U.S. patent application number 10/735118 was filed with the patent office on 2004-12-09 for method for predicting the response to her2-directed therapy.
This patent application is currently assigned to Ventana Medical Systems, Inc.. Invention is credited to Bacus, Sarah S., Smith, Bradley L..
Application Number | 20040248151 10/735118 |
Document ID | / |
Family ID | 46300504 |
Filed Date | 2004-12-09 |
United States Patent
Application |
20040248151 |
Kind Code |
A1 |
Bacus, Sarah S. ; et
al. |
December 9, 2004 |
Method for predicting the response to HER2-directed therapy
Abstract
This invention provides methods for determining or predicting
response to HER2-directed therapy in an individual.
Inventors: |
Bacus, Sarah S.; (Hinsdale,
IL) ; Smith, Bradley L.; (Marblehead, MA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
943041050
|
Assignee: |
Ventana Medical Systems,
Inc.
Cell Signaling Technology
|
Family ID: |
46300504 |
Appl. No.: |
10/735118 |
Filed: |
December 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10735118 |
Dec 11, 2003 |
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10408520 |
Apr 7, 2003 |
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60432942 |
Dec 11, 2002 |
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60370473 |
Apr 5, 2002 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
C07K 16/40 20130101;
G01N 2333/4703 20130101; G01N 2800/52 20130101; G01N 2333/912
20130101; G01N 2440/14 20130101; G01N 33/6893 20130101; C07K
2317/24 20130101; G01N 2333/71 20130101; C07K 16/2863 20130101;
G01N 2333/4756 20130101; G01N 33/5041 20130101; G01N 33/57415
20130101; G01N 33/574 20130101; G01N 2333/9121 20130101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
What we claim is:
1. A method for identifying a mammalian tumor that responds to a
HER2-directed therapy, wherein the mammalian tumor overexpresses
HER2, the method comprising the step of assaying a sample obtained
from the mammalian tumor to detect a pattern of expression,
phosphorylation or both expression and phosphorylation of one or a
plurality of polypeptides consisting of: (a) IGFR polypeptide; (b)
EGFR polypeptide; (c) NDF polypeptide; (d) phosphorylated S6
ribosomal polypeptide; (e) phosphorylated AKT polypeptide; and (f)
phosphorylated ERK polypeptide; wherein the detected pattern of
expression, phosphorylation or both expression and phosphorylation
identifies mammalian tumors that respond to a HER2-directed
therapy.
2. The method of claim 1, wherein the method comprises the step of
assaying a sample obtained from the mammalian tumor to detect a
pattern of expression, phosphorylation or both expression and
phosphorylation of (a) IGFR polypeptide, and one or a plurality of
polypeptides consisting of: (b) EGFR polypeptide; (c) NDF
polypeptide; (d) phosphorylated S6 ribosomal polypeptide; (e)
phosphorylated AKT polypeptide; and (f) phosphorylated ERK
polypeptide.
3. The method of claim 1, wherein the detected pattern is decreased
expression of IGFR polypeptide in the mammalian tumor as compared
to a non-tumor tissue or cell sample.
4. The method of claim 1, wherein the detected pattern is normal or
increased expression of IGFR polypeptide, accompanied by decreased
phosphorylation of AKT polypeptide, decreased phosphorylation of S6
ribosomal polypeptide or both in the mammalian tumor as compared to
a non-tumor tissue or cell sample.
5. The method of claim 1, wherein the detected pattern is normal or
increased expression of EGFR polypeptide, accompanied by decreased
phosphorylation of ERK polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample.
6. The method of claim 1, wherein the detected pattern is decreased
expression of IGFR polypeptide, accompanied by increased
phosphorylation of S6 ribosomal polypeptide in the mammalian tumor
as compared to a non-tumor tissue or cell sample.
7. The method of claim 1, wherein the detected pattern is decreased
expression of IGFR polypeptide, accompanied by increased expression
of NDF polypeptide in the mammalian tumor as compared to a
non-tumor tissue or cell sample.
8. The method of claim 6, wherein the detected pattern further
includes increased phosphorylation of S6 ribosomal polypeptide
9. A method for identifying a mammalian tumor that does not respond
to a HER2-directed therapy, wherein the mammalian tumor
overexpresses HER2, the method comprising the step of assaying a
sample obtained from the mammalian tumor to detect a pattern of
expression, phosphorylation or both expression and phosphorylation
of one or a plurality of polypeptides consisting of: (a) IGFR
polypeptide; (b) EGFR polypeptide; (c) NDF polypeptide; (d)
phosphorylated S6 ribosomal polypeptide; (e) phosphorylated AKT
polypeptide; and (f) phosphorylated ERK polypeptide; wherein the
detected pattern of expression, phosphorylation or both expression
and phosphorylation identifies mammalian tumors that do not respond
to HER2-directed therapy.
10. The method of claim 9, wherein the method comprises the step of
assaying a sample obtained from the mammalian tumor to detect a
pattern of expression, phosphorylation or both expression and
phosphorylation of (a) IGFR polypeptide, and one or a plurality of
polypeptides consisting of: (b) EGFR polypeptide; (c) NDF
polypeptide; (d) phosphorylated S6 ribosomal polypeptide; (e)
phosphorylated AKT polypeptide; and (f) phosphorylated ERK
polypeptide.
11. The method of claim 9, wherein the detected pattern is normal
or increased expression of IGFR polypeptide, accompanied by
increased phosphorylation of AKT polypeptide, increased
phosphorylation of S6 ribosomal polypeptide, or both in the
mammalian tumor as compared to a non-tumor tissue or cell
sample.
12. The method of claim 9, wherein the detected pattern is
decreased expression of EGFR polypeptide and decreased expression
of NDF polypeptide in the mammalian tumor as compared to a
non-tumor tissue or cell sample.
13. The method of claim 9, wherein the detected pattern is
decreased expression of EGFR polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample.
14. The method of claim 9, wherein the detected pattern is
decreased expression of NDF polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample.
15. The method of claim 9, wherein the detected pattern is
decreased expression of EGFR polypeptide and increased
phosphorylation of ERK polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample.
16. The method of claim 9, wherein the detected pattern is normal
or increased expression of IGFR polypeptide and decreased
expression of NDF in the mammalian tumor as compared to a non-tumor
tissue or cell sample.
17. The method of claim 1, wherein the detection of phosphorylation
of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or
both is determined subsequent to contacting the sample obtained
from the mammalian tumor with a HER2-directed therapy.
18. The method of claim 4, wherein the detection of phosphorylation
of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or
both is determined subsequent to contacting the sample obtained
from the mammalian tumor with a HER2-directed therapy.
19. The method of claim 9, wherein the detection of phosphorylation
of AKT polypeptide, phosphorylation of S6 ribosomal polypeptide, or
both is determined subsequent to contacting the sample obtained
from the mammalian tumor with a HER2-directed therapy.
20. The method of claim 11, wherein the detection of
phosphorylation of AKT polypeptide, phosphorylation of S6 ribosomal
polypeptide, or both is determined subsequent to contacting the
sample obtained from the mammalian tumor with a HER2-directed
therapy.
21. The method of claim 1, wherein the HER2-directed therapy
comprises rhuMAb HER2 (HERCEPTIN.RTM.).
22. The method of claim 9, wherein the HER2-directed therapy
comprises rhuMAb HER2 (HERCEPTIN.RTM.).
23. The method of claim 1, wherein the sample obtained from the
mammalian tumor has been contacted with at least one
chemotherapeutic agent.
24. The method of claim 9, wherein the sample obtained from the
mammalian tumor has been contacted with at least one
chemotherapeutic agent.
25. The method of claim 1, wherein the detected pattern of
expression, phosphorylation, or both, of one or a plurality of
polypeptides (a) through (f) is determined using a biodetection
reagent.
26. The method of claim 25, wherein the biodetection reagent is an
antibody.
27. The method of claim 25, wherein the biodetection reagent is a
nucleic acid probe.
28. The method of claim 9, wherein the detected pattern of
expression, phosphorylation, or both, of one or a plurality of
polypeptides (a) through (f) is determined using a biodetection
reagent.
29. The method of claim 28, wherein the biodetection reagent is an
antibody.
30. The method of claim 28, wherein the biodetection reagent is a
nucleic acid probe.
31. The method of claim 1, wherein the detected pattern of
phosphorylated AKT polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated serine residue
at position 473 in SEQ ID NO: 1.
32. The method of claim 9, wherein the detected pattern of
phosphorylated AKT polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated serine residue
at position 473 in SEQ ID NO: 1.
33. The method of claim 1, wherein the detected pattern of
phosphorylated S6 ribosomal polypeptide is determined using an
antibody specific for an epitope comprising a phosphorylated serine
residue at position 235 in SEQ ID NO: 2.
34. The method of claim 9, wherein the detected pattern of
phosphorylated S6 ribosomal polypeptide is determined using an
antibody specific for an epitope comprising a phosphorylated serine
residue at position 235 in SEQ ID NO: 2.
35. The method of claim 1, wherein the detected pattern of
phosphorylated ERK polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated threonine
residue at 202 or a phosphorylated serine residue at position 204
in SEQ ID NO: 3.
36. The method of claim 9, wherein the detected pattern of
phosphorylated ERK polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated threonine
residue at 202 or a phosphorylated serine residue at position 204
in SEQ ID NO: 3.
37. The method of claim 1, wherein the sample obtained from the
mammalian tumor is a paraffin-embedded biopsy sample.
38. The method of claim 9, wherein the sample obtained from the
mammalian tumor is a paraffin-embedded biopsy sample.
39. The method of claim 1, wherein the mammalian tumor is
identified as overexpressing HER2 using an antibody that binds HER2
polypeptide.
40. The method of claim 9, wherein the mammalian tumor is
identified as overexpressing HER2 using an antibody that binds HER2
polypeptide.
41. A method of selecting a subject with cancer for treatment with
a molecule targeting HER2, wherein the cancer overexpresses HER2,
the method comprising the steps of: (a) determining a pattern of
expression, phosphorylation or both expression and phosphorylation
in a cell or tissue sample from the subject of one or a plurality
of polypeptides consisting of: (i) IGFR polypeptide; (ii) EGFR
polypeptide; (iii) NDF polypeptide; (iv) phosphorylated S6
ribosomal polypeptide; (v) phosphorylated AKT polypeptide; and (vi)
phosphorylated ERK polypeptide; and (b) selecting the subject based
on the detected pattern of expression, phosphorylation, or both
expression and phosphorylation.
42. The method of claim 41, wherein step (a) comprises determining
a pattern of expression, phosphorylation or both expression and
phosphorylation in a cell or tissue sample from the subject of (a)
IGFR polypeptide, and one or a plurality of polypeptides consisting
of: (b) EGFR polypeptide; (c) NDF polypeptide; (d) phosphorylated
S6 ribosomal polypeptide; (e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide.
43. The method of claim 41, wherein the subject is selected when
the detected pattern is decreased expression of IGFR polypeptide in
the mammalian tumor as compared to a non-tumor tissue or cell
sample.
44. The method of claim 41, wherein the subject is selected when
the detected pattern is normal or increased expression of IGFR
polypeptide, accompanied by decreased phosphorylation of AKT
polypeptide, decreased phosphorylation of S6 ribosomal polypeptide,
or both in the mammalian tumor as compared to a non-tumor tissue or
cell sample.
45. The method of claim 41, wherein the subject is selected when
the detected pattern is normal or increased expression of EGFR
polypeptide, accompanied by decreased phosphorylation of ERK
polypeptide in the mammalian tumor as compared to a non-tumor
tissue or cell sample.
46. The method of claim 41, wherein the subject is selected when
the detected pattern is decreased expression of IGFR polypeptide,
accompanied by increased phosphorylation of S6 ribosomal
polypeptide in the mammalian tumor as compared to a non-tumor
tissue or cell sample.
47. The method of claim 41, wherein the subject is selected when
the detected pattern is decreased expression of IGFR polypeptide,
accompanied by increased expression of NDF polypeptide in the
mammalian tumor as compared to a non-tumor tissue or cell
sample.
48. The method of claim 47, wherein the subject is selected when
the detected pattern further includes increased phosphorylation of
S6 ribosomal polypeptide
49. The method of claim 41, wherein phosphorylation of AKT
polypeptide, phosphorylation of S6 ribosomal polypeptide, or both
is determined subsequent to contacting the sample obtained from the
mammalian tumor with a HER2-directed therapy.
50. The method of claim 44, wherein phosphorylation of AKT
polypeptide, phosphorylation of S6 ribosomal polypeptide, or both
is determined subsequent to contacting the sample obtained from the
mammalian tumor with a HER2-directed therapy.
51. The method of claim 41, wherein the HER2-directed therapy
comprises rhuMAb HER2 (HERCEPTIN.RTM.).
52. The method of claim 41, wherein the sample obtained from the
mammalian tumor has been contacted with at least one
chemotherapeutic.
53. The method of claim 41, wherein the detected pattern of
expression, phosphorylation, or both of one or a plurality of
polypeptides (i) through (vi) is determined using a biodetection
reagent.
54. The method of claim 53, wherein the biodetection reagent is an
antibody.
55. The method of claim 53, wherein the biodetection reagent is a
nucleic acid probe.
56. The method of claim 41, wherein the detected pattern of
phosphorylated AKT polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated serine residue
at position 473 in SEQ ID NO: 1.
57. The method of claim 41, wherein the detected pattern of
phosphorylated S6 ribosomal polypeptide is determined using an
antibody specific for an epitope comprising a phosphorylated serine
residue at position 235 in SEQ ID NO: 2.
58. The method of claim 41, wherein the detected pattern of
phosphorylated ERK polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated threonine
residue at 202 or a phosphorylated serine residue at position 204
in SEQ ID NO: 3.
59. The method of claim 41, wherein the cell or tissue sample from
the subject is a paraffin-embedded biopsy sample.
60. The method of claim 41, wherein the mammalian tumor is
identified as overexpressing HER2 using an antibody that binds HER2
polypeptide.
61. A method of selecting a subject with cancer to not receive
treatment with a molecule targeting HER2, wherein the cancer
overexpresses HER2, the method comprising the steps of: (a)
determining of a pattern of expression, phosphorylation, or both
expression and phosphorylation in a cell or tissue sample from the
subject of one or a plurality of polypeptides consisting of: (i)
IGFR polypeptide; (ii) EGFR polypeptide; (iii) NDF polypeptide;
(iv) phosphorylated S6 ribosomal polypeptide; (v) phosphorylated
AKT polypeptide; (vi) phosphorylated ERK polypeptide; and (b)
selecting the subject based on the detected pattern of expression,
phosphorylation, or both expression and phosphorylation.
62. The method of claim 61, wherein step (a) comprises determining
a pattern of expression, phosphorylation or both expression and
phosphorylation in a cell or tissue sample from the subject of (a)
IGFR polypeptide, and one or a plurality of polypeptides consisting
of: (b) EGFR polypeptide; (c) NDF polypeptide; (d) phosphorylated
S6 ribosomal polypeptide; (e) phosphorylated AKT polypeptide; and
(f) phosphorylated ERK polypeptide.
63. The method of claim 61, wherein the subject is selected when
wherein the detected pattern is normal or increased expression of
IGFR polypeptide, accompanied by decreased phosphorylation of AKT
polypeptide, decreased phosphorylation of S6 ribosomal polypeptide,
or both in the mammalian tumor as compared to a non-tumor tissue or
cell sample.
64. The method of claim 61, wherein the subject is selected wherein
the detected pattern is decreased expression of EGFR polypeptide
and decreased expression of NDF polypeptide in the mammalian tumor
as compared to a non-tumor tissue or cell sample.
65. The method of claim 61, wherein the subject is selected when
the detected pattern is decreased expression of EGFR polypeptide in
the mammalian tumor as compared to a non-tumor tissue or cell
sample.
66. The method of claim 61, wherein the subject is selected when
the detected pattern is decreased expression of NDF polypeptide in
the mammalian tumor as compared to a non-tumor tissue or cell
sample.
67. The method of claim 61, wherein the subject is selected when
the detected pattern is decreased expression of EGFR polypeptide
and increased phosphorylation of ERK polypeptide in the mammalian
tumor as compared to a non-tumor tissue or cell sample.
68. The method of claim 61, wherein the subject is selected when
the detected pattern is normal or increased expression of IGFR
polypeptide and decreased expression of NDF in the mammalian tumor
as compared to a non-tumor tissue or cell sample.
69. The method of claim 61, wherein phosphorylation of AKT
polypeptide, phosphorylation of S6 ribosomal polypeptide, or both
is determined subsequent to contacting the sample obtained from the
mammalian tumor with a HER2-directed therapy.
70. The method of claim 63, wherein phosphorylation of AKT
polypeptide, phosphorylation of S6 ribosomal polypeptide, or both
is determined subsequent to contacting the sample obtained from the
mammalian tumor with a HER2-directed therapy.
71. The method of claim 61, wherein the HER2-directed therapy
comprises rhuMAb HER2 (HERCEPTIN.RTM.).
72. The method of claim 61, wherein the sample obtained from the
mammalian tumor has been contacted with at least one
chemotherapeutic.
73. The method of claim 61, wherein the detected pattern of
expression, phosphorylation, or both of one or a plurality of
polypeptides (i) through (vi) is determined using a biodetection
reagents.
74. The method of claim 73, wherein the biodetection reagent is an
antibody.
75. The method of claim 73, wherein the biodetection reagent is a
nucleic acid probe.
76. The method of claim 61, wherein the detected pattern of
phosphorylated AKT polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated serine residue
at position 473 in SEQ ID NO: 1.
77. The method of claim 61, wherein the detected pattern of
phosphorylated S6 ribosomal polypeptide is determined using an
antibody specific for an epitope comprising a phosphorylated serine
residue at position 235 in SEQ ID NO: 2.
78. The method of claim 61, wherein the detected pattern of
phosphorylated ERK polypeptide is determined using an antibody
specific for an epitope comprising a phosphorylated threonine
residue at 202 or a phosphorylated serine residue at position 204
in SEQ ID NO: 3.
79. The method of claim 61, wherein the cell or tissue sample is a
paraffin-embedded biopsy sample.
80. The method of claim 61, wherein the mammalian tumor is
identified as overexpressing HER2 using an antibody that binds HER2
polypeptide.
81. A kit for characterizing a mammalian tumor's responsiveness to
a HER2-directed therapy, the kit comprising: (a) an antibody that
binds IGFR polypeptide, and one or more of the following: (b) an
antibody that binds phosphorylated AKT polypeptide; (c) an antibody
that binds phosphorylated S6 ribosomal polypeptide; (d) an antibody
that binds EGFR polypeptide; (e) an antibody that binds HER2
polypeptide; (f) an antibody that binds NDF polypeptide; and (g) an
antibody that binds phosphorylated ERK polypeptide.
82. The kit of claim 81, wherein the antibody of (b) is
immunologically specific for AKT polypeptide having a
phosphorylated serine residue at position 473 in SEQ ID NO: 1;
antibody of (c) is immunologically specific for S6 ribosomal
polypeptide having a phosphorylated serine residue at position 235
in SEQ ID NO: 2; and/or antibody of (f) is immunologically specific
for EKT polypeptide having a phosphorylated threonine residue at
position 202 and a phosphorylated tyrosine at position 204 in SEQ
ID NO: 3.
83. The kit of claim 81, wherein the kit further comprises at least
one secondary antibody that binds to an antibody of subpart (a)
through (g).
Description
[0001] This application claims priority to U.S. provisional
application Ser. No. 60/432,942, filed Dec. 11, 2002.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods for predicting the
response to HER2-directed therapy in an individual.
[0004] 2. Background of the Invention
[0005] Cellular growth and differentiation processes involve growth
factors that exert their actions through specific receptors
expressed in the surfaces of responsive cells. Ligands binding to
surface receptors, such as those that carry an intrinsic tyrosine
kinase activity, trigger a cascade of events that eventually lead
to cellular proliferation and differentiation (Carpenter et al.,
1979, Biochem., 48: 193-216; Sachs et al., 1987, Cancer Res., 47:
1981-1986). Receptor tyrosine kinases can be classified into
several groups on the basis of sequence similarity and distinct
features. One of these groups includes the epidermal growth factor
receptor family, which includes erbB-1 (also termed EGFR or HER-1)
(Carpenter et al., supra); erbB-2 (HER-2/neu) (Semba et al., 1985,
Proc. Natl. Acad. Sci., 82: 6497-6501; Coussens et al., 1985,
Science, 230: 1130-1139; Bargmann et al., 1986, Cell, Vol. 45,
649-657); erbB-3 (HER-3) (Kraus et al., 1989, Proc. Natl. Acad.
Sci., 86: 9193-9197; Carraway et al., 1994, J. Biol. Chem., 269:
14303-14306), and erbB-4 (HER-4) (Plowman et al., 1993, Nature,
366: 473-475; Tzahar et al., 1994, J. Biol. Chem., 269:
25226-25233).
[0006] Most tumors of epithelial origin express multiple erbB (HER)
receptors and co-express one or more EGF-related ligands suggesting
that autocrine-receptor activation plays a role in tumor cellular
proliferation. Because these ligands activate different erbB/HER
receptors, it is possible that multiple erbB receptor combinations
might be active in a tumor, a characteristic that could influence
its response to an erbB-targeted therapeutic. ErbB receptors form
homodimers and heterodimers that can be stimulated by various
ligands leading to downstream signaling events, the extent and
nature of which depend on the combination of specific dimers and
ligands. For example, HER2/neu appears to be the preferred
heterodimerization partner with other members of the epidermal
growth factor receptor family, but ultimately the dimers formed are
determined by the ligand and the erbB receptors expressed in the
cell. Not only may the ligand select the dimerization partners, but
it may also influence the time course of membrane translocation,
activation, and internalization of the receptor. For example,
NDF/Heregulin can stimulate tyrosine phosphorylation of erbB-2
through heterodimerization with either receptors erbB-3 or erbB-4
(Peles et al., 1992, Cell 69, 205-216, Peles et al., 1993, EMBO J.
3, 961-71, Holmes et al., 1992, Science 256, 1205-1210; Tzahar et
al., 1994, Biol. Chem., 269, 25226-25233; Plowman et al., 1993,
Nature 366, 473-475; Pinkas-Kramarski et al., 1994, Proc. Natl.
Acad. Sci. USA, 91, 9387-9391; Pinkas-Kramarski et al., 1996, J.
Biol. Chem., 271, 19029-19032; Pinkas-Kramarski et al., 1998,
Oncogene, 16, 1249-1258). Depending on the cell line studied,
NDF/Heregulin can either elicit a growth arrest and differentiation
phenotype, resulting in morphological changes, induction of lipids,
and expression of intracellular adhesion molecule-1; or it can
induce a mitogenic response (Holmes et al., 1992, Science,
256:1205-1210; Peles et al., 1992, Cell, 69:205-216; Bacus et al.,
1993, Cancer Res. 53:5251-5261).
[0007] Downstream signaling after ligand binding may be determined
by the set of docking proteins that may bind to the activated
receptors. For example, activation of erbB receptor heterodimers is
coupled to and stimulates downstream MAPK-Erk1/2 and PI3K-AKT
growth and survival pathways, whose deregulation in cancer has been
linked to disease progression and refractoriness to therapy (Tzahar
et al., 1996, Mol. Cell. Biol. 16, 5276-5287; Fukazawa et al.,
1996, J. Biol. Chem. 271, 14554-14559, Olayioye et al., 1998, Mol.
Cell. Biol. 18, 5042-5051; Lange et al., 1998, J. Biol. Chem. 273,
31308-31316; Hackel et al., 1999, Curr. Opin. Cell Biol. 11,
184-189). HER-3 is a major docking site for
phosphoinositide-3-kinase (PI3K). In addition, NDF/Heregulin
stimulation causes activation of the PI3K pathway and
phosphorylation of AKT (Altiok et al., 1999, J. Bio. Chem. 274,
32274-32278; Liu et al., 1999, Biochem. Biophys. Res. Comm. 261
897-903; Xing et al., 2000, Nature, Med. 6 189-195). These
observations implicate PI3K/AKT in the signaling cascade that
results from HER-3 heterodimerization with overexpressed HER-2/neu
receptors in breast cancer cells; activation of PI3K/AKT promotes
cell survival and enhanced tumor aggressiveness (Bacus et al.,
2002, Oncogene 21, 3532-3540). In addition, AKT2 was reported to be
activated and overexpressed in HER-2/neu-overexpressing breast
cancers (Id.).
[0008] erbB-2/HER-2 is overexpressed in 20 to 30% of all breast
cancers, and its overexpression is associated with poor prognosis,
suggesting that it could be used as a target for anti-tumor agents
(Slamon et al., 1987; Hudziak et al., 1989; Tagliabue et al.,
1991). In erbB-2-overexpressing breast cancer cells, treatment with
antibodies specific to HER-2/erbB-2 in combination with
chemotherapeutic agents (such as cisplatin, doxoubicin, and taxol)
elicits a higher cytotoxic response than treatment with
chemotherapy alone (Hancock et al., 1991; Arteaga et al., 1994;
Pietras et al., 1994). One possible mechanism by which HER-2/erbB-2
antibodies might enhance cytotoxicity to chemotherapeutic agents is
through the modulation of the HER-2/erbB-2 protein expression
(Bacus et al., 1992 & 1993; Stancovski et al., 1991; Klapper et
al., 1997 & 2000), or by interfering with DNA repair (Arteaga
et al., 1994 & 2001; Pietras et al., 1994).
[0009] Because of the effect of anti-HER-2/erbB-2 antibodies on
cellular growth, a number of approaches have been used to
therapeutically target HER-2/erbB-2- or EGFR-overexpressing
cancers. For clinical use, one approach is to interfere with the
kinase activity of the receptor by using inhibitors that block the
nucleotide binding site of HER-2/neu or EGFR (Bruns et al., 2000;
Christensen et al, 2001, Erlichman et al., 2001, Herbst et al.,
2002; Hidalgo et al, 2001; Moasser et al, 2001; Fujimura et al.,
2002; Normanno et al., 2002). A second approach is using ansamycins
to influence the stability of HER2/neu receptors (Munster et al.,
2002; Basso et al, 2002). Another approach is the use of antibodies
directed to various erbB receptors, specifically EGFR or HER-2/neu
(Alaoui-Jamali et al., 1997; Albanell et al., 2001(a); Baselga et
al., 1994 & 2002; Mendelsohn, 1990). Analysis of various
antibodies to HER-2/neu led to the identification of the murine
monoclonal, 4D5. This antibody recognizes an extracellular epitope
(amino acids 529 to 627) in the cysteine-rich II domain that
resides very close to the transmembrane region. Treatment of breast
cancer cells with 4D5 partially blocks NDF/heregulin activation of
HER-2-HER-3 complexes, as measured by receptor phosphorylation
assays. To allow for chronic human administration, murine 4D5 was
fully humanized to generate trastuzumab/HERCEPTIN.RTM. (Sliwkowski
et al., 1999; Ye et al., 1999; Carter et al, 1992; Fujimoto-Ouchi
et al, 2002; Vogel, et al., 2001 & 2002).
[0010] A number of monoclonal antibodies and small molecule,
tyrosine kinase inhibitors targeting EGFR or erbB-2 have been
developed. For example, HERCEPTIN.RTM. is approved for treating the
25% of women whose breast cancers overexpress erbB-2 protein or
demonstrate erbB-2 gene amplification (Cobleigh et al., 1999, J.
Clin. Oncol. 17, 2639-2648). In addition, several EGFR-targeted
therapies are currently under clinical investigation (Mendelsohn
& Baselga, 2000, Oncogene 19, 6550-6565; Xia et al., 2002,
Oncogene 21, 6255-6263).
[0011] The development of successful oncological drugs has followed
a well-established evaluation process including phases I, II, and
III clinical trial. Phase I studies aim to determine the maximally
tolerated dose of the drug, its optimal schedule of administration
and the dose-limiting toxicities. Historically, cytotoxic cancer
therapies have been developed based on maximum tolerated doses
(MTD), treating patients without understanding the tumor profile
for likely responders. Hence, patients were often subjected to
toxic therapies with limited therapeutic benefit. Recently,
elucidating tumor growth and survival pathways has led to the
development of tumor-targeted therapies. For such targeted
therapeutics that are not expected to produce serious adverse side
effects, relying on a MTD may not be suitable. More relevant may be
the determination of the optimal dose and schedule that is
sufficient to inhibit cellular signaling in patient samples.
Biological assays for signaling biomarkers are needed to establish
such a protocol.
[0012] Preclinically, most erbB-receptor targeted therapies
primarily exert cytostatic anti-tumor effects, necessitating their
chronic administration in clinical practice. Identification of
biologically effective doses (BED), the dose or dose range that
maximally inhibits the intended target, beyond which dose
escalation is likely to add toxicity without benefit, is therefore
essential. Moreover, many of these agents will be used in
combination with cytotoxic therapies, where added toxicity may not
be tolerable, further supporting BED-based dosing.
"Targeted-therapy" implies that populations of likely responders
exists, and can be identified.
[0013] In view of the severe and deleterious consequences of
administering an inappropriate or ineffective therapy to a human
cancer patient, there exists a need in the art for predicting the
response to cancer therapy in an individual. Further, there is a
need to develop diagnostics that are capable of predicting patient
response for the successful development and clinical acceptance of
new HER-2 targeted therapeutics similar to HERCEPTIN.RTM..
SUMMARY OF THE INVENTION
[0014] This invention provides methods for predicting a response of
an individual to a HER2-directed therapy.
[0015] In a first aspect, the invention provides methods for
identifying a mammalian tumor that responds to a HER2-directed
therapy, wherein the mammalian tumor overexpresses HER2, the method
comprising the step of assaying a sample obtained from the
mammalian tumor to detect a pattern of expression, phosphorylation
or both expression and phosphorylation of one or a plurality of
polypeptides consisting of:
[0016] (a) IGFR polypeptide;
[0017] (b) EGFR polypeptide;
[0018] (c) NDF polypeptide;
[0019] (d) phosphorylated S6 ribosomal polypeptide;
[0020] (e) phosphorylated AKT polypeptide; and
[0021] (f) phosphorylated ERK polypeptide;
[0022] wherein the particular combination of polypeptides and
pattern of expression, phosphorylation or both expression and
phosphorylation identifies mammalian tumors that respond to a
HER2-directed therapy.
[0023] In certain embodiments, the pattern that identifies a
mammalian tumor as responding is decreased expression of IGFR
polypeptide in the mammalian tumor as compared to a non-tumor
tissue or cell sample. In other embodiments, the detected pattern
is normal or increased expression of IGFR polypeptide, accompanied
by decreased phosphorylation of AKT polypeptide, decreased
phosphorylation of S6 ribosomal polypeptide or both in the
mammalian tumor as compared to a non-tumor tissue or cell sample.
In further embodiments, the detected pattern is normal or increased
expression of EGFR polypeptide, accompanied by decreased
phosphorylation of ERK polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample. In additional
embodiments, the detected pattern is decreased expression of IGFR
polypeptide, accompanied by increased phosphorylation of S6
ribosomal polypeptide in the mammalian tumor as compared to a
non-tumor tissue or cell sample. In other embodiments, the detected
pattern is decreased expression of IGFR polypeptide, accompanied by
increased expression of NDF polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample; where further the
detected pattern can include increased phosphorylation of S6
ribosomal polypeptide.
[0024] In a second aspect, the invention provides methods for
identifying a mammalian tumor that does not respond to a
HER2-directed therapy, wherein the mammalian tumor overexpresses
HER2, the method comprising the step of assaying a sample obtained
from the mammalian tumor to detect a pattern of expression,
phosphorylation or both expression and phosphorylation of one or a
plurality of polypeptides consisting of:
[0025] (a) IGFR polypeptide;
[0026] (b) EGFR polypeptide;
[0027] (c) NDF polypeptide;
[0028] (d) phosphorylated S6 ribosomal polypeptide;
[0029] (e) phosphorylated AKT polypeptide; and
[0030] (f) phosphorylated ERK polypeptide;
[0031] wherein the particular combination of polypeptides and
pattern of expression, phosphorylation or both expression and
phosphorylation identifies mammalian tumors that do not respond to
a HER2-directed therapy.
[0032] In certain embodiments, the pattern that identifies a
mammalian tumor as not responding is normal or increased expression
of IGFR polypeptide, accompanied by increased phosphorylation of
AKT polypeptide, increased phosphorylation of S6 ribosomal
polypeptide, or both in the mammalian tumor as compared to a
non-tumor tissue or cell sample. In other embodiments, the detected
pattern is decreased expression of EGFR polypeptide and increased
expression of NDF polypeptide in the mammalian tumor as compared to
a non-tumor tissue or cell sample. In further embodiments, the
detected pattern is decreased expression of EGFR polypeptide in the
mammalian tumor as compared to a non-tumor tissue or cell sample.
In other embodiments, the detected pattern is decreased expression
of NDF polypeptide in the mammalian tumor as compared to a
non-tumor tissue or cell sample. In additional embodiments, the
detected pattern is decreased expression of EGFR polypeptide and
increased phosphorylation of ERK polypeptide in the mammalian tumor
as compared to a non-tumor tissue or cell sample. In further
embodiments, the detected pattern is normal or increased expression
of IGFR polypeptide and decreased expression of NDF in the
mammalian tumor as compared to a non-tumor tissue or cell
sample.
[0033] In a third aspect, the invention provides methods of
selecting a subject with cancer for treatment with a molecule
targeting HER2, wherein the cancer overexpresses HER2, the methods
comprising the steps of:
[0034] (a) determining the pattern of expression, phosphorylation
or both expression and phosphorylation in a cell or tissue sample
from the subject of one or a plurality of polypeptides consisting
of:
[0035] (i) IGFR polypeptide;
[0036] (ii) EGFR polypeptide;
[0037] (iii) NDF polypeptide;
[0038] (iv) phosphorylated S6 ribosomal polypeptide;
[0039] (v) phosphorylated AKT polypeptide; and
[0040] (vi) phosphorylated ERK polypeptide; and
[0041] (b) selecting the subject based on the detected pattern of
expression, phosphorylation, or both expression and
phosphorylation. The particular combination of polypeptides and
pattern of expression, phosphorylation or both expression and
phosphorylation is used to select the subjects with cancer for
treatment with a molecule targeting HER2.
[0042] In certain embodiments, the detected pattern for selecting a
subject for treatment with a molecule targeting HER2 is decreased
expression of IGFR polypeptide in the mammalian tumor as compared
to a non-tumor tissue or cell sample. In other embodiments, the
detected pattern is normal or increased expression of IGFR
polypeptide, accompanied by decreased phosphorylation of AKT
polypeptide, decreased phosphorylation of S6 ribosomal polypeptide
or both in the mammalian tumor as compared to a non-tumor tissue or
cell sample. In further embodiments, the detected pattern is normal
or increased expression of EGFR polypeptide, accompanied by
decreased phosphorylation of ERK polypeptide in the mammalian tumor
as compared to a non-tumor tissue or cell sample. In additional
embodiments, the detected pattern is decreased expression of IGFR
polypeptide, accompanied by increased phosphorylation of S6
ribosomal polypeptide in the mammalian tumor as compared to a
non-tumor tissue or cell sample. In other embodiments, the detected
pattern is decreased expression of IGFR polypeptide, accompanied by
increased expression of NDF polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample; where further the
detected pattern can include increased phosphorylation of S6
ribosomal polypeptide.
[0043] In a fourth aspect, the invention provides methods of
selecting a subject with cancer to not receive treatment with a
molecule targeting HER2, wherein the cancer overexpresses HER2, the
methods comprising the steps of:
[0044] (a) determining the pattern of expression, phosphorylation
or both expression and phosphorylation in a cell or tissue sample
from the subject of one or a plurality of polypeptides consisting
of:
[0045] (i) IGFR polypeptide;
[0046] (ii) EGFR polypeptide;
[0047] (iii) NDF polypeptide;
[0048] (iv) phosphorylated S6 ribosomal polypeptide;
[0049] (v) phosphorylated AKT polypeptide; and
[0050] (vi) phosphorylated ERK polypeptide; and
[0051] (b) selecting the subject based on the detected pattern of
expression, phosphorylation, or both expression and
phosphorylation. The particular combination of polypeptides and
pattern of expression, phosphorylation or both expression and
phosphorylation is used to select the subjects with cancer to not
receive treatment with a molecule targeting HER2.
[0052] In certain embodiments, the detected pattern for selecting a
subject not to receive treatment with a molecule targeting HER2 is
normal or increased expression of IGFR polypeptide, accompanied by
increased phosphorylation of AKT polypeptide, increased
phosphorylation of S6 ribosomal polypeptide, or both in the
mammalian tumor as compared to a non-tumor tissue or cell sample.
In other embodiments, the detected pattern is decreased expression
of EGFR polypeptide and increased expression of NDF polypeptide in
the mammalian tumor as compared to a non-tumor tissue or cell
sample. In further embodiments, the detected pattern is decreased
expression of EGFR polypeptide in the mammalian tumor as compared
to a non-tumor tissue or cell sample. In other embodiments, the
detected pattern is decreased expression of NDF polypeptide in the
mammalian tumor as compared to a non-tumor tissue or cell sample.
In additional embodiments, the detected pattern is decreased
expression of EGFR polypeptide and increased phosphorylation of ERK
polypeptide in the mammalian tumor as compared to a non-tumor
tissue or cell sample. In further embodiments, the detected pattern
is normal or increased expression of IGFR polypeptide and decreased
expression of NDF in the mammalian tumor as compared to a non-tumor
tissue or cell sample.
[0053] In various aspects of the invention, including those
mentioned above, the detection of phosphorylation of AKT
polypeptide, phosphorylation of S6 ribosomal polypeptide, or both
can determined subsequent to contacting the sample obtained from
the mammalian tumor with a HER2-directed therapy. Further, the
HER2-directed therapy can be or comprise rhuMAb HER2
(HERCEPTIN.RTM.). In addition, the sample can be contacted with at
least one chemotherapeutic agent. Further, the detected pattern of
expression, phosphorylation, or both, of one or a plurality of
polypeptides (a) through (f) can be determined using a biodetection
reagent. The biodetection reagent can be an antibody or a nucleic
acid probe. Further, the detected pattern of phosphorylated AKT
polypeptide can be determined using an antibody specific for an
epitope comprising a phosphorylated serine residue at position 473,
the detected pattern of phosphorylated S6 ribosomal polypeptide can
be determined using an antibody specific for an epitope comprising
a phosphorylated serine residue at position 235, and/or the
detected pattern of phosphorylated ERK polypeptide can be
determined using an antibody specific for an epitope comprising a
phosphorylated threonine residue at position 202 and a
phosphorylated tyrosine residue at position 204. Further, the
sample obtained from the mammalian tumor can be a paraffin-embedded
biopsy sample. Also, the mammalian tumor can be identified as
overexpressing HER2 using an antibody that binds HER2
polypeptide.
[0054] In a fifth embodiment, the invention provides kits for
characterizing a mammalian tumor's responsiveness to a
HER2-directed therapy, the kit comprising:
[0055] (a) an antibody that binds IGFR polypeptide, and one or more
of the following:
[0056] (b) an antibody that binds phosphorylated AKT
polypeptide;
[0057] (c) an antibody that binds phosphorylated S6 ribosomal
polypeptide;
[0058] (d) an antibody that binds EGFR polypeptide;
[0059] (e) an antibody that binds HER2 polypeptide;
[0060] (f) an antibody that binds NDF polypeptide; and
[0061] (g) an antibody that binds phosphorylated ERK
polypeptide.
[0062] In certain embodiments, the antibody of (b) is
immunologically specific for AKT polypeptide having a
phosphorylated serine residue at position 473; antibody of (c) is
immunologically specific for S6 ribosomal polypeptide having a
phosphorylated serine residue at position 235; and/or the antibody
of (f) is immunologically specific for EKT polypeptide having a
phosphorylated threonine residue at position 202 and a
phosphorylated tyrosine at position 204. In other embodiments, the
kit further comprises at least one secondary antibody that binds to
an antibody of subpart (a) through (g).
[0063] Specific embodiments of the present invention will become
evident from the following more detailed description of certain
preferred embodiments and the claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0064] This invention provides methods for predicting response in
cancer subjects to cancer therapy, including human cancer patients.
In addition, this invention provides predictive biomarkers to
identify the cancer patients for whom the administering a
therapeutic agent will be most effective, including a therapeutic
agent for treating breast cancer. Specifically, this invention
provides predictive biomarkers for assessing the efficacy of
therapeutic agents targeted to Her2/neu, including such agents such
as HERCEPTIN.RTM..
[0065] In contrast to traditional anticancer methods, where
chemotherapeutic drug treatment is undertaken as an adjunct to and
after surgical intervention, neoadjuvant (or primary) chemotherapy
consists of administering drugs as an initial treatment in certain
cancer patients. One advantage of such an approach is that, for
primary tumors of more than 3 cm, it permits the later or
concomitant use of conservative surgical procedures (as opposed to,
e.g., radical mastectomy in breast cancer patients) for the
majority of patients, due to the tumor shrinking effect of the
chemotherapy. Another advantage is that for many cancers, a partial
and/or complete response is achieved in about two-thirds of all
patients. Finally, because the majority of patients are responsive
after two to three cycles of chemotherapeutic treatment, it is
possible to monitor the in vivo efficacy of the chemotherapeutic
regimen employed, in order to identify patients whose tumors are
non-responsive to chemotherapeutic treatment. Timely identification
of non-responsive tumors allows the clinician to limit a cancer
patient's exposure to unnecessary side-effects of treatment and to
institute alternative treatments. Unfortunately, methods present in
the art, including histological examination, are insufficient for
such a timely and accurate identification. The present invention
provides methods for developing more informed and effective regimes
of therapy that can be administered to cancer patients with an
increased likelihood of an effective outcome (i.e., reduction or
elimination of the tumor).
[0066] A cancer diagnosis, both an initial diagnosis of disease and
subsequent monitoring of the disease course (before, during, or
after treatment) is conventionally confirmed through histological
examination of cell or tissue samples removed from a patient.
Clinical pathologists need to be able to accurately determine
whether such samples are benign or malignant and to classify the
aggressiveness of tumor samples deemed to be malignant, because
these determinations often form the basis for selecting a suitable
course of patient treatment. Similarly, the pathologist needs to be
able to detect the extent to which a cancer has grown or gone into
remission, particularly as a result of or consequent to treatment,
most particularly treatment with chemotherapeutic or biological
agents.
[0067] Histological examination traditionally entails
tissue-staining procedures that permit morphological features of a
sample to be readily observed under a light microscope. A
pathologist, after examining the stained sample, typically makes a
qualitative determination of whether the tumor sample is malignant.
It is difficult, however, to ascertain a tumor's aggressiveness
merely through histological examination of the sample, because a
tumor's aggressiveness is often a result of the biochemistry of the
cells within the tumor, such as protein expression or suppression
and protein phosphorylation, which may or may not be reflected by
the morphology of the sample. Therefore, it is important to be able
to assess the biochemistry of the cells within a tumor sample.
Further, it is desirable to be able to observe and quantitate both
gene expression and protein phosphorylation of tumor-related genes
or proteins, or more specifically cellular components of
tumor-related signaling pathways.
[0068] Cancer therapy can be based on molecular profiling of tumors
rather than simply their histology or site of the disease.
Elucidating the biological effects of targeted therapies in tumor
tissue and correlating these effects with clinical response helps
identify the predominant growth and survival pathways operative in
tumors, thereby establishing a pattern of likely responders and
conversely providing a rational for designing strategies to
overcome resistance. Successful diagnostic targeting of a growth
factor receptor must determine if tumor growth or survival is being
driven by the targeted receptor or receptor family, by other
receptors not targeted by the therapy, and whether downstream
signaling suggests that another oncogenic pathway is involved.
[0069] For subjects considered for treatment with HERCEPTIN.RTM.,
it is necessary to consider additional biomarkers beyond the
presence of the target HER-2/neu, at least because the status of
the EGFR and erbB ligands NDF and TGF-.alpha. affect HERCEPTIN.RTM.
therapy response in breast cancer patients. Therefore, considering
HER2/neu expression alone does not necessarily predict overall erbB
oncogenic activity or potential response to erbB inhibitors. In
addition, previous studies have shown that not all tumor cells
respond to inhibition of ErbB receptors, despite exhibiting
aberrant EGFR and/or HER2/neu expression. Examples include the MKN7
and BT474 erbB receptor-overexpressing carcinoma cell lines: BT474
cells respond to HERCEPTIN.RTM. but MKN7 cells do not (Motoyama, et
al., Cancer Research, 62, 3151-3158 (2002)). In addition, the
proliferation block induced as a consequence of decreased EGFR or
HER2 receptor activity, such as by the activity of an
erbB-inhibitor, may be overcome by the presence of EGF-related
ligands such as EGF or NDF/Heregulin (Id). This phenomenon can be
attenuated using a bispecific ErbB-1/ErbB-2 inhibitor, thus
supporting increased antitumor efficacy through simultaneous
inhibition of multiple ErbB receptors.
[0070] In addition, in many cancers NDF/Heregulin or TGF-.alpha.
expression produces an autocrine loop of HER-2/EGFR
heterodimerization. Downregulation of HER-2/neu expression is an
important way of inhibiting signals generated by these
heterodimers. Downregulation can be accomplished by treatment with
HERCEPTIN.RTM., which produces receptor endocytosis. Furthermore,
high levels of phosphorylated ERK (or pAKT) can be a negative
predictor for positive treatment outcome, when observed in
conjunction with the expression of EGFR and the presence of NDF,
suggesting the existence of other pathways that might promote
proliferation of the tumor cellular growth. High pERK is also
associated with resistance to HERCEPTIN.RTM. through downregulation
of p27; this may implicate other signals (such as estrogen
receptor's cross activation of the MAPK and AKT pathways) that may
contribute to high pERK and thus contribute to proliferation of the
tumor cells growth. In addition, phosphorylated AKT has been shown
to be an important part of the response to HERCEPTIN.RTM., as high
pAKT-positive patients had poor response to HERCEPTIN.RTM..
High-phosphorylated AKT has been shown to be associated with high
expression of insulin like growth factor receptors (IGFR-1) as well
as PDGFR and results in resistance to HERCEPTIN.RTM..
Interestingly, data from clinical trials have shown that using a
dual inhibitor (i.e., specific for HER-1/neu and HER-2/neu) has
clinical efficacy in patients when treatment induced downregulation
of pERK and pAKT, but not in patients in which pERK and pAKT levels
didn't diminish after treatment. Thus, in those patients who
overexpressed HER-1 and HER-2, as well as pERK and pAKT, antitumor
activity was dependent on HER-1 and HER-2 receptor activation and a
clinical response was observed. In contrast, in patients for whom
pERK and pAKT activity remained high after treatment with a dual
inhibitor, clinical response didn't occur. Combination therapies
can have clinical significance. A combination of the
ErbB-1-directed monoclonal antibodies mAb 225 and mAb 4D5 inhibited
proliferation of an ovarian tumor cell line more strongly than
either mAb alone (Ye et al., 1999, Oncogene 18: 731-8). In addition
to ErbB-targeted mAbs, a number of different
ErbB-1/ErbB-2-bispecific inhibitors, also referred to as dual
EGFR/erbB-2 kinase inhibitors, have been described recently, such
as GW572016 and PKI166, that are currently in clinical trials
(Motoyama et al., 2002, Cancer Research 62: 3151-3158). Therefore,
response to HERCEPTIN.RTM. is affected by the expression of
multiple erbB receptors and their ligands in tumors.
[0071] Thus, HER-2/neu overexpression alone is not the only
predictor of response to molecules such as HERCEPTIN.RTM.).
HER-2/neu is an orphan, ligandless receptor in need of its partners
EGFR (HER-1) and HER-3 in order to exert its activity. HER-1 and
HER-3 heterodimerization with HER-2 is enhanced by the presence of
EGF or NDF (Tzahar et al., 1996, EMBO J. 15: 254-64, Graus-Porta,
1997, EMBO J, 16 1647-55), and thus HER-2 activity is dependant on
HER-1 or HER-3. Other receptors may also transactivate the erbB
receptors. These receptors may be mediating tumorigenesis through
signaling to downstream proliferative and survival pathways. For
example, the IGFR receptor may mediate patient response to breast
cancer therapies targeting HER2/neu. High IGFR expression combined
with high S6 ribosomal protein phosphorylation correlates with poor
patient response regardless of erb-B expression, indicating that
IGFR acts directly to activate signaling downstream of erb-B
receptors rather than through transactivation of erb-B receptors.
Cell line studies also have suggested a role for IGFR in patient
response. HERCEPTIN.RTM. resistance has been suggested to occur
though activation of IGFR (Lu et al., 2001, J National Cancer
Institute 93: 1852). In addition, co-targeting IGFR as well as
HER2/neu has been shown to produce synergistic inhibition of growth
in breast cancer cells (Camirand et al., 2002, Med Sci Monit. 8:
(12): BR521-6). Therefore, analysis of IGFR expression and
downstream signaling can be critical for an accurate assessment of
potential HERCEPTIN.RTM. response in breast cancer patients.
[0072] Thus, there is no one marker of downstream signaling protein
activation that would integrate multiple upstream signals and
predict response. However, analysis of p-ERK and p-AKT has been
found to be predictive in patients over-expressing EGFR. Therefore,
in the presence of active erbB receptors, high ERK and AKT
signaling indicates that HERCEPTIN.RTM. therapy is less likely to
be effective. AKT activation has been shown to result in
HERCEPTIN.RTM. resistance in breast cancer cell lines (Yakes, et
al., 2002, Cancer Res. 62: 4132-41; Clark et al., 2202, Mol. Cancer
Ther. 1: 707-17). In addition, analysis of S6 ribosomal protein
phosphorylation greatly increased the predictive power of IGFR
expression. In patients with high S6 phosphorylation, positive
response ranged from 8% to 67% based upon IGFR expression.
Approximately 30% of patients with low S6 phosphorylation
responded, regardless of IGFR expression. These results were also
reflected in an analysis of clinical samples, in which only those
patients that lacked active IGFR signaling responded to
HERCEPTIN.RTM. therapy. IGF signaling in breast cancer occurs
through AKT activation (Oh et al., 2002, Neoplasia 4: 204-17;
Dufourny et al., 1997, J. Biol. Chem. 272: 31163-71), which leads
to S6 ribosomal protein phosphorylation. Hence, S6 phosphorylation
can be indicative of active IGF signaling in those tumors
over-expressing IGFR.
[0073] Analysis of down-stream signaling and patient response is
complicated when chemotherapy and radiotherapy therapy is included
in addition to HERCEPTIN.RTM. treatment. AKT and MAP kinase pathway
activation, for example, are known to play a role in response to
DNA-damaging agents (Clark et al., 2002, Mol. Cancer Ther. 1:
707-17; Bacus et al., 2001, Oncogene 20: 147-155). Consideration of
downstream signaling in patients undergoing a combination of
therapies provides additional predictive information not obtained
solely from analysis of receptor or ligand expression levels.
Analysis of patients treated with HERCEPTIN.RTM. as a single agent
therapy can be used to determine which of the identified biomarkers
mediated the response to Herceptin.RTM., as opposed to the
biomarkers that mediate the response to the other therapies.
Nonetheless, the identified biomarkers are useful, among other
things, for designing diagnostics for breast cancer patients
undergoing the common HERCEPTIN.RTM. combination therapies.
[0074] Further, up-regulation of the AKT/mTOR pathway by
Heregulin/NDF is an important predictor for response. pAKT has been
associated with high levels of Cyclin E and low levels of the
cyclin inhibitor p27.
[0075] Before administration of HER2-targeted therapies, a panel of
diagnostics of each tumor is used according to the methods of this
invention to find the best candidate for each therapy. According to
the methods of this invention, treatment by a HER2-targeted
therapy, such as HERCEPTIN.RTM., is effective when a patient's
tumor growth depends on a cellular pathway such as AKT/mTOR that is
driven by the erbB receptors and not by other tyrosine kinases,
such as Insulin-like Growth Factor Receptors (IGFR). When high
levels of activation of these downstream signals occur independent
of erbB receptors, HERCEPTIN.RTM. treatment is not effective. Use
of the methods of this invention permits a clinician to choose a
more effective combination of targeted therapies for cancer
patients.
[0076] The HER2-directed therapies of the present invention can
include, for example, rhuMAb HER2, otherwise known as
HERCEPTIN.RTM.. The samples obtained from the mammalian tumor can
be contacted with at least one chemotherapeutic agent, for example
cisplaint, doxorubicin, or taxol.
[0077] Automated (computer-aided) image analysis systems known in
the art can augment visual examination of tumor samples. In a
representative embodiment, the cell or tissue sample is exposed to
detectably-labeled reagents specific for a particular biological
marker, and the magnified image of the cell is then processed by a
computer that receives the image from a charge-coupled device (CCD)
or camera such as a television camera. Such a system can be used,
for example, to detect and measure expression and activation levels
of EGFR, HER2, HER3, pERK, NDF, TGF-.alpha., IGFR, pS6, and pAKT in
a sample, or any additional diagnostic biomarkers. Thus, the
methods of the invention provide more accurate cancer diagnosis and
better characterization of gene expression in histologically
identified cancer cells, most particularly with regard to
expression of tumor marker genes or genes known to be expressed in
particular cancer types and subtypes (e.g., having different
degrees of malignancy). This information permits a more informed
and effective regimen of therapy to be administered, because drugs
with clinical efficacy for certain tumor types or subtypes can be
administered to patients whose cells are so identified.
[0078] Another drawback of conventional anticancer therapies is
that the efficacy of specific chemotherapeutic agents in treating a
particular cancer in an individual human patient is unpredictable.
In view of this unpredictability, the art is unable to determine,
prior to starting therapy, whether one or more selected agents
would be active as anti-tumor agents or to render an accurate
prognosis of course of treatment in an individual patient. This is
especially important because a particular clinical cancer may
present the clinician with a choice of treatment regimens, without
any current way of assessing which regimen will be most efficacious
for a particular individual. It is an advantage of the methods of
this invention that they are able to better assess the expected
efficacy of a proposed therapeutic agent (or combination of agents)
in an individual patient. The claimed methods are advantageous for
the additional reasons that they are both time- and cost-effective
in assessing the efficacy of chemotherapeutic regimens and are
minimally traumatic to cancer patients.
[0079] Methods of this invention can be used to identify a
mammalian tumor that responds to HER-2 directed therapies. Further,
methods of this invention can be used to select a subject with
cancer for treatment with a molecule targeting HER. Moreover,
methods of this invention can be used to identify a mammalian tumor
that does not respond to HER-2 directed therapies. Further, methods
of this invention can be used to select a subject with cancer to
not receive treatment with a molecule targeting HER2.
[0080] Patterns of expression and phosphorylation of polypeptides
are detected and quantified using methods of the present invention.
More particularly, patterns of expression and phosphorylation of
polypeptides that are cellular components of a tumor-related
signaling pathway are detected and quantified using methods of the
present invention. For example, the patterns of expression and
phosphorylation of polypeptides can be detected using biodetection
reagents specific for the polypeptides. For example, the
biodetection reagents can be antibodies. Alternatively, the
biodetection reagents can be nucleic acid probes. A nucleic acid
probe is defined to be a collection of one or more nucleic acid
fragments whose hybridization to a sample can be detected. The
probe may be unlabeled or labeled so that its binding to the target
or sample can be detected. The probe is produced from a source of
nucleic acids from one or more particular (preselected) portions of
the genome, e.g., one or more clones, an isolated whole chromosome
or chromosome fragment, or a collection of polymerase chain
reaction (PCR) amplification products. The nucleic acid probe may
also be isolated nucleic acids immobilized on a solid surface
(e.g., nitrocellulose, glass, quartz, fused silica slides), as in
an array. The probe may be a member of an array of nucleic acids as
described, for instance, in WO 96/17958. Techniques capable of
producing high density arrays can also be used for this purpose
(see, e.g., Fodor (1991) Science 767-773; Johnston (1998) Curr.
Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092;
Kern (1997) Biotechniques 23: 120-124; U.S. Pat. No. 5,143,854).
One of skill will recognize that the precise sequence of the
particular probes can be modified to a certain degree to produce
probes that are "substantially identical," but retain the ability
to specifically bind to (i.e., hybridize specifically to) the same
targets or samples as the probe from which they were derived. The
term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide in either single- or double-stranded form. The term
encompasses nucleic acids, i.e., oligonucleotides, containing known
analogues of natural nucleotides that have similar or improved
binding properties, for the purposes desired, as the reference
nucleic acid. The term also includes nucleic acids which are
metabolized in a manner similar to naturally occurring nucleotides
or at rates that are improved for the purposes desired. The term
also encompasses nucleic-acid-like structures with synthetic
backbones. One of skill in the art would recognize how to use a
nucleic acid probes for screening of cancer cells in a sample by
reference, for example, to U.S. Pat. No. 6,326,148, directed to
screening of colon carcinoma cells.
[0081] Polypeptides associated with breast cancer can be quantified
by image analysis using a suitable primary antibody against
biomarkers, including but not limited to EGFR, HER-2, HER-3, IGFR,
NDF, TGF-.alpha., p-ERK, pS6, or p-AKT, detected directly or using
an appropriate secondary antibody (such as rabbit anti-mouse IgG
when using mouse primary antibodies) and/or a tertiary avidin (or
Strepavidin) biotin complex ("ABC").
[0082] Examples of reagents useful in the practice of the methods
of the invention as exemplified herein include antibodies specific
for HER2/neu, including but not limited to the mouse monoclonal
antibody CB11, obtained from Ventana Medical Systems, Inc. (VMSI,
Tucson, Ariz.). In addition, reagents useful in the practice of the
methods of the invention include antibodies specific for
phosphorylated AKT, including but not limited to antibodies
specific for a phosphorylated serine residue of position 473,
wherein the sequence of AKT is represented by SEQ ID NO: 1 (Table
8). Further, reagents useful in the practice of the methods of the
invention include antibodies specific for phosphorylated S6,
including but not limited to antibodies specific for a
phosphorylated serine residue of position 235, wherein the sequence
of S6 is represented by SEQ ID NO:2 (Table 8). Also, reagents
useful in the practice of the methods of the invention include
antibodies specific for phosphorylated ERK, including but not
limited to antibodies specific for a phosphorylated threonine
residue at position 202 and a phosphorylated tyrosine residue of
position 204, wherein the sequence of ERK is represented by SEQ ID
NO:3 (Table 8).
[0083] Further, the pattern of expression, phosphorylation, or both
expression and phosphorylation of the predictive polypeptides can
be compared to a non-tumor tissue or cell sample. The non-tumor
tissue or cell sample can be obtained from a non-tumor tissue or
cell sample from the same individual, or alternatively, a non-tumor
tissue or cell sample from a different individual. A detected
pattern for a polypeptide is referred to as decreased in the
mammalian tumor, tissue, or cell sample, if there is less
polypeptide detected as compared to the a non-tumor tissue or cell
sample. A detected pattern for a polypeptide is referred to as
increased in the mammalian tumor, tissue, or cell sample, if there
is more polypeptide detected as compared to the a non-tumor tissue
or cell sample. A detected pattern for a polypeptide is referred to
as normal in the mammalian tumor, tissue, or cell sample, if there
is the same, or approximately the same, polypeptide detected as
compared to the a non-tumor tissue or cell sample.
[0084] The methods of this invention for identifying mammalian
tumors that respond, or that do not respond, to a HER2-directed
therapy comprise the step of assaying a sample obtained from the
mammalian tumor to detect a pattern of expression, phosphorylation
or both of one or a plurality of polypeptides consisting of: (a)
IGFR polypeptide; (b) EGFR polypeptide; (c) NDF polypeptide; (d)
phosphorylated S6 ribosomal polypeptide; (e) phosphorylated AKT
polypeptide; (f) phosphorylated EKT polypeptide. The combination of
polypeptides and pattern of expression, phosphorylation, or both
expression and phosphorylation identifies mammalian tumors that
respond, or that do not respond, to a HER2-directed therapy. The
methods can include the detection of a pattern of expression,
phosphorylation or both of one, two, three, four, five, or all six
of these polypeptides. Further, the methods can, but need not,
include other steps, including steps such as the detection of a
pattern of expression, phosphorylation or both of different
polypeptides.
[0085] The methods of this invention for selecting a subject with
cancer for treatment, or to not receive treatment, with a molecule
targeting HER2, such as, but not limited to treatment with
HERCEPTIN.RTM., comprise the step of determining the pattern of
expression, phosphorylation or both in a cell or tissue sample from
the subject of one or a plurality of polypeptides consisting of:
(a) IGFR polypeptide; (b) EGFR polypeptide; (c) NDF polypeptide;
(d) phosphorylated S6 ribosomal polypeptide; (e) phosphorylated AKT
polypeptide; (f) phosphorylated EKT polypeptide. The combination of
polypeptides and pattern of expression, phosphorylation, or both
expression and phosphorylation is used to select a subject with
cancer for treatment, or to not receive treatment, with a molecule
targeting HER2. The methods can include the detection of a pattern
of expression, phosphorylation or both of one, two, three, four,
five, or all six of these polypeptides. Further, the methods can,
but need not, include other steps, including steps such as the
detection of a pattern of expression, phosphorylation or both of
different polypeptides.
[0086] For example, the pattern that identifies a mammalian tumor
as responding or that can be used to select a subject with cancer
for treatment with a molecule targeted to HER2 is decreased
expression of IGFR polypeptide as compared to a non-tumor tissue or
cell sample. Alternatively, the detected pattern is normal or
increased expression of IGFR polypeptide, accompanied by decreased
phosphorylation of AKT polypeptide, decreased phosphorylation of S6
ribosomal polypeptide or both as compared to a non-tumor tissue or
cell sample. Another potential detected pattern is normal or
increased expression of EGFR polypeptide, accompanied by decreased
phosphorylation of ERK polypeptide as compared to a non-tumor
tissue or cell sample. Further detected patterns include decreased
expression of IGFR polypeptide, accompanied by increased
phosphorylation of S6 ribosomal polypeptide as compared to a
non-tumor tissue or cell sample. In other embodiments, the detected
pattern is decreased expression of IGFR polypeptide, accompanied by
increased expression of NDF polypeptide in the mammalian tumor as
compared to a non-tumor tissue or cell sample; where further the
detected pattern can include increased phosphorylation of S6
ribosomal polypeptide. These identified patterns are understood to
be non-limiting.
[0087] For example, the pattern that identifies a mammalian tumor
as not responding or that can be used to select a subject with
cancer to not receive treatment with a molecule targeted to HER2 is
normal or increased expression of IGFR polypeptide, accompanied by
increased phosphorylation of AKT polypeptide, increased
phosphorylation of S6 ribosomal polypeptide, or both as compared to
a non-tumor tissue or cell sample. Alternatively, the detected
pattern is decreased expression of EGFR polypeptide and increased
expression of NDF polypeptide as compared to a non-tumor tissue or
cell sample. Or, the detected pattern is decreased expression of
EGFR polypeptide as compared to a non-tumor tissue or cell sample.
Further, the detected pattern is decreased expression of NDF
polypeptide as compared to a non-tumor tissue or cell sample. Or,
the detected pattern is decreased expression of EGFR polypeptide
and increased phosphorylation of ERK polypeptide as compared to a
non-tumor tissue or cell sample. Further, the detected pattern is
normal or increased expression of IGFR polypeptide and decreased
expression of NDF as compared to a non-tumor tissue or cell sample.
These identified patterns are understood to be non-limiting.
[0088] In practicing the methods of this invention, staining
procedures can be carried out by a person, such as a technician in
the laboratory. Alternatively, the staining procedures can be
carried out using automated systems. In either case, staining
procedures for use according to the methods of this invention are
preformed according to standard techniques and protocols
well-established in the art.
[0089] By "cell or tissue sample" is meant biological samples
comprising cells, most preferably tumor cells, that are isolated
from body samples, such as, but not limited to, smears, sputum,
biopsies, secretions, cerebrospinal fluid, bile, blood, lymph
fluid, urine and faeces, or tissue which has been removed from
organs, such as breast, lung, intestine, skin, cervix, prostate,
and stomach. For example, a tissue samples can comprise a region of
functionally related cells or adjacent cells.
[0090] The amount of target protein is advantageously quantified by
measuring the average optical density of the stained antigens.
Concomitantly, the proportion or percentage of total tissue area
stained can be readily calculated, for example as the area stained
above a control level (such as an antibody threshold level) in the
second image. Following visualization of nuclei containing
biomarkers, the percentage or amount of such cells in tissue
derived from patients after treatment are compared to the
percentage or amount of such cells in untreated tissue. For
purposes of the invention, "determining" a pattern of expression,
phosphorylation, or both expression and phosphorylation o
polypeptides is understood broadly to mean merely obtaining the
information on such polypeptide(s), either through direct
examination or indirectly from, for example, a contract diagnostic
service.
[0091] Breast cancer tissue sections taken from patients treated
with HERCEPTIN.RTM. and chemotherapy are analyzed, according to the
methods of this invention by immunohistochemistry for expression,
phosphorylation, or expression and phosphorylation of erb-B
ligands, receptors, downstream signaling proteins or any positive
treatment response predictive combination thereof. These
measurements can be accomplished, for example, by using tissue
microarrays. Tissue microarrays are advantageously used in the
methods of the invention, being well-validated method to rapidly
screen multiple tissue samples under uniform staining and scoring
conditions. (Hoos et al., 2001, Am J Pathol. 158: 1245-51). Scoring
of the stained arrays can be accomplished by an automated system
that accurately quantified the staining observed. The results of
this analysis identify biomarkers that best predict patient outcome
following treatment, such as HERCEPTIN.RTM. therapies. Patient
"probability of response" ranging from 0 to 100 percent can be
predicted based upon the expression, phosphorylation or both of a
small set of ligands, receptors, signaling proteins or predictive
combination thereof. Additional samples from breast cancer patients
can be analyzed, either as an alternative to or in addition to
tissue microarray results. For example, analysis of samples from
breast cancer patients can confirm the conclusions from the tissue
arrays, if the patient's responses correlate with a specific
pattern of receptor expression and/or downstream signaling.
[0092] The invention provides, in part, kits for carrying out the
methods of the invention. For example, the method provides kits for
characterizing a mammalian tumor's responsiveness to a
HER2-directed therapy comprising an antibody that binds IGFR
polypeptide, and one or more of the following: an antibody that
binds phosphorylated AKT polypeptide; an antibody that binds
phosphorylated S6 ribosomal polypeptide; an antibody that binds
EGFR polypeptide; an antibody that binds HER2 polypeptide; an
antibody that binds NDF polypeptide; and an antibody that binds
phosphorylated ERK polypeptide. In addition to an antibody that
binds IGFR polypeptide, the kit can include one, two, three, four,
or all five of the following: an antibody that binds phosphorylated
AKT polypeptide; an antibody that binds phosphorylated S6 ribosomal
polypeptide; an antibody that binds EGFR polypeptide; an antibody
that binds HER2 polypeptide; an antibody that binds NDF
polypeptide; and an antibody that binds phosphorylated ERK
polypeptide. Further, the kit can include additional components
other then the above-identified antibodies, including but not
limited to additional antibodies. Such kits may be used, for
example, by a clinician or physician as an aid to selecting an
appropriate therapy for a particular patient, for example, a breast
cancer patient under consideration for HER2-directed therapy.
[0093] Particularly useful embodiments of the present invention and
the advantages thereof can be understood by referring to Examples
1-5. These Examples are illustrative of specific embodiments of the
invention, and various uses thereof. They are set forth for
explanatory purposes only, and are not to be taken as limiting the
invention.
EXAMPLE 1
Staining Procedure for Biomarkers
[0094] Human tumor tissue sections were stained for predictive
biomarkers according to the methods of the invention as follows.
10% Neutral Buffered Formalin Paraffin blocks were sectioned at 4
microns and the sections placed onto coated slides. EGFR and HER2
immunostaining was performed by using the pre-diluted EGFR and HER2
antibodies from Ventana Medical Instruments, Inc. (VMSI, Tucson,
Ariz.). HER3, Heregulin (NDF), and IGFR antibodies were obtained
from NeoMarkers (Fremont, Calif.). TGF-.alpha. antibodies were
obtained from Oncogene Sciences (San Diego, Calif.). EGFR,
HER2/neu, HER3, IGFR, Heregulin, and TGF-.alpha. were immunostained
using the "BenchMark" (VMSI) with I-VIEW (VMSI) detection
chemistry. Antibodies specific for p-ERK (1:100), p-AKT (1:75), and
phospho-S6 ribosomal protein were obtained from Cell Signaling
Technology (Beverly, Mass.), and immunostained using a labeled
streptavidin peroxidase technique. (Vector Elite ABC Kit,
Burlingame, Calif.). Prior to staining, slides for p-S6 ribosomal
protein, p-ERK and p-AKT were antigen retrieved using 0.1 M citrate
buffer, pH 6.0 in the "decloaker" (Biocare Corp.) and the sections
incubated overnight with the primary antibodies at 4.degree. C. The
next day, the slides were placed onto the Autostainer (Dako Corp.)
and the "LSAB2" kit (Dako) was employed as the detection chemistry.
DAB (Dako) was used as the chromogen. After immunostaining, all
slides were counterstained manually with 4% ethyl green
(Sigma).
EXAMPLE 2
Procedure for Western Blot Analysis
[0095] Western blot analysis for detecting expression of predictive
markers was performed as follows. Cells were lysed in ice-cold
buffer (50 mM Tris-HCl (pH 7.5), 137 mM NaCl, 1 mM EDTA, 1% Nonidet
P-40, 0.2% Triton X-100, 10% glycerol, 0.1 mM sodium orthovanadate,
10 mM sodium pyrophosphate, 20 mM .beta.-glycerophosphate, 50 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, 2 .mu.M leupeptin, and 2
.mu.g/ml aprotinin). Protein concentration was determined with a
BioRad Protein Assay Kit (BioRad Laboratories, Hercules, Calif.).
Equal amounts of protein, typically 15 .mu.g protein per lane, were
separated by gel electrophoresis, for example using pre-cast 4-12%
Bis-Tris NuPage gradient gels (Invitrogen) or 7.5% or 4-15%
gradient SDS-PAGE under reducing conditions, and transferred to
membranes, such as HyBond-C nitrocellulose (Amersham Life Science)
or Immobilon-P membranes. Membranes were blocked and then incubated
with primary antibodies, for example antibodies against p-AKT and
p-ERK (Cell Signaling Technology). Antibody incubation was
performed overnight at 4.degree. C. in Tris-buffered saline
containing 3% bovine serum albumin/0.1% Tween 20. Signal was
detected by chemiluminescence (PerkinElmer Life Sciences), or using
a SuperSignal West Femto Maximum sensitivity substrate kit from
Pierce (Rockford, Ill.) as described (Xia et al., 2002, Oncogene
21: 6255-6263).
EXAMPLE 3
Procedure for Immunohistochemistry
[0096] Immunohistochemistry for detecting and measuring predictive
biomarker expression, activation or both was performed as follows.
HER2/neu, EGFR, HER3, IGFR, TGF-.alpha., Heregulin (NDF), p-ERK,
p-AKT, and p-S6 ribosomal protein or phosphorylation levels were
quantified using alkaline phosphatase or peroxidase techniques and
microscope-based image analysis of immunohistochemically stained
slides (as described in Bacus et al., 1997, Analyt. Quant. Cytol.
Histol. 19: 316-328). Quantification was by means of a CAS 200
image analyzer, as previously described (Bacus & Ruby, 1993,
Pathol Annu, 28: 179-204). For the purpose of the analysis, tumors
were classified as negative or positive for each antibody based
upon the level of staining. Statistical analysis was performed
using Systat to quantify frequencies and calculate Pearson
Chi-squared tests of significance for interactions between
variables. In all cases, the p value refers to the significance of
the deviation of the distribution of samples from what would be
expected based upon the overall population distribution.
Comparisons were performed only on samples for which all relevant
data were available. As a result, the number of patients included
in most comparisons was slightly less then the total number of
available samples.
[0097] Quantitative immunohistochemistry (IHC) was performed as
previously described (Bacus et al., 1997, Analyt. Quant. Cytol.
Histol. 19: 316-328). EGFR, and erbB-2 (HER2) immunostaining was
performed using pre-diluted EGFR, and erbB-2 (HER2) antibodies from
Ventana Medical Systems, Inc. (VMSI, Tucson, Ariz.) on the VMSI
automated "BenchMark" staining module as described. The VMSI
"I-View" detection kit was used for all three of the VMSI
pre-diluted primary antibodies according to the manufacturer's
instructions. Antibodies to erbB-3 (1:10), Heregulin (1:25), and
TGF-a (1:20), were also used for immunostaining using the
"BenchMark" with I-VIEW detection chemistry. Antibodies to
Phospho-Erk (1:100) and p-AKT (1:75) were used for immunostaining
using a labeled streptavidin peroxidase technique as described by
the manufacturer. Phospho-Erk and p-AKT slides were antigen
retrieved as described by Bacus et al. (1997, Analyt. Quant. Cytol.
Histol. 19: 316-328). Slides were placed onto the Autostainer (Dako
Corp.) and the "LSAB2" kit (Dako) employed as the detection
chemistry. After staining, all slides were counterstained manually
with 4% ethyl green (Sigma). Investigators preparing and analyzing
tissue sections were blinded to both patient tumor type and
response to therapy.
[0098] For IHC, antibodies to EGFR and erbB-2 were from Ventana
Medical Scientific Instruments (VMSI) (Tucson, Ariz.); anti-p-AKT
(Ser 437) and p-Erk1/2 were from Cell Signaling Technology Inc.
(Beverly, Mass.); antibodies to TGF.alpha., erbB3, heregulin, and
IGFR-1 were from NeoMarkers.
EXAMPLE 4
Analysis of Breast Cancer Tissue Microarrays
[0099] Tissue microarrays derived from 250 breast cancer patients
who received conventional chemotherapy together with HERCEPTIN.RTM.
were obtained from Clinomics Biosciences (Pittsfield, Mass.). The
histology of the tumors varied, with infiltrating ductal carcinoma
being the most common. All patients had received post-surgical
radiotherapy. The tissue samples in the array were taken before
treatment. HER2/neu expression had been determined by using the
HercepTest system (DAKO, Caprintera, Calif.) on the original
biopsies for all patients. Patient response was based upon the case
histories at last follow-up as decided by an independent
pathologist provided by Clinomics.
[0100] Demographics of these patients are reported in Table 1. The
great majority of patients had infiltrating ductal carcinomas and
received anthracycline plus cyclophosphamide. Fifty-seven of the
patients had metastatic diseases. All patients had received a 4
mg/kg HERCEPTIN.RTM. loading dosage and a 2 mg/kg weekly
maintenance dosage.
[0101] From the original tissue arrays of 250 patients,
seventy-five samples were not used in the analysis because of the
lack of clinical data or because the sections did not contain
useable tumor tissue. Overall, 15% of the remaining patients were
disease free or had stable disease after therapy, while 85%
relapsed. Of these remaining one hundred and seventy five patients,
twenty-eight samples lacked HercepTest results and were therefore
also excluded from further analysis. Of the samples for which
HercepTest results were obtained, seventy-seven had a HercepTest
score of +3, and seventy had +2 or less staining intensity (Table
I).
[0102] The HercepTest staining scores were confirmed by analyzing
HER2/neu expression levels using microarrays (data not shown).
HER2/neu expression strongly correlated with patient response; 100%
of the 0 or +1 HER2/neu patients relapsed while only 77% of the +3
patients relapsed. This response rate if similar to what has been
reported previously (see Baselga, 2002, Annuals of Oncology 13:
8-9). Based on these results, further analysis of biomarkers
concentrated on patients that expressed HER2 at the highest (+3)
level. Of the samples that had the highest HercepTest scores (+3),
seventy-four were taken from the primary tumor, two from lymph
nodes, and one from an adrenal metastasis.
[0103] The seventy-seven patients who overexpressed HER2/neu (+3
HercepTest staining score) were analyzed for expression levels of
EGFR, HER3, IGFR, and NDF/Heregulin, and TGF-.alpha., as well as
activated downstream signals p-ERK and p-AKT (phosphorylated forms
of ERK and AKT) and the downstream signal of mTOR, p-S6
(phosphorylated S6 ribosomal protein). The analysis of receptor
kinases reveled that, similar to HER2/neu, EGFR expression also
significantly correlated with patient response (Table 2). Among the
HERCEPTIN.RTM.-treated patients that over-expressed HER-2/neu, 30%
of EGFR-positive patients had stable disease or were disease free,
while only 9% of EGFR-negative patients did not progress. Among the
seventy-seven +3 HER2/neu patients, seventy of them expressed HER3;
however, HER3 expression did not significantly correlate with
patient response (although the low number of HER3-negative patients
limits this comparison in the data set). The growth-factor receptor
HER3 is thought to play an important role in downstream erbB
signaling because it has a P13-Kinase docking site and forms active
heterodimers with the other erbB receptors. The expression of other
growth factor receptors may also mediate patient response, either
through direct stimulation or downstream pathways or through
transactivation of the erbB receptors.
[0104] Expression of erbB ligands, including NDF and TGF-.alpha.,
also varied among patients (see Table 3). Approximately 70% of the
patients expressed high levels of NDF, while approximately 57%
expressed high levels of TGF-.alpha.. A significant correlation was
observed between NDF levels and patient response. A very high
proportion of HER2/neu overexpressing patients who were
NDF-negative relapsed (91%), whereas only 62% of NDF-positive
patients who overexpressed HER2/neu relapsed. In contrast, no
predictive relationship was observed between TGF-.alpha.levels
alone and patient response (see Table 3). The combination of
TGF-.alpha.or NDF expression and EGFR over-expression, however, did
positively correlate with patient response in patients
overexpressing HER2/neu (p=0.02 and p=0.03 respectively) (data not
shown).
[0105] The activation of heterodimers of HER2 with HER3 or EGFR
results in activation of the MAPK and PI3K/AKT pathways. The MAPK
pathway was measured by analyzing the level of activation or
phosphorylation of ERK (pERK). Analysis and comparison of the
levels of activated ERK alone, among patients that overexpressed
HER2/neu and who either had stable disease or who relapsed, failed
to demonstrate any dramatic effect of elevated pERK levels as a
factor for patient response (see Table 4). Similarly, based on this
analysis, AKT activation (p-AKT) alone does not appear to be a
predictive marker for response among HER2-positive patients treated
with HERCEPTIN.RTM. (see Table 4). Also, analysis of S6 ribosomal
protein phosphorylation, which integrates multiple signals through
mTOR and p70 S6 kinase, did not significantly correlate with
patient response for patients that overexpressed HER2/neu (see
Table 4). If consideration of pERK and pAKT expression is limited
to those patients that expressed EGFR and HER2/neu, however, low
expression of either of these signaling molecules was a significant
predictor of positive response to HERCEPTIN.RTM. (Table 5).
[0106] To increase the predicative power of the analysis,
consideration of two or more of the biomarkers were combined in a
multivariate analysis to characterize the tumor. In this analysis,
the observation of the combination of HER2/neu and EGFR expression
with ERK activation significantly predicated response (see Table
5). For example, patients with low EGFR expression and high ERK
phosphorylation failed to respond (0% response), whereas patients
with high EGFR expression and low ERK phosphorylation had a high
response rate (42% response). Similarly, the combination of high
EGFR and HER2/neu with high NDF expression or a combination of high
EGFR and HER2/neu with high TGF-.alpha. expression predicted a
better response compared to patients that had low expression of
EGFR and the NDF ligand (data not shown). This comparison was often
dramatic. For example, while 39% of the patients with high EGFR,
HER2/neu, and NDF expression responded to therapy, none of the
patients with high HER2/neu expression but low EGFR and NDF
expression responded (data not shown).
[0107] The combination of high Her2/neu expression, low IGFR
expression, and high S6 ribosomal protein phosphorylation gave high
patient response (67%, Table 5). This is in contrast to patients
with high HER2/neu and IGFR expression and high S6 ribosomal
protein phosphorylation, a high percentage that did not respond to
therapy. The best combination of markers for predicting whether
patients that overexpressed HER2/neu would respond to
HERCEPTIN.RTM. therapy were high NDF expression, low IGFR
expression, and high S6 phosphorylation (Table 6). In contrast,
none of the patients overexpressed HER2/neu and had low NDF
expression and high IGFR expression responded to therapy,
regardless of S6 status (Table 6). However, these results were
obtained using a small sample population of these patients. In
patients with high NDF, HER2/neu, and EGFR expression levels,
phosphorylation of ERK correlated with a difference in response
from 28% (high p-ERK) to 54% (low p-ERK) (Table 6). Similarly,
those patients with low levels of p-AKT with any other combination
of biomarkers that include the expression of HER2/neu and NDF, did
better than those that over-express this protein (results not
shown). Taken together, this data shows that HER2/neu together with
its ligand and other erbB receptors and ligands, as well as other
growth factor receptors play a role in HERCEPTIN.RTM. response.
Importantly, analysis of a select combination of these proteins
correlated with response rates that varied from 0 to 100%.
EXAMPLE 5
Analysis of Breast Cancer Samples
[0108] Samples from seven breast cancer patients were obtained from
Yale Univeristy. The clinical history of these seven patients
varied, with some given HERCEPTIN.RTM. in combination with
chemotherapy as a first line therapy while others were given
HERCEPTIN.RTM. as an adjuvant therapy. These seven samples were
analyzed for receptor, ligand, and signaling protein expression or
phosphorylation, and the results compared to the results with the
tissue microarray analysis.
[0109] All seven patients over-expressed HER2/neu, as determined at
the time of analysis with the other antibodies immunologically
specific for non-HER2/neu polypeptides. The case histories of the
patients varied. For example, patient #1 was given HERCEPTIN.RTM.
plus docetaxel after relapsing with metastatic disease four years
after initial presentation. This patient had stable disease for
more than a year after commencing combination therapy. Patient #7
was given HERCEPTIN.RTM. plus vinorelbine following the discovery
of a solitary metastasis seven months after initial radiotherapy.
After eight weeks of combination therapy there was progression of
disease. Of the seven patients, three showed response to
HERCEPTIN.RTM. while the other four failed to respond (Table 7).
One of the responders did not express IGFR but did express EGFR and
showed positive downstream signaling. The other one of these
responders expressed IGFR and EGFR but did not show active
downstream signaling in S6 or ERK. All of the non-responders
expressed IGFR and had positive S6 phosphorylation. Two of the
non-responders also expressed EGFR. These results are consistent
with the results obtained from the microarray analysis. Patients
with active IGFR receptors as demonstrated by IGFR expression plus
S6 phosphorylation are unlikely to respond to HERCEPTIN.RTM., while
patients that lack IGFR or have IGFR but no downstream signaling
are more likely to respond.
[0110] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
1TABLE 1 Demographics number of disease-free or patients stable
disease relapse all patients included in study 175 15% 85%
Histology infiltrating ductal carcinoma 109 17% 83% lobular
carcinoma 7 43% 57% medullary carcinoma 3 33% 67% metastatic breast
carcinoma 19 5% 95% papillary carcinoma 3 0% 100% scirrhous
carcinoma 3 100% 0% tubular carcinoma 3 0% 100% treatment following
surgery (followed by Herceptin .RTM.) Doxorubicin 44 2% 98%
anthracycline plus 100 23% 77% cyclophosphamide Paclitaxel 3 100%
0% HER2/neu expression tumor 0 or 1 17 0% 100% 2 53 8% 92% 3 77 30%
70% Demographics of breast cancer patient samples.
[0111]
2 TABLE 2 patient group n % responders % relapse P value EGFR
positive 43 30% 70% 0.002 EGFR negative 23 9% 91% HER3 positive 70
29% 71% 0.43 HER3 negative 7 43% 57% IGFR positive 33 24% 76% 0.16
IGFR negative 35 40% 60%
[0112] Receptor tyrosine kinase expression versus patient response.
Analysis on tissue array samples for which clinical and Herceptest
data was available and who over-expressed HER2/neu.
3TABLE 3 patient group n % responders % relapse P value NDF
positive 55 39% 62% 0.01 NDF negative 22 9% 91% TGF-.alpha.
positive 38 34% 66% 0.56 TGF-.alpha. negative 29 28% 72%
[0113] Receptor tyrosine kinase ligand expression versus patient
response following therapy. Analysis on tissue array samples for
which clinical and Herceptest data was available and who
over-expressed HER2/neu.
4TABLE 4 patient group n % responders % relapse P value p-ERK
positive 36 25% 75% 0.43 p-ERK negative 39 33% 67% p-AKT positive
24 25% 75% 0.53 p-AKT negative 53 32% 68% p-S6 positive 27 33% 67%
0.74 p-S6 negative 44 30% 70%
[0114] Downstream protein activation versus patient response
following therapy. Analysis on tissue array samples for which
clinical and Herceptest data was available and who over-expressed
HER2/neu.
5TABLE 5 patient group n % responders % relapse P value EGFR
pos/p-ERK pos 21 14% 86% 0.04 EGFR pos/p-ERK neg 19 42% 58% EGFR
neg/p-ERK pos 9 0% 100% EGFR neg/p-ERK neg 14 14% 86% EGFR
pos/p-AKT pos 17 18% 82% 0.07 EGFR pos/p-AKT neg 26 38% 62% EGFR
neg/p-AKT pos 5 20% 80% EGFR neg/p-AKT neg 18 6% 94% IGFR pos/p-S6
pos 13 8% 92% 0.01 IGFR pos/p-S6 neg 20 35% 65% IGFR neg/p-S6 pos
12 67% 33% IGFR neg/p-S6 neg 23 26% 74%
[0115] Analysis of receptor and downstream protein activation
versus response in patients following therapy. Analysis on tissue
array samples for which clinical and Herceptest data was available
and who over-expressed HER2/neu.
6TABLE 6 % patient group n responders % relapse P value NDF
neg/p-S6 pos/IGFR neg 2 50% 50% 0.003 NDF neg/p-S6 neg/IGFR neg 9
11% 89% NDF neg/p-S6 neg/IGFR pos 4 0% 100% NDF neg/p-S6 pos/IGFR
pos 4 0% 100% NDF pos/p-S6 pos/IGFR neg 7 100% 0% NDF pos/p-S6
neg/IGFR pos 16 44% 56% NDF pos/p-S6 neg/IGFR neg 14 36% 64% NDF
neg/p-ERK pos/EGFR neg 3 0% 100% 0.08 NDF neg/p-ERK neg/EGFR neg 4
0% 100% NDF neg/p-ERK neg/EGFR pos 10 20% 80% NDF neg/p-ERK
pos/EGFR pos 6 0% 100% NDF pos/p-ERK pos/EGFR neg 5 0% 100% NDF
pos/p-ERK neg/EGFR pos 13 54% 46% NDF pos/p-ERK neg/EGFR neg 6 17%
83% NDF pos/p-ERK pos/EGFR pos 18 28% 72%
[0116] Analysis of ligand and receptor expression and downstream
protein activation versus patient response in patients following
therapy. Analysis on tissue array samples for which clinical and
Herceptest data was available and who over-expressed HER2/neu.
7TABLE 7 Patient IGFR EGFR p-S6 p-AKT p-ERK Response #1 + + - - -
yes #2 - + + + + yes #3 + + - + - yes #4 + - + + + no #5 + + + + -
no #6 + - + + - no #7 + + + + + no
[0117] Receptor tyrosine kinase expression, downstream protein
activation and patient response to therapy in seven breast cancer
patients. Analysis was of whole tissue sections.
8TABLE 8 AKT (NP 005154 GI:4885061) 480 AMINO ACIDS (SEQ ID NO:1)
See, e.g., Staal, S.P., Proc. Natl. Acad. Sci. U.S.A. 84 (14),
5034-5037 (1987). MSDVAIVKEGWLHKRGEYIKTWRPR-
YFLLKNDGTFIGYKERPQDVDQREAPLNNFSVAQCQL (SEQ ID NO:1)
MKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGLKKQEEEEMDFRSGS
PSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVI
VAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSED
RARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGAT- MKTFC
GTPEYLAPEVLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELIL- MEEIRFPRTL
GPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAGIVWQHVYEK- KLSPPFKPQVTSETD
TRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSA- SSTA S6 (NP 001001,
GI:17158044) 249 AMINO ACIDS (SEQ ID NO:2) See, e.g., Pata et al.,
(1992) Gene 121 (2), 387-392.
MKLNISFPATGCQKLIEVDDERKLRTFYEKRMATEVAADALGEEWKGYVVRISGGNDKQGFP (SEQ
ID NO:2) MKQGVLTHGRVRLLLSKGHSCYRPRRTGERKRKSVRGCIVDANLSVLNLV-
IVKKGEKDIPGL TDTTVPRRLGPKRASRIRKLFNLSKEDDVRQYVVRKPLNKEGKKP-
RTKAPKIQRLVTPRVLQ HKRRRIALKKQRTKKNKEEAAEYAKLLAKRMKEAKEKRQE-
QIAKRRRLSSLRASTSKSESSQ K ERK (XP 055766, GI:20562757) 379 AMINO
ACIDS (SEQ ID NO:3) See, e.g., Butch et al., J Biol Chem., 1996.,
271(8): 4230-5. MAAAAAQGGGGGEPRRTEGVGPGV-
PGEVEMVKGQPFDVGPRYTQLQYIGEGAYGMVSSAYDH (SEQ ID NO:3)
VRKTRVAIKKISPFEHQTYCQRTLREIQILLRFRHENVIGIRDILRASTLEAMRDVYIVQDL
METDLYKLLKSQQLSNDHICYFLYQILRGLKYIHSANVLHRDLKPSNLLINTTCDLKICDFG
LARIADPEHDHTGFLTEYVATRWYRAPEIMLNSKGYTKSIDIWSVGCILAEMLSNRPIFPGK
HYLDQLNHILGILGSPSQEDLNCIINMKARNYLQSLPSKTKVAWAKLFPKSDSKALD- LLDRM
LTFNPNKRITVEEALAHPYLEQYYDPTDEPVAEEPFTFAMELDDLPKERLKE- LIFQETARFQ
PGVLEAP
[0118]
Sequence CWU 1
1
3 1 480 PRT Homo sapiens 1 Met Ser Asp Val Ala Ile Val Lys Glu Gly
Trp Leu His Lys Arg Gly 1 5 10 15 Glu Tyr Ile Lys Thr Trp Arg Pro
Arg Tyr Phe Leu Leu Lys Asn Asp 20 25 30 Gly Thr Phe Ile Gly Tyr
Lys Glu Arg Pro Gln Asp Val Asp Gln Arg 35 40 45 Glu Ala Pro Leu
Asn Asn Phe Ser Val Ala Gln Cys Gln Leu Met Lys 50 55 60 Thr Glu
Arg Pro Arg Pro Asn Thr Phe Ile Ile Arg Cys Leu Gln Trp 65 70 75 80
Thr Thr Val Ile Glu Arg Thr Phe His Val Glu Thr Pro Glu Glu Arg 85
90 95 Glu Glu Trp Thr Thr Ala Ile Gln Thr Val Ala Asp Gly Leu Lys
Lys 100 105 110 Gln Glu Glu Glu Glu Met Asp Phe Arg Ser Gly Ser Pro
Ser Asp Asn 115 120 125 Ser Gly Ala Glu Glu Met Glu Val Ser Leu Ala
Lys Pro Lys His Arg 130 135 140 Val Thr Met Asn Glu Phe Glu Tyr Leu
Lys Leu Leu Gly Lys Gly Thr 145 150 155 160 Phe Gly Lys Val Ile Leu
Val Lys Glu Lys Ala Thr Gly Arg Tyr Tyr 165 170 175 Ala Met Lys Ile
Leu Lys Lys Glu Val Ile Val Ala Lys Asp Glu Val 180 185 190 Ala His
Thr Leu Thr Glu Asn Arg Val Leu Gln Asn Ser Arg His Pro 195 200 205
Phe Leu Thr Ala Leu Lys Tyr Ser Phe Gln Thr His Asp Arg Leu Cys 210
215 220 Phe Val Met Glu Tyr Ala Asn Gly Gly Glu Leu Phe Phe His Leu
Ser 225 230 235 240 Arg Glu Arg Val Phe Ser Glu Asp Arg Ala Arg Phe
Tyr Gly Ala Glu 245 250 255 Ile Val Ser Ala Leu Asp Tyr Leu His Ser
Glu Lys Asn Val Val Tyr 260 265 270 Arg Asp Leu Lys Leu Glu Asn Leu
Met Leu Asp Lys Asp Gly His Ile 275 280 285 Lys Ile Thr Asp Phe Gly
Leu Cys Lys Glu Gly Ile Lys Asp Gly Ala 290 295 300 Thr Met Lys Thr
Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val 305 310 315 320 Leu
Glu Asp Asn Asp Tyr Gly Arg Ala Val Asp Trp Trp Gly Leu Gly 325 330
335 Val Val Met Tyr Glu Met Met Cys Gly Arg Leu Pro Phe Tyr Asn Gln
340 345 350 Asp His Glu Lys Leu Phe Glu Leu Ile Leu Met Glu Glu Ile
Arg Phe 355 360 365 Pro Arg Thr Leu Gly Pro Glu Ala Lys Ser Leu Leu
Ser Gly Leu Leu 370 375 380 Lys Lys Asp Pro Lys Gln Arg Leu Gly Gly
Gly Ser Glu Asp Ala Lys 385 390 395 400 Glu Ile Met Gln His Arg Phe
Phe Ala Gly Ile Val Trp Gln His Val 405 410 415 Tyr Glu Lys Lys Leu
Ser Pro Pro Phe Lys Pro Gln Val Thr Ser Glu 420 425 430 Thr Asp Thr
Arg Tyr Phe Asp Glu Glu Phe Thr Ala Gln Met Ile Thr 435 440 445 Ile
Thr Pro Pro Asp Gln Asp Asp Ser Met Glu Cys Val Asp Ser Glu 450 455
460 Arg Arg Pro His Phe Pro Gln Phe Ser Tyr Ser Ala Ser Ser Thr Ala
465 470 475 480 2 249 PRT Homo sapiens 2 Met Lys Leu Asn Ile Ser
Phe Pro Ala Thr Gly Cys Gln Lys Leu Ile 1 5 10 15 Glu Val Asp Asp
Glu Arg Lys Leu Arg Thr Phe Tyr Glu Lys Arg Met 20 25 30 Ala Thr
Glu Val Ala Ala Asp Ala Leu Gly Glu Glu Trp Lys Gly Tyr 35 40 45
Val Val Arg Ile Ser Gly Gly Asn Asp Lys Gln Gly Phe Pro Met Lys 50
55 60 Gln Gly Val Leu Thr His Gly Arg Val Arg Leu Leu Leu Ser Lys
Gly 65 70 75 80 His Ser Cys Tyr Arg Pro Arg Arg Thr Gly Glu Arg Lys
Arg Lys Ser 85 90 95 Val Arg Gly Cys Ile Val Asp Ala Asn Leu Ser
Val Leu Asn Leu Val 100 105 110 Ile Val Lys Lys Gly Glu Lys Asp Ile
Pro Gly Leu Thr Asp Thr Thr 115 120 125 Val Pro Arg Arg Leu Gly Pro
Lys Arg Ala Ser Arg Ile Arg Lys Leu 130 135 140 Phe Asn Leu Ser Lys
Glu Asp Asp Val Arg Gln Tyr Val Val Arg Lys 145 150 155 160 Pro Leu
Asn Lys Glu Gly Lys Lys Pro Arg Thr Lys Ala Pro Lys Ile 165 170 175
Gln Arg Leu Val Thr Pro Arg Val Leu Gln His Lys Arg Arg Arg Ile 180
185 190 Ala Leu Lys Lys Gln Arg Thr Lys Lys Asn Lys Glu Glu Ala Ala
Glu 195 200 205 Tyr Ala Lys Leu Leu Ala Lys Arg Met Lys Glu Ala Lys
Glu Lys Arg 210 215 220 Gln Glu Gln Ile Ala Lys Arg Arg Arg Leu Ser
Ser Leu Arg Ala Ser 225 230 235 240 Thr Ser Lys Ser Glu Ser Ser Gln
Lys 245 3 379 PRT Homo sapiens 3 Met Ala Ala Ala Ala Ala Gln Gly
Gly Gly Gly Gly Glu Pro Arg Arg 1 5 10 15 Thr Glu Gly Val Gly Pro
Gly Val Pro Gly Glu Val Glu Met Val Lys 20 25 30 Gly Gln Pro Phe
Asp Val Gly Pro Arg Tyr Thr Gln Leu Gln Tyr Ile 35 40 45 Gly Glu
Gly Ala Tyr Gly Met Val Ser Ser Ala Tyr Asp His Val Arg 50 55 60
Lys Thr Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr 65
70 75 80 Tyr Cys Gln Arg Thr Leu Arg Glu Ile Gln Ile Leu Leu Arg
Phe Arg 85 90 95 His Glu Asn Val Ile Gly Ile Arg Asp Ile Leu Arg
Ala Ser Thr Leu 100 105 110 Glu Ala Met Arg Asp Val Tyr Ile Val Gln
Asp Leu Met Glu Thr Asp 115 120 125 Leu Tyr Lys Leu Leu Lys Ser Gln
Gln Leu Ser Asn Asp His Ile Cys 130 135 140 Tyr Phe Leu Tyr Gln Ile
Leu Arg Gly Leu Lys Tyr Ile His Ser Ala 145 150 155 160 Asn Val Leu
His Arg Asp Leu Lys Pro Ser Asn Leu Leu Ile Asn Thr 165 170 175 Thr
Cys Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Ile Ala Asp 180 185
190 Pro Glu His Asp His Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg
195 200 205 Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Ser Lys Gly Tyr
Thr Lys 210 215 220 Ser Ile Asp Ile Trp Ser Val Gly Cys Ile Leu Ala
Glu Met Leu Ser 225 230 235 240 Asn Arg Pro Ile Phe Pro Gly Lys His
Tyr Leu Asp Gln Leu Asn His 245 250 255 Ile Leu Gly Ile Leu Gly Ser
Pro Ser Gln Glu Asp Leu Asn Cys Ile 260 265 270 Ile Asn Met Lys Ala
Arg Asn Tyr Leu Gln Ser Leu Pro Ser Lys Thr 275 280 285 Lys Val Ala
Trp Ala Lys Leu Phe Pro Lys Ser Asp Ser Lys Ala Leu 290 295 300 Asp
Leu Leu Asp Arg Met Leu Thr Phe Asn Pro Asn Lys Arg Ile Thr 305 310
315 320 Val Glu Glu Ala Leu Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp
Pro 325 330 335 Thr Asp Glu Pro Val Ala Glu Glu Pro Phe Thr Phe Ala
Met Glu Leu 340 345 350 Asp Asp Leu Pro Lys Glu Arg Leu Lys Glu Leu
Ile Phe Gln Glu Thr 355 360 365 Ala Arg Phe Gln Pro Gly Val Leu Glu
Ala Pro 370 375
* * * * *