U.S. patent application number 16/356698 was filed with the patent office on 2019-09-05 for sh2 domain variants.
The applicant listed for this patent is THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO, THE UNIVERSITY OF WESTERN ONTARIO. Invention is credited to Xuan CAO, Haiming HUANG, Tomonori KANEKO, Shun-Cheng LI, Sachdev Singh SIDHU.
Application Number | 20190271705 16/356698 |
Document ID | / |
Family ID | 49257997 |
Filed Date | 2019-09-05 |
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United States Patent
Application |
20190271705 |
Kind Code |
A1 |
LI; Shun-Cheng ; et
al. |
September 5, 2019 |
SH2 DOMAIN VARIANTS
Abstract
The present invention relates to variant SH2 domains for binding
a phosphotyrosine (pTyr)-containing peptide. The variant SH2
domains of the present invention include a parent SH2 domain having
at least one amino acid substitution in a pre-defined region of 15
amino acid positions of the parent SR2 domain, wherein said at
least one amino acid substitution increases the affinity of the
variant SH2 domain for the pTyr-containing peptide relative to the
parent SH2 domain. The present application relates also to methods
of using the variant SH2 domains in the treatment of protein
kinase-associated disorders, or the diagnosis or prognosis of
protein kinase-associated disorders, for isolating and measuring
the concentration of pTyr-containing molecules, and as reagents in
research.
Inventors: |
LI; Shun-Cheng; (London,
CA) ; KANEKO; Tomonori; (London, CA) ; CAO;
Xuan; (London, CA) ; SIDHU; Sachdev Singh;
(Toronto, CA) ; HUANG; Haiming; (Toronto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNIVERSITY OF WESTERN ONTARIO
THE GOVERNING COUNCIL OF THE UNIVERSITY OF TORONTO |
London
Toronto |
|
CA
CA |
|
|
Family ID: |
49257997 |
Appl. No.: |
16/356698 |
Filed: |
March 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14388592 |
Sep 26, 2014 |
10274499 |
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PCT/CA2013/000279 |
Mar 27, 2013 |
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16356698 |
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61616167 |
Mar 27, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6812 20130101;
G01N 2440/14 20130101; G01N 33/6842 20130101; C07K 14/47 20130101;
A61K 38/1709 20130101; C07K 1/22 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 14/47 20060101 C07K014/47; C07K 1/22 20060101
C07K001/22 |
Claims
1.-19. (canceled)
20. A method for preventing or inhibiting the effects of a tyrosine
kinase in a cell, the method comprising delivering or introducing a
variant SH2 domain into the cell, the variant SH2 domain varying
from a parent SH2 domain, so that relative to the parent SH2
domain, the variant SH2 domain comprises a modified phosphotyrosine
(pTyr) binding region substituted at at least one amino acid
position in a pre-defined region of 15 amino acid positions of the
parent SH2 domain, the substituted amino acids increasing the
binding affinity of the modified pTyr binding region for a
pTyr-containing peptide relative to an unmodified pTyr binding
region of the parent SH2 domain.
21. The method of claim 20, wherein the variant SH2 domain is
provided within a carrier that allows transportation across the
cell.
22. The method of claim 20, wherein the polypeptide is provided as
a fused product comprising the polypeptide and a cell membrane
penetrating molecule.
23. (canceled)
24. A method of assessing the presence of pTyr-containing peptides
in a sample, the method comprising (a) contacting the sample to a
variant SH2 domain, such that a pTyr-containing peptide/variant SH2
domain complex is formed if the pTyr-containing peptides are
present in the sample, the variant SH2 domain varying from a parent
SH2 domain, so that relative to the parent SH2 domain, the variant
SH2 domain comprises a modified phosphotyrosine (pTyr) binding
region substituted at at least one amino acid position in a
pre-defined region of 15 amino acid positions of the parent SH2
domain, the substituted amino acids increasing the binding affinity
of the modified pTyr binding region for a pTyr-containing peptide
relative to an unmodified pTyr binding region of the parent SH2
domain; and (b) detecting the formation of the complex, thereby
detecting the presence of the pTyr-containing peptides in the
sample.
25. (canceled)
26. (canceled)
27. A method for isolating pTyr-containing peptides from a sample,
the method comprising: (a) contacting the sample to a variant SH2
domain, such that a pTyr-containing peptide/variant SH2 domain
complex is formed if the pTyr-containing peptides are present in
the sample, the variant SH2 domain varying from a parent SH2
domain, so that relative to the parent SH2 domain, the variant SH2
domain comprises a modified phosphotyrosine (pTyr) binding region
substituted at at least one amino acid position in a pre-defined
region of 15 amino acid positions of the parent SH2 domain, the
substituted amino acids increasing the binding affinity of the
modified pTyr binding region for a pTyr-containing peptide relative
to an unmodified pTyr binding region of the parent SH2 domain; and
(b) releasing the pTyr-containing peptides from the complex,
thereby isolating the pTyr-containing peptides.
28. The method of claim 27, wherein the method further comprises
determining the concentration of the pTyr-containing peptides in
the sample by measuring the amount of pTyr-containing peptides
released.
29. A method of determining the concentration of pTyr-containing
peptides in a sample comprising: (a) immobilizing a variant SH2
domain on a resin, the variant SH2 domain varying from a parent SH2
domain, so that relative to the parent SH2 domain, the variant SH2
domain comprises a modified phosphotyrosine (pTyr) binding region
substituted at at least one amino acid position in a pre-defined
region of 15 amino acid positions of the parent SH2 domain, the
substituted amino acids increasing the binding affinity of the
modified pTyr binding region for a pTyr-containing peptide relative
to an unmodified pTyr binding region of the parent SH2 domain, (b)
passing the sample through the resin with the bound variant SH2
domain, (c) releasing any pTyr-containing peptide bound to the
resin by adding a solvent that removes the ability for the variant
SH2 domain to bind to the pTyr-containing peptide thereby creating
elution fractions, and (d) determining the concentration of the
pTyr-containing peptides present in the elution fractions.
30. The method of claim 28, wherein the concentration of
pTyr-containing peptides is determined through high performance
liquid chromatography (HPLC).
31. The method of claim 24, wherein the variant SH2 domain is bound
to an affinity column or onto a lateral flow strip.
32. (canceled)
33. A method of manufacturing a variant SH2 domain having enhanced
binding affinity for a pTyr-containing peptide relative to a parent
SH2 domain, the method comprising substituting at least one amino
acid residue in 15 pre-defined amino acid positions of the parent
SH2 domain that correspond to the positions of Arg18 (position 1),
Lys19 (position 2), Ala21 (position 3), Arg38 (position 4), Ser40
(position 5), Glu41 (position 6), Thr42 (position 7), Thr43
(position 8), Ala46 (position 9), Ser48 (position 10), Leu49
(position 11), Ser50 (position 12), Lys63 (position 13), His64
(position 14), and Lys66 (position 15) of SEQ ID NO:1 when the
parent SH2 domain is aligned with SEQ ID NO:1, the substituted
amino acids increasing the binding affinity of the modified pTyr
binding region for the pTyr-containing peptide relative to an
unmodified pTyr binding region of the parent SH2 domain.
34. (canceled)
35. The method of claim 29, wherein the concentration of
pTyr-containing peptides is determined through high performance
liquid chromatography (HPLC).
36. The method of claim 27, wherein the variant SH2 domain is bound
to an affinity column or onto a lateral flow strip.
37. The method of claim 28, wherein the variant SH2 domain is bound
to an affinity column or onto a lateral flow strip.
38. The method of claim 29, wherein the variant SH2 domain is bound
to an affinity column or onto a lateral flow strip.
39. The method of claim 20, wherein the pre-defined region of 15
amino acid positions of the parent SH2 domain correspond to the
positions of Arg18 (position 1), Lys19 (position 2), Ala21
(position 3), Arg38 (position 4), Ser40 (position 5), Glu41
(position 6), Thr42 (position 7), Thr43 (position 8), Ala46
(position 9), Ser48 (position 10), Leu49 (position 11), Ser50
(position 12), Lys63 (position 13), His64 (position 14), and Lys66
(position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned
with SEQ ID NO: 1.
40. The method of claim 24, wherein the pre-defined region of 15
amino acid positions of the parent SH2 domain correspond to the
positions of Arg18 (position 1), Lys19 (position 2), Ala21
(position 3), Arg38 (position 4), Ser40 (position 5), Glu41
(position 6), Thr42 (position 7), Thr43 (position 8), Ala46
(position 9), Ser48 (position 10), Leu49 (position 11), Ser50
(position 12), Lys63 (position 13), His64 (position 14), and Lys66
(position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned
with SEQ ID NO:1.
41. The method of claim 27, wherein the pre-defined region of 15
amino acid positions of the parent SH2 domain correspond to the
positions of Arg18 (position 1), Lys19 (position 2), Ala21
(position 3), Arg38 (position 4), Ser40 (position 5), Glu41
(position 6), Thr42 (position 7), Thr43 (position 8), Ala46
(position 9), Ser48 (position 10), Leu49 (position 11), Ser50
(position 12), Lys63 (position 13), His64 (position 14), and Lys66
(position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned
with SEQ ID NO:1.
42. The method of claim 29, wherein the pre-defined region of 15
amino acid positions of the parent SH2 domain correspond to the
positions of Arg18 (position 1), Lys19 (position 2), Ala21
(position 3), Arg38 (position 4), Ser40 (position 5), Glu41
(position 6), Thr42 (position 7), Thr43 (position 8), Ala46
(position 9), Ser48 (position 10), Leu49 (position 11), Ser50
(position 12), Lys63 (position 13), His64 (position 14), and Lys66
(position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned
with SEQ ID NO:1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/616,167, filed Mar. 27, 2012, the contents of
which are hereby incorporated by reference into the present
disclosure in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to protein tyrosine
kinase signalling, particularly, the present invention relates to
polypeptides with enhanced binding affinity to
phosphotyrosine-containing peptides or proteins, to methods of
using such polypeptides in treating protein tyrosine
kinase-associated disorders such as immunologic and oncologic
disorders, to methods of using such polypeptides for diagnosing
protein tyrosine kinase-associated disorders, to methods of using
such polypeptides to detect, track or monitor tyrosine
phosphorylation events in cells, to methods of using such
polypeptides to enrich or purify phosphotyrosine-containing
peptides or proteins, and to pharmaceutical compositions including
such polypeptides.
BACKGROUND OF THE INVENTION
[0003] Protein tyrosine kinases (PTKs) and their substrates play a
critical role in numerous cellular processes such as proliferation,
differentiation, motility, and apoptosis. Aberrant kinase
activation and the accompanying changes in the phosphotyrosine
(designated also as pTyr or pY) signaling network are hallmarks of
numerous cancers. A primary mechanism used by the cell to interpret
pTyr-mediated signals relies on modular protein domains that bind
specifically to tyrosine-phosphorylated proteins. The Src homology
2 (SH2) domain is the most prevalent of these modular domains, and
plays a central role in PTK signaling pathways. Different pTyr
sites recruit different SH2 domain-containing proteins, which in
turn, activate different signaling pathways.
[0004] PTKs comprise, inter alia, receptor tyrosine kinases,
including members of the epidermal growth factor kinase family.
Enhanced activities of PTKs have been implicated in a variety of
malignant and non-malignant proliferative diseases. In addition,
PTKs are known to play a role in the regulation of cells of the
immune system.
[0005] PTKs are important drug targets for cancer treatment.
Current anti-cancer drugs are largely based on small-molecule
kinase inhibitors or humanized antibodies. These drugs often
display a broad specificity to a group of related kinases, and
patients eventually develop resistance to the drugs after being on
the treatment for a year or so.
[0006] An alternative idea of inhibiting PTK signaling is blockage
of downstream signaling by masking phosphotyrosine of a PTK
substrate. Although phosphotyrosine-specific antibodies have high
affinity to pTyr-containing polypeptides, they cannot be used
inside of cells.
[0007] The pTyr-specific antibody (U.S. Pat. No. 6,824,989) is
widely used to detect pTyr contained in biological specimen.
However, an antibody cannot be used inside of a living cell. An IgG
antibody molecule is heterotetrameric protein with the total
molecular weight of .about.150 kDa that is secreted to the
extracellular space by B cells in the immune system. An antibody
contains disulfide bonds, works outside of a cell in the immune
system, and is not designed to function in cytoplasm or to
penetrate the cell plasma membrane. Therefore, the pTyr-specific
antibody cannot be used as an in vivo agent for interfering with
intracellular signaling events involving protein tyrosine
phosphorylation inside of living cells.
[0008] SH2 domain containing proteins work downstream of PTK
signalling and are points of signal integration. An SH2 domain
contains .about.100 amino acid and is approximately 15 times
smaller than an antibody molecule. Isolated SH2 domains, when
delivered or expressed in cells, can compete with endogenous
signaling proteins that bind to pTyr sites. However, natural SH2
domains are designed to mediate transient interaction with their
cognate binding sites to assure dynamic cellular signaling. In
other words, a natural SH2 domain is inherently designed not to
block PTK signaling pathways in vivo. Because of this feature, a
natural SH2 domain is not usable as a strong inhibitory
reagent.
[0009] U.S. Pat. No. 5,786,454 ("U.S. 454") discloses SH2 domains
that possess an altered binding site that changes sequence
recognition specificity. It has also been reported that
modifications of the target-binding site of an SH2 domain, that
include deletion, substitution, or introduction of unnatural amino
acids, can change sequence recognition specificity of the SH2
domain (Songyang, et al. (1995) J. Biol. Chem., Vol. 270, pp.
26029; Kimber, et al. (2000) Mol. Cell, Vol. 5, pp. 1043; Kaneko,
et al. (2010) Sci. Signal, Vol. 3, pp. ra34; Virdee et al. (2010)
Chemistry & Biology, Vol. 17, pp. 274). SH2 variants created by
this manner exhibit enhanced specificity for their cognate target
polypeptides in some cases. However, these SH2 variants generally
bind to their cognate target polypeptides with similar affinities
as the corresponding natural SH2 domains.
SUMMARY OF THE INVENTION
[0010] The present invention relates variant SH2 domains having
enhanced binding affinity to phosphotyrosine ("pTyr")-containing
peptides or proteins as compared to a parent SH2 domain (including
to a wild-type SH2 domain), to methods of using such variant SH2
domains in treating protein tyrosine kinase-associated disorders
such as immunologic and oncologic disorders, to methods of using
such variant SH2 domains for diagnosing protein tyrosine
kinase-associated disorders, to methods of using such variant SH2
domains to track tyrosine phosphorylation events in cells, to the
use of such variant SH2 domains as affinity or detection reagents
in research, and to pharmaceutical compositions including such
variant SH2 domains.
[0011] The present invention relates also to a general strategy to
enhance binding affinity of an SH2 domain to pTyr-containing
peptides. Residue substitutions have been introduced to the
pTyr-binding region of an SH2 domain and elucidated favourable
substitutions that enhanced binding affinity to pTyr-containing
peptides. Different combinations of substitutions show different
degrees of impacts in affinity increase, and the generated panel of
variant SH2 domains demonstrated an affinity gradient. These
affinity-enhanced variants showed tighter binding to a
pTyr-containing protein compared to the wild type control SH2
domains in in vitro binding assays and in a mammalian cell line.
Therefore, the variant domains function in physiological
environment as well as in vitro conditions.
[0012] In one embodiment the present invention provides for a
variant SH2 domain for binding a phosphotyrosine (pTyr)-containing
peptide. In one embodiment, the variant SH2 domain includes a
parent SH2 domain having at least one amino acid substitution in a
pre-defined region of 15 amino acid positions of the parent SH2
domain that increases the affinity of the variant SH2 domain for
the pTyr-containing peptide relative to the parent SH2 domain.
[0013] In one embodiment of the variant SH2 domain of the present
invention, the pre-defined region of 15 amino acids of the parent
SH2 domain corresponds to Arg18 (position 1), Lys19 (position 2),
Ala21 (position 3), Arg38 (position 4), Ser40 (position 5), Glu41
(position 6), Thr42 (position 7), Thr43 (position 8), Ala46
(position 9), Ser48 (position 10), Leu49 (position 11), Ser50
(position 12), Lys63 (position 13), His64 (position 14), and Lys66
(position 15) of SEQ ID NO:1 when said parent SH2 domain is aligned
with SEQ ID NO:1.
[0014] In another embodiment of the variant SH2 domain of the
present invention, the at least one substitution includes a
substitution to a small or hydrophobic residue at a position in the
parent SH2 domain corresponding to position 10.
[0015] In another embodiment of the variant SH2 domain of the
present invention, the small or hydrophobic residue includes
alanine, isoleucine, leucine or valine.
[0016] In another embodiment of the variant SH2 domain of the
present invention, the at least one substitution includes
substitution to a hydrophobic residue at a position in the parent
SH2 domain corresponding to position 15.
[0017] In another embodiment of the variant SH2 domain of the
present invention, the hydrophobic residue includes isoleucine,
leucine or valine.
[0018] In another embodiment of the variant SH2 domain of the
present invention, the at least one substitution includes
substitutions at positions in the parent SH2 domain corresponding
to positions 10 and 15.
[0019] In another embodiment of the variant SH2 domain of the
present invention, the at least one substitution includes
substitutions at positions in the parent SH2 domain corresponding
to positions 8 and 15.
[0020] In another embodiment of the variant SH2 domain of the
present invention, the at least one substitution includes
substitutions at positions in the parent SH2 domain corresponding
to positions 8, 10 and 15.
[0021] In another embodiment of the variant SH2 domain of the
present invention, the substitution corresponding to position 8
comprises a substitution to a phenylalanine, an isoleucine, a
proline, or a valine.
[0022] In another embodiment of the variant SH2 domain of the
present invention, the variant SH2 domain includes an arginine
residue in position 4, a leucine residue in position 11 and a
serine residue position 12.
[0023] In another embodiment of the variant SH2 domain of the
present invention, the variant SH2 domain includes an amino acid
sequence selected from: SEQ ID NOs:5-17, 19-22.
[0024] In another embodiment of the variant SH2 domain of the
present invention, the parent SH2 domain is eukaryotic.
[0025] The present invention, in one embodiment, also provides for
an isolated DNA sequence encoding the variant SH2 domains according
to any of the above embodiments.
[0026] The present invention, in one embodiment, also provides for
a vector comprising the DNA sequence of the previous
embodiment.
[0027] In one embodiment, the present invention provides for a use
of the variant SH2 domains of the present invention for the
treatment of a pTyr-containing peptide associated disorder.
[0028] In another embodiment, the present invention provides for a
use of the variant SH2 domains of the present invention for
inhibiting or preventing the effects of a tyrosine kinase in a
cell.
[0029] In another embodiment, the present invention provides for a
method for preventing or inhibiting the effects of a tyrosine
kinase in a cell, characterized in that the method includes
delivering or introducing a variant SH2 domain of the above
embodiments into the cell.
[0030] In one aspect of the present invention, the variant SH2
domain is provided within a carrier that allows transportation
across the cell.
[0031] In another aspect of the present invention, the variant SH2
domain is provided as a fused product to a cell membrane
penetrating molecule.
[0032] The present invention, in another embodiment, provides also
for the use of the variant SH2 domain of the above embodiments for
assessing the presence of pTyr-containing peptides in a sample.
[0033] In one embodiment, the present invention provides for method
of assessing the presence of pTyr-containing peptides in a sample,
the method including (a) contacting said sample to a variant SH2
domain of the present invention, such that a pTyr-containing
peptide/variant SH2 domain complex is formed if the pTyr-containing
peptides are present in the sample; and (b) detecting the formation
of the complex, thereby detecting the presence of the
pTyr-containing peptides in the sample.
[0034] The present invention, in another embodiment, provides also
for the use of the variant SH2 domain of the present invention for
the study of the pTyr-containing peptide signalling pathway and/or
for isolating pTyr-containing peptides.
[0035] In one embodiment, the present invention relates to a method
for isolating pTyr-containing peptides from a sample, characterized
in that the method includes: (a) contacting said sample to a
variant SH2 domain of the present invention such that a
pTyr-containing peptide/variant SH2 domain complex is formed if the
pTyr-containing peptides are present in the sample; and (b)
releasing the pTyr-containing peptides from the complex, thereby
isolating the pTyr-containing peptides from the sample.
[0036] In one aspect of the previous method, the method further
includes determining the concentration of the pTyr-containing
peptides in the sample by measuring the amount of pTyr-containing
peptides released.
[0037] In another embodiment, the present invention provides for a
method of determining the concentration of pTyr-containing peptides
in a sample, the method including: (a) immobilizing a variant SH2
domain of the present invention on a resin, (b) passing the sample
through the resin with the bound variant SH2 domain, (c) releasing
any pTyr-containing peptide bound to the resin by adding a solvent
that removes the ability for the variant SH2 domain to bind to the
pTyr-containing peptide thereby creating elution fractions, and (d)
determining the concentration of the pTyr-containing peptides
present in the elution fractions.
[0038] In aspects of the present invention, the concentration of
pTyr-containing peptides is determined through high performance
liquid chromatography (HPLC).
[0039] In aspects of the present invention the variant SH2 domain
is bound to an affinity column or onto a lateral flow strip.
[0040] The present invention provides, in another embodiment, a use
of the variant SH2 domain of the present invention for the binding
or detection of pTyr residue(s) in a peptide or protein in vitro or
in vivo.
[0041] The present invention, in another embodiment, provides for a
method of manufacturing a variant SH2 domain having enhanced
binding affinity for a pTyr-containing peptide relative to a parent
SH2 domain, characterized in that the method includes substituting
at least one amino acid residue in a pre-defined region of 15 amino
acid positions of the parent SH2 domain, the pre-defined region of
15 amino acids of the parent SH2 domain corresponding to Arg18
(position 1), Lys19 (position 2), Ala21 (position 3), Arg38
(position 4), Ser40 (position 5), Glu41 (position 6), Thr42
(position 7), Thr43 (position 8), Ala46 (position 9), Ser48
(position 10), Leu49 (position 11), Ser50 (position 12), Lys63
(position 13), His64 (position 14), and Lys66 (position 15) of SEQ
ID NO:1 when said parent SH2 domain is aligned with SEQ ID
NO:1.
[0042] In one embodiment, the present invention provides for a
polypeptide comprising multiple SH2 domains, at least one of the
multiple SH2 domains in the polypeptide being a variant SH2 domain
of the present invention.
[0043] These and other aspects of the invention will become
apparent from the detailed description by reference to the
following Figures.
BRIEF DESCRIPTION OF THE FIGURES
[0044] The present invention will become more fully understood from
the detailed description given herein and from the accompanying
drawings, which are given by way of illustration only and do not
limit the intended scope of the invention.
[0045] FIG. 1(A) illustrates the position of the 15 residue
positions surrounding pTyr and a sequence alignment utilized for
defining the positions.
[0046] FIG. 1 (B) shows the binding site of pTyr on the human Fyn
SH2 domain. The atomic coordinates are derived from the Protein
Data Bank ID: 1AOT (Mulhern et al. (1997) Structure, Vol. 5, pp.
1313), which describes the structure of the Fyn SH2 domain and a
bound pTyr-containing peptide. In this figure, only the pTyr
residue within the bound peptide is shown for clarity, in stick
representation. 15 SH2 domain residues surrounding the bound pTyr
are shaded in dark gray. The backbone structure of the SH2 domain
is shown as ribbon representation. Locations of the 15 residues are
displayed with ball representation.
[0047] FIG. 2 (a) shows the position of the 15 residue positions
surrounding pTyr and a sequence alignment utilized for defining the
positions. The 15 positions are defined, according to one
embodiment, on the Fyn SH2 domain (residues shaded black on FIG.
2a). The sequence alignment (FIG. 2a) contains the human Fyn SH2
domain (starting from residue W149), the human Src SH2 domain
(starting from residue W151) and the human Grb2 SH2 domain
(starting from residue W60). The 15 positions on an SH2 domain can
be defined from a sequence alignment that includes the human Fyn
SH2 domain (FIG. 2a). FIG. 2 (b) illustrates one embodiment for
determining the 15 positions when an alignment gap exists in an
alignment. According to the embodiment illustrated in FIG. 2(b)
arginine at position 4 is defined first, and then residues at
position 5, 6, 7, and 8 will be identified as the second, third,
fourth, and fifth residues, respectively, C-terminal to the residue
at position 4. FIG. 2 (c) illustrates a comparison between the
numbering of the 15 positions according to the present invention
and the corresponding numbering system defined by Eck et al. (1993,
Nature, Vol. 362, pp. 87).
[0048] FIG. 3 lists pTyr-containing synthetic peptides used for the
phage display screening experiments of the present invention. These
peptides are biotinylated at their N-terminus and amidated at their
C-terminus.
[0049] FIG. 4 is a table showing residues at the 15 positions in 63
variants of the human Fyn SH2 domain, obtained in accordance to one
embodiment of the present invention. Residues substituted from the
wild type are shaded black.
[0050] FIG. 5 is a table showing a list of substituted residues
observed in the 63 variants of FIG. 4.
[0051] FIG. 6 lists pTyr-containing synthetic peptides used for the
fluorescence polarization assay of the present invention. These
peptides are fluorescein-labeled at their N-terminus and amidated
at their C-terminus.
[0052] FIG. 7 shows results of in-solution fluorescence
polarization binding assay that determines affinity of interaction
between the Fyn SH2 domain and peptides listed in FIG. 5. FIG. 7
(a) shows dissociation constant (Kd) values (in M unit) of
interaction between the peptides and variant Fyn SH2 domains that
contain substitutions indicated in the first row. Affinity increase
relative to the wild type for each peptide-variant combination is
calculated and shown in FIG. 7 (b). The variants are sorted from
left to right according to the average affinity increase.
[0053] FIG. 8 shows binding curve and Kd values of the wild-type or
variant Fyn SH2 domain to a peptide, measured by increase of
fluorescein polarization (.DELTA.FP). FIG. 8 (a) shows binding of
the wild-type Fyn SH2 domain to the fluorescein-GpYGG peptide (SEQ
ID NO: 23). FIG. 8 (b) shows binding of the T8V/S10A/K15L variant
Fyn SH2 domain to the fluorescein-GGpYGG peptide (SEQ ID NO: 23).
FIG. 8 (c) shows no apparent signal observed between the
T8V/S10A/K15L variant SH2 domain and a non-phosphorylated
fluorescein-GGYGG peptide (SEQ ID NO: 24).
[0054] FIG. 9 are tables illustrating results of in-solution
fluorescence polarization binding assay that determines affinity of
interaction between the Src SH2 domain and peptides listed in FIG.
5. FIG. 9 (a) is a table showing Kd values (in M unit) of
interaction between pTyr-containing peptides and the wild-type or
variant Src SH2 domains. Affinity increase relative to the wild
type for each peptide-variant combination is calculated and shown
in FIG. 9 (b).
[0055] FIG. 10 (a) is a graph showing binding curve and Kd values
of the wild-type Src SH2 domain to the fluorescein-GGpYGG peptide
(SEQ ID NO: 23). FIG. 10 (b) is a graph showing binding curve and
Kd values of the T8V/C10A/K15L variant Src SH2 domain to the
fluorescein-GGpYGG peptide (SEQ ID NO: 23).
[0056] FIG. 11 shows effects of the 8V/10A/15L-substituted Fyn,
Grb2 and Src SH2 domains (designated as TrM) in comparison with
wild-type (designated as Wt) domains in cellular signalling
downstream of EGFR. SH2 domains are fused with GFP and expressed in
HEK293 cells. FIG. 11 (a) is a photograph of a Western blotting
showing that TrM SH2 domains bind to EGFR much tighter than Wt
domains. IP: immnoprecipitation, IB: immunoblotting. FIG. 11 (b)
shows Erk phosphorylation is significantly reduced in cells that
express TrM SH2 domains. Erk is located downstream of the EGFR
signaling pathway. FIG. 11 (c) is a graph showing quantification of
the band intensity of pErk in FIG. 11 (b), relative to the GFP
empty vector control set as 100%.
[0057] FIG. 12 shows inhibitory effect of the TrM
(8V/10A/15L-substituted) SH2 domains to the growth of HEK293 cells.
The SH2 domains were expressed as a GFP fusion protein in HEK293
cells. FIG. 12 (a) is a graph showing inhibitory effect on cell
viability relative to the GFP empty vector control. FIG. 12 (b) is
a graph showing inhibitory effect of the 8V/10A/15L-substituted SH2
domains to colony formation observed by the soft agar assay,
quantified relative to the GFP empty vector control. FIG. 12 (c)
are example photos of the colonies quantified in FIG. 12(b). In
FIGS. 12(a) and 12(b), black bars represent TrM and white bars
represent Wt. The numbers are calculated relative to GFP empty
vector control sample (set as 100%).
[0058] FIG. 13 are photographs showing transduction of TAT-SH2
protein in cells. Bacterial expressed, purified TAT-FynSH2 domain
is labeled with FITC and incubated with HEK293 cells at the
indicated concentration (columns) and time (rows). Effective
protein transfusion is observed at 2.5 .mu.M SH2 protein after 1 hr
incubation.
[0059] FIG. 14 shows enhanced ability of the Fyn TrM SH2 domain
compared to the Wt SH2 domain for binding to
tyrosine-phosphorylated proteins, as revealed by the glutathione
S-transferase (GST) pulldown assay. IB: immunoblot. 4G10: antibody
against pTyr (Millipore Co.). MW: molecular weight in the unit of
kilodalton. The top panel shows the result of Western blotting
after the pulldown assay. The bottom panel shows loading control of
GST-tagged proteins immobilized on the glutathione sepharose beads
(GE healthcare) as revealed by the Coomassie staining method.
[0060] FIG. 15 Panel A shows a comparison in ability to pull down a
tyrosyl phosphorylated protein between GST-SrcSH2 TrM and an
anti-pTyr mouse monoclonal antibody (Cell Signaling, #9411) Cell
lysate from the H370 cell line, an HEK293 cell line stably
expressing human anaplastic lymphoma kinase (ALK), was used here.
ALK produces a 220 kDa protein which is then cleaved
proteolytically to yield smaller fragments, including one at 140
kDa. ALK is tyrosine phosphorylated upon stimulation with the
antibody mAb46 (described in Moog-Lutz C, Degoutin J, Gouzi J Y,
Frobert Y, Brunet-de Carvalho N, Bureau J, Creminon C, Vigny M. J
Biol Chem. 2005 Jul. 15; 280(28):26039-48. Epub 2005 May 10). The
top panel shows Western blot result revealed with the anti-pTyr
antibody. IP: immunoprecipitation using anti-pTyr antibody and
Protein-G beads. GST-SH2 domain lanes: samples from GST-pulldown
experiments. hSrc: human Src. vSrc: Rous sarcoma virus Src. Panel B
shows the SrcTrM SH2 domain conjugated with HRP (horseradish
peroxidase) can detect phosphorylated ALK protein species on a PVDF
membrane.
[0061] FIG. 16 demonstrates that the Src SH2 TrM can detect
tyrosine-phosphorylated proteins on a membrane. The U937 human
lymphoma cells were treated with pervanadate (+PV), or without it
(-PV, negative control), and 9 .mu.g lysate was loaded on each lane
of an SDS-PAGE gel, and transferred to the PVDF membrane
(Millipore). GST-Src SH2 TrM bound to phosphorylated proteins on
the membrane, and was visualized by rabbit anti-GST antibody-HRP
conjugate (Sigma-Aldrich #A7340).
[0062] FIG. 17 shows that the Fyn and Src SH2 TrM fused to GFP can
be used to monitor localization of tyrosine-phosphorylated proteins
in live cells. Images show green fluorescence in A549 cells
transfected with GFP-fused TrM SH2 domains or the wild-type (WT)
SH2 controls. TrMSrc SH2 and TrM Fyn SH2 domains exhibit similar
subcellular localization patterns. In comparison, the WT Src SH2
and Fyn SH2 domains are distributed more or less evenly in the cell
with a slight enrichment in the nuclear region. Bar=25 .mu.m for
all pictures.
[0063] FIG. 18 shows that the recombinant TrMSrc SH2 domain
delivered by gold nanoparticles into the A549 cells reduces
viability of the cells under the treatment of EGF.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0064] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Also,
unless indicated otherwise, except within the claims, the use of
"or" includes "and" and vice versa. Non-limiting terms are not to
be construed as limiting unless expressly stated or the context
clearly indicates otherwise (for example "including", "having" and
"comprising" typically indicate "including without limitation").
Singular forms including in the claims such as "a", "an" and "the"
include the plural reference unless expressly stated otherwise.
[0065] The following standard one letter and three letter
abbreviations for the amino acid residues may be used throughout
the specification: A, Ala-alanine; R, Arg-Arginine; N,
Asn-Asparagine; D, Asp-Aspartic acid; C, Cys-Cysteine; Q,
Gln-Glutamine; E, Glu-Glutamxic acid; G, Gly-Glycine; H,
His-Histidine; I, Ile-Isoleucine; L, Leu-Leucine; K, Lys-Lysine; M,
Met-Methionine; F, Phe-Phenylalanine; P, Pro-Proline; S,
Ser-Serine; T, Thr-Threonine; W, Trp-Tryptophan; Y, Tyr-Tyrosine;
and V, Val-Valine.
[0066] "pTyr-containing polypeptide" refers to a molecule that
comprises a pTyr-containing peptide fragment.
[0067] The term "parent SH2 domain" includes any eukaryotic SH2
domain or a polypeptide having at least about 50% sequence identity
to an SH2 domain derived from a human protein that contains an SH2
domain. One hundred and eleven (111) human proteins that contain an
SH2 domain are identified in Liu et al. (2011) Science Signaling,
Vol. 4, pp. ra83 (see Table 1). Sequence identity can be determined
by comparing a position in each sequence of about 100 amino acid
residues which may be aligned for purposes of comparison. The
sequence identity between sequences is a function of the number of
matching positions shared by the sequences. As such, the term
"parent SH2" domain includes also artificially made sequences and
viral SH2 domains. For example, one can generate or design
artificial SH2 domain sequences as parent SH2 domains based on one
or more mammalian SH2 domain sequences, which would represent a
quintessential SH2 domain sequence, but would not be identical to
any mammalian SH2. Another example may be v-Strc, encoded by the
Rous Sarcoma virus, which is a viral homolog of human Src with
little sequence deviation.
[0068] The term "fragment" refers to any subject peptide having an
amino acid residue sequence shorter than that of a peptide whose
amino acid residue sequence is shown herein.
[0069] The term "isolated peptide" or "isolated DNA" may be defined
as a peptide or DNA molecule, as the case may be, which is
substantially separated from other cellular components which may
naturally accompany the peptide and DNA. The term includes, without
limitation, recombinant or cloned DNA isolates and chemically
synthesized analogs or analogs biologically synthesized by
heterologous systems.
[0070] The term "ligand" means a molecule that binds another
molecule or target.
[0071] The term "peptide" or "polypeptide" as used herein is
defined as a chain of amino acid residues, usually having a defined
sequence. As used herein the term "peptide" is mutually inclusive
of the terms "polypeptides", "peptides" and "proteins".
[0072] The terms "variant SH2 domain", "SH2 Variant", "SH2
monobody" are used indistinguishably to refer to a parent SH2
domain that incorporates the substitutions for affinity enhancement
of the present invention. The present invention applies to a
variant SH2 domain of a parent SH2 domain, as well as to a variant
of a fragment of a parent SH2 domain that contains a region between
position 1 and position 15 (as this positions are defined below).
In aspects of the present invention, the use of a variant SH2
domain for clinical or diagnostic use in a human, is preferably
designed from a human SH2 domain as a parent SH2 domain, in order
to minimize the possibility of immune response that may be caused
by supplementation of the variant SH2 domain to the body.
[0073] The priority document and all documents referred to in this
application are incorporated herein by reference in their
entirety.
[0074] Overview of the Invention
[0075] The present invention relates in general to variant of SH2
domains and methods of obtaining said variants. The variant SH2(s)
of the present invention may be used to isolate pTyr-containing
molecules, such as peptides, including polypeptides and proteins,
measuring the concentration of pTyr-containing molecules in a
sample, or merely detecting the presence of pTyr-containing
molecules in a sample. The variant SH2 domains of the present
invention may also be used in other applications such as for
therapeutic, diagnosis or as reagents for research purposes.
[0076] SH2 Domain Variant
[0077] Applicants have invented SH2 variant(s) and a strategy to
enhance binding affinity of an SH2 domain to a pTyr-containing
polypeptide. The strategy of the present invention may include
making single or multiple amino acid residue substitutions on a
parent SH2 domain protein sequence.
[0078] The substitutions are applied to pre-defined 15 amino acid
positions on an SH2 domain. An SH2 domain may be used as a
standard. For example, as a standard, these positions may be
defined on the amino acid sequence of the human Fyn SH2 domain as
illustrated in FIG. 1 and FIG. 2 (a). However, a person of ordinary
skill in the art understands that other SH2 domains may be used as
standards for example the Src SH2 domain, the GRB2 SH2 domain and
so forth. With reference to FIG. 1, the 15 positions correspond to
15 amino acid residues surrounding pTyr in the atomic structure.
These positions may be consecutively numbered from position 1 to
position 15 (FIG. 1 A and FIG. 2 (a)). Corresponding 15 positions
on other SH2 domains may be defined by protein sequence alignment.
Positions 1, 2, 3, 4, 9, 10, 11, 12, 13, 14, and 15 on a parent SH2
domain sequence may be directly identified from an alignment, by
referring to the Fyn SH2 domain as a standard (FIG. 2(a)).
Positions 5, 6, 7 and 8 may also be directly identified from the
alignment. In one embodiment, positions 5, 6, 7, and 8 may be
defined counting from position 4 (FIG. 2(b)). This embodiment may
be used, for example, to avoid potential sequence gap problems in
the BC loop region shown in FIG. 1 A. Positions 5, 6, 7, and 8
correspond to four continuous residues, and a residue at position 5
is located two residues C-terminal to the residue at position 4.
These positions correspond to an SH2 domain sequence nomenclature
system defined by Eck et al. (see FIG. 2 (c)).
[0079] FIG. 5 lists amino acid residues within the 15 positions
from which one or multiple residues of substitutions are chosen for
creating a variant SH2 domain. In one embodiment, the variant
SH2(s) of the present invention may include one residue
substitution. For affinity enhancement, in one embodiment, it may
be favourable to substitute a residue at positions 10 to a small or
hydrophobic residue, including alanine, isoleucine, leucine, or
valine. It may also be favourable to substitute a residue at
position 15 to a hydrophobic residue, including isoleucine,
leucine, or valine.
[0080] For further affinity enhancement, in another embodiment, it
may be favourable to employ two substitutions in a variant SH2
domain. For example, a substitution at positions 1 and 2, or 1 and
5, or 1 and 6 or 1 and 7 and any possible combination between any
two positions that would result in a SH2 variant with enhanced pTyr
binding. In one embodiment, it may be favourable to include
substitutions at positions 10 and 15. It may also be especially
favourable to simultaneously substitute residues at positions 8 and
15 to hydrophobic residues. These substitutions include a residue
at position 8 to phenylalanine, isoleucine, proline, or valine, in
combination with a residue at position 15 to isoleucine, leucine,
or valine.
[0081] For further affinity enhancement, in another embodiment, it
may be favourable to employ three substitutions in a variant SH2
domain. For example, a substitution at positions 1, 2 and 5 or 1, 2
and 6, or 1, 2 and 7 or any possible combination between any three
positions that would result in a SH2 variant with enhanced pTyr
binding. In one embodiment, it may be especially desired to
simultaneously employ the three favourable substitutions at
positions 8, 10, and 15 in a variant domain. More than 3
substitutions within the 15 amino acid residues are also covered by
the present disclosure.
[0082] In one embodiment of the present invention a protein
molecule may be designed to contain multiple SH2 domains, in which
at least one of them is a variant SH2 domain. For example, a
protein that comprises multiple SH2 domains, each of which targets
different pTyr-containing binding site, may be designed and
created. Use of a variant SH2 domain in a multi-SH2 domain
construct further increases binding affinity, toward a target
protein that contains multiple pTyr-containing binding sites. SH2
domains are connected by a flexible linker material, preferably a
polypeptide that contains glycine. Variation of the linker length
and composition further changes binding affinity of a multi-SH2
domain protein. A multi-SH2 domain protein may have increased
affinity to a multi-pTyr region such as the ITAM motif of a single
protein. A multi-SH2 domain protein may also serve to bridge
multiple proteins through pTyr sites in target proteins. Inclusion
of a variant SH2 domain to a multi-SH2 domain protein may result in
increased tightness of binding or bridging.
[0083] The affinity of the SH2 variant(s) of the present invention
to a pTyr-containing polypeptide is fine-tunable by optimizing a
combination of substitutions applied to a parent SH2 domain. In
addition, the affinity enhancement substitutions may be combined
with other substitutions that modify sequence recognition
specificity. Therefore, a variant SH2 domain has an advantage of
tunable variability in binding feature to a target pTyr-containing
sequence, including variable binding affinity, variable sequence
recognition specificity, and modularity to connect multiple domains
in tandem. A variant SH2 domain may gain further variability of
function by incorporating an unnatural amino acid within a domain
sequence. For example, incorporation of a photo-crosslinkable amino
acid, p-Trifluoromethyl-diazirinyl-1-phenylalanine, into a natural
SH2 domain has been reported, that aids mass spectroscopic
detection of direct interaction between the SH2 domain and a target
pTyr-containing protein (Hino et al. 2011 J Mol Biol. Vol. 406, pp.
343). Incorporation of a photo-crosslinkable amino acid into the
target-binding site of a variant SH2 domain can help permanent
blocking of the target pTyr-containing binding site.
[0084] The SH2 monobodies of the present invention may be
synthesized by any known method in the art of peptide synthesis
including solid phase synthesis (Merrifield (1964) J. Am. Chem.
Assoc. 65:2149; J. Amer. Chem. Soc. 85:2149 (1963); and Int. J.
Peptide Protein Res. 35:161-214 (1990)) or synthesis in homogenous
solution (Methods of Organic Chemistry, E. Wansch (Ed.) Vol. 15,
pts. I and II, Thieme, Stuttgart (1987) to generate synthetic
peptides.
[0085] Alternatively, the variant SH2 domains of the invention may
be made by the use of recombinant DNA techniques known to one
skilled in the art. Nucleic acid sequences which encode for the
selected peptides of the invention may be incorporated in a known
manner into appropriate expression vectors (i.e. recombinant
expression vectors). Possible expression vectors include (but are
not limited to) cosmids, plasmids, or modified viruses (e.g.
replication defective retroviruses, adenoviruses and
adeno-associated viruses, lentiviruses; herpes viruses,
poxviruses), so long as the vector is compatible with the host cell
used. The expression "vector is compatible with the host cell" is
defined as contemplating that the expression vector(s) contain a
nucleic acid molecule of the invention (hereinafter described) and
attendant regulatory sequence(s) selected on the basis of the host
cell(s) to be used for expression, said regulatory sequence(s)
being operatively linked to the nucleic acid molecule. "Operatively
linked" is intended to mean that the nucleic acid is linked to
regulatory sequence(s) in a manner which allows expression of the
nucleic acid. Suitable regulatory sequences may be derived from a
variety of sources, including bacteria), fungal, or viral genes.
(For example, see the regulatory sequences described in Goeddel,
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, Calif. (1990). Selection of appropriate
regulatory sequence(s) is dependent on the host cell(s) chosen, and
may be readily accomplished by one of ordinary skill in the art.
Examples of such regulatory sequences include the following: a
transcriptional promoter and enhancer, RNA polymerase binding
sequence, or a ribosomal binding sequence (including a translation
initiation signal). Depending on the host cell chosen and the
expression vector employed, other additional sequences (such as an
origin of replication, additional DNA restriction sites, enhancers,
and sequences conferring inducibility of transcription) may be
incorporated into the expression vector.
[0086] It is further contemplated that the invention encompasses
vectors which comprise nucleic acids coding for at least one SH2
monobody.
[0087] The SH2 monobodies of the present invention may be provided
with a cell membrane penetrating peptide, such as a TAT protein
transduction domain, or an Arg-rich peptide, or another peptide, or
liposomes, or nanoparticles, or any other carrier material that
facilitates the delivery of the SH2 monobodies into cells or
tissues. TAT-fusions have been shown to cross cell membranes and,
in some instances, blood barriers. In this regard, Applicants have
confirmed that purified TAT-SH2 domains (labelled with FITC)
penetrate cells and have half-lives of 2-3 days in cell culture
(see FIG. 13).
[0088] The variant SH2 domains of the invention may be labelled
with a label to facilitate their detection in a variety of assays
as is understood by one of skill in the art. Such labels may
include but are not limited to radioactive label, a cytotoxic label
and fluorescent label. The SH2 monobodies of the invention may be
provided with a carrier such as for example couple to bovine serum
albumin (BSA) or keyhole limpet haemocyanin. The peptides may be
covalently or non-covalently coupled to a solid carrier such as a
microsphere of gold or polystyrene, a slide, chip or to a wall of a
microtitre plate. The peptide may be labelled directly or
indirectly with a label selected from but not limited to biotin,
fluorescein and an enzyme such as horseradish peroxidase. For
example, the variant SH2(s) may be preceded by a Biotin N-terminal
sequence that may facilitate peptide concentration determination by
OD280 (of Tyr or Y) measurement (see FIG. 3).
[0089] The present invention also provides pharmaceutical
compositions comprising a variant SH2 capable of treating a protein
tyrosine kinase-associated disorder in an amount effective
therefor, and a pharmaceutically acceptable carrier, vehicle or
diluent. The pharmaceutical composition may be administered to a
subject in a biologically compatible form for administration in
vivo. The peptides of the invention may be provided within DNA
expression vectors as described above that are formulated in a
suitable pharmaceutical composition.
[0090] By "biologically compatible form suitable for administration
in vivo" is meant a form of the substance to be administered in
which any toxic effects are outweighed by the therapeutic effects.
Administration of a therapeutically active amount of the
pharmaceutical compositions of the present invention, or an
"effective amount", is defined as an amount effective at dosages
and for periods of time, necessary to achieve the desired result. A
therapeutically effective amount of a substance may vary according
to factors such as the disease state/health, age, sex, and weight
of the recipient, and the inherent ability of the particular
polypeptide, nucleic acid coding therefor, or recombinant virus to
elicit a desired response. Dosage regima may be adjusted to provide
the optimum therapeutic response. For example, several divided
doses may be administered daily or on at periodic intervals, and/or
the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation. The amount of variant SH2
for administration will depend on the route of administration, time
of administration and varied in accordance with individual subject
responses.
[0091] The variant SH2s may be administered by any suitable means,
for example, orally, such as in the form of tablets, capsules,
granules or powders; sublingually; buccally; parenterally, such as
by subcutaneous, intravenous, intramuscular, intraperitoneal or
intrasternal injection or infusion techniques (e. g., as sterile
injectable aqueous or non-aqueous solutions or suspensions);
nasally such as by inhalation spray; topically, such as in the form
of a cream or ointment; or rectally such as in the form of
suppositories; in dosage unit formulations containing non-toxic,
pharmaceutically acceptable vehicles or diluents. The present
variant SH2 may, for example, be administered in a form suitable
for immediate release or extended release. Immediate release or
extended release may be achieved by the use of suitable
pharmaceutical compositions comprising the present compounds, or,
particularly in the case of extended release, by the use of devices
such as subcutaneous implants or osmotic pumps. The present
compounds may also be administered liposomally.
[0092] The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance (i.e. SH2 variant
peptide) is combined in a mixture with a pharmaceutically
acceptable vehicle. Suitable vehicles are described, for example,
in "Handbook of Pharmaceutical Additives" (compiled by Michael and
Irene Ash, Gower Publishing Limited, Aldershot, England (1995)). On
this basis, the compositions include, albeit not exclusively,
solutions of the substances in association with one or more
pharmaceutically acceptable vehicles or diluents, and may be
contained in buffered solutions with a suitable pH and/or be
iso-osmotic with physiological fluids. In this regard, reference
can be made to U.S. Pat. No. 5,843,456.
[0093] Pharmaceutical acceptable carriers are well known to those
skilled in the art and include, for example, sterile saline,
lactose, sucrose, calcium phosphate, gelatin, dextrin, agar,
pectin, peanut oil, olive oil, sesame oil and water. Other carriers
may be, for example MHC class II molecules. Soluble MHC class II
molecules including monomers, dimers, trimers, tetramers, etc, as
well as citrulline peptide/MHC class II complexes can be made by
methods disclosed in U.S. Pat. No. 5,869,270 (the disclosure of
which is incorporated herein by reference).
[0094] Furthermore the pharmaceutical composition according to the
invention may comprise one or more stabilizers such as, for
example, carbohydrates including sorbitol, mannitol, starch,
sucrose, dextrin and glucose, proteins such as albumin or casein,
and buffers like alkaline phosphates.
[0095] The compositions of the present invention may contain other
therapeutic agents as described below, and may be formulated, for
example, by employing conventional solid or liquid vehicles or
diluents, as well as pharmaceutical additives of a type appropriate
to the mode of desired administration (for example, excipients,
binders, preservatives, stabilizers, flavors, etc.) according to
techniques such as those well known in the art of pharmaceutical
formulation.
[0096] The variant SH2(s) of the present invention may be employed
alone or in combination with each other and/or other suitable
therapeutic agents useful in the treatment of protein tyrosine
kinase-associated disorders such as PTK inhibitors other than those
of the present invention, antiinflammatories, antiproliferatives,
chemotherapeutic agents, immunosuppressants, anticancer agents and
cytotoxic agents.
[0097] Exemplary such other therapeutic agents include the
following: cyclosporins (e. g., cyclosporin A), CTLA4-Ig,
antibodies such as anti-ICAM-3, anti-IL-2 receptor (Anti-Tac),
anti-CD45RB, anti-CD2, anti-CD3 (OKT-3), anti-CD4, anti-CD80,
anti-CD86, monoclonal antibody OKT3, agents blocking the
interaction between CD40 and gp39, such as antibodies specific for
CD40 and/or gp39 (i. e., CD154), fusion proteins constructed from
CD40 and gp39 (CD40Ig and CD8gp39), inhibitors, such as nuclear
translocation inhibitors, of NF-kappa B function, such as
deoxyspergualin (DSG), non-steroidal anti-inflammatory drugs
(NSAIDs) such as ibuprofen, steroids such as prednisone or
dexamethasone, gold compounds, anti-proliferative agents such as
methotrexate, FK506 (tacrolimus, Prograf), mycophenolate mofetil,
cytotoxic drugs such as azathiprine and cyclophosphamide, TNF-oc
inhibitors such as tenidap, anti-TNF antibodies or soluble TNF
receptor such as etanercept (Enbrel), rapamycin (sirolimus or
Rapamune), leflunimide (Arava), and cyclooxygenase-2 (COX-2)
inhibitors such as celecoxib (Celebrex) and rofecoxib (Vioxx), or
derivatives thereof, and the PTK inhibitors.
[0098] Therapeutic Uses
[0099] The variant SH2 of the present invention inhibit the action
of protein tyrosine kinases, especially Src-family kinases such as
Lck, Fyn, Lyn, Src, Yes, Hck, Fgr and Blk, and may thus be useful
in the treatment, including prevention and therapy, of protein
tyrosine kinase-associated disorders such as immunologic and
oncologic disorders. The variant SH2 domains of the present
invention inhibit also the action of receptor tyrosine kinases
including EGFR and may therefore be useful in the treatment of
proliferative disorders such as psoriasis and cancer. The ability
of these variant SH2 to inhibit EGFR and other receptor kinases may
also permit their use as anti-angiogenic agents to treat disorders
such as cancer and diabetic retinopathy. "Protein tyrosine
kinase-associated disorders" are those disorders which result from
aberrant tyrosine kinase activity, and/or which are alleviated by
the inhibition of one or more of these enzymes. For example, Lck
inhibitors are of value in the treatment of a number of such
disorders (for example, the treatment of autoimmune diseases), as
Lck inhibition blocks T cell activation. The treatment of T cell
mediated diseases, including inhibition of T cell activation and
proliferation, is a particularly preferred embodiment of the
present invention. Compounds which selectively block T cell
activation and proliferation may be preferred. Compounds of the
present invention which block the activation of endothelial cell
PTK by oxidative stress, thereby limiting surface expression of
adhesion molecules that induce neutrophil binding, and which
inhibit PTK necessary for neutrophil activation may be useful, for
example, in the treatment of ischemia and reperfusion injury.
[0100] The present invention thus provides methods for the
treatment of protein tyrosine kinase-associated disorders,
comprising the step of administering to a subject in need thereof a
variant SH2 in an amount effective therefor. Other therapeutic
agents such as those described below may be employed with the
inventive compounds in the present methods. In the methods of the
present invention, such other therapeutic agent (s) may be
administered prior to, simultaneously with or following the
administration of the compound (s) of the present invention. In
embodiments of the present invention, the variant SH2 may be
provided as a fused product to a membrane penetrating peptide such
as a TAT protein transduction domain. The variant SH2 may also be
provided within a carrier that allows transportation across a cell
membrane.
[0101] Use of the variant SH2 of the present invention in treating
protein tyrosine kinase-associated disorders is exemplified by, but
is not limited to, treating a range of disorders such as:
transplant (such as organ transplant, acute transplant or
heterograft or homograft (such as is employed in burn treatment))
rejection; protection from ischemic or reperfusion injury such as
ischemic or reperfusion injury incurred during organ
transplantation, myocardial infarction, stroke or other causes;
transplantation tolerance induction; arthritis (such as rheumatoid
arthritis, psoriatic arthritis or osteoarthritis); multiple
sclerosis; chronic obstructive pulmonary disease (COPD), such as
emphysema; inflammatory bowel disease, including ulcerative colitis
and Crohn's disease; lupus (systemic lupus erythematosis); graft
vs. host disease; T-cell mediated hypersensitivity diseases,
including contact hypersensitivity, delayed-type hypersensitivity,
and gluten-sensitive enteropathy (Celiac disease); psoriasis;
contact dermatitis (including that due to poison ivy); Hashimoto's
thyroiditis; Sjogren's syndrome; Autoimmune Hyperthyroidism, such
as Graves' Disease; Addison's disease (autoimmune disease of the
adrenal glands); Autoimmune polyglandular disease (also known as
autoimmune polyglandular syndrome); autoimmune alopecia; pernicious
anemia; vitiligo; autoimmune hypopituatarism; Guillain-Barre
syndrome; other autoimmune diseases; cancers, including cancers
where Lck or other Src-family kinases such as Src are activated or
overexpressed, such as colon carcinoma and thymoma, and cancers
where Src-family kinase activity facilitates tumor growth or
survival; glomerulonephritis; serum sickness; uticaria; allergic
diseases such as respiratory allergies (asthma, hayfever, allergic
rhinitis) or skin allergies; scleracierma; mycosis fungoides; acute
inflammatory responses (such as acute respiratory distress syndrome
and ishchemia/reperfusion injury); dermatomyositis; alopecia
areata; chronic actinic dermatitis; eczema; Behcet's disease;
Pustulosis palmoplanteris; Pyoderma gangrenum; Sezary's syndrome;
atopic dermatitis; systemic schlerosis; and morphea. The present
invention also provides a method for treating the aforementioned
disorders such as atopic dermatitis by administration of any
compound capable of inhibiting protein tyrosine kinase.
[0102] Src-family kinases other than Lck, such as Hck and Fgr, are
important in the Fc gamma receptor responses of monocytes and
macrophages. Variant SH2 domains of the present invention inhibit
the Fc gamma dependent production of TNF alpha in the monocyte cell
line THP-1 that does not express Lck. The ability to inhibit Fc
gamma receptor dependent monocyte and macrophage responses results
in additional anti-inflammatory activity for the present compounds
beyond their effects on T cells. This activity is especially of
value, for example, in the treatment of inflammatory diseases such
as arthritis or inflammatory bowel disease.
[0103] In particular, the present SH2 monobody(ies) may be of value
for the treatment of autoimmune glomerulonephritis and other
instances of glomerulonephritis induced by deposition of immune
complexes in the kidney that trigger Fc gamma receptor responses
leading to kidney damage.
[0104] In addition, Src family kinases other than Lck, such as Lyn
and Src, are important in the Fc epsilon receptor induced
degranulation of mast cells and basophils that plays an important
role in asthma, allergic rhinitis, and other allergic disease. Fc
epsilon receptors are stimulated by IgE-antigen complexes. Variant
SH2s of the present invention inhibit the Fc epsilon induced
degranulation responses, including in the basophil cell line RBL
that does not express Lck. The ability to inhibit Fc epsilon
receptor dependent mast cell and basophil responses results in
additional anti-inflammatory activity for the present compounds
beyond their effect on T cells. In particular, the present
compounds are of value for the treatment of asthma, allergic
rhinitis, and other instances of allergic disease.
[0105] The combined activity of the present variant SH2 towards
monocytes, macrophages, T cells, etc. may be of value in the
treatment of any of the aforementioned disorders.
[0106] By virtue of their ability to inhibit EGFRs, variant SH2 of
the present invention may also be used for the treatment of
proliferative diseases, including psoriasis and cancer. The HER1
receptor kinase has been shown to be expressed and activated in
many solid tumors including non-small cell lung, colorectal, and
breast cancer. Similarly, the HER2 receptor kinase has been shown
to be overexpressed in breast, ovarian, lung and gastric cancer.
Monoclonal antibodies that downregulate the abundance of the HER2
receptor or inhibit signaling by the HER1 receptor have shown
anti-tumor efficacy in preclincal and clinical studies. It is
therefore expected that inhibitors of the HER1 and HER2 kinases
will have efficacy in the treatment of tumors that depend on
signaling from either of the two receptors. These compounds may be
expected to have efficacy either as single agent or in combination
with other chemotherapeutic agents such as placlitaxel (Taxol),
doxorubicin hydrochloride (adriamycin), and cisplatin (Platinol).
See the following documents and references cited therein: Cobleigh,
M. A., Vogel, C. L., Tripathy, D., Robert, N. J., Scholl, S.,
Fehrenbacher, L., Wolter, J. M., Paton, V., Shak, S., Lieberman,
G., and Slamon, D. J., "Multinational study of the efficacy and
safety of humanized anti-HER2 monoclonal antibody in women who have
HER2-overexpressing metastatic breast cancer that has progressed
after chemotherapy for metastatic disease", J. of Clin. Oncol. 17
(9), p. 2639-2648 (1999); Baselga, J., Pister, D., Cooper, M. R.,
Cohen, R., Burtness, B., Bos, M., D'Andrea, G., Seidman, A.,
Norton, L., Gunnett, K., Falcey, J., Anderson, V., Waksal, H., and
Mendelsohn, J., "Phase I studies of anti-epidermal growth factor
receptor chimeric antibody C225 alone and in combination with
cisplatin", J. Clin. Oncol. 18 (4), p. 904-914 (2000).
[0107] The above other therapeutic agents, which is not exhaustive,
when employed in combination with the compounds of the present
invention, may be used, for example, in those amounts indicated in
the Physicians' Desk Reference (PDR) or as otherwise determined by
one of ordinary skill in the art.
[0108] Diagnosis
[0109] According to another embodiment of the invention, provided
is a method for diagnosing a protein tyrosine kinase associated
disorder in a subject.
[0110] In one embodiment a subject's sample may be contacted with a
SH2 variant of the present invention to measure phosphorylated
proteins in the sample. An increase in the amount of phosphorylated
proteins in the sample relative to the amount of phoshphorylated
proteins in a normal control sample, may be indicative of a protein
kinase associated disorder.
[0111] Tissue samples may include tissue lysates, blood, and other
bodily fluids. The tissue samples may be tested for kinase
activation by using the SH2 variant of the present invention to
detect phosphorylated proteins in the tissue sample. The test may
also be done with tissue histology by using fluorescence-labelled
SH2 variants to image phosphorylated proteins on tissue slices;
ELISA-based, combining SH2 variants with an antibody specific for a
target protein to assay its phosphorylation in normal and disease
tissues (or cells), and so forth.
[0112] Another application is in vive imaging. SH2 variant labelled
with an imaging tag used for in vivo imaging of tumours, PET, MRI,
etc. Cancer tissues characterized with aberrant kinase activation
may display enhanced protein phosphorylation relative to normal
tissues, which can be detected and imaged using SH2 variant-based
imaging tools.
[0113] Another embodiment may include SH2 profiling based on Bruce
Mayer's method (U.S. Pat. No. 7,846,746), to compare binding
profiles of normal and disease cell lysates. Yet another embodiment
may include injecting a radiolabeled and maybe TAT-tagged variant
SH2 domain to a cancer patient to detect SH2 accumulation to a
tumor site in the patient's body.
[0114] To detect the SH2 variant in the samples, the variant SH2
domain of the present invention may preferably be labelled with a
probe molecule.
[0115] Detection of pTyr-positive cells may be carried out by a
probe. The probe may include at least a peptide comprising a SH2
variant and an imaging component. Optionally, this probe may be
labelled with a detectable marker which may allow detection of the
location of the pTyr-positive cells. The probe of the present
invention may allow following movement and development of
pTyr-positive cells.
[0116] Methods of preparing probes are well known to those of skill
in the art (see, e.g. Sambrook et al, Molecular Cloning: A
Laboratory Manual (2nd ed.), VoIs. 1-3, Cold Spring Harbor
Laboratory, (1989) or Current Protocols in Molecular Biology, F.
Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New
York (1987)), which are hereby incorporated by reference.
[0117] The imaging component of the probe may generally comprise a
label. Methods of labelling are well known to those of skill in the
art. Preferred labels may be those which are suitable for use in in
vivo imaging. The SH2 monobody probes may be detectably labelled
prior to detection. Alternatively, a detectable label which may
bind to the hybridization product may be used. Such detectable
labels may include, without limitation, any material having a
detectable physical or chemical property and have been
well-developed in the field of immunoassays. A label for use in the
present invention may be any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means.
[0118] Labels which may be used in the present invention include
biotin-based label, magnetic label (e.g. DYNABEADS.TM.),
radioactive label (e.g. .sup.3H, .sup.35S, .sup.32P, .sup.51Cr, or
.sup.125I), fluorescent label (e.g. fluoroscein, rhodamine, Texas
Red, etc.), electron-dense reagents (e.g. gold), enzymes (e.g.
alkaline phosphatase, horseradish peroxidase, or others commonly
used in an ELISA), digoxigenin, or haptens and proteins for which
antisera or monoclonal antibodies may be available (for example the
peptides of the present invention can be made detectable by, for
example, incorporating a radiolabel into the peptide, and used to
detect antibodies specifically reactive with the peptide). The
Variant SH2 of the invention may be provided with a carrier such as
for example coupled to bovine serum albumin (BSA) or keyhole limpet
haemocyanin. The variant SH2 may be covalently or non-covalently
coupled to a solid carrier such as a microsphere of gold or
polystyrene, a slide, chip or to a wall of a microtitre plate. The
variant SH2 may be labelled directly or indirectly with a label
selected from but not limited to biotin, fluorescin and an enzyme
such as horseradish peroxidase.
[0119] The particular label used may not be critical to the present
invention, so long as it does not interfere with the affinity of
the SH2 variant for the pTyr. However, in one embodiment, the
imaging component may be a radionuclide (e.g. .sup.18F, .sup.11C,
.sup.13N, .sup.64Cu, .sup.68Ga, .sup.123I, .sup.111In, .sup.99mTc,
etc.) due to the ease of using such techniques as SPECT, CT and PET
imaging for in vivo detection of SH2 variant-pTyr complexes and
tumor cells. Decision as to appropriate imaging component for
agents used in SPECT or PET imaging may also be determined by
whether the radionuclide is generated by generator or cyclotron or
is an chelator or organic/halide.
[0120] A direct labelled probe, as used herein, may be a probe to
which a detectable label is attached. Because the direct label is
already attached to the probe, no subsequent steps may be required
to associate the probe with the detectable label. In contrast, an
indirect labeled probe may be one which bears a moiety to which a
detectable label is subsequently bound, typically after the SH2
variant peptide is bound with the target pTyr.
[0121] In another embodiment, monoclonal antibodies (mab) which
recognize any of the variant SH2 of the invention may also be made
and used to detect the presence of the variant SH2 in a sample. Mab
may provide a rapid and simple method of testing the compositions
of the invention for their quality. In general, methods for the
preparation of antibodies are well known. For example, methods to
produce mab which specifically recognize the Variant SH2 of the
invention are well known to those of skill in the art. In general,
peptides are injected in Freund's adjuvant into mice. After being
injected 9 times over a three week period, the mice spleens are
removed and re-suspended in phosphate buffered saline (PBS). The
spleen cells may serve as a source of lymphocytes, some of which
may be producing antibody of the appropriate specificity. These may
then fused with a permanently growing myeloma partner cell, and the
products of the fusion may be plated into a number of tissue
culture wells in the presence of a selective agent such as HAT. The
wells may then be screened to identify those containing cells
making useful antibody by ELISA. These may then be freshly plated.
After a period of growth, these wells may again be screened to
identify antibody-producing cells. Several cloning procedures may
be carried out until over 90% of the wells contain single clones
which are positive for antibody production. From this procedure a
stable lines of clones may be established which produce the mab.
The mab may then be purified by affinity chromatography using
Protein A or Protein G Sepharose (see also, U.S. Pat. Nos.
4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117; and
4,720,459).
[0122] Research
[0123] In one embodiment, the variant SH2 domains of the present
invention may be used as reagents. In particular embodiments, a
variant SH2 domain, or a gene that encodes the variant SH2 domain,
may be introduced into a mammalian cell line. A variant SH2 domain
that exhibits super-high affinity to a target pTyr site (Kd value
smaller than about 10 nM) may act to mask the target pTyr site and
may cause severe blocking effects of PTK signalling events
downstream of the pTyr site. Therefore, such variant SH2 domains
may serve as an inhibitory reagent of cellular PTK signalling
pathway. Super-high affinity variant SH2 domains derived from
different natural SH2 domains exhibit distinct sequence recognition
specificity. Consequently, a super-high affinity variant SH2
domain, when introduced in a live cell, may block a specific
signalling pathway, and may be used as a reagent for investigating
physiology of a particular pathway.
[0124] SH2 variants of the present invention having super-high
affinity for pTyr may be used as substitutes for an anti-pTyr
antibody and may be used in research areas where an anti-pTyr
antibody is used, such as, for example, Western blots, IF,
proteomics (enrichment of phosphoproteins/peptides), and so
forth.
[0125] In one embodiment, variant SH2 domains which exhibit
moderately enhanced affinity (variants that show enhanced affinity
compared to the wild type, but preferably with a Kd value grater
than about 10 nM to a target pTyr site) may be produced in
accordance to the present invention. These variant SH2 domains do
not have an ability to completely block a pTyr site and its
downstream signalling, but they may retain inherent sequence
recognition specificity of a parent SH2 domain to which amino acid
substitutions are applied. Therefore, these variant SH2 domains may
be used as tracers of particular tyrosine phosphorylation events in
cells. To detect the tracer SH2 domain in cells, the SH2 domain may
preferably be labelled with a probe molecule, as explained
above.
[0126] Purification, Presence and Concentration of pTyr-Containing
Targets
[0127] Another embodiment of the present invention includes the use
of the variant SH2 polypeptides of the present invention as ligands
for isolation, purification, detecting the presence and/or
determination of the concentration of molecular targets having a
pTyr in a sample. In one embodiment, a method for determining the
presence/concentration of a target having a pTyr in a sample may
comprise: (a) contacting the sample to a variant SH2 peptide of the
present invention (the "SH2 ligand"), such that a target/SH2 ligand
complex is formed if the target is present in the sample; and (b)
determining the concentration of the pTyr-containing target in the
sample by measuring the amount of target/SH2 ligand complex
formed.
[0128] In another embodiment, the present invention provides for a
method for isolating a pTyr-containing target in a sample. The
method may comprise: (a) contacting the sample to a SH2 ligand of
the present invention, such that a pTyr-containing target/SH2
ligand complex is formed if the target is present in the sample;
and (b) releasing the pTyr containing target from the complex,
thereby isolating the pTyr-containing target. The concentration of
the target in the sample may then be obtained by measuring the
amount of pTyr-containing target released.
[0129] In aspects, the SH2 ligand may be immobilized on a resin,
such as an affinity column, and the sample, which may include
fluids such as bodily fluids and extracts, may be passed through
the resin. In aspects, the resin may be washed with a solution free
of target. The pTyr-containing target bound to the SH2 ligand may
be released by adding a solvent that removes the ability for the
SH2 ligand to bind to the target thereby creating elution
fractions. The presence and/or concentration of the target present
in the elution fractions may be determined by any appropriate
method, such as, for example, fluorescence, high performance liquid
chromatography, and so forth. This method may also be used to
isolate a pTyr-containing target from a sample. The SH2 ligand may
also be bound onto a lateral flow strip.
[0130] The presence or concentration of pTyr-containing molecules
such as peptides, including polypeptides and proteins, may be
determined through high performance liquid chromatography (HPLC).
The SH2 ligand may be bound to an affinity column or onto a lateral
flow strip.
[0131] In one embodiment of the present invention the variant SH2
may be used in methods to identify cells with enhanced protein
phosphorylation relative to a control. One such method may comprise
using one or more of the variant SH2 to detect for the presence of
pTyr-positive cells in a sample.
[0132] Advantages
[0133] Advantages of the present invention include:
[0134] (1) Unlike anti-pTyr antibodies, the variant SH2 peptides of
the present invention are single polypeptides with relative smaller
size (.about.12 kDa) than antibody, that are suitable as molecular
drugs or reagents, and with the ability to work inside a live cell,
like natural SH2 domains that function in cytoplasm. In addition,
unlike the pTyr-specific antibody, a variant SH2 peptide of the
present invention is equipped with sequence recognition
specificity, and therefore it can detect only specific
pTyr-containing molecules as targets of intervention.
[0135] (2) Another advantage of using an SH2 domain of the present
invention includes ease of production and modification of the
domain. An SH2 domain comprises .about.100 amino acid residues and
is suitable for recombinant protein production in a standard
expression system including bacterial, yeast, and mammalian cells.
In particular, about two-third out of 120 human SH2 domains were
reportedly produced in Escherichia coli as a recombinant form
(Huang et al. 2008, Mol Cell Proteomics, Vol. 7, pp. 768; Machida
et al. 2007, Mol Cell, Vol. 26, pp. 899). Virdee et al. reported
synthesis of an SH2 domain and incorporation of a non-natural amino
acid by conjugating multiple polypeptide fragments (Virdee et al.
Chemistry & Biology, 2010, Vol. 17, pp. 274). The cell-free
expression system has also been used for production of SH2 domains
(He and Taussig, 2007, Biochem Soc Trans, Vol. 35, pp. 962; Scott
et al. 2004, J Biomol NMR, Vol. 30, pp. 463). The amino acid
substitutions proposed herein are fully compatible to be
incorporated into existing SH2 domain production technologies,
including those mentioned above.
[0136] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific Examples. These Examples are
described solely for purposes of illustration and are not intended
to limit the scope of the invention. Changes in form and
substitution of equivalents are contemplated as circumstances may
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitation.
EXAMPLES
[0137] The examples are described for the purposes of illustration
and are not intended to limit the scope of the invention.
Example 1--Identification of Variant SH2 Domains by the Phage
Display Technology
[0138] The amino acid residues of 15 positions on the human Fyn SH2
domain were randomly substituted to one of 20 natural amino acids
to identify variant SH2 domains that bind to pTyr-containing
peptides. All amino acid residue numbers of the human Fyn SH2
domain are in accordance with the full-length Isoform 1 of the
UniProt database entry FYN_HUMAN. A gene that encodes the wild type
Fyn SH2 domain between Ala139 and Gly249, the amino acid sequence
of which is provided in SEQ ID NO: 1, was subcloned into the
pDEST15 vector (Invitrogen Canada Inc.). The three cysteine
residues in SEQ ID NO:1 were replaced with serine residues by the
QuikChange II site directed mutagenesis kit (Qiagen Inc.). This
mutagenesis generated a gene provided in SEQ ID NO: 2. The gene
shown in SEQ ID NO:2 encodes a protein sequence provided in SEQ ID
NO: 3. SEQ ID NO:3 comprises a fragment of the wild type human Fyn
SH2 domain between Ala139 and Leu238, continued by a polypeptide
sequence SSRLVVPSHKG (SEQ ID NO: 25), in which the three serine
residues were replaced from cysteine residues present in SEQ ID NO:
1. The gene provided in SEQ ID NO: 2 was fused to the gene encoding
the M13 bacteriophage major coat protein. Simultaneous
randomization on the pre-defined 15 amino acid positions, specified
below, was performed with the Kunkel method (Sidhu, et al. 2000,
Method Enzymol. Vol. 328, pp. 333). These 15 positions correspond
to wild-type, full-length human Fyn SH2 residues Arg156 (position
1), Lys157 (position 2), Ala159 (position 3), Arg176 (position 4),
Ser178 (position 5), Glu179 (position 6), Thr180 (position 7),
Thr181 (position 8), Ala184 (position 9), Ser186 (position 10),
Leu187 (position 11), Ser188 (position 12), Lys201 (position 13),
His202 (position 14), and Lys204 (position 15) (FIG. 2a). In SEQ ID
NO:1 these 15 positions are as follows: Arg18 (position 1), Lys19
(position 2), Ala21 (position 3), Arg38 (position 4), Ser40
(position 5), Glu41 (position 6), Thr42 (position 7), Thr43
(position 8), Ala46 (position 9), Ser48 (position 10), Leu49
(position 11), Ser50 (position 12), Lys63 (position 13), His64
(position 14), and Lys66 (position 15). The mutagenesis generated a
library of Fyn SH2 domains that contain randomly substituted amino
acid residues on the 15 positions. The phage display method was
employed to display these Fyn SH2 domains that incorporated the
substitutions, on the surface of M13 bacteriophage. The phages were
screened against 33 immobilized pTyr-containing synthetic peptides
listed in FIG. 3. Each peptide was synthesized on the TentaGel
amide resin (INTAVIS Inc.) and N-terminally labeled with biotin.
Phages that bound to at least one of these peptides were forwarded
to DNA sequencing, to identify sequences of variant SH2 domains
that bound to a peptide. Accordingly, 63 variant Fyn SH2 domains
were identified. The residues on the 15 positions in each variant
SH2 domain are listed in FIG. 4. In each variant, at least one
position contained an amino acid substitution from the wild-type
residue, which are shaded black in FIG. 4. No variant SH2 domain
was identical to the wild-type SH2 domain that has SEQ ID NO:3.
This indicates that all of the variant SH2 domains gained more
favorable sequence composition for enhanced binding to
pTyr-containing peptides compared to the wild-type SH2 domain.
Substitutions observed in the variant SH2 domains are listed
together in FIG. 5. Therefore, we identified amino acid
substitutions, applied to an SH2 domain, that enhance binding to
pTyr-containing polypeptides.
Example 2--Enhanced Binding of Variant Fyn SH2 Domains to
pTyr-Containing Peptides In Vitro
[0139] Single or multiple substitutions were introduced to the
wild-type Fyn SH2 domain and determined degrees of affinity
enhancement derived by introduction of the substitutions. The gene
of SEQ ID NO: 2 was N-terminally fused with a gene coding a
hexa-histidine tag, which resulted in a construct that expresses
SEQ ID NO: 4, which includes a hexa-histidine tag, the wild type
Fyn SH2 domain corresponding to the region between Ala139 and
Leu238, continued by a polypeptide with a sequence SSRLVVPSHKGAAA
(SEQ ID NO: 26). Using this wild-type construct as a template, 13
variant Fyn SH2 domains were constructed, by the directed
mutagenesis method. In following descriptions, position numbers are
used to specify residues to be substituted. For example, T8V
indicates a substitution of Thr at position 8 to Val, applied to
the wild type construct. In another example, T8V/S10A/K15L
indicates a combination of three substitutions, Thr at position 8
to Val, Ser at position 10 to Ala, and Lys at position 15 to Leu,
applied to the wild-type construct. Amino acid sequences between
position 1 and position 15, of the 13 variant SH2 domains are
listed in the SEQUENCE LISTING section. By applying substitutions
to the wild type construct (SEQ ID NO:4), following variant Fyn SH2
domains were constructed: T8V (SEQ ID NO:5), S10V (SEQ ID NO:6),
.DELTA.T8/S10A/K15L (SEQ ID NO:7), S10A (SEQ ID NO:8), K15L (SEQ ID
NO:9), S10V/K15L (SEQ ID NO:10), K2E/T8V/S10A/K15I (SEQ ID NO:11),
T7S/S10A/K15L (SEQ ID NO:12), S10A/K15L (SEQ ID NO:13),
T8V/S10A/K15I (SEQ ID NO:14), T8V/K15L (SEQ ID NO:15), T8/S10A/K15L
(SEQ ID NO:16), and T8V/S10A/K15L (SEQ ID NO:17). .DELTA.T8 denotes
deletion of Thr at position 8.
[0140] A set of peptides that contain pTyr, and one peptide that
does not contain pTyr, were synthesized for binding assay (FIG. 6).
Each peptide was synthesized on the TentaGel amide resin (INTAVIS
Inc.) and N-terminally labeled with fluorescein using
NHS-fluorescein (Thermo Fisher Scientific). Each of the 13 variant
and the wild-type SH2 domain was produced by overexpression in the
Escherichia coli BL21(DE3) strain cultured in LB media (EMD
Chemicals). Expression was induced by 0.3 mM isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG), and the cell culture was
incubated for 5 hours at 37.degree. C. The cells were harvested by
centrifuge, and broken on ice, in buffer solution containing 20 mM
sodium phosphate, 0.1 M NaCl, 20 mM imidazole, 1 mg/ml lysozyme and
1% Triton-X 100, adjusted at pH 7.8. Affinity purification was
performed with the Ni-NTA agarose resin (Qiagen Inc.), according to
the manufacturer's instruction. The purified material was dialyzed
against phosphate-buffered saline (PBS), pH 7.4, at 4.degree. C.
overnight. Measurements of fluorescence polarization were conducted
by titrating the SH2 domain concentration, while keeping the
peptide concentration constant. Kd values were calculated with the
GraphPad Prism software, by assuming the one-site binding model
(GraphPad Software).
[0141] The determined Kd values of interaction between the 14 SH2
domains (including the wild-type) and seven peptides were listed in
FIG. 7(a). Fold increase of affinity enhancement was calculated as
Kd[wild-type] divided by Kd[variant], and listed in FIG. 7(b).
Compared to the wild type Kd values, which range between the orders
of 10.sup.-5 M and 10.sup.+7 M (submicromolar affinity), Kd values
of the T8V/S10A/K15L variant SH2 domain range between the orders of
10.sup.-7 M and 10.sup.-9 M (nanomolar affinity) to the same set of
seven peptides (FIG. 7(a)). In average, this corresponds to
293-fold affinity increase (FIG. 7(b)). In particular, binding to
the EGFR-pY978 peptide showed over 1000-fold affinity increase. The
inventors also compared binding of the wild-type and the
T8V/S10A/K15L variant SH2 domains to a short pTyr-containing
peptide, the GGpYGG peptide (SEQ ID NO: 23). Affinity to this
peptide increased from a Kd value of 67 .mu.M (FIG. 8(a)) to 0.82
.mu.M (FIG. 8(b)). However, the non-phosphorylated peptide, the
GGYGG peptide (SEQ ID NO: 24), did not show detectable binding to
the same variant SH2 domain (FIG. 8(c)). This indicates that the
combination of three substitutions, namely T8V, S10A, and K15L,
contributes to enhancement of binding to the pTyr amino acid
included in the tested peptides. Therefore, the inventors created a
variant SH2 domain that demonstrated significantly enhanced
affinity to pTyr-containing peptides.
[0142] In addition to the abovementioned variant SH2 domain, 12
other variant SH2 domains also showed enhanced affinity to the
seven peptides. Each of these variant SH2 domains contains
different single of multiple substitutions, selected from the amino
acid substitution list appeared in FIG. 5. It was shown that
different substitutions resulted in different degrees of affinity
enhancement. In average, the effect of enhancement ranges between
1.4-fold (the T8V variant) to 86.4-fold (the T8I/S10A/K15L variant)
increase (FIG. 7(b)). Therefore, the inventors obtained a panel of
variant SH2 domain that produced a gradient of affinity enhancement
to pTyr-containing peptides.
Example 3--Affinity Enhancement Observed by Introducing
Substitutions to the Src SH2 Domain
[0143] In this section, the inventors demonstrate that the
substitutions established on the human Fyn SH2 domain also worked
on another SH2 domain for affinity enhancement.
[0144] The gene encoding the human Src SH2 domain between Asp144
and Lys252, residue numbers of which are in accordance with the
UniProt entry SRC_HUMAN, was subcloned into the vector pETM-11
(Dummler, et al. 2005, Microb Cell Fact., Vol. 4, pp. 34). The
resultant construct encodes a protein of SEQ ID NO:18, which
comprises an N-terminal hexa-histidine affinity tag, a Tobacco Etch
Virus protease cleavage site, and the Src SH2 domain. Next, a
sequence alignment that contains the sequences of the human Fyn and
Src SH2 domains was generated using the program PROMALS3D (Pei, et
al., 2008, Nucleic Acids Research, Vol. 36, pp. W30) (FIG. 2a). The
15 positions were identified on the wild-type Src SH2 domain
sequence, based on the aligned positions defined on the Fyn SH2
domain. For example, in the Src SH2 domain sequence, Thr183
corresponds to position 8, Cys188 corresponds to position 10, and
Lys206 corresponds to position 15. Expression constructs for three
variant Src SH2 domains were created by site directed mutagenesis.
The polypeptide sequences for the region between position 8 and
position 15 for each of the three variant Src SH2 domains were
listed in the SEQUENCE LISTING, where SEQ ID NO:19 corresponds to
the K15L variant SH2 domain, SEQ ID NO:20 corresponds to the
T8V/C10A variant SH2 domain, and SEQ ID NO:21 corresponds to the
T8V/C10A/K15L variant SH2 domain. The three amino acid residue
substitutions, a residue at position 8 to Val, a residue at
position 10 to Ala, and a residue at position 15 to Leu, are
derived from the list of favorable substitutions, originally
elucidated from the Fyn SH2 domain phage display experiments (FIG.
5).
[0145] The wild-type and variant Src SH2 domains were expressed in
E. coli BL21(DE3) strain grown in the LB media, by inducing protein
expression with 0.3 mM IPTG, and incubating the cell culture at
30.degree. C. for six hours. Cells were harvested by centrifuge,
and broken on ice in buffer solution containing 20 mM sodium
phosphate, 0.1 M NaCl, 20 mM imidazole, 1 mg/ml lysozyme, 1%
Triton-X 100, adjusted at pH 7.8. Affinity purification was
performed with the Ni-NTA agarose resin, according to the
manufacturer's instruction. The materials eluted from the resin
were dialyzed against buffer solution containing 20 mM Tris-HCl, pH
7.0, 1 mM dithiothreitol (DTT), 0.5 mM ethylendiaminetetraacetic
acid (EDTA), and 50 mM NaCl, at 4.degree. C., overnight. Each of
the dialyzed samples was supplemented with the Tobacco Etch Virus
protease (Tropea, et al. 2009, Methods Mol Biol. Vol. 498, pp.
297), for cleavage of the hexahistidine tag, and incubated at room
temperature, overnight. The samples were further purified with the
Superdex75 size exclusion column (GE Healthcare), with buffer
solution comprising 20 mM Tris-HCl, pH 7.0, 1 mM DTT, and 150 mM
NaCl.
[0146] Fluorescence polarization assay was conducted to determine
Kd values of the interaction between the wild-type or variant Src
SH2 domain and seven fluorescein-labeled pTyr-containing peptides
(FIG. 9a). Fold increase of Kd values was calculated and listed in
FIG. 9b. All the three variant SH2 domains showed affinity increase
compared to the wild type SH2 domain. The T8V/C10A/K15L variant Src
SH2 domain bound to three peptides, namely the EGFR-pY978,
MidT-pY324, and ShcA-pY239 peptides, with Kd values in the order of
10-9 M (nanomolar affinity). This variant Src SH2 domain showed an
average of 238-fold affinity increase from the wild type SH2
domain. This amount of affinity increase is similar to the 293-fold
affinity increase demonstrated for the T8V/S10A/K15L variant Fyn
SH2 domain (FIG. 7(b)). Furthermore, the T8V/C10A/K15L variant Src
SH2 domain showed binding to the GGpYGG peptide (SEQ ID NO: 23)
with a Kd value of 0.51 .mu.M, which indicates much higher affinity
than the wild type Src SH2 domain to the peptide (FIG. 10).
Therefore, the combination of three substitutions, a residue at
position 8 to Val, a residue at position 10 to Ala, and a residue
at position 15 to Leu, applied to different SH2 domains, which
resulted in similar significant effects of affinity enhancement to
pTyr-containing peptides.
Example 4--Binding of Variant S12 Domains to EGFR and Inhibition of
Downstream Signaling in Mammalian Cells
[0147] Binding of the epidermal growth factor (EGF) induces
dimerization of a receptor tyrosine kinase, the EGF receptor
(EGFR), and trans-autophosphorylation on multiple tyrosine residues
in the C-terminal tail. These pTyr-containing sites recruit
downstream proteins that contain an SH2 domain, including Grb2.
Deregulation in the expression or activity of EGFR is associated
with many epithelial cancers. The MAP kinase pathway (the
Ras-Raf-MEK-Erk protein signalling), when activated downstream of
EGFR, leads to cell proliferation; however, when over-activated, it
leads to cellular transformation or invasive behaviour (Kim &
Choi, (2010) Biochimnica et biophysica acta, Vol. 1802, pp. 396).
Here, we further demonstrate that the substitutions defined on the
Fyn SH2 domain worked on other SH2 domains for affinity
enhancement. In addition, the variant SH2 domains created from
different parent SH2 domains, when expressed in mammalian cells,
showed enhanced binding to EGFR. Furthermore, the variant SH2
domains expressed in mammalian cells inhibited cellular signaling
events downstream of EGFR.
[0148] The gene encoding a polypeptide between Met55 and Pro158 of
the human Grb2, where the residue numbers are in accordance with
the UniProt entry GRB2_HUMAN, was subcloned into the pEGFPC2 vector
using the XhoI and BamHI restriction sites. The amino acid sequence
of Grb2 was aligned with the Fyn sequence, to identify positions on
the Grb2 sequence (FIG. 2(a)). Accordingly, amino acid residues at
position 8, 10, and 15 were identified as Ala91, Ser96, and Lys109,
respectively. These residues at the three positions were
substituted with Val, Ala, and Leu, respectively, using the
site-directed mutagenesis method, to generate a construct of the
A8V/S10A/K15L variant Grb2 SH2 domain, the sequence of which,
comprising the region between Met55 and Pro158 with the A8V, S10A,
and K15L substitutions, was shown in SEQ ID NO: 22.
[0149] The genes encoding the wild-type Fyn SH2 domain, the
wild-type Src SH2 domain, the wild-type Grb2 SH2 domain, the
T8V/S10A/K15L variant Fyn SH2 domain, the T8V/C10A/K15L variant Src
SH2 domain, and the A8V/S10A/K15L variant Grb2 SH2 domain were,
respectively, subcloned into the pEGFPC2 vector (Clontech) using
the XboI and BamHI restriction sites.
[0150] The three variant SH2 domains mentioned in the previous
sentence were dubbed as 8V/10A/15L-substituted SH2 domains, or the
TrM SH2 domains, in the following descriptions, as well as in FIG.
11 and FIG. 12. The wild-type (Wt) and TrM SH2 domains, subcloned
into the pEGFPC2 vector, were expressed in the HEK 293 cells as a
GFP-fusion protein. Methods used in this and the following sections
are described below.
[0151] Anti-GFP rabbit polyclonal antibody was purchased from
Sigma-Aldrich. Anti-EGFR rabbit polyclonal antibody was purchased
from Millipore. Anti-p44/42 MAPK (Erk1/2) mouse monoclonal
antibody, and anti-phospho-p44/42 MAPK (Thr202/Tyr204) mouse
monoclonal antibody were purchased from Cell Signaling. HEK 293
cells were grown in Dulbecco's modification of Eagle's medium
(DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum
(FBS, SAFC Biosciences), 50 units/mil penicillin and 50 .mu.g/ml
streptomycin (GIBCO Invitrogen Corp.) in a humidified atmosphere of
5% CO.sub.2 in air at 37.degree. C. EGF (Invitrogen) at a final
concentration of 100 ng/ml was added to the medium at the indicated
time point. Transient transfections were carried out with jet-PEI
(PolyPlus-Transfection; Illkirch, France) according to
manufacturer's instruction.
[0152] HEK 293 cells were transfected with the SH2 domain construct
subcloned in the pEGFPC2 vector, and incubated in serum-containing
full medium for 24 h followed by serum-starvation for 16 h and then
EGF (100 ng/ml) treatment for 10 min, pervanadate treatment for 10
min. The whole cell lysates were prepared and subjected to IB
analysis to detect phosphorylated Erk and total Erk.
[0153] HEK 293 cells were lysed with cold lysis buffer (1% NP-40,
50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10%
Glycerol and protease inhibitor cocktail diluted at 1:1000). After
centrifugation at 13,000*g for 15 min, the supernatants were
collected. After clearance of the lysate with appropriate
pre-immune serum and protein G (Roche), immnuoprecipitation (IP)
and immunoblotting (IB) were performed.
[0154] All TrM SH2 domains tagged with GFP bound significantly to
the EGFR (FIG. 11(a)). The binding was observed with
immunoprecipitation (IP) assay using the anti-GFP and anti-EGFR
antibodies. On the contrary, the Wt Fyn and Src SH2 domains did not
show detectable binding to EGFR. Weak binding of the Wt Grb2 SH2
domain was observed, which is reasonable because EGFR is a
physiological binding target of the Grb2 SH2 domain. Similar to the
TrM Fyn and Src SH2 domain, the TrM Grb2 SH2 domain exhibited
strong binding to EGFR, with significantly higher affinity than the
Wt Grb2 SH2 domain. Therefore, we demonstrated that our strategy of
affinity enhancement worked efficiently by applying it to the Grb2
SH2 domain.
[0155] Next, we observed effects of expression of TrM SH2 domains
towards signaling events downstream of EGFR in mammalian cells. The
phosphorylation level of the downstream protein Erk was observed by
Western blotting with anti-phospho-Erk (pErk) antibody. Significant
reduction of the pErk level was observed for HEK293 cells that
express TrM SH2 domains, compared to Wt SH2 domains (FIG. 11(b),
FIG. 11(c)). This indicates that activation of the MAP kinase
pathway, located downstream of EGFR, was blocked by expression of
the TrM SH2 domain in the cells. Therefore, we demonstrated that
tight binding of a TrM SH2 domain to EGFR blocked activation of
downstream signaling.
Example 5--Inhibitory Effects of TrM SH2 Domains Observed on Cell
Growth and Colony Formation
[0156] To examine effects of the TrM SH2 domains on cell growth,
HEK 293 cells were transfected with the pEGFPC2-SH2 domain
construct plasmid, as described previously, and then incubated in
full medium with EGF (100 ng/ml) for 36 h. Cells were trypsinized
and stained using 0.4% trypan blue (Sigma-Aldrich) and viable and
total cell numbers were counted in haemacytometer with the use of a
microscope according to manufacturer's instruction. FIG. 12(a)
shows the number of viable cells relative to the empty vector
control. Expression of the TrM SH2 domains resulted in reduction of
viable cells, compared cells expressing the Wt SH2 domains. This
observation demonstrated that the TrM SH2 domain has inhibitory
effects that slow down proliferation of cells.
[0157] To examine the effect of the TrM SH2 domains on
anchorage-independent cell growth, which is an indicator of
tumorigenicity, soft agar assay was performed according to the
method described by Howard et al. (Proc Natl Acad Sci, 2003, Vol.
100, pp. 11267). HEK293 cells were transfected with the pEGFPD2-SH2
domain constructs by PEI and grown overnight. On the following day,
cells were trypsinized and plated at a density of 1.times.104 cells
in 0.25% agarose in DMEM (10% FBS) on top of 0.5% agarose in DMEM
(10% FBS) in 60 mm dishes in triplicates. Cells were maintained at
37.degree. C. in 5% CO.sub.2 for 21 days, and stained overnight
with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide.
FIG. 12(b) shows the number of colonies expressing Wt or TrM SH2
domains, relative to the numbers with empty vector control cells.
FIG. 12(c) shows example photos taken from each sample. Therefore,
TrM SH2 domains demonstrated inhibitory effects on colony formation
in soft-agar, which suggests that the TrM SH2 domains can be used
as anti-cancer agents.
Example 6--S12 Variants as In Vivo Probes of EGFR Signalling
[0158] Cellular signalling is highly dynamic.sup.21. However, there
are few experimental tools that allow monitoring of these dynamic
events in a non-invasive manner. Profiling of phosphorylation
dynamics using phospho-specific antibodies.sup.23 or mass
spectrometry (MS).sup.21 can only obtain snap-shots of signal
transduction because cells must be processed for immunostaining or
MS analysis. SH2 domain variants offer an alternative by which to
examine dynamic signalling events in real time and live cells.
[0159] Two important considerations for developing in vivo
signalling probes are specificity and affinity. Natural SH2 domains
bind their cognate pTyr-containing target polypeptides with
moderate specificity and affinity. Using phage displayed libraries
according to the present invention allows obtaining SH2 variants
with desired properties. The objective in this example is to create
a panel of SH2 variants that exhibit defined specificities towards
pTyr sites in the EGFR, and desired affinities with an optimized
pTyr-binding pocket. This panel of "tailor-made" SH2 variants are
expressed in cells as fusions to XFP (i.e. GFP, YFP or CFY).
SH2-XFP fusion can be expressed in A431, a human epithelial
carcinoma line that express high levels of EGFR, and monitor its
localization to the plasma membrane upon EGF stimulation by live
cell imaging.sup.21. The use of one SH2 probe will reveal the
dynamic phosphorylation profile of a single site. The use of
multiple orthogonal probes would permit the simultaneous monitoring
of multiple pTyr sites during EGFR signalling. The studies with the
SH2-XFP probes are complemented by multiple reaction monitoring
(MRM)-MS analysis.sup.22 of site-specific tyrosine phosphorylation
and by Western blots (WB) to examine the activation of the
corresponding signalling pathways. Information on phosphorylation
dynamics obtained in this example can help prioritize which pTyr
sites and SH2 monobodies to test for cancer intervention as
described below.
Example 7--SH2 Monobodies as Specific Inhibitors of ErbB Signalling
and as Therapeutic Agents
[0160] While antibody-based therapies show great promise in
treating cancers, they have to be humanized to avoid fatal
immunoreaction. Most patients become resistant to treatment after
being in the antibody therapy for some time.sup.20, 1-3, making it
necessary to find alternative therapeutic strategies that can be
used alone or in combination with existing therapies.sup.4. SH2
monobodies, which are based on human SH2 domains, are
hypo-immunogenic.sup.5. Hypoimmunogenicity together with their
relatively small size and high specificities towards pTyr sites,
which are often amplified in cancer.sup.6, 7, makes SH2
domain-based monobodies an attractive platform for developing
molecular targeted cancer therapy. The present invention provides
the ability to evolve and design SH2 variants with precise
specificity and ultra-high affinity afford an unprecedented
opportunity to exploit this potential.
[0161] A panel of SH2 variants/monobodies developed in previous
examples is used as therapeutic candidates for breast cancer by
testing the efficacy of an SH2 monobody in inhibiting ErbB receptor
signalling and cell proliferation. Promising candidates are then
evaluated in a 3D cell culture model of breast cancer. Finally, the
best SH2 monobodies are evaluated in tumor xenografts in mice.
Example 8--SH2 Monobodies as Inhibitors of ErbB Signalling
[0162] Because the ErbB family, in particular ErbB1 (EGFR) and
ErbB2, are frequently amplified in breast cancer.sup.20, the super
pTyr-binders developed in the examples herein can be employed to
inhibit ErbB receptor signalling and cell proliferation in a
relevant cell model. To this end, one can take advantage of the
MCF10A-ErbB1 (or 10A-ErbB1) and MCF10A-ErbB2 (or 10A-ErbB2) created
in the Muthuswamy lab.sup.8-11. Specifically, the mammary
gland-derived MCF10A cells were made to stably express chimeric
ErbB1 or ErbB2 receptor whose cytoplasmic domain is linked to the
synthetic ligand-binding domain from FK506-binding protein
(FKBP).sup.8-12. The chimeric ErbB receptors can be dimerized and
thereby, activated by the bivalent FKBP ligand AP1510.sup.8-12. The
MCF10A, 10A-ErbB1 and 10A-ErbB2 cells can be transfected with
plasmids encoding an wt or mutant SH2 domain or treated with an SH2
monobody fused to a TAT protein transduction domain.sup.13-14.
Applicants have confirmed that purified TAT-SH2 domains (labelled
with FITC) penetrate cells and have half-lives of 2-3 days in cell
culture (FIG. 13). Following the stimulation with AP1510, the cells
will be monitored for proliferation by the MTS assay.sup.4 and for
apoptosis by the TUNEL assay.sup.4, 8-10. Immunoprecipitation (IP)
and WB experiments are also carried out to examine the activation
of the Ras/MAPK growth pathway (ie., by measuring the
phosphorylation of MEK1/2 and Erk1/2, respectively) and the
PI3K/Akt survival pathway (i.e. by measuring Akt phosphorylation).
For the group of SH2 monobodies showing an inhibitory effect to
ErbB signalling and ErbB-dependent cell growth, similar studies
will be carried out in other ErbB-overexpressing human breast
tumour cell lines such as BT-474, SKBR-3 and MDA-361.
Example 9--SH2 Monobodies in 3D Culture
[0163] MCF10A cells, when plated on a bed of extracellular matrix,
form 3D acinar structures resemble breast acini in vivo.sup.8.
Breast cancer cells have been shown to form abnormal acinar
structures characterized with aberrant morphology, enhanced
proliferation and reduced apoptosis. These features are
recapitulated by the 10A-ErbB2 cells.sup.8, 10, 15. Therefore, the
MCF10A 3D culture system to further evaluate the SH2 monobodies.
Specifically, MCF10A, 10A-ErbB1 and 10A-ErbB2 cells are cultured on
matrigels in the presence or absence of different concentrations of
AP1510 and/or TAT-SH2 monobodies following established protocols.
The acinar organization at different stages of morphogenesis can
then be determined by confocal analysis of DAPI-labelled
structures. Cell proliferation and survival can be monitored by
immunstaining for the Ki-67 (proliferation marker) and cleaved
caspase-3 (apoptosis) or by TUNEL staining.sup.4, 8, 9, 15, 16.
Example 10--SH2 Monobodies in Mouse Model of Breast Cancer
[0164] The efficacy of SH2 monobodies in inhibiting or slowing down
mammary tumorigenesis can be evaluated in vivo in a mouse model of
human breast cancer. For example by using the Comma-1D cells and
the mammary fat pad transplantation system established in the
Muthuswamy lab.sup.9, 15. CD cells stably expressing Erb1 or Erb2
are already available from the Muthuswamy lab.sup.9; these cells
are injected into the epithelium-cleared mammary fat pad of 3-week
old female BALB/c mice.sup.9. The tumor growth is measured weekly
with digital callipers. Two complementary approaches are used to
evaluate the SH2 monobodies in inhibiting tumour initiation and
growth, respectively. For tumour initiation, CD cells that stably
express both Erb2 (or Erb1) and an SH2 monobody (with a XFP tag for
easy tracking are created. Upon transplantation, the ability of
these cells in initiating mammary tumours is assayed in comparison
to CD cells expressing the Erb2 or Erb1 alone. For tumour
progression, mice transplanted with CD-ErbB2 or CD-ErbB1 cells are
treated with TAT-SH2 monobodies when the tumour reaches a certain
size (eg., .about.150 mm3).sup.4. A TAT-SH2 monobody (or a control
SH2 domain) at different concentrations is injected
intraperitoneally once a day for three weeks. As a control, the
tumours are treated with Trastuzumab (Herceptin), an anti-ErbB2
antibody.sup.4. Tumor sizes are measured daily. At the end of the
treatment, the mice are sacrificed and the tumour tissues fixed and
stained with hematoxylin and eosin to evaluate histological changes
117. TUNEL staining or immunostaining for cleaved caspase-3118 are
used to investigate the apoptosis status of the tumours.
Immunostaining for Ki67 and the proliferating cell nuclear antigen
(PCNA) to assay for tumour proliferation.sup.9, 18. Biochemically,
the tumor lysates are analyzed for the activation of Erk and Akt.
Fyn/Src/Grb-SH2 triple mutants are used to establish the protocols
for evaluating SH2 monobodies using the mouse breast cancer model
and extend the study to include monobodies that exhibit the highest
efficacy in blocking ErbB signalling in 2D and 3D cultures. The
most effective SH2 monobodies can also be further validated in
BT-474 xenografts established in nude mice.sup.4.
Example 11--GST Pulldown Assay for Binding Capacity Comparison of
the TrM with the Wild-Type (WT) SH2 Domain
[0165] GST pulldown assay demonstrated that the TrM SH2 domain
captures more tyrosine-phosphorylated proteins from cell lysate
(FIG. 14).
[0166] The wild-type (Wt) and triple mutant (TrM) Fyn SH2 domains
(Ala.sup.139-Gly.sup.249) were respectively subcloned into the
pETM30 vector (Dummler, et al. Microb. Cell Fact. 4, 34 (2005)).
The Fyn SH2 domain constructs contain a FLAG tag sequence
(DYKDDDDKC) (SEQ ID NO: 27) at the C-terminus. To create the GST
control vector, a stop codon was inserted after the GST tag
sequence of the original pETM30 vector. The GST and GST-SH2
proteins were expressed in E. coli BL21(DE3). HeLa cells were
treated with 50 .mu.M pervanadate for 10 min at 37.degree. C. HeLa
cells were lysed on ice in lysis buffer containing 0.5% NP-40, 50
mM HEPES pH 7.4, 1 mM magnesium chloride, 150 mMKCl, and the
COMPLETE protease inhibitor cocktail (Roche). The GST pulldown
assay was conducted as described (Li et al. J. Biol. Chem. 278,
3852-3859 (2003)). The phosphoproteins were revealed by Western
blot using the 4G10 anti-pTyr antibody (Millipore).
Example 12--Kinase Activation-Dependent Detection of Anaplastic
Lymphoma Kinase (ALK) by the Src SH2 TrM
[0167] Receptor tyrosine kinases are activated by extracellular
stimulation and phosphorylate intracellular substrates, including
the cytoplasmic region of the kinase itself. Anaplastic lymphoma
kinase (ALK) is a receptor tyrosine kinase, which is stimulated by
the activation antibody mAb46. The H370 cell line is an HEK293 cell
line stably expressing ALK. FIG. 15A demonstrates that SrcTrM can
capture a substantially larger amount of phosphorylated proteins
from H370 cell lysate upon stimulation, compared to the Wt.
[0168] The Wt human Src (hSrc) SH2, the TrM hSrc SH2, and Wt Rous
sarcoma virus Src (vSrc) SH2 domains were prepared, respectively,
as GST tag fusion proteins. The vSrc SH2 domain is almost identical
to the hSrc SH2 domain (there are three-residue differences in the
SH2 domain region, and the differences are located outside of the
target-binding surface). 5 .mu.g of the GST-tagged SH2 domain was
incubated with 500 .mu.g of H370 cell lysate treated with or
without the ALK activation antibody mAb-46 at room temperature for
30 min. 20 .mu.l of glutathione sepharose beads was then added for
another 30 minincubation at room temperature. As a positive
control, the anti-pTyr antibody P-Tyr-100 (Cell Signaling, #9411)
was used forimmunoprecipitation. 4 .mu.l of the P-Tyr-100 antibody
was incubated with 500 .mu.g H370 cell lysate treated with or
without the ALK activation antibody mAb-46 for 2 hours at 4.degree.
C. Next, 20 .mu.l protein G beads was added for another incubation
for two hours at 4.degree. C. The glutathione beads or protein G
beads were washed with 1.times.PBS (phosphate buffered saline)
three times. The beads were added to 30 .mu.l 2.times.SDS loading
buffer and boiled for 10 min. The samples were resolved on an 8%
Bis-Tris SDS-PAGE gel. The gel was applied to Western blotting to
transfer the samples onto PVDF membrane (Millipore) and the
proteins were probed with the P-Tyr-100 antibody. The major bands
(220 kDa and 140 kDa) shown on the Western blots are two species of
ALK (FIG. 15A).
[0169] In FIG. 15B, the Src SH2 TrM was further demonstrated to
function as a probe to detect tyrosine-phosphorylated proteins on a
PVDF membrane, as a material conjugated to horseradish peroxidase
(HRP). By directly conjugating HRP to the probe, the step of using
the antibody (so-called the secondary antibody) to detect the probe
can be eliminated.
[0170] The hexahistidine-tagged Src SH2 Wt and TrM proteins were
purified as described in the above sections, and the tag was
cleaved by the Tobacco Etch Virus protease as described in the
above sections. The materials were further purified by liquid
chromatography using the size exclusion column Superdex75 10/300
(GE Healthcare) in the buffer composed of 0.1 M sodium phosphate
and 0.15 M sodium chloride at pH7.2.
[0171] 400 .mu.l SH2 domain protein at a concentration of 0.3
.mu.g/.mu.l was mixed with the lyophilized activated peroxidise
(EZ-Link Plus Activated Peroxidase #31478). Next, 15 .mu.L of
freshly prepared 5M Sodium Cyanoborohydride was added to the
reaction mixture and incubated for 1 hour at room temperature.
Next, 30 .mu.L of Quenching Buffer (3M ethanolamine, pH 9) was
added for reaction at room temperature for 15 minutes. The
HRP-labeled TrMSH2 was dialized against 1L of pH 7.2 Phosphate
Buffered Saline (0.1M sodium phosphate, 0.15M sodium chloride)
overnight.
[0172] 4 .mu.g of the anti-ALK antibody (Santa Cruz) was used to
immunoprecipate ALK from 500 .mu.g of H370 cell lysate (treated
with or without mAb-46). The immunoprecipitation samples were
applied to 8% Bis-Tris SDS-PAGE and the samples were transferred
from the SDS-PAGE gel onto two strips of the PVDF membranes. One
membrane strip was probed with the TrM SH2-HRP conjugate as 1:500
dilution. For comparison, the other membrane strip was incubated
with the primary antibody P-Tyr-100 (Cell Signaling), then
anti-mouseIgG secondary antibody-HRP conjugate (Bio-Rad). Signals
from the HRP-conjugates on the membranes were detected as enhanced
chemiluminescence (Western Lightning Plus-ECL kit, Perkin Elmer)
and detected on the x-ray film (FIG. 15B).
Example 13--Detection of Tyrosine-Phosphorylated Proteins Using the
Src SH2 TrM
[0173] Nollau and Mayer (U.S. Pat. No. 7,846,746) demonstrated that
SH2 domains can detect a subset of tyrosine-phosphorylated proteins
from cell lysate, using the Far-Western blotting method. Because
different SH2 domains in nature are equipped with distinct target
recognition specificity, each SH2 domain binds to a unique subset
of lysate proteins.
[0174] On the contrary, the affinity-enhanced SH2 domain variant
binds to a substantially larger portion of phosphorylated proteins
in cell lysate. By using the Src SH2 TrM for Far-Western blotting
experiments, FIG. 16 demonstrates broad detection of
tyrosine-phosphorylated proteins by the SH2 domain variant.
[0175] The GST protein and GST-Src SH2 TrM were prepared as 3.5
.mu.g/ml concentration in the TBS buffer (50 mM Tris-HCl pH 7.2,
137 mM NaCl). The cultured U937 cells were treated with or without
50 .mu.M pervanadate in PBS for 10 min at 37.degree. C. The cells
were lysed in the buffer (0.5% NP-40, 50 mM HEPES pH 7.4, 1 mM
magnesium chloride, 150 mM KCl) with sonication on ice, and
supernatant was loaded on an SDS-PAGE gel for separation and
following transfer to the PVDF membrane (Millipore). The membrane
was blocked with 5% milk in TBS-T (0.5% Tween-20 in TBS) overnight
at 4.degree. C., and probed with 3.5 .mu.g/ml GST proteins for 1
hour at room temperature. The membrane was washed with TBS-T, and
incubated with anti-GST antibody-HRP (horseradish peroxidase)
conjugate (Sigma, #A7340) for 1 hour at room temperature. The
signal from HRP was detected as enhanced chemiluminescence on an
X-ray film.
Example 14--Monitoring Subcellular Localization of the TrM SH2
Domain to Visualize Tyrosine-Phosphorylated Proteins in Live
Cells
[0176] Different from antibody molecules, including
phosphotyrosine-specific antibodies, the SH2 variants can be used
in live cells as a tool to monitor tyrosine phosphorylation
events.
[0177] Genes encoding Src SH2 Wt, SrcSH2TrM, FynSH2Wt and FynSH2TrM
were inserted into the pEGFP vector (Clontech) to express
functional SH2 variants fused with green fluorescent protein.
Non-small cell lung cancer cell line A549 were grown in Phenol
Red-free Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich)
supplemented with 10% fetal bovine serum (FBS; SAFC Biosciences),
penicillin (50 U/ml), and streptomycin (50 .mu.g/ml) in a
humidified atmosphere containing 5% CO.sub.2 at 37.degree. C. Cells
were transiently transfected by pEGFP constructs with
JetPEIPolyPlus-transfection according to the manufacturer's
protocol. Images were captured in Nikon fluorescent microscope 16
to 20 hours after transfection (FIG. 17).
Example 15--SH2 Variant Selectively Kills EGFR-Expressing Cells
Under EGF Treatment
[0178] Since the TrM SH2 domains inhibited EGFR signaling when the
variant SH2 domains were expressed in cells, the variants of the
present invention may be used as inhibitors for EGFR signaling.
However, the signaling event occurs inside of cells. In order to
use the variant SH2 domain as a protein-based inhibitor material,
the variant needs to be delivered into cells from the outside of
the cell membrane.
[0179] The A549 non-small cell lung cancer cell line expresses a
high level of wild-type epidermal growth factor receptor (EGFR) and
is suitable for monitoring EGFR activation events (PCT Pub. No.
WO/2011/130343). Gold nanoparticles (Zhang et al. Langmuir. 2012
Dec. 11; 28(49):17053-60) were used for delivery of the SH2
variants into cells. FIG. 18 shows that the Src SH2 TrM was
delivered into cells and reduced cell variability upon EGF
treatment, compared with the Wt SH2 domain. For comparison, MCF-7
cells were also tested for this assay. Since MCF-7 cells do not
express the EGF receptor (Ju et al Biochem. J. 2013, 452, 123-134),
EGF stimulation did not significantly affect cell variability after
SH2 variant delivery (FIG. 18). This result indicates that the
TrMSH2 protein exhibits inhibitory effects on cells of which the
growth is dependent on EGF.
[0180] The hexa-histidine-tagged SrcWt and TrM proteins were
purified to homogeneity in 5 mM HEPES buffer (pH 7.6). 1 nM of gold
nanoparticle was mixed with 100 nM Wt or TrM variant protein. The
MCF-7 and A549 cells were starved six hours before treatment. The
cells were then treated with 100 ng/ml EGF for 20 hours. The
nanoparticle-protein mixture was applied to cells. Cells were
trypsinized and stained using 0.4% trypan blue (Sigma-Aldrich) and
viable and total cell numbers were counted in haemacytometer with
the use of a microscope according to manufacturer's
instruction.
TABLE-US-00001 TABLE 1 UniProt Database Entry ID Gene names
3BP2_HUMAN SH3BP2 3BP2 RES4-23 ABL1_HUMAN ABL1 ABL JTK7 ABL2_HUMAN
ABL2 ABLL ARG BCAR3_HUMAN BCAR3 NSP2 SH2D3B UNQ271/PRO308 BLK_HUMAN
BLK BLNK_HUMAN BLNK BASH SLP65 BMX_HUMAN BMX BTK_HUMAN BTK AGMX1
ATK BPK CBLB_HUMAN CBLB RNF56 Nbla00127 CBLC_HUMAN CBLC CBL3 RNF57
CBL_HUMAN CBL CBL2 RNF55 CHIN_HUMAN CHN1 ARHGAP2 CHN CHIO_HUMAN
CHN2 ARHGAP3 BCH CISH_HUMAN CISH G18 CLNK_HUMAN CLNK MIST
CRKL_HUMAN CRKL CRK_HUMAN CRK CSK_HUMAN CSK DAPP1_HUMAN DAPP1 BAM32
HSPC066 FER_HUMAN FER TYK3 FES_HUMAN FES FPS FGR_HUMAN FGR SRC2
FRK_HUMAN FRK PTK5 RAK FYN_HUMAN FYN GRAP2_HUMAN GRAP2 GADS GRB2L
GRID GRAP_HUMAN GRAP GRB10_HUMAN GRB10 GRBIR KIAA0207 GRB14_HUMAN
GRB14 GRB2_HUMAN GRB2 ASH GRB7_HUMAN GRB7 HCK_HUMAN HCK HSH2D_HUMAN
HSH2D ALX ITK_HUMAN ITK EMT LYK JAK1_HUMAN JAK1 JAK1A JAK1B
JAK2_HUMAN JAK2 JAK3_HUMAN JAK3 KSYK_HUMAN SYK LCK_HUMAN LCK
LCP2_HUMAN LCP2 LYN_HUMAN LYN JTK8 MATK_HUMAN MATK CTK HYL
NCK1_HUMAN NCK1 NCK NCK2_HUMAN NCK2 GRB4 P55G_HUMAN PIK3R3
P85A_HUMAN PIK3R1 GRB1 P85B_HUMAN PIK3R2 PLCG1_HUMAN PLCG1 PLC1
PLCG2_HUMAN PLCG2 PTK6_HUMAN PTK6 BRK PTN11_HUMAN PTPN11 PTP2C
SHPTP2 PTN6_HUMAN PTPN6 HCP PTP1C RASA1_HUMAN RASA1 RASA RIN1_HUMAN
RIN1 RIN2_HUMAN RIN2 RASSF4 RIN3_HUMAN RIN3 SH21A_HUMAN SH2D1A DSHP
SAP SH21B_HUMAN SH2D1B EAT2 SH22A_HUMAN SH2D2A SCAP TSAD VRAP
SH23A_HUMAN SH2D3A NSP1 UNQ175/PRO201 SH24A_HUMAN SH2D4A PPP1R38
SH2A SH24B_HUMAN SH2D4B SH2B1_HUMAN SH2B1 KIAA1299 SH2B SH2B2_HUMAN
SH2B2 APS SH2B3_HUMAN SH2B3 LNK SH2D3_HUMAN SH2D3C NSP3
UNQ272/PRO309/PRO34088 SH2D5_HUMAN SH2D5 SH2D6_HUMAN SH2D6
SH2D7_HUMAN SH2D7 SHB_HUMAN SHB SHC1_HUMAN SHC1 SHC SHCA SHC2_HUMAN
SHC2 SCK SHCB SHC3_HUMAN SHC3 NSHC SHCC SHC4_HUMAN SHC4 SHCD
UNQ6438/PRO21364 SHD_HUMAN SHD SHE_HUMAN SHE SHF_HUMAN SHF
SHIP1_HUMAN INPP5D SHIP SHIP1 SHIP2_HUMAN INPPL1 SHIP2 SLAP1_HUMAN
SLA SLAP SLAP1 SLAP2_HUMAN SLA2 C20orf156 SLAP2 SOCS1_HUMAN SOCS1
SSI1 TIP3 SOCS2_HUMAN SOCS2 CIS2 SSI2 STATI2 SOCS3_HUMAN SOCS3 CIS3
SSI3 SOCS4_HUMAN SOCS4 SOCS7 SOCS5_HUMAN SOCS5 CIS6 CISH5 CISH6
KIAA0671 SOCS6_HUMAN SOCS6 CIS4 SOCS4 SOCS7_HUMAN SOCS7 NAP4 SOCS6
SPT6H_HUMAN SUPT6H KIAA0162 SPT6H SRC_HUMAN SRC SRC1 SRMS_HUMAN
SRMS C20orf148 STA5A_HUMAN STAT5A STAT5 STA5B_HUMAN STAT5B
STAP1_HUMAN STAP1 BRDG1 STAP2_HUMAN STAP2 BKS STAT1_HUMAN STAT1
STAT2_HUMAN STAT2 STAT3_HUMAN STAT3 APRF STAT4_HUMAN STAT4
STAT6_HUMAN STAT6 TEC_HUMAN TEC PSCTK4 TENC1_HUMAN TENC1 KIAA1075
TNS2 TENS1_HUMAN TNS1 TNS TENS3_HUMAN TNS3 TEM6 TENS1 TPP
TENS4_HUMAN TNS4 CTEN PP14434 TXK_HUMAN TXK PTK4 RLK TYK2_HUMAN
TYK2 VAV2_HUMAN VAV2 VAV3_HUMAN VAV3 VAV_HUMAN VAV1 VAV YES_HUMAN
YES1 YES ZAP70_HUMAN ZAP70 SRK
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Antitumor protein therapy, application of the protein transduction
domain to the development of a protein drug for cancer treatment.
Breast Cancer 13, 16-26 (2006). [0195] 15. Arias-Romero, L. E. et
al. A Rac-Pak signaling pathway is essential for ErbB2-mediated
transformation of human breast epithelial cancer cells. Oncogene
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the ErbB2 receptor and transforming growth factor beta in induction
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Acad Sci USA 101, 1257-1262 (2004). [0197] 17. Zeller, R. &
Rogers, M. Counterstaining and mounting of autoradiographed in situ
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promotes mammary tumorigenesis and reveals a role for cell polarity
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P., Josephson, L. & Weissleder, R. Tat peptide directs enhanced
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Sequence CWU 1
1
271111PRTHomo sapiensmisc_featureFynSH2wt 1Ala Pro Val Asp Ser Ile
Gln Ala Glu Glu Trp Tyr Phe Gly Lys Leu1 5 10 15Gly Arg Lys Asp Ala
Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg 20 25 30Gly Thr Phe Leu
Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Ser 35 40 45Leu Ser Ile
Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His 50 55 60Tyr Lys
Ile Arg Lys Leu Asp Asn Gly Gly Tyr Tyr Ile Thr Thr Arg65 70 75
80Ala Gln Phe Glu Thr Leu Gln Gln Leu Val Gln His Tyr Ser Glu Arg
85 90 95Ala Ala Gly Leu Cys Cys Arg Leu Val Val Pro Cys His Lys Gly
100 105 1102333DNAArtificial SequenceSynthetic construct
CyslessFynSH2DNA 2gctccagttg actctatcca ggcagaagag tggtactttg
gaaaacttgg ccgaaaagat 60gctgagcgac agctattgtc ctttggaaac ccaagaggta
cctttcttat ccgcgagagt 120gaaaccacca aaggtgccta ttcactttct
atccgtgatt gggatgatat gaaaggagac 180catgtcaaac attataaaat
tcgcaaactt gacaatggtg gatactacat taccacccgg 240gcccagtttg
aaacacttca gcagcttgta caacattact cagagagagc tgcaggtctc
300tcctcccgcc tagtagttcc ctctcacaaa ggg 3333111PRTArtificial
SequenceSynthetic construct CyslessFynSH2Protein 3Ala Pro Val Asp
Ser Ile Gln Ala Glu Glu Trp Tyr Phe Gly Lys Leu1 5 10 15Gly Arg Lys
Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg 20 25 30Gly Thr
Phe Leu Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Ser 35 40 45Leu
Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His 50 55
60Tyr Lys Ile Arg Lys Leu Asp Asn Gly Gly Tyr Tyr Ile Thr Thr Arg65
70 75 80Ala Gln Phe Glu Thr Leu Gln Gln Leu Val Gln His Tyr Ser Glu
Arg 85 90 95Ala Ala Gly Leu Ser Ser Arg Leu Val Val Pro Ser His Lys
Gly 100 105 1104142PRTArtificial SequenceSynthetic construct
HisTagCyslessFynSH2 4Met Lys Ile Glu Glu His His His His His His
Ser Ser Gly Arg Glu1 5 10 15Asn Leu Tyr Phe Gln Gly Gly Ala Ala Gln
Pro Ala Ala Pro Val Asp 20 25 30Ser Ile Gln Ala Glu Glu Trp Tyr Phe
Gly Lys Leu Gly Arg Lys Asp 35 40 45Ala Glu Arg Gln Leu Leu Ser Phe
Gly Asn Pro Arg Gly Thr Phe Leu 50 55 60Ile Arg Glu Ser Glu Thr Thr
Lys Gly Ala Tyr Ser Leu Ser Ile Arg65 70 75 80Asp Trp Asp Asp Met
Lys Gly Asp His Val Lys His Tyr Lys Ile Arg 85 90 95Lys Leu Asp Asn
Gly Gly Tyr Tyr Ile Thr Thr Arg Ala Gln Phe Glu 100 105 110Thr Leu
Gln Gln Leu Val Gln His Tyr Ser Glu Arg Ala Ala Gly Leu 115 120
125Ser Ser Arg Leu Val Val Pro Ser His Lys Gly Ala Ala Ala 130 135
140549PRTArtificial SequenceSynthetic construct T8V 5Arg Lys Asp
Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly1 5 10 15Thr Phe
Leu Ile Arg Glu Ser Glu Thr Val Lys Gly Ala Tyr Ser Leu 20 25 30Ser
Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr 35 40
45Lys649PRTArtificial SequenceSynthetic construct S10V 6Arg Lys Asp
Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly1 5 10 15Thr Phe
Leu Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Val Leu 20 25 30Ser
Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr 35 40
45Lys748PRTArtificial SequenceSynthetic construct deltaT8/S10A/K15L
7Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly1 5
10 15Thr Phe Leu Ile Arg Glu Ser Glu Thr Lys Gly Ala Tyr Ala Leu
Ser 20 25 30Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His
Tyr Leu 35 40 45849PRTArtificial SequenceSynthetic construct S10A
8Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly1 5
10 15Thr Phe Leu Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr Ala
Leu 20 25 30Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys
His Tyr 35 40 45Lys949PRTArtificial SequenceSynthetic construct
K15L 9Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg
Gly1 5 10 15Thr Phe Leu Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala Tyr
Ser Leu 20 25 30Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val
Lys His Tyr 35 40 45Leu1049PRTArtificial SequenceSynthetic
construct S10V/K15L 10Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe
Gly Asn Pro Arg Gly1 5 10 15Thr Phe Leu Ile Arg Glu Ser Glu Thr Thr
Lys Gly Ala Tyr Val Leu 20 25 30Ser Ile Arg Asp Trp Asp Asp Met Lys
Gly Asp His Val Lys His Tyr 35 40 45Leu1149PRTArtificial
SequenceSynthetic construct K2E/T8V/S10A/K15I 11Arg Glu Asp Ala Glu
Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly1 5 10 15Thr Phe Leu Ile
Arg Glu Ser Glu Thr Val Lys Gly Ala Tyr Ala Leu 20 25 30Ser Ile Arg
Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr 35 40
45Ile1249PRTArtificial SequenceSynthetic construct T7S/S10A/K15L
12Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly1
5 10 15Thr Phe Leu Ile Arg Glu Ser Glu Ser Thr Lys Gly Ala Tyr Ala
Leu 20 25 30Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys
His Tyr 35 40 45Leu1349PRTArtificial SequenceSynthetic construct
S10A/K15L 13Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro
Arg Gly1 5 10 15Thr Phe Leu Ile Arg Glu Ser Glu Thr Thr Lys Gly Ala
Tyr Ala Leu 20 25 30Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His
Val Lys His Tyr 35 40 45Leu14111PRTArtificial SequenceSynthetic
construct T8V/S10A/K15I 14Ala Pro Val Asp Ser Ile Gln Ala Glu Glu
Trp Tyr Phe Gly Lys Leu1 5 10 15Gly Arg Lys Asp Ala Glu Arg Gln Leu
Leu Ser Phe Gly Asn Pro Arg 20 25 30Gly Thr Phe Leu Ile Arg Glu Ser
Glu Thr Val Lys Gly Ala Tyr Ala 35 40 45Leu Ser Ile Arg Asp Trp Asp
Asp Met Lys Gly Asp His Val Lys His 50 55 60Tyr Ile Ile Arg Lys Leu
Asp Asn Gly Gly Tyr Tyr Ile Thr Thr Arg65 70 75 80Ala Gln Phe Glu
Thr Leu Gln Gln Leu Val Gln His Tyr Ser Glu Arg 85 90 95Ala Ala Gly
Leu Cys Cys Arg Leu Val Val Pro Cys His Lys Gly 100 105
1101549PRTArtificial SequenceSynthetic construct T8V/K15L 15Arg Lys
Asp Ala Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg Gly1 5 10 15Thr
Phe Leu Ile Arg Glu Ser Glu Thr Val Lys Gly Ala Tyr Ser Leu 20 25
30Ser Ile Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His Tyr
35 40 45Leu16111PRTArtificial SequenceSynthetic construct
T8I/S10A/K15L 16Ala Pro Val Asp Ser Ile Gln Ala Glu Glu Trp Tyr Phe
Gly Lys Leu1 5 10 15Gly Arg Lys Asp Ala Glu Arg Gln Leu Leu Ser Phe
Gly Asn Pro Arg 20 25 30Gly Thr Phe Leu Ile Arg Glu Ser Glu Thr Ile
Lys Gly Ala Tyr Ala 35 40 45Leu Ser Ile Arg Asp Trp Asp Asp Met Lys
Gly Asp His Val Lys His 50 55 60Tyr Leu Ile Arg Lys Leu Asp Asn Gly
Gly Tyr Tyr Ile Thr Thr Arg65 70 75 80Ala Gln Phe Glu Thr Leu Gln
Gln Leu Val Gln His Tyr Ser Glu Arg 85 90 95Ala Ala Gly Leu Cys Cys
Arg Leu Val Val Pro Cys His Lys Gly 100 105 11017111PRTArtificial
SequenceSynthetic construct T8V/S10A/K15L 17Ala Pro Val Asp Ser Ile
Gln Ala Glu Glu Trp Tyr Phe Gly Lys Leu1 5 10 15Gly Arg Lys Asp Ala
Glu Arg Gln Leu Leu Ser Phe Gly Asn Pro Arg 20 25 30Gly Thr Phe Leu
Ile Arg Glu Ser Glu Thr Val Lys Gly Ala Tyr Ala 35 40 45Leu Ser Ile
Arg Asp Trp Asp Asp Met Lys Gly Asp His Val Lys His 50 55 60Tyr Leu
Ile Arg Lys Leu Asp Asn Gly Gly Tyr Tyr Ile Thr Thr Arg65 70 75
80Ala Gln Phe Glu Thr Leu Gln Gln Leu Val Gln His Tyr Ser Glu Arg
85 90 95Ala Ala Gly Leu Cys Cys Arg Leu Val Val Pro Cys His Lys Gly
100 105 11018136PRTArtificial SequenceSynthetic construct
HisTagSrcSH2wt 18Met Lys His His His His His His Pro Met Ser Asp
Tyr Asp Ile Pro1 5 10 15Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Met
Asp Ser Ile Gln Ala 20 25 30Glu Glu Trp Tyr Phe Gly Lys Ile Thr Arg
Arg Glu Ser Glu Arg Leu 35 40 45Leu Leu Asn Ala Glu Asn Pro Arg Gly
Thr Phe Leu Val Arg Glu Ser 50 55 60Glu Thr Thr Lys Gly Ala Tyr Cys
Leu Ser Val Ser Asp Phe Asp Asn65 70 75 80Ala Lys Gly Leu Asn Val
Lys His Tyr Lys Ile Arg Lys Leu Asp Ser 85 90 95Gly Gly Phe Tyr Ile
Thr Ser Arg Thr Gln Phe Asn Ser Leu Gln Gln 100 105 110Leu Val Ala
Tyr Tyr Ser Lys His Ala Asp Gly Leu Cys His Arg Leu 115 120 125Thr
Thr Val Cys Pro Thr Ser Lys 130 1351924PRTArtificial
SequenceSynthetic construct K15L 19Thr Lys Gly Ala Tyr Cys Leu Ser
Val Ser Asp Phe Asp Asn Ala Lys1 5 10 15Gly Leu Asn Val Lys His Tyr
Leu 202024PRTArtificial SequenceSynthetic construct T8V/C10A 20Val
Lys Gly Ala Tyr Ala Leu Ser Val Ser Asp Phe Asp Asn Ala Lys1 5 10
15Gly Leu Asn Val Lys His Tyr Lys 2021109PRTArtificial
SequenceSynthetic construct T8V/C10A/K15L 21Asp Ser Ile Gln Ala Glu
Glu Trp Tyr Phe Gly Lys Ile Thr Arg Arg1 5 10 15Glu Ser Glu Arg Leu
Leu Leu Asn Ala Glu Asn Pro Arg Gly Thr Phe 20 25 30Leu Val Arg Glu
Ser Glu Thr Val Lys Gly Ala Tyr Ala Leu Ser Val 35 40 45Ser Asp Phe
Asp Asn Ala Lys Gly Leu Asn Val Lys His Tyr Leu Ile 50 55 60Arg Lys
Leu Asp Ser Gly Gly Phe Tyr Ile Thr Ser Arg Thr Gln Phe65 70 75
80Asn Ser Leu Gln Gln Leu Val Ala Tyr Tyr Ser Lys His Ala Asp Gly
85 90 95Leu Cys His Arg Leu Thr Thr Val Cys Pro Thr Ser Lys 100
10522104PRTArtificial SequenceSynthetic construct Grb2_triple 22Met
Lys Pro His Pro Trp Phe Phe Gly Lys Ile Pro Arg Ala Lys Ala1 5 10
15Glu Glu Met Leu Ser Lys Gln Arg His Asp Gly Ala Phe Leu Ile Arg
20 25 30Glu Ser Glu Ser Val Pro Gly Asp Phe Ala Leu Ser Val Lys Phe
Gly 35 40 45Asn Asp Val Gln His Phe Leu Val Leu Arg Asp Gly Ala Gly
Lys Tyr 50 55 60Phe Leu Trp Val Val Lys Phe Asn Ser Leu Asn Glu Leu
Val Asp Tyr65 70 75 80His Arg Ser Thr Ser Val Ser Arg Asn Gln Gln
Ile Phe Leu Arg Asp 85 90 95Ile Glu Gln Val Pro Gln Gln Pro
100235PRTArtificial SequenceSynthetic peptide fluorescein-GGpYGG
peptideMOD_RES(3)..(3)PHOSPHORYLATION 23Gly Gly Tyr Gly Gly1
5245PRTArtificial SequenceSynthetic peptide non-phosphorylated
fluorescein-GGYGG peptide 24Gly Gly Tyr Gly Gly1 52511PRTArtificial
SequenceSynthetic peptide 25Ser Ser Arg Leu Val Val Pro Ser His Lys
Gly1 5 102614PRTArtificial SequenceSynthetic peptide 26Ser Ser Arg
Leu Val Val Pro Ser His Lys Gly Ala Ala Ala1 5 10279PRTArtificial
SequenceSynthetic peptide FLAG tag sequence 27Asp Tyr Lys Asp Asp
Asp Asp Lys Cys1 5
* * * * *