HK1011560B - Method of diagnosing and treating epithelioma - Google Patents

Description

The invention relates to methods for the diagnosis and treatment of squamous cell carcinomas based on the expression of the variable exon v6 of the CD44 gene, devices for such methods and their use.

It has recently been shown that the expression of variants of the surface glycoprotein CD44 is necessary and sufficient to induce so-called spontaneous metastatic behaviour in both a non-metastatic pancreatic adenocarcinoma cell line of the rat and a non-metastatic fibrosarcoma cell line of the rat (Günthert et al., 1991). While the smallest CD44 isoform, the standard form CD44s, is ubiquitously expressed in a number of different tissues, including epithelial cells, certain variants of CD44 (CD44v) are expressed only on a subset of epithelial cells.The variants differ in that different amino acid sequences are inserted at a specific site of the extracellular part of the protein. Such variants have been detected in various human tumor cells and in human tumour tissue. For example, the expression of CD44 variants in the course of colorectal carcinogenesis has recently been investigated (Heider et al., 1993). Expression of CD44 variants is absent in normal human colonic epithelium, and only weak expression is detected in the proliferating cells of the malignant cryptosomes., 1993).

Various approaches have been developed to make the differential expression of variants of the CD44 gene in tumours and normal tissues useful for diagnostic and therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851, EP 0531300).

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

The present invention was intended to develop new methods of treatment of squamous cell carcinomas and to provide means for such methods.

This problem was solved by the present invention. It concerns a subject within the scope of claims 1 to 4. It concerns methods for the treatment of squamous cell carcinomas based on the expression of the v6 exon variant of the CD44 gene as a molecular marker or target. In particular, the present invention concerns methods based on the strong and homogeneous expression of v6 in squamous cell carcinomas which have been found to be surprisingly and contrary to the theory as it was known from the state of the art. Antibody molecules with corresponding specificity are particularly suitable as a vehicle for selectively achieving squamous cell carcinomas in vivo.

The preferred method is one which uses an antibody molecule which detects the amino acid sequence WFGNRWHEGYR, in particular the monoclonal antibody BIWA-1 (clone VFF-18), secreted from a hybridome cell line deposited on 7 June 1994 with the DSM-Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany (WO 95/33771), or a derivative thereof.

Other aspects of the present invention are the use of such antibody molecules in the processes of the invention.

The nucleic and amino acid sequence of the v6 variant of the CD44 gene is known (Screaton et al., 1992, Tölg et al., 1993). The existence of degenerate or allele variants is not relevant to the present invention; therefore, such variants are expressly included.

The sequence of exon v6 of the human CD44 gene is:

For example, a peptide of this sequence can be synthesized and used as an antigen in an immunization protocol. Another way is to produce an F protein containing the desired amino acid sequence by producing a nucleic acid (which is synthetic or e.g. produced by polymerase chain reaction (PCR) antigen) by inserting antibodies for these antibodies. In the case of F proteins, the antigen can be integrated into a selective antigen and then used as a selective antigen, and in the case of CD4 and CD4 antibodies, the antigen can be produced in a suitable hybridization process.

However, the method of the invention may also use antibody molecules derived from poly- or monoclonal antibodies, e.g. Fabor F ((ab') 2) fragments of immunoglobulins, recombinant single-chain antibodies (scFv), chimeric or humanized antibodies and other molecules specifically binding to epitopes encoded by exon v6. The complete antibody BIWA-1 (VFF-18) or other antibodies may be produced from the antibody BIWA-1 (VFF-18) or other antibodies, e.g. Fabor F ((ab') 2) fragments or other fragments (Krafman et al., 1993). The expert is also able to produce recombinant antibodies specifically for epitopes encoded by exon v6.Such recombinant antibody molecules may be produced by e.g. humanised antibodies (Shin et al., 1989; Güssow et Seemann, 1991), bispecific or bifunctional antibodies (Weiner et al., 1993; Goodwin, 1989, Featherstone, 1996), single-chain antibodies (FEPv, Johnson et Bird, 1991), complete or fragmentary antibodies (Colobin et al., 1992; Nesbit et al., 1992; Barber et al., 1994; or by humanised antibodies (e.g. CDR239 or CDR400), or by shuffling the chain.Err1:Expecting ',' delimiter: line 1 column 491 (char 490)

It is also within the scope of the average professional to produce, with knowledge of the epitope (see Fig. 1, Fig. 4) of BIWA-1 (VFF-18), equivalent antibodies with the same binding specificity.

For diagnostic purposes, antibody molecules, preferably BIWA-1 antibody molecules, fragments thereof or recombinant antibody molecules of the same idiotype, such as radioactive isotopes such as 125I, 131I, 111In, 99mTc or radioactive compounds (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1988), enzymes such as peroxidase or alkaline phosphatase (Catty and Raykundalia, 1989), fluorescent dyes (Johnson, 1989) or biotin molecules (Guesdon et al., 1979) may be linked together; for therapeutic applications, 153 BiWA6-specific antibody molecules, such as BIWA-171V, 111In, 99mTc or radioactive compounds (Annex 170-1198) or antibodies such as VY-183, FY-183, or radioactive compounds such as BiBiQuad, or similar antibodies (Annex 186-1198, FY-183, FY-183, FY-183, FY-183, FY-183, or FY-183, or similar antibodies, such as BiQuad, or radioactive compounds, such as BiQuad, or radioactive, etc., may be used.The antibody molecule may also be linked to a cell or other immunomodulatory polypeptide, e.g. with lymphoma factor, lymphotoxin (Reukin et al., 1996) or interleukin-2 (Becker et al., 1996), which may also be used in a tumor cell.B. modified with streptavidin or biotin (Goodwin, 1995).

The diagnostic method can be used to detect samples of patients, such as biopsies, in whom platelet carcinoma is suspected or the diagnosis is already present but the tumor is to be characterized more accurately. The detection of variants of CD44 molecules containing an amino acid sequence encoded by the variable exon v6 can be done at the protein level by antibodies or at the nucleic acid level by specific nucleic acid probes or by primary polymerase chain reaction (PCR). For example, tissue layers can be immuno-tochemically examined with antibodies using known methods.

In addition to in vitro diagnostics, antibody molecules with specificity of the invention are also suitable for in vivo diagnosis of squamous cell carcinoma. If the antibody molecule bears a detectable label, detection of the label can be performed for diagnostic purposes, e.g. in vivo imaging of the tumour, or for example for radioguided surgery. For the use of radioisotope conjugated antibodies for immunosynthesis imaging, for example, there are a number of protocols on the basis of which the expert can perform the invention (Siccardi et al., Keenan 1989; et al., 1987; et al., Pimm; et al., 1987; et al., 1987; et al., 1987; and Perkins et al., 1984).

Data obtained by detecting and/or quantifying the expression of the CD44 v6 variant epitope can thus be used for diagnosis and prognosis, and may be useful in combination with other prognostic parameters, such as tumour grade.

Antibody molecules with the specificity of the invention and possibly linked to a cytotoxic agent can be used to benefit the treatment of squamous cell carcinoma, where application may be systemic or topical, e.g. intravenous (as a bolus or continuous infusion), intraperitoneal, intramuscular, subcutaneous, etc. Injection/infusion protocols for administration of conjugated or non-conjugated antibodies (e.g. as complete immunoglobulins, fragments, recombinant humanised molecules, etc.) are the standard technique (Mulshine et al., Larson et al., 1994; Thorpe et al., 1991; Vitaly et al., 1991; Ignace et al., 1991; Weizem et al., 1991; Chather et al., 1985; and Sephora et al., 1989; and others). Antibodies may be used directly for the therapeutic function of a monotypic antibody (Antidote, 1989; and 1989; and similar antibodies may be used for the treatment of a monotypic antibody (Antidote, 1989; and 1982; and 1989; and similar antibodies may be used for the treatment of a monotypic antibody).

A preferred embodiment of therapeutic use is to bind a humanized v6-specific immunoglobulin or an F ((ab') 2 fragment thereof to 90Y (Quadri et al., 1993; Vriesendorp et al., 1995), 131I (Maraveyas et al., 1995a, 1995b; Juweid et al., 1995; Press et al., 1995; Thomas et al., in: Catty 1985, pp. 230-239) 186Re (Breitz et al., 1992, 1995) or another suitable radioisotope and use it for the radiation immunotherapy of platelet carcinomas. For example, the antibody BIWA-1, a humanized version of BIWA-1 or an F ((ab') 2 fragment of BIWA-1 or the human antibody 90Y, can be linked using a chemoprecipitating agent such as mCBD-acetyl-T-Ciocyanate (CBT-Diocyanate, 5 mg/L, 20 mg/L, 5 mg/L, 5 mg/L, 20 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 5 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 mg/L, 6 l, 6 mg/L, 6 mg/L, 6 l, 6 mg/L, 6 l, 6 mg/l, 6 l, 6 mg/l, 6 l, 6 l, 6 mg/l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, 6 l, l, l, l, l, l, l, l, l, l, l,If the antibody molecule is bound to 131I, a possible dosage schedule may be, for example, 2 x 150 mCi at 6 week intervals. The specialist can determine the maximum possible dosages by means of known methods (Maraveyas et al., 1995a, 1995b). With a total protein dose of 2 to 5 mg to be administered, administration may be in the form of a rapid intravenous bolus injection.In the case of monoclonal antibodies, it may be necessary to mix the agent with an excess (e.g. ten-fold molar excess) of the non-radioactive antibody before administration; in this case, administration is better in the form of an intravenous infusion, e.g. over 15 minutes. The application may be repeated. Therapy may be combined with external radiotherapy. It may also be supported by bone marrow transplantation, particularly when a dose of more than 1.6 Gy in the bone marrow is achieved during therapy.

The antibody molecules of the invention can also be used ex vivo for the purification of CD34-positive stem and progenitor cell preparations (immunopurging). Radiation or chemotherapy of squamous cell carcinomas can be supported by autologous bone marrow transplantation. The preparation of hematopoietic stem and progenitor cells must be free of tumour cells. This can be achieved by incubation with antibody molecules of the invention, such as antibody toxin conjugates (Mykust et al., 1994; DE P 196 48 209.7).

The antibody molecules of the invention may also be introduced into the T-cell receptor of T-lymphocytes in the form of recombinant constructs, such reprogrammed T-lymphocytes selectively binding to antigen-expressing tumour cells and exerting cytotoxic effects, so that they can be used in the treatment of squamous cell carcinomas (PCT/EP9604631; Altenschmidt et al., 1996).

The following

Figure 1: Determination of the epitope specificity of BIWA-1 by binding to synthetic peptides derived from the human CD44v6 sequence. The corresponding peptide of rat CD44v6 was tested with the antibody 1.1ASML.

Fig. 2:Immunohistochemical analysis of a carcinoma of the larynx (a) and a liver metastasis of an oesophagus (b) with the CD44v6 specific monoclonal antibody BIWA-1.

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

Fig. 4:Refined epitope mapping of mAb BIWA-1. The binding of BIWA-1 to various overlapping synthetic peptides spanning amino acids 18-32 of the CD44v6 coding region was measured in a competitive ELISA.

The accumulation of the antibody is shown as % ID/g (mean ± SEM) at 4, 24, 48, 120 and 168 h post-injection.

Examples Example 1: Expression of CD44v6 in squamous cell carcinomas Other, of a width of

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

The antibody

The entire region of the HPKII variant of CD44v type (Hofinann et al., 1991) was amplified from human keratinocyte cDNA by polymerase chain reaction (PCR). The two PCR primers 5'-CAGGCTGGGAGCCAAATGAAAAAATG-3', positions 25-52, and 5'-TGATAAGGAGGACATTAGTTG-3', positions 1013-984 of the LCLC97 variant as described by Hofinann et al., contained an EcoRI detector that was used to clone the PCR product directly into the VGEX-2T pctonic sector (Smith et al., 1988). The resulting construct (CD44VX; vKII; vKII; vK10), an affinity protein for HPG, was encoded by Glutathione et al. (SGD.A. et al., 1983) and was transported via the human Glutathione protein (Glutathione et al., 1983) and excreted from the human Glutathione protein (Glutathione et al., 1983).

Female Balb/c mice were immunised intraperitoneally with the affinity purified fusion protein according to the following scheme: Immunisation: 90 μg of fusion protein in complete Friend's adjuvant.

The animals were immunised with 10 μg of fusion protein in PBS for three consecutive days 14 days after the last immunisation, and the next day, mites from an animal with a high antibody titer were fused with P3.X63-Ag8.653 mouse myeloma cells using polyethylene glycol 4000, and the hybrid cells were then selected in micro-titer plates in HAT medium (Köhler et Milstein, 1975; Kearney et al., 1979).

The determination of serum antibody titer and hybridome residues was performed by ELISA. In this test, microtiter plates were first coated with fusion protein (GST-CD44v3-10) or only with glutathione S-transferase. Then, with serial dilutions of serum samples or hybridome residues, the specific antibodies were incubated and detected with peroxidase-conjugated antibodies against mouse immunoglobulin. Hybridomes that reacted only with glutathione S-transferase were rejected. The remaining antibodies were first tested in an ELISA with domain specific fusion proteins (Exon v3, Exon v5, Exon v6, Exon v6, Exon v87, Exon v87, Exon v87, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, Exon v80, and Exon v80, Exon v80, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon v90, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon, Exon

In order to further narrow the epitope of the antibody, various synthetic peptides representing parts of the v6 domains were used in ELISA binding assays (Fig. 1). The 14 amino acid peptide v6D showed the strongest binding. Consequently, the epitope of BIWA-1 lies wholly or partially within the QWFGNRWHYEGRT sequence of the domain encoded by exon v6. This sequence is a binding epitope of the antigen 1.1MLAS, which has been used in a homologous therapeutic council and is specific to the council (CDF4461).

Immunohistochemistry

Before incubation with the primary antibody, paraffin sections (4 μm) were deparaffined in Rotihistol (Roth, Germany) 3 times for 10 minutes and then rehydrated in an ascending alcohol series. The sections were briefly washed with A. dest. and then boiled in a microwave oven (Sharp Model R-6270) 3 times for 10 minutes at 600 watts in 0.01 M Na-citrate buffer. After each microwave incubation, the incubation was isolated for 20 minutes. After the final cooling step, the carriers were washed in PBS and incubated with normal zinc serum (10% in PBS). After 3 PBS investigations, the sections were incubated with BPA (micron-1: 5 μg/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/Ig/I/Ig/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/I/IAs a positive control for the staining response, normal human skin cuts were used, as keratinocytes express a CD44 isoform containing v3-v10. Endogenous peroxidases were blocked with 0.3% H2O2 in PBS, and the cuts were incubated with the biotinated secondary antibody (anti-mouse IgG- ((Fab') 2), DAKO Corp.) for 30 minutes. For staining, the cuts were incubated for 30 minutes with sea cucumber peroxide, which was incubated on biotin as streptovidine biotin peroxidase complex (DAKO Corp.) The cuts were then incubated in 3,3-carbamethyl-9-liptosyl-substrate (sigma-immunohydroxy) for 5-10 minutes, the cuts were stained with F2+ and the reactions were evaluated with a tetracycline, and the cuts were stained with the following chemicals:Only tumour cells with a clear membrane colouration were assessed as positive. The percentage of positive tumour cells in each section was roughly estimated and two groups were formed: focal positive tumours (less than 10% of tumour cells responded with the antibody) and positive tumours (10% or more of tumour cells responded positively). If less than 80% of tumour cells in the positive cells responded with the antibody, the corresponding percentage was indicated.

126 cases of squamous cell carcinoma of various origins were analysed with the CD44v6-specific monoclonal antibody BIWA-1.Expression of CD44v6-containing isoforms was observed in all but one tumor sample.Most samples showed expression of the antigen on 80-100% of tumor cells, staining was limited to the membrane of tumor cells.No response was observed with stromal tissue, lymphocytes, muscle cells or endothelium.

Err1:Expecting ',' delimiter: line 1 column 454 (char 453) Tabelle 1:

46937	86		Primär	Larynx	+++*
4687	90		Primär	Larynx	+++
8372	90		Primär	Larynx	+++
17427	90		Primär	Larynx	+++
27298	90		Primär	Larynx	+++
46908	90		Primär	Larynx	+++
51334	90		Primär	Larynx	+++
51402	91		Primär	Larynx	+++
60414	91		Primär	Larynx	+++
61733	91		Primär	Larynx	+++
12280	92		Primär	Larynx	+++
23140	92		Primär	Larynx	+++
31792	92		Primär	Larynx	+++
32214	92		Primär	Larynx	+++
10209	95		Primär	Larynx	+++
2366	86		Primär	Haut	+++
2574	86		Primär	Haut	+++
9916	86		Primär	Haut	++/+++
2696	87		Primär	Haut	+++
8906	87		Primär	Haut	+++
8191	88		Primär	Haut	+++
8354	88		Primär	Haut	++ 50%
11963	88		Primär	Haut	++
5590	90		Primär	Haut	++/+++
530	92		Primär	Haut	+++
2583	94		Primär	Haut	+++
11337	94		Primär	Haut	+++
10901	95		Primär	Haut	+++
11557	95		Primär	Haut	+++
11744	95		Primär	Haut	+++
11917	95		Primär	Haut	+++
4688	90	I	Lymphknotenmetastase	Larynx	++/+++
4688	90	II	Lymphknotenmetastase	Larynx	-
8374	90		Lymphknotenmetastase	Larynx	+++
17428	90		Lymphknotenmetastase	Larynx	+++
27300	90		Lymphknotenmetastase	Larynx	+++
36942	90		Lymphknotenmetastase	Larynx	+++
46909	90		Lymphknotenmetastase	Larynx	++
51336	90		Lymphknotenmetastase	Larynx	+++
41108	91		Lymphknotenmetastase	Larynx	+++
51398	91		Lymphknotenmetastase	Larynx	+++
60416	91		Lymphknotenmetastase	Larynx	+++
61734	91		Lymphknotenmetastase	Larynx	+++
1318	92	I	Lymphknotenmetastase	Larynx	+++
1318	92	II	Lymphknotenmetastase	Larynx	+++
1318	92	III	Lymphknotenmetastase	Larynx	+++
1318	92	IV	Lymphknotenmetastase	Larynx	+++
2863	92	I	Lymphknotenmetastase	Larynx	+++
2863	92	II	Lymphknotenmetastase	Larynx	+++
5745	92	I	Lymphknotenmetastase	Larynx	+++
5745	92	II	Lymphknotenmetastase	Larynx	+++
8969	92	I	Lymphknotenmetastase	Larynx	+++
8969	92	II	Lymphknotenmetastase	Larynx	+++
8969	92	III	Lymphknotenmetastase	Larynx	++
8969	92	IV	Lymphknotenmetastase	Larynx	+++
8969	92	2/I	Lymphknotenmetastase	Larynx	+++
8969	92	2/II	Lymphknotenmetastase	Larynx	+++
8969	92	2/III	Lymphknotenmetastase	Larynx	++
8969	92	2/IV	Lymphknotenmetastase	Larynx	+/++
9366	92		Lymphknotenmetastase	Larynx	+++
9509	92		Lymphknotenmetastase	Larynx	+++
9566	92		Lymphknotenmetastase	Larynx	+++
12283	92		Lymphknotenmetastase	Larynx	+++
14046	92		Lymphknotenmetastase	Larynx	+++
31787	92		Lymphknotenmetastase	Larynx	+++
49228	92		Lymphknotenmetastase	Larynx	+++ 50%
29228	93		Lymphknotenmetastase	Larynx	+++
29829	93		Lymphknotenmetastase	Larynx	++
29804	95		Lymphknotenmetastase	Larynx	++/+++
15293	91		Lymphknotenmetastase	Lunge	+ 25%
1667	92		Lymphknotenmetastase	Lunge	+ 20%
2757	92	I	Lymphknotenmetastase	Lunge	+++
2757	92	II	Lymphknotenmetastase	Lunge	+++
2757	92	III	Lymphknotenmetastase	Lunge	+++
2757	92	IV	Lymphknotenmetastase	Lunge	+++
4790	92		Lymphknotenmetastase	Lunge	+++
6168	92	I	Lymphknotenmetastase	Lunge	++ 50%
6168	92	II	Lymphknotenmetastase	Lunge	+++
6168	92	III	Lymphknotenmetastase	Lunge	+++
6168	92	IV	Lymphknotenmetastase	Lunge	+++
7206	92		Lymphknotenmetastase	Lunge	+++
7531	92	I	Lymphknotenmetastase	Lunge	+++
7531	92	II	Lymphknotenmetastase	Lunge	+++
7531	92	III	Lymphknotenmetastase	Lunge	++/+++
7531	92	IV	Lymphknotenmetastase	Lunge	+++
10324	92		Lymphknotenmetastase	Lunge	+++
10519	92	II	Lymphknotenmetastase	Lunge	+++
10519	92	RMII	Lymphknotenmetastase	Lunge	+++
10958	92		Lymphknotenmetastase	Lunge	+++
11425	92	I	Lymphknotenmetastase	Lunge	+++
11425	92	II	Lymphknotenmetastase	Lunge	+++
13055	92		Lymphknotenmetastase	Lunge	++/+++
13055	92	II	Lymphknotenmetastase	Lunge	fokal +++
13055	92	III	Lymphknotenmetastase	Lunge	+++
15663	92		Lymphknotenmetastase	Lunge	+++
16713	92		Lymphknotenmetastase	Lunge	+++
14980	91	I	Lymphknotenmetastase	Oesophagus	+++
14980	91	II	Lymphknotenmetastase	Oesophagus	+++
16641	91	I	Lymphknotenmetastase	Oesophagus	+++
16641	91	II	Lymphknotenmetastase	Oesophagus	+++
16641	91	III	Lymphknotenmetastase	Oesophagus	+++
1059	92		Lymphknotenmetastase	Oesophagus	+
1710	92	I	Lymphknotenmetastase	Oesophagus	+++
1710	92	II	Lymphknotenmetastase	Oesophagus	+++
1710	92	III	Lymphknotenmetastase	Oesophagus	+++
11502	92	I	Lymphknotenmetastase	Oesophagus	+++
11502	92	II	Lymphknotenmetastase	Oesophagus	++
202	92		Lymphknotenmetastase	Mundhöhle	++60%
6030	92		Lymphknotenmetastase	Mundhöhle	+/++/+++ 25%
7335	92	I	Lymphknotenmetastase	Mundhöhle	+++
7335	92	II	Lymphknotenmetastase	Mundhöhle	+++
15324	92	II	Lymphknotenmetastase	Mundhöhle	+++ 70%
16164	92	I	Lymphknotenmetastase	Mundhöhle	+++
16164	92	II	Lymphknotenmetastase	Mundhöhle	+++ 50%
16412	92		Lymphknotenmetastase	Mundhöhle	++/+++
16836	92	I	Lymphknotenmetastase	Mundhöhle	+++
16836	92	II	Lymphknotenmetastase	Mundhöhle	+++
16836	92	III	Lymphknotenmetastase	Mundhöhle	+++
6228	92	I	Lymphknotenmetastase	Tonsille	+++
6228	92	II	Lymphknotenmetastase	Tonsille	+++
6618	92		Lymphknotenmetastase	Tonsille	+++
11840	92		Lymphknotenmetastase	Tonsille	++
14172	91	4	Lebermetastase	Oesophagus	+++
14172	91	5	Lebermetastase	Oesophagus	+++
4131	94	1	Lebermetastase	Oesophagus	+/++
8438	94		Lebermetastase	Oesophagus	fokal ++/+++

80-100% of the tumour cells responded positively to BIWA-1.

Example 2: CD44v6 expression in renal cell carcinomas, prostate carcinomas and liver metastases from colon carcinomas Other, of a width of

19 cases of renal cell carcinoma (12 clear cell, 5 chromophilic, 1 chromophobic, 1 oncocytic), 16 primary adenocarcinomas of the prostate and 19 cases of lymph node metastases from prostate cancer, and 30 cases of liver metastases from colon cancer were analysed.

The antibody

The following table shows the information required for the calculation of the CO2 savings:

Immunohistochemistry

The implementation is described in Example 1.

In contrast to squamous cell carcinomas, in the majority of renal cell and prostate carcinomas studied, no or only focal expression of CD44v6 isoforms was detected. In the case of more than focal expression in prostate carcinomas, staining was predominantly diffuse cytoplasmic and weak or heterogeneous compared to normal prostate epithelial staining. In 50% of liver metastases of colon carcinomas studied, more than focal expression of CD44v6 isoforms was detected. The staining in the majority of cases was weak to moderate, with usually less than 100% of the tumor cells in a sample showing a staining with BIWA-1. A summary of the results is given in Table 2. Tabelle 2:

Tumortyp		n	BIWA-1 Reaktivität
			negativ	fokal pos.	positiv
Prostata-Adenokarzinom	Primär	16	8	3	5
Prostata-Adenokarzinom	Lymphknotenmetastasen	19	15	2	2
Nierenzellkarzinom	Primär	19	17	0	2
Kolorektales Karzinom	Lebermetastasen	30	7	8	15

Example 3: Characterisation of CD44v6 specific antibodies Cell line

Human SCC cell line A-431 (spontaneous epidermoid carcinoma of the vulva) was obtained from the American Type Culture Collection (Rockwell MD) and cultured according to the manufacturer's specifications.

Analysis of the kinetic constants

A Glutathione S-transferase CD44 fusion protein containing the region encoded by the exons v3-v10 (GST/ CD44 v3-v10) was immobilized on a CM5 sensor chip, using the amine coupling method as specified by the manufacturer. The antibodies were injected at different concentrations (8-132 nM) in HBS (10 mM pH 7.50 m, sodium, sodium, 3.4 m, 0.5% BTA 0.20 m) The data was calculated using the BIA 0.5% antibody surface area analysis software. The antibodies were observed to have a concentration of 15 μm/ s. The antibodies were analysed using a 5 μm HBS (HIA 0.0 m) controlled analysis software. The antibodies were detected at a concentration of 15 μm/ s. The antibodies were measured at a rate of 5 μm/ s. The data were recorded using a 5 μm HIA controlled analysis of the kinetic energy of the antibodies.

In this way, the antigen affinity of BIWA-1 was compared with other CD44v6-specific mAbs (VFF4, VFF7, BBA-13 (IgG1, R& D Systems, Abingdon, UK). Kinetic and affinity constants of the different antibodies were determined in two independent experiments. Table 3 shows the values of the association rates (ka), dissociation rates (kd) and dissociation constants (Kd) for the 4 mAbs. All mAbs show similar ka and kd, except for BBA-13 which has a 3-fold lower ka and VFF7 which has a significantly higher dissociation rate (factor 5) compared to the other mAbs. This results in lower binding rates (ka), dissociation rates (kd) and dissociation constants (Kd) for VWA-13, and shows the lowest BFF-1 and BIFF-13, compared to all the other mAbs studied. Other Tabelle 3:

VFF4
VFF7
BIWA-1
BBA-13

The test chemical is used to determine the concentration of the active substance in the test chemical.

After a wash with PBS/0.05% Tween 20, the cells were fixed with freezing ethanol for 1 minute, followed by a wash step. Incubation with the primary antibodies (VFF4, VFF7, BIWA-1, BBA-13, 1 ng/ml to 600/ml, each in ng ngsaypu: PBS/ 0.5 % BSA/ 0.05 Tween 20) was performed for 1 hour at room temperature and was followed by 3 steps. A secondary antibody was used as a washing solution. The antibody was diluted with a powder called Perry-Gardner-Gardner-Gardner (Peter-Gardner, USA) in a laboratory called Perry-Gardner-Gardner (Kansas, USA) with a dilution of 1.6 mg/ kg (T-Pack, 1 mg/ ml) in the form of a 1 mg/ ml ELISA.

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

The protein domain encoded by the CD44 exon v6 consists of 45 amino acids (Figure 4). To define more precisely the epitope recognized by BIWA-1, a series of synthetic peptides were used in ELISA assays. Preliminary experiments showed a binding to a centrally localized 14-mer (amino acid residues 18-31; see also Figure 4; Figure 1), but no peptide outside this. A second series of peptides were therefore synthesized and tested in competitive ELISAs (Figure 4). The results show that the peptide region 19-29 (WFGNREGWHYR) represents the minimum structure required for high-level detection.

Example 4: Biodistribution of radioiodine CD44v6 antibodies in xenotransplant-bearing naked mice The following information shall be provided in the following cases:

Eight week old female BALB/c nu/nu naked mice (B & K Universal, Renton, WA) were injected subcutaneously with 5 x 106 cultured A-431 cells (human epidermoid carcinoma of the vulva) into the left median line.

Radioiodine of BIWA-1

G-protein purified mAb BIWA-1 (murinal IgG1) was coupled to streptavidin using the heterobifunctional crosslinker Succinimidyl 4- ((N-maleimido-methyl) cyclohexane-1-carboxylate. Streptavidin lysyl residues were coupled to reduced antibody cysteinyl residues generated by diethiothol pretreatment of the antibody. Obtained 1:1 corvations (> 90%) were further purified by ion exchange chromatography. For biodistribution experiments, BIWA-1/SA was labeled via lysine primates with 125 N-maleimido-methyl cyclohexane.

Biodistribution experiments

Time-lapse biodistribution studies were performed in groups of n=3 animals at 4, 24, 48, 120 and 168 h post-injection. At selected times, mice were weighed, bleed through the retroroorbital plexus and killed by injected localisation. Nine organs and tissues were collected and weighed, including blood, tail, lung, spleen, spleen, kidney, intestine and gamete. The radioactivity in the tissues was calculated in a comparison of the antibody dosage with the dose of the drug injected in a standard instrument (MERGARD, ID/ 80 g/ kg) (Company, CT).

Preliminary experiments had shown that BIWA-1 did not cross-react with murine CD44v6 antigen. Table 4 and Figure 5 show the uptake of radioactivity in tumors and normal tissues. Iodised BIWA-1 showed rapid tumor uptake (7.6% dose/g at 4 h post-injection) which increased to more than 18% ID/g at 48 h and then remained constant for up to 120 h. Seven days post-injection (168 h) the tumour still contained 15.3% ID/g tissue. Tumor:Tissue ratios were calculated for individual time points and shown in BIWA-4. Tabelle 4:

Blut		0.48	1.31	2.60	3.16
Schwanz	1.18	2.62	7.70	12.28	13.06
Lunge	0.40	1.03	2.65	7.04	4.82
Leber	0.94	1.18	2.28	3.57	3.24
Milz	1.40	1.84	4.00	4.86	4.42
Magen	3.89	7.37	19.40	25.56	33.96
Niere	0.82	1.31	2.72	2.79	2.53
Darm	3.54	6.24	11.94	19.24	27.78

Tabelle 4:

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

In an extended study, a total of 544 tumour samples were immunohistochemically tested for CD44v6 expression with the monoclonal antibody BIWA 1 (clone VFF-18) either paraffin-embedded or frozen in liquid nitrogen immediately after surgical removal and stored at -70°C until use. The following tumours were analysed: basal tumors (n=16), adenocarcinomas (AC) of the breast (n=55), AC of the colon (n=83), squamous cell carcinomas (SCC) of the head and neck (n=125), lung cancer (n=120), prostate (n=34), renal cancer (n=27), SCC (n=15) and skin tumours (n=65) The samples were obtained by routine immuno-chemical surgery, such as AC or Magnetobiography.

Table 5 shows an overview of the immunohistochemical analysis of 397 different tumour samples with the mAb BIWA 1. Tabelle 5:

			n	%
Basalioma	Primärtumor	16	10	62
Brust AC	Primärtumor	17	15	88
	Lymphknoten-Metastasen	34	31	91
	Leber-Metastasen	4	4	100
Kolon AC	Lymphknoten-Metastasen	51	21	41
	Leber-Metastasen	26	13	50
	Hirn-Metastasen	6	6	100
Larynx SCC	Lymphknoten-Metastasen	18	18	100
Lunge AC	Primärtumor	35	15	43
Lunge SCC	Primärtumor	9	9	100
Ösophagus SCC	Primärtumor	20	20	100
Prostata AC	Primärtumor	16	5	31
	Lymphknoten-Metastasen	18	0	0
RCC	Primärtumor	27	5	18
SCLC	Primärtumor	31	7	23
Magen AC	Primärtumor	22	15	68
	Lymphknoten-Metastasen	43	16	37
	Leber-Metastasen	4	4	100
		397

Tabelle 5:

*: ≥ 10 % der Tumorzellen positiv AC: Adenokarzinom; RCC: Nierenzellkarzinom SCLC: kleinzelliger Lungenkrebs; SCC: Plattenepithelkarzinom

In small cell lung, renal cell carcinoma and AC of the prostate, no or low reactivity was observed. All other tumour types examined expressed CD44v6-containing isoforms to varying degrees. Most of the ACs examined of the breast showed reactivity to BIWA 1, and the SCCs (larynx, lung and esophagus) examined expressed CD44v6 in 100% of cases.

A total of 185 cases of SCC of different types and classification for BIWA 1 reactivity were examined, including 67 cases 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; oesophagus, n=11; oral cavity, n=6; oropharynx, n=7; hypopharynx, n=10; tonsils, n=4), and 3 samples of liver metastases (esophagus). An overview of the immunohistochemical analysis of all SCCP samples examined is shown in Table 6. Other Tabelle 6:

			n	%	n	%	n	%
Hypopharynx	LNM	10	0	0	0	0	10	100
Oropharynx	PT	3	0	0	0	0	3	100
	LNM	7	0	0	0	0	7	100
Larynx	PT	15	0	0	0	0	15	100
	LNM	30	1	3	0	0	29	97
Lunge	PT	18	2	11	0	0	16	89
	LNM	27	0	0	1	4	26	96
Ösophagus	PT	20	0	0	1	5	19	95
	LNM	11	0	0	0	0	11	100
	LM	3	0	0	0	0	3	100
Mundhöhle	PT	16	0	0	0	0	16	100
	LM	6	0	0	0	0	6	100
Haut	PT	15	0	0	0	0	15	100
Tonsille	LNM	4	0	0	0	0	4	100
Gesamt n		185
Fokal pos.: < 10 % der Tumorzellen positiv; LNM: Lymphknotenmetastasen; PT: Primärtumor; LM: Lebermetastasen

Expression of CD44v6-containing isoforms was found in all but three tumour samples (one case of larynx, two cases of lung). The majority of samples showed expression of the antigen on 80 to 100% of tumour cells within a single incision, with staining concentrated mainly on the membrane of tumour cells. The most homogeneous staining pattern was found in carcinoma of the larynx, esophagus and hypopharynx, with most tumour cells in the incision showing the same staining intensity.

Other

The following is a list of the most commonly used and used antibiotics in the European Union:The results of the study were published in the journal Physiology and Biology in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology and Medicine in the journal Physiology in the journal Physiology and Medicine in the journal Physiology in the journal Physiology and Medicine in the journal Physiology in the journal Physiology and Medicine in the journal Physiology in the journal Physiology in the journal Physiology and Medicine in the journal Physiology in the journal Physiology in the journal Physiology and Medicine in the journal Physiology in the journal Physiology in the journal Physiology in the journal Physiology in the journal Physiology in the journal Physiology in the journal Physiology in the journal Physiology in the journal of the journal Physiology in the journal of the journal Physiology in the journal of the journal Physiology in the journal Physiology in the journal of the journal of the journal Physiology in the journal of the journal of the journal Physiology in the journal of the journal of the journal of the journal Physiology in the journal of the journal of the journal of the journal of the journal Physiology in theThe expression of CD44 in nasopharyngeal carcinoma is unrelated to expression of LMP-1. Am. J. Pathol. 1465): 1102 (1995).Catty, D (Hg). Antibodies Vols. I and II. IRL (1989). D. Rays, C. Rays, C. and related immunoassays: Catty, D. In.In: Catty, D (Hrsg). 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-labeled OC 125 monoclonal antibody intraperitoneally injected into patients operated on for ovarian cancer. Cancer Results: 49: 3087-3094 (1989).Chaudhary V, Batra J, Galdo D, Momas G, G Gingham M, Fitzgerald J, Jericho J. A method of cloning single variable functional antibody region genes in the Willis coli chain as a single nucleus.The study was conducted in the context of a study of the effects of the use of antibodies on the immune system, and was funded by the National Institutes of Health (NIH).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 186Re-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 chemic antibodies. J. Nucl. Med. Biol. 16: 645 (1989).Goodwin. Tumor pretargeting: almost the bottom line. J. Nucl. Med. 365): 876-879 (1995).Gert, S., R., S., R., S., R., S., S., W., M., W., M., M., R., R., S., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R., R.,The following is a list of the most commonly used and used antibodies in the treatment of cancer: Cell 65:13-24 (1991).Guesdon, J. I., Ternynck, T., Avrameas, S. J. Histochem. Cytochem. 27: 1131 (1979).Güssow 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 humanologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps.Heider K. H. Ratschek M. Zatloukal K. Adolf G. R. Expression of CD44 isoforms in human renal cell carcinomas. Virchow Arch. 428: 267-273 (1996a).Heider KH, Sproll M. Susani S. Patzelt E. Beaumier P. Ostermann E. Ahorn H. Adolf GR. Characterization of a high-affinity monoclonal antibody specific for CD44v6 as a candidate for immunotherapy of squamous cell carcinomas. Cancer Immunotherm.: in press. 1996.Hemming AW, Davis NL, Finley RJ. Photodynamic therapy of squamous cell carcinoma: an evaluation of an anti-FREG monoclonal antibody-hyrpropin conjugate. Oncology of Surgical Cancer, 46th Symposium, Los Angeles, March 1993, p. 18-21, oncology of the year.In: Catty, D (Hrsg). Antibodies Vol. II. IRL Press, Oxford (1989), 179-200, s. S. 180-189.Johnson S, Bird R E. Construction of single-chain derivatives of monodonal antibodies and their production in Escherichia coli. Enzyme methods 203: 88-981).Jurcic JG, Scheinberg DA. Recent developments in the current opinion of radioimmunology in cancer.The following is a list of the most commonly used chemicals in the treatment of non-Hodgkins lymphoma: chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals, chemicals,Cancer Res. 47: 6093-6099 (1987).Khazaeli MB, Conry RM, LoBuglio AF. Human immune response to monoclonal antibodies. J. Immunother. 15: 42-52 (1994).Köhler, G., Milstein, C. Continous culture of fused cells secreting antibody of predefined specifity. Nature 265: 495 (1975)Koopman, G., Heider, K.-H., Horts, E., Adolf, G. 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 metastasis-associated variant of CD44.The study was conducted in the United States, Canada, and the United Kingdom, and was funded by the National Institutes of Health (NIH). The study was funded by the National Institutes of Health (NIH). The study was funded by the National Institutes of Health (NIH). The study was funded by the National Institutes of Health (NIH).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 specific 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 (Hrsg.). Biologic therapy of cancer. J. B. Lippincott, based in Philadelphia, 563-588 (1991).Mykust, Godal A, Juell S, Juell A, Pharood O, F. O. Comparison of two methods of antibody elimination from human breast cancer cells.The results of the study were published in the journal Nature and the Journal of Cancer Resolution 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 151: 201-208 (1992).Perkins A C, Pimm M V. A role for gamma scintigraphy in cancer immunology and immunotherapy. Eur. J. Nucl. Med. 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, Kro K A, Fisher D, Porter D, De Kantes, K K, Non P, Thomas E, Bernstein D. Treatment of refractory lymphoma with refractory lymphoma's antibody (CD-13)The study was conducted in the context of a study of the effects of the use of antibodies on the immune system, and was funded by the National Institutes of Health (NIH). The study was conducted in collaboration with the National Institute of Allergy and Infectious Diseases (NIH) and the National Institute of Allergy and Infectious Diseases (NIH), and was funded by the National Institutes of Health (NIH).The study was conducted in the context of a study of the effects of the use of monoclonal antibody on the immune system, and was funded by the National Institutes of Health (NIH).The following is a list of the most commonly used chemicals in the food industry, including chemicals that are used in the production of foodstuffs, and the most commonly used chemicals in foodstuffs, including chemicals that are used in the manufacture of foodstuffs and in the manufacture of foodstuffs.The study was conducted in the context of the European Commission's European Research Council's (ERC) European Research Council (ERC) in the field of the prevention of cancer and the prevention of cancer in the European Union (ESA) in the context of the European Research Council's (ERC) European Research Council (ERC) European Research Council (ERC) in the field of the prevention of cancer and the prevention of cancer in Europe (ESA) in the context of the European Research Council's (ERC) European Research Council (ERC) European Research Council (ERC) European Research Council (ERC) European Research Council (ERC) European Research Council (ERC) European Research Council) and the European Research Council (ERC) European Research Council (ERC) European Research Council (ERC) European Research Council (ERC) European Research Council (ERC) and the European Research Council (ERC) European Research Council (ERC) and the European Research Council (ERC) European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (ERC) and the European Research Council (REC) and the European Research Council (REC) and the European Research Council (REC) and the European Research Council) on the European Research Council (REC) and the European Research Council (REC) and the European Research Council and the European Research Council (REC) on the European Research Council and the European Research Council (REC) and the European Research Council and the European Research Council (REC) on the European Research Council and the European Research Council and the European Research Council on the European Research Council (REC) on the European Research and the European Research Council and the European Research CouncilImmunoscintigraphy of adenocarcinomas by means of radiolabeled 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 polypetides expressed in Escherichia coli as fusions with glutathione S-transferase. 67: 31 (1988).Srivastava S C (Hrsg). Radiolabeled monoclonal antibodies for imaging and therapy. Life Sciences Series A 152, Plenum New York (1988).Tölg, C., Hofmann, M., Herrlich, P., and Ponta, H. Splicing from ten variants selection establishes variability. The nucleotide acids are not required for the production of a pseudotoxin.The study was conducted in the United States, Canada, and the United Kingdom, and was funded by the National Institutes of Health (NIH). The study was funded by the National Institutes of Health (NIH) and the National Institutes of Health (NIH).The study was conducted in the context of a clinical trial of patients with B-cell lymphoma. Cancer Res. 51: 4052-4058 (1991).Vriesendorp H M, Autumn J M, Germack M A, Klein J L, Leichner P K, Loudenslager D M, Order S E. Phase I-II studies of yttrium-labeled antiferritin treatment for end stage Hodgkin's disease, including Radiation Therapy Oncology Group 87-01.The following table shows the results of the analysis of the results of the studies carried out in the framework of the European Monitoring Centre for Disease Prevention and Control (EMCDDA) in the field of the use of the active substance in the control of cancer:The use of the drug in the treatment of infectious diseases is also recommended.

The following shall be added:

(1) GENERAL information: the name of the manufacturer and the name of the manufacturer (i) The applicant: (a) NAME: Boehringer Ingelheim International GmbH (B) NAME: Rheinstrasse (C) NAME: Ingelheim (E) NAME: Germany (F) NAME: Heider, OR-Heinz (G) TELEPHONE: +49-(0) 6132-772770 (H) TELEFACS: +49-(0) 6132-774377 (A) NAME: Research Centre Karlsruhe GmbH (B) NAME: 3640 (C) NAME: Purlsruhe Therapie (F) NAME: Germany (G) NAME: Heider, OR-Heinz (F) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (F) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (F) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (F) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (F) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (F) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (F) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: Heider, OR-Heinz (G) NAME: 721606206206 (F) NAME: OR-Heinz (G) NAME: HEID: HEID: HEIDER (G) NAME: HEIDER (G) NAME: HEIDER (G) HEIDER (G) HEIDER (G) HEIDER, (A) DATENTER: floppy disk (B) COMPUTER: IBM PC compatible (C) Operating system: PC-DOS/MS-DOS (D) Software: Patent in release # 1Err1:Expecting ',' delimiter: line 1 column 332 (char 331)Err1:Expecting ',' delimiter: line 1 column 954 (char 953)Err1:Expecting ',' delimiter: line 1 column 437 (char 436)The Commission shall be assisted by the Member States in the implementation of this Decision. (H) DOCUMENT NUMBER: DE 196 15 074.4 ((I) DATE OF FIRMING: 17-APR-1996 ((xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:)) (2) The following information is provided for the purpose of the SEQ ID: 8: (i) Sequence characteristics: (a) LENGTH: 11 amino acids (b) TYPE: amino acid (c) String shape: single string (d) Topology: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF FIRMING: 06-DEC-1995 (X) Information to be published: The following information is provided for the purpose of the application: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) DATE OF FIRMING: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 8: (ii) The name of the person who is to be notified of the decision: (i) Sequence characteristics: (a) LENGTH: 10 amino acids (b) TYPE: amino acid (c) String shape: single string (d) Topology: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF NOTICE: 06-DEC-1995 (X) PURPOSE: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) DATE OF NOTICE: 17-APR-1996 (xi) Sequence description: SEQ ID No: 9: (I) The information provided by the SEQ ID No: 10: (i) Sequence characteristics: (a) LENGTH: 11 amino acids (b) TYPE: amino acid (c) String shape: single string (d) Topology: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472 The following is a list of the documents which have been submitted to the Commission:The Commission shall be assisted by the Member States in the implementation of this Decision. (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) DATE OF FIRMING: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 10: (ii) The name of the person who is to be notified of the decision: (i) Sequence characteristics: (a) LENGTH: 14 amino acids (b) TYPE: amino acid (c) String shape: single string (d) Topology: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF FIRMING: 06-DEC-1995 (X) Information to be published: The following information is provided for the purpose of the application: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) DATE OF FIRMING: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 11: (ii) The name of the person who is to be notified of the decision: (i) Sequence characteristics: (a) LENGTH: 11 amino acids (b) TYPE: amino acid (c) String shape: single string (d) Topology: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF FIRMING: 06-DEC-1995 (X) Information to be published: The following information is provided for the purpose of the application: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) DATE OF FIRMING: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 12: (I) The following information is provided for the purpose of the SEQ ID No: 13: (i) Sequence characteristics: (A) Length: 11 amino acids,The following information is provided for the purpose of the analysis of the following information: (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF FIRMING: 06-DEC-1995 (X) Information to be published: The following information is provided for the purpose of the application: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) DATE OF FIRMING: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 13: (ii) The name of the person who is to be notified of the decision: (i) Sequence characteristics: (a) LENGTH: 10 amino acids (b) TYPE: amino acid (c) String shape: single string (d) Topology: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF FIRMING: 06-DEC-1995 (X) Information to be published: The following information is provided for the purpose of the application: (H) DOCUMENT NUMBER: DE 196 15 074.4 (I) DATE OF FIRMING: 17-APR-1996 (xi) SEQUENCE DESCRIPTION: SEQ ID No: 14: (ii) The name of the person responsible for the SEQ ID No: 15: (i) Sequence characteristics: (a) LONG: 42 amino acids (b) TYPE: amino acid (c) STRING FORM: single strand (d) TOPOLOGY: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF FIRMING: 06-DEC-1995 (X) Information to be published: The following information is provided for the purpose of the application: (H) DOCUMENT NUMBER: DE 196 15 074. The following is a list of the documents which have been submitted to the Commission:The Commission shall, by means of implementing acts, adopt implementing acts laying down the rules for the application of this Regulation. (i) Sequence characteristics: (a) LENGTH: 14 amino acids (b) TYPE: amino acid (c) String shape: single string (d) Topology: linear (ii) TYPE of molecule: peptide (x) (H) DOCUMENT NUMBER: DE 195 45 472.3 (I) DATE OF FIRMING: 06-DEC-1995 (X) Information to be published: The following information is provided for the purpose of the application: (H) DOCUMENT NUMBER: DE 196 15 074. The following is a list of the documents which have been submitted to the Commission:

Claims (4)

1. Use of an antibody molecule which binds to the amino acid sequence WFGNRWHEGYR, for the preparation of a pharmaceutical composition for the treatment of squamous cell carcinomas.
2. Use according to claim 1, characterised in that the antibody molecule is the monoclonal antibody BIWA-1 (VFF-18), which is formed by the hybridoma cell line with the Accession Number DSM ACC2174, or a derivative of this antibody.
3. Use according to claim 1 or 2, characterised in that the antibody molecule is a monoclonal antibody, a Fab- or F(ab')₂-fragment of an immunoglobulin, an antibody prepared by recombinant methods, a chimeric or humanised antibody prepared by recombinant methods, a bifunctional or a single-chain antibody (scFv).
4. Use according to one of claims 1 to 3, characterised in that the antibody molecule is linked to a radioactive isotope, a photoactivatable compound, a radioactive compound, an enzyme, a fluorescent dye, a biotin molecule, a toxin, a cytostatic, a prodrug, an antibody molecule with a different specificity, a cytokine or another immunomodulatory polypeptide.