SK73698A3 - Use of antibody molecule binding to the amino acid sequence wfgnrwhegyr - Google Patents

Abstract

The invention concerns a method of diagnosing and treating epithelioma, 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.

Description

Disclosed is the use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR for the manufacture of a medicament for treating squamous epithelial carcinomas that is based on binding of an antibody molecule to an epitope encoded by the variable exon v6 of the CD44 gene. In a preferred embodiment, the v6-specific antibody molecule, in particular the monoclonal antibody BIWA-1 (VFF-18), is used in the above method.

-1 Use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR

Field of the invention

The invention relates to the use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR for the manufacture of a medicament for the treatment of squamous epithelial carcinomas which is based on the binding of the antibody molecule to an epitope encoded by the variable exon V6 of the CD44 gene.

BACKGROUND OF THE INVENTION

Recently, it has been shown that expression of CD44 surface glycoprotein variants is necessary and sufficient to induce so-called spontaneous metastasis not only in the non-metastasized rat adenocarcinoma cell line but also in the non-metastasized fibrosarcoma cell line et al (1991). While the smallest CD44 isoform, the wild-type form of CD44, is generally expressed in a variety of tissues, including epithelial cells, certain CD44 graft variants (CD44v) are expressed by only one subset of epithelial cells. Said CD44 isoforms are generated by alternative cleavage such that the 10 exon sequences (v1 to v10) in the CD44 genes are completely excised, but different combinations may occur with higher variants (Screaton et al., 1992; Heider et al., 1993; Hofmann et al., 1991). Variants are distinguished in that different amino acid sequences are inserted at a certain location of extracellular portions of the proteins. Such variants can be detected in various human tumor cells and human tumor tissue. Thus, expression of CD44 variants during colorectal carcinogenesis has recently been identified (heider et al., 1993). Expression of CD44 variants is absent in normal human colon epithelium, and only poor expression in proliferated crypt cells is detectable. In later stages of tumor development, for example in adenocarcinomas, all malignant varieties express CD44 variants.

Recently, expression of CD44 steppe variants in activated lymphocytes as well as in non-Hodgkin's lymphomas has been demonstrated (Koopman et al., 1993).

Attempts have been made to exploit the differences in expression of variant exons of CD44 genes in tumors and normal tissues for diagnostic and therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0 531 300).

The expression of variant CD44 molecules in squamous epithelial carcinomas has also been investigated. Sami et al. (1993) found, with the v6-specific antibody Var3.1, a decrease in v6-expression in tumor cells compared to normal cells. Brooks et al. (1995) achieved a heterogeneous staining of nasopharyngeal carcinoma with the v6-specific antibody 11.9. Only in 2/12 cases strong staining was achieved, while in most cases only weak focal immunohistological evidence could be achieved.

It is therefore an object of the present invention to develop a new method for diagnosing and treating squamous cell carcinoma as well as to provide means for carrying out the method.

SUMMARY OF THE INVENTION

The present invention provides the use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR for the manufacture of a medicament for the treatment of squamous epithelial carcinomas based on binding of the antibody molecule to an epitope encoded by the variable exon V6 of the CD44 gene. Antibody molecules of corresponding specificity are particularly useful as carriers to selectively achieve squamous cell carcinoma in vivo.

Preferred are those characterized by the use of an antibody molecule that recognizes the amino acid sequence QWFGNRWHEGYRQT, more preferably the amino acid sequence WFGNRWHEGYR. The monoclonal antibody BIWA-1 (VFF-18 clone) derived from a hybridoma cell line 7 is particularly preferred. 6. 1994 deposited with DSM (- Deutsche Sammlung fur Microorganismen und Zellkulturen GmbH, Mascheorder Weg 1b, D-38 124

-3Braunschweig, BRD) under accession number DSM ACC2174, or derivatives thereof.

The invention also provides a pharmaceutical composition for the treatment of said diseases.

The nucleic and amino acid sequences of variant exons of the v6 CD44 genes are known (Screaton et. Al., 1992; Tolg et al., 1993). The existence of degenerate or all variants is not essential to the practice of this invention; such variants are therefore expressly included in the invention.

The exon v6 sequence of the human CD44 genes is:

Q A T P WITH WITH T T E E T A T Q TC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAG The 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 WITH H WITH T T G T A ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G.

The invention may be practiced with polyclonal or multiclonal antibodies that are specific for a single epitope encoded by exon v6, particularly for an epitope within the amino acid sequence QWFGNRWHEGYRQT, most preferably within the amino acid sequence WFGNRWHEGYR. Preparation of an antibody against known amino acid sequences can be accomplished by methods known (Catty, 1989). For example, a peptide with this sequence can be synthetically prepared and used as an antigen in an immunization protocol. Alternatively, the preparation can be carried out by preparing a fusion protein containing the desired amino acid sequence by integrating a nucleic acid (synthetic or prepared from a suitable sample, for example, by polymerase chain reaction (PCR)) encoding for said sequence, integrated into a vector expression and fused

The -4 protein is expressed in the host organism. If desired, the purified, fusion protein can then be used as an antigen or as a specific antibody insert in an immunization protocol, or - in the case of a monoclonal antibody - the hybrid that expresses the incorporated specific antibody can be selected appropriately. Such methods are known in the art. Heider et al. (1993, 1996a) and Koopman et al. (1993) described the preparation of an antibody against a variant epitope of CD44.

However, antibody molecules that are derived from poly- or monoclonal antibodies, for example a Fab fragment or F (ab ') ₂ immunoglobulin fragments, a recombinantly produced single chain antibody (scFv), a chimeric or humanized antibody, such as as well as other molecules that specifically bind to an epitope that is encoded by exon v6. The BIWA-1 antibody (VFF-18) or other antibodies, for example Fab or F (ab ') ₂ fragments, can be obtained from complete immunoglobulin (Kreitman et al., 1993). In addition, one of skill in the art is able to generate recombinant v6-specific antibody molecules. By analyzing the amino acid sequence of the BIWA1 antibody (VFF-18) and / or using a hybridoma cell line that produces the antibody, particularly containing genetic information, one of ordinary skill in the art can prepare recombinant antibody molecules of the same idiotype as BIWA-1 (VFF18), means antibody molecules having the same amino acid sequence in the complementarity-determining regions (CDR) region as BIWA-1 (VFF-18). Suitable methods are known in the art. For example, said recombinant antibody molecules may be a humanized antibody (Shin et al., 1989; Gussov et Seemann, 1991), a bispecific or bifunctional antibody (Weiner et al., 1993; Goodwin, 1989; Featherstone, 1996), single chain antibody (scFv, Johnson et Bird, 1991), complete or fragmented immunoglobulin (Coloma et al., 1992; Nesbiot et al., 1992; Barbas et al., 1992), or a chain shuffling antibody prepared (Winter et al., 1994) . The humanized antibody can be prepared, for example, by CDR grafts (EP 0 239 400). Framework regions can also be modified (EP 0 519 596;

^{ζ 1} 5

WO 90 07 861). Methods such as PCR (see for example EP 0 368 684; EP 0 438 310; WO 92 07 075) or computer modeling (see for example WO 92 22 653) are now used to humanize antibodies. Furthermore, fusion proteins can also be prepared and used, for example, a single chain antibody / toxin fusion protein (Chaudhary et al., 1990; Friedman et al., 1993). As used herein, the terms "antibody" and "antibody molecule" include all compounds discussed herein without polyclonal and monoclonal antibodies, as well as other compounds that can be structurally derived from immunoglobulin and are obtainable by known methods.

In the known epitope (cf. FIG. 1, FIG. 4) of BIWA-1 (VFF-18), one of ordinary skill in the art can equally prepare an equivalent antibody with the same binding specificity. Such antibodies are therefore also included within the scope of the invention.

For diagnostic purposes, antibody molecules, preferably BIWA-1 antibody molecules, fragments thereof, or recombinant antibody molecules of the same idiotype can bind to radioactive isotopes such as ¹²⁵ L, ¹³¹ L, ¹¹¹ L, ^99m Tc or radioactive compounds (Larson et al. , 1991; Thomas et al., 1989; Srivastava, 1988), with enzymes such as peroxidase or alkaline phosphatase (Catty and Raykundalia, 1989), with fluorescent dyes (Johnson, 1989), or with biotin molecules (Guesdon et al. , 1979). For therapeutic use, the specific molecule can be N 6 antibody molecule, preferably BIWA-1 (VFF-18), and the VFF-18-derived antibody molecules, e.g., fragments or recombinant antibody molecules with the same idiotype binding with radioisotopes, such as ⁹⁰ Y, ¹³¹ L, ¹⁸⁶ Re, ¹⁵³ Sm, ⁶⁷ Cu, ²¹² Bi, ²¹³ Bi, ¹⁷⁷ Lu (Quadri et al., 1993; Lenhard et al., 1985; Vriesendorp et al., 1991; Wilbur et al., 1989 Maraveyas et al., 1995, Jurcic et Scheinberg, 1994), toxins (Vitetta et al., 19091; Vitetta et Thorpe, 1991; Kreitman et al., 1993 ;, Theuer et al., 1993), cytostatics (Schrappe et al. al., 1992), prodrugs (Wang et al., 1992; Senter et al., 1989), with photoactivatable substances (Hemming et al., 1993), with an antibody molecule of other specificity, or with radioactive compounds. The antibody molecule may further be coupled to a cytokine * 6 or other immunomodulating polypeptide, for example, a tumor necrosis factor, lymphotoxin (Reisfeld et al., 1996), or interleukin-2 (Becker et al., 1996). Antibody molecules for use in pretargeting systems can also be modified, for example, with streptavidin or biotin (Goodwin, 1995).

In the diagnostic methods of the invention, samples taken from patients, for example, from biopsies where squamous cell carcinoma is suspected, or with an existing diagnosis, or with a tumor in which further refinement is required, may advantageously be examined. Detection of variant CD44 molecules containing the amino acid sequence encoded by the variable exon v6 can be performed using an antibody at the protein level or at the nucleic acid level, using specific nucleic acid probes, or by using polymerase chain reaction (PCR) primers. it also relates to antibody and nucleic acid molecules that are suitable for use in said methods as probes or primers, and to the use of such antibodies and nucleic acids for diagnosis and analysis of squamous epithelial carcinomas. For example, tissue sections can be examined by immunohistochemistry with antibodies using known methods. Extracts obtained from tissue samples or body fluids may further be screened by other immunological methods using antibodies, such as western blots, enzyme-linked immunoadsorbent analysis (ELISA, Catty at Raykundalia, 1989), radioimmunoassay (RIA, Catty et Murphy, 1989) ), or similar immunoanalytical methods. Investigations may be performed qualitatively, semi-quantitatively or as quantitative analyzes.

In addition to in vitro diagnostics, antibody molecules with specificity of the invention are also useful for diagnosing squamous epithelial carcinomas in vivo. If the antibody molecule is a carrier of a detectable label, then the detection of the label can be used for diagnostic purposes, for example to visualize the tumor in vivo (Imaging), or, for example, in radioguided surgery. By using radioactive isotopes of the conjugated antibody for immunoscintilography, for example, a number of records are obtained from which one skilled in the art can deduce

7 Conclusions (Siccardi et al., 1989; Keenan et al., 1987; Perkins et Pimm, 1992; Colcherei et al., 1987; Thompson et al., 1984).

Evidence and / or quantification of the increased expression level of variant CD44 v6 epitopes may result in diagnosis and prognosis of the condition. Combinations with other prognostic parameters can be advantageously used, for example in determining the degree of tumor (mit dem Tumorgrad).

Antibody molecules with specificity according to the invention, optionally bound to a cytotoxic agent, can advantageously be used for the treatment of squamous epithelial carcinomas. The use may be systemic or topical, for example intravenously (such as a bolus or infusion), intraperitoneally, intramuscularly, subcutaneously and other injections / infusions. Protocols for administering conjugated or unconjugated antibodies (whether as complete immunoglobulins, fragments, recombinant humanized molecules or the like) are known in the art (Mulshine et al., 1991; Larson et al., 1991; Vitetta et Thorpe, 1991; Vitetta et al. al., 1991; Breitz et al., 1992, 1995; Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989; Sears et al., 1982). For example, the therapeutic use may be similar to that of the 1.1ASML antibodies (Seiter et al., 1993). An unmodified monoclonal antibody can be used therapeutically directly if it is suitable as an effector for a cytotoxic effect, for example, for complement-induced or antibody-induced cellular cytotoxicity (Riethmuller et al., 1994). Suitable monoclonal antibodies for use herein are the mouse IgG2a isotype antibody and the human IgG1 type antibody. Further, unmodified antibodies can be used to induce a patient's own anti-tumor response by an anti-idiotypic mechanism (Baum et al. 1993; Khazaeli et al., 1994).

A preferred embodiment of therapeutic use depends on combining the humanized v6-specific immunoglobulin or its F (ab ') ₂ fragment with ⁹⁰ Y (Quadri et al., 1993; Vriesedorp et al., 1995), ¹³¹ 1 (Maraveyas et al. al., 1995a, 1995b; Juweid et al., 1995; Press et al., 1995; Thomas et al., in: Catty 1985, pages 230-239), ¹⁸⁶ Re (Breitz et al., 1992, 1995), or with another suitable radioisotope and used for radioimmunotherapy of squamous cell carcinomas. For example,

Can bind BIWA-1, or a humanized version of BIWA-1, or F (abj ₂ of BIWA-1 or humanized ⁹⁰ Y antibody) using a chelating linker such as ICTB-DTPA (isothiocyanate-benzyl-diethylenetriaminopentaacetate); to achieve a specific activity of 5 to 20 mCi.mg ^-1 , preferably 10 mCi.mg ^-1 The active ingredient is then administered to a patient with an antigen-positive tumor at doses of 0.1 to 1 mCi.kg ^{-1 of} body weight, preferably 0.3 to 0.5 Mci.kg ^'1 body weight. If the antibody molecule is linked to the ¹³¹ l, may be at a specific activity of 2 mCi.mg' ¹ serving as scheme 2 x 150 mCi at 6 week. one skilled in the art can determine the maximum possible dosage by known methods (Maraveyas et al., 1995a, 1995b) .If a total amount of protein of 2 to 5 mg is to be administered, it can be administered by rapid intravenous injection. The amount of protein administered may be a suitable form of infusion. When administering monoclonal antibodies, it may be necessary for the active ingredient to be mixed with an excess (e.g., a 10-fold molar excess) of the non-radioactive antibody prior to administration; in this case, administration is preferably done as an intravenous infusion, for example over 15 minutes. The use may be repeated. The therapy may be combined with external radiation therapy. Therapy may further be supported by bone marrow transplantation; this is inevitable, in particular, when a dose of 1.6 Gy has been achieved in the bone marrow during therapy.

Furthermore, the antibody molecules of the invention can also be used ex vivo for purification of CD34-positive baseline and continuous cell preparations (Immunopurging). Radiation therapy and chemotherapy of squamous cell carcinomas can be supported by autologous bone marrow transplantation. The hematopoietic baseline and continuous cell preparations used must be free of tumor cells. This can be achieved by incubation with the antibody molecules of the invention, for example by incubation with antibody-toxin conjugates (Myklebust et al., 1994; DE P 196 48 209.7).

The antibody molecules of the invention can further be used in the form of a recombinant construct into which a T cell receptor has been introduced.

-Qlymfocytov. In this way, reprogrammed T lymphocytes selectively bind to tumor cells expressing the antigen and induce a cytotoxic effect; therefore, they can be used in the therapy of squamous epithelial carcinomas (PCT / EP 96 04 631; Altenschmidt et al., 1996).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: Determination of epitope specificity of BIWA-1 by binding to a synthetic peptide derived from a human CD44-v6 sequence. The corresponding rat CD44-v6 peptide was tested with antibody 1.1ASML. Binding was determined by ELISA (enzyme-linked immunoadsorbent analysis) where the peptide was immobilized on a microtiter plate (cf. Heider et al., 1996b, Fig. 2; no binding; +/-: weak binding; +: strong binding).

Fig. 2: Immunohistochemical analysis of squamous cell carcinoma of the larynx (a) and liver metastasis of esophageal carcinoma (b) with the CD44-v6-specific monoclonal antibody BIWA-1. In both cases, the reactivity of the antibody to the tumor cell membrane can be seen. The initial magnification was 40x, counter staining with hematoxylin.

Fig. 3: Comparison of antigen binding of various CD44-v6-specific monoclonal antibodies. Binding of four different CD44-v6-specific mAbs to human SCC A-431 cells was measured by ELISA. The monoclonal antibody BIWA-1 exhibits a higher affinity for tumor cells than other monoclonal antibodies.

Fig. 4: More detailed epitope mapping of mAb BIWA-1. Binding of the BIWA-1 antibody to various overlapping synthetic peptides whose amino acids 18 to 32 of the CD44-v6 coding region are altered was measured by competitive ELISA. The minimum binding sequence (v6 peptide (19-29)) is underlined.

- 10Fig. 5: Biodistribution of ^12S I - BIWA -1 in A-431 xenotransplanted juvenile mice. The accumulation is shown as% ID.g ^-1 (mean ± standard deviation) at 4, 24, 48, 120 and 168 hours after injection.

DETAILED DESCRIPTION OF THE INVENTION

Λ

Example 1: Expression of CD44-v6 in squamous epithelial carcinomas

tissue

A total of 126 paraffin-embedded tumor samples for CD44-v6 expression were analyzed immunohistochemically using mAb BIWA-1 (VFF-18 clone). Samples included 31 cases of primary squamous cell carcinomas (15 cases of larynx, 16 skin cases), 91 cases of lymph node metastases (larynx, n = 38; lung, n = 27; esophagus, n = 11; oral cavity, n = 11; tonsils, n = 4) and 4 cases of liver metastasis.

antibody

The complete HPKII-type variant region was amplified from human keratinocyte-cDNA by the polymerase chain reaction (PCR) of CD44v (Hofmann et al., 1991). Both PCR primers 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3 'at position 25-52, and 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3' at position 1013-984 of the variant LCLC97 region, as described by Hofmann et al. contain an EcoRI recognition site that is necessary for the PCR product to be cloned directly into the pGEX-2T vector (Smith et al., 1988). The resulting construct (pGEX CD44v HPKII, v3-v10) encodes for a fusion protein of about 70 kD, consisting of glutathione S-transferase from Schistosoma japonicum and exons v3 to v10 of human CD44 (Fig. 1; Heider et al., 1993). The fusion protein will be expressed in E. coli and subsequently affinity purified via glutathione agarose (Smith et al., 1988).

Female Balb / c mice were then immunized intraperitoneally with affinity purified fusion protein according to the scheme:

1. immunization: 50 µg of fusion protein in complete Freund's adjuvant;

2nd and 3rd immunizations: 50 µg fusion protein in incomplete Freund's adjuvant.

Immunizations were performed every 4 weeks apart. 14 days after the last immunization, the animals were immunized with 10 µg of fusion protein in PBS for three consecutive days. On the next day, the spleen of the high antibody titer animal was fused with mouse myeloma cells P3.X63-Ag8.653 using polyethylene glycol 4000. The hybridoma cells were then selected on a microtiter plate in HAT medium (Kohler et Milstein, 1975; Kearney et al., 1979).

Determination of serum antibody titers or screening of outdated hybridomas was performed by ELISA. In this assay, a fusion protein coating (GST-CD44v3-v10) or a glutathione-S-transferase only coating was first formed on the microtiter plates. Subsequently, serum and incubated hybridoma samples were incubated in a series of dilutions and the specific anti-mouse immunoglobulin antibody was determined using antibody-conjugated peroxidase. Hybridomas that only react with glutathione-S-transferase have degenerated. The remaining antibody was first characterized by ELISA with domain-specific fusion proteins (exon v3, exon v5 + v6, exon v6 + v7, exon v8-v10) (Koopman et al., 1993). Its immunohistochemical reactivity was tested on sections of human skin.

BIWA-1 (VFF-18; see also WO 95/33771 for preparation and properties) only bound to fusion proteins that contained domains encoded by exon v6. To further limit the epitope of the antibody, various synthetic peptides representing portions of the v6-domain were used in the ELISA (Fig. 1). The 14 amino acid peptide v6D showed the strongest binding. Therefore, the epitope of the BIWA-1I antibody lies partially or totally in the sequence of the QWFGNRWHEGYROT domain that is encoded by exon v6. This sequence is homologous to the binding epitope of antibody 1.1ASML that is

Used in a rat therapeutic model that is specific to rat CD44v6 (Fig. 1).

immunohistochemistry

Prior to incubation with the primary antibody, paraffin sections (4 µm) were dewaxed in Rotihistol (Roth, BRD) three times for 10 minutes each and then rehydrated in an ascending alcohol row. The sections were then rinsed briefly with distilled water and then boiled three times in 0.01 M Na-citrate buffer, each for 10 minutes, in a microwave (Sharp, Model R-6270) at 600 W. After each microwave incubation, the slices were allowed to cool for 20 minutes. After the last boiling, the carrier was rinsed in PBS in preincubation in normal goat serum (10% in PBS). After three washes in PBS, sections were incubated with primary antibody (BIWA-1: 5 pg.mľ ^1, mouse IgG (isotype matching negative control) 5 gg.mľ ^1, in PBS / 1% BSA) for 1 h. Normal human skin sections were used as a positive control in the color reaction since keratinocytes express the v3-v10-containing CD44 isoform. Endogenous peroxidases were blocked with 0.3% H ₂ O ₂ in PBS and sections were incubated with biotinylated secondary antibody (anti-mouse IgG-F (ab ') ₂ , DAK Corp.) for 30 minutes. For staining, sections were incubated for 30 minutes with horseradish peroxidase, which was bound to biotin as a streptavidin-biotin-peroxidase complex. Sections were then incubated in 3,3-amino-9-ethyl-carbazole substrate (Sigma Immunochemicals) for 5-10 minutes, quenched with H ₂ O, and sections were counter-stained with hematoxylin. The color evaluation was performed with a light microscope (Axioskop, Zeiss) and the coloring was expressed as follows: +++, strong expression; ++, mild expression; +, poor expression; - ambiguous or no detectable expression. Only tumor cells with clear membrane staining were evaluated as positive. The percentage of positive tumor cells in each section was determined by rough estimation by dividing the cells into two groups: focal positive tumors (less than 10% of tumor cells reacted with antibody) and positive tumors (10 or more% of positive tumor cells). If less reacted with the antibody

13 as 80% of the tumor cells from the positive cells, then the corresponding percentage is expressed.

Using the CD44-v6-specific monoclonal antibody BIWA-1, 126 cases of squamous epithelial carcinomas of various origins were analyzed. Isoforms expressing CD44-v6 were observed in all but one tumor sample. A large proportion of the samples showed antigen expression in 80-100% of the tumor cells, staining was limited to the tumor cell membrane. No reaction was observed in the tissues of trees, lymphocytes, muscle cells and endothelium.

To quantify the expression of the CD44-v6 molecules of said tumor cells, normal human skin sections were stained in parallel to the tumor sections. Normal skin keratinocytes express a high level of CD44 isoforms and include CD44-v6, the most expressing normal cells as previously described. Therefore, keratinocyte staining is considered comparative and is referred to as "strong" (+++) in this scoring system. In most tumor samples examined, tumor cell staining was comparable or even stronger than skin keratinocyte staining; In only a few cases, tumor staining was poor (3 cases of metastasized lymph nodes) or moderate (2 primary cancers, 10 metastases). The color reaction was very homogeneous in the sections of the tumors, with most tumor cells showing the same staining intensity. No significant differences in CD44-v6 expression were observed between primary tumors and metastases. A detailed summary of the results is given in Table 1, examples of which are shown in FIG. Second

Table 1 Expression of CD44-v6 in squamous epithelial carcinomas

sample Tumor type 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 +++ 322 92 primary larynx +++ 10209 95 primary larynx +++ 2366 86 primary leather +++ 2574 86 primary leather +++ 9916 86 primary leather ++ / +++ 2696 87 primary leather +++ 8906 87 primary leather +++ 8191 88 primary leather +++ 8354 88 primary leather ++ 50% 11963 88 primary leather ++ 5590 90 primary leather ++ / +++ 530 92 primary leather +++ 2583 94 primary leather +++ 11337 94 primary leather +++ 10901 95 primary leather +++ 11557 95 primary leather +++ 11744 95 primary leather +++ 11917 95 primary leather +++ 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 metastasis larynx +++

lymph nodes 2963 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 / Ι lymph node metastasis larynx +++ 8969 92 2 / ΙΙ lymph node metastasis larynx +++ 8969 92 2 / ΙΙΙ lymph node metastasis larynx ++ 8969 .92 2 / IV lymph node metastasis larynx + / ++ 9366 92 lymph node metastasis larynx +++ 9509 92 lymph node metastasis larynx +++ 9566 92 metastasis larynx +++

lymph nodes 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 ++ 298 95 lymph node metastasis larynx ++ / +++ 15293 91 lymph node metastasis lungs + 25% 1667 92 lymph node metastasis lungs + 20% 2757 92 I lymph node metastasis lungs +++ 2757 92 II lymph node metastasis lungs +++ 2757 92 III lymph node metastasis lungs +++ 2757 92 IV lymph node metastasis lungs +++ 4790 92 lymph node metastasis lungs +++ 6168 92 I metastasis lungs ++ 50%

lymph nodes 6168 92 II lymph node metastasis lungs +++ 6168 92 III lymph node metastasis lungs +++ 6168 92 IV lymph node metastasis lungs +++ 7206 92 lymph node metastasis lungs +++ 7531 92 I lymph node metastasis lungs +++ 7531 92 II lymph node metastasis lungs +++ 7531 92 III lymph node metastasis lungs ++ / +++ 7531 92 IV lymph node metastasis lungs +++ 10324 92 lymph node metastasis lungs +++ 10519 92 II lymph node metastasis lungs +++ 10519 92 RMII lymph node metastasis lungs +++ 10958 92 lymph node metastasis lungs +++ 11425 92 I lymph node metastasis lungs +++ 11425 92 II lymph node metastasis lungs +++

13055 92 lymph node metastasis lungs ++ / +++ 13055 92 II lymph node metastasis lungs focal, +++ 13055 92 III lymph node metastasis lungs +++ 15663 92 lymph node metastasis lungs +++ 16713 92 lymph node metastasis lungs +++ 14980 91 I lymph node metastasis esophagus +++ 14980 91 II lymph node metastasis esophagus +++ 16641 91 I lymph node metastasis esophagus +++ 16641 91 II lymph node metastasis esophagus +++ 16641 91 III lymph node metastasis esophagus +++ 1059 92 lymph node metastasis esophagus + 1710 92 l lymph node metastasis esophagus +++ 1710 92 II lymph node metastasis esophagus +++ 1710 92 III lymph node metastasis esophagus +++ 11502 92 I lymph node metastasis esophagus +++

11502 92 II lymph node metastasis esophagus ++ 202 92 lymph node metastasis oral cavity ++ 60% 6030 92 lymph node metastasis oral cavity +/- 25% 7335 92 I lymph node metastasis oral cavity +++ 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 lymph node metastasis oral cavity +++ 50% 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 almonds +++ 6228 92 II lymph node metastasis almonds +++. 6618 92 lymph node metastasis almonds +++

11840 92 lymph node metastasis almonds ++ 14172 91 4 liver metastasis esophagus +++ 14172 91 5 liver metastasis esophagus +++ 4131 94 1 liver metastasis esophagus + / ++ 8438 94 liver metastasis esophagus outbreaks, ++ / +++

* 80-100% of tumor cells reacted positively with BIWA-1. In cases where fewer tumor cells have reacted with the antibody, the corresponding percentage is reported.

Example 2 Expression of CD44-v6 in Renal Carcinoma, Prostate Cancer and Liver Metastases of Colon Carcinoma Cells

tissue

19 cases of kidney cancer cells were analyzed (12 cases purely cellular, 5 cases chromophilic, 1 case chromophobic, 1 oncocytic), 16 primary prostate adenocarcinomas and 19 cases of prostate cancer lymph node metastases, as well as 30 cases of liver metastases of colon cancer.

antibody

BIWA-1 (see Example 1).

immunohistochemistry

Embodiment as in Example 1.

Unlike squamous epithelial carcinomas, in most analyzed cases of kidney and prostate carcinoma cells, there was no or only focal expression of the CD44-v6 isoforms. In prostate cancer, when expression was more extensive than focal compared to normal prostate epithelial staining, staining was predominantly diffuse cytoplasmic and weak, possibly heterogeneous. More than focal expression of CD44-v6 isoforms was found in 50% of the colon carcinoma liver metastases evaluated. In most cases, staining was mild to moderate, with staining with BIWA-1 most often showing less than 100% of the tumor cells of the sample. A summary of the results is given in Table 2.

Table 2 CD44-v6 expression in adenocarcinoma of the prostate, renal carcinoma and liver metastases of colorectal carcinoma

Tumor type n BIWA-1 reactivity negative faecally positive positive Prostate adenocarcinoma primary 16 8 3 5 Prostate adenocarcinoma lymph node metastases 19 15 2 2 Kidney cancer cells primary 19 17 0 2 Colorectal cancer liver metastases 30 7 8 15

Example 3: Characterization of CD44-v6 specific antibodies

Cell line

A human SCC-line A431 (spontaneous epidermoid vulvar carcinoma) was taken from the American Collection (American Type Culture Collection, Rockwell MD, USA) and grown according to the manufacturer's instructions. Surface expression of CD44-v6 containing isoforms was determined by FACS analysis using the FITC-linker mAb BIWA-1.

Analysis of kinetic constants

The affinity and kinetics of the interaction of the monoclonal antibody and CD44-v6 were determined by Surface Plasmon Resonance (SPR) using the BIAcore 2000 system (Pharmacia Biosensor). A glutathione-S-transferase-CD44 fusion protein containing the region encoded by exons v3 to v10 (GST / CD44 v3-v10) was immobilized to the CM5 sensor chip using an amine coupling procedure according to the manufacturer's instructions. The antibody at various concentrations (8-132 nM) in HBS (10 mM Hepes pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% BIAcore surfactant P 20) was injected at 5 pl.min ^'1 on an antigen-specific surface. The interaction was expressed as a change in SPR-signal. Dissociation of the antibody was observed in buffer flow (HBS). The chip surface was regenerated by a single pulse of 15 μΙ of a 30 mM hydrochloric acid solution. Analysis of the data obtained and calculation of kinetic constants was performed using BIA Evaluation Software (version 2.1, Pharmacia Biosensor).

The antigenic affinity of BIWA-1 was compared with other CD44-v6-specific monoclonal antibodies (VFF4, VFF7, BBA-13 (IgG1, R&D Systems, Abingdon, UK). The kinetic and affinity constants of the various antibodies were determined in two independent Table 3 shows the degree of association (k _a ), degree of dissociation (k _d ), and dissociation constants (K _d ) for 3 mAbs, all mAbs showing near k _and if _d except for BBA-13, which had three times

-24 lower k _α and VFF7, which had a significantly higher dissociation grade (5-fold) compared to other mAbs. This results in lower binding activity for VFF7 and BBA-13 compared to VFF4 and BIWA-1. BIWA-1 showed the lowest K i of all antibodies studied.

Table 3: Kinetic and affinity constants of various CD44-v6-specific monoclonal antibodies (mAbs)

antibody k _a / M ' ¹ .s' ¹ k _d / s' ¹ K _d / M VFF4 1.1. 10 ⁵ 2.2.10 ' ⁵ 2,4.10 ^"10 VFF-7 1.1. 10 ⁵ 1,2.W ⁴ 1.1. 10 ' ⁹ BIWA-1 1.3. 10 ⁵ 2.2.10 ' ⁵ 1,7.10- ¹⁰ BBA-13 3,7.10 ⁴ 2.3. 10 ' ⁵ 6.2. 10 ^'10

Analysis of antibody-protein interaction by enzyme-linked immunoadsorbent analysis (ELISA)

A-431 cells expressing CD44-V6 were cultured in a 96 well plate (Falcon Microtest III, Becton Dickinson, Lincoln Park, NJ, USA) at 5 x 10 ⁴ per well, in RPMI 1640 medium with 10% fetal calf serum, via overnight at 37 ° C. After washing with PBS / 0.05% Tween 20, the cells were fixed with ice-cold ethanol for 1 minute and the preparations were rinsed. This was followed by incubation with primary antibodies (VFF4, VFF7, BIWA-1, BBA-13, 1 ng / ml ¹ to 600 ng / ml ⁾ , each in assay buffer: PBS / 0.5% BSA / 0.05% Tween 20 ) for 1 hour at room temperature. The incubation was followed by a triple rinse. A rabbit-anti-mouse-IgG-horseradish peroxidase conjugate antibody (DAK Corporation, Kopenhagen, Denmark; dilution 1: 6,000 in assay buffer) was used as a secondary antibody (1 hour at room temperature). After washing three times, staining with TMB solution (Kirkegaard + Perry, Gaithersburg, USA) was induced. Absorbance was measured using an ELISA-Reader (Hewlett-Packard).

-25Obr. 3 shows that the relative affinities of the antibody, as determined by BIAcore analysis, reflect the interaction with tumor cells, with BIWA1 clearly showing the highest binding affinity.

The protein domain that is encoded by the CD44-exonorn v6 consists of 45 amino acids (Fig. 4). A series of synthetic peptides was used for ELISA analysis to more accurately determine the epitope known to BIWA-1. Preliminary experiments provided binding to the centrally located 14-mer (amino acid residues 18 to 31; Figure 4; cf. also Figure 1), but not to peptides outside this region. Therefore, a second series of peptides was synthesized and the peptides tested by competitive enzyme-linked immunoadsorbent ELISA (Fig. 4). The results show that peptide 19-29 (WFGNRWHEGYR) is the smallest structure required for high affinity binding. Elimination of the C-terminal arginine residue caused more than 100 fold weaker binding.

Example 4: Biodistribution of radioiodinated CD44-v6-antibody in chicks of xenograft mice

Α-431-Xenograft model

Eight week old female BALB / c nu / nu mice (B&K Universal, Renton, WA, USA) were injected subcutaneously in the left midline with cultured 5 x ^{10 6} A-431 cells (human epidermoid vulva carcinoma). Xenotransplanted animals with A-431 tumors were used for biodistribution experiments over 2 weeks (tumor weight: 40-50 mg).

BIODI-1 radio-iodination

The G-purified monoclonal antibody BIWA-1 protein (IgG1 mice) was coupled to streptavidin using a heterobifunctional crosslinking linker of succinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate. The streptavidin-lysyl residue bound to a reduced antibody-cysteinyl residue, which was prepared by pretreating the antibody with dithiothreitol. The obtained 1: 1 conjugate (> 90%) was further purified

-26-ion exchange chromatography. For biodistribution analyzes, BIWA-1 / SA was labeled via the primary amine of lysine. A p-iodophenyl labeling reagent (PIP; NEN Dupont, Wilmington, DE, USA) was used for labeling and the method described by Willbur et al. (1989). Labeling of BIWA-1 with ¹²⁵ I SA avi did not alter its immunoreactivity or antibody pharmacokinetics in mice.

Biodistribution experiments

Young mice xenografted with human A-431 tumors were injected with 5-7 pCi of ¹²⁵ L per 50 pg of BIWA-1 monoclonal antibody (specific activity 0.1 to 0.14 mCi.mg ^-1 ) intravenously into the lateral tail vein. Time biodistribution studies were performed at 4, 24, 48, 120 and 168 hours post-injection in groups of three animals (n = 3). At the selected time, the mice were weighed, bled through the retro-orbital plexus and sacrificed by neck dislocation. Nine organs and tissues were collected and weighed: blood, tail, lung, liver, spleen, stomach, kidney, intestine and tumor, tissue radioactivity from the injected antibody preparation was measured and compared to standard. Measurements were performed with a gamma cintillator (Packard Instrument Company, Meriden, CT, USA), with the energy window set at ¹²⁵ L at 25 to 80 keV. Percentage of injected dose per g tissue (% ID.g ^-1 ) was calculated.

Preliminary experiments have shown that BIWA-1 does not cross-react with CD44-v6antigen mice. Table. 4 and FIG. 5 show the record of radioactivity in tumors and normal tissue. Iodinated BIWA-1: indicates rapid tumor uptake (7.6% injected dose 4 hours after injection), which rises to more than 18% ID.g * ¹ after 48 hours and then remains stable within 120 hours. Seven days after injection (168 hours), the tumor still contained 15.3% ID.g ^-1 tissue. Tumor to tissue ratios are calculated for each time as shown in Table 4. At 24 hours post injection, the tumor: blood ratio was 0.48 and increased to 3.16 on the seventh day. Intake in normal tissue was low and most likely was due to blood background in tissue biopreparations. Selective in vivo targeting of ¹²⁵ L-labeled BIWA-1 to human SCC-xenografts in mice shows that this monoclonal

The antibody has a high potential as a targeted carrier for diagnostic and therapeutic uses in SCC patients.

Table 4: Tumor: tissue ¹²⁵ L - BIWA-1 ratios in tumor-bearing mice (A-431) at different times after injection

Tumor to tumor ratio 4 hours 24 hours 48 hours 120 hours 168 hours blood 0,22 ^a 0.48 1.31 2.60 3.16 tail 1.18 2.62 7.70 12.28 13.06 lungs 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 kidneys 0.82 1.31 2.72 2.79 2.53 intestine 3.54 6.24 11.94 19.24 27,78

^and mean (n = 3); standard deviations were <7%.

Example 5: Different expression of CD44-v6 antibody in a large number of human tumors

In a further investigation, a total of 544 tumor samples for expression of CD44-V6 were evaluated by immunohistochemistry using the monoclonal antibody BIWA-1 (VFF-18 clone). Samples were first dewaxed or frozen in liquid nitrogen immediately after surgery and stored at -70 ° C until use. The following tumors were analyzed: Basalyoma (n = 16), breast adenocarcinoma (AC) (n = 55), AC intestine (n = 83), squamous epithelial carcinoma (SCC) of the head and neck (n = 125), lung carcinoma (n = 120), AC prostate (n = 34), renal cell carcinoma (n = 27), SCC skin (n = 12) and AC stomach (n = 69). Tissues were obtained by routine surgical method or biopsy, normal tissue was delivered with

-28 tumor sample. The immunohistochemistry was performed in the same manner as in Example 1.

Table 5 provides an overview of immunohistochemical analyzes of 397 different tumor samples using the monoclonal antibody BIWA-1.

Table 5: Expression of CD44-v6 in human tumors

Type Total n Positive n examples n Positive '% Basalyóm primary tumor 16 10 62 Breast AC primary tumor 17 15 88 Breast AC lymph node metastases 34 31 91 Breast AC liver metastases 4 4 100 AC intestine lymph node metastases 51 21 41 AC intestine liver metastases 26 13 50 AC intestine brain metastases 6 6 100 SCC larynx lymph node metastases 18 18 100 Pulmonary AC primary tumor 35 15 43 Pulmonary SCC primary tumor 9 9. 100 SCC of the esophagus primary tumor 20 20 100 AC Prostate primary tumor 16 5 31 AC Prostate lymph node metastases 18 0 0 RCC primary: tumor 27 5 18 SCLC primary tumor 31 7 23 AC stomach primary tumor 22 15 68 AC stomach lymph node metastases 43 16 37 AC stomach kidney metastases 4 4 100 Total n 397

% or more positive tumor cells

AC: adenocarcinoma; RCC: renal cell carcinoma; SCLC: small cell lung cancer; SCC: squamous cell carcinoma.

No or limited reactivity was observed with small cell lung carcinomas, renal cell carcinomas and AC / prostate. All other tumor types examined express isoforms containing CD44-v6 to varying degrees.

Most of the investigated breast ACs showed reactivity with BIWA-1 and evaluated

SCC (larynx, lung and esophagus) express CD44-v6 in 100% of all cases.

A total of 185 cases of SCC of different types and classifications were screened for reactivity with BIWA-1. Of these, 67 cases were primary SCC (larynx, n = 15; oral cavity, n = 16; oral pharynx, n = 3; skin, n = 15), 77 samples were lymph node metastases (larynx, n = 12; lung, n = 27; esophagus n = 11; oral cavity, n = 6; oral pharynx, n = 7; pharynx, n = 10; tonsils, n = 4) and 3 liver metastasis samples (from esophageal tumor). An overview of immunohistochemical analyzes of all SCC samples examined is given in Table 6.

Table 6: Expression of CD44-v6 in squamous epithelial carcinomas

Type Total n negative Focal positive positive n % n % n % Podhltan LNM 10 0 0 0 0 10 100 Oral pharynx 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 lungs PT 18 2 11 0 0 16 89 LNM 27 0 0 1 4 26 96 esophagus PT 20 0 0 1 5 19 95 LNM 11 0 0 0 0 11 100 Oral cavity PT 16 0 0 0 0 16 100 LM 6 0 0 0 0 6 100 leather PT 15 0 0 0 0 15 100 almonds LNM 4 0 0 0 0 4 100 Total n 185

(Note: focal positive: <10% positive tumor samples; LNM: lymph node metastasis; PT: primary tumor; LM: liver metastasis.)

-30Expression of isoforms. containing CD44-v6 was found in all but three cases (one case of larynx, 2 cases of lung). Most of the samples showed antigen expression in 80-100% of the tumor cells in the whole section, with the staining being mainly concentrated on the tumor cell membranes. The strongest homogeneously stained pattern was found in the larynx, esophagus, and pharynx tumor, with most tumor cell sections showing the same staining intensity.

-31 Literature

Altenschmidt U, Kahl Mor, Moritz D, Schnierle BS, Gerstmayer B, Wels W, Groner B. Cytoplysis of tumor cells expressing the neu / erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes. Clinical Cancer Res. 2: 1001-1008 (1996).

Barbas C, Bjorling E, Chiodi F, Dunlop N, Cababa D, Jones T M, Zebedee S L, Persson M A A, Nara P L, Norrby E, Burton D R. Recombinant human Fab fragments neutralize human type l immunodeficiency virus in vitro. Proc. Natl. Acad. Sci. U.S.A. 89: 9339-9343 (1992).

Baum RP, Noujaim AA, Nanci A, Moebus A, Hertel A, Niesen A, Donnerstag B, Sykes T, Boniface G, Hor G. Clinical course of ovarian cancer under repeated stimulation of HAMA using MAb OC125 and B43.13. Hybridoma 12 (5): 583-589 (1993).

Becker JC, Varki N., Gillies SD, Furukawa K., Reisfeld RA. An antibody-interleukin 2 fusion protein overcomes tumor heterogeneity by induction of a cellular immune response. Proc. Natl. Acad. Sci. USA 93: 7826-7831 (1996).

Breitz HB, Weiden PL, Vanderheyden JL, Appelbaum JW, Bjorn MJ, Fer MF, Wolf SB, Ratcliff BA, Seiler CA, Foisie DC, Fisher DR, Schroff RW, Fritzberg AR, Abrams P. G. Clinical experience with rhenium-186- labeled monoclonal antibodies for radioimmunotherapy: results of phase I trials. J. Nucl. Med. 33: 1099-1112 (1992).

\

Breitz H B. Durham J S, Fisher D R, 'Weiden P L, DeNardo G L, Goodgold H M, Nelp W B. Phrmacokinetics-and. normal organ dosimeters following intraperitoneal rhenium-186labeled monoclonal antibody. J. Nucl. Med. 36: 754 (1995).

Brooks, L., Niedobitek, G., Agathanggelou, A., Farrell, P.J. The expression of CD44 variant in nasopharyngeal carcinoma is unrelated to the expression of LMP-1. Am. J. Pathol. / 46 (5 / 1102-12 (1995)).

Catty, D (Hrsg). Antibodies Vols. I undll. IRL Press, Oxford (1989).

Catty, D., Raykundalia, C. ELISA and related immunoassays. In: Catty D (Hrsg). Antibodies Vol. II. IRL Press Oxford (1989), 97-152, p. S. 105-109.

'-i

-32 Catty, D., Murphy, G. Immunoassays using radiolabels. In: Catty D (Hrsg). Antibodies Vol. II. IRL Press Oxford (1989) 77-96.

Chatal JF, Saccavini JC, Gesture JF, Thédrez P, Curtet C, Kremer M, Guerreau D, Nolibe D, Fumoleau P, Guillard Y. Biodistribution of indium-111-labeled OC 125 . Cancer Res. 49: 3087-3094 (1989).

Chaudhary V K, Batra J K, Galdo M G, Willingham M C, Fitzgerald D J, Pastan I. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as singlechain immunotoxins. Proc. Natl. ACAD. Sci. U.S.A. 87: 1066 (1990).

Colcher D, Esteban J, Carrasquillo J A, Sugarbaker P, Reynolds J C, Bryant G, Larson S M, Schlom J. Complementation of intracavitary and intravenous administration of monoclonal antibody (B72.3) in patients with carcinoma. Cancer Res. 47: 4218-4224 (1987).

Coloma M J, Hastings A, Wims L A, Morrison S L. Novel vectors for expression of antibody molecules using variable regions generated by polymerase chain reaction. J. Immunol. Methods 152: 89-104 (1992).

Featherstone C. Bispecific antibodies: the new magic bullets. Lancet 348: 536 (1996).

Friedmann P N, McAndrew S J, Gawlak S L, Chace D, Trail P A, Brown J P, Siegall C B. BR96 sFv-PE40, and potent single-chain immunotoxin that selectively kills carcinoma cells. Cancer Res. 53: 334-339 (1993).

Gerretsen M, Visser GWM, Brakenhoff RH, Walsum M van, Snow GB, van Dongen GAMS. Complete ablation of small squamous cell carcinoma xenografts with ¹⁸⁶ Re-labeled monoclonal antibody E48. Cell Biophysics 24/25: 135-141 (1994).

Goodwin D A. A new appoach to the problem of targeting specific monoclonal antibodies to human tumors using anti-hapten chimeric antibodies. J. Nucl. Med. Biol. 16: 645 (1989).

Goodwin DA. Tumor pretargeting: almost the bottom line. J. Nucl. Med. 36 (5): 876-879 (1995).

-33 Gunthert, U., Hofmann, M., Rudy, W., Reber, S., Zoller, M., HauBmann, I., Matzku, S., Wenzel, A., Ponta, H., and Herrlich, P A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell 65: 13-24 (1991).

Guesdon, J.I., Temynck, T., Avrameas, S.J. Histochem. Cytochem. 27: 1131 (1979).

Gussow D, Seemann G. Humanization of monoclonal antibodies. Methods Enzymol. 203: 99-121 (1991).

Heider, K.-H., Hofmann, M., Horst, E., van den Berg, F., Ponta, H., Herrlich, P., and Pals, STA human homologue of rat metastasis-associated variant of CD44 It is expressed in colorectal carcinomas and adenomatous polyps. J. CellBiol. 120: 227-233 (1993).

Heider, K.-H., Mulder, J.-W. R., Ostermann, E., Susani, S., Patzelt, E., Pals, S.T., Adolf, G.R. Splice variants of cell surface glycoprotein CD44 associated with metastatic tumor cells are expressed in normal tissues of humans and cynomolgus monkeys. Eur. J. Cancer 5: 2385-2391 (1995).

Heider K H, Ratschek M, Zatloukal K, Adolf G R. Expression of CD44 isoforms in human renal cell carcinomas. Virchows Árch. 428: 267-273 (1996a).

Heider KH, Sproll M, Susani S, Patzelt E., Beaumier P., Ostermann E., Ahom H., Adolf GR. Characterization of high-affinity monoclonal antibody specific for CD44v6 as a candidate for immunotherapy of squamous cell carcinomas. Cancer Immunol. Immunother .: in press. 1996th

Hemming AW, Davis NL and Finley RJ. Photodynamic therapy of squamous cell carcinoma: evaluation of anti-EGFR monoclonal antibody-prophyrin conjugate. Society of Surgical Oncology, 46th Annual Cancer Symposium. March 18-21, 1993, Los Angeles, CA, p. 67

Hofmann, M., Rudy, W., Zoller, M., Tólg, C., Ponta, H., Herrlich P., and Gunthert, U. CD44 splice variants conferences metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines. Cancer Res. 51: 5292-5297 (1991).

Johnson, G. D. Immunofluorescence. In: Catty D (Hrsg). Antibodies Vol'íl. IRL Press Oxford (1989), 179-200, p. S. 180-189.

-34Johnson S, Bird R E. Construction of single-chain derivatives of monoclonal antibodies and their production in Escherichia coli. MethodsEnzymol. 203: 88-98 (1991).

Jurcic, JG, Scheinberg, DA. Recent Developments in Radioimmunotherapy of Cancer. Current Opinion in Immunology 6: 715-721 (1994).

Juweid M, Sharkey RM, Markowitz A, Behr T, Swayne LC, Dunn R, Hansen HJ, Shevitz J, Leung SO, Rubin AD, Herskovic T, Hanley D, Goldenberg D M. Treatment of NonHodgkins lymphoma with radiolabeled murine, chimeric, or humanized LL2, an anti-CD22 monoclonal antibody. Cancer Res. (Suppl.) 55: 5899s-597s (1995).

Keamey, J.F., Radbruch A., Liesegang B., Rajewski K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits construction of antibody-secreting hybrid cell lines. J. Immunol. 123: 1548 (1979).

Keenan AM, Weinstein JN, JA Carrasquillo, Bunn PA, Reynolds JC, Foon KA et al. Immunolymphoscintigraphy and dose dependence of ^H1 In-labeled.T101 monoclonal antibody in patients with cutaneous T-cell lymphoma. Cancer Res. 47: 6093-6099 (1987).

Khazaeli MB, Conry RM, LoBuglio AF. Human immune response to monoclonal antibodies. J. hnmunother. 15: 42-52 (1994).

Kôhler, G., Milstein, C. Continous culture of fused cells secreting antibody of predefmed specificity. Nature 265: 495 (1975)

Koopman, G., Heider, K.-H., Horts, E., Adolf, GR, van den Berg, F., Ponta, H., Herrlich, P., Pals, ST, Activated Human Lymphocytes and Aggressive Non-Hodgkin's lymphomas express and homologue of rat metastasis-associated variant of CD44. J. Exp. Med. 177: 897-904 (1993).

Kreitman RJ Hansen HJ, Jones AL, FitzGerald DJP, Goldenberg DM, Pastan I. Pseudomonas exotoxin-based immunotoxins containing the antibody LL2 or LL2-Fab 'induced regression of subcutaneous human B-cell lymphoma in mice. Cancer Res. 53: 819-825 (1993).

Larson S M, Cheung N-K V, Leibel S A. Radioisotope Conjugates. In: DeVita T, Hellman S, Rosenberg S A (Hrsg.). Biology therapy of cancer. J. B. Lippincott Comp., Philadelphia, 496-511 (1991).

^ ^en hacd RE, Order SE, Spunberg JJ, Asbell SO, Leibel S. A. Isotopic immunoglobulin. And ⁿ '' ⁱⁿ lstemic therapy for advanced Hodgkin's Disease. J. Clin. Oncol. 3: 1296-1300 ^ BľaVeyas and Myers M, Stafford N, Rowlinson-bus parking G, Stewart JSW, Epenetos A A. ^ ® ^ 01abeled antibody combined with external radiotherapy for the treatment of head and ^c * ⁿ of cancer: Reconstruction of a theoretical phantom of larynx for radiation dose ^C ^ CU | ati _On to local tissues. Cancer Res. 55: 1020-1027 (1995a).

^ µftVeyas A, Stafford N, Rowlinson-Busza G, Stewart JSW, Epenetos A. Pharmacoki ⁿ ®tÍC | _> biodistribution, and dosimetry of specific and control radiolabeled monoclonal anti-patients with primary head and neck squamous cell carcinoma. Cancer Res. 55: ^Q1 60.1O69 (1995b).

^M "llhine JL, JL Magnani, Linnoila RI: Applications of monoclonal antibodies in the ^tre« ment of solid tumors. In: DeVita VT, Hellman S, Rosenberg SA (eds.), Biology ^th ® ^r _o f APY cancer. JB Lippincott Comp., Philadelphia, 563-588 (1991).

Wklebust AT, Godal A Juell S, Pharo A Fodstad O. Comparison of two antibody-based ^met hods for elimination of breast cancer cells from human bone morrow. Cancer Res. ⁵ : 209-214 (1994).

^ * ⁸ blt M, Fu ZF, McDonald-Smith J, Steplewski Z, Curtis P. J. Production of a functional antibody to detect human colorectal carcinoma cells from a baculovirus exP ^f ® ⁸ Mon system. J. Immunol. Methods 151: 201-208 (1992).

^ fklnsAC, Pimm M. V. A role for gamma scintigraphy in cancer immunology and ^Otl Wapy. Eur. J. Nucl. Med. 19: 1054-1063 (1992).

^ ^Γ ®8β OW, eary JF, Badger CC, Martin PJ, Appelbaum FR, Levy R, Miller R, Brown S, ^ ® * PWB, Krohn KA, Fisher D, Desantes K, Porter B, Kidd P, Thomas ED, Bernstein I. Refractory non-Hodgkin's lymphoma with radiolabeled MB-1 (anti-CD37) ^ant * body. Clin. Oncol. 7: 1027-1038 (1989).

^P ® ⁸ Q w ^F , Fary jp, Appelbaum FR. Martin PJ, Nelp WB Glénn S, Fisher DJ, Porter B, ®tthews DC Gooley T, Bernstein I D. Phase II trial of ¹³¹ I-B1 (anti- CD20) antibody-36rapy with autologous stem cell transplantation for relapsed B cell lymphomas. Lancet 346: 336-340 (1995).

Quadri S M, Vriesendorp H M, Leichner P K, Williams J R. Evaluation of indium-111 and yttrium-90 labeled linker immunoconjugates in nude mice and dogs. J. Nucl. Med. 34: 938945 (1993).

Reisfeld, RA, Gillies, SD, Mendelsohn, J, Varki, NM, Becker, JC. Involvement of B lymphocytes in the growth inhibition of human pulmonary melanoma metastases in athymic nu / nu mice by an antibody-lymphotoxin fusion protein. Cancer res. 56: 1707-1712 (1996).

Riethmuller G, Schneider-Gädicke E, Schlimok G, Schmiegel W et al. Randomized trial of monoclonal antibody for adjuvant therapy of resected Dukes' C colorectal carcinoma. Lancet 343: 1177-1183 (1994).

Salmi, M., Gron-Virta, K., Sointu, P., Grenman, R., Kalimo, H., Jalkanen S. Regulated expression of exon v6 containing isoforms of CD44 in man: Downregulation during malignant transformation of squamocellular tumors origin. J. Cell Biol. 122: 431-442 (1993).

Sambrook, J., Fritsch, E. E., Maniatis, I., Molecular cloning. Cold Spring Harbor Laboratory Press (1989).

Schrappe M, Bumol TF, Apelgren LD, Briggs SL, Markpelitz DD, Mueller BM, Reisfeld R A. Long-term growth suppression of human glioma xenografts by chemoimmunoconjugates of 4-desacetylvinblastine-3-carboxyhydrazide and monoclonal antibody 9.2.27 . Cancer Res. 52: 3838-3844 (1992).

Screaton, G.R., Beli, M.V., Jackson, D.G., Cornelis, F.B., Gerth, U., and Beli, J.I. Genomic structure of DNA encoding the CD44 homing receptor reveals at least 12 alternatively spliced exons. Proc. Natl. Acad. Sci. U.S.A. 89: 12160-12164 (1992).

Sears H, Mattis J, Herlyn D, Häyry P, Atkinson B, Ernst C, Steplewski Z, Koprowski H. Phase-I clinical trial of monoclonal antibody in the treatment of gastrointestinal tumors. Lancet 1982 (1): 162-165 (1982).

Seiter, S., Arch, R., Reber, S., Komitowski, D., Hofrnann, M., Ponta, H.: Herrlich, P.,

Matzku, S., Zoller, M. Prevention of tumor metastasis forination by anti-CD44 variant. J.

Exp. Med. 177: 443-455 (1993).

-37Senter P D, Schreiber G J, Hirschberg D L, Ashe S A, Hellstrom K E, Hellstrom I. Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin C and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates. Cancer Res.

49: 5-9-5-92 (1989).

Shin S-U, Morrison S L. Production and properties of chimeric antibody molecules. Meíhods Enzymol. 178: 459-476 (1989).

Siccardi AG, Buraggi GL, Callegaro L., Colella AC, DeFilippi PG et al. Immunoscintigraphy of adenocarcinomas by means of radiolabeled F (abj ₂ fragments of an anti-carcinoembryonic antigen monoclonal antibody: a multicenter study. Cancer Res. 49: 3095-3103 (1989)).

Smith, D. B., Johnson, K.S. Single-step purification of polypetides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 31 (1988).

Srivastava SC (Hrsg). Radiolabeled monoclonal antibodies for imagirig and therapies. Life Sciences Serie A 152, New York Plenum (1988),

Tólg, C., Hofmann, M., Herrlich, P., and Ponta, H. Splicing choice from the variant exons establishes CD44 variability. Nucleic Acids. Res. 21: 1225-1229 (1993).

Theuer C P, Kreitman R J, FitzGerald D J, Pastan I. Immunotoxins made with a recombinant form of pseudomonas exotoxin And that do not require proteolysis for activity. Cancer Res. 53: 340-347 (1993).

Thomas, G. D., Dykes, P.W., Bradwell, A. R. Antibodies for tumor immunodetection and methods for antibody radiolabeling. In: Catty, D. (Hrsg.). Antibodies Vol. II. IRL Press Oxford, 223-244 (1989).

Thompson C H, Stacker A, Salehi N, Lichtenstein M, Leyden M J, Andrews J T. Immunoscintigraphy for detection of lymph node metastases from breast cancer. Lancet 1984 (2): 1245-1247 (1984).

Vitetta E. S, Thorpe P. E. Immunotoxins. In: DeVita T, Heliman S, Rosenberg S A (Hrsg.). Biology therapy of cancer. Lippincott, J. B., Philadelphia, 482-495 (1991)

-38 * ^Does etta ES, Stone M, Amlot P, Fay J, May R, Till M, Newman J, Clark P, Collins R, Cun Sham ⁿ ^ D, Ghetie V, Uhr JW, Thorpe P E. Phase I immunotoxin trial in patients with B ^cell l lymphoma. Cancer Res. 51: 4052-4058 (1991).

^ ^r '®lendorp HM, Herbst JM, Germack MA Klein J, Leichner PK, Loudenslager DM, Ord _<f

With Phase I-II studies of yttrium-labeled antiferritin treatment for end stage Hodgdiesease, including Radiation Therapy Oncology Group 87-01. J. Clin Oncol. 9: ^918-928 (1991).

Lendorp HM, Morton JD, Quadri S M. Review of consecutive studies of radiotobeled immunoglobulin therapy in Hodgkins' Disease. Cancer Res. (Suppl.) 55: 5888s ⁵⁸⁹ 2e (1995).

^ ^and flg SM, Chem JW, Yeh MY, Ng JC, Tung E, Roffler S. R. Specific activation ot Sbicuronide prodrugs by antibody-targeted enzyme conjugates for cancer therapy. Cancer 52: 4484-4491 (1992).

Hitler LM, O'Dwyer J, Kitson J, Comis RL, Frankel AE, Bauer RJ, Kopnrad MS, Gro ^v ® ⁸ B S. Phase I evaluation of anti-breast carcinoma monoclonal antibody 260F9-re ^C ° mbinant ricin A chain immunoconjugate. Cancer Res. 49: 4062-4067 (1989).

^ Ibur DS Hadley, SW, Hylarides, MD, Abrams, PG, beaumier, PA, Morgan, Ac, _t Reno, JM, Fritzberg, AR Development of stable and radioiodinating agent to label ^ OfiOclonal antibodies for radiotherapy of cancer. J. Nucl. Med. 30: 216-226 (1989).

Wfoter, G., Griffith, A. D., Hawkins, R. E., Hoogenboom, H. R. Making antibodies by Pbfige display technology. Ann. Rev. Immunol. 12, 433-455 (1994).

-39 Sequence Protocol (1) General (1) Applicant:

(A) Name: Boehringer Ingelheim International GmbH (B) Street: Rheinstrasse (C) Place: Ingelheim (E) Country: Germany (F) Postcode: 55 216 (G) Telephone: +49 - (0) - 6132 - 772 770 (H) Fax: +49 - (0) - 6132 - 774 377 (A) Name: Forschungzentrum Karlsruhe GmbH (B) Street: Mailbox 3 640 (C) Location: Karlsruhe (E) Country: Germany (F) Postcode: 76 021 (A) Name: Heider, Karl-Heinz (B) Street: Hervicusgasse 4/3/21 (C) Place: Wien (E) Country: Austria (F) Postcode: 1 120 (A) ) Name: Adolf, Guenther (B) Street: Stiftgasse 15-17 / 10 (C) Place: Wien (E) Country: Austria (F) Postcode: 1 070 (A) Name: Ostermann, Elinborg (B) Street : Mauerbachstrasse 56/6 (C) Place: Wien (E) Country: Austria (F) Postcode: 1 140 (A) Name: Patzelt, Erik (B) Street: Hans-Buchmueller-Gasse 8 (C) Location: Purkersdorf (E) Country: Austria (F) Postcode: 3 002 (A) Name: Sproll, Marlies (B) Street: Schwe nkgasse 3 (C) Place: Wien (E) Country: Austria (F) Postcode: 1 120 (ii) Subject of the invention: Method for diagnosis and therapy of squamous cell carcinoma (iii) Number of sequences: 16 (iv) Computer readable frame:

(A) Data carrier: Flexible disk (B) Computer: IBM PC compatible (C) Operating system: PC-DOS / MS -DOS (D) Software: Patentln Release # 1.0, Version # 1.30 (EPA)

(2) SEQ ID NO 1 data:

(i) Sequence designation:

(A) Length: 129 base pairs (B) Species: nucleotide (C) Worm shape: double (D) Topology: double (ii) Molecule type: genomic DNA (ix) Designation:

(A) Name / Key: exon (B) Range 1 .. 129 (D) Special Data: / product = “CD44” / label = v6 / note = “GenBank data base accesion No.

L05411) / citation = ([1]) (ix)

(A) Name / Key: CDS (B) Range: 3 .. 128 (x) Publication Details:

(A) Authors: Screaton, GR

Beli, MV

Jakson, DG

Cornelis, FB

Gerth, U.

Beli, Jl (B) Name: Genomic DNA structure encoding lymphocyte receptor

CD44 reveals at least 12 alternatively cleaved exons.

(C) Magazine: Proc. Natl. Acad. Sci. U.S.A.

(D) Part: 89 (F) Pages: 12 160-12 164 (G) Date: December 1992 (K) Significant residues in SEQ ID NO: 1: from 1 to 129 (x) Publication Details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (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 Glu Glu Thr Ala Thr Gin Lys

1 5 10 15 GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA ACA CCC 95 Glu gin Trp Phe Gly same time Arg Trp His Glu Gly Tyr Arg gin Thr for 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) Details of SEQ ID NO 2:

(i) Sequence designation:

(A) Length: 42 amino acids (B) Species: amino acid (D) Topology: linear (xi) Sequence description: SEQ ID NO: 1 :. .

gin 1 Ala Thr for Ser 5 Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys Glu 10 15 gin Trp Phe Gly same time Arg Trp His Glu Gly Tyr Arg gin Thr for Arg 20 25 30

Glu Asp Ser Ser Ser Thr Thr Gly Thr Ala

40th

-432) SEQ ID NO 3 Data:

(i) Sequence designation:

(A) Length: 14 amino acids (B) Species: amino acid (C) Worm shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 3:

Gin Trp Phe Gly Thr

5 10

2) Data of SEQ ID NO 4:

(i) Sequence designation:

(A) Length: 27 base pairs (B) Species: nucleotide (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: special nucleic acid (A) Description: / dese = “PCR primer” (x) ) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4

-44 (I) Date of Administration: 17-April-1996 (xi) Sequence Description: SEQ ID NO: 4:

CAGGCTGGGA GCCAAATGAA GAAAATG 27

2) Data of SEQ ID NO 5:

(i) Sequence designation:

(A) Length: 30 base pairs (B) Species: nucleotide (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: special nucleic acid (A) Description: / dese = “PCR primer” (x) ) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 5:

TGATAAGGAA CGATTGACAT TAGAGTTGGA 30

2) Data of SEQ ID NO 6:

(i) Sequence designation:

(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Information ο Publication:

(Η) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 6:

Trp Phe Gly Asn Arg

2) Data of SEQ ID NO 7:

(i) Sequence designation:

(A) Length: 43 amino acids (B) Species: amino acid (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 7:

Gin Ala Thr For Ser Ser Thr Thr Glu Thr Ala 'Thr Gin Lys Glu 1 5 10 15

Gin Trp Phe Gly Asn Arg Trp His Glu Gly Thr Arg Gin Thr Pro Arg 20 25 30

Glu Asp Ser Ser Ser Thr Thr Gly Thr Ala Ala

40

2) Data of SEQ ID NO 8:

(i) Sequence designation:

(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 8:

Ser Ser Thr Glu Glu Glu Thr Ala Thr Gin Lys

5 10

2) Data of SEQ ID NO 9:

(i) Sequence designation:

(A) Length: 10 amino acids (B) Species: amino acid (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 9:

Glu Glu Thin Ala Thr Gin Lys Glu Gin Trp

5 10

2) Data of SEQ ID NO 10:

(i) Sequence designation:

(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 10:

Thr Ala Thr Gin Lys Glu Trin Phe Gly Asdn

10

2) Data of SEQ ID NO 11:

(i) Sequence designation:

(A) Length: 14 amino acids (B) Species: amino acid (C) Worm shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 11:

Gin Trp Phe Gly Thr

10

2) Data of SEQ IDNO12:

(i) Sequence designation:

(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 12:

Asn Arg Trp His Glu Gly Tyr

5 10

2) Data of SEQ ID NO 13:

(i) Sequence designation:

(A) Length: 11 amino acids (B) Species: amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

-49 (H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Sequence description: SEQ ID NO: 13:

Glu Gly Tyr Arg

5 10

2) Data of SEQ ID NO 14:

(i) Sequence designation:

(A) Length: 10 amino acids (B) Species: amino acid (C) Screw shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 • (x) Publication date:

(H) File reference: DE 196 15.074.4 (I) Filing date: 17-Apr-1996 (xi) Sequence description: SEQ ID NO: 14: Thr Pro Arg Glu Asp Ser Ser Ser Thr Gly

2) Data of SEQ ID NO 15:

(i) Sequence designation:

(A) Length: 42 amino acids (B) Species: Amino acid (C) Worm form: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4 (I) Filing date: 17-April-1996 (xi) Description of sequence: SEQ ID NO: 15:

Ala Asp 1 Pro Asn Ser 5 Thr Thr Glu Glu Ala Ala 10 Thr Gin Lys 15 Glu Lys Trp Phe Glu same time Glu Trp gin Gly Lys same time for for Thr for Ser 20 25 30 Glu Asp Ser His wall Thr Glu Gly Thr Thr

40

2) Data of SEQ ID NO 16:

(i) Sequence designation:.

(A) Length: 14 amino acids (B) Species: amino acid (C) Worm shape: single stranded (D) Topology: linear (ii) Molecule type: peptide (x) Publication details:

(H) File reference: DE 195 45 472.3 (I) Filing date: 06-December-1995 (x) Publication date:

(H) File number: DE 196 15 074.4

-51 (I) Date of Administration: 17-April-1996 (xi) Sequence Description: SEQ ID NO: 16:

Lys Trp Phe Glu Asn Glu Lys Asn Pro Thr

5 10?! / Tulle ·

Claims (6)

Use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR for the manufacture of a medicament for the treatment of squamous epithelial carcinomas which is based on the binding of the antibody molecule to an epitope encoded by the variable exon v6 of the CD44 gene, Use according to claim 1. characterized in that the monoclonal antibody antibody molecule is BIWA-1 (VFF-18), which is produced by a hybridoma cell line with accession number DSM ACC2174, or a derivative of said antibody. Use according to claim 1 or 2, characterized in that the monoclonal antibody antibody molecule is a Fab- or F (ab ') ₂ -immunoglobulin fragment, a recombinantly prepared antibody, a recombinantly prepared chimeric or humanized antibody, a bifunctional or single chain antibody (scFv). ). Use according to claims 1 to 3, characterized in that the antibody molecule is bound with a radioactive isotope, a photoactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytostate, a prodrug, with an antibody molecule with other specificity. , a cytokine, or other immunomodulatory polypeptides. 5. A pharmaceutical composition comprising an antibody or antibody molecule that binds to the amino acid sequence WFGNRWHEGYR. Use of an antibody molecule that binds to WFGNRWHEGYR for the preparation of a pharmaceutical composition for the treatment of squamous epithelial carcinomas. Human peptide 1/5 sequence binding v6 QATPSSTTEETATQKEQWFGNRWHEGYRQTPREDSHSTTGTAA BIWA-1 V6A SSTTEETATQK V6B EETATQKEQW V6C TATQKEQWFGN v6D QWFGNRWHEGYRQT V6E NRWHEGYRQTP V6F EGYRQTPREDS V6G TPREDSHSTG + / + rat v6 WADPNSTTEEAAŤQKEKWFENEWQGKNPPTPSEDSHVTEGT T 1.1ASML KWFENEWQGKNPPT Fig. 1 Fig. 2 3/5 Absorbance (OD x 1000): Antibody concentration (ng / ml) Fig. 3: 'IRL X ' Peptide v6 (18-31) v6 (18-29) v6 (19-30) v6 (19-29) v6 (19-28) v6 (20-29)