The present invention relates to an isolated domain of G3BP-2 that
mediates binding between G3BP-2 and other proteins, and nucleic
acids encoding same. The invention also relates to a method for
diagnosing, treating and preventing breast cancer including the
step of using a nucleic acid and/or encoded polypeptide for G3BP-2,
or fragment thereof, to detect, treat or prevent breast cancer in
a mammal, preferably human. In one particular form, the invention
relates to an antigen presenting cell, preferably a dendritic cell,
that is capable of presenting G3BP-2 or fragments thereof. The invention
also relates to lymphocytes, in particular cytotoxic T-lymphocytes,
that are G3BP-2 antigen specific.
1. An isolated G3BP-2 protein fragment comprising an NTF2-like
domain, said isolated G3BP-2 protein fragment capable of binding
another protein by way of said NTF2-like domain.
2. The isolated G3BP-2 protein fragment of claim 1 wherein the
isolated G3BP-2 protein fragment comprises isolated G3BP-2a protein
fragment and/or G3BP-2b protein fragment.
3. The isolated G3BP-2 protein fragment of claim 1 wherein said
another protein is selected from the group consisting of: ran nuclear
pore polypeptide, ubiquitin hydrolase and GAP.sup.120.
4. The isolated G3BP-2 protein fragment of claim 3 wherein the
ubiquitin hydrolase is ODE1.
5. The isolated G3BP-2 protein fragment of claim 1 wherein the
NTF2-like domain comprises an amino acid sequence as set forth in
SEQ ID NO: 22.
6. An isolated protein complex comprising a G3BP-2 protein and
a second protein bound to an NTF2-like domain of the G3BP-2 protein.
7. The isolated protein complex of claim 6 wherein said second
protein is selected from the group consisting of: ran nuclear pore
polypeptide, ubiquitin hydrolase and GAP.sup.120.
8. An isolated G3BP-2 protein, inclusive of a fragment, homolog,
variant or derivative used to elicit an immune response in an animal.
9. The isolated G3BP-2 protein of claim 8 wherein said animal is
10. The isolated G3BP-2 protein of claim 8 selected from the group
TABLE-US-00007 (i) KLPNFGFW; [SEQ ID NO:1] (ii) IMFRGEVRL; [SEQ
ID NO:2] and (iii) SATPPPAEPASLPQEPPKPRV [SEQ ID NO:3]
11. An isolated G3BP-2 protein fragment selected from the group
consisting of: TABLE-US-00008 (a) KLPNFGFW; [SEQ ID NO:1] (b) IMFRGEVRL;
[SEQ ID NO:2] and (c) SATPPPAEPASLPQEPPKPRV [SEQ ID NO:3]
12. An isolated nucleic acid encoding a protein of claim 1, inclusive
of fragments, homologs, variants and derivatives of said isolated
13. The isolated nucleic acid of claim 12 encoding a protein comprising
the NTF2-like domain comprising an amino acid sequence as set forth
in SEQ ID NO: 22.
14. The isolated nucleic acid of claim 12 comprising a sequence
set forth in SEQ ID NO: 23.
15. An isolated nucleic acid encoding the G3BP-2 protein fragment
of claim 10.
16. An expression vector comprising the nucleic acid of claim 12
or claim 14.
17. Use of an antagonist to prevent or disrupt binding between
G3BP-2 and another protein.
18. Use of the antagonist of claim 17, whereby said antagonist
prevents or disrupts binding between a NTF2-like domain of G3BP-2
and said another protein.
19. Use of the antagonist of claim 17 wherein said antagonist is
a mimetic of the NTF2-like domain of G3BP-2.
20. Use of the antagonist of claim 17 wherein said antagonist binds
to the NTF2-like domain.
21. Use of the antagonist of claim 17 wherein said antagonist is
22. Use of the antagonist of claim 21 wherein said protein comprises
an Src homology 3 (SH3) domain.
23. Use of the antagonist of claim 22 wherein said protein comprises
an amino acid sequence as set forth in SEQ ID NO: 6.
24. Use of the antagonist of claim 17 wherein said antagonist is
a non-peptide compound.
25. An isolated antigen presenting cell which has been contacted
with a G3BP-2 protein, fragment, homolog, variant or derivative
26. An isolated antigen presenting cell which has been transfected
with a nucleic acid encoding a G3BP-2 protein, inclusive of fragments,
homologs, variants and derivatives thereof.
27. The isolated antigen presenting cell of claim 25 or claim 26
wherein said cell is a dendritic cell.
28. The isolated antigen presenting cell of claim 25 or claim 26
wherein said G3BP-2 protein, inclusive of a fragment, a homolog,
a variant and a derivative thereof comprises an amino acid sequence
as set forth in SEQ ID NO: 5.
29. The isolated antigen presenting cell of claim 25 or claim 26
wherein said G3BP-2 fragment comprises an amino acid sequence selected
from the group consisting of: KLPNFGFVV [SEQ ID NO: 1] and IMFRGEVRL
[SEQ ID NO: 2].
30. An isolated lymphocyte that is G3BP-2 antigen specific.
31. The isolated lymphocyte of claim 30, wherein said isolated
lymphocyte is a cytotoxic T-lymphocyte.
32. The isolated lymphocyte cell of claim 30, wherein said G3BP-2
antigen is a protein, inclusive of fragments, homologs, variants
and derivatives thereof, comprises an amino acid sequence as set
forth in SEQ ID NO: 5.
33. The isolated lymphocyte of claim 30, wherein said G3BP-2 protein
fragment comprises an amino acid sequence as set forth in SEQ ID
NO: 1 or SEQ ID NO: 2.
34. A pharmaceutical composition comprising at least one active,
wherein the active is selected from the group consisting of: a protein
of any one of claims 1, 6, 11; a nucleic acid of any one of claims
12, 15, 16 or an isolated antigen presenting cell or lymphocyte
of any one of claims 25, 26, 30.
35. A method for preventing or treating breast cancer in a mammal
including the step of administering to said mammal a pharmaceutical
composition comprising at least one active, wherein the active is
selected from the group consisting of: a protein of any one of claims
1, 6, 11; a nucleic acid of any one of claims 12, 15, 16, a mimetic
of the NTF2-like domain of G3BP-2; an antagonist that prevents or
disrupts binding between a NTF2-like domain of G3BP-2 and another
protein; or isolated cell of any one of claims 25, 26, 30.
36. The method of claim 35 wherein said mammal is human.
37. A method for modulating cell proliferation including the step
of administering to an animal or isolated cell, an active which
prevents or disrupts binding between G3BP-2 and another protein.
38. The method of claim 37 wherein said animal is human.
39. A method for isolating a molecule that binds G3BP-2, including
the step of determining if one or more candidates in a sample bind
to the NTF2-like domain of G3BP-2.
40. The method of claim 39 wherein said molecule is an antagonist.
41. The method of claim 40 wherein said antagonist is a protein
or a non-protein molecule.
42. A method for diagnosing breast cancer in a mammal including
the steps of comparing G3BP-2 protein expression in a test sample
obtained from the mammal with G3BP-2 in a reference sample, wherein
if the expression of G3BP-2 in the test sample is different than
the reference sample, the mammal is diagnosed with an increased
likelihood of having breast cancer.
43. The method of claim 42 when G3BP-2 protein expression is detected
using an antibody.
44. The method of claim 43 wherein said antibody binds to a G3BP-2
protein, inclusive of a fragment, a homolog, a variant and a derivative
thereof, comprising an amino acid sequence as set forth in SEQ ID
45. The method of claim 44 wherein said G3BP-2 fragment comprises
a NTF2-like domain.
46. The method of claim 43 wherein said antibody binds to a G3BP-2
protein fragment comprising an amino acid sequence SATPPPAEPASLPQEPPKPRV
[SEQ ID NO: 3].
47. The method of claim 42 wherein said mammal is human.
48. The method of claim 48 wherein said test sample is breast tissue.
49. A method for diagnosing breast cancer in a mammal including
the step of detecting a G3BP-2 nucleic acid or fragment thereof
in a test sample obtained from the mammal.
50. The method of claim 48 wherein said mammal is human.
51. A method of immunising a mammal against breast cancer, including
the step of administering to said mammal an immunogenic agent comprising
at least one active selected from the group consisting of: (1) a
G3BP-2 protein; (2) a fragment, a homolog, a variant or a derivative
of (1); (3) a G3BP-2 nucleic acid; (4) a fragment, a homolog, a
variant or a derivative of (3); (5) an isolated antigen presenting
cell that has been contacted with (1) or (2); and (6) an isolated
antigen presenting cell that has been transfected with a nucleic
acid of (3) or (4).
52. The method of claim 51 wherein said G3BP-2 protein fragment
is selected from the group consisting of: KLPNFGFVV [SEQ ID NO:
1] and IMFRGEVRL [SEQ ID NO: 2].
53. The method of claim 51 wherein said antigen presenting cell
is a dendritic cell.
54. The method of claim 51 wherein said mammal is human.
FIELD OF THE INVENTION
 THIS INVENTION relates to an isolated domain of G3BP-2 that
mediates binding between G3BP-2 and other proteins, and nucleic
acids encoding same. More particularly, a method for diagnosing,
treating and preventing breast cancer including the step of using
a nucleic acid and/or encoded polypeptide for G3BP-2 to detect,
treat or prevent breast cancer in a mammal.
BACKGROUND OF THE INVENTION
 ras-GTPase-Activating Protein SH3-Domain-binding Proteins
(G3BPs) are a family of proteins which comprise SH3 domain-binding
motifs which have been shown to specifically bind the ras-GTPase-activating
protein, rasGAP.sup.120 (Parker et al., 1996; Kennedy et al, 1997).
Furthermore, this family of proteins have been shown to be RNA-binding
proteins (Kennedy et al., 1997) that may have an RNAase activity
on c-myc transcripts (Gallouzi et al., 1998). Both the ras-GAP signalling
pathway and c-myc have been implicated in oncogenic activity (Bos,
1989; Facchini and Penn, 1998). The evidence presented suggests
that the G3BP family of proteins are members of a novel signal transduction
mechanism that utilises components of previously described pathways
to regulate mRNA stability and through these pathways may regulate
oncogenic signals or factors. These activities may be modulated
through their SH3 domain-binding activity (Parker et al., 1996),
their RNAase activity (Gallouzi et al., 1998) or their helicase
activity (Costa et al., 1999). G3BPs have recently been shown to
be upregulated at the transcriptional level in some cancers (Guitard
et al., 2001).
 rasGAP.sup.120 is an important regulator of signal transduction
(Pomerance et al., 1996) as it sits at the nexus of positive and
negative control of the oncogene ras. rasGAP.sup.120 itself stimulates
the hydrolysis of GTP bound ras (reviewed in Tocque et al., 1997)
and thereby regulates the activity of ras. The amino-terminus of
rasGAP.sup.120 comprises a Scr homology (SH3) domain (Tocque et
al. 1997) which has been implicated in an effector-like activity
(Duchesne et al., 1993).
 Human G3BP-1 was first identified by its co-immunoprecipitation
with rasGAP.sup.120 using an antibody raised to the carboxy-terminal
domain of rasGAP.sup.120 (Parker et al., 1996). G3BP was the first
protein shown to bind the rasGAP.sup.120 SH3 domain, however, other
rasGAP.sup.120 SH3 binding proteins have since been reported, including
a 14 kDa protein (Hu and Settleman 1997) and the huntingtin protein
(Liu et al., 1997). Genetic studies in Drosophila support a role
for G3BP in ras signaling (Pazman et al., 2000).
 The inventors previously cloned and sequenced mouse G3BP-2
as part of a general screening for RNA Recognition motif (RRM)-containing
proteins (Kennedy et al., 1997). Primary sequence analysis of G3BPs
also indicated that they contain an RNA Recognition Motif (RRM)
(Nagai et al., 1995), an RGG domain (Burd and Dreyfuss 1994; Siomi
and Dreyfuss 1997) and a Nuclear Transport Factor 2-like (NTF2-like)
domain (Suyama et al., 2000). The proposed structure of the RRM
in G3BP has been reported elsewhere (Kennedy et al. 1997). The G3BPs
also contain acid-rich and RGG domains which are often considered
auxiliary domains for RRM-type RNA-binding proteins (Burd and Dreyfuss
1994; Siomi and Dreyfuss 1997). These structural motifs are consistent
with a recent finding that G3BP-1 is implicated in RNA metabolism
by acting in vitro as a cleavage factor for c-myc transcripts (Gallouzi
et al. 1998).
 NTF2 polypeptide is involved in nuclear transport of polypeptides
and appears to be facilitated by binding RanGDP in the cytoplasm.
Once NTF2/RanGDP is bound to a cargo the complex is imported to
the nucleus where it is released and the Ran nucleotide exchange
factor, RCC1, converts RanGDP to RanGTP. This signals export of
NTF2 to the cytoplasm where RanGTP is hydrolysed by Ran GTPase activating
protein (RanGAP) and the system is reset (reviewed in Macara 1999).
The NTF2-like domain of G3BP-2 may target G3BP-2 to the nuclear
envelope, although a mechanism for this activity is unclear (Prigent
et al., 2000).
 RNA processing is an integral part of cellular metabolism
controlled through pre-mRNA splicing, RNA transport and RNA stability
(Dreyfuss et al., 1996). Regulation of RNA metabolism has been shown
to play an important role in development. Recently there has been
increased interest in the control of mRNA translation mediated by
RNA-binding proteins, in particular the role of these proteins in
5' UTR interactions that influence elongation factors (Svitkin et
al., 1996) as well as 3' interactions involving translational activity
(Dreyfuss et al. 1996) and degradation (Gallouzi et al., 1998).
It is important to characterise the mechanisms that allow RNA-binding
proteins to respond to environmental and developmental signals through
transduction cascades in order to understand their role in human
 SH3 domains were initially characterised in signal transduction
proteins such as Src, Fyn and Grb as well as rasGAP.sup.120. Typically
these domains interact with proline rich motifs with a minimum consensus
of PxxP (Urquhart et al., 2000 and papers cited therin). It has
also been shown that the acidic and PxxP domains, and not the RNA-binding
domain nor the NTF2-like domain of G3BP-2, are sufficient to mediate
binding to I.kappa.B.alpha. (Prigent et al; 2000). Primary sequence
analysis of alternatively spliced homologues of human G3BP-2a and
G3BP-2b reveals that they respectively comprise five and six minimal
potential SH3 domain-binding motifs (Lee et al., 1996). As G3BP-2a
and G3BP-2b comprise PxxP sequences, it was predicted that these
proline-rich motifs would bind with SH3 domains of polypeptides.
 Major advances have been achieved in the early diagnosis
(screening mammography) and treatment (adjuvant therapy) of breast
cancer and this has translated into a significant reduction in the
mortality generated by this disease (Chlebowski, 2002). However,
breast cancer still accounts for 26% of the cancers diagnosed in
Australia in 1999, and in 1996 there were 9,556 new cases diagnosed
with 2,619 deaths ((AIHW), 1999). An additional important input
into the better management of the disease has been the characterization
of genetic predisposing markers BRCA1 and 2 (reviewed in (Nathanson
et al., 2001)), allowing the prediction of a proportion (10%) of
women at risk to develop breast cancer. Considering the reduced
adjuvant therapeutic options currently available (Pritchard et al.,
2002) and the risks involved with their use, novel strategies are
urgently needed that could prevent the development of the disease
at early stages of the disease and/or in those with higher genetic
SUMMARY OF THE INVENTION
 Although G3BPs have been shown to bind the SH3 domain of
GAP.sup.120 the inventors were surprised to discover that this binding
is mediated through the N-terminal NTF2-like domain of G3BP and
not facilitated by a proline-rich motif (PxxP) contained within
G3BP-2 as the prior art would suggest. The smallest G3BP truncated
protein that was capable of binding to the SH3 domain of rasGAP.sup.120
did not contain any of the predicted PxxP motifs normally associated
with SH3 binding. The unexpected results clearly showed that the
N-terminal NTF2-like domain of G3BP is responsible for the binding
interactions with N-terminal rasGAP.sup.120. This finding has led
to novel uses of G3BP, in particular the NTF2-like domain thereof,
as described herein for identifying and producing potential reagents
for diagnosing, treating or preventing breast cancer.
 In a first aspect, the invention provides an isolated G3BP-2
protein fragment comprising an NTF2-like domain, said isolated G3BP2
protein fragment capable of binding another protein by way of said
 Preferably, the G3BP-2 protein comprises G3BP-2a and/or
 Preferably, the another protein is selected from the group
consisting of: ran nuclear pore polypeptide, ubiquitin hydrolase
 More preferably, the ubiquitin hydrolase is ODE1.
 The NTF2-like domain preferably is encoded by amino acid
residues 1 to 146 as set forth in SEQ ID NO: 22.
 In a second aspect the invention provides an isolated protein
complex comprising a G3BP-2 protein having an NTF2-like domain and
another protein bound to the NTF2-like domain, selected from the
group consisting of: ran nuclear pore polypeptide, ubiquitin hydrolase
 In a third aspect, the invention provides an isolated G3BP-2
protein, inclusive of a fragment, homolog, variant or derivative
thereof capable of eliciting an immune response in an animal.
 Preferably, the animal is human.
 Preferably, the G3BP-2 fragment is selected from the group
consisting of: TABLE-US-00001 (i) KLPNFGFW; [SEQ ID NO:1] (ii) IMFRGEVRL;
[SEQ ID NO:2] and (iii) SATPPPAEPASLPQEPPKPRV [SEQ ID NO:3]
 In a fourth aspect, the invention provides an isolated G3BP-2
protein fragment selected from the group consisting of: TABLE-US-00002
(a) KLPNFGFW; [SEQ ID NO:1] (b) IMFRGEVRL; [SEQ ID NO:2] and (c)
SATPPPAEPASLPQEPPKPRV [SEQ ID NO:3]
 In a fifth aspect, the invention provides an isolated nucleic
acid encoding a protein of the first aspect, inclusive of fragments,
homologs, variants and derivatives thereof, each capable of binding
another protein by way of said NTF2-like domain.
 In one form, the isolated nucleic acid encodes a protein
comprising the NTF2-like domain comprising an amino acid sequence
as set forth in SEQ ID NO: 22, said NTF2-like domain being encoded
by amino acid residues 1 to 146, wherein amino acid residue 1 is
the first methionine (M).
 In another form, the isolated nucleic acid comprises a nucleotide
sequence set forth in SEQ ID NO: 23.
 In a sixth aspect, the invention provides an isolated nucleic
acid encoding a G3BP-2 protein fragment of the fourth aspect.
 In a seventh aspect, the invention provides an expression
vector comprising a nucleic acid of any one of the abovementioned
 In an eighth aspect, the invention relates to use of an
antagonist to prevent or disrupt binding between G3BP-2 and another
 In one form, the antagonist of the eighth aspect prevents
or disrupts binding between a NTF2-like domain of G3BP-2 and said
 In another form, the antagonist is a mimetic of the NTF2-like
domain of G3BP-2.
 In yet another form, the antagonist binds to the NTF2-like
 The antagonist may be a protein.
 In one form, the protein comprises an Src homology 3 (SH3)
 Preferably, the protein comprises an amino acid sequence
as set forth in SEQ ID NO: 6.
 The antagonist may be a non-peptide compound.
 In a ninth aspect, the invention provides an isolated antigen
presenting cell which has been in contact with a G3BP-2 protein,
fragment, homolog, variant or derivative thereof, wherein contact
includes pulsing or loading the antigen presenting cell with G3BP-2
protein, fragment, homolog, variant or derivative thereof.
 In a tenth aspect, the invention provides an isolated antigen
presenting cell which has been transfected with a nucleic acid encoding
G3BP-2 protein, inclusive of fragments, homologs, variants and derivatives
 The isolated antigen presenting cell of the ninth and tenth
aspects is preferably a dendritic cell.
 The G3BP-2 protein, inclusive of a fragment, a homolog,
a variant and a derivative thereof of the ninth and tenth aspects
preferably comprises an amino acid sequence as set forth in SEQ
ID NO: 5.
 The G3BP-2 fragment of the ninth and tenth aspects preferably
comprises an amino acid sequence selected from the group consisting
of: KLPNFGFVV [SEQ ID NO: 1] and IMFRGEVRL [SEQ ID NO: 2].
 In an eleventh aspect, the invention provides an isolated
lymphocyte cell that is G3BP-2 antigen specific.
 Preferably, the isolated lymphocyte cell is a cytotoxic
 Preferably, the lymphocyte cell is G3BP-2 antigen specific
for a protein, inclusive of fragments, homologs, variants and derivatives
thereof, comprising an amino acid sequence as set forth in SEQ ID
 Preferably, the G3BP-2 protein fragment comprises an amino
acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
 In a twelfth aspect, the invention provides a pharmaceutical
composition comprising at least one active, wherein the active is
selected from the group consisting of: a protein, a nucleic acid
or an isolated cell of any one of the above aspects.
 In a thirteenth aspect, the invention provides a method
for preventing or treating breast cancer in a mammal including the
step of administering to said mammal a pharmaceutical composition
comprising at least one active, wherein the active is selected from
the group consisting of: a protein, a nucleic acid, a mimetic of
the NTF2-like domain of G3BP-2, an antagonist that prevents or disrupts
binding between a NTF2-like domain of G3BP-2 and another protein
or isolated cell of any one of the above-mentioned aspects.
 Preferably the mammal is human.
 In a fourteenth aspect, the invention provides a method
for modulating cell proliferation including the step of administering
to an animal or isolated cell, an active which prevents or disrupts
binding between G3BP-2 and another protein.
 Preferably the animal is human.
 In a fifteenth aspect, the invention provides a method for
isolating a molecule that binds G3BP-2, including the step of determining
if one or more candidates in a sample bind to the NTF2-like domain
 In one form, the molecule is an antagonist.
 The antagonist may be a protein or a non-protein molecule.
 In a sixteenth aspect, the invention provides a method for
diagnosing breast cancer in a mammal including the steps of comparing
G3BP-2 protein expression in a test sample obtained from the mammal
with G3BP-2 in a reference sample, wherein if the expression of
G3BP-2 in the test sample is different than the reference sample,
the mammal is diagnosed with an increased likelihood of having breast
 G3BP-2 protein expression may be detected using an antibody.
 The antibody may bind to a G3BP-2 protein, inclusive of
a fragment, a homolog, a variant and a derivative thereof, comprising
an amino acid sequence as set forth in SEQ ID NO: 5.
 In one form, the antibody binds to a G3BP-2 protein fragment
comprising an amino acid sequence SATPPPAEPASLPQEPPKPRV [SEQ ID
 The G3BP-2 fragment may comprise a NTF2-like domain.
 Preferably, the mammal is human.
 In a seventeenth aspect, the invention provides a method
for diagnosing breast cancer in a mammal including the step of detecting
a G3BP-2 nucleic acid or fragment thereof in a test sample obtained
from the mammal.
 Preferably, the test sample is breast tissue.
 Preferably, the mammal is human.
 In an eighteenth aspect, the invention provides a method
of immunising a mammal against breast cancer, including the step
of administering to said mammal an immunogenic agent comprising
at least one active selected from the group consisting of: 
(1) a G3BP-2 protein;  (2) a fragment, a homolog, a variant
or a derivative of (1);  (3) a G3BP-2 nucleic acid; 
(4) a fragment, a homolog, a variant or a derivative of (3); 
(5) an isolated antigen presenting cell that has been contacted
with (1) or (2); and  (6) an isolated antigen presenting cell
that has been transfected with a nucleic acid of (3) or (4).
 The immunisation may be preventative or as a treatment for
an animal with breast cancer.
 The G3BP-2 protein fragment is preferably selected from
the group consisting of: KLPNFGFVV [SEQ ID NO: 1] and IMFRGEVRL
[SEQ ID NO: 2].
 The antigen presenting cell is preferably a dendritic cell.
 Preferably, the mammal is human.
 The isolated protein and nucleic acid, and methods according
to the aforementioned aspects of the invention are useful in therapeutic
or prophylactic treatments of breast cancer and diagnosis thereof.
As will be described in more detail hereinafter, disruption of interactions
between G3BP-2 and another protein or endogenous binding partner
may inhibit tumour proliferation.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
 FIG. 1 is a nucleotide sequence of HsaG3BP-2a [SEQ ID NO:
4] and encoded amino acid sequence [SEQ ID NO: 5]; and
 FIG. 2 is an amino acid sequence [SEQ ID NO: 6] of the N-terminus
of rasGAP.sup.120, which includes a SH3 domain;
 FIG. 3 shows a clustal alignment of the G3BP family of proteins,
mouse MmuG3BP2a [SEQ ID NO:7], human HsaG3BP2a [SEQ ID NO:5], mouse
MmuG3BP2b [SEQ ID NO:8], human HsaG3PB2b [SEQ ID NO:9], human HsaG3BP1
[SEQ ID NO:10] and mouse MmuG3BP1 [SEQ ID NO:11];
 FIG. 4 shows analysis of protein-protein interactions between
the SH3 domain of N-terminal rasGAP.sup.120 and G3BP;
 FIG. 5 shows Western blot analysis of G3BP expression in
 FIGS. 6A and 6B shows immunohistochemistry of adult mouse
tissues probed with anti-G3BP-1 and anti-G3BP-2 antibodies;
 FIGS. 7A and 7B show G3BP-2 immunohistochemistry of two
human breast cancers. Panel A is an in situ ductal tumour and panel
B shows an infiltrating cancer;
 FIG. 8A-T shows immunohistochemical staining of breast tumour
sections and immunofluorescence of synchronised NIH 3T3 cells with
antibodies specific for G3BPs. Panels A-C are breast tumour sections
stained using an antibody specific for G3BP-1. Panels A and B represent
stained IDC while. Panel C shows a small section of stained normal
ducts (ND). Panels D-O show immunohistochemistry of G3BP-2 in human
breast tumours. Panel D shows a normal lobe, Panels E and F show
normal ducts cut transverse and longitudinally, respectively (CT
denotes connective tissue). Panels G and H show a normal duct adjacent
to an IDC (DC denotes ductal carcinoma). Panel I shows an IDC which
does not express G3BP-2. Panel J shows an IDC adjacent to normal
connective tissue. The arrow indicates cells within the connective
tissue which stain positive for G3BP-2 in the nucleus. Panel K shows
a lower magnification of an IDC (left side) adjacent to normal connective
tissue. Panels L-O illustrate a variety of G3BP-2 subcellular localisations
in human breast cancer. All panels are ductal carcinomas from different
patients. Panel L shows cytoplasmic localisation of G3BP-2. Panels
M and N show nuclear localisation of G3BP-2 in two different cases
of breast cancer; cytoplasmic expression is also observed in these
sections. Panel O shows G3BP-2 expression around the nuclear envelope
region; cytoplasmic staining is also observed. Panels P-T show the
immunofluorescence of synchronised NIH 3T3 cells. Panel P shows
the sub-cellular localisation of G3BP-2 in cells in G0 phase (time=0).
The time after serum stimulation and hence cell cycle commencement
is 2 hours, 5 hours, 9 hours and 12 hours for Panels Q, R, S and
 FIG. 9 illustrates antibody specificity of G3BP-2 antibodies.
The breast cancer cell line MDA-MB-435 and the cervical cancer cell
line HeLa (labeled 435 and HeLa respectively) were lysed and equal
amounts of protein were resolved by SDS-PAGE. The samples were transferred
to a membrane and probed with either the polyclonal G3BP-2 antibody
or the commercial G3BP-1 antibody as indicated (Panel A). Purified
recombinant G2BP-2b (lane 1), G3BP-1 (lane 2) and G3BP-2a (lane
3) were resolved by SDS-PAGE along with four different truncations
of G3BP-2a, N1 (lane 4), C1 (lane 5), C2 (lane 6), N2 (lane 7).
These samples were transferred to a membrane and probed with the
G3BP-2 antibody (Panel B). Panel C is a schematic representation
of G3BP-2a and the recombinant G3BP-2a truncations (N1, N2, C1,
C2). It also illustrates the region in which the G3BP-2 polyclonal
antibody binds. Inset shows the sub-domains within G3BP-2a.;
 FIG. 10 shows G3BP-2 peptide binding to HLA-A*0201 measured
by T2 binding assay; and
 FIG. 11 is a graph showing data for a chromium release cytotoxic
assay using different effector cell: target cell ratios.
 Table 1: shows exemplary conservative substitutions in the
polypeptide; and  Table 2 summarises expression of G3BP-2
in 58 breast tumour sections. DCIS=ductal carcinoma in situ, IDC=infiltrating
ductal carcinoma, LCIS=lobular carcinoma in situ, ILC=infiltrating
lobular carcinoma. Grade is assigned according to a range of factors--a
well-differentiated tumour is generally assigned a grade 1 while
a poorly differentiated tumour is assigned a grade 3. ER=oestrogen
receptor status. Node status refers to the presence (+) or absence
(-) of tumour in the lymph nodes. NG=not graded. ND=not determined.
The column labelled cytoplasm indicates the level of expression
of G3BP-2 in the cytoplasm of cells (1+=low, 2+=medium, 3+=high).
Unless stated otherwise, staining was present in greater than 75%
of cell population. The column labelled nucleus indicates the presence
(+) or absence (-) of G3BP-2 in the nucleus of cancer cells.
 Table 3 summarises of G3BP-1 in 24 breast tumour sections.
DCIS=ductal carcinoma in situ, IDC=infiltrating ductal carcinoma,
LCIS=lobular carcinoma in situ, ILC=infiltrating lobular carcinoma.
Grade is assigned according to a range of factors--a well-differentiated
tumour is generally assigned a grade 1 while a poorly differentiated
tumour is assigned a grade 3. ER=oestrogen receptor status. Node
status refers to the presence (+) or absence (-) of tumour in the
lymph nodes. NG=not graded. ND=not determined. The column labelled
cytoplasm indicates the level of expression of G3BP-1 in the cytoplasm
of cells (1+=low, 2+=medium, 3+=high). Unless stated otherwise,
staining was present in greater than 75% of cell population. The
column labelled nucleus indicates the presence (+) or absence (-)
of G3BP-1 in the nucleus of cells.
DETAILED DESCRIPTION OF THE INVENTION
 Unless defined otherwise, all technical and scientific terms
used herein have a meaning as commonly understood by those of ordinary
skill in the art to which the invention belongs. Although any method
and material similar or equivalent to those described herein can
be used in the practice or testing of the present invention, preferred
methods and materials are described. For the purpose of the present
invention, the following terms are defined below.
 For the purposes of this invention, by "isolated"
is meant material that has been removed from its natural state or
otherwise been subjected to human manipulation. Isolated material
may be substantially or essentially free from components that normally
accompany it in its natural state, or may be manipulated so as to
be in an artificial state together with components that normally
accompany it in its natural state. Isolated material includes material
in native and recombinant form. For example, G3BP-2 nucleic acids
and encoded polypeptides (inclusive of HsaG3BP-2a, HsaG3BP-2b isolated
from human; and MmuG3BP-2a and MmuG3BP-2b isolated from mouse) have
been respectively isolated from human and mouse.
 By "endogenous" nucleic acid or polypeptide is
meant a nucleic acid or polypeptide which may be found in a native
cell, tissue or animal in isolation or otherwise.
Polypeptide or Protein
 By "polypeptide" is also meant "protein",
either term referring to an amino acid polymer, comprising natural
and/or non-natural amino acids as are well understood in the art.
For example, G3BP-2 may be referred to as both a protein or polypeptide.
"Protein" may refer to a peptide, polypeptide, or fragments
thereof. "G3BP-2" protein encompasses isoforms thereof,
including G3BP-2a and G3BP-2b and all other isoforms, unless a specific
isoform is referred to.
 A "peptide" is a protein having no more than fifty
(50) amino acids.
 In one embodiment, a "fragment" includes an amino
acid sequence which constitutes less than 100%, but at least 20%,
preferably at least 30%, more preferably at least 80% or even more
preferably at least 90% of said polypeptide.
 The fragment may also include a "biologically active
fragment" which retains biological activity of a given polypeptide
or peptide. For example, a biologically active fragment of G3BP-2
comprises a NTF2-like fragment which is associated with binding
a SH3 domain. The NTF2-like domain includes amino acid residues
1 to 146 as shown in FIG. 1. It is understood that the fragment
may be derived from either a native or a recombinant polypeptide
or peptide. The biologically active fragment constitutes at least
greater than 1% of the biological activity of the entire polypeptide
or peptide, preferably at least greater than 10% biological activity,
more preferably at least greater than 25% biological activity and
even more preferably at least greater than 50% biological activity.
 In another embodiment, a "fragment" is a small
peptide, for example of at least 6, preferably at least 10 and more
preferably at least 20 amino acids in length, which comprises one
or more antigenic determinants or epitopes. Larger fragments comprising
more than one peptide are also contemplated, and may be obtained
through the application of standard recombinant nucleic acid techniques
or synthesized using conventional liquid or solid phase synthesis
techniques. For example, reference may be made to solution synthesis
or solid phase synthesis as described, for example, in Chapter 9
entitled "Peptide Synthesis" by Atherton and Shephard
which is included in a publication entitled "Synthetic Vaccines"
edited by Nicholson and published by Blackwell Scientific Publications.
Alternatively, peptides can be produced by digestion of a polypeptide
of the invention with a suitable proteinases. The digested fragments
can be purified by, for example, high performance liquid chromatographic
 In one form, the invention provides a protein that comprises
an NTF2-like domain. The term "comprises" refers to the
protein at least having the NTF2-like domain and any additional
amino acid sequence.
 In another form, the invention provides a protein that consists
essentially of the NTF2-like domain. The term "consists essentially
of" in relation to a protein refers to a protein that in addition
to the stated portion thereof, eg. the NTF2-like domain, consists
of no more than 30 additional amino acids located the amino and/or
carboxyl terminal end(s) thereof. Preferably, the protein consists
of no more than 20 additional amino acids. More preferably, the
protein consists of between 1-10 additional amino acids. The additional
amino acids or "additions" may comprise a fusion protein,
for example those well known in the art including GST and (6.times.-HIS)-tag
as described hereinafter.
 In another form, the invention provides a protein that "consist
of" the NTF2-like domain. This means a protein comprising an
amino acid sequence of only the NTF2-like domain.
 The NTF2-like domain is set forth in SEQ ID NO: 5, referring
to amino acid residues 1 to 146, wherein amino acid residue 1 is
the first methionine (M).
 As used herein, "variant" polypeptides are polypeptides
of the invention in which one or more amino acids have been replaced
by different amino acids. It is well understood in the art that
some amino acids may be changed to others with broadly similar properties
without changing the nature of the activity of the polypeptide (conservative
substitutions). Exemplary conservative substitutions in the polypeptide
may be made according to Table 1.
 Substantial changes in function are made by selecting substitutions
that are less conservative than those shown in Table 1. Other replacements
would be non-conservative substitutions and relatively fewer of
these may be tolerated. Generally, the substitutions which are likely
to produce the greatest changes in a polypeptide's properties are
those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted
for, or by, a hydrophobic residue (e.g. Leu, Ile, Phe or Val); (b)
a cysteine or proline is substituted for, or by, any other residue;
(c) a residue having an electropositive side chain (e.g., Arg, His
or Lys) is substituted for, or by, an electronegative residue (e.g.,
Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe
or Trp) is substituted for, or by, one having a smaller side chain
(e.g., Ala, Ser) or no side chain (e.g., Gly).
Polypeptide and Nucleic Acid Sequence Comparison
 Terms used herein to describe sequence relationships between
respective nucleic acids and polypeptides include "comparison
window", "sequence identity", "percentage of
sequence identity" and "substantial identity". Because
respective nucleic acids/polypeptides may each comprise (1) only
one or more portions of a complete nucleic acid/polypeptide sequence
that are shared by the nucleic acids/polypeptides, and (2) one,
or more portions which are divergent between the nucleic acids/polypeptides,
sequence comparisons are typically performed by comparing sequences
over a "comparison window" to identify and compare local
regions of sequence similarity. A "comparison window"
refers to a conceptual segment of typically at least 6 contiguous
residues that is compared to a reference sequence. The comparison
window may comprise additions or deletions (i.e., gaps) of about
20% or less as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the respective
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by computerised implementations of algorithms
(for example ECLUSTALW and BESTFIT provided by WebAngis GCG, 2D
Angis, GCG and GeneDoc programs, incorporated herein by reference)
or by inspection and the best alignment (i.e., resulting in the
highest percentage homology over the comparison window) generated
by any of the various methods selected.
 The ECLUSTALW program is used to align multiple sequences.
This program calculates a multiple alignment of nucleotide or amino
acid sequences according to a method by Thompson, J. D., Higgins,
D. G. and Gibson, T. J. (1994). This is part of the original ClustalW
distribution, modified for inclusion in EGCG. The BESTFIT program
aligns forward and reverse sequences and sequence repeats. This
program makes an optimal alignment of a best segment of similarity
between two sequences. Optimal alignments are determined by inserting
gaps to maximize the number of matches using the local homology
algorithm of Smith and Waterman. ECLUSTALW and BESTFIT alignment
packages are offered in WebANGIS GCG (The Australian Genomic Information
Centre, Building JO3, The University of Sydney, N.S.W 2006, Australia).
 Reference also may be made to the BLAST family of programs
as for example disclosed by Altschul et al., 1997, Nucl. Acids Res.
25 3389, which is incorporated herein by reference.
 A detailed discussion of sequence analysis can be found
in Chapter 19.3 of Ausubel et al, supra.
 The term "sequence identity" is used herein in
its broadest sense to include the number of exact nucleotide or
amino acid matches having regard to an appropriate alignment using
a standard algorithm, having regard to the extent that sequences
are identical over a window of comparison. Thus, a "percentage
of sequence identity" is calculated by comparing two optimally
aligned sequences over the window of comparison, determining the
number of positions at which the identical nucleic acid base (e.g.,
A, T, C, G, U) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage
of sequence identity. For example, "sequence identity"
may be understood to mean the "match percentage" calculated
by the DNASIS computer program (Version 2.5 for windows; available
from Hitachi Software engineering Co., Ltd., South San Francisco,
 As generally used herein, a "homolog" shares a
definable nucleotide or amino acid sequence relationship with a
nucleic acid or polypeptide of the invention as the case may be.
 "Polypeptide homologs" share at least 80%, preferably
at least 90% and more preferably at least 95% sequence identity
with the amino acid sequences of polypeptides of the invention as
hereinbefore described. Polypeptide homologs include, for example
G3BP-1. Also included are G3BP-2 isoforms G3BP-2a and G3BP-2b.
 Included within the scope of homologs are "orthologs",
which are functionally-related polypeptides and their encoding nucleic
acids, isolated from other organisms. For example G3BP-2 polypeptides
isolated from human (eg. HsaG3BP-2a, HsaG3BP-2b) and mouse (eg.
 With regard to polypeptide variants, these can be created
by mutagenising a polypeptide or by mutagenising an encoding nucleic
acid, such as by random mutagenesis or site-directed mutagenesis.
Examples of nucleic acid mutagenesis methods are provided in Chapter
9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., supra
which is incorporated herein by reference.
 It will be appreciated by the skilled person that site-directed
mutagenesis is best performed where knowledge of the amino acid
residues that contribute to biological activity is available. In
many cases, this information is not available, or can only be inferred
by molecular modeling approximations, for example.
 In such cases, random mutagenesis is contemplated. Random
mutagenesis methods include chemical modification of proteins by
hydroxylamine (Ruan et al., 1997, Gene 188 35), incorporation of
dNTP analogs into nucleic acids (Zaccolo et al., 1996, J. Mol. Biol.
255 589) and PCR-based random mutagenesis such as described in Stemmer,
1994, Proc. Natl. Acad. Sci. USA 91 10747 or Shafikhani et al.,
1997, Biotechniques 23 304, each of which references is incorporated
herein. It is also noted that PCR-based random mutagenesis kits
are commercially available, such as the Diversify.TM. kit (Clontech).
 As used herein, "derivative" polypeptides are
polypeptides of the invention which have been altered, for example
by conjugation or complexing with other chemical moieties or by
post-translational modification techniques as would be understood
in the art. Such derivatives include amino acid deletions and/or
additions to polypeptides of the invention, or variants thereof.
 "Additions" of amino acids may include fusion
of the peptide or polypeptides or variants thereof with other peptides
or polypeptides. Particular examples of such peptides include amino
(N) and carboxyl (C) terminal amino acids added for use as "tags".
Use of an N-terminal 6.times.-His tag for isolating an expressed
fusion polypeptide is described herein.
 N-terminal and C-terminal tags include known amino acid
sequences which bind a specific substrate, or bind known antibodies,
preferably monoclonal antibodies. pRSET B vector (ProBond.TM.; Invitrogen
Corp.) is an example of a vector comprising an N-terminal 6.times.-His-tag
which binds ProBond.TM. resin.
 Other derivatives contemplated by the invention include,
modification to side chains, incorporation of unnatural amino acids
and/or their derivatives during peptide or polypeptide synthesis
and the use of cross linkers and other methods which impose conformational
constraints on the polypeptides, fragments and variants of the invention.
Examples of side chain modifications contemplated by the present
invention include modifications of amino groups such as by acylation
with acetic anhydride; acylation of amino groups with succinic anhydride
and tetrahydrophthalic anhydride; amidination with methylacetimidate;
carbamoylation of amino groups with cyanate; pyridoxylation of lysine
with pyridoxal-5-phosphate followed by reduction with NaBH.sub.4;
reductive alkylation by reaction with an aldehyde followed by reduction
with NaBH.sub.4; and trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene
sulphonic acid (TNBS).
 The carboxyl group may be modified by carbodiimide activation
via O-acylisourea formation followed by subsequent derivitization,
by way of example, to a corresponding amide.
 The guanidine group of arginine residues may be modified
by formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
 Sulphydryl groups may be modified by methods such as performic
acid oxidation to cysteic acid; formation of mercurial derivatives
using 4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;
2-chloromercuri-4-nitrophenol, phenylmercury chloride, and other
mercurials; formation of a mixed disulphides with other thiol compounds;
reaction with maleimide, maleic anhydride or other substituted maleimide;
carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation
with cyanate at alkaline pH.
 Tryptophan residues may be modified, for example, by alkylation
of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl
halides or by oxidation with N-bromosuccinimide.
 Tyrosine residues may be modified by nitration with tetranitromethane
to form a 3-nitrotyrosine derivative.
 The imidazole ring of a histidine residue may be modified
by N-carbethoxylation with diethylpyrocarbonate or by alkylation
with iodoacetic acid derivatives.
 Examples of incorporating unnatural amino acids and derivatives
during peptide synthesis include, use of 4-amino butyric acid, 6-aminohexanoic
acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic
acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine,
sarcosine, 2-thienyl alanine and/or D-isomers of amino acids.
 Polypeptides in relation to the invention such as those
exemplified in FIG. 1 (inclusive of fragments, variants, derivatives
and homologs in general) may be prepared by any suitable procedure
known to those of skill in the art.
 For example, the polypeptide may be prepared by a procedure
including the steps of:  (i) preparing an expression construct
which comprises a recombinant nucleic acid of the invention, operably
linked to one or more regulatory nucleotide sequences, for example
a T7 promoter;  (ii) transfecting or transforming the expression
construct into a suitable host cell, for example E. coli; and 
(iii) expressing the polypeptide in said host cell.
 Preferably, the recombinant nucleic acid of the invention
encodes a polypeptide as shown in FIG. 1, or fragment thereof.
 Recombinant proteins may be conveniently expressed and purified
by a person skilled in the art using commercially available kits,
for example "ProBond.TM. Purification System" available
from Invitrogen Corporation, Carlsbad, Calif., USA, herein incorporated
by reference. Alternatively, standard molecular biology protocols
may be used, as for example described in Sambrook, et al., MOLECULAR
CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated
herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons,
Inc. 1995-1999), incorporated herein by reference, in particular
Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds.
Coligan et al., (John Wiley & Sons, Inc. 1995-1999) which is
incorporated by reference herein, in particular Chapters 1, 5, 6
 The term "nucleic acid" as used herein designates
single or double stranded mRNA, RNA, cRNA and DNA, said DNA inclusive
of cDNA and genomic DNA.
 The term "isolated nucleic acid" as used herein
refers to a nucleic acid subjected to in vitro manipulation into
a form not normally found in nature. Isolated nucleic acid include
both native and recombinant (non-native) nucleic acids. For example,
a nucleic acid isolated from human or mouse.
 A "polynucleotide" is a nucleic acid having eighty
(80) or more contiguous nucleotides, while an "oligonucleotide"
has less than eighty (80) contiguous nucleotides.
 The term "consists essentially of in relation to a
nucleic acid refers to a nucleic acid having no more than 90 nucleotides
located at the 5' and/or 3' thereof. Preferably, the nucleic acid
consist of no more than 60 additional nucleic acids, more preferably
the nucleic acid consist of between 1-30 nucleotides.
 A "probe" may be a single or double-stranded oligonucleotide
or polynucleotide, suitably labeled for the purpose of detecting
complementary sequences in Northern or Southern blotting, for example.
 A "primer" is usually a single-stranded oligonucleotide,
preferably having 20-50 contiguous nucleotides, which is capable
of annealing to a complementary nucleic acid utemplate" and
being extended in a template-dependent fashion by the action of
a DNA polymerase such as Taq polymerase, RNA-dependent DNA polymerase
or Sequenase.TM.. For example, the following primers were used for
chromosomal mapping of human G3BP-1 and G3BP-2.
 HsaG3BP-1 Specific Primers: TABLE-US-00003 5'GGAGGCATGGTGCAGAAACCA;
[SEQ ID NO:12] and 5'CAGGAAAGGGAAGAGAGGGAG [SEQ ID NO:13]
 HsaG3BP-2 Specific Primers: TABLE-US-00004 5'GTCTTGGCAGTGGTACATTAT;
[SEQ ID NO:14] and 5'AGTTCACTTTGTCGTAGATAGTTTAAG [SEQ ID NO:15]
 For the purposes of host cell expression, the recombinant
nucleic acid is operably linked to one or more regulatory sequences
in an expression vector, for example a T7 promoter.
 An "expression vector" may be either a self-replicating
extra-chromosomal vector such as a plasmid, or a vector that integrates
into a host genome. An example of an expression vector is pRSET
B (Invitrogen Corp.) and derivations thereof.
 By "operably linked" is meant that said regulatory
nucleotide sequence(s) is/are positioned relative to the recombinant
nucleic acid of the invention to initiate, regulate or otherwise
 Regulatory nucleotide sequences will generally be appropriate
for the host cell used for expression. Numerous types of appropriate
expression vectors and suitable regulatory sequences are known in
the art for a variety of host cells.
 Typically, said one or more regulatory nucleotide sequences
may include, promoter sequences, leader or signal sequences, ribosomal
binding sites, transcriptional start and termination sequences,
translational start and termination sequences, and enhancer or activator
 Constitutive or inducible promoters as known in the art
are contemplated by the invention. The promoters may be either naturally
occurring promoters, or hybrid promoters that combine elements of
more than one promoter. For example, the lac promoter is inducible
 The expression vector may further comprise a selectable
marker gene to allow the selection of transformed host cells. Selectable
marker genes are well known in the art and will vary with the host
cell used. For example, an ampicillin resistance gene for selection
of positively transformed host cells when grown in a medium comprising
 The expression vector may also include a fusion partner
(typically provided by the expression vector) so that the recombinant
polypeptide of the invention is expressed as a fusion polypeptide
with the fusion partner. An advantage of fusion partners is that
they assist identification and/or purification of the fusion polypeptide.
Identification and/or purification may include using a monoclonal
antibody or substrate specific for the fusion partner, for example
a 6.times.-His tag or GST. A fusion partner may also comprise a
leader sequence for directing secretion of a recombinant polypeptide,
for example an alpha-factor leader sequence.
 Well known examples of fusion partners include hexahistidine
(6.times.-HIS)-tag, N-Flag, Fc portion of human IgG, glutathione-5-transferase
(GST) and maltose binding protein (MBP), which are particularly
useful for isolation of the fusion polypeptide by affinity chromatography.
For the purposes of fusion polypeptide purification by affinity
chromatography, relevant matrices for affinity chromatography may
include nickel-conjugated or cobalt-conjugated resins, fusion polypeptide
specific antibodies, qlutathione-conjugated resins, and amylose-conjugated
resins respectively. Some matrices are available in "kit"
form, such as the ProBond.TM. Purification System (Invitrogene Corp.)
which incorporates a 6.times.-His fusion vector and purification
using ProBond.TM. resin.
 In order to express the fusion polypeptide, it is necessary
to ligate a nucleic acid according to the invention into the expression
vector so that the translational reading frames of the fusion partner
and the nucleotide sequence of the invention coincide.
 The fusion partners may also have protease cleavage sites,
for example enterokinase (available from Invitrogen Corp. as EnterokinaseMax.TM.),
Factor X.sub.a or Thrombin, which allow the relevant protease to
digest the fusion polypeptide of the invention and thereby liberate
the recombinant polypeptide of the invention therefrom. The liberated
polypeptide can then be isolated from the fusion partner by subsequent
 Fusion partners may also include within their scope "epitope
tags", which are usually short peptide sequences for which
a specific antibody is available.
 As hereinbefore, polypeptides of the invention may be produced
by culturing a host cell transformed with an expression construct
comprising a nucleic acid encoding a polypeptide, or polypeptide
homolog, of the invention. The conditions appropriate for polypeptide
expression will vary with the choice of expression vector and the
host cell. For example, a nucleotide sequence of the invention may
be modified for successful or improved polypeptide expression in
a given host cell. Modifications include altering nucleotides depending
on preferred codon usage of the host cell. Alternatively, or in
addition, a nucleotide sequence of the invention may be modified
to accommodate host specific splice sites or lack thereof. These
modifications may be ascertained by one skilled in the art.
 Host cells for expression may be prokaryotic or eukaryotic.
 Useful prokaryotic host cells are bacteria.
 A typical bacteria host cell is a strain of E. coli.
 Useful eukaryotic cells are yeast, SF9 cells that may be
used with a baculovirus expression system as described herein, and
other mammalian cells.
 The recombinant polypeptide may be conveniently prepared
by a person skilled in the art using standard protocols as for example
described in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual
(Cold Spring Harbor Press, 1989), incorporated herein by reference,
in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY Eds. Ausubel et al., (John Wiley & Sons, Inc. 1995-1999),
incorporated herein by reference, in particular Chapters 10 and
16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al.,
(John Wiley & Sons, Inc. 1995-1999) which is incorporated by
reference herein, in particular Chapters 1, 5 and 6.
 In one embodiment, nucleic acid homologs encode polypeptide
homologs of the invention, inclusive of variants, fragments and
 In another embodiment, nucleic acid homologs share at least
60%, preferably at least 70%, more preferably at least 80%, and
even more preferably at least 90% sequence identity with the nucleic
acids of the invention.
 In yet another embodiment, nucleic acid homologs hybridise
to nucleic acids of the invention under at least low stringency
conditions, preferably under at least medium stringency conditions
and more preferably under high stringency conditions.
 "Hybridise and Hybridisation" is used herein to
denote the pairing of at least partly complementary nucleotide sequences
to produce a DNA-DNA, RNA-RNA or DNA-RNA hybrid. Hybrid sequences
comprising complementary nucleotide sequences occur through base-pairing.
 In DNA, complementary bases are:  (i) A and T; and
 (ii) C and G.
 In RNA, complementary bases are:  (i) A and U; and
 (ii) C and G.
 In RNA-DNA hybrids, complementary bases are:  (i)
A and U;  (ii) A and T; and  (iii) G and C.
 Modified purines (for example, inosine, methylinosine and
methyladenosine) and modified pyrimidines (thiouridine and methylcytosine)
may also engage in base pairing.
 "Stringency" as used herein, refers to temperature
and ionic strength conditions, and presence or absence of certain
organic solvents and/or detergents during hybridisation. The higher
the stringency, the higher will be the required level of complementarity
between hybridizing nucleotide sequences.
 "Stringent conditions" designates those conditions
under which only nucleic acid having a high frequency of complementary
bases will hybridize.
 Reference herein to low stringency conditions includes and
encompasses:--  (i) from at least about 1% v/v to at least
about 15% v/v formamide and from at least about 1 M to at least
about 2 M salt for hybridisation at 42.degree. C., and at least
about 1 M to at least about 2 M salt for washing at 42.degree. C.;
and  (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M
NaHPO.sub.4 (pH 7.2), 7% SDS for hybridization at 65.degree. C.,
and (i) 2.times.SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM
NaHPO.sub.4 (pH 7.2), 5% SDS for washing at room temperature.
 Medium stringency conditions include and encompass:-- 
(i) from at least about 16% v/v to at least about 30% V/v formamide
and from at least about 0.5 M to at least about 0.9 M salt for hybridisation
at 42.degree. C., and at least about 0.5 M to at least about 0.9
M salt for washing at 42.degree. C.; and  (ii) 1% Bovine Serum
Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS for
hybridization at 65.degree. C. and (a) 2.times.SSC, 0.1% SDS; or
(b) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 5% SDS for
washing at 42.degree. C.
 High stringency conditions include and encompass:-- 
(i) from at least about 31% v/v to at least about 50% v/v formamide
and from at least about 0.01 M to at least about 0.15 M salt for
hybridisation at 42.degree. C., and at least about 0.01 M to at
least about 0.15 M salt for washing at 42.degree. C.;  (ii)
1% BSA, 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS for hybridization
at 65.degree. C., and (a) 0.1.times.SSC, 0.1% SDS; or (b) 0.5% BSA,
1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 1% SDS for washing at a temperature
in excess of 65.degree. C. for about one hour; and  (iii)
0.2.times.SSC, 0.1% SDS for washing at or above 68.degree. C. for
about 20 minutes.
 In general, the T.sub.m of a duplex DNA decreases by about
1.degree. C. with every increase of 1% in the number of mismatched
 Notwithstanding the above, stringent conditions are well
known in the art, such as described in Chapters 2.9 and 2.10 of
Ausubel et al., supra, which are herein incorporated by reference.
A skilled addressee will also recognize that various factors can
be manipulated to optimize the specificity of the hybridization.
Optimization of the stringency of the final washes can serve to
ensure a high degree of hybridization.
 Typically, complementary nucleotide sequences are identified
by blotting techniques that include a step whereby nucleotides are
immobilized on a matrix (preferably a synthetic membrane such as
nitrocellulose), a hybridization step, and a detection step. Southern
blotting is used to identify a complementary DNA sequence; Northern
blotting is used to identify a complementary RNA sequence. Dot blotting
and slot blotting can be used to identify complementary DNA/DNA,
DNA/RNA or RNA/RNA polynucleotide sequences. Such techniques are
well known by those skilled in the art, and have been described
in Ausubel et al., supra, at pages 2.9.1 through 2.9.20, herein
incorporated by reference.
 According to such methods, Southern blotting involves separating
DNA molecules according to size by gel electrophoresis, transferring
the size-separated DNA to a synthetic membrane, and hybridizing
the membrane bound DNA to a complementary nucleotide sequence.
 In dot blotting and slot blotting, DNA samples are directly
applied to a synthetic membrane prior to hybridization as above.
 An alternative blotting step is used when identifying complementary
nucleic acids in a cDNA or genomic DNA library, such as through
the process of plaque or colony hybridisation. Other typical examples
of this procedure are described in Chapters 8-12 of Sambrook et
al., supra which are herein incorporated by reference.
 Typically, the following general procedure can be used to
determine hybridisation conditions. Nucleic acids are blotted/transferred
to a synthetic membrane, as described above. A nucleotide sequence
of the invention is labeled as described above, and the ability
of this labeled nucleic acid to hybridise with an immobilized nucleotide
 A skilled addressee will recognise that a number of factors
influence hybridisation. The specific activity of radioactively
labeled polynucleotide sequence should typically be greater than
or equal to about 10.sup.8 dpm/.mu.g to provide a detectable signal.
A radiolabeled nucleotide sequence of specific activity 10.sup.8
to 10.sup.9 dpm/.mu.g can detect approximately 0.5 pg of DNA. It
is well known in the art that sufficient DNA must be immobilised
on the membrane to permit detection. It is desirable to have excess
immobilised DNA, usually 10 pg. Adding an inert polymer such as
10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000
during hybridisation can also increase the sensitivity of hybridisation
(see Ausubel et al., supra at 2.10.10).
 To achieve meaningful results from hybridisation between
a nucleic acid immobilised on a membrane and a labeled nucleic acid,
a sufficient amount of the labeled nucleic acid must be hybridised
to the immobilised nucleic acid following washing. Washing ensures
that the labeled nucleic acid is hybridised only to the immobilised
nucleic acid with a desired degree of complementarity to the labeled
 Methods for detecting labeled nucleic acids hybridised to
an immobilised nucleic acid are well known to practitioners in the
art. Such methods include autoradiography, chemiluminescent, fluorescent
and colourimetric detection.
 Nucleic acid homologs of the invention may be prepared according
to the following procedure:  (i) obtaining a nucleic acid
extract from a suitable host, for example a bacterial species; 
(ii) creating primers which are optionally degenerate wherein each
comprises a portion of a nucleotide sequence of the invention; and
 (iii) using said primers to amplify, via nucleic acid amplification
techniques, one or more amplification products from said nucleic
 As used herein, an "amplification product" refers
to a nucleic acid product generated by nucleic acid amplification
 Suitable nucleic acid amplification techniques are well
known to the skilled addressee, and include PCR as for example described
in Chapter 15 of Ausubel et al. supra, which is incorporated herein
by reference; strand displacement amplification (SDA) as for example
described in U.S. Pat. No. 5,422,252 which is incorporated herein
by reference; rolling circle replication (RCR) as for example described
in Liu et al., 1996, J. Am. Chem. Soc. 118 1587 and International
application WO 92/01813; and Lizardi and Caplan, International Application
WO 97/19193, which are incorporated herein by reference; nucleic
acid sequence-based amplification (NASBA) as for example described
by Sooknanan et al., 1994, Biotechniques 17 1077, which is incorporated
herein by reference; ligase chain reaction (LCR) as for example
described in International Application WO89/09385 which is incorporated
herein by reference; and Q-.beta. replicase amplification as for
example described by Tyagi et al., 1996, Proc. Natl. Acad. Sci.
USA 93 5395 which is incorporated herein by reference. Preferably,
amplification is by PCR using primers disclosed herein.
G3BP-2 and Breast Cancer
 G3BP-2 protein was found to be in breast cancers that are
derived from epithelial tissues and non-proliferative tissues. This
was an unexpected result, at least partly because it was not known
that expression would be shown to be specific to tumours and that
up-regulation of G3BP-2 would occur so early in the development
of the tumour. G3BP-2 is up-regulated in approximately 80% of breast
cancers studied. This compares to genes that are well recognised
as causing breast cancers such as Brca1, which is found to be mutated
in 15% of familial breast cancers (percentages depend on the country
of the study). Familial breast cancers only represent 15-30% of
total breast cancers, which means that Brca1 is causative of only
2-5% of all breast cancers.
 The inventors have proposed a model that suggests G3BP-2
migrates to the nucleus of the cell to pick up transcripts and export
them to the ribosome so that translation of selected gene transcripts
can be regulated at the level of the ribosome. The inventors have
proposed this model because of a surprising finding that G3BP-2
can be immunoprecipitated with ribosomal proteins normally associated
with polysomes. This suggests that a possible method of action may
be to export the transcripts of oncogenes that regulate cell cycle
to the ribosome to up-regulate their transcription and thereby enhancing
cancer progression. Interestingly it also suggests that G3BP-2 may
represent a connection between signal transduction and RNA-processing.
 G3BP-2 may not actually be causative of breast cancer, but
may be required by the cancer to cause proliferation and thereby
account for its up-regulation in 80% of breast cancers. G3BP-2 is
specifically localised to sub-cellular compartments in a cell cycle
dependent manner, moving into the nucleus during proliferation and
then back out to the cytoplasm. G3BP-2 appears to "short-circuit"
normal ras-GAP.sup.120 signalling by receiving messages directly
from GAP.sup.120 and moving into the cell nucleus. In the nucleus
G3BP-2 is most likely binding with transcript targets (eg. c-myc)
and exporting them from the nucleus to the ribosomes in the cytoplasm.
The inventors have co-immunoprecipitated G3BP-2 with a series of
polypeptides normally associated with translational active ribosomes.
Accordingly, G3BP-2 may be facilitating increased proliferation
of breast cancers by allowing the up-regulation of oncogenic gene
transcripts (eg. c-myc). Therapeutics designed to block the activity
of G3BP-2, in particular at the N-terminal NTF2-like domain, may
limit cancer progression.
Cloning, Sequence Homology and Structural Homology
 The inventors previously reported cloning MmuG3BP-2 (MMU65313)
in a general PCR-based screening for RRM-containing proteins (Kennedy
et al. 1997). This was achieved by using degenerate primers designed
to consensus sequences within the RRM (Birney et al., 1993) and
using the amplified PCR product to screen a late-primitive-streak
stage mouse embryo cDNA library to isolate a full-length cDNA. Due
to the conserved sequence homology between the G3BP genes, the coding
region of the MmuG3BP-2 cDNA can be used as a useful tool to recognise
both human G3BP-2 and MmuG3BP-1 in Northern analysis and library
screening. The inventors used the coding region of MmuG3BP-2 to
isolate and clone MmuG3BP-1 (MMAB1927) and human G3BP-2 (HsaG3BP-2)
from the late-primitive-streak stage mouse embryo cDNA library and
a foetal human brain cDNA library respectively. The clones were
sequenced and analysed to identify Met start codons, open reading
frames and stop codons. Protein sequence comparison (FIG. 3) between
HsaG3BP-2 and MmuG3BP-2 show 98.5% identity, HsaG3BP-2 and HsaG3BP-1
(HS3251910) show 65% identity and HsaG3BP-1 shares 94.4% sequence
identity with MmuG3BP-1. In FIG. 3, amino acids are shown in single
letter format and grouped in blocks of 10. Numbering at the end
of the line indicates the amino acid position within the indicated
protein. Dashes within the aligned sequences indicate conserved
amino acids with respect to HsaG3BP-2a, amino acid changes between
proteins have been indicated by the appropriate substitution. Spaces
within the aligned proteins indicate gaps inserted into the sequences
to maintain co-linearity. Boxes represent proline rich sequences
(PxxP). Sequences in italics indicate the acid-rich domain. Ovals
represent components of an RGG domain, note that the RGG domains
of G3BP-1 and G3BP-2 are divergent. Underlined and double underlined
sequences indicate RNP-2 and RNP-1 respectively.
 Screening of the libraries also revealed that at least two
different G3BP-2 isoforms are produced in both mouse and human.
The alternative splicing event, which is 100% conserved between
mouse and human, deletes 99 nucleotides from the coding region and
does not introduce any stop codons nor a frame shift. Both these
transcripts are translated in vivo as was confirmed by Western blot
analysis and recombinant protein expression (see below). The longer
isoform has been designated G3BP-2a (human G3BP-2a accession number
AF145285, mouse G3BP-2a accession number AF145285). The shorter
protein isoform is referred to as G3BP-2b (human G3BP-2b accession
number AF053535 and AF051311 and mouse G3BP-2b accession number
MMU65313) differs from G3BP-2a by a 33 amino acid deletion from
the central region of the primary structure (FIG. 3). A third MmuG3BP-2
transcript was detected and cloned (referred to herein as G3BP-2c);
however, sequence analysis indicated that translation of this transcript
would lead to a truncated gene product and so far no corresponding
protein has been detected in cells or tissues suggesting that it
may not be translated.
 All G3BPs share highly conserved RNP-1 and RNP-2 sequences,
which are consensus motifs of RRMs. The most notable difference
between G3BP-2 and G3BP-1 RRMs is a Val to Ile substitution in the
RNP-2 consensus sequence (FIG. 3). The structure of the G3BP RRM
has been reported elsewhere (Kennedy et al. 1997). In addition,
the G3BPs contain a conserved acid-rich domain and an RGG domain
(Birney et al. 1993), both of which are commonly found in RNA-binding
proteins. It should be noted that there are considerable differences
in the RGG domain structure between G3BP-1 and G3BP-2 (see FIG.
3) and this may result in a different RNA target specificity. Although
acid-rich domains are found in association with a variety of RNA-binding
proteins such as hnRNP C and nucleolin, the function of this domain
remains unclear and maybe involved in protein-protein interactions.
The most significant difference between the G3BP-1 and G3BP-2 proteins
lies in the number of potential SH3 domain-binding motifs (PxxP,
where x represents any amino acid) (Lee et al. 1996). The G3BP-2a
protein comprises a cluster of four PxxP motifs between the acid-rich
and RRM domains whereas G3BP-2b contains five in the homologous
region (FIG. 3). The additional proline-rich PxxP motif in G3BP-2b
is generated by the 33 amino acids spliced out of G3BP-2a. In contrast
to the multiple PxxP clusters found in G3BP-2s, G3BP-1 contains
only one such motif in the homologous region of the protein (FIG.
3). Furthermore, both G3BP-1 and G3BP-2 comprise a conserved PxxP
motif (PGGP) within their non-conserved RGG domains (FIG. 3). The
specific conservation of the minimal SH3 domain-binding motif within
a region of the protein, which is generally not conserved, may suggest
a retained function although this remains to be determined. The
overall differences in the number of potential SH3 domain-binding
motifs between the G3BPs may indicate that in vivo they may bind
different SH3 domain-containing partners or have different affinities
for the same protein.
Protein Expression in Insect Cells
 MmuG3BP-2a and 2b cDNAs encode proteins whose predicted
sizes are 58.2 kDa and 52 kDa respectively. The expressed recombinant
proteins have apparent molecular weights of 68.5 kDa and 62 kDa
respectively, as determined by SDS-PAGE. These differences in predicted
and apparent masses are consistent with an increase in mass due
to post translational modifications and are similar to those reported
for HsaG3BP-1 (predicted mass of 52, observed apparent mass of 68
kDa) (Parker et al. 1996).
Mapping of G3BP-rasGAP.sup.120 Interactions
 Interactions between G3BP-1 and rasGAP.sup.120 have been
reported and show that G3BP-1 specifically interacts with the SH3
domain of rasGAP.sup.120 (Parker et al. 1996) implicating G3BP in
the rasGAP.sup.120 signal transduction pathway (see also Pazman
et al. 2000). However, the region within the G3BPs responsible for
the observed interaction with rasGAP.sup.120 has not been thoroughly
investigated. Initially it was presumed that the interactions would
be facilitated through proline rich motifs that are known to interact
with SH3 domains (Lee et al. 1996). To further map the interactions
between G3BPs and the SH3 domain of rasGAP.sup.120 several GST-G3BP
peptide fusions were expressed (FIG. 4) and probed in bead binding
assays with His-tagged N-terminal rasGAP.sup.120 peptides. The G3BP
peptide constructs were designed to represent truncated proteins
containing single or multiple domains as well as peptides that would
contain isolated proline rich motifs (FIG. 4). In the assays GST-G3BP
peptides are bound to glutathione beads and His-tagged N-terminal
rasGAP.sup.120 is added to the different constructs. Specific protein-protein
interactions between the G3BP peptides and the SH3 domain of rasGAP.sup.120
are detected by running the bound proteins on a Western blot and
identifying His-tagged rasGAP.sup.120 by probing with an anti-His
antibody. The results show (FIG. 4) that the interaction between
the SH3 domain of rasGAP.sup.120 maps to the NTF2-like domain contained
within the N-terminal domain of the G3BPs but not the proline rich
motifs. No interactions were detected with the other domains of
G3BP including the Acid-Rich domain, the RRM or the RGG-rich domain
 FIG. 4A shows a schematic representation of sub-domains
and motifs contained within G3BP-2a and G3BP-1 proteins and includes
the N-terminal NTF2-like, Acid-rich, RGG and proline-rich domains
as well as the RNA-recognition motif (see insert for details). Below
the respective full length proteins are shown various truncated
peptides that were expressed as GST-fusion proteins to map interactions
with the N-terminal SH3 domain containing region of rasGAP.sup.20.
The numbering corresponds to the amino acid at the site of the peptide
truncation (ie. .delta.-2a-N146 represents the truncated G3BP-2a
peptide including the N-terminal amino acids 1 to 146), the relative
position of these truncations is shown to approximate scale in the
full length proteins. FIGS. 4B and 4C show Western blot analysis
of Glutathione beads bound with various GST-G3BP peptides and probed
with the His-tagged N-terminal SH3 domain containing region of rasGAP.sup.120.
 G3BP-rasGAP.sup.120 interactions were determined by probing
the Western blots with anti-His antibodies. The results show that
the N-terminal SH3 domain containing region of rasGAP.sup.120 interacted
with the NTF2 like domain of G3BP-2a (.delta.-2a-N146, panel B)
and any peptide of G3BP-2a that comprised the NTF2 like domain (.delta.-2a-N205,
.delta.-2a-N257, .delta.-2a-N329 and full length G3BP-2a, panel
B). The results obtained from G3BP-1 are consistent with these results
from G3BP-2a and show that full length G3BP-1, .delta.-1-N229 and
.delta.-1-N309 interact with the N-terminal SH3 domain containing
region of rasGAP.sup.120. Truncated peptides of either G3BP-2a or
G3BP-1 that did not contain an intact NTF2-like domain failed to
bind with the SH3 region of rasGAP.sup.120.
 The results reported herein were confirmed using far-Western
protocols (data not shown) and are consistent with the data obtained
from the bead binding assays.
G3BPs have a Tissue Specific Expression
 Antibodies raised against isoform specific synthetic peptides
determined from G3BP-1 and G3BP-2 sequences were used to probe western
blots of total cell lysates from adult mouse tissues (FIG. 5). FIG.
5, panel A shows tissues probed with anti-G3BP-1 polyclonal antibodies
whereas panel B shows a collage of tissues probed with anti-G3BP-2
polyclonal antibodies. Some tissues are shown to express both isoforms
of G3BP-2 (FIG. 5, panel B) including lung, liver, kidney, stomach
and colon (also pancreas and testis, data not shown). Other tissues
are restricted to only expressing G3BP-2a (upper band in FIG. 5,
panel B) including brain, muscle (small amount of G3BP-2b expression
is seen and is presumably caused by the presence of different cell
populations within the sample), and heart. Small intestine expresses
only MmuG3BP-2b (lower band FIG. 5, panel B) and spleen does not
express either protein at detectable levels. Although the general
expression of G3BP-1 appears lower than that of G3BP-2 some tissues
express abundant levels of G3BP-1 and include lung, kidney and colon.
Heart, liver and spleen also express low levels of G3BP-1.
 FIG. 6 shows immunohistochemistry results of adult mouse
tissues probed with anti-G3BP-1 and anti-G3BP-2 antibodies. Panels
A to E are probed with an anti-G3BP-1 antibody whereas panels F
to J are probed with an anti-G3BP-2 antibody. All tissue staining
was visualised with horse radish peroxidase and sections were counterstained
with haematoxylin. Panels A and F are brain (Ne denotes a neurone,
GI denotes a glial cell), B and G are kidney (Gm denotes a glomerulus,
Tu denotes a tubule), C and H are colon (Ig denotes an intestinal
gland) D and I are small intestine (V denotes a villi) and E and
J are stomach (Ep denotes epithelial mucus secreting cells and Pg
denotes a pyloric gland). All photographs are taken at 100.times.
magnification (bars represent 100 .mu.m) with the exception of stomach
(panels E and J), which is displayed at 50.times. magnification
(bars represent 100 .mu.m).
Chromosomal Location of G3BPs
 Sequence data obtained from the library clones was used
to design G3BP-1 and G3BP-2 specific primers, which were subsequently
used on the GeneBridge hybridisation panel to determine the chromosomal
localisation of these genes. G3BP-1 mapped to chromosome 5 at 1.51
cR from FB25D10 (lod>3.0) which places the gene between 5q33.1-5q33.3.
G3BP-2 mapped to chromosome 4 at 3.36 cR from WI-5565 (lod>3.0)
which places the gene between 4q12-4q24. A plasmid artificial chromosome
(PAC) library (kindly donated by Dr. P. Ioannou, The Murdoch Institute,
Australia) was subsequently screened and isolated clones used to
perform fluorescent in-situ hybridisation. The results of the FISH
confirmed the genetic location of these genes. In addition to screening
the GeneBridge hybridisation panel and the FISH analysis, the human
genome sequences in the NCBI databases (http://ncbi.nlm.nih.gov/genome/seq/)
were BLAST searched with the cDNA sequences of human G3BP-1 and
G3BP-2. The results indicated that there are several chromosomes
with matches to either G3BP-1 or G3BP-2, which may represent gene
duplications or pseudo-genes. The BLAST analysis on its own was
not sufficient to map the G3BPs, however, in conjunction with the
FISH and the GeneGridge hybridisation panel results the chromosomal
localisation of these genes as indicated above has been confirmed.
 A search of the genomic databases (available at http://gdbwww.gdb.org/gdbreports/GeneByChromosome.4.alpha.html
did not reveal any candidate loci for diseases that may represent
genetic defects/polymorphisms in this family of proteins although,
there does appear to be some clustering of RNA-binding protein genes
on chromosome 5q including, hnRNP A/B (GDB:128837), hnRNP H1 (GDB:5428597),
ribosomal protein L7 pseudo gene (GDB:277889), ribosomal protein
S14 (GDB:119572), ribosomal protein S17a-like 1 (GDB:119573), ribosomal
protein S20A (GDB:119575) and ribosomal protein S20B (GDB:119576).
Chromosome 4q also contains several RNA-binding proteins including
EIF4EL1 (GDB:126371), G-rich RNA sequence binding factor 1 (GDB:696354),
hnRNP D (GDB:9391694), RNA polymerase II polypeptide B (GDB:135034)
and ribosomal protein L34 (GDB:9863242). Segment deletions and breakpoint
analysis of regions overlapping the chromosomal location of G3BP-1
suggest that this region may be involved in myeloid leukemias (Horrigan
et al., 1999) whereas similar studies for chromosome 4q suggest
the region containing the G3BP-2 gene may be involved in colorectal
adenoma (Wong et al., 1999) and Hodgkin's disease (Atkin 1998).
However, there is insufficient data to suggest that any G3BPs are
involved in human pathologies that are linked to these regions of
the human chromosomes.
Antibodies to G3BP-2
 The invention also relates to antibodies against the isolated
G3BP-2 polypeptide, fragments, variants and derivatives thereof.
A peptide fragment of G3BP-2 may comprise amino acid sequence SATPPPAEPASLPQEPPKPRV
[SEQ ID NO: 3], as herein described. Antibodies of the invention
may be polyclonal or monoclonal. Well-known protocols applicable
to antibody production, purification and use may be found, for example,
in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY
(John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane,
D. Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring
Harbor Laboratory, 1988, which are both herein incorporated by reference.
 Generally, antibodies of the invention bind to or conjugate
with a polypeptide, fragment, variant or derivative of the invention.
For example, the antibodies may comprise polyclonal antibodies.
Such antibodies may be prepared for example by injecting a polypeptide,
fragment, variant or derivative of the invention into a production
species, which may include mice or rabbits, to obtain polyclonal
antisera. Methods of producing polyclonal antibodies are well known
to those skilled in the art. Exemplary protocols which may be used
are described for example in Coligan et al., CURRENT PROTOCOLS IN
IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.
 In lieu of the polyclonal antisera obtained in the production
species, monoclonal antibodies may be produced using the standard
method as for example, described in an article by Kohler & Milstein,
1975, Nature 256, 495, which is herein incorporated by reference,
or by more recent modifications thereof as for example, described
in Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing
spleen or other antibody producing cells derived from a production
species which has been inoculated with one or more of the polypeptides,
fragments, variants or derivatives of the invention.
 The invention also includes within its scope antibodies,
which comprise Fc or Fab fragments of the polyclonal or monoclonal
antibodies referred to above. Alternatively, the antibodies may
comprise single chain Fv antibodies (scFvs) against the peptides
of the invention. Such scFvs may be prepared, for example, in accordance
with the methods described respectively in U.S. Pat. No. 5,091,513,
European Patent No 239,400 or the article by Winter & Milstein,
1991, Nature 349 293, which are incorporated herein by reference.
 The antibodies of the invention may be used for affinity
chromatography in isolating natural or recombinant polypeptides
of the invention. For example, reference may be made to immunoaffinity
chromatographic procedures described in Chapter 9.5 of Coligan et
al., CURRENT PROTOCOLS IN IMMUNOLOGY, supra.
 Antibodies may be purified from a suitable biological fluid
of the animal by ammonium sulfate fractionation, affinity purification
or by other methods well known in the art. Exemplary protocols for
antibody purification are given in Sections 10.11 and 11.13 of Ausubel
et al., supra, which are herein incorporated by reference.
 Immunoreactivity of the antibody against the native or parent
polypeptide may be determined by any suitable procedure such as,
for example, Western blot.
Mimetics, Agonists and Antagonists
 G3BP-2 offers a unique possibility for therapeutics because
of its interaction with oncogenic pathways and its unique features,
which appear to regulate cell cycle. Of particular interest is an
N-terminal NTF2-like domain, which has a surprising host of activities:
 (1) The NTF2-like domain appears to regulate nuclear localisation
through an interaction with the ran nuclear pore protein. 
(2) The expression of a known oncogene, NF.kappa.B, through interactions
with a Ubiquitin hydrolase, ODE1.  (3) Signal transduction
through interactions with GAP.sup.120
 The inventors have also determined that G3BP-2 receives
its messages from GAP.sup.120 through the NTF2-like domain. The
NTF2-like domain of G3BP-2 is highly conserved to the entire NTF2
protein. NTF2 is a nuclear pore protein that shuttles into the nucleus
through energy dependent interactions with ran. The inventors speculate
that G3BP-2 shuttles in and out of the nucleus using the same mechanisms
as NTF2 and to this extent they have shown that G3BP-2 interacts
with ran. The inventors have also determined that the NTF2-like
domain of G3BP-2 interacts with ODE1, a ubiquitin hydrolase and
that this interaction can increase the gene expression of NF.kappa.B,
another protein implicated in tumour progression.
 Targeting the NTF2-like domain for anti-cancer therapeutics
may inhibit tumour cell proliferation. This would be achieved by
blocking ability of G3BP-2 to receive signals from GAP.sup.120,
to block its ability to shuttle mRNA transcripts from the nucleus
to the cytoplasm and to block its ability to cause the up-regulation
 The invention contemplates agents which may prevent or disrupt
formation of polypeptide complexes comprising G3BP-2 and a native
or endogenous target polypeptide. Such an agent may be a mimetic,
which antagonizes or mimics one or more biological activities of
G3BP-2 polypeptides, or homologs thereof.
 It will be appreciated that G3BP-2 comprises several recognisable
sub-domains (an acid-rich domain, an RNA-recognition motif, an arginine-glycine
rich motif and a proline-rich motif). A key to its biological activity
as a polypeptide that can facilitate cancer progression may lie
in the G3BP-2 N-terminal NTF2-like domain.
 The NTF2-like domain is considered to be preferred target
for the screening or design of potential G3BP-2 mimetics.
 The term "mimetic" is used herein to refer to
molecules that are designed to resemble particular functional regions
of proteins or peptides, and includes within its scope the terms
"agonist", "analogue" and "antagonist"
as are well understood in the art.
 An antagonist may be a competitive antagonist or non-competitive
 It is contemplated that mimetics could be engineered which
disrupt or prevent formation of polypeptide complexes between G3BP-2
and endogenous target polypeptides. A mimetic preferably disrupts
or prevents formation of a complex between the NTF2-like domain
of G3BP-2 and an endogenous target peptide, for example rasGAP.sup.120.
In particular, a polypeptide fragment of rasGAP.sup.120 comprising
the SH3 domain having amino acid sequence [SEQ ID NO: 6] (NCBI accession
number: P20936):  VRAILPY TKVPDTDEIS FLKGDMFIVH NELEDGWMWV
TNLRTDEQGL IVEDLVEEVG REED
 Conversely, it is contemplated that an analogue of the NTF2-like
domain of G3BP-2 could be engineered which enables formation of
a complex between the analogue and an endogenous native target polypeptide
of G3BP-2, thereby competing with G3BP-2 for binding of the endogenous
native target. Suitably, the analogue would also bind an endogenous
target polypeptide of G3BP-2.
 The aforementioned mimetics may be peptides, polypeptides
or other organic molecules, preferably small organic molecules,
with a desired biological activity and half-life.
 Mimetics may be identified by way of screening libraries
of molecules such as synthetic chemical libraries, including combinatorial
libraries, by methods such as described in Nestler & Liu, 1998,
Comb. Chem. High Throughput Screen. 1 113 and Kirkpatrick et al.,
1999, Comb. Chem. High Throughput Screen 2 211.
 It is also contemplated that libraries of naturally-occurring
molecules may be screened by methodology such as reviewed in Kolb,
1998, Prog. Drug. Res. 51 185.
 More rational approaches to designing mimetics may employ
computer assisted screening of structural databases, computer-assisted
modelling, or more traditional biophysical techniques which detect
molecular binding interactions, as are well known in the art.
 Computer-assisted structural database searching is becoming
increasingly utilized as a procedure for identifying mimetics. Database
searching methods which, in principle, may be suitable for identifying
mimetics, may be found in International Purification WO 97/418232
(directed to producing HIV antigen mimetics), U.S. Pat. No. 5,752,019
and International Publication WO 97/41526 (directed to identifying
EPO mimetics), each of which is incorporated herein by reference.
 Other methods include a variety of biophysical techniques,
which identify molecular interactions. These allow for the screening
of candidate molecules according to whether said candidate molecule
affects formation of G3BP-2:endogenous target polypeptide complexes,
for example. Methods applicable to potentially useful techniques
such as competitive radioligand binding assays (see Upton et al.,
1999, supra for relevant methods), analytical ultracentrifugation,
microcalorimetry, surface plasmon resonance and optical biosensor-based
methods are provided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN
SCIENCE Eds. Coligan et al., (John Wiley & Sons, 1997) which
is incorporated herein by reference.
 A further feature of the invention is use of the polypeptide,
fragment, variant or derivative thereof as actives in a pharmaceutical
composition. The actives may be "immunogenic agents" which
are capable of eliciting an immune response in an animal. An immunogenic
agent may comprise a protein, nucleic acid, vaccine or antigen presenting
cell loaded or pulsed with an antigen, or any combination of these
agents. The antigen presenting cell may be loaded or pulsed with
antigen by contacting the cell with an antigen, for example a protein,
polypeptide, fragment, variant or derivative of the invention. The
antigen may be internalised within the antigen presenting cell by
any suitable means including for example, phagocytosis, micro-injection,
engulfing, and the like. The antigen may be combined with any suitable
carrier, for example a latex bead, fusion protein, or any other
delivery particle commonly used in the art. The antigen presenting
cell may be, for example a dendritic cell or any other antigen presenting
cell as known in the art of immunology.
 A pharmaceutical composition may also comprise an antagonist,
which prevents binding between the NTF2-like domain of G3BP-2 and
an endogenous binding partner thereof.
 Suitably, the pharmaceutical composition comprises a pharmaceutically-acceptable
carrier. By "pharmaceutically-acceptable carrier, diluent or
excipient" is meant a solid or liquid filler, diluent or encapsulating
substance that may be safely used in systemic administration. Depending
upon the particular route of administration, a variety of carriers,
well known in the art may be used. These carriers may be selected
from a group including sugars, starches, cellulose and its derivatives,
malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic
oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers,
isotonic saline, and pyrogen-free water.
 Any suitable route of administration may be employed for
providing a patient with the pharmaceutical composition of the invention.
For example, oral, rectal, parenteral, sublingual, buccal, intravenous,
intra-articular, intramuscular, intra-dermal, subcutaneous, inhalational,
intraocular, intraperitoneal, intracerebroventricular, transdermal
and the like may be employed. Intra-muscular and subcutaneous injection
is appropriate for administration of immunogenic agents of the present
 Dosage forms include tablets, dispersions, suspensions,
injections, solutions, syrups, troches, capsules, suppositories,
aerosols, transdermal patches and the like. These dosage forms may
also include injecting or implanting controlled releasing devices
designed specifically for this purpose or other forms of implants
modified to act additionally in this fashion. Controlled release
of the therapeutic agent may be effected by coating the same, for
example, with hydrophobic polymers including acrylic resins, waxes,
higher aliphatic alcohols, polylactic and polyglycolic acids and
certain cellulose derivatives such as hydroxypropylmethyl cellulose.
In addition, the controlled release may be effected by using other
polymer matrices, liposomes and/or microspheres.
 Pharmaceutical compositions of the present invention suitable
for administration may be presented as discrete units such as vials,
capsules, sachets or tablets each containing a pre-determined amount
of one or more immunogenic agent of the invention, as a powder or
granules or as a solution or a suspension in an aqueous liquid,
a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil
liquid emulsion. Such compositions may be prepared by any of the
methods of pharmacy but all methods include the step of bringing
into association one or more immunogenic agents as described above
with the carrier, which constitutes one or more necessary ingredients.
In general, the compositions are prepared by uniformly and intimately
admixing the agents of the invention with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping
the product into the desired presentation.
 The above compositions may be used as a therapeutic or prophylactic
vaccines comprising a polypeptide and/or nucleic acid of the invention,
or respective fragments thereof. In one embodiment, the vaccine
comprises an immunogenic agent as described above. Preferably, the
vaccine prevents or treats breast cancer.
 Accordingly, the invention extends to the production of
vaccines comprising as actives one or more of the immunogenic agents
of the invention. Any suitable procedure is contemplated for producing
such vaccines. Exemplary procedures include, for example, those
described in NEW GENERATION VACCINES (1997, Levine et al., Marcel
Dekker, Inc. New York, Basel Hong Kong) which is incorporated herein
 An immunogenic agent according to the invention can be mixed,
conjugated or fused with other antigens, including B or T cell epitopes
of other antigens. In addition, it can be conjugated to a carrier
as described below.
 When a haptenic peptide of the invention is used (i.e.,
a peptide which reacts with cognate antibodies, but cannot itself
elicit an immune response), it can be conjugated with an immunogenic
carrier. Useful carriers are well known in the art and include for
example: thyroglobulin; albumins such as human serum albumin; toxins,
toxoids or any mutant cross reactive material (CRM) of the toxin
from tetanus, diptheria, pertussis, Pseudomonas, E. coli, Staphylococcus,
and Streptococcus; polyamino acids such as poly(lysine:glutamic
acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis
B virus core protein; hepatitis B virus recombinant vaccine and
the like. Alternatively, a fragment or epitope of a carrier protein
or other immunogenic polypeptide may be used. For example, a haptenic
peptide of the invention can be coupled to a T cell epitope of a
bacterial toxin, toxoid or CRM. In this regard, reference may be
made to U.S. Pat. No. 5,785,973 which is incorporated herein by
 The vaccines can also contain a physiologically-acceptable
carrier, diluent or excipient such as water, phosphate buffered
saline and saline.
 The vaccines and immunogenic agents may include an adjuvant
as is well known in the art. Suitable adjuvants include, but are
not limited to adjuvants for use in human for example SBAS2, SBAS4,
QS21 or ISCOMs.
 The immunogenic agents of the invention may be expressed
by attenuated viral hosts. By "attenuated viral hosts"
is meant viral vectors that are either naturally, or have been rendered,
substantially avirulent. A virus may be rendered substantially avirulent
by any suitable physical (e.g., heat treatment) or chemical means
(e.g., formaldehyde treatment). By "substantially avirulent"
is meant a virus whose infectivity has been destroyed. Ideally,
the infectivity of the virus is destroyed without affecting the
polypeptides that carry the immunogenicity of the virus. From the
foregoing, it will be appreciated that attenuated viral hosts may
comprise live viruses or inactivated viruses.
 Attenuated viral hosts which may be useful in a vaccine
according to the invention may comprise viral vectors inclusive
of adenovirus, cytomegalovirus and preferably pox viruses such as
vaccinia (see for example Paoletti and Panicali, U.S. Pat. No. 4,603,112
which is incorporated herein by reference) and attenuated Salmonella
strains (see for example Stocker, U.S. Pat. No. 4,550,081 which
is herein incorporated by reference). Live vaccines are particularly
advantageous because they lead to a prolonged stimulus that can
confer substantially long-lasting immunity.
 Multivalent vaccines can be prepared from one or more different
epitopes of G3BP-2.
 A recombinant vaccinia virus may be prepared to express
a nucleic acid according to the invention. Upon introduction into
a host, the recombinant vaccinia virus expresses the immunogenic
agent, and thereby elicits a host CTL response. For example, reference
may be made to U.S. Pat. No. 4,722,848, incorporated herein by reference,
which describes vaccinia vectors and methods useful in immunization
 A wide variety of other vectors useful for therapeutic administration
or immunization with the immunogenic agents of the invention will
be apparent to those skilled in the art from the present disclosure.
 The nucleic acid of the invention may be used as a vaccine
in the form of a "naked DNA" vaccine as is known in the
art. For example, an expression vector of the invention may be introduced
into a mammal, where it causes production of a polypeptide in vivo,
against which the host mounts an immune response as for example
described in Barry, M. et al., (1995, Nature, 377:632-635) which
is hereby incorporated herein by reference.
Dendritic Cell Therapy
 "Dendritic cells" (DC) are antigen presenting
cells capable of initiating an antigen-specific T-cell response
in an animal. DC's may be isolated from various locations of an
animal's body, including peripheral blood. Methods for in vitro
proliferation and expansion of DC precursors have been described,
for example in U.S. Pat. No. 5,994,126, incorporated herein by reference.
Also described is a method for producing mature dendritic cells
in culture from proliferating dendritic cell precursors.
 "Dendritic cell therapy" refers to therapeutic
cancer vaccines or cellular vaccines used for tumour immunotherapy
as a method for treating cancer. Dendritic cell therapy typically
involves isolating DC from a patient, culturing the isolated DC
in the presence of a tumour-associated antigen (TAA) thereby contacting
the DC with a TAA ("antigen loading or pulsing"), and
administering the antigen loaded DC's to the patient. Other methods
for antigen loading an isolated DC include transfecting, micro-injecting,
calcium phosphate transfection, DEAE-transfection, electroporation
or otherwise introducing an isolated nucleic acid encoding a tumour-associated
antigen into the isolated DC. Common method for introducing nucleic
acids into a cell are described in Chapter 9 of CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY Eds. Ausubel et al., (John Wiley & Sons,
Inc. 1995-1999), incorporated herein by reference. Preferably, the
TAA is G3BP-2, fragment, variant, homolog or derivative thereof.
The nucleic acid may be DNA or RNA. The nucleic acid may be transiently
or stably express the TAA as is known in the art.
 Methods for loading or pulsing dendritic cells with an antigen
are described in (Meidenbauer et al, 2001, Biol Chem 4 507; Rains,
et al, 2001, Hepatogastroenterology 38 347; Nestle, 2000, Oncogene
56 6673; Gilboa and Lyerly, 1998, Cancer Immunotherapy 46 82) incorporated
herein by reference.
 U.S. Pat. No. 5,788,963, incorporated herein by reference,
describes methods and compositions for use of human dendritic cells
to activate T-cells for immunotherapeutic responses against primary
and metastatic prostate cancer. In one embodiment isolated DC are
exposed in vitro to a prostate cancer antigen before administration
to a patient.
 In one embodiment of the present invention, a pharmaceutical
composition comprises DC's antigen load with G3BP-2 polypeptide,
fragment, variant or derivative thereof in accordance with the invention.
In another embodiment of the present, the DC's are transfected with
a nucleic acid encoding a G3BP-2 polypeptide, fragment, variant
or derivative thereof. Suitable G3BP-2 protein fragments for use
with DC cell therapy are set forth as SEQ ID NOS: 1 and 2. It will
be appreciated by a skilled person that antigen presenting cells
other than DC may be used and that use of DC is merely preferred.
Preparation of Immunoreactive Fragments
 The invention also extends to a method of identifying an
immunoreactive fragment of a polypeptide, variant or derivatives
according to the invention. This method essentially comprises generating
a fragment of the polypeptide, variant or derivative, administering
the fragment to a mammal; and detecting an immune response in the
mammal. Such response may include production of elements which specifically
bind G3BP-2, respective variant, or derivative thereof, including
NTF2-like domain, which may provide a protective effect against
 Prior to testing a particular fragment for immunoreactivity
in the above method, a variety of predictive methods may be used
to deduce whether a particular fragment can be used to obtain an
antibody that cross-reacts with the native antigen. These predictive
methods may be based on amino-terminal or carboxy-terminal sequence
as for example described in Chapter 11.14 of Ausubel et al., supra.
Alternatively, or in addition, these predictive methods may be based
on predictions of hydrophilicity as for example described by Kyte
& Doolittle 1982, J. Mol. Biol. 157 105 and Hopp & Woods,
1983, Mol. Immunol. 20 483) which are incorporated by reference
herein, or predictions of secondary structure as for example described
by Choo & Fasman, 1978, Ann. Rev. Biochem. 47 251), which is
incorporated herein by reference.
 Generally, a peptide fragment consisting of 10 to 15 residues
provides optimal results. Peptides as small as 6 or as large as
20 residues have worked successfully. Such peptide fragments may
then be chemically coupled to a carrier molecule such as keyhole
limpet hemocyanin (KLH) or bovine serum albumin (BSA) as for example
described in Sections 11.14 and 11.15 of Ausubel et al., supra).
 The peptides may be used to immunize an animal as for example
discussed above. Antibody titers against the native or parent polypeptide
from which the peptide was selected may then be determined by, for
example, radioimmunoassay or ELISA as for instance described in
Sections 11.16 and 11.14 of Ausubel et al., supra.
 Immunoreactive protein fragments in the context of the Major
Histocompatibility Complex (MHC) may be determined using methods
well known in the art including those described by Schultze and
Vonderheide, 2001, incorporated herein by reference.
 The present invention also provides a kit for detection
of G3BP-2 in a biological sample. A kit will contain one or more
particular agents described above depending upon the nature of the
test method employed. In this regard, the kits may include one or
more of a polypeptide, fragment, variant, derivative, antibody,
antibody fragment or nucleic acid according to the invention. The
kits may also optionally include appropriate reagents for detection
of labels, positive and negative controls, washing solutions, dilution
buffers and the like. For example, an antibody-based detection kit
may include (i) a polypeptide, or fragment or variant thereof according
to the invention (which may be used as a positive control), (ii)
an antibody according to the invention (preferably a monoclonal
antibody) which binds to G3BP-2 or fragment thereof in (i), and
(iii) a suitable means for detecting a complex formed between a
target (eg. G3BP-2 in a sample) and the antibody in (ii), the detection
means may include, for example colloidal gold. Suitable antibodies
for use in a detection kit include those described herein in relation
to Western blots and immunohistochemistry.
 A detection kit may also be nucleic acid based. Such a detection
kit may include the step of amplifying a nucleic acid from the test
sample obtained from an animal using techniques such as Polymerase
Chain Reaction (PCR) or other known amplification method known in
the art. Useful PCR primers may include those set forth herein as
SEQ ID NOS: 16-21. The nucleic acid may be RNA or DNA. The test
sample is preferably breast tissue isolated from a patient.
 Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises"
and "comprising" will be understood to imply the inclusion
of a stated integer or group of integers but not the exclusion of
any other integer or group of integers.
 In order that the invention may be readily understood and
put into practical effect, particular preferred embodiments will
now be described by way of the following non-limiting examples.
Cloning, Sequence Homology and Structural Homology
PCR and Subcloning
 PCR reactions to amplify probes or for hybrid mapping were
carried out using 1.1 units of Tth Plus DNA Polymerase fragment
(Biotech International) and buffer supplied by the manufacturer
(Biotech International) and contained 100 ng template DNA and 50
pmol of each appropriate primer. Cycling conditions were: denaturation
of DNA at 94.degree. C. for 1 min, annealing at 65.degree. C. for
1 min and extension at 72.degree. C. for 1 min for 25 cycles. Primers
that can be used to amplify either full length G3BPs or the NTF2-like
domain of the G3BPs:
 Full length human G3BP-1 is amplified using primers G3BP-2met
with G3BP-1stop; full length human G3BP-2 is amplified using primers
G3BP-2met with G3BP-2stop; the NTF2-like domain of human G3BP-1
is amplified using primers G3BP-1 met with G3BP-1 ntf; and the NTF2-like
domain of human G3BP-2 is amplified using primers G3BP-2met with
 Primer Sequences: TABLE-US-00005 G3BP-1met atggtgatggagaagcctagtcccctgctggt
[SEQ ID NO:16] G3BP-2met atggttatggagaagcccagtccg [SEQ ID NO:17]
G3BP-2met atcaagttcaggctcagaatcacc [SEQ ID NO:18] G3BP-1met ctgaggctcagtgacaaacccaac
[SEQ ID NO:19] G3BP-2stop gcttcagcgacgctgtcctgtgaagc [SEQ ID NO:20]
G3BP-1stop ctgccgtggcgcaagcccccttcc [SEQ ID NO:21]
Library Screening and Phage Isolation
 Plasmid preparations, to be used as probes, library screening
or sequencing, were made as described by Sambrook et al., 1989 (Sambrook
et al., 1989). The libraries, late-primitive-streak stage mouse
embryo cDNA (Kennedy et al. 1997) and a foetal human brain (cDNA
kindly donated by Dr. T. Cox, University of Adelaide, Australia)
were plated out at approximately 25 000 pfu/130 mm plate and duplicate
filters (Hybond-N, Amersham) made from 21 plates. Library screening
was performed as described by Sambrook et al., 1989 (Sambrook et
al. 1989) with radioactive probes prepared using Amersham's Megaprime
DNA labeling system according to the manufacturer's instructions.
Hybridization conditions were as follows: 50% formamide, 5.times.SSC,
20 mM Tris.HCl pH 7.6, 1.times. Denhardt's solution and 0.1% SDS
at 42.degree. C. A minimum of 3.times.20 min washes of hybridized
filters were performed in 0.2.times.SSC, 0.1% SDS at 65.degree.
C. cDNAs from purified plaques were subcloned into pBluescript SK
Protein Expression in Insect Cells
Sub Cloning and Expression of Proteins Using Baculovirus
 Full length cDNAs encoding for MmuG3BP-2a and 2b were excised
from pBluescript using EagI (-85 bp from met start codon) and AccI
(+140 bp from the stop codon) and subsequently end filled using
Klenow fragment polymerase. These cDNAs were subcloned into SmaI
linearised pBacPAK9 (Clontech) and transformed into competent DH5'.alpha.
E. coli cells. The orientation of the inserts were checked by PCR.
Plasmids containing the inserts in the correct orientation were
transfected into Spodoptera frugiperda IPLB-Sf21 (Sf21) cells with
Bsu36I digested BacPAK6 viral expression vector according to the
manufacturers instructions (Clontech #K1601-1). Recombinant virus
plaques were selected from an Sf21 monolayer and once again screened
by PCR. Virus containing the coding cDNAs were amplified in Sf21
cells and total cell lysates visualised on polyacrylamide gels for
expression of proteins.
Mapping of G3BP-rasGAP.sup.120 Interactions
Fusion Protein Constructs and Expression
 Truncated cDNAs representing either N-terminal or C-Terminal
sequences of G3BP-1 and G3BP-2a (FIG. 2) were subcloned into bacterial
GST-fusion expression vectors (pGex, Pharmacia) so that the recombinant
fusion proteins could be expressed and used in bead binding assays
(see below) to identify the sub-domains of G3BP that interact with
the SH3 domain of rasGAP.sup.120. The vectors containing the recombinant
fusion protein construct were transformed into competent DH5' E.
Coli and expressed by IPTG induction. To collect the recombinant
proteins the cells were washed and resuspended in PBS and sonicated
to release the fusion proteins.
 The N-terminal domain of rasGAP.sup.120 (kindly provided
by Prof. I.G. Macara, University of Virginia) containing amino acids
1-356 which includes the full SH3 domain of rasGAP.sup.120 was subcloned
into proEX HT bacterial expression vector (Life Technologies) and
expressed as described above.
Bead Binding Assays
 Glutathione columns (Pharmacia) were blocked overnight at
4.degree. C. in 1.times. binding buffer A (50 mM HEPES pH 7.4, 100
mM NaCl, 5 mM MgCl.sub.2, 50 mg/ml BSA, 1 mM DTT, 1 mM PMFS and
protease inhibitors (Protease inhibitor cocktail, Roche Diagnostics,
Australia). The following day the columns were washed twice with
binding buffer A. Bacterial lysates containing either expressed
GST alone or GST fusion proteins were diluted 1:1 with 2.times.
binding buffer A and added to the equilibrated GST columns, the
columns were gently rocked overnight at 4.degree. C. To remove excess
proteins the columns were washed twice in 1.times. binding buffer
A. Bacterial lysates containing the His-tagged N-terminal of rasGAP.sup.120
protein were combined with an equal volume of 2.times. binding buffer
A containing 200 mg/ml BSA and 0.1% Tween. In addition to the high
concentration of BSA, which is used to block non-specific protein-protein
interactions, Ethidium bromide was added to a final concentration
of 25 ng/ml to abrogate non-specific interactions caused by excess
bacterial genomic DNA and bacterial RNA. This mix was then added
to the pre-bound GST columns (above) and allowed to bind for 2 hrs
at 4.degree. C. The columns were then washed three times with binding
buffer B (50 mM HEPES pH 7.4, 200 mM NaCl, 5 mM MgCl.sub.2, 1 mM
DTT, 1 mM PMFS and protease inhibitors).
Western Analysis of Protein-Protein Interactions
 One .mu.l of the GST beads complexed with proteins, as described
above, was added to an equal volume of 2.times.PAGE loading dye,
heated for 5 mins at 95.degree. C. and loaded onto a 7.5% Polyacrylamide
gel and run at 100V for 3 hours. The proteins were transferred to
Nitrocellulose and probed with anti-His antibodies (Tetra-His antibody,
Qiagen). Columns, which were bound with GST alone, were used as
negative controls to ensure no non-specific His-tagged N-terminal
rasGAP.sup.120 remained associated with the columns. To ensure that
the GST columns had been saturated with the appropriate GST-G3BP
peptides all experiments were probed with anti-GST to confirm that
the columns contained the "bait" peptide (data not shown).
 500 ml of Sf21 cells at a concentration of 1.2 to 2.0.times.10.sup.6
cells/ml cells were infected with 60 ml of baculovirus (6.6.times.10.sup.8
to 1.6.times.10.sup.9 pfu/ml) containing cDNAs for expression of
proteins and incubated at 28.degree. C. for 4 days in an orbital
incubator. The cells were harvested and washed twice at 4.degree.
C. with PBS, lysed in 50 ml of HNTG lysis buffer by 30 sec vortexing
and gentle rocking for 30 min at 4.degree. C. and cleared by centrifugation
at 9500 rpm.times.10 min at 4.degree. C. The final salt concentration
was adjusted to 30 mM NaCl by addition of NaCl free and triton X-100
free HNTG lysis buffer (containing protease inhibitors) and incubated
overnight on a rotating mixer at 4.degree. C. with pre-equilibrated
heparin sepharose CL-6B (Pharmacia biotech #17-0467-01) at a concentration
of 15 mg of protein/ml of heparin sepharose. The gel was washed
in 50 mM Hepes pH 7.5, 10% glycerol and packed into a column (Pharmacia
XK-26). The column was subjected to a 30 mM to 1.0 M NaCl gradient
over 120 min at a flow rate of 0.83 ml/min using a Pharmacia FPLC
system. 1.5 ml samples were collected and assayed for MmuG3BP by
separation on polyacrylamide gels and visualised by coomassie blue
staining or Western blot analysis using the 663 antibody.
 Fractions containing the recombinant G3BP proteins were
pooled and again diluted to a final 60 mM NaCl concentration. The
pooled samples were incubated with agarose-polyribouridylic acid
AGPoly(U), type 6 (Pharmacia biotech #27-5535) at a concentration
of 1.5 mg of protein/ml of gel overnight at 4.degree. C. on a rotating
mixer. The following morning the gel was washed with 50 mM Hepes
pH 7.5, 10% glycerol, packed into a glass column (Pharmacia XK-16)
and subjected to a 60 mM to 1 M NaCl gradient at a flow rate of
0.33 ml/min as described above. 0.5 ml fractions were collected,
assayed and pooled as described above. Pooled samples were diluted
to 30 mM NaCl as described above and loaded onto a mono S HR 5/5
ion exchanger column (Pharmacia #17-0547-01) at a flow rate of 0.3
ml/min and subjected to a 30 mM to 1 M NaCl gradient. 0.5 ml fractions
were collected and assayed as described above.
Polyclonal Antibody Production
 Affinity purified antibodies to G3BP-2 were obtained from
an antiserum raised against an internal peptide sequence (SATPPPAEPASLPQEPPKPRV).
Polyclonal antibodies raised against G3BP-1 have been described
elsewhere (Parker et al. 1996) and a monoclonal G3BP-1 antibody
is commercially available as (BD Biosciences, Sydney, AUS).
G3BP-2 Antibody Specificity
 The specificity of the G3BP-2 antibody was assessed by testing
its ability to bind human recombinant GST fusion G3BP-1, G3BP-2a,
G3BP-2b and G3BP-2a truncations. LB/Amp agar plates were inoculated
with E. coli transformed with pGEX vectors (Amersham Biosciences,
GST gene fusion system) containing four alternative truncations
of G3BP-2a (N1, N2, C1 and C2), as well as full length G3BP-1, 2a
and 2b, as previously described in Kennedy et al. (2001). One colony
from each plate was used to inoculate 5 ml of LB/Amp broth, which
was incubated at 37.degree. C. overnight. Isolation of the GST fusion
proteins was performed by glutathione sepharose affinity chromatography
from IPTG-induced cultures as per the manufacturer's instructions
(Amersham Biosciences). Following elution with glutathione, the
solutions were spun at 5000 rpm for 5 minutes and the supernatant
was dialysed in PBS.
 Purified recombinant G3BP-1, G3BP-2a, G3BP-2b, and the four
truncations of G3BP-2a were resolved by 12% SDS-PAGE and transferred
to PVDF (Millipore) and incubated with anti-G3BP-2 antibody. Proteins
were visualised by HRP conjugated anti-rabbit antibodies using an
ECL system (Amersham Biosciences).
Cell Extracts, SDS-PAGE and Western Blotting
 The expression of G3BP in human cell lines was examined
by Western blot. Cells were maintained in vitro in DMEM supplemented
with 10% FCS and harvested by trypsinisation, washed twice with
PBS and resuspended in HNTG buffer (50 mM Hepes, pH 7.5, 150 mM
NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl.sub.2, 1 mM EGTA,
1 mM Na.sub.3VO.sub.4, 10 mM Na.sub.4P.sub.2O.sub.7, 10 mM NaF,
1 mM PMSF and 1.times. mammalian protease inhibitor cocktail # P8340
(Sigma, Castle Hill, AUS). Lysates were cleared by centrifugation
at 15 000 rpm for 10 minutes and the protein concentration was determined
using the Pierce BCA Protein Assay (Rockford, USA). A total of 75
.mu.g of protein was resolved by 8% SDS-PAGE and transferred to
an Immobilon-P PVDF membrane (Millipore, Sydney, AUS) for Western
analysis using the antibodies described above. Proteins were visualised
by horseradish peroxidase (HRP) conjugated anti-rabbit or mouse
antibodies using an ECL system (Amersham Biosciences, Sydney, AUS).
Antibodies Used for Immunohistochemistry were Highly Specific
 To assess antibody specificity and the expression of G3BPs
in a range of cell types, the breast cancer cell line, MDA-MB-435
and the cervical cancer cell line, HeLa were lysed and equal amounts
of protein were resolved by SDS-PAGE. The samples were transferred
to a membrane and analysed by Western blotting. The commercial monoclonal
G3BP-1 antibody (BD Biosciences) was used to assess G3BP-1 expression,
while the polyclonal G3BP-2 antibody (Kennedy et al. 2001) was used
to examine the expression of G3BP-2 in these cell lines. As illustrated
in FIG. 9, Panel A, it is apparent that both cell lines express
significant levels of G3BP-1, present as a single distinct band,
and G3BP-2, present as two distinct bands, representing the two
different isoforms. The same expression patterns were seen in the
immortalised human cell line HEK 293T (data not shown). There did
not appear to be any significant cross-reactivity. However, there
was some variation between the relative masses of G3BP-1, 2a and
2b according to amino acid sequence, and the apparent masses of
the proteins as determined by PAGE. According to the data, G3BP-2a
should resolve at a point just above G3BP-1, but it actually appears
just below G3BP-1.
 The specificity of the polyclonal G3BP-2 antibody was further
examined. It was tested against recombinant GST-fusion G3BP-1, 2a
and 2b as well as several different truncated forms of G3BP-2. As
presented in FIG. 9, Panel B, the anti-G3BP-2 antibody specifically
binds to recombinant G3BP-2a and G3BP-2b (lanes 3 and 1, respectively)
while it does not bind to G3BP-1 (lane 2). The antibody bound two
of the recombinant G3BP-2a truncations (lanes 5 and 7) but it did
not bind the short C-terminal or N-terminal truncations (Lanes 4
and 6). This indicated that the region that the antibody binds to
is the central domain as shown in FIG. 9, Panel C. It was found
that the G3BP-2 antibody is specific for G3BP-2a and 2b and binds
the central region of the protein only. Despite excessively high
protein loads (3 .mu.g of recombinant protein per lane), the antibody
did not cross-react with G3BP-1 or the shorter of the G3BP-2 N-terminal
(N1) or C-terminal (C2) truncations.
G3BPs have a Tissue Specific Expression
 Proteins were fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (7.5% gel) using the method of Laemmli and transferred
onto polyvinylidene difloride (PVDF) membrane (Millipore Corp.)
in a Bio-Rad Trans-blot Cell using a transfer buffer containing
25 mM Tris pH 8.3, 192 mM Glycine and 15% methanol. Electroblotting
was carried out at 100 volts overnight at 4.degree. C. The blot
was blocked by incubation in 20 mM Tris-HCl pH 7.5, 150 mM NaCl,
0.1% Tween-20 containing 10% skim milk (blocking solution) at room
temperature for 1 hour, followed by incubation with the primary
antibody, anti-G3BP-1 (diluted 1:500) or anti-G3BP-2 (diluted 1:4000)
in blocking solution, at 4.degree. C. overnight. The blot was washed
3 times in blocking solution for 10 minutes and subsequently incubated
for 2 hours at 37.degree. C. in the secondary antibody which was
an anti-rabbit conjugated with horseradish peroxidase (BioRad),
diluted 1:10000 in blocking solution.
Total Protein Cell and Tissue Lysates
 Cells and tissues were washed twice with ice cold phosphate-buffered
saline (PBS) and solubilised or homogenised with a dounce homogeniser
in HNTG lysis buffer consisting of 50 mM HEPES, pH 7.5, 150 mM NaCl,
1% Triton X-100, 10% glycerol, 1 mM MgCl.sub.2, 1 mM EGTA, phosphatase
inhibitors (1 mM Na.sub.3VO.sub.4, 10 mM Na.sub.4P.sub.2O.sub.7
and 10 mM NaF) and protease inhibitors (1 .mu.g of leupeptin per
ml, 1 .mu.g of trypsin inhibitor per ml, 1 .mu.g of pepstatin A,
2 .mu.g of aprotinin per ml, 10 .mu.g benzamidine per ml, 1 mM phenylmethylsulfonyl
floride, 1 .mu.g of antipain per ml, 1 .mu.g of chymostatin per
ml). Lysates were cleared by centrifugation at 15 000 rpm for 10
min and the protein concentration determined by the Bradford dye-binding
procedure using Bio-Rads Protein Assay (# 500-0001).
 Immunohistochemistry was used to analyse the degree of cell
specificity of the G3BP-1 and G3BP-2 expression. Until isoform specific
antibodies are raised against G3BP-2a and G3BP-2b it is not possible
to distinguish these isoforms in immunohistochemistry, however,
in some tissues a it can be determined which specific isoform is
being expressed by comparison to the Western blot data (as herein
 FIGS. 6A and 6B show a cross section of results from some
of the tissues studied. Panels A to E are probed with anti-G3BP-1
antibodies whereas panels F to J are probed with anti-G3BP-2 antibodies.
Panels A and F show a comparison of adult mouse brain. As determined
by Western analysis (FIG. 5, panel A), brain does not express G3BP-1
(FIG. 6, panel A) however, a sub-population of cells express G3BP-2a
 The inventors determined by cell morphology and double staining
with a neural marker (data not shown) that the G3BP-2a positive
cells are neural cells (panel F, labeled Ne) and that the negative
cells are glial cells (panel F, labeled GI). In the kidney (panels
B and C) G3BP-1 appears to be expressed in interstitial cells or
a sub-population of tubules (panel B) whereas G3BP-2 is expressed
at low levels in all tubules (labeled Tu in panel G). Neither G3BP-1
nor G3BP-2 are expressed in the glomerulus (labeled Gm in panel
G). The colon (panels C and H) shows that G3BP-1 is expressed at
the periphery of the intestinal glands or possibly in interstitial
cells whereas G3BP-2 is expressed in the lumen of the intestinal
glands (labeled Ig in panels H and I). G3BP-2 is also expressed
at high levels in the villi of the small intestine (FIG. 6 panel
I) whereas G3BP-1 (FIG. 6 panel D) was not detected at levels above
the background staining of the negative control. Once again no detectable
staining was observed for G3BP-1 in stomach (FIG. 6 panel E) whereas
G3BP-2 (presumably G3BP-2b only from the Western blot data) appears
to be expressed in the mucus secreting cells of the stomach lumen
(labeled Ep in panel J) and the internal surface of the pyloric
glands (labeled Pg in panel J). Other tissues examined by immunohistochemistry
include heart, liver and spleen (data not shown). Heart and liver
showed a general low level expression of G3BP-1 and G3BP-2 whereas
spleen was negative for G3BP-2 and showed a cell specific staining
of G3BP-1. It is still to be determined what types of cells constitute
the G3BP-1 expressing islands observed within the spleen.
 Frozen mouse tissue sections (10 .mu.m thickness) were fixed
to Histogrip treated slides (SuperFrost Plus microscope slides,
Menzel-Glaser, Germany) and air-dried overnight at room temperature.
Sections were fixed for 5 minutes in 50% Chloroform, 50% Acetone,
air dried and rehydrated in Tris-buffered saline (TBS) (25 mM Tris,
137 mM NaCl, pH 7.4). Nonspecific antibody binding was inhibited
by incubation with TBS containing 4% skim milk powder for 15 minutes
followed by an additional 20 min incubation in TBS containing 10%
normal goat serum (Gibco). Sections were then incubated overnight
with either anti-G3BP-1 (diluted 1:300) or an anti-G3BP-2 (diluted
1:2000). Excess antibody was removed by washing in TBS (3.times.5
min) and prediluted horseradish peroxidase (HRP) labeled anti-rabbit
immunoglobulins (Envision) was applied for 30 minutes. Sections
were then washed with TBS (3.times.5 min) and colour was developed
in 3,3'-diaminobenzidine with H.sub.2O.sub.2 (Zymed) as a substrate
for 2 minutes. Sections were washed with gently running tap water
for 10 minutes to remove excess chromogen, lightly counterstained
in Mayers' haematoxylin, dehydrated through ascending graded alcohols,
cleared in xylene, then mounted using DPX (Harlow and Lane 1988).
Chromosomal Location of G3BPs
Fluorescence In Situ Hybridisation of PACs
 Fluorescence in situ hybridisation (FISH) was performed
on peripheral human metaphase chromosomes. PAC DNA was biotin-14dATP-labelled
by nick translation using the BioNick labeling system (Life Technologies).
Chromosome preparation and FISH conditions were as described previously
(Wicking et al., 1995). Slides were analysed using an Olympus BH2
 Two HsaG3BP-1 specific primers, 5' GGAGGCATGGTGCAGAAACCA
[SEQ ID NO: 12] and 5' CAGGAAAGGGAAGAGAGGGAG [SEQ ID NO: 13] and
two HsaG3BP-2 specific primers, 5' GTCTTGGCAGTGGTACATTAT [SEQ ID
NO: 14] and 5' AGTTCACTTTGTCGTAGATAGTTTAAG [SEQ ID NO: 15] were
used to amplify specific templates from human genomic DNA and were
subsequently used on the Genebridge4 Hybrid panel to identify positives.
This data was then processed by the online mapping software available
through the Whitehead Institute/MIT Center for Genome research (http://carbon.wi.mit.edu).
Antagonists of G3BP-2
 An antagonist which prevents or disrupts G3BP-2 binding
with its endogenous target may be useful in preventing or treating
breast cancer. An antagonist may mimic either the NTF2-like domain
of G3BP-2 or mimic an endogenous target which binds to the NTF2-like
domain. For example, the SH3 domain of rasGAP.sup.120 could be used
as a peptide antagonist for blocking the activity of G3BP-2 in breast
cancers. The amino acid sequence of the SH3 domain of rasGAP.sup.120
is: TABLE-US-00006 VRAILPY TKVPDTDEIS FLKGDMFIVH [SEQ ID NO:6] NELEDGWMWV
TNLRTDEQGL IVEDLVEEVG REED
The NCBI accession number: P20936 for the above sequence. The antagonists
may be a polypeptide, but may also be a non-peptide molecule which
is capable of acting as an antagonist.
 Mimetics may be identified by way of screening libraries
of molecules such as synthetic chemical libraries, including combinatorial
libraries, by methods such as described in Nestler & Liu, 1998,
supra and Kirkpatrick et al., 1999, supra. Libraries of naturally-occurring
molecules may be screened by methodology such as reviewed in Kolb,
 Three-dimensional (3D) structural modelling by homology
can be used to assign a 3D structure to the NTF2-like domain of
G3BP-2 based on structural information from the known crystal structure
of the NTF-2 polypeptide. Methods for 3D structural modelling by
homology is described in Blundell et al, 1987, Nature 326 347, herein
incorporated by reference. An antagonist that interacts with the
NTF2-like domain may be designed based on structural modelling by
 Mimetics may be designed using computer assisted screening
of structural databases, computer-assisted modelling, or more traditional
biophysical techniques which detect molecular binding interactions,
as are well known in the art.
 Other methods include a variety of biophysical techniques
which identify molecular interactions. These methods may screen
candidate molecules according to whether the candidate molecule
affects formation of G3BP-2:endogenous target polypeptide complexes.
Methods applicable to potentially useful techniques such as competitive
radioligand binding assays (see Upton et al., 1999, supra for relevant
methods), analytical ultracentrifugation, microcalorimetry, surface
plasmon resonance and optical biosensor-based methods are provided
in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan
et al., (John Wiley & Sons, 1997) which is incorporated herein
Diagnosis of Breast Cancer
 G3BP-2 may be useful for diagnosing breast cancer in an
individual. In one embodiment, the method may include the steps
of: (i) assaying a test sample obtained from the mammal for expression
of G3BP-2 polypeptide; (ii) comparing G3BP-2 expression from the
test sample with expression in a normal sample from a normal mammal;
and (iii) diagnosing the mammal with a likelihood of breast cancer
if the expression of G3BP-2 in the test sample is different than
the normal sample. The term different refers to at least a detectable
difference either by aided or unaided means. For example, an unaided
means includes a person visually comparing a difference in relative
apparent abundance of protein, such as a thicker or darker "band"
on a Western blot or darker well of an ELISA. Aided means includes
for example, use of a microscope to assess antibody binding of a
tissue section or an apparatus that is capable is detecting and
measuring a difference in protein amount, for example an ELISA plate
reader or FACS. The method for diagnosing breast cancer may include
the step of detecting a G3BP-2 polypeptide, or fragment thereof,
in the test sample using an antibody which binds to the G3BP-2 polypeptide,
or fragment thereof. The antibody described herein, for example
as used in FIGS. 7-9, may be useful in a diagnostic kit. The antibody
may be a polyclonal or monoclonal antibody.
 The method for diagnosing breast cancer may include methods
of detecting a polypeptide, for example Western blot analysis, ELISA,
FACS analysis and immunohistochemistry as is commonly known in the
art. Examples of Western blot analysis and immunohistochemistry
as described herein may be useful in detecting G3BP-2 polypeptide,
fragment, homolog or derivative thereof. Western blot analysis is
useful in determining expression of different isoforms of G3BP-2,
ie. G3BP-2a and G3BP-2b, which are distinguishable by size, as shown
for example in FIG. 5. Immunohistochemistry is useful in determining
cellular and subcellular localisation of G3BP-2, as shown for example
in FIGS. 6A and 6B. An antibody which specifically binds to either
G3BP-2a or G3BP-2b is useful in determining expression respective
Detection of G3BP-2 Protein in Human Cancer by Immunohistochemistry
 Fifty-nine cases of invasive breast carcinoma diagnosed
at the Department of Pathology, Royal Brisbane Hospital, between
1981 and 1990 were randomly selected. Archival paraffin blocks were
accessed subject to ethics approval from the Royal Brisbane Hospital
and had been previously characterised as part of a larger study
of MUC1 epithelial mucin expression (McGuckin et al. 1995). Histological
classification and grading of the tumours was performed in accordance
with the Nottingham modification of the Bloom and Richardson system
(Elston & Ellis 1990). Data including nodal status and oestrogen
receptor (ER) status as determined by biochemical dextran-coated
charcoal method were obtained from clinical charts and pathology
Immunohistochemistry of Breast Cancer Sections
 Breast tumour sections (3-4 .mu.m) were affixed to adhesive
slides and dried at 37.degree. C. overnight. The sections were dewaxed
and rehydrated through descending graded alcohols to deionised water
using standard protocols. Sections to be stained for G3BP-1 were
subjected to antigen heat retrieval by autoclaving at 120.degree.
C. for 20 minutes in 1 mM EDTA, pH 8.0. G3BP-2 samples were subjected
to antigen heat retrieval by boiling in 0.1M Tris-HCl, pH 9.0-9.2,
for 5 minutes in a microwave, and repeating the process using fresh
Tris-HCl buffer. All sections were allowed to cool to room temperature
(20-30 min) and then washed in Tris buffered saline, pH 7.4 (TBS).
Endogenous peroxidase activity was blocked by incubating the section
in 1.0% H2O2, 0.1% NaN.sub.3 in TBS for 10 minutes. Sections were
washed in TBS and subsequently incubated in 4% non-fat skim milk
powder in TBS for 15 minutes. Sections were rinsed briefly in TBS
and then incubated with 10% normal goat serum (NGS) for 20 minutes
in a humidified chamber. Excess normal serum was decanted and primary
antibody (or TBS as negative control) was applied overnight at room
temperature. Sections were washed in TBS and then incubated with
secondary antibody (DAKO, Glostrup Denmark, EnVision Kit) for 45
minutes. Sections were washed in TBS and colour was developed in
3,3-diaminobenzidine (DAB) with H.sub.2O.sub.2 as substrate. Sections
were washed in gently running tap water then lightly counterstained
in Mayers' haemotoxylin, dehydrated through ascending graded alcohols,
cleared in xylene, and mounted in DPX mounting medium.
G3BP-2 is Over-Expressed in 88% of Breast Tumours
 In addition to determining expression of G3BP-2, expression
of G3BP-1 was also examined in 24 breast tumour cases by immunohistochemistry
as shown in FIG. 8. Of these, 22 sections were infiltrating ductal
carcinomas (IDC) and two were cases of infiltrating lobular carcinoma
(ILC). All sections were counterstained with haematoxylin, which
stains nuclei blue and the expression of G3BP-1 was visualised using
horseradish peroxidase seen as brown staining (See FIG. 8, Panels
A-C). Most normal cells exhibited detectable cytoplasmic expression
of G3BP-1 (see FIG. 8, Panel C). Two normal ducts (ND) are seen
in FIG. 8, Panel C, and cytoplasmic expression of G3BP-1 is apparent
as seen by the distinct brown Panel A and B, but the adjacent connective
tissue (CT) does not express G3BP-1 at detectable levels. The tumour
cells in FIG. 8, Panels A and B appear to express higher levels
of G3BP-1 in the cytoplasm as compared to that seen in the normal
ducts of FIG. 8, Panel C.
 In many cases tumour staining was heterogeneous and in some
cases G3BP-1 appeared to localise more prominently to one side of
the cell. This can be seen quite clearly in some of the tumour cells
in FIG. 8, Panel A (indicated by the arrow). There was no nuclear
staining present in any normal cells, although two of the 24 tumour
cases contained distinct nuclear staining in less than 10% of tumour
 In summary, most normal breast cells expressed G3BP-1, but
all of the tumours examined appeared to over-express G3BP-1 to some
extent (see Table 3). No significant relationship was found between
G3BP-1 over-expression and clinicopathological parameters of breast
cancer such as lymph node involvement, hormone receptor status or
nuclear or histological grade.
 A total of 58 breast tumour cases were examined by immunohistochemistry
for altered G3BP-2 expression. Of these, 54 tumours were IDCs and
four were ILCs. As with G3BP-1, all sections were counterstained
with haematoxylin and the expression of G3BP-2 was visualised using
horseradish peroxidase (See FIG. 7 and FIG. 8, Panels D-O). Unlike
that for G3BP-1, the immunohistochemistry showed no detectable expression
of G3BP-2 in normal lobes of the breast (See FIG. 8, Panel D) including
the lobular and ductal epithelium and surrounding connective tissue.
Panels E and F of FIG. 8 show a higher magnification of two different
ducts, Panel E shows a transverse section of a duct and Panel F
shows a longitudinal section. As can be seen, there is no detectable
expression of G3BP-2 in normal ducts of the breast or within cells
of the surrounding connective tissue.
 Immunohistochemistry revealed that G3BP-2 is over-expressed
in breast tumours. FIG. 8, Panel G shows a normal duct adjacent
to an IDC. As can be seen by the brown staining, G3BP-2 is highly
expressed in the tumour but not expressed in the normal duct. This
can be seen more clearly in Panel H which shows an IDC at higher
magnification adjacent to a normal duct. Again, the normal duct
does not express G3BP-2 and the IDC highly expresses G3BP-2.
 The over-expression of G3BP-2 in breast tumours was not
seen in all breast tumours examined (12%). Panel I of FIG. 8 shows
one case of IDC that does not express G3BP-2. Another interesting
observation noted when examining the expression of G3BP-2 in human
breast tumours was that in some cases G3BP-2 is expressed in the
nucleus of normal cells within the connective tissues lying between
the tumours (marked by the arrow), but not in the cells within connective
tissue away from the tumour (See FIG. 8, Panel J).
 FIG. 8, Panel K shows an example of a lower magnification
of a tumour and adjacent connective tissue. As can be seen, G3BP-2
is expressed in cells within the connective tissue peripheral to
the tumour and the expression becomes lower the further away the
cells are from the tumour. These cells are most likely infiltrating
lymphocytes as there seems to be a greater population of these cells
around the tumours. This could suggest that G3BP-2 expression is
induced in response to a factor secreted by some tumours or that
G3BP-2 produces a chemotaxis-like effect.
 Table 2 shows the results of all breast tumours examined
for G3BP-2 expression. Also listed is the available information
on each of the breast tumours including its oestrogen receptor status,
tumour grade and stage. In summary, 88% of all tumours examined
over-express G3BP-2 and no significant relationship was found between
G3BP-2 over-expression and clinicopathological parameters of breast
cancer such as stage, hormone receptor status or nuclear or histological
 In the majority of human breast tumours that were screened,
G3BP-2 is over-expressed and in many cases shows a distinct nuclear
localisation (See FIG. 8, Panel M to O). Panels L to O show four
different cases of IDC with three different sub-cellular localisations.
Panel L is an example of a breast tumour where G3BP-2 is exclusively
cytoplasmic. Panel M and N are two examples of breast tumours where
G3BP-2 is found in the nucleus and in the cytoplasm. Panel O shows
G3BP-2 localised around the nuclear envelope region. These tumours
also show a cytoplasmic distribution for G3BP-2. This is the first
case in which G3BP-2 has been found in the nucleus in situ. Approximately
50% of all tumours that express G3BP-2 have G3BP-2 in the nucleus,
although it should be noted that it is possible that nuclear staining
was not observed in some cells where G3BP-2 is expressed at low
levels, due to masking by the haematoxylin counterstain. The nuclear
staining varied between cases. Some cases had G3BP-2 in the nucleus
of all cells, whereas others had less than 10% of cells with nuclear
staining. The percentage of cells which express G3BP-2 in the nucleus
does not correlate with the grade of the tumour or level of metastasis.
G3PB-2 Expression During the Cell Cycle
 Interestingly, G3BP-2 can shuttle into the nucleus and its
movement appears to be cell cycle dependent (See FIG. 8P-8T). In
serum starved resting cells G3BP-2 localises to the cytoplasm (FIG.
8P); however, within 2 hrs of releasing cells from G.sub.0 G3BP-2
can be seen to move into the nucleus (FIG. 8Q-8T) and at 5 hrs appears
to be almost totally nuclear (FIG. 8R). After this time G3BP can
be seen in both compartments consistent with it shuttling between
the nucleus and the cytoplasm (FIGS. 8S and 8T).
 FIG. 8P-8T show the immunofluorescence of synchronised NIH
3T3 cells. Cells were synchronised by serum starvation and subsequently
induced to enter the cell cycle by serum stimulation. Cells were
stained for G3BP-2 using immunofluorescent technique at several
time intervals following serum starvation. FIG. 8P shows the sub-cellular
localisation of G3BP-2 in cells in G.sub.0 phase (time=0). The time
after serum stimulation and hence cell cycle commencement is 2 hours,
5 hours, 9 hours and 12 hours for FIGS. 8Q, 8R, 8S and 8T, respectively.
Cell Cycle Synchronisation of NIH 3T3 Cells
 Cell cycle synchronisation of NIH 3T3 cells was performed
using the serum deprivation method (Tobey et al. 1988). NIH 3T3
cells were seeded onto coverslips at sub-confluent conditions in
10% FCS. Following 24 hours, the cells were washed 3 times with
PBS and serum free medium was then replaced with medium containing
10% FCS. Coverslips were removed from the media during serum starvation,
and at 2, 5, 9 and 12 hours after serum stimulation. The coverslips
were then processed for immunofluorescence to examine the expression
Immunofluorescence of Cultured Cells
 NIH 3T3 cells were grown on coverslips, treated as described
above, and washed 3.times.2 min with PBS and dried overnight at
room temperature. The cells were fixed with 100% cold acetone for
5 min, allowed to dry, then rehydrated by washing the coverslips
with PBS 3.times.5 min. The cells were then permeabilised by incubating
the coverslips in 0.1% Triton-X 100 in PBS for 5 min. The detergent
was then removed by washing the coverslips 3.times.5 min with PBS.
Primary antibody, diluted to the appropriate concentration in PBS,
was applied and left overnight at 4.degree. C. The following morning
the coverslips were washed 3.times.5 min in 1% normal goat serum
(NGS)/1% bovine serum albumin (BSA) in PBS. Secondary antibody was
then applied and incubated for 1 hour at room temperature. The secondary
antibody was anti-rabbit IgG conjugated with either a FITC or Rhodamine
fluorescent tag (Molecular Probes, Eugene, USA) and was diluted
in 0.1% Triton-X 100 in PBS at the dilution specified by the manufacturer.
The coverslips were then washed 2.times.5 min in 0.1% Triton-X 100
in PBS followed by 2.times.5 min PBS. Finally, coverslips were mounted
onto slides with 50% glycerol/50% PBS and sealed with nail polish.
Images were generated using an Olympus Provis AX-70 and captured
in digital format with a DAGE-MTI CCD camera using Scion Image 1.62
frame grabber software. Images were analysed using Adobe Photoshop
5 image-processing software (Adobe systems incorporated, Eastman
Kodak Company, 1996).
Dendritic Cell Therapy
 The invention provides pharmaceutical compositions and methods
for preventing or treating breast cancer in an animal. The pharmaceutical
composition comprises an isolated antigen presenting cell which
has been in contact with G3BP-2 polypeptide, fragment, homolog or
derivative thereof, thereby loading or pulsing the cell with antigen.
The isolated antigen presenting cell is preferably a dendritic cell
isolated from a patient which is undergoing dendritic cell therapy.
The antigen presenting cell may also be a precursor dendritic cell.
The antigen presenting cell may be cultured in vitro to expand or
increase the number of cells before or after antigen loading. Alternatively,
or in addition, to loading the antigen presenting cell with G3BP-2
polypeptide, fragment, homolog or derivative thereof, the antigen
presenting cell may be transfected with a nucleic acid encoding
G3BP-2 polypeptide, fragment, homolog or derivative thereof. The
nucleic acid may be DNA or RNA.
 The method of preventing or treating breast cancer in an
animal includes the step of administering to the animal a pharmaceutical
composition comprising antigen presenting cells which have been
loaded or pulsed with G3BP-2 polypeptide, fragment, homolog, or
derivative thereof; or cells which have been transfected with a
nucleic acid encoding G3BP-2, fragment, homolog, variant or derivative
 In one embodiment, the method includes the steps of: (a)
isolating antigen presenting cells from an animal; (b) contacting
the isolated cells with G3PB-2 polypeptide, fragment, homolog, or
derivative thereof, thereby antigen loading or pulsing the isolated
cells; and (c) administering the loaded or pulsed isolated cells
to the animal. The cells are preferably autologous dendritic cells
isolated from an animal which is administered the pharmaceutical
 In another embodiment, the invention method includes the
steps of: (a) isolating antigen presenting cells from an animal;
(b) transfecting the isolated cells with a nucleic acid encoding
G3PB-2 polypeptide, fragment, homolog, or derivative thereof; and
(c) administering the transfected cells to the animal. The cells
are preferably autologous dendritic cells isolated from an animal
which is administered the pharmaceutical composition.
 In either or both embodiments, the method may further include
the step of expanding the isolated antigen presenting cells in culture
before step (c).
G3BP-2 and New Immunotherapy for Breast Cancer
 Immuno-prevention is a very attractive therapy for breast
cancer due to the minimal tumor load and an ideal target for the
intervention by the immune system. One of the most promising strategies
for immuno-prophylaxic therapy is based on the use of dendritic
cells (DC), the most potent antigen presenting cell (APC) of the
immune system, responsible for the initiation of the immune response
(Hart, 1997) and vital link between the innate and adaptive immunity
(Clark et al., 2000). Researchers have investigated the important
role of DC in health and disease (Ho et al., 2001) and established
mechanisms to optimize their APC capacity (Ho et al., 2002). Their
potent capacity to induce cytotoxic T-lymphocyte (CTL) responses
has been harnessed with success for the treatment of various tumors
(reviewed in (Lopez and Hart, 2002)). Furthermore, it has been demonstrated
that it is possible to obtain large number of DC from the blood
(Lopez et al., 2002). This very promising field of research still
awaits critical inputs before being applied in a generic form.
 In breast cancer research, the most important hurdle is
the availability of tumor-associated antigens (TAA) that may elicit
strong enough immune responses. An ideal TAA would have the characteristics
of being crucial for the development of cancer cells, over-(or selectively)
expressed in cancer cells, intracellularly localized and recognized
by the immune system (Pardoll, 2002). Only two firm TAA candidates
are available and they are currently being tested. MUC-1, (CD227)
is a transmembrane mucin molecule normally polarised to the apical
surface of epithelial cells characterized by a large extracellular
domain of GC-rich random repeats (Gendler et al., 1991); it is highly
expressed in breast cancer cells and over-expressed in more than
90% of patients with breast cancer (Hadden, 1999). Her2/neu is a
transmembrane glycoprotein, homologous to the epidermal growth factor
receptor that is over expressed in 20-30% of patients with breast
cancer (Wang et al., 2001). It is correlated to the aggressiveness
of the disease and an indicator of poor prognosis. Both cellular
and humoral immune responses to this protein have been detected
in patients (Wang and Hung, 2001). Additionally, TAA shared with
other malignancies, eg. MAGE 1 and 3 (melanoma antigens) are expressed
in 20% and 26% of breast cancers respectively (Mashino et al., 2001;
Otte et al., 2001; Russo et al., 1995). Finally, newer antigens
such as the carbohydrate antigen globo H (Gilewski et al., 2001)
have emerged lately, and are currently being evaluated. None of
the available TAA candidates fulfill the abovementioned criteria
for an optimal TAA in breast cancer. The present researcher's recent
data, however, indicate that G3BP proteins represent excellent candidate
 Immunogenicity of G3BP proteins within the context of the
HLA-A*0201 molecule have been evaluated, the most commonly found
allele of the Major Histocompatibility Complex (MHC), following
the genomic/immunogenic approach (Schultze and Vonderheide, 2001),
incorporated herein by reference. Predicted HLA binding sequences
were identified applying web-based algorithms (for example as provided
by SYPEITHI (http://svfpeithi.bmi-heidelberg.com/) and BIMAS (http://bimas.dcrt.nih.gov/molbio/hla_bind/)
and synthetic peptides produced for further evaluation. From the
peptides generated, MHC/peptide binding assays demonstrated a very
strong binding to the HLA-A*0201 molecule, as shown in FIG. 10.
T2 cells were incubated with peptide dilutions shown and HLA expression
measured by flow cytometry. Two peptides (peptide 1 and 2) of G3BP-2
were tested and control matrix protein influenza peptide 58-66 was
used as a reference. Peptide 1 has an amino acid sequence of KLPNFGFVV
[SEQ ID NO:1] and peptide 2 has an amino acid sequence of IMFRGEVRL
[SEQ ID NO:2]. Peptides 1 and 2 bound to the HLA-A*0201 molecule
with an affinity equal to that of the control influenza MP 58-66
peptide, a well-defined CTL epitope.
 The various peptides were evaluated for the generation of
CTL responses induced by DC in peripheral blood mononuclear cells
(PBMC) from healthy individuals and tested in ELISPOT (Gonzalez
et al, 2000). T2 cells (target) pulsed with Peptide 1 or no peptide
were labelled with .sup.51Cr and incubated with clone IH7 (effector)
at various ratios. After 4 hours, .sup.51Cr released from lysed
cells was measured and the percentage of specific lysis calculated
with the formula: 100.times.(experimental release-spontaneous release)/(total
release-spontaneous release). Total release was obtained with detergent
lysis of labelled target.
 Strong responses were efficiently generated for Peptide
1, demonstrated by high frequencies of IFN-.gamma. producing peptide
specific CD8+ lymphocytes obtained after one (and two) round of
stimulation in 2 separate donors. These results indicate that, indeed,
the Peptide 1 epitope is included in the T-cell receptor (TCR) repertoire
and is, therefore, immunogenic. Confirming this finding, we generated
Peptide 1 specific CTL clones capable of identifying peptide-loaded
targets in a classical chromium release assay (FIG. 11), the desired
profile for an efficient anti-tumor response (Lopez and Hart, 2002),
incorporated herein by reference. These clones will be instrumental
in the evaluation of the expression of this antigen in cancer cells,
allowing the formal evaluation of this molecule as a TAA.
 Throughout the specification the aim has been to describe
the preferred embodiments of the invention without limiting the
invention to any one embodiment or specific collection of features.
It will therefore be appreciated by those of skill in the art that,
in light of the instant disclosure, various modifications and changes
can be made in the particular embodiments exemplified without departing
from the scope of the present invention.
 The disclosure of each patent and scientific document, computer
program and algorithm referred to in this specification is incorporated
by reference in its entirety.