NO319903B1 - Use of an antibody molecule for the preparation of a pharmaceutical composition for the treatment of squamous cell carcinoma. - Google Patents

Description

The invention relates to the use of an antibody molecule for the production of a pharmaceutical preparation for the treatment of squamous cell carcinomas. Methods for the diagnosis and treatment of squamous cell carcinomas which depend on the expression of the variable exon v6 of the CD44 gene, means for such methods and their use are described.

It has recently been shown that the expression of variants of the surface glycoprotein CD44 is necessary and sufficient to trigger so-called spontaneous metastatic conditions both in a non-metastatic rat pancreatic adenocarcinoma cell line and a non-metastatic rat fibrosarcoma cell line (Gunthert eta !., 1991). While the smallest CD44 isoform, the standard form CD44s, is expressed ubiquitously in a number of different tissues, including epithelial cells, certain splice variants of CD44 (CD44v) are expressed only in a subset of epithelial cells. The CD44 isoforms are formed by alternative splicing so that the sequence of 10 exons <vl-v10) in CD44s is completely excised, but in the larger variants can still occur in different combinations (Screaton et al., 1992; Heider et al., 1993; Hofmann era/., 1991). The variants differ from each other in that different amino acid sequences have been inserted in certain places of the extracellular part of the protein. Such variants can be detected in different human tumor cells and in human tumor tissue. Thus, the expression of CD44 variants in the course of colorectal carcinogenesis has recently been investigated (Heider et al., 1993). The expression of CD44 variants is missing in normal human colonic epithelium, and only a weak expression can be detected in the proliferating cells of the crypts. In later stages of tumor development, e.g. in adenocarcinomas, all malignancies express variants of CD44. Moreover, expression of CD44 splice variants has recently been demonstrated in activated lymphocytes, as well as in non-Hodgkin's lymphomas (Koopman et al, 1993).

Various attempts have been published to make use of the expression differences of exon variants of the CD44 gene in tumors and normal tissues for diagnostic and therapeutic procedures (WO 94/02633, WO 94/12631, WO 95/00658,

WO 95/00851, EP 0531300).

The expression of variants of CD44 molecules in squamous cell carcinomas has also already been investigated. Salmi et al., (1993) thus found with the v6-specific antibody Var3.1. a reduction in v6 expression in tumor cells compared to normal cells. Brooks et al., (1995) obtained a heterogeneous (inheritance of nasopharyngeal carcinomas with the v6-specific antibody 11.9. Only in 2 of 12 cases was a strong staining obtained, while in most of the cases only a weak staining could be detected immunohistologically focal v6 expression.

The task of the present invention has been to develop new methods for the diagnosis and therapy of squamous cell carcinomas, as well as the provision of means for such methods.

This task was solved through the present invention as defined in claims 1-4. Methods are described for the diagnosis and therapy of squamous cell carcinomas depending on the expression of the exon variant v6 of the CD44 gene, which respectively molecular marker or target. In particular, the present invention relates to applications in connection with the strong and homogeneous expression of v6 in squamous cell carcinomas and which, surprisingly enough and contrary to what has previously been assumed, could be determined. Antibody molecules with similar specificity are particularly suitable as a carrier to selectively reach squamous cell carcinomas in vivo.

One aspect of the present invention comprises the use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR, for the production of a pharmaceutical preparation for the treatment of squamous cell carcinomas. Particularly preferred is a use according to the present invention of the monoclonal antibody BIWA-1 (clone VFF-18) which is excised from a hybridoma cell line deposited on June 7, 1994, under accession number DSM ACC2174 at DSM-Deutsche Sammlung fur Mikroorganismen und Zellkulturen GmbH , Mascheroder Weg 1b, D-38124 Braunschweig, Germany (WO 95/33771) or derivatives of this antibody.

Another particularly preferred application according to the present invention is an application where the antibody molecule is a monoclonal antibody, a Fab or F(ab')2* fragment of an immunoglobulin, an antibody produced by recombinant methods, a chimeric or humanized antibody produced by recombinant methods , a bifunctional or single-chain antibody (scFv). A further particularly preferred use according to the present invention is a use according to one of claims 1 to 3, where the antibody molecule is connected to a radioactive isotope, a photoactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytostatic drug, a prodrug, an antibody molecule with a different specificity, a cytokine or another immunomodulatory polypeptide. The nucleic and amino acid sequence of the exon variant v6 in the CD44 gene is known (Screaton et al, 1992, Tolg et al., 1993). The existence of degenerate variants or allelic variants has no significance for the performance of the invention, and such variants are thus expressly included. ;The sequence of exon v6 of the human CD44 gene is: ;;The invention can be carried out with polyclonal or monoclonal antibodies specific for an epitope encoded by exon v6, in particular an epitope within the amino acid sequence WFGNRWHEGYR. The preparation of antibodies against known amino acid sequences can be carried out according to known methods {Catty, 1989). For example, a peptide with this sequence can be produced synthetically and used as an antigen according to an immunization protocol. Another way is the production of a fusion protein containing the desired amino acid sequence, where a nucleic acid (which can be produced synthetically or by PCR (polymerase chain reaction) from a suitable probe) which codes for this sequence, is integrated into an expression vector, and where the fusion protein is expressed in a host organism. The possibly purified fusion protein can then be included as an antigen in an immunization protocol, and insert-specific antibodies, or in the case of monoclonal antibodies, hybridomas expressing insert-specific antibodies, are selected using suitable methods. Such methods are currently known. Heider et al., (1993), 1996a) and Koopman et al., (1993) thus describe the production of antibodies against epitope variants of CD44. For the application according to the invention, however, antibody molecules which are derived from poly- or monoclonal antibodies can also be used, e.g. "Fab or F(ab')2 fragments of immunoglobulins, recombinantly produced single-chain antibodies (scFv), chimeric or humanized antibodies, as well as other molecules that specifically bind to epitopes encoded by exon v6. Of the complete immunoglobulin to the antibody BIWA-1 (VFF-18) or other antibodies, for example Fab or F(ab')2 fragments or other fragments can be produced (Kreitman et ai, 1993). The person skilled in the art is also able to produce recombinant v6-specific In particular, after analyzing the amino acid sequence of the antibody BIWA-1 (VFF-18) and/or using the hybridoma cell line that produces this antibody, especially the genetic information it contains, the skilled person will be able to produce recombinant antibody molecules with the same idiotype as BIWA -1 (VFF-18), i.e. antibody molecules which in the region of the antigen-binding site (complementarity determining regions, CDR) exhibit the same amino acid sequence as antisto ffet BIWA-1 (VFF-18). Similar methods are known today. Such recombinant antibody molecules can e.g. be humanized antibodies (Shin et al., 1989; Gussow et Seemann, 1991), bispecific or bifunctional antibodies (Weiner et ai, 1993; Goodwin, 1989, Featherstone, 1996), "s/nge/-c/7a/n" antibodies (scFv, Johnson et Bird, 1991), complete or fragmentary immunoglobulins (Coloma et ai, 1992; Nesbit et al., 1992; Barbas ef ai, 1992) or antibodies produced by "chain shuffling<*>(Winter ef ai, 1994 Humanized antibodies can for example be produced by CDR grafting

(EP 0239400). Framework regions can also be modified (EP 0519596;

WO 9007861). For the humanization of antibodies, methods such as PCR (see e.g. EP 0368684; EP 0438310; WO 9207075) or computer modeling (see e.g. WO 9222653) can be used today. Fusion proteins can also be produced and used, for example "s/ny/e-cr>a/n" antibody Aoxin fusion proteins (Chaudhary et al, 1990; Friedman et al, 1993). In addition to polyclonal and monoclonal antibodies, the collective terms "antibody" and "antibody molecules" also include all the compounds discussed in this section, as well as other compounds - which structurally can be derived from immunoglobulins and which can be produced according to known methods.

With knowledge of the epitope (cf. Fig. 1, Fig. 4) of BIWA-1 (VFF-18), it is also within the expert's knowledge to produce equivalent antibodies with the same binding specificity that can be used in the present invention.

For diagnostic procedures, antibody molecules, preferably BIWA-1 - antibody molecules, fragments thereof or recombinant antibody molecules with the same idiotype, can be associated, for example, with radioactive isotopes such as 125l,1311,111ln, <99m>Tc or radioactive compounds (Larson et ai, 1991; Thomas ef ai, 1989; Srivastava, 1988), with enzymes such as peroxidase or alkaline phosphatase (Catty ef Raykundalia, 1989), with fluorescent dyes (Johnson, 1989) or biotin molecules (Guesdon ef ai, 1979). For therapeutic applications, v6-specific antibody molecules, preferably BIWA-1 (VFF-18) antibody molecules or VFF-18-derived antibody molecules, e.g. fragments thereof or recombinant antibody molecules of the same idiotype, associate with radioisotopes such as 90Y,<131>1, <186>Re, 18<8>Re, 1<63>Sm, ^Cu, <212>Bi, <213>Bi, <177>Lu (Quadri ef ai, 1993; Lenhard ef ai, 1985; Vriesendorp ef ai, 1991; Wilbur et ai, 1989, Maraveyas etat., 1995a, Jurcic ef Scheinberg, 1994), toxins (Vitetta ef ai, 1991, Vitetta efThorpe, 1991; Kreitman ef ai, 1993; Theuer ef ai, 1993), cytostatics (Schrappe et ai, 1992), prodrugs (Wang ef ai, 1992; Senter et ai, 1989), photoactivatable substances (Hemming et al., 1993), an antibody molecule with a different specificity or with radioactive compounds. The antibody molecule can also be associated with a cytokine or another immunomodulating polypeptide, e.g. with tumor necrosis factor, lymphotoxin (Reisfeld, et al, 1996) or interleukin-2 (Becker et al, 1996). For use in a "pretargeting system", the antibody molecules can e.g. also modified with streptavidin or biotin (Goodwin, 1995).

Using the diagnostic methods, samples from patients, for example from biopsies where squamous cell carcinoma is suspected, or where this diagnosis already exists, but where the tumor needs to be characterized more precisely, can be examined. The detection of CD44 molecular variants containing an amino acid sequence encoded by the variable exon v6 can be done at the protein level using antibodies, or at the nucleic acid level using specific nucleic acid probes or primers for PCR (polymerase chain reaction). For example, tissue sections can be examined immunohistochemically with antibodies using methods known per se. Extracts, extracted from tissue samples or body fluids, can also be examined with other immunological methods using antibodies, for example by Western blot, ELISA (enzyme-linked immunosorbent assay)

(Catty ef Raykundalia, 1989), radioimmunoassays (RIA, Catty et Murphy, 1989) or related immunoassays. The investigations can be carried out qualitatively, semi-quantitatively or quantitatively.

In addition to in vitro diagnostics, the antibody molecules with specificity according to the invention are also suitable for in vivo diagnosis of squamous cell carcinoma. If the antibody molecule carries a detectable label, the label can be detected for diagnostic purposes, e.g. visualization of the tumor in vivo (imaging) or, for example, for radioactively supported surgery (radioguided surgery). For the use of antibodies conjugated with radioactive isotopes for immunoscintigraphy (imaging) there are a number of protocols on the basis of which the person skilled in the art can carry out the invention (Siccardi et al., 1989; Keenan et al., 1987; Perkins ef Pimm, 1992; Colcher et al. et al. 1987; Thompson et al., 1984).

Data obtained by detecting and/or quantifying the expression of the CD44 epitope variant v6 can thus be included in the diagnosis and prognosis. The combination with other prognostic parameters such as e.g. the tumor grade, can therefore be advantageous.

Antibody molecules with the specificity according to the invention, and optionally associated with a cytotoxic agent, can be suitably used for the treatment of squamous cell carcinoma. The application can take place systemically or locally, for example by intravenous (as a bolus or continuous infusion), intraperitoneal, intramuscular, subcutaneous or similar injection/infusion. Protocols for the administration of conjugated or non-conjugated antibodies (such as complete immunoglobulins, fragments, recombinant humanized molecules or the like) are known today (Mulshine et al., 1991; Larson et al., 1991; Vitetta et Thorpe, 1991; Vitetta et al., 1991; Breitz et al., 1992,1995; Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989; Sears et al., 1982). A therapeutic application can, for example, take place analogously to the use of the antibody 1.1ASML (Seifer et al., 1993). Unmodified monoclonal antibodies can be used directly therapeutically when they exhibit a suitable natural effector function for a cytotoxic effect, for example for complement-induced or antibody-induced cellular cytotoxicity (Riethmuller et al., 1994). Suitable monoclonal antibodies for this application are mouse antibody of isotype IgG2a or antibody of human IgG1 type. Furthermore, unmodified antibodies can be administered via an anti-idiotypic mechanism to induce a patient-specific anti-tumor response (Baum ef ai, 1993; Khazaeli et al, 1994).

A preferred therapeutic application consists in connecting a humanized v6-specific immunoglobulin or an F(ab')2 fragment thereof with <90>Y (Quadri et al,

1993; Vriesendorp et ai, 1995).<131>i (Maraveyas et ai, 1995a, 1995b; Juweid ef a/., 1995; Press et a/., 1995; Thomas et ai, in: Catty 1985, pp. 230-239 ), <186>Re (Breitz ef ai 1992,1995) or another suitable radioactive isotope and use this for radio-immunotherapy of squamous cell carcinomas. For example, the antibody BIWA-1, a humanized version of BIWA-1 or an F(ab')2 fragment of BIWA-1 or the humanized antibody can be linked to "Y using a chelating linker such as ITCB-DTPA (isothiocyanate-benzyl-diethylenetriamine pentaacetate), whereby a specific activity of 5-20 mCi/mg, preferably 10 mCt/mg, should be achieved This agent can then be administered to a patient with an antigen-positive tumor at a dosage of 0.1 to 1 mCi/kg body weight, preferably 0.3 to 0.5 mCi/kg body weight.If the antibody molecule is associated with <131>1, a possible dosage regimen at a specific activity of 2 mCi/mg may, for example, be 2 x 150 mCi at 6-week intervals. The person skilled in the art can determine the maximum possible dosage using methods known per se-(Maraveyas et ai, 1995a, 1995b). When administering a total amount of protein of 2 to 5 mg, this can be done in in the form of a rapid intravenous bolus injection.In the case of larger amounts of protein, an infusion may be more beneficial form of administration. When using monoclonal antibodies, it may be necessary to mix this agent with an excess (eg with a tenfold molar excess) of the non-radioactive antibody before administration. In that case, it is better to carry out the administration in the form of an intravenous infusion, e.g. within 15 minutes. The application can be repeated. The therapy can be combined with external radiation therapy. It can also be supplemented with bone marrow transplantation, which is particularly necessary when the therapy achieves a dose of more than 1.6 Gy in the bone marrow.

Antibody molecules can also be used ex wVo for purification of CD34-positives

stem and precursor cell preparations (Immunopurging). The radiation treatment or chemotherapy of squamous cell carcinomas can be supported through autologous bone marrow transplantation. The thereby applied preparation of hematopoietic stem and precursor cells must be free of tumor cells. This can be achieved by incubation with antibody molecules, for example antibody-toxin conjugates (Myklebust et al., 1994; DEP 19648209.7).

Antibody molecules can also be introduced in the form of recombinant constructs into the T-cell receptor of T-lymphocytes. Such reprogrammed T-lymphocytes bind selectively to the antigen-expressing tumor cells and exert a cytotoxic effect so that they can be used for the treatment of squamous cell carcinomas (PCT/EP9604631; Altenschmidt et al., 1996).

Figures

Fig. 1: Determination of the epitope specificity of BIWA-1 through binding to synthetic peptides derived from the human CD44v6 sequence. The corresponding peptide from rat CD44v6 was tested with the antibody 1.1ASML The binding was determined in an ELISA, as the peptides were immobilized on microtiter plates (cf. Heider et al., 1996b, Fig. 2). -: no binding, +/-: weak binding, +: strong binding. Fig. 2: Immunohistochemical analysis of a squamous cell carcinoma of the larynx (a) and a liver metastasis of an esophageal carcinoma (b) with the CD44v6-specific monoclonal antibody BIWA-1.

In both cases, the reactivity of the antibody with the tumor cell's membrane can be seen. Original magnification 40x, counterstaining hematoxylin.

Fig. 3: Comparison of the antigen binding of different CD44v6-specific mAbs.

The binding of four different CD44v6-specific mAbs to human SCC A-431 cells was measured in a Celte ELISA. mAb BIWA-1 exhibits a higher affinity for the tumor cells than the other mAbs.

Fig; 4: Improved epitope mapping of mAb BIWA-1.

The binding of BIWA-1 to various overlapping synthetic peptides spanning from amino acids 18-32 of the CD44v6 coding region was measured in a competitive ELISA. The minimal binding sequence (peptide v6 (19-29)) is underlined.

Fig. 5: Biodistribution of <12>SI-BIWA-1 in A-431 xenotransplanted nude mice

The accumulation of the antibody is indicated as %ID/g (mean value ± SEM) at 4, 24, 48, 120 and 168 hours after injection.

Examples

Example 1: Expression of CD44v6 in squamous cell carcinomas

Loom

A total of 126 paraffin-embedded tumor samples were immunohistochemically examined for expression of CD44v6 with mAb BIWA-1 (clone VFF-18). The samples included 31 cases of primary squamous cell carcinomas (15 cases larynx, 16 cases skin), 91 cases of lymph node metastases (larynx, n=38; lung, n=27; esophagus, n=11; oral cavity, n=11; tonsils, n= 4) and four cases of liver metastases (oesophagus).

Antibody

The entire HPKII-type variable region of CD44v (Hofmann et al., 1991) was amplified from human keratinocyte cDNA by PCR. Both PCR primers 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3\ positions 25-52, and 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3\ positions 1013-984, of the LCLC97 variable region, contained as described by Hofmann et al., an EcøRI recognition site that was used to directly clone the PCR product into the vector pGEX-2T (Smith et al., 1988). The resulting construct (pGEX CD44v HPKII, v3-v10) encodes a fusion protein of -70 kD, consisting of glutathione-S-transferase of Schistosoma japonicum and exons v3-v10 of human CD44 (Fig. 1; Heider et al., 1993). The fusion protein was expressed in E. colt and then affinity purified over glutathione-agarose (Smith ef a/., 1988).

Balb/c female mice were immunized intraperitoneally with the affinity-purified fusion protein according to the following schedule:

1. Immunization: 90 µg fusion protein in Freund's complete adjuvant

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

The immunizations took place 4 weeks apart. 14 days after the last immunization, the animals were further immunized with 10 µg of fusion protein in PBS on three consecutive days. The day after, myrtle cells from an animal with a high antibody titer were fused with P3.X63-Ag8.653 mouse myeldm cells using polyethylene glycol 4000. The hybridoma cells were then selected in microtiter plates in HAT medium (Kohler ef Milstein, 1975; Kearney et al. , 1979).

The determination of the antibody titer in serum, resp. the screening of the hybridoma supernatant was carried out by means of an ELISA. In this test, microtiter plates were first coated with fusion protein (GST-CD44v3-10) or only with glutathione-S-transferase. These were then incubated with serial dilutions of serum samples, resp. hybridoma supernatants, and the specific antibody detected with peroxidase-conjugated antibody against mouse immunoglobulin. Hybridomas that just. reacted with glutathione-S-transferase, was discarded. Remaining antibodies were then characterized with domain-specific fusion proteins (exon v3, exon v5 + v6,

exon v6 + v7, exon v8 - v10) by means of an ELISA (Koopman ef a/., 1993). Their immunohistochemical reactivity was tested on human skin sections.

BIWA-1 (VFF-18; regarding manufacture and properties, see also

WO 95/33771) bound only to fusion proteins containing a domain encoded by exon v6. To further delineate the antibody's epitope, different synthetic peptides representing parts of the v6 domain were used in an ELISA binding test (Fig. 1). The 14 amino acid peptide v6D showed the strongest binding. Accordingly, the epitope of BIWA-1 lies wholly or partially within the sequence QWFGNRWHEGYRQT, the domain encoded by exon v6. This sequence is homologous to the binding epitope of the antibody 1.1 AS ML, which was used in a therapeutic rat model, and which is specific for rat CD44v6 (Fig. 1).

Immunohistochemistry

Before the incubation with the primary antibody, paraffin sections (4 µm) were deparaffinized three times for 10 min in Rotihistol (Roth, Germany) and then rehydrated in an ascending alcohol series. The sections were given a brief wash with distilled water, and then boiled in 0.01 M Na-citrate buffer in a microwave oven (Sharp Model R-6270) three times for 10 minutes at 600 Watts. After each microwave incubation, the sections were cooled for 20 minutes. After the last cooling step, the carriers were washed in PBS and preincubated with normal goat serum (10% in PBS). After three washes in PBS, the sections were incubated for 1 hour with primary antibody (BIWA-1: 5 ug/mL; mouse IgG (isotype - corresponding negative control) 5 ug/mL in PBS/1% BSA). As a positive control for the color reaction, normal human skin sections were used, as keratinocytes express a CD44 isoform containing v3-v10. Endogenous peroxidases were blocked with 0.3% H 2 O 2 in PBS, and the sections were incubated with the biotinylated secondary antibody (anti-mouse IgG-F(ab')2, DAKO Corp.) for 30 minutes. For color development, the sections were incubated for 30 minutes with horseradish peroxidase linked to biotin as streptavidin-biotin-peroxidase complex (DAKO Corp.). The sections were then incubated in 3,3-amino-9-ethylcarbazole substrate (Sigma Immunochemicals) for 5-10 minutes, after which the reaction was stopped with H 2 O and the sections counterstained with hematoxylin. The assessment of (heirs was performed with a Zeiss-Axioskop light microscope, and the staining intensities quantified as follows: +++, strong expression; ++, moderate expression; +, weak expression; -, ambiguous or no detectable expression. Tumor cells only with a clear membrane staining were considered positive. The percentage of positive tumor cells in each section was roughly estimated and two groups were created: focally positive tumors (less than 10% of the tumor cells reacted with the antibody) and positive tumors (10% or more of the tumor cells were When less than 80% of the tumor cells in the positive cells reacted with the antibody, the corresponding percentage was indicated.

126 cases of squamous cell carcinomas of various origins were analyzed with the CD44v6-specific monoclonal antibody BIWA-1. Expression of CD44v6-containing isoforms was observed in all but one tumor sample. The majority of the samples showed an expression of the antigen on 80-100% of the tumor cells, and the inheritance was limited to the tumor cell membrane. With stromal tissue, lymphocytes, muscle cells or endothelium, no reaction was observed.

To quantify the expression of CD44v6 molecules on these tumor cells, sections of normal human skin were stained parallel to the tumor sections. Normal skin keratinocytes express high levels of CD44 isoforms and are considered among the strongest CD44v6-expressing normal cells described to date. The keratinocyte staining was therefore chosen as a reference and classified as "strong" (+++) in our grading system. In most of the examined tumor samples, the staining of the tumor cells was comparable, or even stronger, than the staining of skin keratinocytes, and only a few cases showed weak (3 cases of lymph node metastases) or moderate (2 primary carcinomas, 10 metastases) tumor staining. The staining reaction within a given tumor section was very homogeneous, as most of the section's tumor cells showed the same staining intensity. No significant differences were observed in the CD44v6 expression pattern between primary tumors and metastases. A detailed summary of the results is shown in Table 1, and examples are shown in Fig. 2.

80-100% of the tumor cells reacted positively with BIWA-1.1 in the cases where fewer tumor cells reacted with the antibody, the corresponding percentage is indicated.

Example 2: Expression of CD44v6 in renal cell carcinomas, prostate carcinomas and liver metastases of colon carcinomas

Loom

19 cases of renal cell carcinomas were analyzed (12 cases of clear cells, 5 cases of chromophilic, 1 case of chromophobe, 1 oncocytoma), 16 primary adenocarcinomas of the prostate and 19 cases of lymph node metastases of prostate carcinomas, as well as 30 cases of liver metastases of colon carcinomas.

Antibody

BIWA-1 (see Example 1)

Immunohistochemistry

Execution, see Example 1.

In contrast to the squamous cell carcinomas, no, or only a focal, expression of CD44v6 isoforms could be detected in most of the examined renal cell and prostate carcinomas. In the presence of a more than focal expression of the prostate carcinomas, the staining was predominantly diffuse cytoplasmic and weak or heterogeneous compared to the staining of normal prostate epithelium. In 150% of the examined liver metastases of colon carcinomas, a more than focal expression of CD44v6 isoforms was detected. The staining of the majority of the cases was weak to medium, as often less than 100% of the tumor cells in a sample showed staining with BIWA-1. A summary of the results is given in Table 2.

Example 3: characterization of CD44v6-specific antibodies

Cell line

The human SCC cell line A-431 (spontaneous epidermoid vulvar carcinoma) was obtained from the American Type Culture Collection (Rockwell MD) and cultured according to the manufacturer's instructions. The surface expression of CD44v6-holdigé isoforms was determined by FACS analysis, using a FITC-linked mAb BIWA-1.

Analysis of the kinetic constants

The determinations of the affinity and kinetics of the monoclonal antibody-CD44v6 interaction were made by SPR (Surface Plasmon Resonance), using a BIAcore 2000 system (Pharmacia Biocensor). A glutathione-S-transferase-CD44 fusion protein containing the region coding for exon v3-v10 (GST/CD44v3-vtO) was immobilized on a CM5 Sensor-Chip, during which the amine coupling method was performed according to the manufacturer's instructions. Antibodies at various concentrations (8-132 nM) in HBS (10 mM Hepes, pH 7.4, 150 mM sodium chloride, 3.4 mM EDTA, 0.05% BIAcore surfactant P20) were injected over the antigen-specific surface at a flow rate of 5 pl/min. The interaction was plotted as a change in the SPR signal. The dissociation of the antibodies was observed in buffer flow (HBS) for 5 minutes. The chip surface was regenerated with a single pulse of 15 µL of 30 mM HCl. Analysis of the data and calculation of the kinetic constants was carried out with the Pharmacia Biosensor BIA Evaluation Software, version 2.1.

In this way, the antigen affinity of BIWA-1 was compared with other CD44v6-specific mAbs (VFF4, VFF7, BBA-13 (IgGI, R&D Systems, Abingdon, UK)). Kinetic constants and affinity constants for the different antibodies were each time determined in 2 independent experiments. Table 3 shows the values of the association rates (M, the dissociation rates (kd) and the dissociation constant (Kd) for the 4 mAbs. All mAbs showed similar ka and kd with the exception of BBA-13, which has a 3-fold lower ka. and VFF7, which exhibits a significantly higher dissociation rate (factor 5) compared to the other mAbs. This results in a lower binding affinity for VFF7 and BBA-13 compared to VFF4 and BIWA-1. BIWA-1 exhibits the lowest Kd of all antibodies examined.

Analysis of antibody-protein interaction using ELISA

CD44v6-expressing A-431 cells were grown in 96-well plates (Falcon Microtest 111, Becton Dickinson, Lincoln Park, NJ) at a number of 5 x 10<4> per well in RPM11640 with 10% fetal calf serum at 37° C overnight. After a wash with PBS/0.05% Tween 20, the cells were fixed with ice-cold ethanol for 1 minute, after which a washing step followed. The incubation with the primary antibodies (VFF4, VFF7, BIWA-1, BBA-13.1 ng/mL to 600 ng/mL, each time in determination buffer: PBS/0.5% BSA/0.05% Tween 20) took place for 1 hour at room temperature and was followed by 3 washing steps. A rabbit-anti-mouse-IgG-horseradish-peroxidase-conjugated antibody (DAKO Corporation, Copenhagen, Denmark; dilution 1:6000 in assay buffer) was used as secondary antibody (1 hour/RT). After 3 washing steps, larval development was carried out with TMB solution (Kirkegaard + Perry, Gaithersburg, USA). Extinction was measured with a Hewlett-Packard ELISA-Reader.

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

The protein domains encoded by CD44 exon v6 consist of 45 amino acids (Figure 4). To more precisely define the epitope recognized by BIWA-1, a number of synthetic peptides were used in ELISA assays. Previous experiments showed a binding to a centrally located 14-mer (amino acid residues 18-31; Figure 4; cf. also Figure 1), but not to peptides outside this region. Another series of peptides was therefore synthesized and tested in competitive ELISA determinations (Figure 4). The results show that peptide 19-29 (WFGNRWHEGYR) represents the minimal structure required for high-affinity binding. The elimination of the C-terminal arginine residue produced a more than 100-fold weaker binding.

Example 4: Biodistribution of radioiodinated CD44v6 antibody in nude mice that were carriers of xenograft

A-431 xenograft model

Eight-week-old female Balb/c nu/nu nude mice (B & K Universal, Renton, WA) were subcutaneously injected in the left side in the midline with 5 x 10 6 cultured A-431 cells (human epidermoid vulvar carcinoma). Xenotransplanted animals that were carriers of A-431 tumors were used for the biodistribution experiments during 2 weeks (tumor weight 40-50 mg).

Radioiodination of BIWA-1

Protein G-purified mAb BIWA-1 (murine IgG1) was coupled to streptavidin, using the heterobifunctional cross-linker succinimidyl-4-(N^maleimido-methyl)cyclohexane-1-carboxylate. Streptavidin lysyl residues were associated with reduced antibody cysteinyl residues which had been produced by dithiothreitol pretreatment of the antibody. Obtained 1:1 conjugates (>90%) were purified by ion-exchange chromatography. For the biodistribution experiments, BIWA-1/SA was labeled via primary amines of lysine with <125>l, using p-iodophenyl labeling reagent (PIP; NEN Dupont, Wilmington, DE), following the procedure of Wilbur et al., ( 1989). Labeling of BIWA-1 with SÅ or <126>l did not change the antibody's immunoreactivity or pharmacokinetics in mice.

Biodistribution experiments

Nude mice xenografted with human A-431 tumors were intravenously (i.v.) injected with 5-7 µl Ci <125>l of 50 µg mAb BIWA-1 (specific activity 0.1-0.14 mCi/mg) through the lateral tail veins. Time-course biodistribution studies were performed in groups of n=3 animals per time point, 4, 24, 48, 120 and 168 hours after injection. At selected times, blood samples were taken from weighed mice via the retro-orbital plexus and the animals euthanized by cervical dislocation. Nine organs and tissues, namely blood, tail, lung, liver, spleen, stomach, kidney, intestine and tumor, were removed and weighed. Radioactivity in tissue was counted in a gamma scintillation counter (Packard Instrument Company, Meriden, CT) and compared to standards of the injected antibody preparation, during which the energy window was set at 25-80 keV for <125>l. The percentage of injected dose/g of the tissue was calculated (% ID/g).

Previous experiments had shown that BIWA-1 did not cross-react with murine CD44v6 antigen. Table 4 and Figure 5 show the absorption of radioactivity in tumors and normal tissue. Iodinated BIWA-1 showed a rapid tumor uptake (7.6% injected dose/g four hours after injection) which increased to more than 18% ID/g at 48 hours and then remained constant up to 120 hours. 7 days after injection (168 hours) the tumor still contained 15.3% ID/g tissue. Tumor tissue ratios were calculated for individual time points and are shown in Table 4. At 24 hours after injection, the tumor blood ratio was 0.48 and increased to 3.16 on day 7. Uptake in normal tissue was low and

was most likely caused by blood supply background in the tissue biopsies. Selective in vivo targeting of human SCC xenografts in nude mice with <12S>1-labeled BIWA-1 demonstrated that these monoclonal antibodies exhibit high potential as targeting carriers for diagnostic and therapeutic application in SCC patients.

Example 5: Differential expression of CD44v6 in a large number of human tumors

In an extended investigation, a total of 544 tumor samples were examined immunohistochemically with the monoclonal antibody BIWA-1 (clone VFF-18) for the expression of CD44v6. The samples were either paraffin-embedded or immediately after surgical removal they were frozen in liquid nitrogen and stored at -70°C until use. The following tumors were analyzed: basal cell carcinomas (n=16), adenocarcinomas (AC) of the breast (n=55), AC of the colon (n=83), squamous cell carcinoma (SCC) of the head and neck (n=125), lung carcinoma (n =120), prostate AC (n=34), renal cell carcinoma (n=27),

SCC in skin (n=15) and AC of stomach (n=69). The tissue was taken during routine surgery or biopsy, as normal tissue was the tissue that accompanied the tumor samples. The immunohistochemical examination was performed as in Example 1.

Table 5 shows an overview of the immunohistochemical analysis of 397 different tumor samples with mAb BIWA1.

In small cell lung carcinomas, renal cell carcinomas and AC of the prostate, no or only weak reactivity was observed. All other tumor types examined expressed CD44v6-rich isoforms to varying degrees. Most of the examined AC from the breast showed reactivity with BIWA 1, and the tested SCC (larynx, lung and esophagus) expressed CD44v6 in 100% of the cases.

A total of 185 cases of SCC of different types and classification on reactivity with BIWA 1 were examined. Among these, 67 cases were of primary SCC (larynx, n«15; oral cavity, n=16, oropharynx, n=3; skin, n =15), 77 samples of lymph node metastases (larynx, n=12; lung, n=27; esophagus, n=11; oral cavity, n=6, oropharynx, n=7; hypopharynx, n=10; tonsils, n=4 ) and three samples of liver metastases (oesophagus). Table 6 shows an overview of the immunohistochemical analysis of all examined SCC samples.

Focal pos.: < 10% of tumor cells positive; LNM: lymph node metastasis;

PT: primary tumor; LM: liver metastases

Expression of CD44v6-containing isoforms was found in all but three tumor samples (one larynx case, 2 lung cases). Most samples showed an expression of the antigen on 80 to 100% of the tumor cells within a single section, where the staining was mainly concentrated to the tumor cell membrane. The strongest homogeneous staining pattern was found in carcinomas of the larynx, esophagus and hypopharynx, where most of the section's tumor cells showed the same staining intensity.

Literature

Altenschmidt U, Kahl R, 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. Clinicat Cancer Res. 2:1001-1008 (1996).

Barbas C F, Bjoding E, Chiodi F, Dunlop N, Cababa Dt 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 1 immunodeficiency virus in vhro. Pro. Nati. Acad. Pollock. U.S.A. 89:9339-9343 (1992).

Baum RP, Noujaim AA, Nanci A, Moebus V, Hertel A, Niesen A, Donnerstag B, Sykes T, Boniface G, Hor G. Clinical course of ovarian cancer patients 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. Pro. nati. Acad. Pollock. USA 53:7826-7831 (1996).

Breitz H B, Weiden P L, Vanderheyden J-L, Appelbaum J W, Bjom M J, Fer M F, Wolf S B, Ratcliff B A, Seiler C A, Foisie D C, Fisher D R, Schroff R W, Fritzberg A R, Abrams P G. Clinical experience with rhenium-186- labeled monoclonal antibodies for radioimmunotherapy: results of phase I trials. J. Nucl. With. 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 dosimetry following intraperitoneal rhenium-186-labeled monoclonal antibody. J. Nucl. Med 36:754 (1995).

Brooks, L., Niedobitek, G., Agathanggelou, A., Farrell, P.J. The expression of variant CD44 in nasopharyngeal carcinoma is unrelated to expression of LMP-1. Am. J. Pathol. 746Y5>: 1102-12 (1995).

Catty, D (Ed.). Antibodies Vols. I and II. IRL Press Oxford (1989).

Catty, D., Raykundalia, C. ELISA and related immunoassays. in: Catty, D (Ed.). Antibodies Vol. II. IRL Press Oxford (1989), 97-152, pp. 105-109.

Catty, D., Murphy, G. Immunoassays using radiolabels. in: Catty, D<Ed.). Antibodies Vol. II. IRL Press Oxford (1989), 77-96.

Chatal J-F, Saccavini J-C, Gestin J-F, Thédrez P, Curtet C, Kremer M, Guerreau D, Nolibé D, Fumoleau P, Guillard Y. Biodistribution of indium-111-labeledOC 125 monoclonal antibody intraperitoneally injected into patients operated on for ovarian carcinomas. 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 donating functional variable-region antibody genes in Escherichia eoli as single-chain immunotoxins. Pro. Nati. Acad. Pollock. U.S.A. 87:1066 (1990). Colcher 0, Esteban J, Carrasquillo J A, Sugarbaker P, 'Reynolds J C, BryantG, Larson S M, Schlom J. Complementation of intracavrtary and intravenous administration of a 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 the 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, a potent single-chain immunotoxin that selectively kills carcinoma cells. Cancer Res. 53:334-339 (1993).

Gerretsen M, Visser GWM, Brakenhoff RH, van Walsum M, 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 approach to the problem of targeting specific monoclonal antibodies to human tumors using anti-hapten chimeric antibodies. J. Nucl. With. Biol. 16:645 (1989).

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

Gunthert, U., Hofmann, M., Rudy, W., Reber, S., Zdller, 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. Celt 65:13-24 (1991).

Guesdon, J.I., Ternynck, T., Avrameas, S.J. Histochem. Cytocheny. 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, S.T.A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps. J. Cell Biol. 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 the cell surface glycoprotein CD44 associated with metastatic tumor cells are expressed in normal tissues of humans and cynomolgus monkeys. Eur. J. Cancer 3M. 2385-2391 (1995).

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

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

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

Hofmann, M., Rudy, W., Zoller, M., Tokj, C, Ponta, H, Herrlich P., and Gunthert, U. CD44 splice variants confer 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 (Editor). Antibodies Vol. II. IRL Press Oxford (1989), 179-200, pp. 180-189.

Johnson S, Bird R E. Construction of single-chain derivatives of monoclonal antibodies and their production in Escherichia coli. Methods Enzymol. 203:88-98

(1991).

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

Juweid M, Sharkey R M, MarkowHz A, Behr T, Swayne L C, Dunn R, Hansen H J, Shevitz J, Leung S-O, Rubin A D, Herskovic T, Hanley D, Goldenberg D M. Treatment of Non-Hodgkins's lymphoma with radiolabeled murine, chimeric, or humanized LL2, an anti-CD22 monoclonal antibody. Cancer Res. ( Suppl.) 55:5899s-5907s (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 A M, Weinstein J N, Carrasquillo J A, Bunn P A, Reynolds J C, Foon K A et al. Immunolymphoscintigraphy and the dose dependence of<111> ln-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. Immunother. 15:42-52 (1994).

Kohler, G., Milstein, C. Continuous culture of fused cells secreting antibody of predefined specificity. Nature 265:495 (1975)

Koopman, G., Heider, K.-H., Horts, E., Adolf,<3. R., van den Berg, F., Ponta, H., Herrlich, P., Pals, S. T. Activated human lymphocytes and aggressive Non-Hodgkin's lymphomas express a homologue of the rat metastasis-associated variant of CD44. J. Exp. Med. 777:897-904 (1993).

Kreitman R J Hansen H J, Jones A L, FitzGerald D J P, Goldenberg D M, Pastan I. Pseudomonas exotoxin-based immunotoxins containing the antibody LL2 or LL2-Fab1 induce 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 V T, Hellman S, Rosenberg S A (Eds.). Biological therapy of cancer. J.B. Lippincott Comp., Philadelphia, 496-511 (1991).

Lenhard R E, Order S E, Spunberg J J, Asbell S O, Leibel S A. Isotopic immunoglobulin. A new systemic therapy for advanced Hodgkit's Disease. J. Clin.

Oncol. 3:1296-1300 (1985).

Maraveyas A, Myers M, Stafford N, Rowlinson-Busza G, Stewart J S W, Epenetos A A. Radiolabeled antibody combined with extemal radiotherapy for the treatment of head and neck cancer: Reconstruction of a theoretical phantom of the larynx for radiation dose calculation to local tissues. Cancer Res. 55:1020-1027 (1995a).

Maraveyas A, Stafford N, Rowlinson-Busza G, Stewart J S W, Epenetos A A. Pharmacokinetics, biodistribution, and dosimetry of specrfic and control radiolabeled monoclonal antibodies in patients with primary head and neck squamous-cell carcinoma. Cancer Res. 55:1060-1069 (1995b).

Mulshine J L, Magnani J L, Linnoila R I: Applications of monoclonal antibodies in the treatment of solid tumors, In: DeVita V T, Hellman S, Rosenberg S A (Eds.). Biological therapy of cancer. J.B. Lippincott Comp., Philadelphia, 563-588 (1991).

Myklebust AT, Godal A, Juell S, Pharo A, Fodstad O. Comparison of two antibody based methods for elimination of breast cancer cells from human bone marrow. Cancer Res. 54.-209-214 (1994).

Nesbit M, Fu Z F, McDonald-Smith J, Steplewski Z, Curtis P J. Production of a functional monoclonal antibody recognizing human colorectal carcinoma cells from a baculovirus expression system. J. Immunol. Methods 757:201-208 (1992).

Perkins A C, Pimm M V. A role for gamma scintigraphy in cancer immunology and immunotherapy. Eur. J. Nucl. With. 19:1054-1063 (1992).

Press O W, Eary J F, Badger C C, Martin P J, Appelbaum F R, Levy R, Miller R, Brown S, Nelp W B, Krohn K A, Fisher D, DeSantes K, Porter B, Kidd P, Thomas E

D, Bernstein I D. Treatment of refractory Non-Hodgkir<Y>s lymphoma with radiolabelled MB-1 (anti-CD37) antibody. J. Clin. Oncol. 7:1027-1038 (1989).

Press O W, Eary J F, Appelbaum F R .Martin P J, Nelp W B Glenn S, Fisher D J, Porter B, Matthews D C Gooley T, Bernstein I D. Phase II trial of<131>1-B1 (anti-CD20) antibody therapy 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.

With. 54:938-945 (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-Gådicke 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 tumors of squamocellular origin.

J. Cell Biol. 122:431-442 (1993).

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

SchrappeM, BumolTF, ApelgrenLD, BriggsSL, KoppelG A, MarkowitzDD, Mueller B M, 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., Bell, M.V., Jackson, D.G., Comelis, F.B., Gerth, U., and Bell, J.I. Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Pro. Night. Acad. Pollock. U. S. A. 69:12160-12164

(1992).

Sears H F, Mattis J, Herlyn D, Håyry P, Atkinson B, Ernst C, Steplewski Z, Koprowski H. Phase-I clinical trial of monoclonal antibody in treatment of gastrointestinal tumours. Lancet 1982 (1): 762-765 (1982).

Seiter, S., Arch, R., Reber, S., Komitowski, D., Hofmann, M., Ponta, H:, Herrlich, P., Matzku, S., Zoller, M. Prevention of tumor metastasis formation by anti-variant CD44. J. Exp. Med. 177:443-455 (1993).

Senter P D, Schreiber G J, Hirschberg D L, Asne S A, Hellstrøm K €, Hellstrøm I. En-hancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin

C and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates. Cancer Res. 49:5789-5792 (1989).

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

Siccardi A G, Buraggi G L, Catlegaro L, Colella A C, DeFilippi P G et al. Immunoscintigraphy of adenocarcinomas by means of radiolabelled F(ab')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 polypeptides expressed in Escherichia coli fusions with glutathione S-transferase. Gene 67:31 (1988).

Srivastava S C (Eds.). Radiolabelled monoclonal antibodies for imaging and therapy. Life Sciences Series A 152, Plenum New York (1988).

Tolg, C, Hofmann, M., Herrlich, P., and Ponta, H. Splicing choice from ten 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 A 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. (Ed.). Antibodies Vol. It. IRL Press Oxford, 223-244 (1989).

Thompson C H, Stacker S 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 V T, Hellman S, Rosenberg S A (Eds.). Biological therapy of cancer. J.B. Lippincott Comp., Philadelphia, 482-495

(1991) Vitetta E S, Stone M, Amlot P, Fay J, May R, Till M, Newman J, Clark P, Collins R, Cunningham D, Ghetie V, Uhr J W, Thorpe P E. Phase I immunotoxin trial in patients with B-cell lymphoma. Cancer Res. 57:4052-4058 (1991).

Vriesendorp H M, Herbst J M, Germack M A, Klein J L, Leichner P K, Loudenslager D M, Order S E. Phase l-ll studies of yttrium-labeled antiferritin treatment for end stage Hodgkir's disease, including Radiation Therapy Oncology Group 87-01. J. Clin Oncol. 9: 918-928 (1991).

Vriesendorp H M, Morton J D, Quadri S M. Review of five consecutive studies of radiolabelled immunoglobulin therapy in Hodgkins's Disease. Cancer Res. ( Supt.) 55:5888s-5892s (1995). Wang S-M, Chem J-W, Yeh M-Y, Ng J C, Tung E, Roffler S R. Specific activation of glucuronide prodrugs by antibody-targeted enzyme conjugates for cancer therapy. Cancer Res. 52:4484-4491 (1992). Weiner L M, 0'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-recombinant ricin A chain immunoconjugate. Cancer Res. 45:4062-4067

(1989).

Wilbur, D.S., Hadley, S.W., Hylandes, M.D., Abrams, P.G., Beaumier, P.A., Morgan, A.C., Reno, J.M., Fritzberg, A.R. Development of a stable radioiodinating agent to label monoclonal antibodies for radiotherapy of cancer. J. Nucl. With. 30: 216-226 (1989).

Winter, G., Griffith, A.D., Hawkins, R.E., Hoogenboom, H.R. Making antibodies by phage display technology. Ann. Fox. Immunot. 12,433-455 (1994).

Claims (4)

1. Use of an antibody molecule that binds to the amino acid sequence WFGNRWHEGYR, for the production of a pharmaceutical preparation for the treatment of squamous cell carcinomas. 2. Use according to claim 1, where the antibody molecule is the monoclonal antibody BIWA-1 (VFF-18) which is produced by the hybridoma cell line with the accession number DSM ACC2174, or a derivative of this antibody. 3. Use according to any one of claims 1 or 2, where the antibody molecule is a monoclonal antibody, a Fab or F(ab')2* fragment of an immunoglobulin, an antibody produced by recombinant methods, a chimeric or humanized antibody produced by recombinant methods, a bifunctional or a single-chain antibody (scFv). 4. Use according to one of claims 1 to 3, wherein the antibody molecule is associated with a radioactive isotope, a photoactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytostatic agent, a prodrug, an antibody molecule with a other specificity, a cytokine or another immunomodulatory polypeptide.