A-431 (spontaneous epidermoid carcinoma of the vulva) was obtained from the American Type Culture Collection (Rockwell MD) and cultured m accordance with the manufacturer's instructions The surface expression of CD44v6-containing 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 CD44v6-mteraction was determined by Surface Plasmon Resonance (SPR), using a BIAcore 2000 system (Pharmacia Biocensor) A glutathione-S-transferase-CD44-fiision protein which contained the region coded by the exons v3-vl0 (GST/CD44 v3-vl0) was immobilised on a CM5 Sensor Chip, the amine coupling method being earned out m accordance with the manufacturer's instructions Antibodies in various concentrations (8-132 nM) m HBS (10 mM HEPES pH 7 4,150 mM sodium chloride, 3 4 mM EDTA, 0 05% BIA core surfactant P20) was injected over the antigen-specific surface at a flow rate of 5 |il/mm 18 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 jj.1 30 mM HC1 The data analysis and calculation of the kinetic constants were earned out using the Pharmacia Biocensors BIA Evaluation 5 Software, Version 2 1 In this way, the antigen affinity of BIWA-1 was compared with other CD44v6-specificmAbs (VFF4, VFF7,BBA-13 (IgGl, R&D Systems, Abmgdon, UK)) Kinetic and affinity constants of the vanous antibodies were determined m two mdependent 10 experiments Table 3 shows the values of the association rates (ka), 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 15 BIWA-1 shows the lowest Kd of all the antibodies investigated Table 3 Kinetic and affinity constants of vanous CD44v6-specific mAbs Antibody ka (M'V1) Ms"1) Kd (M) VFF4 1 1 x 105 2 6 x 10-5 2 4 x 10"10 VFF7 1 1 x 105 1 2 x lO-4 1 1 x 10'9 BIWA-1 1 3 x 10s 2 2 x 10"5 1 7x lO-10 BBA-13 3 7x 104 2 3 x 10'5 6 2 x 10"10 Analysis of the antibody-protem interaction using ELISA ( <A CD44v6 expressing A-431 cells were cultivated in 96-well plates (Falcon Microtest III, Becton Dickinson, Lincoln Park, NJ) m numbers of 5 x 104 per well in RPMI1640 with 25 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, m each case in assay buffer PBS/0 5% BSA/0 05% Tween 20) was earned out for 1 hour at ambient temperature and followed by 3 washing steps The secondary antibody used was a 30 rabbit-antimouse-IgGhorseradish peroxidase-conjugated antibody (DAKO Corporation, & 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 5 Figure 3 shows that the relative affinities of the antibodies as determined by BIAcore analysis are reflected m their interaction with the tumour cells, with BIWA-1 clearly showing the highest binding affinity I 10 The protein domain which is coded by the CD44-exon v6 consists of 45 ammo acids (Figure 4). 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-nier (amino acid groups 18-31, Figure 4, cf also Figure 1) but not to peptides outside this region A second senes of 15 peptides were therefore synthesised and tested in competitive ELISAs (Figure 4) The results show that the peptide 19-29 (WFGNRWHEGYR) represents the minimum structure required for high affinity bmdmg Elimination of the C-terminal argimne groups resulted in a more than 100 times weaker bmdmg 20 Example 4 Biodistnbution of radio-iodinated CD44v6-antibodies in xenotransplant-carrying nude mice A-431-Xenotransplant model 25 8 week old female BALB/c nu/nu nude mice (B & K Universal, Renton, WA) were subcutaneously injected m 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 biodistnbution experiments within two weeks (weight of tumours 40-50 mg) 30 Radio-iodmation of BIWA-1 Protein G-punfied mAb BIWA-1 (murine IgGl) was coupled to streptavidin, using the heterobifunctional crosslinker succinimidyl 4-(N-maleimido-methyl)cyclohexane-l- 35 carboxylate Streptavidin-lysyl groups were linked to reduced antibody-cystemyl groups produced by preliminary treatment of the antibody with dithiothreitol The 1 1 conjugates obtained (> 90%) were further purified by ion exchange chromatography For biodistnbution experiments, BIWA-1/SA was labelled via pnmary amines of lysine with 9 20 125I, using p-iodophenyl labelling reagent (PEP, NEN Dupont, Wilmington, DE), followed by the process of Willbur et al (1989) Labelling BIWA-1 with SA or 125I did not affect the unmunoreactivity or the pharmacokinetics of the antibody in mice 5 Biodistnbution experiments Nude mice which were xenotransplanted with human A-431 tumours were intravenously injected through the lateral caudal vem with 5-7 jiCi 125I on 50 fig of mAb BIWA-1 (specific activity 0 1-014 mCi/mg) Time-biodistnbution studies were earned 10 out m groups of n=3 animals for each point of time at 4, 24,48,120 and 168 hours post-mjection 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, intestme and tumour The radioactivity in the tissues was counted m a gamma-scintillation counter (Packard 15 Instrument Company, Menden, CT) by companson with standards of the injected antibody preparation, the energy window being set at 25-80 keV for 125I The percentage of mjected dose per gram of tissue was calculated (% IB/g) Preliminary experiments had shown that BIWA-1 did not cross-react with munne 20 CD44v6-antigen Table 4 and Figure 5 show the absorption of radioactivity in tumours and , normal tissue Iodmated BIWA-1 showed a rapid tumour absorption (7 6% mjected 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 25 times and these are shown m 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 m the tissue biopsies Selective in vivo targeting of human SCC-xenotransplants in nude mice with 125I-labelled BIWA-1 shows that this monoclonal antibody has a high potential as a targeting vehicle for diagnostic and 30 therapeutic use in SCC patients 21 Table 4 Tumour tissue ratios of 125I-BIWA-1 m A-431 -tumour-carrying nude mice at vanous times post-injection Ratio of tumour to 4h 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 140 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 5 a mean values (n=3), 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 10 expression of CD44v6 The samples were either embedded m paraffin or frozen m liquid nitrogen immediately after surgical removal and stored at -70°C until required The following tumours were analysed basalioma (n=16), adenocarcinoma (AC) of the breast (n=55), AC of the colon (n=83), squamous cell carcinomas (SCC) of the head and neck (n=125), lung carcinomas (n=120), prostate AC (n=34), kidney cell carcinomas (n=27), 15 SCC of the skm (n=l 5) and AC of the stomach (n=69) The tissues were obtamed by routine surgery or biopsy and normal tissue was obtained to accompany the tumour samples The immunohistochemical investigations were earned out as m Example 1 Table 5 shows a summary of the immunohistochemical analysis of 397 different 20 types of tumour with the mAb BIWA 1 ' <0 22 Table 5: Expression of CD44v6 in human tumours Type Total number Positive cases* n % Basalioma Pnmary 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 50 Brain metastases 6 6 100 Larynx SCC Lymph node metastases 18 18 100 Lung AC Primary tumour 35 15 43 Lung SCC Pnmary tumour 9 9 100 Oesphagus SCC Pnmary tumour 20 20 100 Prostate AC Pnmary tumour 16 5 31 Lymph node metastases 18 0 0 RCC Pnmary tumour 27 5 18 SCLC Pnmary tumour 31 7 23 < Stomach AC Pnmary tumour < 22 15 68 Lymph node metastases 43 16 37 Liver metastases 4 4 100 Total number 397 * > 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-contaimng isoforms to varying degrees The majonty 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 vanous types and classification were investigated for their reactivity with BIWA 1 These included 67 cases of pnmary SCC (larynx, n=15, oral cavity, n=1 6, oropharynx, n=3, skm, n=15), 77 samples of lymph node metastases 23 (larynx, n=12, lung, n=27, oesphagus, n=ll; oral cavity, n=6, oropharynx, n=7, hypopharynx, n=10, tonsil, n=4), and 3 samples of liver metastases (oesphagus) Table 6 summarises the immunohistochemical analysis of all the SCC samples investigated 5 Table 6: Expression of CD44v6 in squamous cell carcinomas i Type Total number Negative Focal dos ' 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 95 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 Skm 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 m all but three tumour 10 samples (one case of larynx, 2 cases of lung) The majonty of the samples showed expression of the antigen on 80 to 100% of the tumour cells within a smgle section, the staining being concentrated chiefly on the membrane of the tumour cells The most homogeneous staining pattern was found m carcinomas of the larynx, oesphagus and hypopharynx, with the majonty of tumour cells in 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antibody-targeted enzyme conjugates for cancer therapy Cancer Res 52 4484-4491 (1992) 20 Werner L M, O'Dwyer J, Kitson J, Comis R L, Frankel A E, Bauer R J, Kopnrad M S, Groves E S Phase I evaluation of an anti-breast carcinoma monoclonal antibody 260F9-recombmant ricin A chain immunoconjugate Cancer Res 49 4062-4067(1989) 25 Wilbur, D S , Hadley, S W, Hylandes, M D , Abrams, P G, Beaumier, P A, Morgan, A C , Reno, J M, Fntzberg, A R Development of a stable radioiodinating agent to label monoclonal antibodies for radiotherapy of cancer J Nucl Med 30 216-226(1989) Winter, G , Gnffith, A D , Hawkins, R E, Hoogenboom, H R Making antibodies by 30 phage display technology Ann Rev Immunol 12,433-455(1994) 32 SEQUENCE LISTING GENERAL DATA: (l) APPLICANT: (A) NAME: Boehringer Ingelheim International GmbH (B) STREET: Rhemstrasse (C) TOWN: Ingelheim (E) COUNTRY- Germany (F) POSTCODE: 55216 (G) TELEPHONE: +49-(0)-6132-772*770 (H) FAX- +49-(0)-6132-774377 (A) NAME: Forschungszentrum Karlsruhe GmbH (B) STREET. Postfach 3640 (C) TOWN: Karlsruhe (E) COUNTRY: Germany (F) POSTCODE. 76021 (A) NAME: Heider, Karl-Hemz (B) STREET. Hervicusgasse 4/3/21 (C) TOWN: Vienna (E) COUNTRY: Austria (F) POSTCODE. 1120 (A) NAME- Adolf, Guenther (B) STREET Stiftgasse 15-17/10 (C) TOWN: Vienna (E) COUNTRY: Austria (F) POSTCODE: 1070 (A) NAME: Ostermann, Elinborg (B) STREET: Mauerbachstr 56/6 (C) TOWN: Vienna (E) COUNTRY: Austria 33 (F) POSTCODE: 1140 (A) NAME: Patzelt, Erik (B) STREET. Hans-Buchmueller-Gasse 8 (C) TOWN: Purkersdorf (E) COUNTRY: Austria (F) POSTCODE: 3002 (A) NAME: Sproll, Marlies (B) STREET. Schwenkgasse 3 (C) TOWN: Vienna (E) COUNTRY: Austria (F) POSTCODE: 1120 (ii) TITLE OF THE INVENTION: Process for the diagnosis and treatment of squamous cell carcinomas \ 1 i (in) NUMBER OF SEQUENCES: 16 (lv) COMPUTER-READABLE VERSION: (A) DATA CARRIER: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1 0, Version #1.30 (EPO) i (2) DATA RELATING TO SEQ ID NO: 1: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 129 base pairs (B) NATURE: Nucleotide (C) STRAND FORM: both (D) TOPOLOGY: both (11) NATURE OF THE MOLECULE: Genome DNA (xx) FEATURE. (A) NAME/KEY: exon (B) POSITION:1 .129 (D) OTHER DATA-/product= "CD4 4" /label= v6 /note= "GenBank data base accession No. L05411" /citation= ([1]) (ix) FEATURE: (A) NAME/KEY- CDS (B) POSITION-3 .128 (x) PUBLICATION INFORMATION: (A) AUTHORS- Screaton, GR Bell, MV Jackson, DG Cornells, FB - Gerth, U Bell, JI (B) TITLE: Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons (C) JOURNAL: Proc Natl. Acad. Sci. U S.A. (D) VOLUME: 89 (F) PAGES: 12160-12164 (G) DATE. December-1992 (K) IMPORTANT GROUPS IN SEQ ID NO 1 FROM 1 TO 129 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER- DE 196 15 074.4 (I) FILING DATE: 17-APR-1996 I \ 9 9 35 (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 15 10 15 GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA ACA CCC 95 Glu Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro 20 25 30 AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G 129 Arg Glu Asp Ser His Ser Thr Thr/^Gly Thr Ala 35 ^ 40 (2) DATA RELATING TO SEQ ID NO: 2: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH 42 amino acids (B) NATURE* ammo acid (D) TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Gin Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys Glu 1 ' 5 10 15 Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro Arg 20 25 30 Glu Asp Ser His Ser Thr Thr Gly Thr Ala 35 40 Q ' > i 36 jf Jl (2) DATA RELATING TO SEQ ID NO: 3: (l) 'SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 ammo acids (B) NATURE, ammo acid (C) STRAND FORM: single strand (D) TOPOLOGY: linear (n) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER* DE 195 45 472 3 (I) 'FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION. (H) DOCUMENT NUMBER: DE 196 15 074 4 (I) FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION. SEQ ID NO: 3: Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr 15 10 (2) DATA RELATING TO SEQ ID NO: 4: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) NATURE* Nucleotide (C) STRAND FORM: single strand (D) TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: other nucleic acid (A) DESCRIPTION: /desc = "PCR primer" 37 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO 4: CAGCCTGGGA GCCAAATGAA GAAAATG (2) DATA RELATING TO SEQ ID NO 5 (l) SEQUENCE CHARACTERISTICS- (A) LENGTH: 30 base pairs (B) NATURE- Nucleotide (C) STRAND FORM single strand (D) TOPOLOGY- linear (n) NATURE OF THE MOLECULE: other nucleic acid (A) DESCRIPTION /desc = "PCR primer" (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE- 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TGATAAGGAA CGATTGACAT TAGAGTTGGA 38 (2) DATA RELATING TO SEQ ID NO: 6* (l) SEQUENCE CHARACTERISTICS- (A) LENGTH: 11 amino acids (B) NATURE: ammo acid (C) STRAND FORM: single strand <D) TOPOLOGY: linear (n) NATURE OF THE MOLECULE peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472 3 (I) FILING DATE 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074 4 (I) FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION. SEQ ID NO 6 Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg 15 10 (2) DATA RELATING TO SEQ ID NO* 7 (l) SEQUENCE CHARACTERISTICS.' (A) LENGTH: 43 amino acids (B) NATURE- amino acidr (C) STRAND FORM: single strand (D) TOPOLOGY: linear (n) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE'195 45 472 3 % 9 39 (I) FILING DATE- 06-DEC-1995 (x) PUBLICATION INFORMATION: , (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) 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 15 10 15 Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro Arg 20 25 30 I Glu Asp Ser His Ser Thr Thr Gly Thr Ala Ala 35 40 (2) DATA RELATING TO SEQ ID NO- 8: (l) SEQUENCE CHARACTERISTICS. (A) LENGTH- 11 amino acids (B) NATURE: amino acid (C) STRAND FORM: single strand (D) TOPOLOGY linear (n) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE: 17-APR-1996 9 40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys 15 10 (2) DATA RELATING TO SEQ ID NO. 9. (l) SEQUENCE CHARACTERISTICS: (A) LENGTH. 10 amino acids (B) NATURE: amino acid (C) STRAND FORM: single strand (D) TOPOLOGY: linear (n) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472 3 (I) FILING~DATE 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE. 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Glu Glu Thr Ala Thr Gin Lys Glu Gin Trp 1 5 10 (2) DATA RELATING TO SEQ ID NO: 10: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) NATURE: ammo acid £ (C) STRAND FORM* single strand 41 (D) TOPOLOGY: linear (11) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE: 17-APR-1996 i- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Thr Ala Thr Gin Lys Glu Glh Trp Phe Gly Asn 15 10 (2) DATA RELATING TO SEQ ID NO: 11 (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) NATURE: amino acid (C) STRAND FORM: single strand (D) TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: p (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 42 Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr 15 10 (2) DATA (i) (ii) (x) (x) (xi) Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro 15 10 (2) DATA PELATING TO SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS' (A) LENGTH: 11 amino acids (B) NATURE: ammo acid (C) STRAND FORM: single strand (D) TOPOLOGY: linear (n) NATURE OF THE MOLECULE: peptide RELATING TO SEQ ID NO: 12: SEQUENCE CHARACTERISTICS (A) LENGTH: 11 ammo acids (B) NATURE, ammo acid (C) STRAND FORM: s'ingle strand (D) TOPOLOGY: linear NATURE OF THE MOLECULE: peptide PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472 3 (I) FILING DATE: 06-DEC-1995 PUBLICATION"INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE: 17-APR-1996 SEQUENCE DESCRIPTION: SEQ IJD NO: 12. I A W 43 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) 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 10 (2) DATA RELATING TO SEQ ID NO 14 (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) NATURE" amino acid (C) STRAND FORM: single strand (D) TOPOLOGY- linear (ii) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION- (H) DOCUMENT NUMBER: DE 196 15 074 4 (I) FILING DATE: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Thr Pro Arg Glu Asp Ser His Ser Thr Gly 1 ' 5 10 ft @ 44 (2) DATA RELATING TO SEQ ID NO: 15: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH* 42 amino acids (B) NATURE: amino acid (C) STRAND FORM: single strand (D) TOPOLOGY: linear (ii) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATE 17-APR-3 996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO 15* Trp Ala Asp Pro Asn Ser Thr Thr Glu Glu Ala Ala Thr Gin Lys Glu 15 10 15 Lys Trp Phe Glu Asn Glu Trp Gin Gly Lys Asn Pro Pro Thr Pro Ser 20 25 30 Glu Asp Ser His Val Thr Glu Gly Thr Thr 35 40 (2) DATA RELATING TO SEQ ID NO: 16 (l) SEQUENCE CHARACTERISTICS: (A) LENGTH* 14 amino acids (B) NATURE* ammo acid 69 45 (C) STRAND FORM: single strand (D) TOPOLOGY: linear (il) NATURE OF THE MOLECULE: peptide (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) FILING DATE: 06-DEC-1995 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) FILING DATEi 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Lys Trp Phe Glu Asn Glu Trp Gin Gly Lys Asn Pro Pro Thr 15 10 -V , </div>

Claims

<div class="application article clearfix printTableText" id="claims">



J 

32 4314 

46 

What We Claim Is: 

1 Use of an antibody molecule which binds to the amino acid sequence WFGNRWHEGYR, for the preparation of a pharmaceutical composition for the treatment of squamous cell carcinomas
2 Use according to claim 1, wherein the antibody molecule is the monoclonal antibody BIWA-1 (VFF-18), which is formed by the hybndoma cell line with the Accession Number DSM ACC2174, or a derivative of this antibody
3 Use according to claim 1 or 2, wherein the antibody molecule is a monoclonal antibody, a Fab- or F(ab')2- fragment of an immunoglobulin, an antibody prepared by recombmant methods, a chimeric or humanised antibody prepared by recombinant methods, a Afunctional or a single-chain antibody (scFv)
4 Use accordmg to one of claims 1 to 3, wherem the antibody molecule is linked to a radioactive isotope, a photactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotrn molecule, a toxin, a cytostatic, a prodrug, an antibody molecule with a different specificity, a cytokine or another immunomodulatory polypeptide
5 Use of an antibody molecule according to any one of claims 1 to 4 substantially as described herem with reference to the Figures and/or Examples 

END OF CLAIMS 



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