Note: Descriptions are shown in the official language in which they were submitted.
CA 02239709 1998-06-04
FIL~, ~ I r ~ T ~ r ~
'i E~ Tj~~ Lr/~ N
S016704J.200
Boehringer Ingelheim International GmbH Case 12/178,181
Forschungszentrum Karlsruhe GmbH
Process for the diagnosis and treatment of
squamous cell carcinomas
The invention relates to processes for the diagnosis and therapy of squamous cell
o carcinomas which are based on the ~x~lession of the variable exon v6 of the CD44 gene,
agents for these processes and the use thereof.
It has recently been shown that the ~x~res~ion of variants of the surface
glycoprotein CD44 is necessary and sufficient to trigger so-called spontaneous metastatic
behaviour both in a non-metast~ in~ pancreas-adenocarcinoma cell line of the rat and
also in a non-metastasising fibrosarcoma cell line of the rat (Gunthert et al., 1991).
Whereas the smallest CD44-isoform, the standard form CD44s, are expressed ubiquitously
in a series of dirrer~lll tissues, including epithelial cells, certain splice variants of CD44
(CD44v) are expressed only in a subgroup of epithelial cells. The CD44-isoforms 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 dirr~l~lll combinations in the larger
variants (Screaton et al., 1992; Heider et al., 1993; Hofmann et al.; l99l2. The variants
differ in that different amino acid sequences are inserted at a specific site of the
extracellular part of the protein. Such variants can be detected in dirreiell~ human tumour
cells and in human tumour tissue. Thus, the ~x~ression of CD44-variants in the course of
colorectal carcinogenesis has recently been investi~;~ted (Heider et al., 1993). There is no
expression of CD44-variants in normal human colon epithelium and only slight expression
can be detected in the proliferating cells of the cavities. At later stages of the tumour
progression, e.g. in adenocarcinomas, all m~lignzlnt degenerations express variants of
CD44. Moreover, the t;x~lession of CD44-splice variants has recently been demonstrated
in acti.vated lymphocytes and in non-Hodgkin's lymphomas (Kooprnan et al., 1993).
Various approaches have been adopted for making use of the differential expression
of variant exons of the CD44-gene in tumours and normal tissue for diagnostic and
therapeutic purposes (WO 94/02633, WO 94/12631, WO 95/00658, WO 95/00851,
EP 0531300).
The expression of variant CD44-molecules in squamous cell carcinomas has also
CA 02239709 1998-06-04
been investig~ted. Salmi et a/. (1993) found, with the v6-specific antibody Var3.1, that
there was a reduction in the v6-expression in tumour cells compared with normal cells.
With the v6-specific antibody 11.9, Brooks et a/. (1995) obtained a heterogeneous staining
of nasopharyngeal carcinomas. Strong staining was achieved in only 2/12 cases, whilst in
s the majority of cases only a slight focal v6-expression could be detected by
immunohistology.
The aim of the present invention was to develop new methods for the diagnosis and
therapy of squamous cell carcinomas and to provide agents for such methods.
This objective has been achieved by means of the present invention. It relates to
processes for the diagnosis and therapy of squamous cell carcinomas which-are based on
the expression of the variant exon v6 of the CD44 gene as a molecular marker or target. In
particular, the present invention relates to processes based on the powerful homogeneous
5 expression of v6 in squamous cell carcinomas, which was surprisingly detected, in
contradiction to the teaching known from the prior art. Antibody molecules of
corresponding specificity are particularly suitable as vehicles for selectively re~rhing
squamous cell carcinomas in vivo.
The preferred processes are those characterised in that an antibody molecule is used
which recognises the arnino acid sequence QWFGNRWHEGYRQT, more preferably the
amino acid sequence WFGNRWHEGYR. The monoclonal antibody BIWA-1 (clone
VFF- 18) secreted by a hybridoma cell line which was deposited on 7.6.1994 underAccession No. DSM ACC2174 at the DSM-Deutsche Sammlung fhr Mikroorganismen und
Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany (WO
95/33771), and derivatives of this antibody, are particularly ple~ d.
Other aspects of the present invention are the use of antibody molecules of this kind
in the processes according to the invention and agents for carrying out these processes.
The nucleic and amino acid sequence of the variant exon v6 of the CD44-gene is
known (Screaton et a/., 1992, Tolg et al., 1993). The existence of degenerate or allelic
variants is of no significance to the implementation of the invention; such variants are
therefore expressly included.
The sequence of exon v6 of the human CD44-gene is:
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Q A T P S S T T E E T A T Q
TC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAG
K E Q W F G N R W H E G Y R Q
S AAG GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA
T P R E D S H S T T G T A
ACA CCC AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G.
lo The invention may be carried out with polyclonal or monoclonal antibodies which
are specific to an epitope coded by exon v6, particularly an epitope within the amino acid
sequence QWFGNRWHEGYRQT, most preferably within the amino acid sequence
WFGNRWHEGYR. The preparation of antibodies against known amino acid sequences
may be carried out according to methods knownper se (Catty, 1989). For example, a
peptide of this sequence may be prepared synthetically and used as an antigen in an
immunisation procedure. Another method is to prepare a fusion protein which contains the
desired amino acid sequence, by integrating a nucleic acid (which may be prepared
synthetically or, for example, by polymerase chain reaction (PCR) from a suitable probe)
which codes for this sequence into an expression vector and expressing the fusion protein
20 in a host org~ni~m. The fusion protein, optionally purified, 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 then be selected by
suitable methods. Processes of this kind are known in the art. Heider et al. (1993, 1996a)
and Koopman et a/. (1993) describe the plepaL~lion of antibodies against variant epitopes
25 of CD44.
However, for the process according to the invention, it is also possible to use other
antibody molecules, which are derived from poly- or monoclonal antibodies, e.g. Fab- or
F(ab')2-fragments of immunoglobulins, single-chain antibodies (scFv) prepared by the
30 recombinant method, chimeric or hllm~ni~ed antibodies and other molecules which bind
specifically to epitopes coded by exon v6. Fab- or F(ab')2-fragments or other fragments
may be produced, for example, from the complete immunoglobulin of the antibody
BIWA-1 (VFF-18) or other antibodies (Kreitman et a/., 1993). The skilled person is also in
a position to produce recombinant v6-specific antibody molecules. In particular, after
35 analysing the amino acid sequence of the antibody BIWA- 1 (VFF- 18) and/or using the
hybridoma cell line which produces this antibody, particularly the genetic information
contained therein, he can produce recombinant antibody molecules having the sameidiotype as BIWA-1 (VFF-18), i.e. antibody molecules which have the same amino acid
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sequence in the region of the antigen binding site (complementari~-determining regions,
CDR) as the antibody BIWA-1 (VFF-18). Such processes are known in the art.
Recombinant antibody molecules of this kind may be, for example, humanised antibodies
(Shin et al., 1989; Gussow and Seemann, 1991), bispecific or bifunctional antibodies
s (Weiner et al., 1993; Goodwin, 1989, Featherstone, 1996), single-chain antibodies (scFv,
Johnson and Bird, 1991), complete or fragmentary immunoglobulins (Coloma et al., 1992;
Nesbit et al., 1992; Barbas et al., 1992) or antibodies produced by chain shuffling (Winter
et al., 1994). H--m~ni~ed antibodies may be prepared, for example, by CDR-grafting
(EP 0239400). Framework regions can also be modified (EP 0519596; WO 9007861). For
o hllm~nising antibodies, methods such as PCR (see for example EP 0368684; EP 0438310;
WO 9207075) or computer-modelling (see for example WO 9222653) may be used
nowadays. It is also possible to prepare and use fusion proteins, e.g. single~ehain
antibody/toxin fusion proteins (Ch~ lh~ry et al., 1990; Friedman et al., 1993). The terms
"antibodies" and "antibody molecules" are intended to cover not only polyclonal and
monoclonal antibodies but all the compounds discussed in this paragraph which can be
structurally derived from immunoglobulins and may be prepared by methods knownper se.
.
It is also within the capabilities of the average skilled person with a knowledge of
the epitope (cf. Fig. 1, Fig. 4) of BIWA-1 (VFF-18), to produce equivalent antibodies with
20 the same binding specificit,v. Such antibodies are therefore also included in the invention.
For diagnostic purposes, antibody ~nolecules, preferably BIWA-1 antibody
molecules, fragments thereof or recombinant antibody molecules with the same idiotype,
may be linked for example with radioactive isotopes such as 125I, 131I, l l lIn 99mTc or
25 radioactive compounds (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1988),
enzymes such as peroxidase or ~lk~line phosphatase (Catty and Rayklm(l~ 1989), with
fluorescent dyes (Johnson, 1989) or biotin molecules (Guesdon et al., 1979). Fortherapeutic purposes v6-specific antibody molecules, preferably BIWA-1 (VFF-18)-antibody molecules or VFF- 18-derived antibody molecules, e.g. fragments thereof or
30 recombinant antibody molecules with the same idiotype, may be linked to radioisotopes
such as 90y 131I 186Re 188Re, 153Sm, 67cu, 212si, 213Bi, 177Lu (Quadri et al.,
1993; Lenhard et al., 1985, Vriesendorp et al., 1991; Wilbur et al., 1989, Maraveyas et al.,
1995a, Jurcic et Scheinberg, 1994), toxins (Vitetta et al., 1991; Vitetta et Thorpe, 1991;
Kreitman et al., 1993; Theuer et al., 1993), cytostatics (Schrappe et al., 1992), prodrugs
35 (Wang et al., 1992; Senter et al., 1989), photoactivatable substances (Hemming et al.,
1993), an antibody molecule with a different specificity or radioactive compounds. The
antibody molecule may also be linked to a cytokine or some other immunomodulatory
polypeptide, e.g. with tumour necrosis factor, lymphotoxin (Reisfeld et al., 1996) or
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interleukin-2 (Becker et al., 1996). The antibody molecules may also be modified for use in
a pl~l~g~lling system, e.g. with streptavidin or biotin (Goodwin, 1995).
Advantageously, the diagnostic process according to the invention may be used to5 investigate samples from patients, e.g. from biopsies, where there is a suspicion of
squamous cell carcinoma or the diagnosis has already been made but the tumour needs to
be further characterised. The detection of variant CD44-molecules which contain an amino
acid sequence coded by the variable exon v6 may be carried out at the protein level using
antibodies or at the nucleic acid level using specific nucleic acid probes or primers for
o polymerase chain reaction (PCR). The invention consequently also relates to antibody
molecules and nucleic acids which are suitable as probes or primers for such processes, and
the use of these antibodies and nucleic acids for diagnosing and analysing squamous cell
carcinomas. For example, tissue sections can be examined immunohistochemically using
antibodies by methods knownper se. Extracts taken from tissue samples or body fluids
5 may also be investigated by other immunological methods using antibodies, e.g. in Western
blots, enzyme-linked immunosorbent assays (ELISA, Catty and Raykundalia, 1989),
radioimmunoassays (RIA, Catty and Murphy, 1989) or related immunoassays. The
investigations may be qualitative, semi-qua~ ive or quantitative.
Apart from in vitro diagnosis, antibody molecules with specificity according to the
invention are also suitable for the in vivo diagnosis of squamous cell carcinomas. If the
antibody molecule carries a detectable label, the label can be detected for diagnostic
purposes, e.g. for im~ging the tumour in vivo or for radioguided surgery, for example. For
using antibodies conjugated with radioactive isotopes for immunoscintigraphy (im~ging),
for example, there are numerous procedures which can be used by the skilled person as the
basis for performing the invention (Siccardi et al., 1989; Keenan et al., 1987, Perkins and
Pimm, 1992; Colcher et al., 1987, Thompson et al., 1984).
Data obtained by the detection and/or quantifying of the expression of the variant
CD44-epitope v6 can thus be used for diagnosis and prognosis. It may be advantageous to
combine these data with other prognostic parameters, e.g. the grade of tumour.
Antibody molecules having the specificity according to the invention, optionallycombined with a cytotoxic agent, may advantageously be used for the treatment ofsquamous cell carcinomas. They may be ~lmini~tered systemically or topically, e.g. by
intravenous route (as a bolus or continuous infusion) or by intraperitoneal, intramuscular,
subcutaneous or other injection/infusion. Procedures for ~(lmini~tering conjugated or non-
conjugated antibodies (be they in the form of complete immunoglobulins, fragments,
CA 02239709 1998-06-04
recombinant hllm~nised 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, 1995
Press et al., 1989; Weiner et al., 1989; Chatal et al., 1989; Sears et al., 1982). They may be
used therapeutically, for example, in the same way as the antibody 1. lASML (Seiter et al.,
s 1993). Unmodified monoclonal antibodies can be used directly for therapeutic purposes if
they have the intrinsic effector function suitable for a cytotoxic effect, e.g. for complement-
induced or antibody-induced cell cytotoxicity (Riethmuller et al. 1994). Suitable monoclo-
nal antibodies for this application are murine antibodies of isotype IgG2a or antibodies of
the human IgG1-type. Unmodified antibodies may also be ~(lmini.ctered in order to induce
o the patient's own antitumoral reaction through an anti-idiotypic mech~ni~m (Baum et al.,
1993; Khazaeli et al., 1994).
According to a plerelled embodiment of a therapeutic application, a humanised v6-
specific immunoglobulin or a F(ab')2 fragment thereof is linked with 90Y (Quadri et al.,
1993; Vriesendorp etal., 1995), 131I (Maraveyas etal., 1995a, 1995b; Juweid etal., 1995;
Press et al., 1995; Thomas et al., in: Catty 1985, p. 230-239) 186Re (Breitz et al., 1992,
1995) or another suitable radioisotope and used for radioimmunotherapy of squamous cell
carcinomas. For example, the antibody BIWA-1, a hllm~ni~ed version of BIWA-1 or a
F(ab')2 fragment of BIWA-1 or the hllm~ni~ed antibody may be linked to 90Y using a
20 chelating linker such as ITCB-DTPA (isothiocyanatobenzyl-
diethylenetri~minepentaacetate), achieving a specific activity of 5-20 mCi/mg, preferably
10 mCi/mg. This agent can then be ~tlministered to a patient with an antigen-positive
tumour in a dose of 0.1 to 1 mCi/kg of body weight, preferably 0.3 to 0.5 mCi/kg of body
weight. If the antibody molecule is linked to 131I, a possible dosage plan might be, for
25 example, 2 x 150 mCi at 6 week intervals for a specific activity of 2 mCi/mg. The skilled
person can determine the ma~illlul~l possible dosages using methods known per se(Maraveyas et al., 1995a, 1995b). When the total quantity of protein to be ~imini~tered is 2
to 5 mg it may be given in the form of a fast intravenous bolus injection. In the case of
larger amounts of protein an infusion might be a more appropriate method of
30 ~lmini.~tration. With monoclonal antibodies it may be necessary to mix the agent with an
excess (e.g. a tenfold molar excess) of the non-radioactive antibody before ~lmini~tration;
in this case, it is better to ~lmini~ter the agent in the form of an intravenous infusion, e.g.
over 15 minutes. This can be repeated. The therapy may be combined-with externalradiation therapy. It may also be backed up by bone marrow transplantation; this is
35 particularly necessary when a dosage of more than 1.6 Gy is reached in the bone marrow
during therapy.
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Antibody molecules according to the invention may also be used ex vivo to purifyCD34-positive stem and precursor cell preparations (immunopurging). Radiation orchemotherapy of squamous cell carcinomas may also be backed up with autologous bone
marrow transplantation. The plep~lion of h~em~topoietic stem and precursor cells thus
5 ,q~lmini~tered must be free from tumour cells. This can be achieved by incubation with
antibody molecules according to the invention, e.g. antibody-toxin conjugates (Myklebust
et al., 1994; DE P 196 48 209.7).
Antibody molecules according to the invention may also be introduced into the T-cell
o receptor of T-lymphocytes in the form of recombinant constructs. Reprogrammed T-
lymphocytes of this kind bind selectively to the antigen-expressing tumour cells and
develop a cytotoxic activity, with the result that they can be used for the treatment of
squamous cell carcinomas (PCT/EP9604631; Altenschmidt et al., 1996).
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Figures
Fig. 1: Determining fhe epitope specificity of BIWA-1 by binding to synthefic
s peptides derivedfrom the human CD44v6 sequence. The corresponding peptide from rat
CD44v6 was tested with the antibody 1. lASML. Binding was determined in an ELISA in
which the peptides were immobilised on microtitre plates (cf. Heider et al., 1996b, Fig. 2).
-: no binding, +/-: slight binding, +: strong binding.
o Fig. 2: Immunohistochemical analysis of a squamous cell carcinoma of the larynx
(a) and a liver metastasis of a carcinoma of the oesophagus (b) with the CD44v6-specific
monoclonal antibody BIVVA-1. In both cases the reactivity of the antibody v~th the
membrane of the tumour cells can be observed. Original m~gnification 40x,
counterstaining haematoxylin.
Fig. 3: Comparison of antigen binding of various CD44v6-specific mAbs. The
binding of four different CD44v6-specific mAbs to human SCC A-431-cells was measured
in a cell ELISA. MAb BIWA-l shows a higher affinity for the tumour cells than the other
mAbs.
Fig. 4: Refined epitope mapping of the mAb BIVVA-1. The binding of BIWA-1 to
various overlapping synthetic peptides which span the amino acids 18-32 of the CD44v6-
coded region was measured by competitive ELISA. The minimum binding sequence
(peptide v6 (19-29)) is underlined.
Fig. 5: Biodistribution of 125I-BIWA-l in A-431 xenotransplanted nude mice. The
accumulation of the antibody is given as % ID/g (mean + SEM) at 4, 24, 48, 120 and 168
hours post injection.
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Examples
Example 1: Expression of CD44v6 in squamous cell carcinoma
5 Tissue
A total of 126 cases of paldrr~ embedded tumour samples were analysed
immunohistochemically with the mAb BIWA-1 (clone VFF-18) for expression of CD44v6.
The samples included 31 cases of primary squamous cell carcinomas (15 cases larynx, 16
lo cases 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 (oesophagus).
Antibodies
The total variant region of the HPKII type 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 ofthe
LCLC97-variant region as described by Hofmann et al., contained an EcoRI recognition
site which was used in order to clone the PCR product directly into the vector pGEX-2T
(Smith et al., 1988). The resulting construct (pGEX CD44v HPKII, v3-v10) codes for a
fusion protein of ~70 kD, consisting of glutathione-S-transferase from Schistosoma
japonicum and the exons v3-v10 of human CD44 (Fig. 1; Heider et al., 1993). The fusion
protein was expressed in E. coli and then subjected to affinity purification over glutathione-
2s agarose (Smith et al., 1988).
Female Balb/c mice were intraperitoneally illullullised with the affinity-purified
fusion protein according to the following plan:
1 st immunisation: 90 ~lg of fusion protein in complete Freund's
adjuvant
2nd and 3rd immunisations: 50 ~lg of fusion protein in incomplete Freund's
adjuvant.
3s The immunisations were given at intervals of 4 weeks. 14 days after the last
immunisation the ~nim~ls were immunised on three successive days with 10 llg of fusion
protein in PBS. On the following day, the spleen cells from an animal with a high antibody
titre were fused with P3.X63-Ag8.653 mouse myeloma cells using polyethyleneglycol
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4000. The hybridoma cells were then selected in microtitre plates in HAT-medium
(Kohler and Milstein, 1975; Kearney et al., 1979).
Measurement of the antibody titre in the serum or screening of the hybridoma
5 supern~t:mt~ were carried out using an ELISA. In this test, first of all microtitre plates
were covered with fusion protein (GST-CD44v3- 10) or only with glutathione-S-
transferase. They were then incubated with serial dilutions of serum samples or hybridoma
supern:~t~nt~ and the specific antibodies were detected using peroxidase-conjugated
antibodies against mouse immunoglobulin. Any hybridomas which reacted only with
o glutathione-S-transferase were discarded. The rem~ining antibodies were first
characterised in an ELISA with domain-specific fusion proteins (exon v3, exon v5 + v6,
exon v6 + v7, exon v8 - vlO) (Koopman et al., 1993). Their immunohistochemical
reactivity was tested on human skin sections.
BIWA-1 (VFF-18; for plepa-~lion and properties see also WO 95/33771) bound
only to fusion proteins which contained a domain coded by the exon v6. In order to further
restrict the epitope of the antibody, various synthetic peptides which represented parts of
the v6 domain were used in ELISA binding assays (Fig. 1). The 14 amino acid peptide
v6D showed the strongest binding. Consequently, the epitope of BIWA-1 is wholly or
partly within the sequence QWFGNRWHEGYRQT of the domain coded by exon v6. This
sequence is homologous to the binding epitope of the antibody 1. lASML, which was used
in a therapeutic rat model and which is specific to rat CD44v6 (Fig: 1).
Immunohistochemistry
Before incubation with the primary antibody, paraffin sections (4 ~lm) in Rotihistol
(Roth, Germany) were depar~ffin~ted 3 times for 10 minutes each time and then rehydrated
in a rising alcohol series. The sections were briefly washed with distilled water and then
cooked in a microwave oven (Sharp Model R-6270) 3 times for 10 minutes each time at
600 Watts in 0.01 M Na-citrate buffer. After each microwave incubation the sections were
cooled for 20 minutes. After the final cooling stage the carriers were washed in PBS and
pre-incubated with normal goat serum (10% in PBS). After 3 washes in PBS the sections
were incubated with primary antibody (BIWA-1: 5 ~lg/ml; murine IgG (isotype-
corresponding negative control) 5 ,ug/ml in PBS/1% BSA) for 1 hour. The positive control
3s used for the staining reaction consisted of normal human skin sections, as keratinocytes
express a CD44-isoform which contains v3-vlO. Endogenous peroxidases were blocked
with 0.3% H2O2 in PBS, and the sections were incubated for 30 minutes with the
biotinylated secondary antibody (anti-mouse IgG-F(ab')2, DAKO Corp.). In order to
CA 02239709 1998-06-04
develop the stain, the sections were incubated for 30 minutes with horseradish peroxidase
which was coupled to biotin as a streptavidin-biotin-peroxidase complex (DAKO Corp.).
The sections were then incubated for 5-10 minutes in 3,3-amino-9-ethyl-carbazolesubstrate (Sigma Immunochemicals), the reaction was stopped with H2O and the sections
5 were counterstained with haematoxylin. The stains were evaluated using a Zeiss Axioskop
Light Microscope and the colour intensities were quantified as follows: +++, strong
expression; ++, moderate expression; +, weak expression; -, unclear or no expression
detected. Only tumour cells with a clear membrane stain were evaluated as positive. The
percentage of positive tumour cells in each section was roughly estimated and two groups
0 were formed: focally positive tumours (less than 10% of the tumour cells reacted with the
antibody) and positive tumours (10 or more % of the tumour cells positive). If fewer than
80% of the tumour cells in the positive cells reacted with the antibody, the corresponding
percentage was indicated.
1S 126 cases of squamous cell carcinomas of various origins were analysed using the
CD44v6-specific monoclonal antibody BIWA-l. The expression of isoforms cont~ining
CD44v6 was observed in all but one tumour sample. The majority of the samples
exhibited expression ofthe antigen on 80-100% ofthe tumour cells and the staining was
restricted to the membrane of the tumour cells. No reaction was observed with stroma
20 tissue, lymphocytes, muscle cells or endothelium.
In order to quantify the ~xl)les~ion of CD44v6-molecules on these tumour cells,
sections of normal human skin were stained parallel to the tumour sections. Normal skin
keratinocytes express high levels of CD44-isoforms and are among the strongest expressors
25 of CD44v6 of the normal cells which have been described hitherto. Consequently,
keratinocyte staining was used as the reference and classified as "strong" ( I I I ) in our
system of evaluation. In the majority of the tumour samples examined, the staining of the
tumour cells was comparable with or even greater than the staining of the skin
keratinocytes, with only a few cases showing weak tumour staining (3 cases of lymph node
30 metastasis) or moderate tumour staining (2 primary carcinomas, 10 metastases). The
staining reaction was very homogeneous within a given tumour section, with the majority
of tumour cells in the section having the same stain intensity. No significant dirreLences
were observed in the CD44v6-~Lession pattern between the primary tumours and
metastases. A detailed summary of the results is shown in Table 1 with the Examples
35 shown in Fig. 2.
CA 02239709 1998-06-04
Table 1: Expression of CD44v6 in squamous cell carcinomas
Sample Type of tumour BIWA-1
Reactivity
46937 86 Primary Larynx +++*
4687 90 Primary Larynx +++
8372 90 Primary Larynx +++
17427 90 Primary Larynx +++
27298 90 Primary Larynx +++
46908 90 Primary Larynx +++
51334 90 Primary Larynx +++
51402 91 Primary Larynx +++
60414 91 Primary Larynx +++
61733 91 Primary Larynx +++
12280 92 Primary Larynx +++
23140 92 Primary Larynx +++
31792 92 Primary Larynx +++
32214 92 Primary Larynx +++
10209 95 Primary Larynx +++
2366 86 Primary Skin +++
2574 86 Primary Skin +++
9916 86 Primary Skin ++/11 1
2696 87 Primary Skin +++
8906 87 Primary Skin +++
8191 88 Primary Skin +++
8354 88 Primary Skin ++50%
11963 88 Primary Skin ++
5590 90 Primary Skin ++/+++
530 92 Primary Skin -+++
2583 94 Primary Skin +++
11337 94 Primary Skin +++
10901 95 Primary Skin l l I
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11557 95 Primary Skin +++
11744 95 Primary Skin +++
11917 95 Primary Skin +++
4688 90 I Lymph node metastasisLarynx ++/+++
4688 90 II Lymph node metastasisLarynx
8374 90 Lymph node metastasisLarynx +++
17428 90 Lymph node metastasisLarynx +++
27300 90 Lymph node metastasisLarynx +++
36942 90 Lymph node metastasisLarynx +++
46909 90 Lymph node metastasisLarynx ++
51336 90 Lymph node metastasisLarynx +++
41108 91 Lymph node metastasisLarynx +++
51398 91 Lymph node metastasisLarynx +++
60416 91 Lymph node metastasisLarynx +++
61734 91 Lymph node metastasisLarynx +++
1318 92 I Lymph node metastasisLarynx +++
1318 92 II Lymphnodemetastasis Larynx l l I
1318 92 III Lymphnodemetastasis Larynx +++
1318 92 IV Lymph node metastasisLarynx +++
2863 92 I Lymph node metastasisLarynx
2863 92 II Lymph node metastasisLarynx +++
5745 92 I Lymph node metastasisLarynx +++
5745 92 II Lymphnodemetastasis Larynx
8969 92 I Lymphnodemetastasis Larynx l l I
8969 92 II Lymph node metastasisLarynx +++
8969 92 III Lymph node metastasisLarynx ++
8969 92 IV Lymph node metastasisLarynx +++
8969 92 2/I Lymph node met~t~ Larynx +++
8969 92 2/II Lymph node metastasisLarynx +++
8969 92 2/III Lymph node metastasisLarynx ++
8969 92 2/IV Lymph node metastasisLarynx +/++
9366 92 Lymph node metastasisLarynx +++
CA 02239709 1998-06-04
9509 92 Lymphnodemet~.~t~ Larynx l l I
9566 92 Lymph node metastasis Larynx +++
12283 92 Lymph node metastasis Larynx +++
14046 92 Lymphnodemet~.ct~ Larynx l l l
31787 92 Lymph node metastasis Larynx +++
49228 92 Lymph node metastasis Larynx +++ 50%
29228 93 Lymph node metastasis Larynx +++
29829 93 Lymph node metastasis Larynx ++
29804 95 Lymph node metastasis Larynx ++/+++
15293 91 Lymph node metastasis Lung + 25%
1667 92 Lymph node metastasis Lung + 20%
2757 92 I Lymphnodemetastasis Lung
2757 92 II Lymphnodemet~t~i.c Lung
2757 92 III Lymphnodemet~t~ - Lung l l l
2757 92 IV Lymph node met~t~ Lung +++
4790 92 Lymph node met~t~ Lung +++
6168 92 I Lymph node met~t~ Lung ++ 50%
6168 92 II Lymph node met~t~ Lung +++
6168 92 III Lymph node metastasis Lung +++
6168 92 IV Lymphnodemet~t~ Lung l l I
7206 92 Lymph node met~t~ Lung +++
7531 92 I Lymph node metastasis Lung +++
7531 92 II Lymph node met~t~i.c Lung +++
7531 92 III Lymph node metastasis Lung ++/+++
7531 92 IV Lymphnodemetastasis Lung l l l
10324 92 Lymph node met~et~ Lung +++
10519 92 II Lymphnodemet~t~ Lung
10519 92 RM Lymphnodemetastasis Lung
II
10958 92 Lymphnodemet~et~ei~ Lung
11425 92 I Lymphnodemetastasis Lung l l I
11425 92 II Lymph node metastasis Lung +++
CA 02239709 1998-06-04
13055 92 Lymph node metastasisLung ++/+++
13055 92 II Lymph node metastasisLung focal +++
13055 92 III Lymphnodemetastasis Lung l l I
15663 92 Lymph node metastasisLung +++
16713 92 Lymph node metastasisLung +++
14980 91 I Lymph node met~t~ Oesophagus +++
14980 91 II Lymph node metastasisOesophagus +++
16641 91 I LymphnodemetastasisOesophagus l l I
16641 91 II Lymph node metastasisOesophagus +++
16641 91 III Lymph node metastasisOesophagus +++
1059 92 Lymph node metastasis Oesophagus +
1710 92 I Lymphnodemetastasis Oesophagus
1710 92 II Lymphnodemetastasis Oesophagus l l I
1710 92 III Lymphnodemetastasis Oesophagus l l l
11502 92 I Lymph node metastasis Oesophagus +++
11502 92 II Lymph node metastasis Oesophagus ++
202 92 Lymph node metastasis Oral cavity ++ 60%
6030 92 - Lymph node metastasis Oral cavity +!++/+++ 25%
7335 92 I Lymph node metastasis Oral cavity +++
7335 92 II Lymph node metastasis Oral cavity +++
15324 92 II LymphnodemetastasisOralcavity I l l 70%
16164 92 I LymphnodemetastasisOralcavity +++
16164 92 II LymphnodemetastasisOralcavity I I 1 50%
16412 92 LymphnodemetastasisOralcavity ++/+++
16836 92 I LymphnodemetastasisOralcavity
16836 92 II LymphnodemetastasisOralcavity
16836 92 III LymphnodemetastasisOralcavity ~ l l
6228 92 I Lymphnodemet~t~ci~ Tonsil r I I --
6228 92 II Lymphnodemetastasis Tonsil
6618 92 Lymphnodemetastasis Tonsil
11840 92 Lymph node metastasisTonsil ++
CA 02239709 1998-06-04
16
14172 91 4 Livermetastasis Oesophagus +++
14172 91 5 Livermetastasis Oesophagus l l I
4131 94 1 Liver metastasis Oesophagus +/++
8438 94 Liver metastasis Oesophagus focal ++/+++
80- 100% of the tumour cells reacted positively with BIWA- 1. In cases where fewer
tumour cells reacted with the antibody, the percentage obtained is given.
5 Example 2: Expression of CD44v6 in kidney cell carcinomas, prostate carcinomas and liver metastases of colon carcinomas
Tissue
0 19 cases of kidney cell carcinomas (12 cases of clear cell, 5 cases of chromophilic,
1 case of chromophobic, 1 oncocytoma), 16 primary adenocarcinomas of the prostate and
19 cases of lymph node metastases of prostate carcinoma, and 30 cases of liver metastases
of colon carcinomas were analysed.
Antibody
BIWA-1 (see Example 1).
Immunohistochemistry
For method see Example 1.
In contrast to the squamous cell carcinomas, no or only focal expression of
CD44v6-isoforms could be detected in the majority of the kidney cell and prostate
carcinomas investig~tr.A In the case of a more than focal expression in the prostate
carcinomas the staining was predomin~ntly diffusely cytoplasmic and weak or
heterogeneous, compared with the staining of normal prostate epithelium. In 50% of the
liver metastases of colon carcinomas investigated, a more than focal expression of CD44v6
isoforms was detected. The staining in the majority of cases was faint to moderate, but
generally fewer than 100% of the tumour cells in a sample showed any staining with
BIWA-1. The results are summarised in Table 2.
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Table 2: Expression of CD44v6 in prostate adenocarcinomas, kidney cell carcinomas and
liver metastases of colorectal carcinomas
Type oftumour n BIWA-l Reactivity
negative focal pos. positive
Prostate Primary 16 8 3 5
adenocarcinoma
Prostate Lymph node 19 15 2 2
adenocarcinoma metastases
Kidney cell Primary 19 17 0 2
carcinoma
Colorectal Liver metastases 30 7 8 15
carcmoma
Example 3: Characterisation of CD44v6-specific antibodies
Cell line
lo The human SCC cell line A-431 (spontaneous epidermoid carcinoma of the vulva)
was obtained from the American Type Culture Collection (Rockwell MD) and cultured in
accordance with the manufacturer's instructions. The surface e~lession of CD44v6-
cont:~ining isoforms was determined by FACS analysis, using an FITC-linked mAb
BIWA- 1.
Analysis of the kinetic constants
The affinity and kinetics of the monoclonal antibody CD44v6-interaction was
determined by Surface Plasmon Resonance (SPR), using a BIAcore 2000 system
20 (Pharmacia Biocensor). A glutathione-S-transferase-CD44-fusion protein which contained
the region coded by the exons v3-vl O (GST/CD44 v3-vl O) was immobilised on a CM5
Sensor Chip, the amine coupling method being carried out in accordance with the
manufacturer's instructions. Antibodies in various concentrations (8-132 nM) in HBS
(10 mM HEPES pH 7.4, 150 mM sodium chloride, 3.4 mM EDTA, 0.05% BIA core
25 surfactant P20) was injected over the antigen-specific surface at a flow rate of 5 ~ll/min.
CA 02239709 1998-06-04
The interaction was recorded as a change in the SPR signal. Dissociation of the antibody
was observed for 5 minutes in the buffer flow (HBS). The surface of the chip wasregenerated with a single pulse of 15 ~11 30 mM HCl. The data analysis and calculation of
the kinetic constants were carried out using the Pharmacia Biocensors BIA Evaluation
5 Software, Version 2.1.
In this way, the antigen affinity of BIWA-1 was compared with other CD44v6-
specific mAbs (VFF4, VFF7, BBA-13 (IgG1, R&D Systems, Abingdon, U.K.)). Kinetic
and affinity constants of the various antibodies were determined in two independent
o experiments. Table 3 shows the values of the association rates (ka), dissociation rates (kd)
and dissociation constants (Kd) for the 4 mAbs. A11 the mAbs showed similar ka and kd,
with the exception of BBA- 13, which has a 3-times lower ka and VFF7, whieh has a
significantly higher dissociation rate (factor 5) compared with the other mAbs. This results
in a lower binding affinity for VFF7 and BBA- 13 compared with VFF4 and BIWA- 1.s BIWA-1 shows the lowest Kd of all the antibodies investigated.
Table 3: Kinetic and affinity constants of various CD44v6-specific mAbs
Antibody k (M-lS-1) kd(s-l) Kd (M)
VFF4 1.1 x 105 2.6 x 10-5 2.4 x 10-1~
VFF7 1.1 x 105 1.2 x 10-4 l.i X 10-9
BIWA-1 1.3 x 105 2.2 x 10-5 1.7 x 10-1~
BBA-13 3.7 x 104 2.3 x 10-5 6.2 x 10-1~
Analysis of the antibody-protein interaction using ELISA.
CD44v6 expressing A-431 cells were cultivated in 96-well plates (Falcon Microtest
III, Becton Dickinson, Lincoln Park, NJ) in numbers of 5 x 104 per well in RPMI 1640 with
10% foetal calf serum overnight at 37~C. After washing with PBS/0.05% Tween 20 the
cells were fixed for 1 rninute with ice cold ethanol, followed by a washing step. Incubation
with the primary antibodies (VFF4, VFF7, BIWA-1, BBA-13, 1 ng/ml to 600 ng/ml, in
each case in assay buffer: PBS/0.5% BSA/0.05% Tween 20) was carried out for 1 hour at
ambient temperature and followed by 3 washing steps. The secondary antibody used was a
rabbit-antimouse-IgG horseradish peroxidase-conjugated antibody (DAKO Corporation,
CA 02239709 1998-06-04
19
Copenhagen, J)enmark; dilution 1 :6000 in assay buffer) (1 hour at room temperature).
After 3 washing steps the colour was developed using TMB solution (Kirkegaard and
Perry, Gaithersburg, USA). The extinction was measured using a Hewlett-Packard ELISA
Reader.
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.
o The protein domain which is coded by the CD44-exon v6 consists of 45 amino
acids (Figure 4). In order to define more accurately the epitope which is recognised by
BIWA-l, a series of synthetic peptides were used in ELISA assays. Preliminary
experiments showed binding to a centrally located 14-mer (amino acid groups 18-31;
Figure 4; cf. also Figure 1) but not to peptides outside this region. A second series of
peptides were therefore synthesised and tested in competitive ELISAs (Figure 4). The
results show that the peptide l 9-29 (WFGNRWHEGYR) represents the lllh~ lulll structure
required for high affinity binding. Flimin~tion of the C-terminal arginine groups resulted
in a more than 100 times weaker binding.
Example 4: Biodistribution of radio-iodinated CD44v6-antibodies in xenotransplant-
carrying nude mice
A-431 -Xenotransplant model
8 week old female BALB/c nu/nu nude mice (B & K Universal, Renton, WA) were
subcutaneously injected in the left-hand median line with 5 x 106 cultivated A-431 cells
(human epidermoid carcinoma of the vulva). Xenotransplanted ~nim~l~ carrying A-431
tumours were used for biodistribution experiments within two weeks (weight of tumours:
40 50 mg)
Radio-iodination of BIWA-1
Protein G-purified mAb BIWA-1 (murine IgG1) was coupled to streptavidin, using
the heterobifunctional crosslinker succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-
carboxylate. Streptavidin-lysyl groups were linked to reduced antibody-cysteinyl groups
produced by preliminary treatment of the antibody with dithiothreitol. The 1: 1 conjugates
obtained (~ 90%) were further purified by ion exchange chromatography. For
biodistribution experiments, BIWA-1/SA was labelled via primary amines of lysine with
CA 02239709 1998-06-04
l25I, using p-iodophenyl labelling reagent (PIP; NEN Dupont, Wilmington, DE), fnllowed
by the process of Willbur et al. (1989). Labelling BIWA-1 with SA or l2sI did not affect
the immunoreactivity or the ph~ cokinetics of the antibody in mice.
Biodistribution experiments
Nude mice which were xenotransplanted with human A-431 tumours were
intravenously injected through the lateral caudal vein with 5-7 IlCi l2sI on 50 ~g of mAb
BIWA-1 (specific activity 0.1-0.14 mCi/mg). Time-biodistribution studies were carried
o out in groups of n=3 ~nim~l~ for each point of time at 4, 24, 48, 120 and 168 hours post-
injection. At selected times, mice were weighed, blood was extracted from the retro-orbital
plexus and they were killed by cervical dislocation. Nine organs and tissues-were collected
and weighed: blood, tail, lung, liver, spleen, stomach, kidney, intestine and tumour. The
radioactivity in the tissues was counted in a gamma-scintillation counter (Packard
Instrument Company, Meriden, CT) by comparison with standards of the injected antibody
preparation, the energy window being set at 25-80 keV for l2sI. The percentage of injected
dose per gram of tissue was calculated (% ID/g).
Preliminary experiments had shown that BIWA-1 did not cross-react with murine
20 CD44v6-antigen. Table 4 and Figure S show the absorption of radioactivity in tumours and
normal tissue. Iodinated BIWA-1 showed a rapid tumour absorption (7.6% injected dose/g
at 4 hours post-injection) which increased to more than 18% ID/g a~fter 48 hours and then
remained constant for up to 120 hours. Seven days post-injection (168 hours) the tumour
still contained 15.3% ID/g of tissue. Tumour:tissue ratios were calculated for individual
25 times and these are shown in Table 4. At 24 hours post-injection the tumour:blood ratio
was 0.48 and increased to 3.16 on day 7. The uptake in normal tissue was low and most
probably caused by blood-pool background in the tissue biopsies. Selective in vivo
targeting of human SCC-xenotransplants in nude mice with 12sI-labelled BIWA- 1 shows
that this monoclonal antibody has a high potential as a targeting vehicle for diagnostic and
30 therapeutic use in SCC patients.
CA 02239709 1998-06-04
Table 4: Tumour:tissue ratios of l2sI-BIWA- 1 in A-431 -tumour-carrying nude mice at
various times post-injection
Ratio of tumour to4h 24h 48h 120h168h
Blood 0.22a 0.48 1.31 2.60 3.16
Tail 1.18 2.62 7.70 12.28 13.06
Lung 0.40 1.03 2.65 7.04 4.82
Liver 0.94 1.18 2.28 3.57 3.24
Spleen 1.40 1.84 4.00 4.86 442
Stomach 3.89 7.37 19.40 25.56 33.96
Kidney 0.82 1.31 2.72 2.79 2.53
- Intestine 3.54 6.24 11.94 19.24 27.78
5 a mean values (n=3), SD are <7%
Example 5: Different ~ ession of CD44v6 in a large number of human tumours
In a wider investigation, a total of 544 tumour samples were examined
immunohistochemically with the monoclonal antibody BIWA 1 (clone VFF-18) for the10 expression of CD44v6. The samples were either embedded in pal~rm or frozen in liquid
nitrogen immediately after surgical removal and stored at -70~C until required. The
following tumours were analysed: basalioma (n= 16), adenocarcinoma (AC) of the breast
(n=55), AC of the colon (n=83), squamous cell carcinomas (SCC) of the head and neck
(n=125), lung carcinomas (n=120), prostate AC (n=34), kidney cell carcinomas (n=27),
5 SCC of the skin (n=15) and AC of the stomach (n=69). The tissues were obtained by
routine surgery or biopsy and normal tissue was obtained to accompany the tumour- samples. The immunohistochemical investigations were carried out as in Example 1.
Table 5 shows a summary of the immunohistochemical analysis of 397 different
20 types of tumour with the mAb BIWA 1.
CA 02239709 1998-06-04
Table 5: Expression of CD44v6 in hurnan turnours
Type Total number Positive cases*
n %
Basalioma Primary tumour 16 10 62
Breast AC Primary tumour 17 15 88
Lymph node metastases 34 31 91
Liver metastases 4 4 100
ColonAC Lymphnodemetastases 51 21 41
Liver metastases 26 13 50
Brain metastases 6 6 100
LarynxSCC Lymphnodemetastases 18 18 _ 100
Lung AC Primary tumour 35 15 43
Lung SCC Primary tumour 9 9 100
Oesphagus Primary turnour 20 20 100
SCC
Prostate AC Primary tumour 16 5 31
Lymphnodemetastases - 18 0 0
RCC Primary tumour 27 5 18
SCLC Primary tumour 31 7 23
Stomach AC Primary tumour 22 - 15 68
Lymph node metastases - 43 16 37
Liver metastases 4 - 4 100
Total number 397
*: 2 10% of tumour cells positive
AC: adenocarcinoma; RCC: renal cell carcinoma
SCLC: small cell lung cancer; SCC: squarnous cell carcinoma
In small cell lung cancers, renal cell carcinomas and AC of the prostate, no or only
little reactivity was observed. All the other types of tumour investigated expressed
CD44v6-cont:~ining isoforms to varying degrees. The majority of the AC of the breast
investigated showed reactivity with BIWA 1, and the SCC tested (larynx, lung andoesophagus) expressed CD44b6 in 100% of all cases.
185 additional cases of SCC of various types and classification were investig~te~l
for their reactivity with BIWA 1. These included 67 cases of primary SCC (larynx, n=15;
oral cavity, n=16, oropharynx, n=3; skin, n=15), 77 samples of lymph node metastases
CA 02239709 1998-06-04
(larynx, n=12; lung, n=27; oesphagus, n=11; oral cavity, n=6; oropharynx, n=7;
hypopharynx, n=10; tonsil, n=4), and 3 samples of liver metastases (oesphagus).- Table 6
summarises the immunohistochemical analysis of all the SCC samples investigated.
5 Table 6: Expression of CD44v6 in squamous cell carcinomas
Type Totalnumber Negative Focal~os. Positive
n % n % n %
Hypopharynx LNM 10 0 0 0 0 10 100
Oropharynx PT 3 0 0 0 0 3 100
LNM 7 0 0 0 0 7 100
Larynx PT 15 0 0 0 0 15 100
LNM 30 1 3 0 0 29 97
Lung PT 18 2 11 0 0 16 89
LNM 27 0 0 1 4 26 96
Oesphagus PT 20- 0 0 1 5 19 95
LNM 11 0 0 0 0 11 100
LM 3 0 0 0 0 3 100
Oral cavity PT 16 0 0 0 0 16 100
LM 6 0 0 0 0 6 100
Skin PT 15 0 0 0 0 15 100
Tonsil LNM 4 0 0 ~ 0 4 100
Total number 185
Focal pos.: <10% of tumour cells positive; LNM: lymph node metastases;
PT: primary tumour; LM: liver metastases
Expression of CD44v6 co~ g isoforms was found in all but three tumour
10 samples (one case of larynx, 2 cases of lung). The majority of the samples showed
expression of the antigen on 80 to 100% of the tumour cells within a single section, the
staining being concentrated chiefly on the membrane of the tumour cells. Thè most
homogeneous staining pattern was found in carcinomas of the larynx, oesphagus and
hypopharynx, with the majority of tumour cells in the section having the same intensity of
5 staining.
CA 02239709 1998-06-04
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CA 02239709 1998-06-04
SEQUENCE LISTING
(1) GENERAL DATA:
(i) APPLICANT:
(A) NAME: Boehringer Ingelheim International GmbH
(B) STREET: Rheinstrasse
(C) TOWN: Ingelheim
(E) COUNTRY: Germany
(F) POSTCODE: 55216
(G) TELEPHONE: +49-(0)-6132-772770
(H) FAX: +49-(0)-6132-774377
(A) NAME: Forschungszentrum Karlsruhe GmbH
(B) STREET: Postfach 3640
(C) TOWN: Karlsruhe
(E) COUNTRY: Germany
(F) POSTCODE: 76021
(A) NAME: ~eider, Karl-Heinz
(B) STREET: Hervicusgasse-4/3/21
(C) TOWN: Vienna
(E) COUNTRY: Austria
(F) POSTCODE: 1120
(A) NAME: Adolf, Guenther
(B) STREET: Stiftgasse 15-17/10
(C) TOWN: Vienna
(E) COUNTRY: Austria
(F) POSTCODE: 1070
(A) NAME: Ostermann, Elinborg
(B) STREET: Mauerbachstr. 56/6
(C) TOWN: Vienna
(E) COUNTRY: Austria
CA 02239709 1998-06-04
(F) POSTCODE: 1140
(A) NAME: Patzelt, Erik
(B) STREET: Hans-Buchmueller-Gasse 8
(C) TOWN: Purkersdorf
(E) COUNTRY: Austria
(F) POSTCODE: 3002
(A) NAME: Sproll, Marlies
(B) STREET: Schwenkgasse 3
(C) TOWN: Vienna
(E) COUNTRY: Austria
(F) POSTCODE: 1120
(ii) TITLE OF THE INVENTION: Process for the diagnosis and
treatment of squamous cell carcinomas
(iii) NUMBER OF SEQUENCES: 16
(iv) C~MPUTER-READABLE VERSION:
(A) DATA CARRIER: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM:-PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)
(2) DATA RELATING TO SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 129 base pairs
(B) NATURE: Nucleotide
(C) STRAND FORM: both
(D) TOPOLOGY: both
(ii) NATURE OF THE MOLECULE: Genome DNA
CA 02239709 1998-06-04
34
(ix) FEATURE:
(A) NAME/KEY: exon
(B) POSITION:1..129
(D) OTHER DATA:/product= "CD44"
/label= v6
/note= "GenBank data base accession No. L05411"
/citation= ([1])
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) POSITION:3..128
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Screaton, GR
Bell, MV
Jackson, DG
Cornelis, FB
Gerth, U
Bell, JI
(B) TITLE: Genomic structure of DNA encoding the lympho-
cyte homing receptor CD44 reveals at least 12 alternatively
spliced exons
(C) JOURNAL: Proc. Natl. Acad. Sci. U.S.A.
(D) VOLUME: 89
(F) PAGES: 12160-12164
(G) DATE: December-1992
(K) IMPORTANT GROUPS IN SEQ ID NO: 1: FROM 1 TO 129
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
CA 02239709 1998-06-04
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
TC CAG GCA ACT CCT AGT AGT ACA ACG GAA GAA ACA GCT ACC CAG AAG 47
Gln Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys
1 5 10 15
GAA CAG TGG TTT GGC AAC AGA TGG CAT GAG GGA TAT CGC CAA ACA CCC 95
Glu Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro
20 25 30
AGA GAA GAC TCC CAT TCG ACA ACA GGG ACA GCT G 129
Arg Glu Asp Ser His Ser Thr Thr Gly Thr Ala
35 40
(2) DATA RELATING TO SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) NATURE: amino acid
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: Protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gln Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys Glu
1 5 10 15
~ln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro Arg
Glu Asp Ser His Ser Thr Thr Gly Thr Ala
CA 02239709 1998-06-04
36
(2) DATA RELATING TO SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr
1 5 10
(2) DATA RELATING TO SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) NATURE: Nucleotide
(C) STRAND FORM: single strand
(D) ~OPOLOGY: linear
(ii) NATURE OF THE MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
CA 02239709 1998-06-04
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CAGGCTGGGA GCCAAATGAA GAAAATG 27
(2) DATA RELATING TO SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) NATURE: Nucleotide
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "PCR primer"
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TGATAAGGAA CGATTGACAT TAGAGTTGGA 30
! CA 02239709 1998-06-04
(2) DATA RELATING TO SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg
1 - 5 io
(2) DATA RELATING TO SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
CA 02239709 1998-06-04
39
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Gln Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys Glu
1 5 10 15
Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro Arg
20 25 30
Glu Asp Ser His Ser Thr Thr Gly Thr Ala Ala
(2) DATA RELATING TO SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 11 amino acids
(B) NATURE: amino acid
- (C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
CA 02239709 1998-06-04
; (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Ser Ser Thr Thr Glu Glu Thr Ala Thr Gln Lys
1 5 10
(2) DATA RELATING TO SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Glu Glu Thr Ala Thr Gln Lys Glu Gln Trp
1 5 10
(2) DATA RELATING TO SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
CA 02239709 1998-06-04
41
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Thr Ala Thr Gln Lys Glu Gln Trp Phe Gly Asn
1 5 10
(2) DATA RELATING TO SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: sin~le strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CA 02239709 1998-06-04
42
;
Gln Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gln Thr
1 5 10
(2) DATA RELATING TO SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Asn Arg Trp His Glu Gly Tyr Arg Gln Thr Pro
1 5 10
(2) DATA RELATING TO SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
CA 02239709 1998-06-04
43
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Glu Gly Tyr Arg Gln Thr Pro Arg Glu Asp Ser
1 5 10
(2) DATA RELATING TO SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Thr Pro Arg Glu Asp Ser His Ser Thr Gly
1 5 10
CA 02239709 1998-06-04
(2) DATA RELATING TO SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) NATURE: amino acid
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Trp Ala Asp Pro Asn Ser Thr Thr Glu Glu Ala Ala Thr Gln Lys Glu
1 5 10 15
Lys Trp Phe Glu Asn Glu Trp Gln Gly Lys Asn Pro Pro Thr Pro Ser
20 25 30
Glu Asp Ser His Val Thr Glu Gly Thr Thr
(2) DATA RELATING TO SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) NATURE: amino acid
CA 02239709 1998-06-04
(C) STRAND FORM: single strand
(D) TOPOLOGY: linear
(ii) NATURE OF THE MOLECULE: peptide
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 195 45 472.3
(I) FILING DATE: 06-DEC-1995
(x) PUBLICATION INFORMATION:
(H) DOCUMENT NUMBER: DE 196 15 074.4
(I) FILING DATE: 17-APR-1996
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Lys Trp Phe Glu Asn Glu Trp Gln Gly Lys Asn Pro Pro Thr
1 5 10
