CA2168988A1

CA2168988A1 - Polypeptides coded by exon v5 of the cd44 gene as targets for immunotherapy and immunoscintigraphy of tumours

Info

Publication number: CA2168988A1
Application number: CA002168988A
Authority: CA
Inventors: Gunther Adolf; Helmut Ponta; Karl-Heinz Heider; Peter Herrlich; Steven T. Pals; Erik Patzelt
Original assignee: Individual
Current assignee: Boehringer Ingelheim International GmbH; Forschungszentrum Karlsruhe GmbH
Priority date: 1993-08-07
Filing date: 1994-07-21
Publication date: 1995-02-16
Also published as: WO1995004547A1; EP0713398A1; AU7459894A; DE4326573A1

Abstract

The present invention relates to agents for the treatment and in vivo diagnosis of tumours based on the selective bonding of antibodies to epitopes coded by variant exons of the CD44 gene. Special preference is hereby given to exon v5 of the human CD44 gene. Exon v5 is expressed in normal tissues only very slightly or not at all, whereas even at early stages of tumour progression it is expressed in large quantities. Therefore v5-coded epitopes are very suitable as targets for the immunotherapy and immunoscintigraphy of tumours.

Description

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S015314J.62 Polypeptides coded by exon v5 of the CD44 gene as targets for immunotherapy and immunoscintigraphy of tumours The invention relates to the use of antibodies against epitopes which are coded by variant exons of the CD44 gene, for the treatment or in vivo diagnosis of tumours, agents which are suitable for the treatment or in vivo diagnosis of tumours, and the use of polypeptides which contain sequences coded by the variant exon v5 of the CD44 gene, or v5-specific antiidiotypic antibodies as tumour vaccines.
Since the introduction of hybridoma technology in 1975 (Kohler and Milstein) there has been an intensive search for tumour-associated or -specific antigens. By definition, a tumour-specific antigen is expressed in a specific malignant disease, whereas it is absent from other types of tumours and in normal adult and foetal tissue. The second aspect, the absence in normal tissue, is critical to the use of tumour-specific antibodies in tumour therapy and for the imaging of tumours. Up till now, no tumour-specific marker has been found which completely fits the definition. A
whole series of antibodies against tumour-associated antigens have been developed up till now and used in clinical trials for the treatment of tumours or for tumour imaging (Mulshine et al., 1991; Vitetta and Thorpe, 1991; Larson et al., 1991). The principle is that the antibody as a probe binds specifically to tumour cells and destroys the tumour cell either directly or via an agent coupled thereto or can be used to show up the tumour by the detection of a label linked to the antibody. The agents linked to the antibody may themselves be cytotoxic, e.g. radioactive isotopes, bacterial or plant toxins or cytostatic factors, they 2168~88 _ may have an immunomodulatory effect, such as cytokines, or they may be enzymes, for example, capable of converting a precursor molecule (prodrug) into a cytotoxic agent. Radiolabelled antibodies may be used in the visual representation of tumours in vivo (imaging); for some time, nuclear spin resonance methods have also been developed which use, for example, antibodies linked to chelated trivalent cations as contrast agents (Brasch, 1992; Hider and Hall, 1991;
Saccavini et al., 1988). However, up till now, no antigen/antibody pair has been found which is suitable for wide use in tumour therapy and in vivo diagnosis.
The search for tumour-associated antigens as suitable targets for antibody-aided methods of tumour therapy and diagnosis therefore continues unabated.
It has recently been shown that the expression of variants of the surface glycoprotein CD44 is necessary and sufficient to trigger so-called spontaneous metastatic behaviour both in a non-metastasising pancreas-adenocarcinoma cell line in the rat and also in a non-metastasising fibrosarcoma cell line in the rat (Gunthert et al., 1991). Whereas the smallest CD44-isoform, the standard form CD44s, is expressed ubiquitously in a number of various tissues, including epithelial cells, specific splice variants of CD44 (CD44v) are expressed only in a subgroup of epithelial cells. The CD44 variants are produced by alternative splicing in such a way that the sequences of 10 exons (vl-vlO) in CD44s are excised completely, but may occur in the larger variants in various combinations (Screaton et al ., 1992; Tolg et al ., 1993; Hofmann et al ., 1991).
The variants differ in that different amino acid sequences are inserted at a specific point of the extracellular part of the protein. Such variants may be detected in various human tumour cells and in human tumour tissue. Thus, recently, the expression of CD44 variants in the course of colorectal carcinogenesis was _ investigated (Heider et al ., 1993). The expression of CD44 variants does not take place in normal human colon epithelium and only slight expression can be detected in the proliferating cells of the crypts. At later stages of the tumour progression, e.g. in adenocarcinomas, all malignant degenerations express variants of CD44.
Tissue expression of variant CD44 at a high level has also been demonstrated in aggressive non-Hodgkin's lymphomas ~Koopman et al ., 1993).
The objective of the present invention was to provide new agents for the treatment or in vivo diagnosis of cancers, based on the principle of the immunological recognition of tumour-associated antigens.
This objective has been achieved with the present invention. The agents according to the invention and their use are based on antibodies against epitopes which are coded by variant exons of the CD44 gene, particularly antibodies against epitopes which are coded by the variant exon v5 of the human CD44 gene, and on v5-specific antiidiotypic antibodies. The antibodies may be polyclonal or monoclonal, complete immunoglobulins, Fab- or F(ab')2-fragments of immunoglobulins or other derivatives, bispecific, chimeric or humanised antibodies or recombinantly produced antibodies, e.g. single-chain antibodies (scFv), Fab-fragments, other fragments or complete immunoglobulins. The antibodies may be administered without an additional agent or with a radioactive substance, a cytotoxic agent, an immunomodulating agent, a substance by means of which a cytotoxic agent can be produced locally, a nuclear spin resonance contrast agent or some other detectable label. The invention further relates to the use of polypeptides which contain sequences coded by the variant exon v5 of the CD44 gene, and v5-specific antiidiotypic antibodies as tumour vacclnes .
If the antibody carries a detectable label, the - 2168~88 label may be detected for diagnostic purposes, e.g. for visualising the tumour in vivo (imaging), or for (radioguided surgery), for example.
The nucleic and amino acid sequence of the variant part of the CD44 gene is known (Hofmann et al., 1991;
Screaton et al ., 1992 Tolg et al ., 1993). The existence of degenerate or allelic variants is of no significance to the performing of the invention; such variants are therefore expressly included. The sequence of exon v5 D V D R N G T T A Y E G N W N
GAT GTA GAC AGA AAT GGC ACC ACT GCT TAT GAA GGA AAC TGG AAC
P E A H P P L I H H E H H E E
CCA GAA GCA CAC CCT CCC CTC ATT CAC CAT GAG CAT CAT GAG GAA
E E T P H S T S T
GAA GAG ACC CCA CAT TCT ACA AGC ACA A

is particularly preferred. As agents for carrying out the invention it is possible to use antibodies, particularly those which are directed against epitopes within the sequence of exon v5, or v5-specific antiidiotypic antibodies. Monoclonal antibodies are particularly preferred. However, for the process according to the invention, it is also possible to use polyclonal antibodies, Fab- or F(ab')2-fragments of immunoglobulins, recombinantly produced antibodies or fragments, e.g. single-chain antibodies (scFv), bispecific, chimeric or humanised antibodies or equivalent molecules which specifically bind exon-v5-coded epitopes. Antibodies against known amino acid sequences may be prepared using methods known per se (Catty, 1989). For example, a peptide of this sequence may be produced synthetically and used as an antigen in an immunisation procedure. Another method is the production of a fusion protein which contains the desired amino acid sequence, by integrating a nucleic acid (which may be prepared synthetically or, for example, by polymerase chain reaction (PCR) from a 2168~88 suitable sample), which codes for this sequence, into an expression vector and expressing the fusion protein in a host organism. The optionally purified fusion protein can then be used as an antigen in an immunisation procedure and insert-specific antibodies or, in the case of monoclonal antibodies, hybridomas which express insert-specific antibodies can be selected by suitable methods (Wunderlich et al., 1992). The complete CD44 gene or a fragment thereof (e.g. the extracellular component) may also be expressed in a suitable system without the expressed sequences being fused with other peptides, then isolated and used as antigens in immunisation procedures. Such methods are known in the art. Heider et al., (1993) and Koopman et al. (1993) describe the production of antibodies against variant epitopes of CD44.
Instead of an intact immunoglobulin molecule it is also possible to use Fab- or F(ab')2-fragments or other fragments (Kreitman et al., 1993). Chimeric antibodies may also be used, e.g. hl~m~n;sed mouse antibodies (Shin et al., 1989; Gussow and Seeman, 1991), bispecific antibodies (Weiner et al., 1993; Goodwin, 1989) or single-chain antibodies/toxin-fusion proteins (Friedman et al., 1993). The antibodies may be isolated from sera or from hybridoma supernatants or obtained by recombinant expression as single-chain antibodies (scFv, Johnson and Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992, Barbas et al., 1992). The antibodies may be used on their own or combined with an agent. If the antibody is to be used therapeutically or for immunoscintigraphy, it may be combined with a suitable radioactive isotope, e g 131I 125I 111In, 136Re, 90Y, 99mTc or 211At. This combining may be carried out directly or using a linker molecule, e.g. a chelating agent. Methods of radiolabelling antibodies are known in the art (Larson et al., 1991; Thomas et al., 1989; Greiner et al., 1993;

- 6 2168~88 Srivastava, 1988; Rhodes et al., 1986). For therapeutic use the antibody may also be linked to a radioactive cytotoxic agent. This may be a cytostatic (Schrappe et al., 1992) or a cytotoxic polypeptide, e.g. a bacterial or plant toxin (Vitetta et al., 1991; Kreitman et al., 1993). A cytotoxic polypeptide of this kind may be linked covalently, e.g. via disulphide bridges, to the antibody (Theuer et al., 1993) or combined with an antibody in the form of a fusion protein in a single-chain immunotoxic (Chaudhary et al., 1990; Friedman et al., 1993). The antibody may also be linked to a cytokine or another immunomodulatory polypeptide, e.g.
with tumour necrosis factor or interleukin-2. The antibody may also be linked to an agent which is not itself cytotoxic but is capable of producing a cytotoxic substance, e.g. an enzyme which catalyses the conversion of an inactive precursor molecule (prodrug) into a cytostatic (Wang et al., 1992; Senter et al., 1989).
The recombinant expression of polypeptides which contain v5-coded sequences as well as the production of v5-specific antiidiotypic antibodies may be carried out using methods known in the art (Sambrook et al., 1989;
Briles and Kearney, 1985).
The use of the antibodies against variant CD44 epitopes, according to the invention, may be carried out by systemic or topical administration, e.g.
intravenously (as a bolus or continuous infusion), by intraperitoneal, intramuscular, subcutaneous or other injection or infusion. It is also possible to perfuse individual organs or limbs. Procedures for the administration of conjugated or non-conjugated antibodies (be they in the form of complete immunoglobulins, fragments, recombinant chimeric molecules or the like) are known in the art (Mulshine et al., 1991; Larson et al., 1991; Vitetta and Thorpe, 1991; Vitetta et al., 1991; Breitz et al., 1992; Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989;

~_ - 7 -Sears et al ., 1982).
In addition to the therapeutic treatment of cancers, the agents according to the invention are suitable for in vivo diagnosis of tumours. For the use of antibodies conjugated with radioactive isotopes for immunoscintigraphy (imaging) there is also a series of procedures on which the person skilled in the art can base his method for carrying out the invention (Siccardi et al ., 1989; Keenan et al ., 1987; Perkins and Pimm, 1992; Colcher et al., 1987; Thompson et al., 1984).
Recombinant polypeptides which contain amino acid sequences coded by exon v5, as well as v5-specific antiidiotypic antibodies, may be used as tumour vaccines. Antiidiotypic antibodies recognise epitopes within the variable regions of immunoglobulins (idiotopes). By v5-specific antiidiotypic antibodies are meant the antibodies directed against idiotopes of antibodies which recognise v5-coded amino acid sequences. If v5-specific antiidiotypic antibodies are used as antigens in an immunisation procedure, antibodies are formed against these antigens and these antibodies largely also bind to v5-coded epitopes.
Polypeptides which contain v5-coded sequences, as well as v5-specific antiidiotypic antibodies, may thus be used for ;mmlln;sing tumour patients. The antibodies formed against v5-coded epitopes in the patient's body recognise tumour cells which express these epitopes and thus aid the immunological fight against the tumour by the body.
Variant CD44 molecules are tumour-associated antigens which are highly suitable as targets for immunotherapy and immunoscintigraphy of cancers.
Variant CD44 molecules which contain the amino acid sequence coded by the variable exon v5, an allelic variant or a fragment of this sequence, as well as v5-specific antiidiotypic antibodies are particularly suitable.

- 2168~88 _ Immunohistochemical trials with monoclonal antibodies against various CD44 variants on human normal and tumour tissue show that many tumours express CD44v strongly, whereas the corresponding normal tissue does only slightly or not at all.
Fine analysis of the expression of various CD44 variants shows that not all exons are overexpressed in tumours in the same way. Table 1 shows a trial on gastric tumours in a total of 42 patients. In 42/42 cases the expression of variant CD44 molecules can be detected (polyclonal antiserum against the amino acid sequences of the exons v3 to vlO). Investigations with exon-specific monoclonal antibodies then show that exons v3/v4, v7 and v8-vlO are not expressed and v6 is expressed only in 26/42 tumours (62~). v5, on the other hand, is expressed in 39/42 tumours (93~). In contrast to v6, using v5-specific antibodies it was possible to detect gastric tumours of both the diffuse type (14/17) and also of the intestinal type (25/25). In tumours which express both exons, the expression of v5 is generally stronger than that of v6 (Table 2).
Figure 3 shows the results of a study on 39 colorectal carcinomas. In contrast to all the other exons, v5 is not expressed at all in normal tissue but in tumour tissue it is expressed even at the earliest tumour stage (early adenoma) in over 80~ of the tumours.
v3 expression was not detected at all, v6 expression was detected to be increasing significantly only at the late tumour stages and v8-10 were expressed even in normal tissue. Even at later stages of the colorectal tumour progression, v5 is expressed more frequently and to a greater extent than v6.
An investigation of breast tumours (62 invasive carcinomas, 4 in si tu carcinomas, 9 local recurrences, 16 lymph node metastases) also showed that v5 is very frequently expressed in these tumours (82-100~), more frequently than v3 or v6 (Table 3).

2168~)88 g For processes of immunotherapy and scintigraphy it is important that the tumour-associated antigen which is the target for the antibody is expressed in the tumour but not in normal tissue, but that in the largest possible number of tumours it is expressed as highly as possible at the earliest possible tumour stage, in order to allow widespread and reliable use. Surprisingly, CD44 variants which contain amino acid sequences coded by exon v5 satisfy these requirements to a high degree.
The agents and uses according to the invention are thus exceptionally suitable for immunotherapy and in vivo diagnosis/immunoscintigraphy of tumours, particularly carclnomas .

Table 1: Expression of variant CD44 epitopes on the cell surfaces of gastric tumours Seruml Monoclonal antibodies AdenocarcinomasaCD44v VFF11 VFF8 VFF4VFF9 VFF14 (v3-v10) (v3/4) (v5) (v6)(v7) (v8-v10) Diffilse type17/172 0/17 14/17 3/170/17 0/17 Intestinal type25/25 0/25 25/25 23/250/25 0/25 Overall 42/42 0/42 39142 261420/42 0/42 1 polyclonal antiserum 2 number of positive tumours/number of tumours investigated _ Table 2: Expression of variant CD44 epitopes in primary tumours of the stomach and the corresponding lymph node metastases Antibody specificity polyclonal serum monoclonal antibody antiCD44v . VFF8(vS) VFF4(v6) ¦ Adenocarcinoma3 ¦ Intensity ¦ pos. cells ¦ Intensity ¦ pos. cells ¦ Intensity ¦ pos. cells Dif~se type 645/89 ++41005/1006 ++ 20/90 - 0/0 12589/89 ++ 40/50 ++ . 80/90 - 0/0 12924/89 ++ 70/80 ++ 40/30 - 0/0 25501/89 ++ 90/70 ++ 70/70 ++ 30/30 33383/89 ++ 80/30 ++ 40/20 - 0/0 Intestinal type 32761/88 ++ 60/10 + 10/20 + 20/90 33295/88 ++ 90/10 ++ 80/60 + 40/40 9891/89 l l l100/80 ++ 70/70 ++ 20/5 18352/89 l l l90/90 +++ 90/80 ++ 70/50 9069/90 ++ 90/90 ++ 60/60 + 20/80 3 The numbers relate to tumours and the corresponding lymph node metastases (not given separately). The tumours are contained in the summary given in Table 1.

4 Intensity (because there was no difference in intensity between primary tumour and lymph node metastasis, here the same grading is used: - negative, + weak, ++ moderate, +++ strong 5 Percentage of positive tumour cells in the primary tumour 6 Percentage of positive tumour cells in a lymph node metastasis in the same patient 2168~88 Table 3: Expression of CD44 variants in primary in si tu and invasive breast tumours, recurrences and lymph node metastases (number of positive samples).

Antibody Carcinoma Invasive Local Lymph node in situ carcinoma recurrence metastases (n=4) (n=62) (n=9) (n= 16) a-CD44v (v3-vlO)4 (100%) 57 (92%) 8 (89%) 16 (100%) a-DI (v3) 2 (50%) 33 (53%) 4 (44%) 16 (100%) a-DIII (v6,v7)4 (100%) 51 (82%) 9 (100%) 16 (100%) VFF8 (v5) 4 (100%) 51 (82%) 8 (89%) 16 (100%) VFF7 (v6) 4 (100%) 46 (74%) 6 (66%) 15 (100%)7 - 2168~88 Figures Fig. 1: Diagrammatic representation of a CD44 splice variant. This variant by way of example carries all the variant exon sequences at the single insertion site. Dark grey boxes represent CD44 standard sequences (CD44s). The location of the epitopes of the monoclonal antibodies VFF4, VFF7, VFF8, VFF9, VFF11, VFF14, VFF16 and VFF18 are indicated by bars. All the monoclonal antibodies are exon-specific.
Fig. 2: Immunohistochemistry of normal mucosa and adenocarcinomas of the stomach. A focally emphasised anti-CD44v-positive reaction is found in tumour cells of a moderately differentiated adenocarcinoma (intestinal type according to Lauren) of the stomach (a) and in a regional lymph node metastasis (b). In normal mucosa of the stomach with chronic gastritis, the foci of intestinal metasplasias react positively with mAb VFF4 (c, arrows) and with mAb VFF8 (d, arrows), accompanied by an additional reaction on the mucoid surface and the foveolar epithelium (d, arrow heads). Almost all beaker cells carcinomas of the stomach (diffuse type according to Lauren) show a negative reaction with mAb VFF4 (e), and unlike adenocarcinomas of the intestinal type, the normal mucoid epithelium is negative (e, arrow heads).
In most cases a positive reaction with mAb VFF8 (f) is found in these beaker cell carcinomas and the remaining normal mucoid epithelium also exhibits immunoreactivity (f, arrow heads).
(ABC Method a, b: anti-CD44v polyclonal serum, 140 x; c: VFF4, 80x; d: VFF8, 80x; e: VFF4, 210x; f:
VFF8, 210x; counterstaining with haematoxylin).
Fig. 3: Expression of variant CD44 exons at various stages of colorectal tumour progression. Results from immunohistochemical stainings of tissue sections (Example 2). Dark grey bars indicate the percentage of positive tumours. Light grey bars indicate samples with only focal staining.

2168~88 Examples Tumours and tissue Colon: Normal and pathological tissue were taken from the supplies of the Pathology Department of the Academic Medical Centre, Amsterdam University, Netherlands. Colorectal carcinomas (n=39) were divided into stages according to the Dukes Classification (1937, 1980), into Dukes A (n=9) in which the disease is restricted to the intestinal wall, Dukes B (n=14), spread beyond the layer of muscle without metastasis;
Dukes C/D (n=16), tumours with regional or remote metastases. Adenomas were subdivided into early adenomas (diameter < 1 cm, n=ll) and late adenomas (diameter > 1 cm, n=12) and were graded as poorly or highly differentiated using standard criteria.
Stomach: Tumour samples and normal tissue were selected from the supplies of the Pathology Department of the University of Wurzburg, Germany. The samples were flash-frozen immediately after surgical removal and stored at -80C until ready for use. Normal tissue was taken from twelve different tumour patients, from both the corpus and antrum region of the stomach.
Pathological tissue was obtained from a total of 47 patients with an average age of 63. Of the primary carcinomas, 29 were of the intestinal type and 18 of the diffuse type according to Lauren (1965). The tumour stages ranged from localised (pTl) to widespread (pT4) and the histological grading ranged from well differentiated (Gl) to poorly differentiated (G3) adenocarcinomas.
Breast: Frozen tissue (stored at -70C) was obtained from the Womens Clinic at the University of Heidelberg, Germany. It included 62 samples of primary breast cancers, 9 local recurrences of breast cancers, 4 cases of pure in situ carcinomas and 16 axillary lymph node metastases (taken from the same patients as the primary tumours~. The cases were selected at random and included a representative selection of histological tumour types, stages and gradings. As a comparison, samples of normal breast tissue, ductal hyperplasias and fibroadenomas were selected.

Example 1: Preparation of the antibodies against epitopes coded by variant exon sequences of the CD44 gene Cloning of pGEX fusion proteins The entire variant region of the HPKII types of CD44v (Hofmann et al ! 1991) was amplified from human keratinocyte cDNA by polymerase chain reaction (PCR). The two PCR primers 5'-CAGGCTGGGAGCCAAATGAAGAAAATG-3', positions 25-52, and 5'-TGATAAGGAACGATTGACATTAGAGTTGGA-3', positions 1013-984 of the LCLC97-variant region, as described by Hofmann et al., contained an EcoRI
recognition site which was used to clone the PCR product directly into the vector pGEX-2T (Smith et al., 1988).
The resulting construct (pGEX CD44v HPKII, v3-vlO) codes for a fusion protein of approximately 70 kD. The fusion protein was expressed in E. coli and then purified by affinity over glutathione-agarose (Smith et al., 1988).
In order to obtain subclones of the variant regions which were able to be used for affinity purification and Western blot analyses, fragments were cloned containing DI (v3), DII/III (v5, v6) and DIII (v6, v7), using the appropriate restriction cutting sites. Fusion protein DI contains the CD44 sequence described by Stamenkovic et al., (1989), from position 744 to position 142 of the sequence of variant CD44, as described by Hofmann et al.
(1991). Fusion protein DII/III contains the variant sequence of position 290-460, fusion protein DIII the _ 2168~88 variant sequence from position 378-638 (Hofmann e t al ., 1991). The fragments containing DI and DIII were cloned into the pGEX vector system and the DII/III fragment into the pATH vector (Angel et al., 1988).

Pol ycl onal an ti serum The preparation and purification of the polyclonal antiserum against the variant region of the CD44 molecule is described in the literature (Heider et al., 1993).

Monocl onal antibodies Female BALB/c mice were immunised with affinity-purified fusion protein obtained from pGEX CD44v HPKII
(exons v3-vlO) as described above. Spleen cells from an animal with a high antibody titre were fused with P3X63Ag8.653 myeloma cells using polyethyleneglycol 4000. Hybridomas were selected in HAT medium (Kearney et al ., 1979). The measurement of the antibody titre in the serum and the antibody screening were carried out using ELISA. The microtitre plates were coated with fusion protein, incubated with serial dilutions of serum samples or hybridoma supernatants, and specific antibodies were detected with peroxidase-coupled antibodies against murine IgG. Hybridomas which reacted with glutathione-transferase were eliminated. The antibodies rem~'n;ng were further characterised by means of ELISA tests, using fusion proteins of the variable domains DI (exon v3), DII/III (exons v5, v6), DIII
(exons v6, v7), DI-IV (exons v3-v8), DIII-VI (exons v7-vlO) and v6 (exon v6) [v5 v6 v7]. The reactivity of the antibodies with human skin keratinocytes was investigated immunohistochemically.
The exon specificity of various monoclonal antibodies used (VFF4, VFF7, VFF8, VFF9, VFF11, VFF14, - 2168~88 VFF16 and VFF18) is shown in Fig. 1.

Example 2: Immunohistochemistry Frozen sections were fixed in ice-cooled methanol for 10 minutes, washed in PBS (8 g/l NaCl, 0.2 g/l KCl, 1.44 g/l Na2HPO4, 0.24 g/l KH2PO4, pH 7.4) and pre-incubated with normal goat serum (10~ in PBS). It was then washed 3x with PBS and incubated for 1 hour with the primary antibody (in PBS, 1~ BSA). Endogenous peroxidase was blocked with 0.3~ H202 in methanol and the sections were incubated with biotinylated second antibodies (either anti-mouse or anti-rabbit F(ab') 2 DAKO Corp., Santa Barbara, CA, USA, depending on the primary antibody used). The immune complex was visualised with horse-radish peroxidase coupled to Biotin as a streptavidine-biotin-peroxidase complex (DAKO). After thirty minutes' incubation with the streptavidine-biotin-peroxidase complex the sections were developed for 5 to 10 minutes with 3,3-amino-9-ethyl-carbazole (Sigma Chemicals, Deisenhofen, Germany) and the reaction was stopped with H20. The cells were counterstained with haematoxylin, coated with glycerol-gelatine and studied under the microscope.
Tumours were termed "positive" if more than 10~ of the tumour cells were stained. If fewer than 10~ of the tumour cells were stained this was termed "focal".

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Claims

1. Use of an antibody which binds exclusively to an epitope which is coded by the variant exon v5 of the CD44 gene, for selective binding to tumours in vivo.

2. Use of an antibody according to claim 1, characterised in that the exon v5 codes for the amino acid sequence DVDRNGTTAYEGNWNPEAHPPLIHHEHHEEEETPHSTST

or an allelic variant or a fragment of this sequence.

3. Use of an antibody according to claim 2, characterised in that the exon v5 contains the nucleic acid sequence or a degenerate or allelic variant or a fragment of this sequence.

4. Use of an antibody according to claims 1 to 3, characterised in that the antibody is monoclonal.

5. Use of an antibody according to claims 1 to 3, characterised in that the antibody is the Fab- or F(ab')2-fragment of an immunoglobulin, a recombinantly produced antibody or a recombinantly produced antibody fragment, a recombinantly produced single-chain antibody (svFv), a chimeric, bispecific or humanised antibody.

6. Use of an antibody according to claims 1 to 5, characterised in that the antibody is linked to a cytotoxic agent.

7. Use of an antibody according to claim 6, characterised in that the antibody is linked to a radioactive isotope.

8. Use of an antibody according to claim 6, characterised in that the antibody is linked to a toxin.

9. Use of an antibody according to claim 6, characterised in that the antibody is linked to a cytostatic factor.

10. Use of an antibody according to claim 6, characterised in that the antibody is linked to a cytokine.

11. Use of an antibody according to claim 6, characterised in that the antibody is linked to an enzyme capable of catalysing the conversion of a precursor molecule (prodrug) into a cytotoxic agent.

12. Use of an antibody according to claims 1 to 11 for the immunotherapy of tumours.

13. Use of an antibody according to claims 1 to 5 or 7 for the in vivo diagnosis of tumours.

14. Use of an antibody according to claims 1 to 13, characterised in that the tumours are carcinomas.

15. Use of an antibody according to claim 14, characterised in that the carcinomas are adenocarcinomas.

16. Use of an antibody according to claim 14, characterised in that the carcinomas are gastric carcinomas.

17. Use of an antibody according to claim 14, characterised in that the carcinomas are colon carcinomas.

18. Use of an antibody according to claim 14, characterised in that the carcinomas are mammary carcinomas.

19. Use of an antibody which binds exclusively to an epitope by the variant exon v5 of the CD44 gene, for preparing a pharmaceutical composition for treating tumours.

20. Use of an antibody which binds exclusively to an epitope which is coded by the variant exon v5 of the CD44 gene, for preparing an in vivo tumour diagnostic agent.

21. Use of a polypeptide which contains sequences coded by exon v5 of the CD44 gene, or a v5-specific antiidiotypic antibody as tumour vaccine.

22. Agent for treating tumours, characterised in that it contains an antibody which binds exclusively to an epitope which is coded by the variant exon v5 of the CD44 gene.

23. Agent for the in vivo diagnosis of tumours, characterised in that it contains an antibody which binds exclusively to an epitope which is coded by the variant exon v5 of the CD44 gene.

24. Agent for producing antibodies against variant CD44 molecules in vivo, characterised in that it contains one or more v5-specific antiidiotypic antibodies.

25. Agent according to claims 22 to 24, characterised in that the antibody is monoclonal.

26. Agent according to claims 22 to 24, characterised in that the antibody is the Fab- or F(ab')2-fragment of an immunoglobulin, a recombinantly produced antibody or a recombinantly produced antibody fragment, a recombinantly produced single-chain antibody (scFv), a chimeric, bispecific or humanised antibody.

27. Agent according to claims 22, 25 or 26, characterised in that the antibody is linked to a radioactive isotope, a toxin, a cytostatic factor, a cytokine or some other cytotoxic or immunomodulatory agent or to an enzyme which is capable of catalysing the conversion of a precursor molecule into a cytotoxic agent.

28. Agent according to claim 23, characterised in that the antibody is linked to a radioactive isotope or to a contrast agent for nuclear spin resonance spectroscopy.

29. Antiidiotypic antibody, characterised in that it contains an idiotope of an antibody which binds variant CD44 molecules which contain sequences coded by exon v5.