IE83303B1 - IGF-1 receptor interacting proteins (IIPs), genes coding thereof and uses thereof - Google Patents
IGF-1 receptor interacting proteins (IIPs), genes coding thereof and uses thereofInfo
- Publication number
- IE83303B1 IE83303B1 IE1999/1020A IE991020A IE83303B1 IE 83303 B1 IE83303 B1 IE 83303B1 IE 1999/1020 A IE1999/1020 A IE 1999/1020A IE 991020 A IE991020 A IE 991020A IE 83303 B1 IE83303 B1 IE 83303B1
- Authority
- IE
- Ireland
- Prior art keywords
- igf
- iip
- nucleic acid
- receptor
- protein
- Prior art date
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- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
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- 235000020183 skimmed milk Nutrition 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
Description
PATENTS ACT, 1992
991020
ICE-1 RECEPTOR TNTERACTING PROTEINS (TIPS), GENES CODING
THEREFOR AND USES THEREOF
The present invention relates to lGF—1 receptor interacting proteins (llPs), nucleic acids
coding therefor, their use for diagnostics and therapeutics, especially in the field of cancer.
In particular, the invention relates to the diagnosis of said genes in mammalian cells,
especially in malignant tumor cells, to gene therapy methods for inhibiting the interaction
between IGF-1 receptor and HPS, methods of screening for potential cancer therapy agents,
and cell lines and animal models useful in screening for and evaluating potential useful
pharmaceutical agents inhibiting the interaction between llPs and IGF-1 receptor.
The present invention relates in particular to the cloning and characterization of the gene
1lP—10 and the gene products thereof. Said gene products (polypeptides, mRNA) are
especially characterized as having the ability to modulate the lGF—1 receptor signaling
pathway. The function of the gene products according to the invention is therefore to
modulate signal transduction of the IGF-1 receptor. Forced activation of llPs therefore
correlates with increased tumor cell proliferation, survival and escape of apoptosis.
The lGF—1 receptor signaling system plays an important role in tumor proliferation and
survival and is implicated in inhibition of tumor apoptosis. In addition and independent of
its mitogenic properties, lGF—1R activation can protect against or at least retard
programmed cell death in vitro and in vivo (Harrington et al., EMBO I. 13 (1994)
3286-3295; Sell et al., Cancer Res. 55 (1995) 303-305; Singleton et al., Cancer Res. 56 (1996)
4522-4529). A decrease in the level of IGF-1R below wild type levels was also shown to
cause massive apoptosis of tumor cells in vivo (Resnicoff et al., Cancer Res. 55 (1995)
2463-2469; Resnicoff et al., Cancer Res. 55 (1995) 3739-3741). Overexpression of either
ligand (IGF) and/or the receptor is a feature of various tumor cell lines and can lead to
tumor formation in animal models. Overexpression of human lGF—1R resulted in ligand-
dependent anchorage-independent growth of NIH 3T3 or Rat-1 fibroblasts and inoculation
of these cells caused a rapid tumor formation in nude mice (Kaleko et al., Mol. Cell. Biol.
(1990) 464-473; Prager et al., Proc. Natl. Acad. Sci. USA 91 (1994) 2181-2185).
Transgenic mice overexpressing IGF-ll specifically in the mammary gland develop
mammary adenocarcinorna (Bates et al., Br. I. Cancer 72 (1995) 1189-1193) and transgenic
mice overexpressing IGF-II under the control of a more general promoter develop an
elevated number and wide spectrum of tumor types (Rogler et al., 1. Biol. Chem. 269 (1994)
13779-13784). One example among many for human tumors overexpressing lGF—l or IGF-
II at very high frequency (>800/o) are Small Cell Lung Carcinomas (Quinn et al., 1. Biol.
Chem. 271 (1996) 11477-11483). Signaling by the IGF system seems to be also required for
the transforming activity of certain oncogenes. Fetal fibroblasts with a disruption of the
IGF-1R gene cannot be transformed by the SV40 T antigen, activated Ha-ras, a
combination of both (Sell et al., Proc. Natl. Acad. Sci. USA 90 (1993) 11217-11221; Sell et
al., Mol. Cell. Biol. 14 (1994) 3604-3612), and also the E5 protein of the bovine papilloma
virus is no longer transforming (Morrione et al., J. Virol. 69 (1995) 5300-5303).
Interference with the IGF/IGF-1R system was also shown to reverse the transformed
phenotype and to inhibit tumor growth (Trojan et al., Science 259 (1993) 94-97; Kalebic et
al., Cancer Res. 54 (1994) 5531-5534; Prager et al., Proc. Natl. Acad. Sci. USA 91 (1994)
2181-2185; Resnicoff et al., Cancer Res. 54 (1994) 2218-2222; Resnicoff et al., Cancer Res.
54 (1994) 4848-4850; Resnicoff et al., Cancer Res. 55 (1995) 2463-2469). For example,
mice injected with rat prostate adenocarcinoma cells (PA-Ill) transfected with IGF-1R
antisense cDNA (729 bp) develop tumors 90% smaller than controls or remained tumor-
free after 60 days of observation (Burfeind et al., Proc. Natl. Acad. Sci. USA 93 (1996)
7263-7268). IGF-1R mediated protection against apoptosis is independent of de-novo gene
expression and protein synthesis. Thus, IGF-1 likely exerts its anti-apoptotic function via
the activation of preformed cytosolic mediators.
Some signaling substrates which bind to the IGF-1R (e.g. IRS-1, SHC, p85 PI3 kinase etc.,
for details see below) have been described. However, none of these transducers is unique to
the IGF-1R and thus could be exclusively responsible for the unique biological features of
the IGF-1R compared to other receptor tyrosine kinase including the insulin receptor. This
indicates that specific targets of the IGF-1R (or at least the IGF-receptor subfamily) might
exist which trigger survival and counteract apoptosis and thus are prime pharmaceutical
targets for anti-cancer therapy.
By using the yeast two-hybrid system it was shown that p85, the regulatory domain of
phosphatidyl inositol 3 kinase (PI3K), interacts with the IGF-1R (Lamothe, B., et al., FEBS
Lett. 373 (1995) 51-55; Tartare-Decker, S., et al., Endocrinology 137 (1996) 1019-1024).
However binding of p85 is also seen to many other receptor tyrosine kinases of virtually all
families. Another binding partner of the IGF-1R defined by two-hybrid screening is SHC
which binds also to othertyrosine kinases as trk, met, EGF-R and the insulin receptor
(Tartare-Deckert, S., et al., I. Biol. Chem. 270 (1995) 23456-23460). The insulin receptor
substrate 1 (IRS-1) and insulin receptor substrate 2 (IRS-2) were also found to interact
both with the IGF-1R as well as the insulin receptor (Tartare-Deckert, S., et al., 1. Biol.
Chem. 270 (1995) 23456-23460; He, W., et al., J. Biol. Chem. 271 (1996) 11641-11645; Dey,
R.B., et al., Mol. Endocrinol. 10 (1996) 631-641). Grb 10 which interacts with the IGF-1R
also shares many tyrosine kinases as binding partners, e.g. met, insulin receptor, kit and abl
(Dey, R.B., et al., Mol. Endocrinol. 10 (1996) 631-641; Morrione, A., et al., Cancer Res. 30
(1996) 3165-3167). The phosphatase PTP1D (syp) shows also a very promiscuous binding
capacity, i.e. binds to IGF-1R, insulin receptor, met and others (Rocchi, S., et al.,
Endocrinology 137 (1996) 4944-4952). More recently, mSH2-B and vav were described as
binders of the IGF-1R, but interaction_is also seen with other tyrosine kinases, e.g. mSH2-B
also bind to ret and the insulin receptor (Wang, 1., and Riedel, 1-1., J. Biol. Chem. 273
(1998) 3136-3139). Taken together, the so far described IGF-1R binding proteins represent
relatively unspecific targets for therapeutic approaches, or are in the case of the insulin
receptor substrates (IRS-1, IRS-2) indispensable for insulin-driven activities.
It is an object of the invention to provide novel genes encoding binding proteins of 1GF- 1R
as well as the corresponding polypeptides which are the basis for new cancer therapy based
on the modulation (preferably, inhibition) of the interaction between IGF-1R and 1lPs
according to the invention.
The invention comprises a nucleic acid encoding a protein binding to IGF-1
receptor (IIP-10) selected from the group comprising
a) the nucleic acids shown in SEQ ID NO:5 or a nucleic acid sequence which is
complementary thereto,
b) nucleic acids which hybridize under stringent conditions with one of the nucleic acids
from a) encoding a polypeptide showing at least 75% homology with the polypeptide
ofSEQ ID NO:6 or
c) sequences that due to the degeneracy of the genetic code encode IIP-10 polypeptides
having the amino acid sequence of the polypeptides encoded by the sequences of a)
and b).
The cDNA of llP~10 codes for a new protein of 226 aa with a calculated molecular weight
of 25.697. 1lP-10 is a lysine rich protein (11%). 1113-10 contains an N-glycosylation site,
several N-myristoylation sites, Cl<2 and PKC phosphorylation sites, one tyrosine kinase
phosphorylation site and one putative nuclear localization signal (Fig. 5). The CDNA
sequence of IIP-10 shows 65% homology to the CDNA sequence of the Gallus Gallus
thymocyte protein cthy28kD (EMBL accession number: GG34350). The amino acid
sequences of IIP-10 and cthy28kD show 70% identity. Nt 383 to 'nt 584 of the HP-10 CDNA
are 94% identical to a partial CDNA described in W0 95/ 14772 (human gene signature
HUMGSO6271; accession number T24253). By immunofluorescence flag-tagged IlP—1O
shows both a cytoplasmic and a nuclear localization in NIH3T3 cells overexpressing the
IGF—1 receptor. Further yeast two-hybrid analysis revealed that llP-10 interacts in a
phosphorylation dependent manner with the IGF-l receptor. llP-10 does not interact with
the insulin receptor. Deletion analysis of l1P-10 revealed that aa 19 to aa 226 are sufficient
for binding to the IGF-1 receptor.
,,lnteraction or binding between M310 and the IGF-1 receptor“ means a specific binding of
the llP10 polypeptide to the IGF-1 receptor but not to control proteins such as lamin in the
yeast two hybrid system. Specific binding to the IGF-1 receptor can be demonstrated using
glutathion-S-transferase (GST)—IlP fusion proteins expressed in bacteria and lGF—1
receptors expressed in mammalian cells. Furthermore, an association between a Flag tagged
HP-10 fusion protein (cf. Weidner, M., et al., Nature 384 (1996) 173-176) and the IGF—1
receptor can be monitored in mammalian cell systems. For this purpose eukaryotic
expression vectors are used to transfect the respective cDNAs. Interaction between the
proteins is visualized by coimmunoprecipitation experiments or subcellular localization
studies using anti-Flag or anti~lGF-1 receptor antibodies.
Probes and primers for the genes according to the invention as well as nucleic acids which
encode antigenic determinants of the» gene include nucleic acids with preferably 10 to 50, or
more preferably, 10 to 20 consecutive nucleotides out of the disclosed sequences.
The term "nucleic acid" denotes a polynucleotide which can be, for example, a DNA, RNA,
or derivatized active DNA or RNA. DNA and mRNA molecules are preferred, however.
The term "hybridize under stringent conditions" means that two nucleic acid fragments are
capable of hybridization to one another under standard hybridization conditions described
in Sambrook et al., Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor
Laboratory Press, New York, USA.
More specifically, ,,stringent conditions“ as used herein refers to hybridization in 5.0 x SSC,
x Denhardt, 7% SDS, 0.5 M phosphate buffer pH 7.0, 10% dextran sulfate and,100 pg/ml
salmon sperm DNA at about 50°C-68°C, followed by two washing steps with 1 X SSC at
68°C. In! addition, the temperature in the wash step can be increased from low stringency
conditions at room temperatures, about 22°C, to high stringency conditions at about 68°C.
The invention further comprises recombinant expression vectors which are suitable for the
expression of IIP-10, recombinant host cells transfected with such expression vectors, as
well as a process for the recombinant production of a protein which is encoded by the
llP-10 gene.
The invention further comprises synthetic and recombinant polypeptides which are
encoded by the nucleic acids according to the invention, and preferably encoded by the
DNA sequence shown in SEQ ID NO:5 as well as peptidomimetics based thereon. Such
peptidomimetics have a high affinity for cell membranes and are readily taken up by the
cells. Peptidomimetics are preferably compounds derived from peptides and proteins, and
are obtained by structural modification using unnatural amino acids, conformational
restraints, isosterical placement, cyclization, etc. They are based preferably on 24 or fewer,
preferably 20 or fewer, amino acids, a basis of approximately 12 amino acids being
particularly preferred.
The polypeptides and peptidomimetics can be defined by their corresponding DNA
sequences and by the amino acid sequences derived therefrom. The isolated HP polypeptide
can occur in natural allelic variations which differ from individual to individual. Such
variations of the amino acids are usually amino acid substitutions. However, they may also
be deletions, insertions or additions of amino acids to the total sequence leading to
biologically active fragments. The HP protein according to the invention - depending, both
in respect of the extent and type, on the cell and cell type in which it is expressed- can be in
glycosylated or non-glycosylated form. Polypeptides with tumoricidic and/or metastatic
activity can easily be identified by a tumor progression inhibition assay using carcinoma
cells expressing said polypeptides and measuring the proliferation capacity and apoptosis in
relation to carcinoma cells not expressing said polypeptides.
"Polypeptide with IlP—10 activity or lIP-10" therefore means proteins with minor amino
acid variations but with substantially the same activity as IIP-10. Substantially the same
means that the activities are of the same biological properties and the polypeptides show at
least 75% homology (identity) in amino acid sequences with llP-10. More preferably, the
amino acid sequences are at least 90% identical. Homology according to the invention can
be determined with the aid of the computer programs Gap or BestFit (University of
Wisconsin; Needleman and Wunsch, J. Biol. Chem. 48 (1970) 443-453; Smith and
Waterman, Adv. Appl. Math. 2 (1981) 482-489).
- 5 _
Other llPs according to, and used by, the invention are in particular:
IIP-1
A cDNA encoding an IGF—1 receptor interacting protein which was named IIP-1
(SEQ ID NO:1) was isolated. The cDNA of IIP-1 codes for a new protein of 333 aa with a
calculated molecular weight of 35,727. IIP-1 is a glycine rich protein (13%). I1P—1 contains
several N—myristoylati0n sites, PKC and Cl<2 phosphorylation sites and two putative
nuclear localization signals. A second isoform, IIP-1 (p26), of 236 aa in length with a
calculated molecular weight of 26,071 was identified which was generated most likely by
alternative splicing (Fig. 3). Both isoforms bind to the IGF-1 receptor.
cDNA sequences of IIP-1 have been reported previously (Database EMBL Nos. AF089818
and AFO61263; DeVries, L., et al., Proc. Natl. Acad. Sci. USA 95 (1998) 12340-12345). Two
overlapping cDNA clones (Fig. 4) were identified which show high homology to the human
TIP-2 partial cDNA (GenBank accession number: AF028824) (Rousset, R., et al., Oncogene
16 (1998) 643-654) and were designated as IIP—1a and IIP-lb. The IIP—1a cDNA
corresponds to nt 117 to 751 of Tll?-2. The IIP—1b cDNA shows besides TIP-2 sequences
(nt 1 to 106) additional 5’ sequences which are homologous to sequence Y2H35 of WO
97/27296 (nt 25 to 158).
IIP-1a and IIP-lb both share the sequence coding for the PDZ domain of TIP-2 (nt 156 to
410) which is a known protein-protein interaction domain (Ponting, C.P., et al., Biolissays
19 (1997) 469-479). By deletion analysis the PDZ domain was determined as the essential
and sufficient IGF_-1 receptorpbinding domain of IIP-1 (Fig. 4).
Further yeast two—hybrid analysis revealed that binding of the IIP—1 protein to the IGF-1
receptor is specific for this receptor tyrosine kinase. No interaction was seen to the insulin
receptor or Ros. Receptor tyrosine kinases of other families did not interact with I1P—1 (e.g.
Met, Ret, Kit, Frns, Neu, EGF receptor). Thus, HP-1 most likely is the first interaction
protein shown to be specific for the IGF-1 receptor tyrosine kinase. I1P—1 also binds to the
kinase inactive mutant of the IGF-1 receptor.
' up-2
HP-2 was identified as a new binder of the cytoplasmic part of the IGF-1 receptor which
corresponds to human APS (EMBL accession number: HSAB520). APS has been previously
isolated in a yeast two-hybrid screen using the oncogenic c-kit kinase domain as bait
(Yokouchi, M., et al., Oncogene 15 (1998) 7-15). IIP-2 interacts with the IGF-1 receptor in
a kinase dependent manner. Binding of I1P—2 was also observed to other members of the
insulin receptor family (insulin receptor, Ros), but not to an unrelated receptor tyrosine
kinase (Met). The region of IIP—2 which was found to interact with the IGF-1 receptor
corresponds to human APS (nt 1126 to 1674, EMBL Acc No. AB000520) contains the SH2
domain of APS (nt 1249 to 1545).
IIP—3
lIP—3 was isolated as a new IGF-1 receptor interacting protein and is identical to PSM
(GenBank accession number: AF020526). PSM is known as a PH and SH2 domain
containing signal transduction protein which binds to the activated insulin receptor
(Riedel, H., et al., I. Biochem. 122 (1997) 1105-1113). A variant of PSM has also been
described (Riedel, H., et al., J. Biochem. 122 (1997) 1105-1113). Binding of IIP-3 to the
IGF-1 receptor is dependent on tyrosyl phosphorylation of the receptor.
A CDNA clone corresponding to nt 1862 to 2184 of the variant form of PSM was identified.
The isolated CDNA clone turned out to code for the IGF-1 receptor binding region. The
SH2 domain of PSM (nt 1864 to 2148, EMBL Acc No. AF020526) is encoded by the
sequence of the IIP-3 partial cDNA clone isolated.
IIP-4
HP-4 was isolated as a new interacting protein of the cytoplasmic domain of the lGF—l
receptor. HP-4 corresponds to p59fyn, a src-like tyrosine kinase (EMBL accession number:
MMU70324 and human fyn EM_HUM1:HS66I-I14) (Cooke, M.P., and Perlmutter, R.M.,
New Biol. 1 (1989) 66-74). HP-4 binds in a kinase dependent manner to the IGF-1 receptor
and to several other receptor tyrosine kinases as to the insulin receptor and Met. The region
of IIP-4 interacting with the IGF-1 receptor (nt 665 to 1044) contains the SH2 domain of
p59fyn (EMBL Acc No. U70324).
HP-5
IIP-5 was isolated as a new IGF-1 receptor interacting protein. IlP—5 shows a high
homology to the zinc finger protein Zfp38 (EMBL accession number: MMZFPTA) and is at
least 80%) homologous to the corresponding human gene. Zfp-38 is known as a
transcription factor (Chowdhury, K., et al., Mech. Dev. 39 (1992) 129-142). IIP-5 interacts
exclusively with the activated and phosphorylated IGF-1 receptor, but not with a kinase
inactive mutant. In addition to binding of lIP—5 to the IGF-1 receptor interaction of HP-5
with receptor tyrosine kinases of the insulin receptor family (insulin receptor, Ros) was
observed. IIP-5 does not bind to the more distantly related receptor tyrosine kinase Met.
One cDNA clone binding to the IGF-1 receptor which codes for nt 756 to 1194 of Zfp38
(EMBL Acc No. MMZFPTA) and contains the first zinc finger (nt 1075 to 1158) was
isolated. This domain is sufficient for binding to the activated IGF-1 receptor.
IIP—6
IIP—6 was identified as a new IGF-1 receptor interacting protein. IIP—6 shows weak
similarity to the zinc finger domain of Zfp29 (EMBL accession number: MMZFP29). Zfp29
consists of a N—terminal transcriptional activation domain and 14 C—terminal Cys2His2 zinc
fingers (Denny, P., and Ashworth, A., Gene 106 (1991) 221-227). Binding of IIP—6 to the
IGF-1 receptor depends on phosphorylation of the IGF-1 receptor kinase. IIP—6 also binds
to the insulin receptor, but does not interact with Met. The region of IIP—6 found to
interact with the IGF-1 receptor (SEQ ID NO:3, SEQ ID NO:4) contains two zinc finger
domains of the Cys2His2 type.
IIP-7
IIP-7 was isolated as a new IGF-1 receptor interacting protein which corresponds to Pax-3
(EMBL accession number: MMPAX3R and human Pax3 EM-HUM2:S69369). Pax-3 is
known as a DNA-binding protein being expressed during early embryogenesis (Goulding,
M.D., et al., EMBO I. 10 (1991) 1135-1147). IIP-7 binds in a phosphorylation dependent
manner to the IGF-1 receptor. IIP-7 also interacts with the insulin receptor and Met. A
partial HP-7 CDNA clone turned out to code for the IGF-1 receptor binding domain of
Pax3 (nt 815 to 1199, EMBL Acc No. MMPAX3R). This region contains the Pax-3 paired
damain octapeptide (nt 853 to 876) and the paired-type homeodomain (nt 952 to 1134).
IIP-8
llP—8 codes for the full—length cDNA of Grb7 (EMBL accession number: MMGRB7P,
human Grb7 EM_HUM1:AB008789). Grb7, a PH domain and a SH3 domain containg
signal transduction protein, was first published as an EGF receptor—binding protein
(Margolis, B.L., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 8894-8898). HP-8 does not
interact with the kinase inactive mutant of the lGF—1 receptor. Binding of HP-8 to several
other receptor tyrosine kinases (e.g. insulin receptor, Ros and Met) was also observed.
IIP-9
HP-9 was identified as a new IGF-1 receptor interaction protein. 1113-9 is identical to nck-
beta (EMBL Acc No. AF043260). Nck is a cytoplasmic signal transduction protein
consisting of SH2 and SH3 domains (Lehmann,-).M., et al., Nucleic Acids Res. 18 (1990)
1048). lIP~9 interacts with the lGF—1 receptor in a phosphorylation dependent manner. nck
binds to the juxtamembrane region of the IGF-1 receptor. Apart from binding of llP—9 to
the lGF~1 receptor, interaction with the insulin receptor but not with Ros or Met was seen.
A preferred object of the invention are polypeptides that are homologous, and more
preferably, polypeptides that are substantially identical to the polypeptides of SEQ ID NO:6
(lIP—10). Homology can be examined by using the FastA algorithm described by Pearson,
W.R., Methods in Enzymology 183 (1990) 63-68, Academic Press, San Diego, US. By
,,substantially identical“ is meant an amino acid sequence which differs only by
conservative amino acid substitutions, for example substitutions of one amino acid for
another of the same class (e.g. valine for glycine, arginine for lysine, etc.) or by one or more
non-conservative amino acid substitution, deletions or insertions located at positions of the
amino acid sequence which do not destroy the biological function of the polypeptide. This
includes substitution of alternative covalent peptide bonds in the polypeptide. By
,,polypeptide“ is meant any chain of amino acids regardless of length or posttranslational
modification (e.g., glycosylation or phosphorylation) and can be used interchangeably with
the term ,,protein“.
According to the invention by ,,biologically active fragment“ is meant a fragment which can
exert a physiological effect of the full—length naturally—occurring protein (e.g., binding to its
biological substrate, causing an antigenic response, etc.). Such fragments can be
antigenic fragments.
The term ,,antigenic“ as used herein refers to fragments of the protein which
can induce a specific immunogenic response, e.g. an immunogenic response which yields
antibodies which specifically bind to the protein according to the invention. The fragments
are preferably at least 8 amino acids, and preferably up to 25 amino acids, in length. In one
preferred embodiment, the fragments include the domain which is responsible for the
binding of the HPS to the IGF-1 receptor (i.e., the PDZ domain of HP-1. By ,,domain“ is
meant the region of amino acids in a protein directly involved in the interaction with its
binding partner. PDZ domains are approximately 90—residue repeats found in a number of
proteins implicated in ion-, channel and receptor clustering and the linking of receptors to
effector enzymes. Such PDZ are described in general by Cabral, I.H., et al., Nature 382
(1996) 649-652.
The invention further comprises a method for producing a protein according to the
invention whose expression or activation is correlated with tumor proliferation, by
expressing an exogenous DNA in prokaryotic or eukaryotic host cells and isolation of the
desired protein or expressing said exogeneous DNA in vivo for pharmaceutical means,
wherein the protein is coded preferably by a DNA sequence coding for HP-10, preferably
the DNA sequence shown in SEQ ID NO:5.
The polypeptides according to the invention can also be produced by recombinant means,
or synthetically. Non-glycosylated IIP-10 polypeptide is obtained when it is produced
recombinantly in prokaryotes. With the aid of the nucleic acid sequences provided by the
invention it is possible to search for the IIP-10 gene or its variants in genomes of any
desired cells (e.g. apart from human cells, also in cells of other mammals), to identify these
and to isolate the desired gene coding for the HP-10 protein. Such processes and suitable
hybridization conditions (see also above, ,,stringent conditions“) are known to a person
skilled in the art and are described, for example, by Sambrook et al., Molecular Cloning: A
laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA, and
Hames, B.D., Higgins, S.G., Nucleic acid hybridisation - a practical approach (1985) IRL
Press, Oxford, England. In this case the standard protocols described in these publications
are usually used for the experiments.
The use of recombinant DNA technology enables the production of numerous active
IIP-10 derivatives. Such derivatives can, for example, be modified in individual or several
amino acids by substitution, deletion or addition. The derivatization can, for example, be
carried out by means of site directed mutagenesis. Such variations can be easily carried out
by a person skilled in the art (I. Sambrook, B.D. Hames, loc. cit.). It merely has to be
ensured by means of the below-mentioned tumor cell growth inhibition assay that the
characteristic properties of HP-10 are preserved.
With the aid of such nucleic acids coding for an HP-10 protein, the protein according to
the invention can be obtained in a reproducible manner and in large amounts. For
expression in prokaryotic or eukaryotic organisms, such as prokaryotic host cells or
eukaryotic host cells, the nucleic acid is integrated into suitable expression vectors,
according to methods familiar to a person skilled in the art. Such an expression vector
preferably contains a regulatable/inducible promoter. These recombinant vectors are then
introduced for the expression into suitable host cells such as, e.g., E. coli as a prokaryotic
host cell or Saccharomyces cerevisiae, teratocarcinoma cell line PA-1 sc 9117 (Biittner et al.,
Mol. Cell. Biol. 11 (1991) 3573-3583), insect cells, CHO or COS cells as eukaryotic host
cells and the transformed or transduced host cells are cultured under conditions which
allow expression of the heterologous gene. The isolation of the protein can be carried out
according to known methods from the host cell or from the culture supernatant of the host
cell. Such methods are described for example by Ausubel 1., Frederick M., Current
Protocols in Mol. Biol. (1992), John Wiley and Sons, New York. Also in vitro reactivation
of the protein may be necessary if it is not found in soluble form in the cell culture.
The protein can be isolated from the cells or the culture supernatant and purified by
chromatographic means, preferably by ion exchange chromatography, affinity
chromatography and/or reverse phase HPLC.
HP-10 can be purified after recombinant production by affinity chromatography using
known protein purification techniques, including immunoprecipitation, gel filtration, ion
exchange chromatography, isoelectric selective
chromatofocussing, focussing,
precipitation, electrophoresis, or the like.
Diagnostic methods:
The invention further comprises a method for detecting a nucleic acid molecule encoding
an IIP-gene, comprising incubating a sample (e.g., body fluids such as blood, cell lysates)
with a nucleic acid molecule according to the invention and determining hybridization
under stringent conditions of said nucleic acid molecule to a target nucleic acid molecule
for determination of presence of a nucleic acid molecule which is said IIP gene and
therefore a method for the identification of IGF-1R activation or inhibition in mammalian
cells or body fluids.
Therefore the invention also includes a method for the detection of the proliferation
potential of a tumor cell comprising
a) incubating a sample of body fluid of a patient suffering from cancer, a sample of
cancer cells, or a sample of a cell extract or a cell culture supernatant of said cancer
cells, whereby said sample contains nucleic acids with a nucleic acid probe which is
selected from the group consisting of
(i) the nucleic acids shown in SEQ ID NOS:1, 3 or 5 or a nucleic acid which is
complementary thereto and g
(ii) nucleic acids which hybridize under stringent conditions with one of the
nucleic acids from (i) and
b) detecting hybridization by means of a further binding partner of the nucleic acid of
the sample and/or the nucleic acid probe or by X-ray radiography.
Hybridization between the probe and nucleic acids from the sample indicates the presence
of the RNA of such proteins. Such methods are known to a person skilled in the art and are
described, for example, in W0 89/06698, EP-A 0 200 362, USP 2915082, EP-A O 063 879,
EP-A 0173 251, EP-A 0128 018.
In a preferred embodiment of the invention the coding nucleic acid of the sample is
amplified before the test, for example by means of the known PCR technique. Usually a
derivatized (labeled) nucleic acid probe is used within the framework of nucleic acid
diagnostics. This probe is contacted with a denatured DNA or RNA from the sample which
is bound to a carrier and in this process the temperature, ionic strength, pH and other
buffer conditions are selected - depending on the length and composition of the nucleic
acid probe and the resulting melting temperature of the expected hybrid - such that the
labeled DNA or RNA can bind to homologous DNA or RNA (hybridization see also Wahl,
G.M., et al., Proc. Natl. Acad. Sci. USA 76 (1979) 3683-3687).
membranes or carrier materials based on nitrocellulose (e.g., Schleicher and Schtill, BA 85,
Suitable carriers are
Amersham Hybond, C.), strengthened or bound nitrocellulose in powder form or nylon
membranes derivatized with various functional groups (e.g., nitro groups) (e.g., Schleicher
and Schiill, Nytran; NEN, Gene Screen; Amersham Hybond M.; Pall Biodyne).
Hybridizing DNA or RNA is then detected by incubating the carrier with an antibody or
antibody fragment after thorough washing and saturation to prevent unspecific binding.
The antibody or the antibody fragment is directed towards the substance incorporated
‘during derivatization into the nucleic acid probe. The antibody is in turn labeled. However,
it is also possible to use a directly labeled DNA. After incubation with the antibodies it is
washed again in order to only detect specifically bound antibody conjugates. The
determination is then carried out according to known methods by means of the label on
the antibody or the antibody fragment.
The detection of the expression can be carried out for example as:
in situ hybridization with fixed whole cells, with fixed tissue smears and isolated
metaphase chromosomes, H
— colony hybridization (cells) and plaque hybridization (phages and viruses),
— Southern hybridization (DNA detection),
— Northern hybridization (RNA detection),
— serum analysis (e.g., cell type analysis of cells in the serum by slot—blot analysis),
— after amplification (e.g., PCR technique).
Preferably the nucleic acid probe is incubated with the nucleic acid of the sample and the
hybridization is detected optionally by means of a further binding partner for the nucleic
acid of the sample and/or the nucleic acid probe.
The nucleic acids according to the invention are hence valuable prognostic markers in the
diagnosis of the metastatic and progression potential of tumor cells of a patient.
Screening for antagonists and agonists of IIPs or inhibitors
According to the invention antagonists of IIP—1O or inhibitors for the expression of HP
(e.g., antisense nucleic acids) can be used to inhibit tumor progression and cause massive
apoptosis of tumor cells in vivo, preferably by somatic gene therapy.
Therefore, the present invention also relates to methods of screening for potential
therapeutics for cancer, diabetes, neurodegenerative disorders, bone diseases, to methods of
treatment for disease and to cell lines and animal models useful in screening for and
evaluating potentially useful therapies for such disease. Therefore another object of the
invention are methods for identifying compounds which have utility in the treatment of
the afore-mentioned and related disorders. These methods include methods for
modulating the expression of the polypeptides according to the invention, methods for
identifying compounds which can selectively bind to the proteins according to the
invention and methods of identifying compounds which can modulate the activity of said
polypeptides. These methods may be conducted in vitro and in vivo and may employ the
transformed cell lines and transgenic animal models of the invention.
An antagonist of IIPs or an inhibitor of HP is defined as a substance or compound which
inhibits the interaction between IGF-1R and HP, preferably IIP-10. Therefore the biological
activity of IGF-IR decreases in the presence of such a compound. In general, screening
procedures for IIP antagonists involve contacting candidate substances with lIP—bearing
host cells under conditions favorable for binding and measuring the extent of decreasing
receptor mediated signaling (in the case of an antagonist). Such an antagonist is useful as a
pharmaceutical agent for use in tumor therapy. For the treatment of diabetes, neural
diseases, or bone diseases, stimulation of the signaling pathway is required, i.e., screening
for agonists is useful.
HP activation may be measured in several ways. Typically, the activation is apparent by a
change in cell physiology such as an increase or decrease in growth rate or by a change in
the differentiation state or by a change in cell metabolism which can be detected in
standard cell assays, for example MTT or XTT assays (Roche Diagnostics GmbH, DE).
The nucleic acids and proteins according to the invention could therefore also be used to
identify and design drugs which interfere with the interaction of IGF-1R and IlPs. For
instance, a drug that interacts with one of the proteins could preferentially bind it instead
of allowing binding its natural counterpart. Any drug which could bind to the IGF-1
receptor and, thereby, prevent binding of an IIP or, vice versa, bind to an IIP and, thereby,
prevent binding of the IGF-.1 receptor. In both cases, signal transduction of the IGF-1
receptor system would be modulated (preferably inhibited). Screening drugs for this facility
occurs by establishing a competitive assay (assay standard in the art) between the test
compound and interaction of HP and the IGF-1 receptor and using purified protein or
fragments with the same properties as the binding partners.
The protein according to the invention is suitable for use in an assay procedure for the
identification of compounds which modulate the activity of the proteins according to the
invention. Modulating the activity as described herein includes the inhibition or activation
of the protein and includes directly or indirectly affecting the normal regulation of said
protein activity. Compounds which modulate the protein activity include agonists,
antagonists and compounds which directly or indirectly affect the regulation of the activity
of the protein according to the invention. The protein according to the invention may be
obtained from both native and recombinant sources for use in an assay procedure to
identify modulators. In general, an assay procedure to identify modulators will contain the
IGF receptor, a protein of the present invention, and a test compound or sample which
contains a putative modulator of said protein activity. The test compounds or samples may
be tested directly on, for example, purified protein of the invention, whether native or
recombinant, subcellular fractions of cells producing said protein, whether native or
recombinant, and/or whole cells expressing said protein, whether native or recombinant.
The test compound or sample may be added to the protein according to the invention in
the presence or absence of known modulators of said protein. The modulating activityiof
the test compound or sample may be determined by, for example, analyzing the ability of
the test compound or sample to bind to said protein, activate said protein, inhibit its
activity, inhibit or enhance the binding of other compounds to said protein, modifying
receptor regulation or modifying intracellular activity.
The identification of modulators of the protein activity are useful in treating disease states
involving the protein activity. Other compounds may be useful for stimulating or
inhibiting the activity of the protein according to the invention. Such compounds could be
of use in the treatment of diseases in which activation or inactivation of the protein
according to the invention results in either cellular proliferation, cell death, non-
proliferation, induction of cellular neoplastic transformations, or metastatic tumor growth
and hence could be used in the prevention and/or treatment of cancers such as, for
example, prostate and breast cancer. The isolation and purification of a DNA molecule
encoding the protein according to the invention would be useful for establishing the tissue
distribution of said protein as well as establishing a process for identifying compounds
which modulate the activity of said protein and/or its expression.
Therefore a further embodiment of the invention is a method for screening a compound
that inhibits the interaction between lGF—1R and IIP-1 or HP-10, comprising
a) combining IGF-1R and IIP-1 or IIP-10 polypeptide with a solution containing a
candidate compound such that the lGF—1R and HP-l or IIP-10 polypeptide are
capable of forming a complex and '
b) determining the amount of complex relative to the predetermined level of binding in
the absence of said candidate compound and therefrom evaluating the ability of said
candidate compound to inhibit binding of IGF-1R to HP-1 or —llP-10 polypeptide.
Such a screening assay is preferably performed as an ELISA assay whereby IGF-1R or IIP-1
or IIP-I0 is bound on a solid phase.
A further embodiment of the invention is a method for the production of a therapeutic
agent for the treatment of carcinomas in a patient comprising combining a therapeutically
effective amount of a compound which inhibits the interaction between IGF-IR and HP in
biochemical and/or cellular assays to an extent of at least 50%. Biochemical assays are
preferably ELISA-based assays or homogeneous assays. In the case of the ELISA system
antibodies specific 172‘ the two binding partners are used for detection of the complexes. In
the case of the homogenous assay at least one binding partner is labeled with tluorophores
which allows analysis of the complexes. Cellular assays are preferably assays whereby tumor
cells or cells transfected with expression constructs of the IGF-IR and the respective
binding proteins are treated with or without drugs and complex formation between the two
components is then analyzed using standard cell assays.
A preferred embodiment of the invention is a method for the production of a therapeutic
agent for the treatment of carcinomas in a patient combining a
pharmaceutically acceptable carrier with a therapeutically effective amount of a compound
which inhibits the interaction between IGF—1R and IIP~I or lIP—10 in a cellular assay,
whereby in said cellular assay tumor cells or cells transfected with expression constructs of
IGF-1R and of the respective HP are treated with said compound, and complex formation
between lGF—lR and said respective HP is analyzed, and the extent of said complex
formation in the case of inhibition does not exceed 50% referred to 100% for complex
formation without said compound in said same cellular assay.
Using the methods of the invention it is possible to produce a compound
for use in a method of treating a patient suffering from a carcinoma with
a therapeutically effective amount of a compound which inhibits the
interaction between lGF—1R and IIP-I or IIP—10 in a cellular assay, whereby in said cellular
assay tumor cells or cells transfected with expression constructs of IGF-1R and of the
respective IIP are treated with said compound, and complex formation between IGF-IR
and said respective IIP is analyzed, and the extent of said complex formation in the case of
inhibition does not exceed 50% referred to 100% for complex formation without said
compound in said same cellular assay.
A further embodiment of the invention is an antibody against IIP—1 or IIP-10 according to
the invention.
Antibodies were generated from the human, mouse, or rat polypeptides. Antibodies
specifically recognizing IIP-1 or llP-10 are encompassed by the invention. Such antibodies
are raised using standard immunological techniques. Antibodies may be polyclonal or
monoclonal or may be produced recombinantly such as for a humanized antibody. An
antibody fragment which retains the ability to interact with HP-1 or IIP—10 is also provided.
Such a fragment can be produced by proteolytic cleavage of a full-length antibody or
produced by recombinant DNA procedures. Antibodies of the invention are useful in
diagnostic and therapeutic applications. They are used to detect and quantitate llP—1 or
HP-10 in biological samples, particularly tissue samples and body fluids. They are also used
to modulate the activity of I1P—1 or IlP—10 by acting as an agonist or an antagonist.
The following examples, references, sequence listing and drawing are provided to aid the
understanding of the present invention, the true scope of which is set forth in the appended
claims. It is understood that modifications can be made in the procedures set forth without
departing from the spirit of the invention.
Description of the Figures and Sequences
Figure 1 Domain structure of yeast two-hybrid baits which were used to screen
CDNA libraries for cytoplasmic binding proteins of the IGF-1 receptor.
The LexA DNA binding domain was fused to the cytoplasmic (cp)
domain (nt 2923 to 4154) of the wildtype IGF-1 receptor (a) or the kinase
inactive mutant (K/A mutation at aa 1003) (b) (Ullrich, A., et al,, EMBO
I. 5 (1986) 2503-2512; Weidner, M., et al., Nature 384 (1996) 173-176).
The nucleotide and amino acid sequence of two different linkers inserted
between the LexA DNA-binding domain and the receptor domain are
shown below. The 11 (wt IGF-1 receptor) and K1 (kinase inactive mutant
IGF-1 receptor) constructs contain an additional proline and glycine
compared to the 12 and K2 constructs.
Modification of the yeast two-hybrid LexA/IGF—1 receptor bait construct.
a) Schematic illustration of cytoplasmic binding sites of the IGF-1
Figure 2
receptor. The on-subunits of the IGF-1 receptor are linked to the B-chains
via disulfid bonds. The cytoplasmic part of the B-chain contains binding
sites for substrates in the juxtamembrane and C—terminal domain.
b) Domain structure of the two-hybrid bait containing only the
juxtamembrane IGF-1 receptor binding sites. The juxtamembrane
Figure 3
Figure 4
domain of the IGF—1 receptor (nt 2923 to 3051) (Ullrich, A., et al., EMBO
J. 5 (1986) 2503-2512) was fused to the kinase domain oftprmet (nt 3456
to 4229) (GenBank accession number: HSU19348).
c) Domain structure of the two-hybrid bait containing only the C-
terminal IGF-1 receptor binding sites. The C-terminal domain of the
IGF-1 receptor (nt 3823 to 4149) (Ullrich, A., et al., EMBO I. 5 (1986)
2503-2512) was fused to the kinase domain of tprmet (nt 3456 to 4229)
(GenBank accession number: HSU19348).
lsoforms of IIP-1.
a) Delineation of the CDNA sequences of HP-1 and IlP—1 (p26).
Nucleotides are numbered above. The potential translation initiation site
within the IIP-1 CDNA is at position 63. The first ATG as potential
translation initiation site in the alternative splice variant HP-1 (p26) is at
position 353. Both cDNAs contain a stop codon at position 1062.
b) Domain structure of HP-1 and IIP—1 (p26). Amino acid positions are
indicated above. In comparison to IIP-1 (p26) IIP-1 contain additional
97 amino acids at the N-terminus. Both isoforms of IIP-1 contain a PDZ
domain spanning a region between amino acids 129 and 213.
Delineation of the IGF—1 receptor binding domain of IIP—1.
Full-length HP-1, its partial cDNA clones (IIP-la and HP-lb) and
deletion mutants (HP—1a/mul, IlP—1a/mu2, HP-1a/mu3, HP-1b/mul)
were examined for interaction with the IGF-1 receptor in the yeast two-
hybrid system. Yeast cells were cotransfected with a LexA IGF-1 receptor
fusion construct and an activation plasmid coding for HP-1 or the
different HP-1 mutants fused to the VP16 activation domain. Interaction
between HP-1 or its mutants and the IGF-1 receptor was analyzed by
monitoring growth of yeast transfectants plated out on histidine deficient
medium and incubated for 6d at 30°C (diameter of yeast colonies: +++, >
1 mm in 2d; ++, > 1 mm in 4d; +, > 1 mm in 6d; -, no detected growth).
The PDZ domain can be defined as essential and sufficient for mediating
the interaction with the IGF—1 receptor. Nucleotide positions with respect
to full length IIP-1 are indicated above.
Figure 5
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
SEQ ID NO:4
SEQ ID NO:5
SEQ ID NO:6
SEQ ID NO:7
SEQ ID N028
SEQ ID NO:9
SEQ ID NO:10
Example 1
_ 19 -
Protein sequence motifs of IIP—10.
The amino acid sequence of IIP-10 was analyzed using the computer
program ,,Motifs“ which looks for protein motifs by searching protein
sequences for regular expression patterns described in the PROSITE
Dictionary.
Nucleotide sequence of IIP—1 (CDNA).
Predicted amino acid sequence of IIP-1.
Nucleotide sequence of the IIP-6 partial CDNA clone.
Deduced amino acid sequence of the IIP-6 partial CDNA clone. Cysteine
and histidine residues of the two Cys2His2 Zinc finger domains are amino
acids 72, 75, 88, 92, 100, 103, I16, and 120.
Nucleotide sequence of IIP-IO (CDNA).
Deduced amino acid sequence of IIP—l0.
Primer TIP2c-s.
Primer TIP2b-r.
Primer Hcthy-s.
Primer I-Icthy-r.
Isolation and characterization of IGF— 1R binding proteins
' The yeast two—hybrid system (Fields, 8., and Song, 0., Nature 340 (1989) 245-246) was
used to isolate unknown cytosolic IGF—1 receptor binding proteins. For screening a
modified version of the yeast two-hybrid system was used which allows interchain
tyrosylphosphorylation of the receptors in yeast.
The yeast two-hybrid bait plasmid (BTM116—cp1GF-1 receptor) was constructed by fusing
the cytoplasmic domain of the B-subunit of the IGF-1 receptor (nt 2923 to 4154) (Ullrich,
A., et a1., EMBO J. 5 (1986) 2503-2512) to the LexA DNA—binding domain which forms
dimers and mimics the situation of the activated wildtype receptor (cf. Weidner, M., et al.,
Nature 384 (1996) 173-176). By introducing a proline-glycine spacer between the LexA
DNA—binding domain and the receptor domain the ability of the bait to bind known
substrates of the 1GF—1 receptor was remarkably increased in comparision to other spacer
amino acids (Fig. 1).
Alternatively a bait was constructed containing only the juxtamembrane or C—terminal
region of the IGF—1 receptor (nt 2923 to 3051 or nt 3823 to 4146) (Ullrich, A., et al., EMBO
J. 5 (1986) 2503-2512) fused to the kinase domain of an unrelated, very potential receptor
tyrosine kinase. Here the kinase domain of tpr met (nt 3456 to 4229) (GenBank accession
number: HSU19348) (Fig. 2) was used. In this way it is possible to delineate the region of
the IGF—1 receptor which mediates binding to downstream effectors.
The lGF—1 receptor bait plasmid was used to screen activation domain cDNA libraries (e.g.
VP16- or Gal4 based activation domain) (cf. Weidner, M., et al., Nature 384 (1996)
173-176). The bait and prey plasmids were co-transfected into Saccharomyces cerevisiae
strain L40 containing a HIS3 and lacZ reporter gene. Library plasmids were isolated from
yeast colonies growing on histidine deficient medium, were sequenced and reintroduced
into yeast strain L40. By co-transfecting experiments with different test baits, i.e. BTM116
plasmids coding for a kinase inactive mutant of the IGF-1 receptor (LlO33A) or the
cytoplasmic domain of receptor tyrosine kinases of the insulin receptor family (insulin
receptor, Ros) and of unrelated receptor tyrosine kinase families (Met, EGF receptor, Kit,
Fms, Neu) the specificity of the putative bait-prey interactions was evaluated. Several
cDNAs were identified which code for previously unknown IGF-1 receptor interacting
proteins (1IPs). In addition binding domains of known substrates of the 1GF-1 receptor
such as the C-terminal SH2 domain of p85P13K and the SH2 domain of Grb10 were found.
The results are shown in Table 1.
&1
IIP wt IGF-IR mu IGF-1R IR Ros Met
IIP—1 + + — — —
HP-2 + — + + —
HP-3 + — +
IIP—4 + — + nd
HP-5 + —- + + -
IIP-6 + — + nd —
HP-7 + — + nd
IIP-8 + — + +
IIP—9 + ~ + — —
IIP-10 + - — nd nd
Delineation of the binding specificity of the lIPs with respect to different receptor tyrosine
kinases tested in the yeast two-hybrid system. Yeast cells were cotransfected with a LexA
fusion construct coding for the different receptor tyrosine kinases and an activation
plasmid coding for the different lIPs fused to the VP16 activation domain. Interaction
between the IlPs and the different receptor tyrosine kinases was analyzed by monitoring
growth of yeast transfectants plated out on histidine deficient medium and incubated for
3d at 30°C (wt lGF—1R, kinase active IGF-1 receptor; mu IGF—1 R, kinase inactive mutant
lGF—1 receptor; IR, insulin receptor; Ros, Ros receptor tyrosine kinase; Met, Met receptor
tyrosine kinase; +, growth of yeast transfectants within 3 days larger than 1mm in diameter;
-, no detected growth; nd, not determined).
Example 2
Assay systems:
A) In-vitro/biochernical assays:
ELISA-based assay/homogenous assay
IGF-1R and the binding proteins (HPs) are expressed with or without Tag—enzymes in
E.coli or eucaryotic cells and purified to homogeneity. Interaction of IGF-1R and the
respective binding proteins are analyzed in the presence or absence of drugs. Compounds
which either inhibit or promote binding of IGF-1R and the respective binding proteins are
selected. In the case of the ELISA system antibodies specific for the two binding partners
are used for detection of the complexes. In the case of the homogenous assay at least one
binding partner is labeled with fluorophores which allows analysis of the complexes.
Alternatively, anti-Tag-antibodies are used to monitor interaction.
B) Cellular assays:
Tumor cells or cells transfected with expression constructs of the IGF—1R and the respective
binding proteins are treated with or without drugs and complex formation between the two
components is then analyzed using standard assays.
Example 3
cDNA cloning of IIP—1 and IIP-10 (and RT-PCR-assay)
The nucleotide sequence of full length IIP-1 was aligned using database information (ESTs)
and sequences of the partial cDNA clones of IlP—1 (IIP-1a, IIP-lb). cDNA cloning of full
length IIP-1 was performed by RT PCR on total RNA isolated from a MCF7ADRbreast cell
line. PT PCR with two oligonucleotide primers: TlP2c-s (SEQ ID NO:7) and TlP2b—r
(SEQ ID NO:8) resulted in amplification of two DNA fragments of 1.0 kb (HP-1) and
0.7 kb (HP-1(p26)).
The nucleotide sequence of full length IIP-10 was aligned using database information
(ESTs) and the sequence of the partial cDNA clone of HP-10. cDNA cloning of IIP-10 was
performed on total RNA isolated from the colon cancer cell line SW480. RT PCR with two
oligonucleotide primers: Hcthy-s (SEQ ID NO:9) and Hcthy-r (SEQ ID NO:10) resulted in
amplification of a cDNA fragment of 676bp (IlP—10).
DNA sequencing was performed using the dideoxynucleotide chain termination method
on an ABI 373A sequencer using the Ampli Taqo FS Dideoxyterminator kit (Perkin Elmer,
Foster City, CA). Comparison of the cDNA and deduced protein sequences was performed
using Advanced Blast Search (Altschul, S.F., et al., ]. Mol. Biol. 215 (1990) 403-410;
Altschul, S.F., et al., Nucleic Acids Res. 25 (1997) 3389-3402).
Example 4
Western blot analysis of 1113-1 and IIP-10
Total cell lysates were prepared in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl,
1% NP40, 0.5 % deoxycholic acid, 0.1 % SDS, and 1 mM EDTA pH and cleared by
centrifugation for 15 min at 4° C. The protein concentration of the supernatants was
measured using the Micro BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL)
according to the manufacturer's manual. IGF-1 receptors were immunoprecipitated using
anti-IGF-1 receptor antibodies (Santa Cruz). Proteins were fractionated by SDS-PAGE and
electrophoretically transferred to nitrocellulose filters. Nitrocellulose filters were
preincubated with 10 % (w/v) fat-free milk powder in 20 mM Tris pH 7.5, 150 mM NaCl,
0.2% Tween*—20. Binding of a mouse monoclonal antibody directed against the flag epitope
was detected by horseradish peroxidase~labeled goat-anti-mouse lgG antiserum (Biorad,
Munich, DE) and visualized using an enhanced chemoluminescence detection system,
ECU“ (Arnersham, Braunschweig, DE).
Example 5
Overexpression of IIP-1 to IIP-10 in mammalian cells by liposome-mediated transfection
The cDNAs for IIP—1 to -10 were cloned into the NotI site of pBATflag or pcDNA3flag
(Weidner, M., et al., Nature 384 (1996) 173~l76; Behrens, 1., et al., Nature 382 (1996) 638-
642; Behrens, I., et al., Science 280 (1998) 596-599). NIH3T3 cells or other recipient cells
were transfected with pcDNAflagllP-1 to -10 or alternatively with pBATflag IIP-1 to -10
using PuGENE6 (Roche Biochemicals) as transfection agent. Cells were selected in
0.4 mg/ml G418. Single clones were picked and analzyed for expression of HP-1 to -10 and
functionally characterized with respect to proliferation.
Northern blot analysis
Human and murine mRNA multiple tissue Northern blots were purchased from Clontech
(Palo Alto, CA, US). A CDNA probe spanning IlP—10 nt343-nt676 of the coding region was
labeled with DIG—dUTP using the PCR DIG Labeling Mix (Roche Diagnostics GmbH, DE).
A digoxygenin labeled actin RNA probe was purchased from Roche Diagnostics GmbH,
DB. Hybridization was performed using the DIG Easy}-lyb hybridization solution (Roche
Diagnostics GmbH, DE), IIP-10 mRNA was detected with DIG-specific antibodies
conjugated to alkaline phosphatase and the CSPD substrate (Roche Diagnostics Gmbl-l,
DE).
* Trade Mark
Example 6
Detection of mRNA in cancer cells
In order to detect Whether proteins are expressed in cancer cells which are coded by nucleic
acids which hybridize with SEQ ID NO:1 or SEQ ID N025 or the complementary sequence
and consequently whether mRNA is present, it is possible on the one hand to carry out the
established methods of nucleic acid hybridization such as Northern hybridization, in—situ
hybridization, dot or slot hybridization and diagnostic techniques derived therefrom
(Sambrook et al., Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor
Laboratory Press, New York, USA; Hames, B.D., Higgins, S.G., Nucleic acid hybridisation -
a practical approach (1985) IRL Press, Oxford, England; W0 89/06698; EP-A0 200 362;
EP—A 0 063 879; EP—A0 173 251; EP—A 0 128 018). On the other hand it is possible to use
methods from the diverse repertoire of amplification techniques using specific primers
(PRC Protocols - A Guide to Methods and Applications (1990), publ. M.A. Innis, D.H.
Gelfand, 1.]. Sninsky, T.]. White, Academic Press Inc.; PCR - A Practical Approach (1991),
publ. M.]. McPherson, P. Quirke, G.R. Taylor, IRL Press).
The RNA for this is isolated from the cancer tissue by the method of Chomcszynski and
Sacchi, Anal. Biochem. 162 (1987) 156-159. 20 pg total RNA was separated on a 1.2%
agarose formaldehyde gel and transferred onto nylon membranes (Amersham,
Braunschweig, DE) by standard methods (Sambrook et al., Molecular Cloning: A
laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA. The
DNA sequence SEQ ID NO:1 or SEQ ID N025 was radioactively labeled as probes
(Feinberg, A.P., and Vogelstein, B., Anal. Biochem. 137 (1984) 266-267). The hybridization
was carried out at 68°C in 5 x SSC, 5 x Denhardt, 7% SDS/0.5 M phosphate buffer pH 7.0,
% dextran sulfate and 100 pg/ml salmon sperm DNA. Subsequently the membranes were
washed twice for one hour each time in 1 x SSC at 68°C and then exposed to X-ray film.
Example 7
Procedure for identification of modulators of the activity of the protein according to the
invention
The expression vector of Example 5 (either for IIP-1 or IIP-10 10 pg/106 cells) is transferred
into NIH 3T3 cells by standard methods known in the art (Sambrook et al.). Cells which
have taken up the vector are identified by their ability to grow in the presence of the
selection or under selective conditions (0.4 mg/ml G418). Cells which express DNA
encoding IIP produce RNA which is detected by Northern blot analysis as described in
Example 5. Alternatively, cells expressing the protein are identified by identification of the
protein by Western blot analysis using the antibodies described in Example 4. Cells which
express the protein from the expression vector will display an altered morphology and/or
enhanced growth properties.
Cells which express the protein and display one or more of the altered properties described
above are cultured with and without a putative modulator compound. By screening of
chemical and natural libraries, such compounds can be identified using high throughput
cellular assays monitoring cell growth (cell proliferation assays using as chromogenic
substrates the tetrazolium salts WST-1, MTT, or XTT, or a cell death detection ELISA using
bromodesoxyuridine (BrdU); cf. Boehringer Mannheim Gmbl-I, Apoptosis and Cell
Proliferation, 2nd edition, 1998, pp. 70-84).
The modulator compound will cause an increase or a decrease in the cellular response to
the HP protein activity and will be either an activator or an inhibitor of lGF—receptor
function, respectively.
Alternatively, putative modulators are added to cultures of tumor cells, and the cells display
an altered morphology and/or display reduced or enhanced growth properties. A putative
modulator compound is added to the cells with and without HP protein and a cellular
response is measured by direct observation of morphological characteristics of the cells
and/or the cells are monitored for their growth properties. The modulator compound will
cause an increase or a decrease in the cellular response to IIP protein and will be either an
activator or an inhibitor of IGF-1 receptor activity, respectively.
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Claims (16)
1. A nucleic acid (IIP—lO) encoding a protein binding to the IGE—l receptor selected from the group comprising a) the nucleic acids shown in SEQ ID NO:5 or a nucleic acid sequence which is complementary thereto, b) nucleic acids which hybridize under stringent conditions with one of the nucleic acids from a) encoding a polypeptide showing at least 75% homology with the polypeptide of SEQ ID NO:6, or c) sequences that due to the degeneracy of the genetic code encode IIP—lO polypeptides having the amino acid sequence of the polypeptides encoded by the sequences of a) and b).
2. A nucleic acid according to claim I, wherein said nucleic acid has a sequence in accordance with SEQ ID NO:5.
3. A recombinant expression vector comprising a nucleic acid molecule as defined in claim 1 suitable for the expression of said nucleic acid molecule.
4. A host cell transformed by a nucleic acid of claims I to 2.
5. A recombinant polypeptide which binds to the 25 lGE—l receptor encoded by a nucleic acid according to claims 1 to 2.
6. A method for the production of a protein which binds to the lGF—l receptor, by expressing an exogenous DNA in prokaryotic or eukaryotic host cells and isolation of the desired protein, wherein the protein is coded by the DNA sequence shown in SEQ ID NQ:5 or a nucleic acid which hybridizes under stringent conditions with a nucleic acid complementary to the nucleic acid shown in SEQ ID NO:5.
7. A method for the detection of the proliferation potential of a cancer cell comprising a) incubating a sample of body fluid of a patient suffering from cancer, of tumor cells, or of a cell extract or a cell culture supernatant of said tumor cells, whereby said sample contains nucleic acids with a nucleic acid probe which is selected from the group consisting of (i) the nucleic acid shown in SEQ ID NOS:l, 3 or 5 or a nucleic acid which is complementary thereto and (ii) nucleic acids which hybridize under stringent conditions with one of the nucleic acids from (i) and b) detecting the hybridization by means of a further binding partner of the nucleic acid of the sample and/or the nucleic acid probe.
8. The method of claim 7, wherein hybridization is effected at least with the nucleic acid fragment of SEQ ID NO:l or SEQ ID N025 or the complementary fragment.
9. The method of claim 7 or 8, wherein the nucleic acid to be detected is amplified before the detection.
10. A method for screening a compound that inhibits the interaction between lGF—lR and IlP—lO or lIP~l comprising a) combining IGF—lR and said IIP polypeptide with a solution containing a candidate compound such that the lGF—lR and said IIP polypeptide are capable of forming a complex and b) determining the amount of complex relative to the predetermined level of binding in the absence of the compound and therefrom evaluating the ability of the compound to inhibit binding of IGF—lR to said llP.
ll. A method for the production of a therapeutic agent for the treatment of carcinomas in a patient comprising combining a pharmaceutically acceptable carrier with a therapeutically effective amount of a compound which modulates the interaction between IGF—lR and llP—lO in a cellular assay, whereby in said cellular assay tumor cells or cells transfected with expression constructs of IGF—lR and of said IIP are treated with said compound, and complex formation between IGF—lR and said respective IIP is analyzed, and the extent of said complex formation in the case of inhibition does not exceed 50% referred to l00% for complex formation without said compound in said same cellular assay.
12. A method for the production of a therapeutic agent for the treatment of carcinomas in a patient comprising combining a pharmaceutically acceptable carrier with a therapeutically effective amount of a compound which modulates the interaction between IGF—lR and IIP—l in a cellular assay, whereby in said cellular assay tumor cells or cells transfected with expression constructs of IGF-lR and of said II? are treated with said compound, and complex formation between lGF—lR and said respective ll? is analyzed, and the extent of said complex formation in the case of inhibition does not exceed 50% referred to 100% for complex formation without said compound in said same cellular assay.
l3. A method according to claim ll or l2, wherein the compound inhibits the interaction.
14. An antibody specifically recognizing lIP—l or IIP—l0.
15. A therapeutic agent produced by the method of any one of Claims ll to l3 for use in a method of treating a patient suffering from carcinoma.
l6. Use of a therapeutic agent produced by the method of any one of Claims 11 to 13 for the manufacture of a medicament for the treatment of Carcinoma. E. R. KELLY & CO., AGENTS FOR THE APPLICANTS
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EPEUROPEANPATENTOFFICE(EPO)03/12/1 | |||
EP98122992A EP1006184A1 (en) | 1998-12-03 | 1998-12-03 | IGF-1 receptor interacting proteins (IIPs) genes coding therefor and uses thereof |
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IE83303B1 true IE83303B1 (en) | 2004-02-11 |
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