Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02208318 1997-06-19
WO96/20211 PCT~S95/16988
"PHOSPHOTYROSINE ASSAY AND PEPTIDES FOR USE THEREIN"
This invention relates to an assay for molecules
which are able to interact with cytokine receptor
intracellular peptideg, in particular receptors of the non-
protein tyrosine kinase domain type, and to peptidessuitable for use in this assay. In a particularly
preferred embodiment, the assay relates to detection of
molecules which are able to bind to cytokine receptors.
Backqround and Prior Art
Cellular proliferation and differentiation in
multicellular organisms requires precise coordination and
regulation. Bin~;ng of extracellular signalling molecules,
such as cytokines and growth factors, to specific cell
surface receptors provides one major mechanism by which
this is achieved. These receptors span the cell membrane,
and are classified as to whether or not they possess an
intrinsic protein tyrosine kinase domain in their
intracellular region. The tyrosine kinase domain
phosphorylates tyrosine residues in many intracellular
proteins, providing an important step in signal
transduction. Receptors which have an intrinsic protein
tyrosine kinase are known as receptor tyrosine kinases.
With the advent of the Polymerase Chain Reaction
(PCR) many novel members of the receptor protein tyrosine
kinase family (RTR; growth factor receptors) have been
identified via the highly conserved structural elements
within their catalytic ~o~n~. Even though these
receptors share a common cytoplasmic protein tyrosine
kinase (PTR) domain, they have distinctive structural
elements in their extracellular domains. These structural
elements can be used to classify the RTKs into a given
subfamily, and demonstrate the structural diversity that
exists within the RTK family (Wilks, 1989).
The signals which are received by the
transmembrane RTKs are transmitted further downstream, and
eventually into the nucleus of the cell, by other signal
CA 02208318 1997-06-19
WO 96120211 PCI~/US9S/16g88
transduction molecules which have SRC Homology 2 (SH2)
domains. These SH2 domains are regions of protein
sequences (modules) of approximately 100 amino acids which
bind independently to phosphotyrosine residues. The
b;n~;ng demonstrates high affinity and amino acid sequence
specificity. The direct, sequence-specific interaction of
SH2 domains with extracellular signal-induced
phosphotyrosine residues has been recognised as a unifying
theme in intracellular signal transduction.
The provision of high affinity and specificity in
this interaction appears to be the most important factor
defining the intracellular outcome of extracellular
interactions. For cell-surface RTKs which are member~ of
the protein tyrosine kinase (PTR) family, it is now well
documented that recruitment of substrates to the activated
receptor is mediated by the phosphorylation of tyrosine
residues in the intracellular domain of the receptor. The
specific amino acid context in which these phosphotyrosines
are embedded defines which "second-wave" substrates are
bound to the receptor and become substrates for its PTK
activity.
Thus these phosphotyrosine residues form a series
of molecular tags to which proteins contA; n; ng SH2 domains
are attracted. Association of the SH2-bearing signal
transducers with the activated receptors leads in turn to
the activation of the transducers, often due to tyro~ine
phosphorylation by the RTR. This provides a mechanism
whereby a particular receptor can select a specific subset
of SH2-containing proteins to be bound, and subsequently
modified and activated. Thus all signal transduction
processes triggered by the activation of a RTR are
coordinated by the SH2 ~n~-; n -phosphotyrosine nexus.
The JAR family of PTKs (Just Another Rinase or
JAnus ~inase) were discovered by polymerase chain reaction-
based screening using degenerate oligonucleotide primersbased on highly conserved PTR catalytic domain motifs. See
for example Australian Patent Application No. 88229/91.
CA 02208318 1997-06-19
WO96120211 PCT~S95/16988
For many receptors that are not themselves PTRs,
members of the JAR family of PTRs are employed as receptor
"~ chAin~"~ and these molecules then act in much the same
way as do the intrinsic PTR domains of the RTR family.
While JAR PTRs do not possess obvious SH2 and SH3 domains,
they do have a PTR domain (JHl) and a kinase-related domain
(JH2), as well as 5 other JH domains.
The cell surface receptors for a large proportion
of haemopoietic growth factors (lymphok;nes), including
IL-3 and IL-6, while not themselves members of the
PTR family, can be grouped together with the receptors for
growth hormone, prolactin, leukaemia inhibitory factor,
erythropoietin and ciliary neurotrophic factor, and other
hormone6 and growth factors such as oncostatin M, to form
an extended family of cytokine receptors. Members of this
family are characterised by the presence of four highly
conserved cysteine residues in the extracellular domain,
and, except for the growth hormone receptor, the
characteristic sequence motif -WSRWS-, usually close to a
single transm~mbrane domain. Although the cytokine
receptors have little significant amino acid sequence
homology within their intracellular ~- ~; n, they are
clearly an evolutionarily-related set of cell surface
receptors. They do not possess any protein kinase-related
sequences, but it is well known that interaction of these
receptors with their respective ligands triggers a ca~cade
of tyrosine phosphorylation in the target cell. Thus while
these receptors are not themselves members of the
PTK family, recruitment of PTR activity is an important
step in propagation of intracellular signalling pathways
downstream of cytokine receptors, and in fact it has been
shown that members of the JAK family of PTKs act as
downstream transduction factors for these receptors.
These non-PTR cytokine receptors fall into two
classes. Type I cytokine receptors either require ligand-
induced homodimerization for signal transduction, for
example erythropoietin receptor and growth hormone
CA 02208318 1997-06-19
WO 96/20211 PCI~/US95/16988
receptor, or require recruitment of at least two distinct
sub-units for effective high-affinity ligand b;n~;ng and
signal transduction. In the latter group, in many cases
several different ligand-specific ~ sub-units are capable
of combining with the same ~ sub-unit. For example, the
sub-units of the receptors for IL-3, I~-5 and GM-CSF all
combine with the same ~ sub-unit.
The Type II cytokine receptors include the
receptor for interferon-~ and interferon-~ (the IFN-~/~
receptor) and the interferon-~ receptor. While
interferon-~ and interferon-~ appear to share a common
receptor, interferon-~ binds to a separate receptor, and
there is little or no sequence homology between the two
groups of interferons. However, the cellular response
elicited by the two receptor types shares common features,
suggesting that they may recruit very similar intracellular
signal transduction pathways.
Stimulation of cells by interferon molecules
leads to activation of latent cytoplasmic transcription
factors which migrate to the nucleus and bind specific
recognition sequences located within the promoter regions
of various interferon-inducible genes. In the case of
IFN-~, the cytoplasmic transcription factor is designated
interferon-stimulated gene factor 3~ (ISGF-3~), which in
turn binds to an interferon-stimulated response element
(ISRE), which is required for induction of transcription.
The ISGF-3~ complex consists of three
polypeptides, formerly designated pll3, p91 and p84, but
now known respectively as STAT2, STATl~ and STATl~, where
STAT stands for Signal Transducers and Activators of
Transcription. The STAT molecules are a family of related
transcription factors. So far, 5 genes have been described
in the literature, although about 10 are thought to exist.
STATl has two alternatively spliced forms,
previously designated p91 or STAT91 and p84 or STAT84
respectively. They are now called STATl~ and STATl~
respectively. STAT2 is encoded by a different gene, and
CA 02208318 1997-06-19
W096/20211 PCT~S95/16988
the protein was previously known as pll3 or STAT113.
STAT3, STAT4, STAT5 and STAT6 have been
identified, and encode proteins that are also involved in
an analogous manner to STATl~, STAT1~ and STAT2 in signal
transcription from various cytokine receptors.
This subject has recently been reviewed by two of
the present inventors (Wilks and Harpur, 1994).
As discussed in that review, a great many growth
factors and cytokines utilise common intracellular
signalling molecules to generate a nuclear response.
Pathways activated by EGF, PDGF, IFN-~, IFN-~, CSF-1 and
IL-6 utilise homologous DNA promoter sequences and common
transcription factor components. While the IFN-~ receptor
utilises STAT2, STATl~ and STAT1~, the receptors for IFN-~,
EGF, PDGF and CSF-1 use STAT1~ and other as yet
unidentified phosphoproteins. There is also re~-~n~ncy or
overlap of the DNA response elements involved. For
example, a number of cytokines cause activation of DNA
b;n~;ng proteins which recognise a single region,
designated the IFN-~ response region (GRR), located within
the promoter of the high-affinity Fc-~ receptor gene. Thus
IL-3, IL-5, IL-10 and GM-CSF all ultimately activate
transcription factors which bind to sequences within a
single element, further demonstrating the overlap and
degeneracy involved in intracellular signal transduction.
The specific phosphotyrosine-cont~in;ng motifs on
cytokine receptors which attract members of the STAT family
via their SH2 domains are at present poorly defined. One
exception is the STAT bi n~i ng site of the IFN-~ receptor.
The tyrosine located at amino acid position 440 in the
human sequence has been shown to be the most important
residue in defining the binding of STAT1~, and therefore in
the commitment of the IFN-~ treated cell to respond to this
cytokine in the appropriate way.
As a model, a 16-amino acid peptide was
synthesized, designated Y440, whose sequence corresponds to
part of the sequence of the IFN-~ receptor
CA 022083l8 l997-06-l9
WO96/20211 PCT~S95/16988
(RAPTSF~Y~k~hv~VD; SEQ. ID. NO. l), a biosensor (BIAcore )-
based assay using the interaction of STATl from crude
cellular extracts with Y440 on a BIAcore chip was
developed. The assay is simple (it can be used to assay
crude cellular extracts, with Y440 on a BIAcore chip),
reproducible (the signal is strong and easily read), and
dependent upon the presence of phosphotyrosine in the
target peptide. It has been further demonstrated that the
b;n~;ng is exclusively due to STATl, and that the b;n~ing
i8 both specific and of high affinity. Thus, a robust
assay for agonists, antagonists, modulators and mimics of
the STATl/Y440-type interaction has been devised.
Essentially the signal transduction pathway of
IFN-~ has been broken down into a series of protein-protein
interactions, and the most specific of them has been
targeted. This assay therefore provides a means of
rational drug design and/or screening for agonists,
antagonists, modulators or mimics of the effects of IFN-
~on cell metabolism, including but not limited to screening
of natural products.
The same basic mechanism is predicted to operate
for all cytokines which act through the JAR kinase-STAT
nexus. For example, the correspon~;ng STAT2 b;n~;ng site
on the ~ chain of the IFN ~/~ receptor has been identified.
This i6 IFN-a R ~Y466p], correspo~;ng to residues 460-474
of the IFN-~ receptor (FLR~lNYvrr~SLRP; SEQ. ID. NO. 2).
An IFN ~/~, IL-2, IL-4, IL-5, IL-9, IL-13, IL-15, GM-CSF,
growth hormone or prolactin specific drug screen is
envisaged along the same lines.
Antagonists of IL-4 and IL-5 are useful as anti-
atopy drugs in the treatment of conditions such as asthma
and psoriasis. Antagonists of GM-CSF are useful in the
treatment of inflammatory conditions such as rheumatoid
arthritis. Antagonists of interferon are useful in the
treatment of autoimmune diseases such as insulin-dependent
diabetes mellitus. Agonists of interferon and other immune
mediators are useful in the treatment of neoplastic
CA 02208318 1997-06-19
WO 96/20211 P~ 9!ill6g88
disease, including leukaemias. Antagonists of haemopoietic
growth factors are useful in the treatment of blood
dysplasias involving overproduction of blood cells, e.g.
erythropoietin antagonists for treatment of overproduction
of red cells, and antagonists of GM-CSF or CSF-1 for
treatment of overproduction of white cells.
SummarY of the Invention
According to a firgt aspect the invention
provides a peptide correspon~;ng to a tyrosine-cont~;n;ng
region of the sequence of an intracellular domain of a
cytokine or hormone receptor or of a JAK protein tyrosine
kinase, wherein said peptide comprises a tyrosine residue
which is able to be phosphorylated, and in it~
phosphorylated form is able to bind a cytoplasmic
transcription factor of the STAT family.
It will be clearly understood that the invention
is applicable to any cytokine and its cognate STAT
molecule. For example some cytokines or hormones
preferentially interact with STAT1~, some with STAT1~, and
some with STAT2. The skilled person will be able to
identify the most appropriate STAT molecule to use with
each desired cytokine receptor phosphopeptide, using
methods known in the art or described herein.
Preferably the peptide, referred to herein as the
receptor peptide, is derived from a receptor for a protein
selected from the group consisting of growth hormone,
prolactin, leukaemia inhibitory factor, erythropoietin,
ciliary neurotrophic factor, interferon-~, interferon-~,
interferon-~, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9,
IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, GM-CSF, CSF-1,
erythropoietin, EGF, PDGF and Oncostatin M.
More preferably the peptide is derived from a
receptor for a protein selected from the group consisting
of growth hormone, prolactin, interferon-~, interferon-~,
interferon-~, IL-2, IL-4, IL-5, GM-CSF and erythropoietin.
CA 02208318 1997-06-19
WO 96/20211 ~ 9!i/16988
In one particularly preferred embodiment, the
invention provides a peptide having the sequence
RAPTSF~Y~K~v~VD (SEQ. ID. N0. 1), correspo~;ng to
residues Lys433 to Asp448 of the amino acid sequence of the
interferon-~ receptor protein, which in its phosphorylated
form has the ability to bind STAT1. The peptide and its
phosphorylated form are referred to herein as
IFN-~ R [Y440] and IFN-~ R [Y440p] receptor.
In a second particularly preferred embodiment,
the peptide has the sequence FLR~lNYvrr~SLKP (SEQ. ID. N0.
2), correspon~;ng to residues Phe460 to Pro474 of the
interferon-~ receptor protein, which in its phosphorylated
form has the ability to bind STAT2. This peptide and its
phosphorylated form are referred to herein as
IFN-~ R [Y466] and IFN-~ R [Y466p] respectively.
It will be clearly understood that homologues or
variants of these peptide sequences are within the scope of
the invention, provided that the relevant tyrosine is still
present and able to be phosphorylated, and provided that
the ability of the phosphorylated peptide to bind a STAT
molecule is retained.
The peptides of the invention may be prepared by
any convenient method; for example, they may be synthesised
chemically, u~ing conventional solid-phase techniquec~ they
may be produced by recombinant DNA method~, or they may be
prepared by cleavage of the appropriate receptor protein
using appropriate enzyme techniques. The receptor protein
may be prepared by any convenient method.
The peptides of the invention are useful in the
screening of molecules for their ability to interact with
the receptor proteins. For example the peptides of the
invention may be used to screen for inhibitors of
interferon-~, interferon-~, interferon-~ or of other
cytokines, such as interleukins, especially interleukin-4,
interleukin-5 and interleukin-6, GM-CSF, G-CSF, other
haemopoietins, including but not re~tricted to leukaemia
inhibitory factor, and ciliary neurotrophic factor, and
CA 02208318 1997-06-19
W096/20211 PCT~S95/16988
hormones and growth factors, such as growth hormone and
EGF.
All cytokines which use the same basic signal
transduction mechanisms as those of interferon-~ are
suitable for use with the peptides of this invention,
especially IFN-~ R [Y440p]. Thus any molecule whose
activity is mediated by a STAT molecule or by the JAK-STAT
pathway is within the scope of the invention. The person
skilled in the art will readily be able to test whether the
receptor for a given protein belongs in this class, to
identify a suitable phosphorylation site for use in the
assay of the invention, and to prepare an appropriate
peptide, using methods already known in the art.
The complete amino acid sequences for many
cytokine or hormone receptors are already known, or can be
determined using routine methods. From the sequence,
tyrosine residues susceptible to phosphorylation can
readily be identified. Tyrosine(s) which are important to
interaction with the cognate STAT molecule can be rapidly
'20 identified using routine site-directed mutagenesis. Once
the relevant tyrosine residue is identified, conveniently-
sized peptides can rapidly be synthesized and tested.
Accordingly, in a second aspect the invention
provides a method of screening of molecules for their
ability to interact with a receptor peptide, comprising the
step of exposing a receptor peptide to a molecule to be
screened, and measuring the degree of interaction between
the peptide and the molecule.
According to a third aspect, the invention also
provides a method of measuring the ability of a molecule to
inhibit or promote interaction between a receptor peptide
and a molecule known to be able to bind to said receptor,
comprising the step of exposing the receptor peptide to a
known molecule having the ability to bind to said receptor
in the presence of a putative inhibitor or promoter, and
measuring the ability of the putative inhibitor to inhibit
or promote Raid bin~in~.
CA 02208318 1997-06-19
WO 96/20211 PCI~/US95/16988
- 10 -
For the purpo~es of this specification, the term
"interact" is to be understood to encompass promoting or
inhibiting the activity of the receptor peptide. Thus a
molecule which interacts with a receptor peptide may act ac
an agonist or antagonist of the receptor, may mimic the
activity of the receptor, or may modulate its activity
directly or indirectly. Moreover, the interaction may take
place either during the screening process, or may take
place prior to screening. Thus this aspect of the
invention includes within its scope detection of
interactions which take place in vitro or in vivo, before
preparation of the sample which is actually subjected to
the assay, as well as interactions t~k; ng place within the
sample. It is particularly envisaged that cell or tissue
cultures whose medium is to be assayed, or an animal from
which a tissue sample or biological fluid is to be assayed,
may be pretreated with the molecule to be screened for its
ability to modulate the interaction between a receptor
peptide with its cognate STAT.
In both the second and third aspects of the
invention, the molecules to be screened may be of either
synthetic, recombinant or natural origin, and may be of a
wide ~ariety of structures.
In both the second and third aspects, the binding
may be measured by any con~enient means. For example,
either the receptor peptide or the molecule to be tested
for b;n~;ng may be labelled with a detectable marker, ~uch
as a radioactive label, a fluorescent label, or a marker
detectable by way of an enzyme reaction. Many suitable
detection systems are known in the art. Thus assay system~
which are suitable for use in the methods of the second and
third aspects of the invention include, but are not limited
to, immunoassays such as enzyme linked immunosorbent assay
(ELISA); affinity-type assays such as those using coated
microtitre plates, beads or slides, or affinity
chromatography; fluorescence-acti~ated cell sorting;
biosensor assays; and bioassays.
CA 02208318 1997-06-19
W096/20211 PCT~S95/16988
One particularly rapid and convenient test system
uses an optical biosensor, such as the BIAcore
(Pharmacia Biosensor AB, Uppsala, Sweden), which enables
the use of proteins or peptides immobilized to a sensor
chip. The biosensor assay is very simple, reproducible and
rapid, while having high specificity. The biosensor assay
enables a very high throughput of samples, and is Am~n~hle
to automation, and to use with relatively crude samples,
such as biological fluids, or cell or tissue extracts. It
0 i8 therefore suitable for screening of natural products for
their ability to inhibit or promote b;n~;ng, or for
activity as agonists or antagonists of cytokines. It is
also suitable for use with biological fluids, for example
in clinical assays, or culture media, including medium from
recombinant bacterial or cell cultures.
The BIAcore~ assay of the invention permits
qualitative or quantitative analysis of the effects of
putative modulators, including agonists and antagonists, on
the kinetics of the STAT/STAT and STAT/receptor peptide
interaction. Potentiation of STAT b;n~;ng or decrease in
affinity for STAT are characteristics which may be
desirable in a given potential therapeutic agent.
Detailed DescriPtion of the Invention
Abbreviations used herein as are as follows:
25 BSA bovine serum albumin
FCS foetal calf serum
GM-CSF granulocyte-macrophage colony-stimulating factor
HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid
30 HPLC high performance liquid chromatography
IFN interferon
IL interleukin
MAb monoclonal antibody
PTK protein tyrosine kinase
35 RTK receptor tyrosine kinase
RU response units
CA 02208318 1997-06-19
W096/20211 PCT~S95/16988
STAT signal transducers and activators of
transcription.
The invention will now be described by way of
reference only to the following non-limiting example~, and
to the figures.
Figure l shows a sensorgram profile of
immunological detection of the phosphorylated tyrosine-
cont~in;ng peptide IFN-y R [Y440p] immobilized to a
BIAcore sensor chip, using a phosphotyrosine-specific MAb
0 A l ~g/ml antiphosphotyrosine monoclonal antibody,
IFN-~ R ~Y440p]-derivatised sensor chip;
0 1 ~g/ml anti-phosphotyro~ine MAb and peptide
IFN-~ R [Y440]-derivatised sensor chip;
0 l ~g/ml anti-STATl monoclonal antibody, peptide
IFN-~ R [Y440p]-derivatised sensor chip.
Figure 2 shows the relative response of
IFN-~ R [Y440p] peptides to crude lysates of HeLa 3 cells.
Figure 3 shows the correlation between the
relative response of an IFN-~ R [Y440p]-coupled biosensor
chip with the presence of immunoreactive STATl in the
cytosolic fraction of a HeLa S3 cell extract.
Figure 4 shows the effect of depletion of STATl
from a nuclear extract of HeLa S3 cells on the response of
a biosensor coupled to IFN-~ R [Y440p].
Figure 5 shows a sensorgram demonstrating
immunodetection of STATl b;n~;ng to an IFN-~ R [RY440p]-
coupled sensor chip.
Figure 6 shows
A: Immunoblot detection of STATl in cytosolic
extracts of cell lines expressing STATl,
but not in a mutant cell line which doeQ
not express this protein;
B: Sensorgrams showing immunodetection of
IFN-~ R [Y440p] b; n~; ng activity of these
cell extracts;
C: Comparison between binding of
IFN-~ R [Y440p] and IFN-~ R [Y466p] by the
CA 02208318 1997-06-19
WO96/20211 PCT~S95/16988
cell extracts; and
D: Immunoblot detection of STAT1 in peptide-
affi agarose treated samples of cell
extract6 by anti-STATl MAb.
5 Example 1 Immunoloqical detection of IFN-~ receptor
(R) PePtides, immobilized on to activated
BIAcore Sensor chiPs
The 16 amino acid peptide IFN-~ R [Y440], having
the sequence RAPTSFGYDKPHVLVD correspo~; ng to residues
Lys433-Asp448 within the cytoplasmic ~ -; n of the IFN
receptor, and its tyrosine-phosphorylated homologue IFN-~
R [Y440p], encompassing the functionally important residue
Tyr440 of the IFN-~ receptor ~ chain (Farrar et al, 1992;
Greenlund et al , 1994), were each synthesised by
conventional solid-phase Fmoc chemistry, purified by either
precipitation and by preparative reversed phase (RP)-HPLC,
and their identity confirmed by amino acid analysis, ion
spray mass spectrometry (Nash et al, 1993) and microbore
RP-HPLC with on-line W spectroscopy.
The application of the BIAcore biosensor for the
analysis of biomolecular interactions (Fagerstam, 1991;
Jonsson et al , 1991; Jonsson and Malmqvist, 1992), in
particular for the analysis of phosphopeptide/protein
interactions, has been described elsewhere (Felder et al,
1993; End et al, 1992; Panayotou et al, 1993) and in
application notes issued by Pharmacia. Unless otherwise
stated, the r~nn;ng buffer used in the BIAcore experiments
was 20 mM HEPES, pH 7.4, 150 mM NaCl, 2.8 mM EDTA,
0.005% Tween 20. For peptide derivatisation of BIAcore
sensor chips (CM5, Pharmacia), carboxyl groups of the
carboxymethylated dextran matrix were activated with a 1:1
mixture of N-hyd ox~ccinimide (NHS) and
200 mM N-ethyl-N"-(3-diethylaminopropyl)-carbodiimide
(EDC), both from Pharmacia, as previously described (Nice
et al , 1993), and 45 ~1 of purified peptides (~98% purity
by microbore RP-HPLC, ion spray mass spectrometry) at
CA 02208318 1997-06-19
W096/20211 PCT~S95/16988
- 14 -
2 mg/ml in 50 mM HEPES, pH 7.5, contA;n;ng 0.15 M NaCl were
injected on to the matrix at 2 ~l/min. Excess reactive NHS
groups were blocked with 30 ~l 1.0 M ethanolamine at
2.0 ~l/min. Following the immobilization the baseline
response level had increased by 450 and 400 Relative
Response unit~ (RU) for IFN-~ R [Y440] and IFN-~ R [Y440p],
respectively, suggesting surface concentrations of 0.45 and
0.4 ng/mm2.
The phosphotyrosine-specific MAb and the
anti-STAT1 MAb were diluted into BIAcore rllnn;ng buffer
at 1 ~g/ml prior to injection. The anti-STAT1 MAb was
provided at 0.25 mg/ml in a solution contA; n; ng
1.0 mg/ml BSA and 50% glycerol as stabilizers. The
presence of these additives does not have an effect on the
immunoreactivity of the MAb (see Example 5), but iB evident
in the sensorgram as a substantial refractive index change.
A 35 ~l sample contA; n; ng 1 ~g/ml of a
phosphotyrosine-specific MAb (Upstate Biotechnology
Incorporated, Lake Placid, NY, USA. Cat. No. 05-321) was
passed at 5 ~l/min over a BIAcore sensor chip which had
been derivatised with approximately 0.5 ng of IFN-
~receptor-derived peptide [Y440]. Following the sample
pulse the chip surface was washed for 750 8 with rllnn; ng
buffer followed by injection of 30 ~l of 20 mM
phenylphosphate to desorb phosphopeptide-bound MAb. The
sensorgram profile is shown in Figure 1. Injection of
sample, end of injection and injection of desorption
solution are indicated with ~1, ~2, ~3, respectively. In a
concurrent experiment an identical sample was injected over
a parallel channel of the same sensor chip derivatised with
the correspo~; ng tyrosine-phosphorylated peptide
IFN-~ R [Y440p]. Furthermore, a non-relevant MAb
(anti-STAT1 MAb, Transduction Laboratories, Lexington, RY,
USA. Cat. No. G16920) was injected over the
IFN-~ R [Y440p]-derivatised channel to provide a control
for the BIAcore response in this and a ~ubsequent
experiment (Example 5).
CA 02208318 1997-06-19
W096~0211 PCT~S95/16988
- 15 -
Example 2 BIAcore responses of immobilized
IFN-~ R [Y440] to crude HeLa lysates
Cell lysates were prepared as follows. Hela S3
cells were grown in RPMI +10% FCS under 5% CO2 in a
3L spinner flask (Bellco Biotechnology) to a density of
5-8x105 cells.mL~1. Cells were-stimulated for 12-16 hours
(priming) or 15 minutes (acute) with 5 ng.mL~l human
recombinant IFN-~ (Genentech). Cytoplasmic extracts were
prepared after appropriate stimulation as described by
Lock et al (1991), with the following modifications. Cells
were washed in ice-cold PBS, then resuspended in ice- cold
hypotonic lysis buffer [5 mM Tris.HCl pH 7.5, 2.5 mM RCl,
1 mM MgCl2, 1 mM dithiothreitol, protease inhibitors
(1 mM phenylmethyl sulphonyl fluoride (PMSF) ,
10 ~g/ml aprotinin, 10 ~g/ml leupeptin, 1 ~M pepstatin,
1 mM 1,10-ph~nAnthroline, 5 ~M E64) and tyrosine
phosphatase inhibitor (0.1 mM sodium orthovanadate) at a
density of lxlO7/ml, and then disrupted by Dounce
homogenisation (to 80% disruption). Insoluble material was
removed by centrifugation at 1000 g for 4 minute~. The
supernatant was then diluted 2x with hypotonic lysis buffer
contA;n;ng 20% sucrose, and clarified by centrifugation at
100,000 g for 30 minuteR.
Clarified supernatant was then concentrated 7 to
10-fold in an Amicon Centricon concentrator (Amicon,
Cat. No. 4304). Prior to the b;n~;ng experiments, 2.5 ml
aliquot~ of concentrated cell extracts were buffer
eYchAnged into an assay buffer containing 50 mM HEPES,
pH 7.0, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, protea~e
inhibitors (10 ~M each of leupeptin and pepstatin,
5 ~M E64, 1 mM 1,10-phen~nthroline, 1 mM PMSF, 1 mM EDTA)
and phosphatase inhibitors (1 mM sodium orthovanadate,
10 mM NaF) using PD10 or NAP5 desalting cartridges
(Pharmacia), with approximately 95% efficacy (according to
manufacturer's notes).
The freeze-dried phosphopeptide IFN-~ R [Y440p]
wa~ dissolved in a volume of 700 ~1 at 2 mg/ml into DMSO
CA 02208318 1997-06-19
W096t20211 PCT~S95/16988
- 16 -
and incubated with 500 ~1 of Affi-lO NHS-activated agarose
(BioRad) for 8 hours at room temperature. Non-reacted NHS
groups of the affinity resin were blocked by incubation
with 1 M ethanolamine-HCl, pH 8.5. The immobilization of
peptide was monitored by microbore RP-HPLC (with on-line
W -spectroscopy) of samples taken prior to and following
the coupling and blocking steps, and coupling efficiency
was estimated from the peak area (absorbance at 214 nm)
obtained for the analysed peptide solutions. The coupling
efficiency under these conditions was 20%, yielding
280 ~g peptide/ml of agarose.
For b;n~ing studies, 1.5 ml of 7-fold
concentrated HeLa extract was incubated on an end-over-end
rotator for 30 minutes at room temperature with 0.5 ml
IFN-~ R [Y440p~-coupled agarose. Samples (100 ~1) were
taken prior to the incubation and after 10, 20 and
30 minutes, and passed over an IFN-~ R [Y440p]-derivatised
sensor chip at 5 ~l/minute. The BIAcore rllnn;ng buffer
for these experiments was similar to the assay buffer, but
did not contain protease inhibitors and glycerol.
Samples of 10-fold concentrated cytosolic
extracts of HeLa cells, or dilutions thereof, were
incubated either with free IFN-~ R [Y440p] phosphopeptide
or with this peptide immobilized to NHS-activated agarose
(Affi-gel 10, BioRad). Aliquots of the former samples and
of 100,000 x g supernatants (s/n) aspirated at indicated
times off the IFN-~ R [Y440p]-Affi-gel 10, or dilutions
thereof, were injected at 5 ~l/minute on to BIAcore sensor
chips, derivatised on parallel ~h~nnels with IFN-~ R [Y440]
or IFN-~ R [Y440p] as described in Example 1. The increase
of BIAcore responses following injection of the sample
plugs (Relative Response, RU) were recorded at a defined
time point (after position ~2, as indicated in Example 1)
and are shown in Figure 2. After each cycle, any material
r: -;n;ng bound to the surface was removed by injection of
a 20 ~l-pulse of 10 mM HCl containing 0.5 M NaCl.
CA 022083l8 l997-06-l9
WO 96/20211 PCT~S95/16988
- 17 -
Example 3 Correlation of IFN-~ R [Y440p] BIAcore
response with immunoreactive STAT1 in
- SE-HPLC fractions of crude cYtosolic HeLa
extracts
Ali~uots (0.5 ml) of 10-fold concentrates of the
cytosolic fractions of HeLa cells, which had been cultured
for 16 hours with 5 ng/ml IFN-~ and then stimulated with an
additional 5 ng/ml IFN-~ 15 minutes prior to harvest, were
fractionated at 0.25 ml/minute in assay buffer (without
glycerol, protease inhibitors or NaF) on a size exclusion
HPLC column which had been calibrated with various proteins
of known molecular radius (Nr)~
Size exclusion-HPLC was performed at
0.25 ml/minute on a Superose-12 column (lOx300 mm,
Pharmacia) using a Waters Nodel 626 Protein purification
system. Protease inhibitors and NaF were omitted from the
HPLC r~lnn;ng buffer, since they resulted in critically
increased backpressure during the separation. Calibration
of the size exclusion-HPLC column with a mixture of
stAn~Ard proteins yielded a linear correlation (r=0.9)
between the log Mr and retention time for the molecular
weight range between 17 and 670 Kd. Coll~n fractions which
were not tested immediately were stored at 4~C overnight or
at -70~C for extended periods, without 1088 in b;n~;ng
activity.
Analysis on the biosensor was performed in the
modified assay buffer as described in Example 2. The
sample rack of the instrument was kept at 14 ~ C during the
course of the analysis.
Samples (35 ~1) of each eluting 1 minute fraction
were passed over parallel channels of the same sensor chip
derivatised with IFN-~ R [Y440p] and IFN-~ R [Y440]
peptides, respectively. The relative responses (as defined
in Example 2) in each fraction are shown in Figure 3A. No
response to the immobilized, non-phosphorylated
IFN-~ R peptide [Y440] was seen in any of the fractions.
Remaining sample (48 ~1) in fractions 41-47, which showed
CA 02208318 1997-06-19
WOg6/20211 PCT~S95116988
- 18 -
maximal BIAcore responses, was analysed by SDS-PAGE on a
12% gel, followed by immunodetection of electroblotted
proteins using an anti-STATl MAb. Proteins were
electrophoretically transferred to PVDF (Immobilon,
Millipore Waters. Cat. No. IPVH 000 10) membrane, and then
immunodetection with STATl antibody was performed under
conditions specified by the manufacturer, using detection
by ~nh~nced chemiluminescence (Amersham Cat. No. RPN 2109).
The results are shown in Figure 3B.
The results show that treatment of the cells with
IFN-~ causeR both a net increase in IFN-~ R [Y440p] b;n~;ng
(RU) and a shift of the b;n~;ng factor to an apparently
higher M~, as determined by size exclusion-HPLC. This
change correlates with an increased quantity and apparent
Mr of immunoreactive STATl under non-denaturing conditions
during size exclusion HPLC, suggesting its identity with
the b;n~;ng factor.
Example 4 Decrease of IFN-~ R ~Y440P] reactivitY in
STATl-depleted HeLa nuclear extracts
HeLa cells were cultured as described in
Example 2, but activated with IFN-~ only 15 minutes prior
to extraction. Nuclear extracts were prepared as described
by Dignam et al (1983), with the following modifications.
The insoluble material removed after homogenization was
resuspended in High-salt Buffer [20 mM Hepe~ pH 7.9,
25% glycerol, 1.5 mM MgCl2, 1.2 M KCl, 0.2 mM EDTA,
0.2 mM PMSF, 0.5 mM DTT], and allowed to incubate for
30 minutes on ice. Nuclear matrices were removed by
centrifugation for 20 minutes at 25,000 g. Nuclear extract
and the anti-STATl affinity eluate were exch~nged into
assay buffer, as described in Example 2. The dilution of
the extract prior to affinity absorption was chosen to
yield a BIAcore response within the same range a~ the
other samples.
The concentrated nuclear extract of HeLa cells,
~timulated for 15 minutes with IFN-~ (IFN-~ activated
CA 02208318 1997-06-19
WOg6nO211 PCT~S9S/16988
cell~) prior to cell ly~is and fractionation, was absorbed
on to an anti-STAT1 MAb affinity resin (Transduction
- Laboratorie~. Following a wA~h;ng cycle with assay buffer
the affinity coll~m~ was eluted with a buffer cont~;n;ng
3 M MgCl2, 0.075 M HEPES pH 6.5, 25% ethyleneglycol. The
relative BIAcore respon~es to i_mobilised IFN-y R ~Y440p]
peptide in dilutions of the extract before and after the
affinity extraction, and of the col~mn eluate, were
determined. The rem~;n~er of the samples used for BIAcore
analysis was analysed on a 12% SDS-PAGE gel followed by
immunodetection of electroblotted proteins using an
anti-STAT1 MAb as described in Example 3. In addition, an
aliquot of the anti-STAT1 affinity matrix after
MgCl2-elution was analysed on the same gel. The results
are shown in Figure 4. Panel A summarizes the BIAcore
responses; Panel B ~hows the immunoblot results.
Example 5 Immunodetection of STAT1~-b;n~;n~ to an
IFN-y R [Y440p] derivatised sensor chiP
The nuclear extract of IFN-~ activated HeLa cells
wa~ purified on an IFN-y R ~Y440p] affinity resin and the
phenylphosphate eluate of the affinity matrix was ~Ych~nged
into the appropriate assay buffer, as described in
Example 2. The eluate wa~ injected either directly (A, B)
or after incubation with anti-STAT1 MAb (C) over a sensor
chip derivatised with IFN-~ R ~Y440p]. In a second
injection immediately following the fir~t sample plug and
prior to the start of the dissociation phase, either buffer
(A, C) or anti-STAT1 MAb (B) was injected on to the same
channel of the sensor chip. Figure 5 illustrates an
overlay of BIAcore responses normalised to the same
baseline. The individual injections are marked with an
arrow labelled "1" (first injection) and "2" (second
injection). The response levels at the start of the second
injection are indicated.
Activated HeLa cells were fractionated as
described in Example 4, and a 4 ml sample of the nuclear
CA 02208318 1997-06-19
WO 96/20211 PCI~/US95/16988
- 20 -
fraction was extracted on 0.5 ml IFN-~ R [Y440p]-agaro~e
for 30 minutes at 0~C as de~cribed in Example 2. The
affinity resin was washed quickly with 8 column volumes of
as~ay buffer and eluted with one column volume of
phenylphosphate (20 mM), collected in a single 0.5 ml
fraction. The breakthrough from this column was loaded
again under identical conditions. This second b; n~; ng step
increased recovery from the lysate to 90%. Both eluates
were pooled, and a 90 ~1 aliquot incubated for 30 minutes
at room temperature with 10 ~1 of anti-STATl MAb
(200 ~g/ml) diluted into as~ay buffer, or with the same
volume of assay buffer. Aliquots (35 ~1) of these sample~
were passed over a IFN-~ R [Y440p]-derivatised sensor chip
at 5 ~l/minute and immediately after each sample plug had
left the flow cell were followed by a second 35 ~1
injection of either rllnn;ng buffer or anti-STATl MAb at
1 ~g/ml.
Thus either preincubation of cellular extracts
with anti-STATl MAb prior to BIAcore analysis or injection
of MAb on to IFN-~ R [Y440p] sensor chip-bound factor
resulted in an increased BIAcore response. In contrast,
direct injection of the MAb on to the IFN-~ R [Y440p]-
derivatised sensor chip yielded no signal (Example 1), thus
indicating that the primary antibody-enhanced b; n~; ng
response is due to STAT1.
Exam~le 6 Absence of INF-~ R [Y440] Peptide B; n~; n~
Component in Crude Cell Extracts, Detected
by BIAcore and Immunotechnoloqy Correlates
with the Absence of STAT1 Protein
Parental (2fTGH) and mutant (U2A, U3A, U4A) human
fibroblasts [as described elsewhere (Pellegrini et al,
1989: McKendry e t al, 1991)] were grown in Dulbecco'~
Modified Eagle' 8 Medium (DMEM) supplemented with 10%
(vol./vol.) heat-inactivated foetal calf serum and 5 ~M
L-glutamine. The cells were grown to 80% confluency, and
harvested, following 2 washes in ice cold PBS supplemented
CA 022083l8 l997-06-l9
W096/20211 PCT~S95/16988
with 0.1 mM Na3V04, in 1 ml trypsin-versene. Following
trypsinisation, cells were washed twice in DMEM plu8 10%
foetal calf serum, and twice in ice-cold PBS supplemented
with 0.1 mM Na3V04. HeLa S3 cells were grown as described
in Example 2. Cytoplasmic extracts were then prepared as
described in Example 2.
The presence of STAT1 in samples (48 ~1) of
10-fold concentrated cytosolic extracts, prepared as
described above, was analysed by SDS-PAGE on a 7.5% gel
followed by immuno-detection of electroblotted proteins
using an anti-STAT1 Mab (as described in Example 3). This
i~ presented in Figure 6A, and shows that STAT1 is not
present in line U3A.
BIAcoreTM detection of IFN-~ R [Y440p] b; n~; ng
activity in the above samples was carried out as follow~.
Samples of 10-fold concentrated cytosolic extracts,
prepared as described above, were incubated either with
IFN-~ R [Y440p] affi agarose or IFN-~ R [Y440] affi
agarose, aspirated after 30 minute~ at room temperature,
and then injected at 5 ~l/minute~ onto BIAcoreTM sensor
chips, derivatised on parallel channels with IFN-~ R [Y440]
or IFN-~ R [Y440p] as described in Example 1.
BIAcoreTM detection of IFN-~ R [Y440p] b; n~; ng
activity in samples of 10-fold concentrated cytosolic
extracts, prepared as de6cribed above, was carried out on
sensor chips, derivatised on parallel channels with
IFN-~ R [Y440] or IFN-~ R [Y440p] as described in
Example 1. The response sensorgrams of samples aspirated
after 30 minutes incubation at room temperature with
IFN-~ R [Y440p] affi agarose, subtracted (BIAevaluation
software ver.2.1) from the response in samples incubated
with IFN-~ R [Y440] affi agarose are indicated in
Figure 6B. No response to any of the extracts was detected
on the IFN-~ R [Y440] derivatised channel.
Identical experiments were carried out
substituting the IFN-~ R [Y440] peptide with a
phosphopeptide (IFN-~ R [Y466p]) correspo~;ng to residues
CA 02208318 1997-06-19
WO 96/20211 PCrlUS9S/16988
- 22 -
460-474 of the IFN-~ R (FLRClN~v~SL~P; SEQ. ID. NO. 2)
and synthesised as described in Example 1. The relative
BIAcore responses following injection of each cell extract
were determined after 575 seconds (indicated by the arrow
in Figure 6B). Figure 6C compares the responses of each
cell extract to both phosphopeptides, plotted in relative
response units.
Parallel aliquots of the peptide affi-agarose
treated samples analysed in Figure 6B were examined by
SDS-PAGE on a 7.5% gel followed by immunodetection of
electroblotted proteins using an anti-STAT1 MAb as
described in Example 3. The results, shown in Figure 6D,
confirm that the approach taken above is valid, in that
treatment of these extracts with phosphopeptide
affi-agarose effectively depletes STATl protein.
Furthermore, the absence of STAT1 in U3A extracts
correlates with the lack of BIAcore response depicted in
the sensorgram of Figure 6B.
ExamDle 7 Other Potential STAT B; n~; n~ Sites
Tyro6ine-cont~;n;ng amino acid sequences in which
the tyrosine can be phosphorylated are present in a number
of proteins which have been reported in the literature.
Some of these phosphotyrosine residues are located upon
cytokine receptors, whereas others are found towards the C-
2 5 terminal end of the STATs themselves. The putative STAT
b;n~;ng sites are summarized in Table 1, with the location
of the phosphotyrosine identified in brackets. This
location uses sequence numbering as indicated by mutation
analysis.
CA 02208318 1997-06-19
WO 96/20211 r~ l6988
- 23 -
::Ln Ln Ln Ln
_ILn ~ ~ ~ Ln Ln
~a 0
0
~ h ~ h
0 P~
h ,~ 0 ~ h ,~
~ n ~D ~ Ln ~~
m n n n n n n n n
al
_I
.~, ~ ~ ~ Ln
t~ Ln o ~ ~
~ D O ~'CO ~1 ~ O
a) ~7 Ln ~ c~
U ~ ~ ~ ~--~ Ln ~ --~~ ~O ~ ~ O
~ Ln ~ ~
": V ~ -- ~~ ~ ~
r~ O~ O ~-- O E-l O ~ O 1~1 0 0 0 V. O
n; ~ Z,e Z U. V 4
a~ ~ ~ v
p H ,~3 H ~ H ~ H H P~ H . H ~:1 H Y H
a Y ~ ~ a Y ~ -
~a ~Ja~a ~a ~a ~:a. a ~a ~a
,3 rn ~i3 V~ W ~ ~ a ~ ~ ~
YVI ~ V~ V V~ V u~ :C V~ avn ~ V~ :~ Vl ~ Vl
-- ~--V-- V-- ~ --~-- ~-- Y --
I r~ h ~d
u~
X
j~
o
~r ~ I vn
o
~ ~ P4 H I _~
li3 H P4 tJ~ ~ V n
CA 02208318 1997-06-19
WO 96/20211 PCTIUS95/16988
- 24 -
U U ~
~~ U
,, o~ o o ~ ~
o~ o
u
p n~ ~ ~ X
U
~ U
~.~ ~~J ~,1 , ~,1
o ~, ~n
r.~ ~4 _14 _I V c4 ~1
~0
.
m
-
~,
~d ~
E~ C
~J
~ o ~ o~
O _ _
~ a~ . . . _ . _ .
~o Or~ o 4 O O O
Z -I ~ Z ~ Z;
rJ~ 4 _1 1_
~ ~ ~ ~ ~ 01 ~ E- -
a
H H r H 4 H ~ H L H
> O
01 P4 0l ~ Ol
rn ~ rn ~ rn > rn V rn
-- rn-- ~ ¢~ a--
1 ~,
E~ .,
rn ~ ~
P_ ~ ~ X ~ ~ o
-- -- ~ ~ 0
U 4 ~1
o
rn n rn n n ~4 P: H
li3 ~4 ~
CA 022083l8 l997-06-l9
WO 96/20211 P~ 9~i/16g88
- 25 -
Only the STAT1~ Y701 i8 demonstrably a STAT
b;n~;ng site, while the other sites have been shown to be
~ strong candidates for STAT b;nA;ng by virtue of the fact
that either phenylalanine substitution of the tyrosine
results in 1088 of STAT bin~;ng (for example in the case of
the EPO-R Y343 or the PRL-R Y580), or the sites lie in a
region which appears to be required for signal transduction
down-stream of a particular cytokine receptor (for example
in the case of the LIF-R and gpl30). A further class of
predicted sites has been derived on the basis of what is
known about STAT b;n~;ng site~, coupled with what iB known
in the field of cytokine receptor-mediated signal
transduction (usually denoted as Harpur, 1995).
The u~e of specific components of the IFN-
~signalling cascade and synthetic analogues thereof in an
BIAcore biosensor-based assay to study initial steps of
this pathway in detail has been described herein. As a
m;n;m~l, functionally critical portion of the ~-chain of
the cytokine receptor, a peptide sequence surro~n~;ng a
tyrosine residue (Tyr440) which in its phosphorylated form
is essential for biological responsiveness (Farrar et al,
1991, Greenlund et al, 1994) was immobilized onto the
sensor chip of the biosensor. The efficacy of the coupling
reaction and the integrity and accessibility of the
phosphorylated tyrosine was demonstrated by monitoring, in
real time, the binding of an anti-phosphotyrosine MAb to
the peptide (Example 1). HeLa cell extracts and fibroblast
extracts (2fTGH, U2A and U4A) were shown to contain a
component that bound specifically to the sensor chip-
immobilized, phosphorylated form of the receptor peptide.
Specificity was demonstrated by:
i) competition with free IFN-~ R [Y440p]
peptide
ii) lack of response to IFN-~ R [Y440] peptide
iii) depletion of the IFN-~ R [Y440p] b;n~;ng
CA 02208318 1997-06-19
WO 96/20211 PCI~/US95/16988
factor from the crude extract with
immobilized receptor peptide (Example 2).
iv) $ailure of extracts of U3A mutant cells,
which lack STAT1, to generate a b;n~;ng
response to IFN-~ ~Y440p] peptide
(Example 6).
Treatment of the cells with IFN-~ causes both a
net increase in IFN-~ R [Y440p] b;n~;ng (RU) and a shift of
the b;n~;ng factor to an apparently higher Mr as determined
by size exclusion-HPLC. This change correlates with an
increased quantity and apparent Mr ~f immunoreactive STAT1,
suggesting its identity with the bi nA; ng factor
(Example 3).
Three approaches were taken to verify this
identity. First, immunodepletion of cellular extract with
an anti-STAT1 affinity resin resulted in a titratable net
reduction in IFN-y R [Y440p] b; n~; ng (RU). The successful
retention of STAT1 on the affinity resin was confirmed by
western analysis (Example 4; Figure 4 lane 4), which also
revealed "bleeding" of MAb together with antigen from the
affinity resin (lanes 2, 5-7). Elution of the affinity
col-~mn with 3M MgCl2 yielded a small but titrable BIAcore
response, suggesting unusually high avidity of the
anti-STAT1 MAb.
Second, either preincubation of cellular extracts
with anti-STAT1 MAb prior to BIAcore analysis or injection
of MAb on to IFN-~ R [Y440p] sensor chip-bound factor
resulted in an increased BIAcore response. In contrast,
injection of the MAb on to the IFN-~ R [Y440p]-derivatised
sensor chip gave no signal.
Finally, application of cellular extracts from
the mutant fibroblast cell line U3A, which completely lacks
STAT1~ or STAT1~ proteins, failed to generate a b;n~;ng
response to the IFN-~ R [Y440p] peptide. Thus it is clear
that the presence of STAT1 is required for the b; n~; ng
CA 02208318 1997-06-19
W096/20211 PCT~S95116988
response detected towards the IFN-~ R [Y440p] peptide.
Taken together, these f;n~;ngs demonstrate that
- the local sequence of relatively short peptide~ contains
sufficient information to enable their use as
physiologically relevant affinity reagent~. Thus a wide
range of biomolecular interactions can be analysed using
the approach presented herein.
It will be apparent to the person skilled in the
art that while the invention has been described in some
detail for the purposes of clarity and understAn~; ng,
various modifications and alteration6 to the embodiments
and methods described herein may be made without departing
from the scope of the inventive concept disclosed in this
specification.
References cited herein are listed on the
following pages, and are incorporated herein by thi~
reference.
CA 02208318 1997-06-19
WO96r20211 PCT~S95/16988
- 28 -
REFERENCES
Damen, J.E., Wakao, I.I., Miyajima, A., Rrosl, J.,
Humphries, R.K., Cutler, R.L. and Crystal, G
"Tyrosine 343 in the erythropoietin receptor positively
regulates erythropoietin-induced cell proliferation and
STAT5 activation"
EMB0, 1995 14 5557-5568
Dignam, J.D., Lebovitz, R.M., and Roeder, R.G.
"Accurate transcription initiation by RNA polymerase II in
a soluble extract from isolated mammalian nuclei"
Nucl. Acid. Res., 1983 11 1475-1489.
End, P., Panayotou, G., Entwistle, A., Waterfield, M.D.,
and Chiquet, M.
"Tenascin: a modulator of cell growth"
Eur. J. Biochem., 1992 209 1041-1051.
Fagerstam, L.
"Techniques in protein chemistry II"
New York: Academic press, 1991
Farrar, M.A., Campbell, J.D., and Schreiber, R.D.
"Identification of a functionally important sequence in the
C terminus of the interferon-gamma receptor"
Proc. Natl. Acad. Sci. U.S.A., 1992 89 11706-11710.
Felder, S., Zhou, M., Hu, P., Urena, J., Ullrich, A.,
Chaudhuri, M., White, M., Shoelson, S.E., and
Schlessinger, J.
"SH2 domains exhibit high-affinity b;n~ing to tyrosine-
phosphorylated peptides yet also exhibit rapid dissociation
and exchange"
Mol. Cell Biol., 193 13 1449-1455.
CA 02208318 1997-06-19
W096/20211 PCT~S9S/16988
- 29 -
Gouilleux, F., Wakao, H., Mimdr, M. and Groner, B.
"Prolactin induces phsophorylation of Tyr694 of STAT5
- (MGF), a prerequisite for DNA b;n~;ng and induction of
transcription"
EMBO, 1994 13 4361-4369
Greenlund, A.C., Farrar, M.A., Viviano, B.L., and
Schreiber, R.D.
"Ligand-induced IFNg receptor tyrosine phosphorylation
couples the receptor to its signal transduction system
(p91)"
EMBO, 1994 13 1591-1600.
Harpur, A.G.
PhD Thesis, University of Melbourne, 1995
Hou, J., Sc~;n~ler, U., Henzel, W.J., Ho. T.C.,
Brasseur, M. and McKnight, S.L.
"An interleukin-4-induced transcription factor IL-4 STAT"
Science, 1994 265 1710-1706
Jons~on, U., Fagerstam, L., Ivarsson, B., Johnsson, B.,
Karlsson, R., Lun &, R., Lofas, S., Persson, B., Roos, H.,
Ronnberg, I., and et al
"Real-time biospecific interaction analysis using surface
plasmon resonance and a sensor chip technology"
Biotechniques, 1991 11 620-627.
Jonsson, U. and Malmqvist, M. (1992).
Advances in Biosen~or, Vol.2. (T.on~nn: JAI Press Ltd.).
Lebrum, I.J., Ali. S., Goffin. V., Ullrich, A. and
Relly, P.A.
"A single phosphotyrosine residue of the prolactin receptor
is responsible for activation of gene transcription"
Proc. Natl. Acad. Sci. USA, 1995 92 4031-4035
CA 02208318 1997-06-19
W096/20211 PCT~S95/16988
- 30 -
Lock, P., Ralph, S., Stanley, E., Boulet, I., Ramsay, R.,
and Dunn, A.R.
"Two isoform6 of murine hck, generated by utilization of
alternative translational initiation codons, exhibit
different patterns of subcellular localization"
Mol. Cell Biol., 1991 11 4363-4370.
McK~n~ry, R., John, J., Flavell, D., Muller, M.,
Kerr, I.M., and Stark, G.R.
"High-frequency mutagenesis of human cells and
characterization of a mutant unresponsive to both alpha and
gamma interferons"
Proc. Natl. Acad. Sci. U.S.A., 1991 88 11455-11459.
Nash, K., Feldmuller, M., de Jersey, J., Alewood, P., and
Hamilton, S. (1993).
"Continuous and discontinuous assays for phosphotyrosyl
protein phosphatase activity using phosphotyrosyl peptide
substrates"
Anal. Biochem., 1993 213 303-309.
Nice, E., Lackm~nn, M., Smyth, F., Fabri, L., and Burgess,
A.W.
"Synergies between micropreparative high-performance liquid
chromatography and an instrumental optical biosensor"
J. Chromatogr. A., 1994 660 169-185.
Panayotou, G., Gish, G., End, P., Truong, 0., Gout, I.,
Dhand, R., Fry, M.J., Hiles, I., Pawson, T., and
Waterfield, M.D.
"Interactions between SH2 domains and tyrosine-
phosphorylated platelet-derived growth factor beta-receptor
sequences: analysis of kinetic parameters by a novel
biosensor-based approach"
Mol. Cell Biol., 1993 13 3567-3576.
CA 02208318 1997-06-19
WO96/20211 PCT~S9S/16988
- 31 -
Pellegrini, S., John, J., Shearer, M., Rerr, I.M., and
Stark, G.R.
"Use of a selectable marker regulated by alpha interferon
to obtain mutations in the signaling pathway"
Mol. Cell Biol., 1989 2 4605-4612.
Shuai, R. Stark, G.R., Rerr, I.M. and Darnell, J.E.
"A single phogphortyrosine residue of STAT91 is required
for gene activation of Interferon-~ n
Science, 1993 261 1744-1746
Stahl, N., Parruggella, T.J., Boulton, T.G., Zhong, Z.
Darnell, J.E.J and Yancopoulos, G.D.
"Choice of STATs and other substrates specified by modular
tyrosine-based motifs in cytokine receptors"
Science, 1995 267 1349-1353
Wilks, A.F.
Proc. Natl. Acad. Sci. U.S.A., 1989 86 1603-1607
Wilks, A.F. and Harpur, A.G.
"Cytokine Signal Transduction and the JAK Family of Protein
Rinases"
BioEssays, 1994 16 313-320
Zhong, Z. Wen. Z and Darnell, J.E.J.
"Stat3 and Stat4 members of the family of signal
transducers and activators of transcription"
Proc. Natl. Acad. Sci. USA, 1994 91 4806-4810
CA 02208318 1997-06-19
WO 96120211 PCI~/US9Stl6988
SEO~N~ LISTING
( 1 ) ÇT~T~T- INFORMATION:
(i) APPLICANT: LUDWIG lN~lllul~ FOR CANCER
RESEARCH
(ii) TITLE: ASSAY AND ~Ll~ES FOR USE THEREIN
(iii) NUMBER OF SEQu~N~S: 16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSESS: Felfe & Lynch
(B) STREET: 805 Third Avenue
(C) CITY: New York
(D) STATE: New York
(E) CO~N1K~: U.S.A.
(F) POSTCODE: 10022
(v) COM~ul~K RT~'~n~RTT~' FORM:
(A) MEDIUM TYPE: 3.5 1.44 Mb storage diskette
(B) COM~u~:K: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect
(vi) PRIOIR APPLICATION DATE:
(A) APPLICATION NO.: PN 0249
(B) FILING DATE: 23 DECT~RT~'R 1995
(vii) CURRENT APPLICATION DATA:
(A) APPLICATION NO.: Not yet assigned
(B) FILING DATE: Not yet assigned
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Patricia A. Pasqualini
(B) REFERENCE NO.: LUD-5399-PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200
(B) TELEFAX: ~212) 838-3881
CA 02208318 1997-06-19
WO96120211 PCT~S95/16988
(2) INFORMATION FOR SEQ ID NO. l:
(i) SEQ~N~ CHARACTERISTICS:
- (A) LENGTH: 16
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)- MOLECULE TYPE: peptide
(iii) HYPOln~LlCAL: no
(v) FRA~-M~T TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: l
LYB Ala Pro Thr Ser Phe Gly Tyr ABP Lys Pro His Val Leu Val ABP
5 l0 15
(2) INFORMATION FOR SEQ ID NO. 2:
(i) S~Qu~N~ ~ARACTERISTICS:
(A) LENGTH: 15
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOLn~llCAL: no
(v) FRA~.M~NT TYPE: internal
(xi) SEQ~:N~ DESCRIPTION: SEQ ID NO: 2
Phe Leu Arg Cy8 Ile Asn Tyr Val Phe Phe Pro Ser Leu LYB Pro
5 l0 15
(2) INFORMATION FOR SEQ ID NO. 3:
(i) SEQ~ CHARACTERISTICS:
(A) LENGTH: 13
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOLn~llCAL: no
(v) FRA~-M~NT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
CA 02208318 1997-06-19
WO96/20211 PCT~S95/16988
- 34 -
Ala Gln A~p Thr Tyr Leu Val Leu Asp Glu Trp Leu Leu
(2) INFORMATION FOR SEQ ID NO. 4:
(i) SE~u~N~ C~ARA~TERISTICS:
(A) LENGTH: 13
(B) TYPE: amino acid
- (C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOL~LlCAL: no
(v) F~A~-MRNT TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 4
Pro Ala Gly Gly Tyr Gln Glu Phe Val Gln Ala Val Ly~
(2) INFORMATION FOR SEQ ID NO. 5:
(i) S~Q~N~ ~ARACTERISTICS:
(A) LENGTH: 14
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOL~hl-lCAL: no
(~) FRAGMENT TYPE: internal
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO: 5
Gly Gly Pro Gly Try Ly~ Ala Phe Ser Ser Leu Leu Ser Ser
(2) INFORMATION FOR SEQ ID NO. 6:
(i) SEQu~N~ C~AR~CTERISTICS:
(A) LENGTH: 12
(B) TYPE: amino acid
(C) STRANDEDNESS: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOL~LlCAL: no
CA 02208318 1997-06-19
WO 96/20211 PCI/'US9S116g88
(v) F~r-M~NT TYPE: internal
(xi) SEQ~:N~ DESCRIPTION: SEQ ID NO: 6
Gly Gly Leu Asp Tyr Leu Asp Pro Ala Cys Phe Thr
(2) INFORMATION FOR SEQ ID NO. 7:
(i) SEQ~N~ C~ARA.~TERISTICS:
(A) LENGTH: 14
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPGL~-LlCAL: no
(v) F~GM~NT TYPE: internal
(xi) SEQu~:N~ DESCRIPTION: SEQ ID NO: 7
Val His Ser Gly Tyr Arg His Gln Val Pro Ser Val Gln Val
(2) INFORMATION FOR SEQ ID NO. 8:
(i) SEQ~N~ ~ARACTERISTICS:
(A) LENGTH: 13
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) NOLECULE TYPE: peptide
(iii) HYPG~ CAL: no
(v) F~A~-M~NT TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 8
Val Gln Ser Met Tyr Gln Pro Gln Ala Ly~ Pro Glu Glu
(2) INFORMATION FOR SEQ ID NO. 9:
(i) SEQu~:N~ C~ARACTERISTICS:
(A) LENGTH: 11
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02208318 1997-06-19
W O 96/20211 PC~rtUS95tl6988
(ii) MOLECULE TYPE: peptide-
(iii) HYPOl~hllCAL: no
(v) F~A~.M~NT TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 9
Gly Gly Ala Gly Tyr Lys Pro Gln Met His Leu
(2) INFORMATION FOR SEQ ID NO. 10:
(i) SEQ~N~ CRARACTERISTICS:
(A) LENGTH: 11
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOL~llCAL: no
(~) F~A~-M~T TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 10
Leu Val Gln Ala Tyr Val Leu Gln Gly Pro Asp
(2) INFORMATION FOR SEQ ID NO. 11:
(i) SEQ~N~ CR~RACTERISTICS
(A) LENGTH: 13
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOLn~llCAL: no
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11
Lys Gly Thr Gly Tyr Ile Lys Thr Glu Leu Ile Ser Val
(2) INFORMATION FOR SEQ ID NO. 12:
(i) SEQUENCE CHARACTERISTICS:
CA 02208318 1997-06-19
WO96t20211 PCTtUS95tl6988
(A) LENGTH: 8
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOl-~hLlCAL: no
(v) F~M~NT TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 12
Tyr Leu Lys His Arg Leu Ile Val
(2) INFORMATION FOR SEQ ID NO. 13:
(i) SEQu~N~ CHARACTERISTICS:
(A) LENGTH: 12
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii), HYPOTHETICAL: no
(v) F~r-M~NT TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 13
Ser Ala Ala Pro Tyr Leu Lys Thr Lys Phe Ile Cys
(2) INFORMATION FOR SEQ ID NO. 14:
(i) SEQUENCE C~CTERISTICS:
(A) LENGTH: 12
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: no
(v) F~-M~NT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
Gly Asp Lys Gly Tyr Val Pro Ser Val Phe Ile Pro
CA 02208318 1997-06-19
WO96120211 PCT~S95116988
- 38 -
(2) INFORMATION FOR SEQ ID NO. l5:
(i) SEQ~ r~R~CTERISTICS:
(A) LENGTH: ll
(B) TYPE: amino acid
(C) STRANDEDNESS: 8 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOLnhllCAL: no
(v) F~ M~NT TYPE: internal
(xi) SEQ~:N~ DESCRIPTION: SEQ ID NO: 15
Ala Val A~p Gly Tyr Val Lys Pro Gln Ile LYB
(2) INFORMATION FOR SEQ ID NO. 16:
(i) SEQ~N~ C~CTERISTICS:
(A) LENGTH: ll
(B) TYPE: amino acid
(C) STRANDEDNESS: 8 ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOLnh~-lCAL: no
(v) F~r-M~NT TYPE: internal
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO: 16
Asp Gly Arg Gly Tyr Val Pro Ala Thr Ile Lys
