U.S. patent application number 13/675255 was filed with the patent office on 2013-05-30 for methods of treating cancer.
The applicant listed for this patent is Thomas Harding, Kevin Hestir, Li Long, Servando Palencia. Invention is credited to Thomas Harding, Kevin Hestir, Li Long, Servando Palencia.
Application Number | 20130136740 13/675255 |
Document ID | / |
Family ID | 48430074 |
Filed Date | 2013-05-30 |
United States Patent
Application |
20130136740 |
Kind Code |
A1 |
Harding; Thomas ; et
al. |
May 30, 2013 |
METHODS OF TREATING CANCER
Abstract
Methods of treating cancers comprising FGFR1 gene amplification
are provided. In some embodiments, the methods comprise
administering a fibroblast growth factor receptor 1 (FGFR1)
extracellular domain (ECD) and/or an FGFR1 ECD fusion molecule. In
some embodiments, the methods comprise administering a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) and/or
an FGFR1 ECD fusion molecule in combination with at least one
additional therapeutic agent.
Inventors: |
Harding; Thomas; (San
Francisco, CA) ; Palencia; Servando; (San Francisco,
CA) ; Long; Li; (Lafayette, CA) ; Hestir;
Kevin; (Kensington, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Harding; Thomas
Palencia; Servando
Long; Li
Hestir; Kevin |
San Francisco
San Francisco
Lafayette
Kensington |
CA
CA
CA
CA |
US
US
US
US |
|
|
Family ID: |
48430074 |
Appl. No.: |
13/675255 |
Filed: |
November 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61559259 |
Nov 14, 2011 |
|
|
|
61616761 |
Mar 28, 2012 |
|
|
|
Current U.S.
Class: |
424/134.1 ;
514/9.1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 25/00 20180101; C07K 14/71 20130101; C07K 2319/30 20130101;
A61K 45/00 20130101; A61P 35/00 20180101; C12Q 1/6813 20130101;
A61K 2300/00 20130101; A61K 45/06 20130101; A61K 39/395 20130101;
C12Q 1/686 20130101; A61K 38/179 20130101; A61K 38/179 20130101;
G01N 33/5005 20130101 |
Class at
Publication: |
424/134.1 ;
514/9.1 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 45/00 20060101 A61K045/00; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating cancer having an FGFR1 gene amplification
in a subject, wherein an FGFR1 gene amplification is indicative of
therapeutic responsiveness by the cancer to a fibroblast growth
factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1
ECD fusion molecule, comprising: administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject.
2. A method of treating cancer in a subject, comprising:
administering a therapeutically effective amount of a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) or an
FGFR1 ECD fusion molecule to the subject, wherein, prior to
administration of the FGFR1 ECD or FGFR1 ECD fusion molecule, at
least a portion of the cells of the cancer have been determined to
have an FGFR1 gene amplification, and wherein an FGFR1 gene
amplification in a cancer is indicative of therapeutic
responsiveness by the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule.
3. The method of claim 1 or claim 2, wherein at least a portion of
the cells of the cancer having an FGFR1 gene amplification comprise
at least three copies of the FGFR1 gene.
4. The method of claim 3, wherein at least a portion of the cells
of the cancer having an FGFR1 gene amplification comprise at least
four copies of the FGFR1 gene.
5. The method of claim 3, wherein at least a portion of the cells
of the cancer having an FGFR1 gene amplification comprise at least
five copies of the FGFR1 gene.
6. The method of claim 3, wherein at least a portion of the cells
of the cancer having an FGFR1 gene amplification comprise at least
six copies of the FGFR1 gene.
7. The method of claim 3, wherein at least a portion of the cells
of the cancer having an FGFR1 gene amplification comprise at least
eight copies of the FGFR1 gene.
8. The method of claim 1 or claim 2, wherein at least a portion of
the cells of the cancer having an FGFR1 gene amplification have a
ratio of FGFR1 gene to chromosome 8 centromere of at least 1.5.
9. The method of claim 8, wherein the ratio of FGFR1 gene to
chromosome 8 centromere is at least 2.
10. The method of claim 8, wherein the ratio of FGFR1 gene to
chromosome 8 centromere is at least 2.5.
11. The method of claim 8, wherein the ratio of FGFR1 gene to
chromosome 8 centromere is at least 3.
12. The method of claim 8, wherein the ratio of FGFR1 gene to
chromosome 8 centromere is at least 3.5.
13. The method of claim 8, wherein the ratio of FGFR1 gene to
chromosome 8 centromere is at least 4.
14. The method of claim 2, wherein FGFR1 gene amplification was
determined by a method selected from fluorescence in situ
hybridization, array comparative genomic hybridization, DNA
microarray, spectral karyotyping, quantitative PCR, southern
blotting, or sequencing.
15. The method of any one of the preceding claims, wherein the
cancer overexpresses at least one, at least two, at least three, at
least four, or at least five markers selected from FGFR1,
FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4.
16. The method of any one of the preceding claims, wherein the
cancer overexpresses at least one, at least two, at least three, or
at least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
and FGF18.
17. The method of any one of claims 1 to 15, wherein the cancer
overexpresses ETV4.
18. The method of claim 15, wherein the cancer overexpresses at
least two, at least three, at least four, or at least five markers
selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4.
19. The method of any one of claims 15, 16, and 18, wherein FGFR1
is FGFR1IIIc.
20. A method of treating cancer that overexpresses at least one, at
least two, at least three, at least four, or at least five markers
selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 in a
subject, wherein overexpression of at least one marker selected
from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 is indicative of
therapeutic responsiveness by the cancer to a fibroblast growth
factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1
ECD fusion molecule, comprising: administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject.
21. A method of treating cancer in a subject, comprising:
administering a therapeutically effective amount of a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) or an
FGFR1 ECD fusion molecule to the subject, wherein, prior to
administration of the FGFR1 ECD or FGFR1 ECD fusion molecule, at
least a portion of the cells of the cancer have been determined to
overexpress at least one, at least two, at least three, at least
four, or at least five marker selected from FGFR1, FGFR3IIIc, FGF2,
DKK3, FGF18, and ETV4, and wherein overexpression of at least one
marker selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4
in a cancer is indicative of therapeutic responsiveness by the
cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule.
22. The method of claim 20 or claim 21, wherein the cancer
overexpresses at least two, at least three, at least four, or at
least five markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4.
23. The method of any one of claims 20 to 22, wherein the cancer
overexpresses ETV4.
24. The method of claim 20 or claim 21, wherein the cancer
overexpresses at least one, at least two, at least three, or at
least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3, and
FGF18.
25. A method of treating cancer that overexpresses FGF2, wherein
FGF2 overexpression is indicative of therapeutic responsiveness by
the cancer to a fibroblast growth factor receptor 1 (FGFR1)
extracellular domain (ECD) or an FGFR1 ECD fusion molecule,
comprising: administering a therapeutically effective amount of an
FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject.
26. A method of treating cancer in a subject, comprising:
administering a therapeutically effective amount of a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) or an
FGFR1 ECD fusion molecule to the subject, wherein, prior to
administration of the FGFR1 ECD or FGFR1 ECD fusion molecule, at
least a portion of the cells of the cancer have been determined to
overexpress FGF2, and wherein FGF2 overexpression is indicative of
therapeutic responsiveness by the cancer to an FGFR1 ECD or FGFR1
ECD fusion molecule.
27. The method of claim 25 or claim 26, wherein the cancer does not
have an FGFR1 gene amplification.
28. The method of any one of claims 20 to 27, wherein the
overexpression is protein overexpression.
29. The method of claim 28, wherein protein overexpression is
determined using immunohistochemistry.
30. The method of any one of claims 20 to 27, wherein the
overexpression is mRNA overexpression.
31. The method of claim 30, wherein mRNA overexpression is
determined using quantitative RT-PCR.
32. The method of any one of claims 20 to 31, wherein the cancer
has an FGFR1 gene amplification.
33. The method of claim 32, wherein at least a portion of the cells
of the cancer having an FGFR1 gene amplification comprise at least
three, at least four, at least five, at least six, at least seven,
or at least eight copies of the FGFR1 gene.
34. The method of any one of the preceding claims, wherein the
method further comprises administering at least one additional
therapeutic agent.
35. The method of claim 34, wherein at least one additional
therapeutic agent is selected from docetaxel, paclitaxel,
vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin,
5-fluorouracil (5-FU), leucovorin, pemetrexed, etoposide,
topotecan, sorafenib, a VEGF antagonist, a VEGF trap, an anti-VEGF
antibody, and bevacizumab.
36. The method of any one of the preceding claims, wherein the
method comprises administering an FGFR1 ECD.
37. The method of claim 36, wherein the FGFR1 ECD comprises an
amino acid sequence selected from SEQ ID NOs: 1 to 4.
38. The method of any one of claims 1 to 35, wherein the method
comprises administering an FGFR1 ECD fusion molecule.
39. The method of claim 38, wherein the FGFR1 ECD fusion molecule
comprises an FGFR1 ECD and a fusion partner, and wherein the fusion
partner is Fc.
40. The method of claim 39, wherein the FGFR1 ECD fusion molecule
comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
6.
41. The method of any one of the preceding claims, wherein the
cancer is selected from lung cancer, renal cancer, colon cancer,
liver cancer, breast cancer, ovarian cancer, endometrial cancer,
esophageal cancer, head and neck cancer, glioblastoma, and prostate
cancer.
42. The method of claim 41, wherein the cancer is lung cancer,
breast cancer, head and neck cancer, renal cancer, or esophageal
cancer.
43. The method of claim 42, wherein the cancer is lung cancer.
44. The method of claim 43, wherein the lung cancer is non-small
cell lung cancer.
45. The method of claim 44, wherein the lung cancer is small cell
lung cancer.
46. A method of identifying a subject with cancer who may benefit
from administration of an FGFR1 ECD or FGFR1 ECD fusion molecule,
comprising determining the number of copies of an FGFR1 gene in at
least a portion of the cancer cells in a sample obtained from the
subject, wherein greater than 2 copies of the FGFR1 gene in a cell
indicates that the cell has FGFR1 gene amplification, and wherein
FGFR1 gene amplification is indicative of therapeutic
responsiveness by the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule.
47. A method of identifying a subject with cancer who may benefit
from administration of an FGFR1 ECD or FGFR1 ECD fusion molecule,
comprising determining the ratio of FGFR1 gene to chromosome 8
centromere in at least a portion of the cancer cells in a sample
obtained from the subject, a ratio of greater than 1 in a cell
indicates that the cell has FGFR1 gene amplification, and wherein
FGFR1 gene amplification is indicative of therapeutic
responsiveness by the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule.
48. The method of claim 46 or claim 47, wherein the number of
copies of an FGFR1 gene or the ratio of FGFR1 gene to chromosome 8
centromere is determined by a method selected from fluorescence in
situ hybridization, array comparative genomic hybridization, DNA
microarray, spectral karyotyping, quantitative PCR, southern
blotting, or sequencing.
49. The method of any one of claims 46 to 48, further comprising
determining the level of at least one, at least two, at least
three, at least four, or at least five proteins or mRNAs selected
from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 in at least a
portion of the cancer cells in a sample obtained from the subject,
wherein overexpression of at least one protein or mRNA selected
from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 is indicative of
therapeutic responsiveness by the cancer to an FGFR1 ECD or FGFR1
ECD fusion molecule.
50. The method of claim 49, wherein the method further comprises
determining the level of at least one, at least two, at least
three, or at least four proteins or mRNAs selected from FGFR1,
FGFR3IIIc, FGF2, DKK3, and FGF18.
51. The method of claim 49, wherein the method further comprises
determining the level of ETV4.
52. A method of identifying a subject with cancer who may benefit
from administration of an FGFR1 ECD or FGFR1 ECD fusion molecule,
comprising determining the level of at least one, at least two, at
least three, at least four, or at least five proteins or mRNAs
selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 in at
least a portion of the cancer cells in a sample obtained from the
subject, wherein overexpression of at least one protein or mRNA
selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 is
indicative of therapeutic responsiveness by the cancer to an FGFR1
ECD or FGFR1 ECD fusion molecule.
53. The method of claim 52, wherein the method comprises
determining the level of at least one, at least two, at least
three, or at least four proteins or mRNAs selected from FGFR1,
FGFR3IIIc, FGF2, DKK3, and FGF18.
54. The method of claim 52, wherein the method comprises
determining the level of ETV4.
55. The method of claim 52 or claim 53, wherein FGFR1 is
FGFR1IIIc.
56. A method of identifying a subject with cancer who may benefit
from administration of an FGFR1 ECD or FGFR1 ECD fusion molecule,
comprising determining the level of FGF2 in at least a portion of
the cancer cells in a sample obtained from the subject, wherein
overexpression of FGF2 is indicative of therapeutic responsiveness
by the cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule.
57. The method of claim 56, further comprising determining that the
cancer does not have an FGFR1 gene amplification.
58. The method of any one of claims 49 to 57, wherein the level of
one or more proteins is determined.
59. The method of claim 58, wherein the level of protein is
determined using immunohistochemistry.
60. The method of any one of claims 49 to 57, wherein the level of
one or more mRNAs is determined.
61. The method of claim 60, wherein the level of mRNA is determined
using quantitative RT-PCR.
62. The method of any one of claims 49 to 56, further comprising
determining the number of copies of an FGFR1 gene in at least a
portion of the cancer cells in a sample obtained from the subject,
wherein greater than 2 copies of the FGFR1 gene in a cell indicates
that the cell has FGFR1 gene amplification, and wherein FGFR1 gene
amplification is indicative of therapeutic responsiveness by the
cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule.
63. The method of any one of claims 49 to 56, further comprising
determining the ratio of FGFR1 gene to chromosome 8 centromere in
at least a portion of the cancer cells in a sample obtained from
the subject, a ratio of greater than 1 in a cell indicates that the
cell has FGFR1 gene amplification, and wherein FGFR1 gene
amplification is indicative of therapeutic responsiveness by the
cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule.
64. The method of any one of claims 46 to 63, wherein the FGFR1 ECD
comprises an amino acid sequence selected from SEQ ID NOs: 1 to
4.
65. The method of any one of claims 46 to 63, wherein the FGFR1 ECD
fusion molecule comprises an FGFR1 ECD and a fusion partner, and
wherein the fusion partner is Fc.
66. The method of claim 65, wherein the FGFR1 ECD fusion molecule
comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
6.
67. The method of any one of claims 46 to 66, wherein the cancer is
selected from lung cancer, renal cancer, colon cancer, liver
cancer, breast cancer, ovarian cancer, endometrial cancer,
esophageal cancer, head and neck cancer, glioblastoma, and prostate
cancer.
68. The method of claim 67, wherein the cancer is lung cancer,
breast cancer, renal cancer, head and neck cancer, or esophageal
cancer.
69. The method of claim 68, wherein the cancer is lung cancer.
70. The method of claim 69, wherein the lung cancer is non-small
cell lung cancer.
71. The method of claim 69, wherein the lung cancer is small cell
lung cancer.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/559,259, filed Nov. 14, 2011; and U.S.
Provisional Application No. 61/616,761, filed Mar. 28, 2012, which
are incorporated herein by reference in their entireties for any
purpose.
BACKGROUND
[0002] Soluble forms of Fibroblast Growth Factor Receptor 1 (FGFR1)
have been shown to inhibit tumor cell growth in vitro and in vivo.
See, e.g., U.S. Pat. No. 7,678,890. The efficacy of anti-cancer
therapies is, in some instances, dependent on the genetic makeup of
the cancer being targeted.
SUMMARY OF THE INVENTION
[0003] The inventors have demonstrated that certain cancers that
comprise FGFR1 gene amplification are, in some embodiments, more
responsive to therapies involving administration of a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) or
FGFR1 ECD fusion molecule, than cancers that do not comprise an
FGFR1 gene amplification. In some embodiments, cancers that have
FGFR1 overexpression are more responsive to therapies involving
administration of FGFR1 ECD or FGFR1 ECD fusion molecules, than
cancers that do not have FGFR1 overexpression. In some embodiments,
FGFR1 is FGFR1IIIc. In some embodiments, cancers that have
fibroblast growth factor receptor 3 isoform IIIc (FGFR3IIIc)
overexpression are more responsive to therapies involving
administration of FGFR1 ECD or FGFR1 ECD fusion molecules, than
cancers that do not have FGFR31IIc overexpression. In some
embodiments, cancers that have fibroblast growth factor 2 (FGF2)
overexpression are more responsive to therapies involving
administration of FGFR1 ECD or FGFR1 ECD fusion molecules, than
cancers that do not have FGF2 overexpression. In some embodiments,
cancers that have dickkopf-related protein 3 (DKK3) overexpression
are more responsive to therapies involving administration of FGFR1
ECD or FGFR1 ECD fusion molecules, than cancers that do not have
DKK3 overexpression. In some embodiments, cancers that have ETS
translocation variant 4 (ETV4) overexpression are more responsive
to therapies involving administration of FGFR1 ECD or FGFR1 ECD
fusion molecules, than cancers that do not have ETV4
overexpression. In some embodiments, cancers that have FGF18
overexpression are more responsive to therapies involving
administration of FGFR1 ECD or FGFR1 ECD fusion molecules, than
cancers that do not have FGF18 overexpression.
[0004] In some embodiments, methods of treating a cancer having an
FGFR1 gene amplification, wherein an FGFR1 gene amplification is
indicative of therapeutic responsiveness by the cancer to a
fibroblast growth factor receptor 1 (FGFR1) extracellular domain
(ECD) or an FGFR1 ECD fusion molecule, comprise administering a
therapeutically effective amount of an FGFR1 ECD or an FGFR1 ECD
fusion molecule to the subject. In some embodiments, methods of
treating cancer in a subject comprise administering a
therapeutically effective amount of a fibroblast growth factor
receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1 ECD
fusion molecule to the subject, wherein, prior to administration of
the FGFR1 ECD or FGFR1 ECD fusion molecule, at least a portion of
the cells of the cancer have been determined to have an FGFR1 gene
amplification, and wherein an FGFR1 gene amplification in a cancer
is indicative of therapeutic responsiveness of the cancer to an
FGFR1 ECD or FGFR1 ECD fusion molecule.
[0005] In some embodiments, methods of treating a lung cancer
having an FGFR1 gene amplification, wherein an FGFR1 gene
amplification is indicative of therapeutic responsiveness by the
lung cancer to a fibroblast growth factor receptor 1 (FGFR1)
extracellular domain (ECD) or an FGFR1 ECD fusion molecule,
comprise administering a therapeutically effective amount of an
FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject. In some
embodiments, methods of treating lung cancer in a subject comprise
administering a therapeutically effective amount of a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) or an
FGFR1 ECD fusion molecule to the subject, wherein, prior to
administration of the FGFR1 ECD or FGFR1 ECD fusion molecule, at
least a portion of the cells of the lung cancer have been
determined to have an FGFR1 gene amplification, and wherein an
FGFR1 gene amplification in a cancer is indicative of therapeutic
responsiveness of the lung cancer to an FGFR1 ECD or FGFR1 ECD
fusion molecule. In some embodiments, the lung cancer is small cell
lung cancer. In some embodiments, the lung cancer is non-small cell
lung cancer.
[0006] In some embodiments, at least a portion of the cells of the
cancer comprise at least three, at least four, at least five, at
least six, at least eight, or at least ten copies of the FGFR1
gene. In some embodiments, at least a portion of the cells of the
cancer have a ratio of FGFR1 gene to chromosome 8 centromere of at
least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, or
at least 4.
[0007] In some embodiments, including any of the foregoing
embodiments, the cancer may overexpress at least one, at least two,
at least three, at least four, or at least five markers selected
from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4. In some
embodiments, the cancer may overexpress at least one, at least two,
at least three, at least four, or five markers selected from FGFR1,
FGFR3IIIc, FGF2, DKK3, and FGF18. In some embodiments, the cancer
may overexpress ETV4. In some embodiments, including any of the
foregoing embodiments, the cancer may overexpress Gene 1 and Gene 2
from any line in Table 10 below, or any comination thereof. In some
embodiments, FGFR1 is FGFR1IIIc. In some embodiments, including any
of the foregoing embodiments, the FGFR1 gene may be amplified.
[0008] In some embodiments, methods of treating a cancer that
overexpress at least one, at least two, at least three, or at least
four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and
ETV4 are provided. In some embodiments, overexpression of at least
one, at least two, at least three, or at least four markers
selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 is
indicative of therapeutic responsiveness by the cancer to a
fibroblast growth factor receptor 1 (FGFR1) extracellular domain
(ECD) or an FGFR1 ECD fusion molecule. In some embodiments, a
method comprises administering a therapeutically effective amount
of an FGFR1 ECD or an FGFR1 ECD fusion molecule to a subject with
cancer that overexpress at least one, at least two, at least three,
or at least four markers selected from FGFR1, FGFR3IIIc, FGF2,
DKK3, FGF18, and ETV4. In some embodiments, methods of treating
cancer in a subject comprise administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject, wherein, prior to administration of the FGFR1 ECD or
FGFR1 ECD fusion molecule, at least a portion of the cells of the
cancer have been determined to overexpress at least one, at least
two, at least three, or at least four markers selected from FGFR1,
FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4, and wherein overexpression
of at least one, at least two, at least three, or at least four
markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4
in a cancer is indicative of therapeutic responsiveness of the
cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule. In some
embodiments, the cancer also has an FGFR1 gene amplification. In
some embodiments, at least a portion of the cells of the cancer
having an FGFR1 gene amplification comprise at least three, at
least four, at least five, at least six, at least seven, or at
least eight copies of the FGFR1 gene. In some embodiments, the
overexpression is mRNA overexpression. In some embodiments, mRNA
overexpression is determined by quantitative RT-PCR. In some
embodiments, the overexpression is protein overexpression. In some
embodiments, protein overexpression is determined by
immunohistochemistry. In some embodiments, FGFR1 is FGFR1IIIc.
[0009] In some embodiments, methods of treating a cancer having
FGFR1 overexpression, wherein FGFR1 overexpression is indicative of
therapeutic responsiveness by the cancer to a fibroblast growth
factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1
ECD fusion molecule, comprise administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject. In some embodiments, methods of treating cancer in a
subject comprise administering a therapeutically effective amount
of an FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject,
wherein, prior to administration of the FGFR1 ECD or FGFR1 ECD
fusion molecule, at least a portion of the cells of the cancer have
been determined to have FGFR1 overexpression, and wherein FGFR1
overexpression in a cancer is indicative of therapeutic
responsiveness of the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule. In some embodiments, the cancer does not have an FGFR1
gene amplification. In some embodiments, the FGFR1 overexpression
is mRNA overexpression. In some embodiments, FGFR1 mRNA
overexpression is determined by quantitative RT-PCR. In some
embodiments, the FGFR1 overexpression is protein overexpression. In
some embodiments, FGFR1 protein overexpression is determined by
immunohistochemistry. In some embodiments, FGFR1 is FGFR1IIIc.
[0010] In some embodiments, methods of treating a cancer having
FGFR3IIIc overexpression, wherein FGFR3IIIc overexpression is
indicative of therapeutic responsiveness by the cancer to a
fibroblast growth factor receptor 1 (FGFR1) extracellular domain
(ECD) or an FGFR1 ECD fusion molecule, comprise administering a
therapeutically effective amount of an FGFR1 ECD or an FGFR1 ECD
fusion molecule to the subject. In some embodiments, methods of
treating cancer in a subject comprise administering a
therapeutically effective amount of an FGFR1 ECD or an FGFR1 ECD
fusion molecule to the subject, wherein, prior to administration of
the FGFR1 ECD or FGFR1 ECD fusion molecule, at least a portion of
the cells of the cancer have been determined to have FGFR3IIIc
overexpression, and wherein FGFR3IIIc overexpression in a cancer is
indicative of therapeutic responsiveness of the cancer to an FGFR1
ECD or FGFR1 ECD fusion molecule. In some embodiments, the cancer
does not have an FGFR1 gene amplification. In some embodiments, the
FGFR3IIIc overexpression is mRNA overexpression. In some
embodiments, FGFR3IIIc mRNA overexpression is determined by
quantitative RT-PCR. In some embodiments, the FGFR3IIIc
overexpression is protein overexpression. In some embodiments,
FGFR3IIIc protein overexpression is determined by
immunohistochemistry. In some embodiments, the cancer having
FGFR3IIIc overexpression is selected from bladder cancer, renal
cell carcinoma, head-and-neck squamous carcinoma, and colorectal
cancer.
[0011] In some embodiments, methods of treating a cancer having
FGF2 overexpression, wherein FGF2 overexpression is indicative of
therapeutic responsiveness by the cancer to a fibroblast growth
factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1
ECD fusion molecule, comprise administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject. In some embodiments, methods of treating cancer in a
subject comprise administering a therapeutically effective amount
of an FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject,
wherein, prior to administration of the FGFR1 ECD or FGFR1 ECD
fusion molecule, at least a portion of the cells of the cancer have
been determined to have FGF2 overexpression, and wherein FGF2
overexpression in a cancer is indicative of therapeutic
responsiveness of the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule. In some embodiments, the cancer does not have an FGFR1
gene amplification. In some embodiments, the FGF2 overexpression is
mRNA overexpression. In some embodiments, FGF2 mRNA overexpression
is determined by quantitative RT-PCR. In some embodiments, the FGF2
overexpression is protein overexpression. In some embodiments, FGF2
protein overexpression is determined by immunohistochemistry. In
some embodiments, the cancer having FGF2 overexpression is selected
from glioblastoma, renal cell carcinoma, and hepatocellular
carcinoma.
[0012] In some embodiments, methods of treating a cancer having
DKK3 overexpression, wherein DKK3 overexpression is indicative of
therapeutic responsiveness by the cancer to a fibroblast growth
factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1
ECD fusion molecule, comprise administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject. In some embodiments, methods of treating cancer in a
subject comprise administering a therapeutically effective amount
of an FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject,
wherein, prior to administration of the FGFR1 ECD or FGFR1 ECD
fusion molecule, at least a portion of the cells of the cancer have
been determined to have DKK3 overexpression, and wherein DKK3
overexpression in a cancer is indicative of therapeutic
responsiveness of the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule. In some embodiments, the DKK3 overexpression is mRNA
overexpression. In some embodiments, DKK3 mRNA overexpression is
determined by quantitative RT-PCR. In some embodiments, the DKK3
overexpression is protein overexpression. In some embodiments, DKK3
protein overexpression is determined by immunohistochemistry. In
some embodiments, the cancer having DKK3 overexpression is selected
from pancreatic cancer, prostate cancer, renal cell carcinoma, lung
adenocarcinoma, hepatocellular cancer, and colorectal cancer.
[0013] In some embodiments, methods of treating a cancer having
FGF18 overexpression, wherein FGF18 overexpression is indicative of
therapeutic responsiveness by the cancer to a fibroblast growth
factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1
ECD fusion molecule, comprise administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject. In some embodiments, methods of treating cancer in a
subject comprise administering a therapeutically effective amount
of an FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject,
wherein, prior to administration of the FGFR1 ECD or FGFR1 ECD
fusion molecule, at least a portion of the cells of the cancer have
been determined to have FGF18 overexpression, and wherein FGF18
overexpression in a cancer is indicative of therapeutic
responsiveness of the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule. In some embodiments, the FGF18 overexpression is mRNA
overexpression. In some embodiments, FGF18 mRNA overexpression is
determined by quantitative RT-PCR. In some embodiments, the FGF18
overexpression is protein overexpression. In some embodiments,
FGF18 protein overexpression is determined by
immunohistochemistry.
[0014] In some embodiments, methods of treating a cancer having
ETV4 overexpression, wherein ETV4 overexpression is indicative of
therapeutic responsiveness by the cancer to a fibroblast growth
factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1
ECD fusion molecule, comprise administering a therapeutically
effective amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to
the subject. In some embodiments, methods of treating cancer in a
subject comprise administering a therapeutically effective amount
of an FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject,
wherein, prior to administration of the FGFR1 ECD or FGFR1 ECD
fusion molecule, at least a portion of the cells of the cancer have
been determined to have ETV4 overexpression, and wherein ETV4
overexpression in a cancer is indicative of therapeutic
responsiveness of the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule. In some embodiments, the ETV4 overexpression is mRNA
overexpression. In some embodiments, ETV4 mRNA overexpression is
determined by quantitative RT-PCR. In some embodiments, the ETV4
overexpression is protein overexpression. In some embodiments, ETV4
protein overexpression is determined by immunohistochemistry.
[0015] In some embodiments, methods of treating a lung cancer
having FGFR1 overexpression, wherein FGFR1 overexpression is
indicative of therapeutic responsiveness by the lung cancer to a
fibroblast growth factor receptor 1 (FGFR1) extracellular domain
(ECD) or an FGFR1 ECD fusion molecule, comprise administering a
therapeutically effective amount of an FGFR1 ECD or an FGFR1 ECD
fusion molecule to the subject. In some embodiments, methods of
treating lung cancer in a subject comprise administering a
therapeutically effective amount of an FGFR1 ECD or an FGFR1 ECD
fusion molecule to the subject, wherein, prior to administration of
the FGFR1 ECD or FGFR1 ECD fusion molecule, at least a portion of
the cells of the lung cancer have been determined to have FGFR1
overexpression, and wherein FGFR1 overexpression in a cancer is
indicative of therapeutic responsiveness of the lung cancer to an
FGFR1 ECD or FGFR1 ECD fusion molecule. In some embodiments, the
cancer does not have an FGFR1 gene amplification. In some
embodiments, the lung cancer is small cell lung cancer. In some
embodiments, the lung cancer is non-small cell lung cancer. In some
embodiments, FGFR1 is FGFR1IIIc.
[0016] In some embodiments, methods of treating a lung cancer
having FGF2 overexpression, wherein FGF2 overexpression is
indicative of therapeutic responsiveness by the lung cancer to a
fibroblast growth factor receptor 1 (FGFR1) extracellular domain
(ECD) or an FGFR1 ECD fusion molecule, comprise administering a
therapeutically effective amount of an FGFR1 ECD or an FGFR1 ECD
fusion molecule to the subject. In some embodiments, methods of
treating lung cancer in a subject comprise administering a
therapeutically effective amount of an FGFR1 ECD or an FGFR1 ECD
fusion molecule to the subject, wherein, prior to administration of
the FGFR1 ECD or FGFR1 ECD fusion molecule, at least a portion of
the cells of the lung cancer have been determined to have FGF2
overexpression, and wherein FGF2 overexpression in a cancer is
indicative of therapeutic responsiveness of the lung cancer to an
FGFR1 ECD or FGFR1 ECD fusion molecule. In some embodiments, the
cancer does not have an FGFR1 gene amplification. In some
embodiments, the lung cancer is small cell lung cancer. In some
embodiments, the lung cancer is non-small cell lung cancer. In some
embodiments, the lung cancer does not have an FGFR1 gene
amplification.
[0017] In some embodiments, a method of treating a cancer having an
FGFR1 gene amplification comprises administering an FGFR1 ECD or
FGFR1 ECD fusion molecule and at least one additional therapeutic
agent. In some embodiments, a method of treating a cancer that
overexpresses at least one, at least two, at least three, or at
least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4 comprises administering an FGFR1 ECD or FGFR1 ECD
fusion molecule and at least one additional therapeutic agent. In
some embodiments, at least one additional therapeutic agent is
selected from docetaxel, paclitaxel, vincristine, carboplatin,
cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),
leucovorin, pemetrexed, etoposide, topotecan, sorafenib, a VEGF
antagonist, a VEGF trap, an anti-VEGF antibody, and bevacizumab. In
some embodiments, the at least one additional therapeutic agent is
docetaxel. In some embodiments, the cancer is non-small cell lung
cancer. In some embodiments, the FGFR1 is FGFR1IIIc.
[0018] In some embodiments, a method of treating a cancer having an
FGFR1 gene amplification comprises administering an FGFR1 ECD or
FGFR1 ECD fusion molecule and at least two additional therapeutic
agents. In some embodiments, a method of treating a cancer that
overexpresses at least one, at least two, at least three, or at
least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4 comprises administering an FGFR1 ECD or FGFR1 ECD
fusion molecule and at least two additional therapeutic agents. In
some embodiments, at least two additional therapeutic agents are
selected from docetaxel, paclitaxel, vincristine, carboplatin,
cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),
leucovorin, pemetrexed, etoposide, topotecan, sorafenib, a VEGF
antagonist, a VEGF trap, an anti-VEGF antibody, and bevacizumab. In
some embodiments, the two additional therapeutic agents are
paclitaxel and carboplatin. In some embodiments, the two additional
therapeutic agents are doxorubicin and paclitaxel. In some
embodiments, the two additional therapeutic agents are cisplatin
and etoposide. In some embodiments, the two additional therapeutic
agents are oxaliplatin and 5-FU. In some embodiments, the two
additional therapeutic agents are 5-FU and leucovorin. In some
embodiments, the two additional therapeutic agents are 5-FU and
bevacizumab. In some embodiments, the two additional therapeutic
agents are paclitaxel and bevacizumab. In some embodiments, the
cancer is non-small cell lung cancer.
[0019] In some embodiments, a method of treating a cancer having an
FGFR1 gene amplification comprises administering an FGFR1 ECD or
FGFR1 ECD fusion molecule and at least three additional therapeutic
agents. In some embodiments, a method of treating a cancer that
overexpresses at least one, at least two, at least three, or at
least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4 comprises administering an FGFR1 ECD or FGFR1 ECD
fusion molecule and at least three additional therapeutic agents.
In some embodiments, at least three additional therapeutic agents
are selected from docetaxel, paclitaxel, vincristine, carboplatin,
cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),
leucovorin, pemetrexed, etoposide, topotecan, sorafenib, a VEGF
antagonist, a VEGF trap, an anti-VEGF antibody, and bevacizumab. In
some embodiments, the three additional therapeutic agents are
oxaliplatin, 5-FU and leucovorin. In some embodiments, the three
additional therapeutic agents are bevacizumab, 5-FU and
leucovorin.
[0020] In some embodiments, methods of treating a cancer having an
FGFR1 gene amplification and/or that overexpresses at least one, at
least two, at least three, or at least four markers selected from
FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 comprise
administering an FGFR1 ECD. In some such embodiments, the FGFR1 ECD
comprises an amino acid sequence selected from SEQ ID NOs: 1 to 4.
In some embodiments, methods of treating a cancer having an FGFR1
gene amplification and/or FGFR1 overexpression and/or FGF2
overexpression and/or DKK3 overexpression and/or FGF18
overexpression and/or ETV4 overexpression comprise administering an
FGFR1 ECD fusion molecule, wherein the FGFR1 ECD fusion molecule
comprises an FGFR1 ECD and at least one fusion partner. In some
embodiments, at least one fusion partner is selected from an Fc,
albumin, and polyethylene glycol. In some embodiments, at least one
fusion partner is an Fc. In some embodiments, the Fc comprises an
amino acid sequence selected from SEQ ID NOs: 8 to 10. In some
embodiments, the FGFR1 ECD fusion molecule comprises a sequence
selected from SEQ ID NO: 5 and SEQ ID NO: 6. In some embodiments,
the at least one fusion partner is an Fc and polyethylene glycol.
In some embodiments, the at least one fusion partners is
polyethylene glycol. In some embodiments, the fusion molecule
comprises a linker between the FGFR1 ECD and one or more fusion
partners. In some embodiments, the FGFR1 ECD fusion molecule is
FGFR1 ECD.339-Fc.
[0021] In some embodiments, an FGFR1 ECD or FGFR1 ECD fusion
molecule is glycosylated and/or sialylated. In some embodiments, an
FGFR1 ECD or the polypeptide portion of the FGFR1 ECD fusion
molecule is expressed in Chinese hamster ovary (CHO) cells. In some
embodiments, an FGFR1 ECD comprises an amino acid sequence selected
from SEQ ID NO: 1 and SEQ ID NO: 3.
[0022] In some embodiments, the FGFR1 ECD or FGFR1 ECD fusion
molecule is an amount in the range of about 0.5 mg/kg body weight
to about 30 mg/kg body weight, such as an amount in the range of
about 8 to about 16 mg/kg body weight. In some embodiments, the
therapeutically effective amount of the FGFR1 ECD or FGFR1 ECD
fusion molecule is a dose of about 8 mg/kg body weight. In some
embodiments, the therapeutically effective amount of the FGFR1 ECD
or FGFR1 ECD fusion molecule is a dose of about 16 mg/kg body
weight. In some embodiments, the therapeutically effective amount
of the FGFR1 ECD or FGFR1 ECD fusion molecule is a dose of about 20
mg/kg body weight. In some embodiments, dosages may be administered
twice a week, weekly, every other week, at a frequency between
weekly and every other week, every three weeks, every four weeks,
or every month.
[0023] In certain embodiments, the cancer is prostate cancer,
breast cancer, colorectal cancer, lung cancer, brain cancer,
ovarian cancer, endometrial cancer, esophageal cancer, head and
neck cancer, laryngeal cancer, liver cancer, renal cancer,
glioblastoma, or pancreatic cancer. In certain embodiments, the
cancer is breast cancer, esophageal cancer, renal cancer, head and
neck cancer, or lung cancer. In certain embodiments, the cancer is
lung cancer. In some embodiments, the lung cancer is non-small cell
lung cancer. In some embodiments, the lung cancer is small cell
lung cancer. In some embodiments, the lung cancer is squamous cell
carcinoma. In some embodiments, the cancer is head and neck cancer.
In some embodiments, the head and neck cancer is squamous cell
carcinoma of the head and neck.
[0024] In some embodiments, methods of identifying a subject with
cancer who may benefit from administration of an FGFR1 ECD or FGFR1
ECD fusion molecule are provided. In some embodiments, a method
comprises determining whether at least a portion of the cancer
cells in a sample obtained from the subject comprise an FGFR1 gene
amplification, wherein FGFR1 gene amplification is indicative of
therapeutic responsiveness by the cancer to an FGFR1 ECD or FGFR1
ECD fusion molecule. In some embodiments, FGFR1 gene amplification
is determined by a method selected from fluorescence in situ
hybridization, array comparative genomic hybridization, DNA
microarray, spectral karyotyping, quantitative PCR, southern
blotting, or sequencing.
[0025] In some embodiments, methods of identifying a subject with
cancer who may benefit from administration of an FGFR1 ECD or FGFR1
ECD fusion molecule are provided. In some embodiments, a method
comprises determining whether at least a portion of the cancer
cells in a sample obtained from the subject overexpress at least
one, at least two, at least three, at least four, or at least five
markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and
ETV4, wherein overexpression is indicative of therapeutic
responsiveness by the cancer to an FGFR1 ECD or FGFR1 ECD fusion
molecule. In some embodiments, the method comprises determining
whether at least a portion of the cancer cells in a sample obtained
from the subject overexpress at least one, at least two, at least
three, or at least four markers selected from FGFR1, FGFR3IIIc,
FGF2, DKK3, and FGF18. In some embodiments, the method comprises
determining whether at least a portion of the cancer cells in a
sample obtained from the subject overexpress ETV4. In some
embodiments, including any of the foregoing embodiments, the method
comprises determining whether at least a portion of the cancer
cells in a sample obtained from the subject overexpress Gene 1 and
Gene 2 from any line in Table 10 below, or any comination thereof.
In some embodiments, FGFR1 is FGFR1IIIc. In some embodiments, the
overexpression is mRNA overexpression.
[0026] In some embodiments, mRNA overexpression is determined by
quantitative RT-PCR. In some embodiments, the overexpression is
protein overexpression. In some embodiments, protein overexpression
is determined by immunohistochemistry. In some embodiments,
including any of the foregoing embodiments, the method comprises
determining whether at least a portion of the cancer cells in a
sample obtained from the subject have an FGFR1 gene
amplification.
[0027] In some embodiments, methods of identifying a subject with
cancer who may benefit from administration of an FGFR1 ECD or FGFR1
ECD fusion molecule are provided. In some embodiments, a method
comprises determining whether at least a portion of the cancer
cells in a sample obtained from the subject overexpress FGF2,
wherein overexpression is indicative of therapeutic responsiveness
by the cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule. In some
embodiments, the overexpression is mRNA overexpression. In some
embodiments, mRNA overexpression is determined by quantitative
RT-PCR. In some embodiments, the overexpression is protein
overexpression. In some embodiments, protein overexpression is
determined by immunohistochemistry. In some embodiments, the cancer
is determined not to have an FGFR1 gene amplification. In some
embodiments, the cancer is lung cancer. In some embodiments, the
cancer is non-small cell lung cancer or small cell lung cancer.
[0028] Any embodiment described herein or any combination thereof
applies to any and all methods of the invention described
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows cell number in a culture of (A) NCI-H1581, (B)
NCI-H520, (C) DMS53, and (D) DMS114 tumor cells grown in the
presence or absence of FGFR1-ECD.339-Fc, with varying amounts of
serum, as described in Example 1.
[0030] FIG. 2 shows thymidine incorporation by (A) NCI-H1581, (B)
NCI-H520, (C) DMS53, and (D) DMS114 tumor cells grown in the
presence or absence of FGFR1-ECD.339-Fc, with varying amounts of
serum, as described in Example 1.
[0031] FIG. 3 shows a plot of average % decrease in cell number in
various FGFR1 gene amplified lung cancer cell lines and various
FGFR1 gene non-amplified lung cancer cell lines grown in the
presence of FGFR1-ECD.339-Fc, as described in Example 1.
[0032] FIG. 4 shows a plot of average % reduction in 3H-thymidine
incorporation in various FGFR1 gene amplified lung cancer cell
lines and various FGFR1 gene non-amplified lung cancer cell lines
grown in the presence of FGFR1-ECD.339-Fc, as described in Example
1.
[0033] FIG. 5 shows mean tumor volume at various time points in
mice implanted with DMS53 cells and treated with FGFR1-ECD.339-Fc
or albumin, as described in Example 2.
[0034] FIG. 6 shows mean tumor volume at various time points in
mice implanted with DMS114 cells and treated with FGFR1-ECD.339-Fc
or albumin, as described in Example 3.
[0035] FIG. 7 shows mean tumor volume at various time points in
mice implanted with NCI-H1581 cells and treated with
FGFR1-ECD.339-Fc or albumin, as described in Example 4.
[0036] FIG. 8 shows mean tumor volume at various time points in
mice implanted with NCI-H520 cells and treated with
FGFR1-ECD.339-Fc or albumin, as described in Example 5.
[0037] FIG. 9 shows % tumor growth inhibition by FGFR1-ECD.339-Fc
in mouse xenografts of tumor cells having FGFR1 gene amplification
and tumor cells having a non-amplified FGFR1 gene, as described in
Example 6.
[0038] FIG. 10 shows a scatter plot of FGFR1 mRNA expression in
lung cancer cell lines with and without FGFR1 gene amplification,
as described in Example 7.
[0039] FIG. 11 shows graphs of (A) average luminescence in the
CellTiterGlo.RTM. assay and (B) counts per minute in the tritiated
thymidine incorporation assay carried out on NCI-H226 cells grown
with varying amounts of serum and in the presence or absence of
FGFR1-ECD.339-Fc, as described in Example 7.
[0040] FIG. 12 shows a scatter plot of FGFR1 mRNA expression in
lung cancer xenografts with and without FGFR1 gene amplification,
as described in Example 7.
[0041] FIG. 13 shows mean tumor volume at various time points in
mice implanted with PDX D35087 cells and treated with
FGFR1-ECD.339-Fc or albumin, as described in Example 7.
[0042] FIG. 14 shows (A) FGF2 mRNA (normalized to GUSB) and (B)
FGF2 protein expression (normalized to total protein) in
FGFR1-ECD.339-Fc responder and non-responder xenografts, as
described in Example 8.
[0043] FIG. 15 shows DKK3 mRNA expression (normalized to GUSB) in
FGFR1-ECD.339-Fc responder and non-responder xenografts, as
described in Example 9.
[0044] FIG. 16 shows anti-tumor activity of FGFR1-ECD.339-Fc in (A)
a Caki-1 renal cell carcinoma xenograft model, and (B) a MSTO-211H
mesothelioma xenograft model, as described in Example 8.
[0045] FIG. 17 shows (A) FGFR1 and (B) FGFR3IIIc mRNA expression in
FGFR1-ECD.339-Fc responsive and non-responsive xenograft models, as
described in Example 8.
[0046] FIG. 18 shows (A) plasma FGFR1-ECD.339-Fc levels over time
in rats administered weekly doses of FGFR1-ECD.339-Fc, and (B)
serum phosphate levels after 24 hours and 168 hours in rats
administered FGFR1-ECD.339-Fc or FGFR kinase inhibitor PD173074, as
described in Example 10.
[0047] FIG. 19 shows FGFR1-ECD.339-Fc mediated inhibition of FGF-2
and VEGF-A induced angiogenesis in a matrigel plug assay, as
described in Example 11.
[0048] FIG. 20 shows that FGFR1-ECD.339-Fc does not inhibit VEGF-A
induced human umbilical vein endothelial cell (HUVEC)
proliferation, as described in Example 11.
[0049] FIG. 21 shows inhibition of tumor angiogenesis (as assessed
by CD31 immunostaining) in Caki-1 renal cell carcinoma xenograft
model mice administered FGFR1-ECD.339-Fc, as described in Example
12.
[0050] FIG. 22 shows FGFR1-ECD.339-Fc mediated inhibition of FGFR1
signaling in a JIMT-1 breast cancer xenograft, as described in
Example 13.
DETAILED DESCRIPTION
[0051] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
Definitions
[0052] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular.
[0053] Certain techniques used in connection with recombinant DNA,
oligonucleotide synthesis, tissue culture and transformation (e.g.,
electroporation, lipofection), enzymatic reactions, and
purification techniques are known in the art. Many such techniques
and procedures are described, e.g., in Sambrook et al. Molecular
Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989)), among other
places. In addition, certain techniques for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients are also known in the art.
[0054] In this application, the use of "or" means "and/or" unless
stated otherwise. In the context of a multiple dependent claim, the
use of "or" refers back to more than one preceding independent or
dependent claim in the alternative only. Also, terms such as
"element" or "component" encompass both elements and components
comprising one unit and elements and components that comprise more
than one subunit unless specifically stated otherwise.
[0055] As used herein, all numbers are approximate, and may be
varied to account for measurement error and the rounding of
significant digits. The use of "about" before certain measured
quantities includes variations due to sample impurities,
measurement error, human error, and statistical variation, as well
as the rounding of significant digits.
[0056] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0057] The terms "nucleic acid molecule" and "polynucleotide" may
be used interchangeably, and refer to a polymer of nucleotides.
Such polymers of nucleotides may contain natural and/or non-natural
nucleotides, and include, but are not limited to, DNA, RNA, and
PNA. "Nucleic acid sequence" refers to the linear sequence of
nucleotides that comprise the nucleic acid molecule or
polynucleotide.
[0058] The terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues, and
are not limited to a minimum length. Such polymers of amino acid
residues may contain natural or non-natural amino acid residues,
and include, but are not limited to, peptides, oligopeptides,
dimers, trimers, and multimers of amino acid residues. Both
full-length proteins and fragments thereof are encompassed by the
definition. The terms also include post-expression modifications of
the polypeptide, for example, glycosylation, sialylation,
acetylation, phosphorylation, and the like. Furthermore, for
purposes of the present invention, a "polypeptide" refers to a
protein which includes modifications, such as deletions, additions,
and substitutions (generally conservative in nature), to the native
sequence, as long as the protein maintains the desired activity.
These modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to PCR
amplification. When a polypeptide "consists of" a particular amino
acid sequence, it may still contain post-translational
modifications, such as glycosylation and sialylation.
[0059] The term "FGFR1 extracellular domain" ("FGFR1 ECD") includes
full-length FGFR1 ECDs, FGFR1 ECD fragments, and FGFR1 ECD
variants. As used herein, the term "FGFR1 ECD" refers to an FGFR1
polypeptide that lacks the intracellular and transmembrane domains,
with or without a signal peptide. In some embodiment, the FGFR1 ECD
is a human full-length FGFR1 ECD having an amino acid sequence
selected from SEQ ID NOs: 1 and 2. The term "full-length FGFR1
ECD", as used herein, refers to an FGFR1 ECD that extends to the
last amino acid of the extracellular domain, and may or may not
include an N-terminal signal peptide. As defined herein, the last
amino acid of the full-length FGFR1 ECD is at position 353. Thus, a
human full-length FGFR1 ECD may consist of the amino acid sequence
corresponding to SEQ ID NO.: 2 (mature form) or to SEQ ID NO.: 1
(with the signal peptide). As used herein, the term "FGFR1 ECD
fragment" refers to an FGFR1 ECD having one or more residues
deleted from the N and/or C terminus of the full-length ECD and
that retains the ability to bind to FGF-2. The FGFR1 ECD fragment
may or may not include an N-terminal signal peptide. In some
embodiments, the FGFR1 ECD fragment is a human FGFR1 ECD fragment
having an amino acid sequence corresponding to SEQ ID NO.: 4
(mature form) or to SEQ ID NO.: 3 (with the signal peptide).
[0060] As used herein, the term "FGFR1 ECD variants" refers to
FGFR1 ECDs that contain amino acid additions, deletions, and
substitutions and that remain capable of binding to FGF-2. Such
variants may be at least 90%, 92%, 95%, 97%, 98%, or 99% identical
to the parent FGFR1 ECD. The % identity of two polypeptides can be
measured by a similarity score determined by comparing the amino
acid sequences of the two polypeptides using the Bestfit program
with the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman, Advances in
Applied Mathematics 2:482-489 (1981) to find the best segment of
similarity between two sequences. In some embodiments, an FGFR1 ECD
variant is at least 95% identical to the sequence of SEQ ID NO:
4.
[0061] A polypeptide having an amino acid sequence at least, for
example, 95% identical to a reference amino acid sequence of an
FGFR1 ECD polypeptide is one in which the amino acid sequence of
the polypeptide is identical to the reference sequence except that
the polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the reference polypeptide.
In other words, to obtain a polypeptide having an amino acid
sequence at least 95% identical to a reference amino acid sequence,
up to 5% of the amino acid residues in the reference sequence may
be deleted or substituted with another amino acid, or a number of
amino acids, up to 5% of the total amino acid residues in the
reference sequence, may be inserted into the reference sequence.
These alterations of the reference sequence may occur at the N- or
C-terminal positions of the reference amino acid sequence or
anywhere between those terminal positions, interspersed either
individually among residues in the reference sequence, or in one or
more contiguous groups within the reference sequence.
[0062] As a practical matter, whether any particular polypeptide is
at least 70%, 80%, 90%, or 95% identical to, for instance, an amino
acid sequence or to a polypeptide sequence encoded by a nucleic
acid sequence set forth in the Sequence Listing can be determined
conventionally using known computer programs, such the Bestfit
program. When using Bestfit or other sequence alignment program to
determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present
invention, the parameters are set, of course, that the percentage
of identity is calculated over the full length of the reference
amino acid sequence and that gaps in homology of up to 5% of the
total number of amino acid residues in the reference sequence are
allowed.
[0063] As used herein, the terms "hFGFR1-ECD.353" and "hFGFR1.353"
may be used interchangeably to refer to the full-length human FGFR1
ECD corresponding to SEQ ID NO: 1 (with signal peptide) or to SEQ
ID NO: 2 (without signal peptide; mature form).
[0064] As used herein, the terms "hFGFR1-ECD.339" and "hFGFR1.339"
may be used interchangeably to refer to the human FGFR1 ECD
corresponding to SEQ ID NO: 3 (with signal peptide) or to SEQ ID
NO: 4 (without signal peptide; mature form).
[0065] Additional hFGFR1 ECDs are described, for example, in U.S.
Pat. No. 7,678,890, which is incorporated by reference herein in
its entirety for any purpose.
[0066] The term "FGFR1 ECD fusion molecule" refers to a molecule
comprising an FGFR1 ECD, and one or more "fusion partners." In some
embodiments, the FGFR1 ECD and the fusion partner are covalently
linked ("fused"). If the fusion partner is also a polypeptide ("the
fusion partner polypeptide"), the FGFR1 ECD and the fusion partner
polypeptide may be part of a continuous amino acid sequence, and
the fusion partner polypeptide may be linked to either the N
terminus or the C terminus of the FGFR1 ECD. In such cases, the
FGFR1 ECD and the fusion partner polypeptide may be translated as a
single polypeptide from a coding sequence that encodes both the
FGFR1 ECD and the fusion partner polypeptide (the "FGFR1 ECD fusion
protein"). In some embodiments, the FGFR1 ECD and the fusion
partner are covalently linked through other means, such as, for
example, a chemical linkage other than a peptide bond. Many known
methods of covalently linking polypeptides to other molecules (for
example, fusion partners) may be used. In other embodiments, the
FGFR1 ECD and the fusion partner may be fused through a "linker,"
which is comprised of at least one amino acid or chemical
moiety.
[0067] In some embodiments, the FGFR1 ECD polypeptide and the
fusion partner are noncovalently linked. In some such embodiments,
they may be linked, for example, using binding pairs. Exemplary
binding pairs include, but are not limited to, biotin and avidin or
streptavidin, an antibody and its antigen, etc.
[0068] Exemplary fusion partners include, but are not limited to,
an immunoglobulin Fc domain, albumin, and polyethylene glycol. The
amino acid sequences of some exemplary Fc domains are shown in SEQ
ID NOs: 8 to 10. In some embodiments, an FGFR1 ECD fused to an Fc
is referred to as an "hFGFR1 ECD-Fc." In some embodiments, the Fc
domain is selected from an IgG1 Fc, an IgG2 Fc, an IgG3 Fc, and an
IgG4 Fc.
[0069] As used herein, the terms "hFGFR1-ECD.339-Fc" and
"hFGFR1.339-Fc" may be used interchangeably to refer to an amino
acid sequence selected from SEQ ID NO: 6 (without signal peptide,
mature form) and SEQ ID NO: 5 (with signal peptide). Nonlimiting
exemplary cancers that may be treated with hFGFR1-ECD.339-Fc
include, but are not limited to, lung cancer, colon cancer, breast
cancer, gastric cancer, head and neck cancer, prostate cancer,
endometrial cancer, sarcoma, small cell lung cancer, ovarian
cancer, Kaposi's sarcoma, Hodgkin's disease, leukemia,
non-Hodgkin's lymphoma, neuroblastoma (brain cancer),
rhabdomyosarcoma, Wilms' tumor, acute lymphoblastic leukemia, acute
lymphoblastic leukemia, bladder cancer, testicular cancer,
lymphomas, germ cell tumors, cancers of the colon and rectum,
gastrointestinal cancers, thyroid cancer, multiple myeloma,
pancreatic cancer, mesothelioma, malignant pleural mesothelioma,
hematological/lymphatic cancers, malignant peritoneal mesothelioma,
esophageal cancer, renal cell carcinoma, glioblastoma multiforme,
and liver cancer.
[0070] The term "signal peptide" refers to a sequence of amino acid
residues located at the N terminus of a polypeptide that
facilitates secretion of a polypeptide from a mammalian cell. A
signal peptide may be cleaved upon export of the polypeptide from
the mammalian cell, forming a mature protein. Signal peptides may
be natural or synthetic, and they may be heterologous or homologous
to the protein to which they are attached. Exemplary signal
peptides include, but are not limited to, FGFR1 signal peptides,
such as, for example, the amino acid sequence of SEQ ID NO: 7.
Exemplary signal peptides also include signal peptides from
heterologous proteins. A "signal sequence" refers to a
polynucleotide sequence that encodes a signal peptide. In some
embodiments, an FGFR1 ECD lacks a signal peptide. In some
embodiments, an FGFR1 ECD includes at least one signal peptide,
which may be a native FGFR1 signal peptide or a heterologous signal
peptide.
[0071] The term "vector" is used to describe a polynucleotide that
may be engineered to contain a cloned polynucleotide or
polynucleotides that may be propagated in a host cell. A vector may
include one or more of the following elements: an origin of
replication, one or more regulatory sequences (such as, for
example, promoters and/or enhancers) that regulate the expression
of the polypeptide of interest, and/or one or more selectable
marker genes (such as, for example, antibiotic resistance genes and
genes that may be used in colorimetric assays, e.g.,
.beta.-galactosidase). The term "expression vector" refers to a
vector that is used to express a polypeptide of interest in a host
cell.
[0072] A "host cell" refers to a cell that may be or has been a
recipient of a vector or isolated polynucleotide. Host cells may be
prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells
include mammalian cells, such as primate or non-primate animal
cells; fungal cells; plant cells; and insect cells. Exemplary
mammalian cells include, but are not limited to, 293 and CHO cells,
and their derivatives, such as 293-6E and DG44 cells,
respectively.
[0073] The term "isolated" as used herein refers to a molecule that
has been separated from at least some of the components with which
it is typically found in nature. For example, a polypeptide is
referred to as "isolated" when it is separated from at least some
of the components of the cell in which it was produced. Where a
polypeptide is secreted by a cell after expression, physically
separating the supernatant containing the polypeptide from the cell
that produced it is considered to be "isolating" the polypeptide.
Similarly, a polynucleotide is referred to as "isolated" when it is
not part of the larger polynucleotide (such as, for example,
genomic DNA or mitochondrial DNA, in the case of a DNA
polynucleotide) in which it is typically found in nature, or is
separated from at least some of the components of the cell in which
it was produced, e.g., in the case of an RNA polynucleotide. Thus,
a DNA polynucleotide that is contained in a vector inside a host
cell may be referred to as "isolated" so long as that
polynucleotide is not found in that vector in nature.
[0074] The term "anti-neoplastic composition" refers to a
composition useful in treating cancer comprising at least one
active therapeutic agent, e.g., an "anti-cancer agent." Examples of
therapeutic agents (anti-cancer agents) include, but are not
limited to, e.g., chemotherapeutic agents, growth inhibitory
agents, cytotoxic agents, agents used in radiation therapy,
anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and
other agents to treat cancer, such as anti-VEGF antibodies (e.g.,
bevacizumab, AVASTIN.RTM.), anti-HER-2 antibodies (e.g.,
trastuzumab, HERCEPTIN.RTM.) anti-CD20 antibodies (e.g., rituximab,
RITUXAN.RTM.), an epidermal growth factor receptor (EGFR)
antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFR
inhibitors (e.g., erlotinib, TARCEVA.RTM.), platelet derived growth
factor inhibitors (e.g., GLEEVEC.RTM., imatinib mesylate)), COX-2
inhibitors (e.g., celecoxib), interferons, cytokines, antagonists
(e.g., neutralizing antibodies) that bind to one or more of the
following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL,
BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive and
organic chemical agents, etc. Combinations thereof are also
included in the invention.
[0075] A "chemotherapeutic agent" refers to a chemical compound
useful in the treatment of cancer. Examples of chemotherapeutic
agents include alkylating agents such as thiotepa and
cyclophosphamide (CYTOXAN.RTM.); alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gammall and calicheamicin omegall (see, e.g., Nicolaou et al.,
Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral
alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.), liposomal doxorubicin TLC D-99
(MYOCET.RTM.), pegylated liposomal doxorubicin (CAELYX.RTM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.RTM.), pemetrexed (ALIMTA.RTM.);
tegafur (UFTORAL.RTM.), capecitabine (XELODA.RTM.), an epothilone,
and 5-fluorouracil (5-FU); folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogs such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.); dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel
(TAXOL.RTM.), albumin-engineered nanoparticle formulation of
paclitaxel (ABRAXANE.TM.), and docetaxel (TAXOTERE.RTM.);
chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum
agents such as cisplatin, oxaliplatin (e.g., ELOXATIN.RTM.), and
carboplatin; vincas, which prevent tubulin polymerization from
forming microtubules, including vinblastine (VELBAN.RTM.),
vincristine (ONCOVIN.RTM.), vindesine (ELDISINE.RTM.,
FILDESIN.RTM.), and vinorelbine (NAVELBINE.RTM.); etoposide
(VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid, including bexarotene (TARGRETIN.RTM.);
bisphosphonates such as clodronate (for example, BONEFOS.RTM. or
OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic
acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.),
pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or
risedronate (ACTONEL.RTM.); troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); antisense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1
inhibitor (e.g., LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.);
BAY439006 (sorafenib, NEXAVAR.RTM.; Bayer); SU-11248 (sunitinib,
SUTENT.RTM., Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib
or etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib
(VELCADE.RTM.); CCI-779; tipifarnib (R11577); orafenib, ABT510;
Bc1-2 inhibitor such as oblimersen sodium (GENASENSE.RTM.);
pixantrone; EGFR inhibitors (see definition below); tyrosine kinase
inhibitors (see definition below); serine-threonine kinase
inhibitors such as rapamycin (sirolimus, RAPAMUNE.RTM.);
farnesyltransferase inhibitors such as lonafarnib (SCH 6636,
SARASAR.TM.); and pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone; and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.RTM.) combined with 5-FU and
leucovorin.
[0076] Chemotherapeutic agents as defined herein include
"anti-hormonal agents" or "endocrine therapeutics" which act to
regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer. They may be hormones themselves,
including, but not limited to: anti-estrogens with mixed
agonist/antagonist profile, including, tamoxifen (NOLVADEX.RTM.),
4-hydroxytamoxifen, toremifene (FARESTON.RTM.), idoxifene,
droloxifene, raloxifene (EVISTA.RTM.), trioxifene, keoxifene, and
selective estrogen receptor modulators (SERMs) such as SERM3; pure
anti-estrogens without agonist properties, such as fulvestrant
(FASLODEX.RTM.), and EM800 (such agents may block estrogen receptor
(ER) dimerization, inhibit DNA binding, increase ER turnover,
and/or suppress ER levels); aromatase inhibitors, including
steroidal aromatase inhibitors such as formestane and exemestane
(AROMASIN.RTM.), and nonsteroidal aromatase inhibitors such as
anastrazole (ARIMIDEX.RTM.), letrozole (FEMARA.RTM.) and
aminoglutethimide, and other aromatase inhibitors include vorozole
(RIVISOR.RTM.), megestrol acetate (MEGASE.RTM.), fadrozole, and
4(5)-imidazoles; lutenizing hormone-releasing hormone agonists,
including leuprolide (LUPRON.RTM. and ELIGARD.RTM.), goserelin,
buserelin, and tripterelin; sex steroids, including progestins such
as megestrol acetate and medroxyprogesterone acetate, estrogens
such as diethylstilbestrol and premarin, and androgens/retinoids
such as fluoxymesterone, all transretinoic acid and fenretinide;
onapristone; anti-progesterones; estrogen receptor down-regulators
(ERDs); anti-androgens such as flutamide, nilutamide and
bicalutamide; and pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above.
[0077] An "angiogenic factor or agent" refers to a growth factor
which stimulates the development of blood vessels, e.g., promote
angiogenesis, endothelial cell growth, stability of blood vessels,
and/or vasculogenesis, etc. For example, angiogenic factors,
include, but are not limited to, e.g., VEGF and members of the VEGF
family (VEGF-B, VEGF-C and VEGF-D), PlGF, PDGF family, fibroblast
growth factor family (FGFs), TIE ligands (Angiopoietins), ephrins,
delta-like ligand 4 (DLL4), del-1, fibroblast growth factors:
acidic (aFGF) and basic (bFGF), follistatin, granulocyte
colony-stimulating factor (G-CSF), hepatocyte growth factor
(HGF)/scatter factor (SF), interleukin-8 (IL-8), leptin, midkine,
neuropilins, placental growth factor, platelet-derived endothelial
cell growth factor (PD-ECGF), platelet-derived growth factor,
especially PDGF-BB or PDGFR-beta, pleiotrophin (PTN), progranulin,
proliferin, transforming growth factor-alpha (TGF-alpha),
transforming growth factor-beta (TGF-beta), tumor necrosis
factor-alpha (TNF-alpha), etc. It would also include factors that
accelerate wound healing, such as growth hormone, insulin-like
growth factor-I (IGF-I), VIGF, epidermal growth factor (EGF), CTGF
and members of its family, and TGF-alpha and TGF-beta. See, e.g.,
Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit
and Detmar (2003) Oncogene 22:3172-3179; Ferrara & Alitalo
(1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003)
Oncogene 22:6549-6556 (e.g., Table 1 listing known angiogenic
factors); and, Sato (2003) Int. J. Clin. Oncol. 8:200-206.
[0078] An "anti-angiogenic agent" or "angiogenesis inhibitor"
refers to a small molecular weight substance, a polynucleotide
(including, e.g., an inhibitory RNA (RNAi or siRNA)), a
polypeptide, an isolated protein, a recombinant protein, an
antibody, or conjugates or fusion proteins thereof, that inhibits
angiogenesis, vasculogenesis, or undesirable vascular permeability,
either directly or indirectly. It should be understood that the
anti-angiogenic agent includes those agents that bind and block the
angiogenic activity of the angiogenic factor or its receptor. For
example, an anti-angiogenic agent is an antibody or other
antagonist to an angiogenic agent as defined above, e.g., fusion
proteins that binds to VEGF-A such as ZALTRAP.TM. (Aflibercept),
antibodies to VEGF-A such as AVASTIN.RTM. (bevacizumab) or to the
VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR
inhibitors such as GLEEVEC.RTM. (Imatinib Mesylate), small
molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284,
SU6668, SUTENT.RTM./SU11248 (sunitinib malate), AMG706, or those
described in, e.g., international patent application WO
2004/113304). Anti-angiogenic agents also include native
angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See,
e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol. 53:217-39;
Streit and Detmar (2003) Oncogene 22:3172-3179 (e.g., Table 3
listing anti-angiogenic therapy in malignant melanoma); Ferrara
& Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al.
(2003) Oncogene 22:6549-6556 (e.g., Table 2 listing known
anti-angiogenic factors); and, Sato (2003) Int. J. Clin. dOncol.
8:200-206 (e.g., Table 1 listing anti-angiogenic agents used in
clinical trials).
[0079] The term "VEGF" or "VEGF-A" as used herein refers to the
165-amino acid human vascular endothelial cell growth factor and
related 121-, 189-, and 206-amino acid human vascular endothelial
cell growth factors, as described by Leung et al. (1989) Science
246:1306, and Houck et al. (1991) Mol. Endocrin, 5:1806, together
with the naturally occurring allelic and processed forms thereof.
The term "VEGF" also refers to VEGFs from non-human species such as
mouse, rat or primate. Sometimes the VEGF from a specific species
are indicated by terms such as hVEGF for human VEGF, mVEGF for
murine VEGF, and etc. The term "VEGF" is also used to refer to
truncated forms of the polypeptide comprising amino acids 8 to 109
or 1 to 109 of the 165-amino acid human vascular endothelial cell
growth factor. Reference to any such forms of VEGF may be
identified in the present application, e.g., by "VEGF (8-109),"
"VEGF (1-109)," "VEGF-A.sub.109" or "VEGF165." The amino acid
positions for a "truncated" native VEGF are numbered as indicated
in the native VEGF sequence. For example, amino acid position 17
(methionine) in truncated native VEGF is also position 17
(methionine) in native VEGF. The truncated native VEGF has binding
affinity for the KDR and Flt-1 receptors comparable to native
VEGF.
[0080] A "VEGF antagonist" refers to a molecule capable of
neutralizing, blocking, inhibiting, abrogating, reducing or
interfering with VEGF activities including, but not limited to, its
binding to one or more VEGF receptors. VEGF antagonists include,
without limitation, anti-VEGF antibodies and antigen-binding
fragments thereof, receptor molecules and derivatives which bind
specifically to VEGF thereby sequestering its binding to one or
more receptors, anti-VEGF receptor antibodies, VEGF receptor
antagonists such as small molecule inhibitors of the VEGFR tyrosine
kinases (e.g., pazopanib) and immunoadhesins that binds to VEGF
such as VEGF trap (e.g., aflibercept). The term "VEGF antagonist,"
as used herein, specifically includes molecules, including
antibodies, antibody fragments, other binding polypeptides,
peptides, and non-peptide small molecules, that bind to VEGF and
are capable of neutralizing, blocking, inhibiting, abrogating,
reducing or interfering with VEGF activities. Thus, the term "VEGF
activities" specifically includes VEGF mediated biological
activities of VEGF.
[0081] The term "VEGF trap" as used herein means a protein, such as
a fusion molecule, that binds to VEGF and is capable of
neutralizing, blocking, inhibiting, abrogating, reducing or
interfering with VEGF activities. An example of a VEGF trap is
aflibercept.
[0082] The term "anti-VEGF antibody" or "an antibody that binds to
VEGF" refers to an antibody that is capable of binding to VEGF with
sufficient affinity and specificity that the antibody is useful as
a diagnostic and/or therapeutic agent in targeting VEGF. Anti-VEGF
neutralizing antibodies suppress the growth of a variety of human
tumor cell lines in nude mice (Kim et al., Nature 362:841-844
(1993); Warren et al., J. Clin. Invest. 95:1789-1797 (1995);
Borgstrom et al., Cancer Res. 56:4032-4039 (1996); Melnyk et al.,
Cancer Res. 56:921-924 (1996)) and also inhibit intraocular
angiogenesis in models of ischemic retinal disorders. Adamis et
al., Arch. Ophthalmol. 114:66-71 (1996). For example, the anti-VEGF
antibody can be used as a therapeutic agent in targeting and
interfering with diseases or conditions wherein the VEGF activity
is involved. See, e.g., U.S. Pat. Nos. 6,582,959, 6,703,020;
WO98/45332; WO 96/30046; WO94/10202, WO2005/044853; EP 0666868B1;
US Patent Applications 20030206899, 20030190317, 20030203409,
20050112126, 20050186208, and 20050112126; Popkov et al., Journal
of Immunological Methods 288:149-164 (2004); and WO2005012359. The
antibody selected will normally have a sufficiently strong binding
affinity for VEGF. For example, the antibody may bind hVEGF with a
K.sub.a value of between 100 nM-1 pM. Antibody affinities may be
determined by a surface plasmon resonance based assay (such as the
BIAcore assay as described in PCT Application Publication No.
WO2005/012359); enzyme-linked immunoabsorbent assay (ELISA); and
competition assays (e.g. RIA's), for example. The antibody may be
subjected to other biological activity assays, e.g., in order to
evaluate its effectiveness as a therapeutic. Such assays are known
in the art and depend on the target antigen and intended use for
the antibody. Examples include the HUVEC inhibition assay; tumor
cell growth inhibition assays (as described in WO 89/06692, for
example); antibody-dependent cellular cytotoxicity (ADCC) and
complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No.
5,500,362); and agonistic activity or hematopoiesis assays (see WO
95/27062). An anti-VEGF antibody will usually not bind to other
VEGF homologues such as VEGF-B, VEGF-C, VEGF-D or VEGF-E, nor other
growth factors such as PlGF, PDGF or bFGF.
[0083] In one embodiment, anti-VEGF antibodies include a monoclonal
antibody that binds to the same epitope as the monoclonal anti-VEGF
antibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant
humanized anti-VEGF monoclonal antibody (see Presta et al. (1997)
Cancer Res. 57:4593-4599), including but not limited to the
antibody known as "bevacizumab" also known as "rhuMAb VEGF" or
AVASTIN.RTM.. AVASTIN.RTM. is presently commercially available.
Nonlimiting exemplary cancers that may be treated with bevacizumab
include non-small cell lung cancer, colorectal cancer, breast
cancer, renal cancer, ovarian cancer, glioblastoma multiforme,
pediatric osteosarcoma, gastric cancer and pancreatic cancer.
Bevacizumab comprises mutated human IgG.sub.1 framework regions and
antigen-binding complementarity-determining regions from the murine
antibody A.4.6.1 that blocks binding of human VEGF to its
receptors. Bevacizumab and other humanized anti-VEGF antibodies are
further described in U.S. Pat. Nos. 6,884,879, and 7,169,901.
Additional anti-VEGF antibodies are described in PCT Application
Publication Nos. WO2005/012359 and WO2009/073160; U.S. Pat. Nos.
7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/45332; WO
96/30046; WO94/10202; EP 0666868B1; U.S. Patent Application
Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317,
20030203409, and 20050112126; and Popkov et al., Journal of
Immunological Methods 288:149-164 (2004).
[0084] The terms "subject" and "patient" are used interchangeably
herein to refer to a mammal. In some embodiments, the subject or
patient is a human. In other embodiments, methods of treating other
mammals, including, but not limited to, rodents, simians, felines,
canines, equines, bovines, porcines, ovines, caprines, mammalian
laboratory animals, mammalian farm animals, mammalian sport
animals, and mammalian pets, are also provided.
[0085] The term "sample" or "patient sample" as used herein, refers
to a composition that is obtained or derived from a subject of
interest that contains a cellular and/or other molecular entity
that is to be characterized and/or identified, for example based on
physical, biochemical, chemical and/or physiological
characteristics. For example, the phrase "disease sample" and
variations thereof refers to any sample obtained from a subject of
interest that would be expected or is known to contain the cellular
and/or molecular entity that is to be characterized. By "tissue or
cell sample" is meant a collection of similar cells obtained from a
tissue of a subject or patient. The source of the tissue or cell
sample may be solid tissue as from a fresh, frozen and/or preserved
organ or tissue sample or biopsy or aspirate; blood or any blood
constituents; bodily fluids such as cerebral spinal fluid, amniotic
fluid, peritoneal fluid, or interstitial fluid; cells from any time
in gestation or development of the subject. The tissue sample may
also be primary or cultured cells or cell lines. Optionally, the
tissue or cell sample is obtained from a disease tissue/organ. The
tissue sample may contain compounds which are not naturally
intermixed with the tissue in nature such as preservatives,
anticoagulants, buffers, fixatives, nutrients, antibiotics, or the
like.
[0086] A "reference sample", "reference cell", or "reference
tissue", as used herein, refers to a sample, cell or tissue
obtained from a source known, or believed, not to be afflicted with
the disease or condition for which a method or composition of the
invention is being used to identify. In some embodiments, a
reference sample, reference cell or reference tissue is obtained
from a healthy part of the body of the same subject or patient in
whom a disease or condition is being identified using a composition
or method of the invention. In some embodiments, a reference
sample, reference cell or reference tissue is obtained from a
healthy part of the body of one or more individuals who are not the
subject or patient in whom a disease or condition is being
identified using a composition or method of the invention.
[0087] "Cancer" and "tumor," as used herein, are interchangeable
terms that refer to any abnormal cell or tissue growth or
proliferation in an animal. As used herein, the terms "cancer" and
"tumor" encompass solid and hematological/lymphatic cancers and
also encompass malignant, pre-malignant, and benign growth, such as
dysplasia. Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular non-limiting examples of such cancers include squamous
cell cancer, small-cell lung cancer, pituitary cancer, esophageal
cancer, astrocytoma, soft tissue sarcoma, non-small cell lung
cancer, adenocarcinoma of the lung, squamous carcinoma of the lung,
cancer of the peritoneum, hepatocellular cancer, gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, renal cancer, liver
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, brain cancer, endometrial cancer, testis cancer,
cholangiocarcinoma, gallbladder carcinoma, gastric cancer,
melanoma, and various types of head and neck cancer.
[0088] The term "lung cancer," as used herein, refers to both small
cell lung cancer and non-small cell lung cancers. Non-small cell
lung cancer includes, but is not limited to, squamous cell lung
cancer, adenocarcinoma, large-cell lung carcinoma, sarcomatoid
carcinoma, carcinoid tumors, pulmonary pleomorphic carcinoma, and
adenosquamous carcinoma and bronchioloalveolar carcinoma. Small
cell lung cancer may, in some embodiments, be referred to as
"oat-cell" cancer, and includes, but is not limited to, combined
small-cell carcinoma, which comprises a mixture of small cell and
non-small cell carcinomas.
[0089] A "cell with FGFR1 gene amplification" refers to a cell that
comprises more than two copies of the FGFR1 gene. In some
embodiments, a cell with FGFR1 gene amplification refers to a cell
that has a ratio of FGFR1 gene to chromosome 8 centromere of
greater than 1. In some embodiments, the ratio is determined by
fluorescence in situ hybridization. "Cancer with FGFR1 gene
amplification," as used herein, refers to a cancer in which at
least a portion of the cancer cells have FGFR1 gene amplification.
In some embodiments, a cancer with FGFR1 gene amplification refers
to a cancer in which at least a portion of the cancer cells
comprise at least four copies of the FGFR1 gene. In some
embodiments, a cancer with FGFR1 gene amplification refers to a
cancer in which at least a portion of the cancer cells have an
FGFR1 gene:chromosome 8 centromere ratio of greater than 1. An
exemplary FGFR1 gene sequence can be found, e.g., NCBI Reference
Sequence: NG.sub.--007729.1 dated 25 Mar. 2012.
[0090] In some embodiments, a cell with FGFR1 gene amplification
comprises at least 3 copies, at least 4 copies, at least 5 copies,
at least 6 copies, at least 8 copies, or at least 10 copies of the
FGFR1 gene. In some embodiments, a cell with FGFR1 gene
amplification comprises at least 4 copies. In some embodiments, a
cell with FGFR1 gene amplification has a ratio of FGFR1
gene:chromosome 8 centromere of at least 1.5, at least 2, at least
2.5, at least 3, at least 3.5, or at least 4. In some embodiments,
a cell with FGFR1 gene amplification has a ratio of FGFR1
gene:chromosome 8 centromere of at least 2. In some embodiments,
each copy of the FGFR1 gene in a cell with FGFR1 gene amplification
need not be a complete copy of the FGFR1 gene. In some embodiments,
a cell with FGFR1 gene amplification has elevated levels of FGFR1
(i.e., in some embodiments, a cell with FGFR1 gene amplification is
also a cell with FGFR1 overexpression).
[0091] A "cell with FGFR1 overexpression" or a "cell that
overexpresses FGFR1" refers to a cell that has at least a 2-fold
greater level of FGFR1 mRNA or protein than a reference cell. A
"cancer with FGFR1 overexpression" or a "cancer that overexpresses
FGFR1" refers to a cancer in which at least a portion of the cells
have at least a 2-fold greater level of FGFR1 mRNA or protein than
a reference cell. In some embodiments, a cell with FGFR1
overexpression has at least 3-fold, at least 4-fold, at least
5-fold, at least 7-fold, or at least 10-fold greater level of FGFR1
mRNA or protein than a reference cell. The level of FGFR1 mRNA or
protein can be determined by any suitable method including, but not
limited to, the methods described herein. In some embodiments,
FGFR1 is FGFR1IIIc. An exemplary human FGFR1 protein sequence can
be found, e.g., at UniProtKB/Swiss-Prot Reference Sequence: P11362
(FGFR1_HUMAN) dated Mar. 21, 2012. An exemplary human FGFR1 mRNA
sequence can be found, e.g., at NCBI Reference Sequence:
NM.sub.--023110.2 dated 24 Mar. 2012. An exemplary human FGFR1IIIc
protein sequence can be found, e.g., at NCBI Reference Sequence:
NP.sub.--075598.2 dated 24 Mar. 2012. An exemplary human FGFR1IIIc
mRNA sequence can be found, e.g., at NCBI Reference Sequence:
NM.sub.--023110.2 dated 24 Mar. 2012.
[0092] A "cell with FGFR3IIIc overexpression" or a "cell that
overexpresses FGFR3IIIc" refers to a cell that has at least a
2-fold greater level of FGFR3IIIc mRNA or protein than a reference
cell. A "cancer with FGFR3IIIc overexpression" or a "cancer that
overexpresses FGFR3IIIc" refers to a cancer in which at least a
portion of the cells have at least a 2-fold greater level of
FGFR3IIIc mRNA or protein than a reference cell. In some
embodiments, a cell with FGFR3IIIc overexpression has at least
3-fold, at least 4-fold, at least 5-fold, at least 7-fold, or at
least 10-fold greater level of FGFR3IIIc mRNA or protein than a
reference cell. The level of FGFR3IIIc mRNA or protein can be
determined by any suitable method including, but not limited to,
the methods described herein. An exemplary human FGFR3IIIc protein
sequence can be found, e.g., at NCBI Reference Sequence:
NP.sub.--000133.1 dated 12 Feb. 2012. An exemplary human FGFR3IIIc
mRNA sequence can be found, e.g., at NCBI Reference Sequence:
NM.sub.--000142.4 dated 12 Feb. 2012.
[0093] A "cell with FGF2 overexpression" or a "cell that
overexpresses FGF2" refers to a cell that has at least a 2-fold
greater level of FGF2 mRNA or protein than a reference cell. A
"cancer with FGF2 overexpression" or a "cancer that overexpresses
FGF2" refers to a cancer in which at least a portion of the cells
have at least a 2-fold greater level of FGF2 mRNA or protein than a
reference cell. In some embodiments, a cell with FGF2
overexpression has at least 3-fold, at least 4-fold, at least
5-fold, at least 7-fold, or at least 10-fold greater level of FGF2
mRNA or protein than a reference cell. The level of FGF2 mRNA or
protein can be determined by any suitable method including, but not
limited to, the methods described herein. An exemplary human FGF2
protein sequence can be found, e.g., at NCBI Reference Sequence:
NP.sub.--001997.5 dated 12 Feb. 2012. An exemplary human FGF2 mRNA
sequence can be found, e.g., at NCBI Reference Sequence:
NM.sub.--002006.4 dated 12 Feb. 2012.
[0094] A "cell with DKK3 overexpression" or a "cell that
overexpresses DKK3" refers to a cell that has at least a 2-fold
greater level of DKK3 mRNA or protein than a reference cell. A
"cancer with DKK3 overexpression" or a "cancer that overexpresses
DKK3" refers to a cancer in which at least a portion of the cells
have at least a 2-fold greater level of DKK3 mRNA or protein than a
reference cell. In some embodiments, a cell with DKK3
overexpression has at least 3-fold, at least 4-fold, at least
5-fold, at least 7-fold, or at least 10-fold greater level of DKK3
mRNA or protein than a reference cell. The level of DKK3 mRNA or
protein can be determined by any suitable method including, but not
limited to, the methods described herein. An exemplary human DKK3
protein sequence can be found, e.g., at NCBI Reference Sequence:
NP.sub.--001018067.1 dated 22 Jan. 2012. An exemplary human DKK3
mRNA sequence can be found, e.g., at NCBI Reference Sequence:
NM.sub.--001018057.1 dated 22 Jan. 2012.
[0095] A "cell with FGF18 overexpression" or a "cell that
overexpresses FGF18" refers to a cell that has at least a 2-fold
greater level of FGF18 mRNA or protein than a reference cell. A
"cancer with FGF18 overexpression" or a "cancer that overexpresses
FGF18" refers to a cancer in which at least a portion of the cells
have at least a 2-fold greater level of FGF18 mRNA or protein than
a reference cell. In some embodiments, a cell with FGF18
overexpression has at least 3-fold, at least 4-fold, at least
5-fold, at least 7-fold, or at least 10-fold greater level of FGF18
mRNA or protein than a reference cell. The level of FGF18 mRNA or
protein can be determined by any suitable method including, but not
limited to, the methods described herein. An exemplary human FGF18
protein sequence can be found, e.g., at NCBI Reference Sequence:
NP.sub.--003853 dated 27 Jun. 2012. An exemplary human FGF18 mRNA
sequence can be found, e.g., at NCBI Reference Sequence:
NM.sub.--003862.2 dated 27 Jun. 2012.
[0096] A "cell with ETV4 overexpression" or a "cell that
overexpresses ETV4" refers to a cell that has at least a 2-fold
greater level of ETV4 mRNA or protein than a reference cell. A
"cancer with ETV4 overexpression" or a "cancer that overexpresses
ETV4" refers to a cancer in which at least a portion of the cells
have at least a 2-fold greater level of ETV4 mRNA or protein than a
reference cell. In some embodiments, a cell with ETV4
overexpression has at least 3-fold, at least 4-fold, at least
5-fold, at least 7-fold, or at least 10-fold greater level of ETV4
mRNA or protein than a reference cell. The level of ETV4 mRNA or
protein can be determined by any suitable method including, but not
limited to, the methods described herein. An exemplary human ETV4
protein sequence can be found, e.g., at NCBI Reference Sequence:
NP.sub.--001977.1 dated 8 Sep. 2012. An exemplary human ETV4 mRNA
sequence can be found, e.g., at NCBI Reference Sequence:
NM.sub.--001986.2 dated 8 Sep. 2012.
[0097] "Treatment," as used herein, includes any administration or
application of a therapeutic for condition in a mammal, including a
human, and includes inhibiting the condition or progression of the
condition, inhibiting or slowing the condition or its progression,
arresting its development, partially or fully relieving the
condition, or curing the condition, for example, by causing
regression, or restoring or repairing a lost, missing, or defective
function; or stimulating an inefficient process. In some
embodiments, "treatment" refers to clinical intervention in an
attempt to alter the natural course of the individual or cell being
treated, and can be performed either for prophylaxis or during the
course of clinical pathology. Desirable effects of treatment
include preventing occurrence or recurrence of disease, alleviation
of symptoms, diminishment of any direct or indirect pathological
consequences of the disease, preventing metastasis, decreasing the
rate of disease progression, amelioration or palliation of the
disease state, and remission or improved prognosis.
[0098] An "effective amount" or "therapeutically effective amount"
of a molecule or a combination of molecules means an amount that is
sufficient to treat a condition and/or to inhibit growth of tumor
cells in at least a subset of subjects when given alone or in
combination with other treatments. In certain embodiments, a
therapeutically effective amount refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result. A therapeutically effective
amount of FGFR1 fusion protein of the invention may vary according
to factors such as the disease state, age, sex, and weight of the
individual, and the ability of FGFR1 fusion protein to elicit a
desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the
FGFR1 fusion proteins are outweighed by the therapeutically
beneficial effects. In the case of cancer, the effective amount of
the drug may reduce the number of cancer cells; reduce the tumor
size; inhibit (i.e., slow to some extent and typically stop) cancer
cell infiltration into peripheral organs; inhibit (i.e., slow to
some extent and typically stop) tumor metastasis; inhibit, to some
extent, tumor growth; allow for treatment of the tumor, and/or
relieve to some extent one or more of the symptoms associated with
the disorder. To the extent the drug may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic.
[0099] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0100] The terms "inhibition" or "inhibit" refer to a decrease or
cessation of any phenotypic characteristic or to the decrease or
cessation in the incidence, degree, or likelihood of that
characteristic. Nonlimiting exemplary inhibition includes
inhibition of tumor growth.
[0101] The terms "benefit", "clinical benefit", "responsiveness",
and "therapeutic responsiveness" as used herein in the context of
benefiting from or responding to administration of a therapeutic
agent, can be measured by assessing various endpoints, e.g.,
inhibition, to some extent, of disease progression, including
slowing down and complete arrest; reduction in the number of
disease episodes and/or symptoms; reduction in lesion size;
inhibition (i.e., reduction, slowing down or complete stopping) of
disease cell infiltration into adjacent peripheral organs and/or
tissues; inhibition (i.e. reduction, slowing down or complete
stopping) of disease spread; decrease of auto-immune response,
which may, but does not have to, result in the regression or
ablation of the disease lesion; relief, to some extent, of one or
more symptoms associated with the disorder; increase in the length
of disease-free presentation following treatment, e.g.,
progression-free survival; increased overall survival; higher
response rate; and/or decreased mortality at a given point of time
following treatment.
[0102] Administration "in combination with" one or more further
therapeutic agents includes concurrent (including simultaneous) and
consecutive (i.e., sequential) administration in any order.
[0103] A "pharmaceutically acceptable carrier" refers to a
non-toxic solid, semisolid, or liquid filler, diluent,
encapsulating material, formulation auxiliary, or carrier
conventional in the art for use with a therapeutic agent that
together comprise a "pharmaceutical composition" for administration
to a subject. A pharmaceutically acceptable carrier is non-toxic to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation. The
pharmaceutically acceptable carrier is appropriate for the
formulation employed. For example, if the therapeutic agent is to
be administered orally, the carrier may be a gel capsule. If the
therapeutic agent is to be administered subcutaneously, the carrier
ideally is not irritable to the skin and does not cause injection
site reaction.
Therapeutic Compositions and Methods
[0104] Methods of Treating Cancer Having FGFR1 Gene Amplifications
using FGFR1 ECDs and/or FGFR1 ECD Fusion Molecules
[0105] In some embodiments, the invention provides methods of
treating cancers in which at least a portion of the cancer cells
have FGFR1 gene amplification. Such cancers have been found, in
some embodiments, to be particularly responsive to treatment with a
fibroblast growth factor receptor 1 (FGFR1) extracellular domain
(ECD) or FGFR1 ECD fusion molecule. Accordingly, in some
embodiments, a method of treating cancer having an FGFR1 gene
amplification comprises administering a therapeutically effective
amount of an FGFR1 ECD or an FGFR1 ECD fusion molecule to the
subject. In some embodiments, a method of treating cancer in a
subject comprises administering a therapeutically effective amount
of a fibroblast growth factor receptor 1 (FGFR1) extracellular
domain (ECD) or an FGFR1 ECD fusion molecule to the subject,
wherein, prior to administration of the FGFR1 ECD or FGFR1 ECD
fusion molecule, at least a portion of the cells of the cancer have
been determined to have an FGFR1 gene amplification. In such
methods, an FGFR1 gene amplification in a cancer is indicative of
therapeutic responsiveness by the cancer to an FGFR1 ECD or FGFR1
ECD fusion molecule.
[0106] In some embodiments, the invention provides methods of
treating cancers in which at least a portion of the cancer cells
have overexpression of at least one, at least two, at least three,
or at least four markers selected from FGFR1, FGFR3IIIc, FGF2,
DKK3, FGF18, and ETV4. In some embodiments, FGFR1 is FGFR1IIIc. In
some embodiments, the overexpression is mRNA overexpression. In
some embodiments, the overexpression is protein overexpression. In
some embodiments, a method of treating cancer that overexpresses at
least marker selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and
ETV4 comprises administering a therapeutically effective amount of
an FGFR1 ECD or an FGFR1 ECD fusion molecule to the subject. In
some embodiments, a method of treating cancer in a subject
comprises administering a therapeutically effective amount of a
fibroblast growth factor receptor 1 (FGFR1) extracellular domain
(ECD) or an FGFR1 ECD fusion molecule to the subject, wherein,
prior to administration of the FGFR1 ECD or FGFR1 ECD fusion
molecule, at least a portion of the cells of the cancer have been
determined to have overexpression of at least marker selected from
FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4. In such methods,
FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and/or ETV4 overexpression in
a cancer is indicative of therapeutic responsiveness by the cancer
to an FGFR1 ECD or FGFR1 ECD fusion molecule. In some embodiments,
FGFR1 is FGFR1IIIc.
[0107] In some embodiments, in a cancer with an FGFR1 gene
amplification, at least a portion of the cancer cells comprise at
least four copies of the FGFR1 gene. In some embodiments, in a
cancer with an FGFR1 gene amplification, at least a portion of the
cancer cells comprise at least five, at least six, at least 8, or
at least 10 copies of the FGFR1 gene. Determination of the FGFR1
gene copy number can be carried out by any suitable method in the
art. Certain nonlimiting exemplary methods are discussed herein. In
some embodiments, in a cancer with an FGFR1 gene amplification, at
least a portion of the cancer cells have a ratio of FGFR1 gene to
chromosome 8 centromere of at least 2. In some embodiments, in a
cancer with an FGFR1 gene amplification, at least a portion of the
cancer cells have a ratio of FGFR1 gene to chromosome 8 centromere
of at least 2.5, at least 3, at least 3.5, or at least 4.
Determination of such a ratio can be carried out by any suitable
method in the art. Certain nonlimiting exemplary methods are
discussed herein.
[0108] In some embodiments, the cancer is selected from prostate
cancer, breast cancer, colorectal cancer, lung cancer, brain
cancer, ovarian cancer, endometrial cancer, head and neck cancer,
laryngeal cancer, liver cancer, renal cancer, glioblastoma, and
pancreatic cancer. In certain embodiments, the cancer is selected
from breast cancer, esophageal cancer, and lung cancer. In some
embodiments, the cancer is lung cancer. In some embodiments, the
lung cancer is selected from non-small cell lung cancer and small
cell lung cancer. In some embodiments, the lung cancer is squamous
cell carcinoma. In some embodiments, the cancer is head and neck
cancer. In some embodiments, the head and neck cancer is squamous
cell carcinoma of the head and neck.
[0109] In some embodiments, the FGFR1 ECD has an amino acid
sequence selected from SEQ ID NOs: 1 to 4. In some embodiments, the
FGFR1 ECD has an amino acid sequence selected from SEQ ID NOs: 2
and 4. In some embodiments, the FGFR1 ECD fusion molecule has an
amino acid sequence selected from SEQ ID NOs: 5 and 6. In some
embodiments, the FGFR1 ECD fusion molecule is FGFR1 ECD.339-Fc with
an amino acid sequence of SEQ ID NO: 6.
[0110] In some embodiments, an FGFR1 ECD or FGFR1 ECD fusion
molecule is administered with one or more additional anti-cancer
therapies. Examples of the additional anti-cancer therapies
include, without limitation, surgery, radiation therapy
(radiotherapy), biotherapy, immunotherapy, and chemotherapy or a
combination of these therapies. In addition, cytotoxic agents,
anti-angiogenic and anti-proliferative agents can be used in
combination with the FGFR1 ECD or FGFR1 ECD fusion molecule. In
certain aspects of any of the methods and uses, the invention
provides treating cancer in which at least a portion of the cancer
cells comprise an FGFR1 gene amplification and/or overexpress at
least one, at least two, at least three, or at least four markers
selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4, by
administering therapeutically effective amounts of an FGFR1 ECD
and/or FGFR1 ECD fusion molecule and one or more chemotherapeutic
agents to a subject. In some embodiments, the subject's cancer has
not previously been treated. A variety of chemotherapeutic agents
may be used in the combined treatment methods and uses of the
invention. An exemplary and non-limiting list of chemotherapeutic
agents contemplated is provided herein under "Definitions" and in
the "Summary of the Invention." In some embodiments, the invention
provides methods of treating cancer, by administering
therapeutically effective amounts of an FGFR1 ECD and/or FGFR1 ECD
fusion molecule and one or more anti-angiogenic agent(s) to a
subject. In some embodiments, the invention provides treating
cancer, by administering therapeutically effective amounts of an
FGFR1 ECD and/or FGFR1 ECD fusion molecule and one or more VEGF
antagonists to a subject. In some embodiments, the invention
provides treating cancer, by administering therapeutically
effective amounts of an FGFR1 ECD and/or FGFR1 ECD fusion molecule
and one or more VEGF antagonists in combination with one or more
chemotherapeutic agents to a subject. In some embodiments, the one
or more VEGF antagonists are anti-VEGF antibodies and/or VEGF
traps.
[0111] In some embodiments, methods of treating cancer comprising
administering to a subject an FGFR1 ECD and/or FGFR1 ECD fusion
molecule in combination with at least one additional therapeutic
agent selected from docetaxel, paclitaxel, vincristine,
carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil
(5-FU), leucovorin, pemetrexed, sorafenib, etoposide, topotecan, a
VEGF antagonist, an anti-VEGF antibody, a VEGF trap, and
bevacizumab are provided. In another example, methods of treating
cancer comprising administering to a subject an FGFR1-ECD.339-Fc in
combination with at least one additional therapeutic agent selected
from docetaxel, paclitaxel, vincristine, carboplatin, cisplatin,
oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,
pemetrexed, sorafenib, etoposide, topotecan, a VEGF antagonist, an
anti-VEGF antibody, a VEGF trap, and bevacizumab are provided. In
some embodiments, methods of treating cancer comprising
administering to a subject an FGFR1-ECD.339-Fc and docetaxel are
provided.
[0112] Pharmaceutical compositions comprising FGFR1 ECD and/or
FGFR1 ECD fusion molecules (e.g., FGFR1-ECD.339-Fc) are
administered in a therapeutically effective amount for the specific
indication. The therapeutically effective amount is typically
dependent on the weight of the subject being treated, his or her
physical or health condition, the extensiveness of the condition to
be treated, and/or the age of the subject being treated. In
general, an FGFR1 ECD and/or FGFR1 ECD fusion molecule (e.g.,
FGFR1-ECD.339-Fc) is to be administered in an amount in the range
of about 50 .mu.g/kg body weight to about 100 mg/kg body weight per
dose. Optionally, the FGFR1 ECD and/or FGFR1 ECD fusion molecule
(e.g., FGFR1-ECD.339-Fc) can be administered in an amount in the
range of about 100 .mu.g/kg body weight to about 30 mg/kg body
weight per dose. Further optionally, the FGFR1 ECD and/or FGFR1 ECD
fusion molecule (e.g., FGFR1-ECD.339-Fc) can be administered in an
amount in the range of about 0.5 mg/kg body weight to about 20
mg/kg body weight per dose. In certain embodiments, the FGFR1 ECD
and/or FGFR1 ECD fusion molecule (e.g., FGFR1-ECD.339-Fc) is
administered at a dose of about 8 mg/kg body weight to about 20
mg/kg body weight. In some embodiments, the FGFR1 ECD and/or FGFR1
ECD fusion molecule (e.g., FGFR1-ECD.339-Fc) is administered at a
dose of about 8 mg/kg body weight to about 16 mg/kg body weight (or
about 10 mg/kg body weight to about 20 mg/kg body weight when
calculated using an extinction coefficient of 1.11 mL/mg*cm). In
some embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion molecule
(e.g., FGFR1-ECD.339-Fc) is administered at a dose of about 8 mg/kg
body weight, about 10 mg/kg body weight, about 11 mg/kg body
weight, about 12 mg/kg body weight, about 13 mg/kg body weight,
about 14 mg/kg body weight, about 15 mg/kg body weight, about 16
mg/kg body weight, about 17 mg/kg body weight, about 18 mg/kg body
weight, about 19 mg/kg body weight, or about 20 mg/kg body weight.
In some embodiments, the FGFR1 fusion protein is administered at a
dose of about 10 mg/kg body weight as calculated using an
extinction coefficient of 1.11 mL/mg*cm. In other embodiments, the
FGFR1 fusion protein is administered at a dose of about 20 mg/kg
body weight as calculated using an extinction coefficient of 1.11
mL/mg*cm. The FGFR1 ECD and/or FGFR1 ECD fusion molecules may also
be administered at ranges from one of the above doses to another.
In some embodiments, dosages may be administered twice a week,
weekly, every other week, at a frequency between weekly and every
other week, every three weeks, every four weeks, or every
month.
[0113] In certain embodiments, dosages of the FGFR1 ECD and/or
FGFR1 ECD fusion molecules can be calculated in two ways depending
on the extinction coefficient (EC) used. The extinction coefficient
differs depending on whether the glycosylation of the proteins is
taken into account. In one embodiment, the extinction coefficient
based on the amino acid composition of FGFR1-ECD.339-Fc, for
example, is 1.42 mL/mg*cm. In another embodiment, when the
carbohydrate portion as well as the amino acid portion of
FGFR1-ECD.339-Fc is accounted for, the extinction coefficient is
1.11 mL/mg*cm. Calculation of the FGFR1-ECD.339-Fc dose using an EC
of 1.11 mL/mg*cm increases the calculated dose by 28%, as shown in
Table 1. Although the doses calculated using the two extinction
coefficients are different, the molar concentrations, or the actual
amounts of drug administered, are identical. Unless otherwise
noted, the doses disclosed herein are each calculated using the
extinction coefficient that does not take account of glycosylation.
How these dosages compare to those calculated using the extinction
coefficient that takes account of glycosylation for
FGFR1-ECD.339-Fc is shown in Table 1. As can be seen from Table 1,
a dosage of about 8 mg/kg (e.g., 7.8 and 8.0) using an EC of 1.42
mL/mg*cm herein corresponds to a dosage of about 10 mg/kg (e.g.
10.0 and 10.2) when calculated using an EC of 1.11 mL/mg*cm. A
dosage of about 16 mg/kg (e.g. 15.6 and 16.0 mg/kg) using an EC of
1.42 mL/mg*cm herein corresponds to a dosage of about 20 mg/kg
(e.g. 20.0 and 20.5) when calculated using an EC of 1.11 mL/mg*cm.
As noted in the "Definitions" section above, measured numbers
provided herein are approximate and encompass values having
additional significant digits that are rounded off. For instance, 8
mg/kg encompasses values with two significant digits such as 7.6,
7.8, 8.0, 8.2, 8.4, and 8.45, each of which round to 8. Likewise, a
value such as 16 mg/kg encompasses values with three significant
digits that round to 16, such as, for example 15.6 and 16.0.
TABLE-US-00001 TABLE 1 Conversion of FGFR1-ECD.339-FC Dose
Dose.sup.a Dose.sup.a EC = 1.42 mL/mg * cm EC = 1.11 mL/mg * cm 0.5
0.6 0.75 1.0 1.0 1.3 2.0 2.6 3.0 3.8 4.0 5.1 5.0 6.4 6.0 7.7 7.0
9.0 7.8 10.0 8.0 10.2 9.0 11.5 10.0 12.8 11.0 14.1 12.0 15.4 13.0
16.6 14.0 17.9 15.0 19.2 15.6 20.0 16.0 20.5 17.0 21.8 18.0 23.0
19.0 24.3 20.0 25.6 30.0 38.4 .sup.aDoses shown in mg/kg.
[0114] The pharmaceutical compositions comprising FGFR1 ECDs, FGFR1
ECD fusion molecules, and/or at least one additional therapeutic
agent can be administered as needed to subjects. In certain
embodiments, an effective dose of a therapeutic molecule is
administered to a subject one or more times. In various
embodiments, an effective dose of a therapeutic molecule is
administered to the subject at least once every two months, at
least once a month, at least twice a month, once a week, twice a
week, or three times a week. In various embodiments, an effective
dose of a therapeutic molecule is administered to the subject for
at least a week, at least a month, at least three months, at least
six months, or at least a year.
[0115] In certain embodiments, the combined administration of an
FGFR1 ECDs, FGFR1 ECD fusion molecule and at least one additional
therapeutic agent includes concurrent administration, including
simultaneous administration, using separate formulations or a
single pharmaceutical formulation, as well as consecutive
administration in any order. Optionally there is a time period
while both (or all) active agents simultaneously exert their
biological activities. Therapeutically effective amounts of
therapeutic agents administered in combination with the FGFR1 ECD
and/or FGFR1 ECD fusion molecule (e.g., FGFR1-ECD.339-Fc) will be
at the physician's or veterinarian's discretion. Dosage
administration and adjustment is done to achieve maximal management
of the conditions to be treated. The dose will additionally depend
on such factors as the type of therapeutic agent to be used, the
specific patient being treated, the stage of the disease, and the
desired aggressiveness of the treatment regime.
[0116] In certain embodiments, a patient is treated with a
combination of the FGFR1 ECD and/or FGFR1 ECD fusion molecule
(e.g., FGFR1-ECD.339-Fc) and a VEGF antagonist. In some
embodiments, the VEGF antagonist is a VEGF trap (e.g.,
aflibercept). In some embodiments, the VEGF antagonist is a
tyrosine kinase inhibitor (e.g., pazopanib). In some embodiments,
the VEGF antagonist is an anti-VEGF antibody. In some embodiments,
the VEGF antibody is bevacizumab. One exemplary dosage of
bevacizumab is in the range from about 0.05 mg/kg to about 20
mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0
mg/kg, 7.5 mg/kg, 10 mg/kg or 15 mg/kg (or any combination thereof)
may be administered to the patient. Such doses may be administered
intermittently, e.g., every week, every two, or every three
weeks.
[0117] In some embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion
molecule (e.g., FGFR1-ECD.339-Fc) is administered in combination
with another therapeutic agent, such as chemotherapeutic agent or
anti-angiogenic agent, at the recommended or prescribed dosage
and/or frequency of the therapeutic agent.
[0118] In some embodiments, an additional therapeutic agent is
administered at a dosage approved by an agency responsible for
approving therapeutic treatments, such as the Food and Drug
Administration, or at the manfacturer's recommended dosage.
[0119] Routes of Administration and Carriers
[0120] In some embodiments, an FGFR1 ECD and/or FGFR1 ECD fusion
molecule can be administered intravenously and/or subcutaneously.
In some embodiments, an FGFR1 ECD and/or FGFR1 ECD fusion molecule
can be administered by another route, such as intra-arterial,
parenteral, intranasal, intramuscular, intracardiac,
intraventricular, intratracheal, buccal, rectal, intraperitoneal,
intradermal, topical, transdermal, or intrathecal, or otherwise by
implantation or inhalation. In various embodiments, at least one
additional therapeutic agent can be administered in vivo by a
variety of routes, including intravenous, intra-arterial,
subcutaneous, parenteral, intranasal, intramuscular, intracardiac,
intraventricular, intratracheal, buccal, rectal, intraperitoneal,
intradermal, topical, transdermal, and intrathecal, or otherwise by
implantation or inhalation. Each of the subject compositions can be
formulated alone or in combination into preparations in solid,
semi-solid, liquid, or gaseous forms, such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, enemas,
injections, inhalants, and aerosols.
[0121] In various embodiments, compositions comprising an FGFR1
ECD, FGFR1 ECD fusion molecule, and/or at least one additional
therapeutic agent are provided in formulation with pharmaceutically
acceptable carriers, a wide variety of which are known in the art
(see, e.g., Gennaro, Remington: The Science and Practice of
Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed.
(2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery
Systems, 7.sup.th ed., Lippencott Williams and Wilkins (2004);
Kibbe et al., Handbook of Pharmaceutical Excipients, 3.sup.rd ed.,
Pharmaceutical Press (2000)). Various pharmaceutically acceptable
carriers, which include vehicles, adjuvants, carriers, and
diluents, are available to the public. Moreover, various
pharmaceutically acceptable auxiliary substances, such as pH
adjusting and buffering agents, tonicity adjusting agents,
stabilizers, wetting agents and the like, are also available to the
public. Certain non-limiting exemplary carriers include saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof. In some embodiments, a therapeutic agent is
formulated as the brand-name drug indicated above in the
Definitions section, or a generic equivalent. In some embodiments,
docetaxel is formulated as Taxotere.RTM. (Sanofi Aventis) or a
generic equivalent.
[0122] In various embodiments, compositions comprising FGFR1 ECDs,
FGFR1 ECD fusion molecules, and/or at least one additional
therapeutic agent can be formulated for injection by dissolving,
suspending, or emulsifying them in an aqueous or nonaqueous
solvent, such as vegetable or other oils, synthetic aliphatic acid
glycerides, esters of higher aliphatic acids, or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers
and preservatives. In various embodiments, the compositions may be
formulated for inhalation, for example, using pressurized
acceptable propellants such as dichlorodifluoromethane, propane,
nitrogen, and the like. The compositions may also be formulated, in
various embodiments, into sustained release microcapsules, such as
with biodegradable or non-biodegradable polymers. A non-limiting
exemplary biodegradable formulation includes poly lactic
acid-glycolic acid polymer. A non-limiting exemplary
non-biodegradable formulation includes a polyglycerin fatty acid
ester. Certain methods of making such formulations are described,
for example, in EP 1 125 584 A1.
[0123] Pharmaceutical dosage packs comprising one or more
containers, each containing one or more doses of an FGFR1 ECD, an
FGFR1 ECD fusion molecule, and/or at least one additional
therapeutic agent are also provided. In certain embodiments, a unit
dosage is provided wherein the unit dosage contains a predetermined
amount of a composition comprising an FGFR1 ECD, an FGFR1 ECD
fusion molecule, and/or at least one additional therapeutic agent
with or without one or more additional agents. In certain
embodiments, such a unit dosage is supplied in single-use prefilled
syringe for injection. In various embodiments, the composition
contained in the unit dosage may comprise saline, sucrose, or the
like; a buffer, such as phosphate, or the like; and/or be
formulated within a stable and effective pH range. Alternatively,
in certain embodiments, the composition may be provided as a
lyophilized powder that can be reconstituted upon addition of an
appropriate liquid, for example, sterile water. In certain
embodiments, a composition comprises one or more substances that
inhibit protein aggregation, including, but not limited to, sucrose
and arginine. In certain embodiments, a composition of the
invention comprises heparin and/or a proteoglycan.
[0124] In some embodiments, a dosage pack comprises instructions to
determine whether a cancer comprises an FGFR1 gene amplification
and/or overexpresses at least one, at least two, at least three, or
at least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4 prior to administering an FGFR1 ECD and/or an FGFR1
ECD fusion molecule. In some embodiments, FGFR1 is FGFR1IIIc. In
some such embodiments, the instructions indicate that the presence
of an FGFR1 gene amplification and/or overexpression of at least
one, at least two, at least three, or at least four markers
selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 in at
least a portion of the cancer cells is indicative of therapeutic
responsiveness to an FGFR1 ECD and/or an FGFR1 ECD fusion molecule.
In some embodiments, the instructions indicate that the presence of
at least four copies of an FGFR1 gene in at least a portion of the
cancer cells is indicative of therapeutic responsiveness to an
FGFR1 ECD and/or an FGFR1 ECD fusion molecule. In some embodiments,
the instructions indicate that the presence of at least four, at
least six, at least eight, or at least ten copies of an FGFR1 gene
in at least a portion of the cancer cells is indicative of
therapeutic responsiveness to an FGFR1 ECD and/or an FGFR1 ECD
fusion molecule. In some embodiments, the instructions indicate
that a ratio of FGFR1 gene to chromosome 8 centromere of at least 2
in at least a portion of the cancer cells is indicative of
therapeutic responsiveness to an FGFR1 ECD and/or an FGFR1 ECD
fusion molecule. In some embodiments, the instructions indicate
that a ratio of FGFR1 gene to chromosome 8 centromere of at least
2.5, at least 3, at least 3.5, or at least 4 in at least a portion
of the lung cancer cells is indicative of therapeutic
responsiveness to an FGFR1 ECD and/or an FGFR1 ECD fusion
molecule.
[0125] In some embodiments, a dosage pack comprises instructions to
determine whether a lung cancer comprises an FGFR1 gene
amplification and/or overexpresses at least one, at least two, at
least three, or at least four markers selected from FGFR1,
FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4 prior to administering an
FGFR1 ECD and/or an FGFR1 ECD fusion molecule. In some embodiments,
FGFR1 is FGFR1IIIc. In some such embodiments, the instructions
indicate that the presence of an FGFR1 gene amplification and/or
overexpression of at least one, at least two, at least three, or at
least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4 in at least a portion of the lung cancer cells is
indicative of therapeutic responsiveness to an FGFR1 ECD and/or an
FGFR1 ECD fusion molecule. In some embodiments, the instructions
indicate that the presence of at least four copies of an FGFR1 gene
in at least a portion of the lung cancer cells is indicative of
therapeutic responsiveness to an FGFR1 ECD and/or an FGFR1 ECD
fusion molecule. In some embodiments, the instructions indicate
that the presence of at least four, at least six, at least eight,
or at least ten copies of an FGFR1 gene in at least a portion of
the lung cancer cells is indicative of therapeutic responsiveness
to an FGFR1 ECD and/or an FGFR1 ECD fusion molecule. In some
embodiments, the instructions indicate that a ratio of FGFR1 gene
to chromosome 8 centromere of at least 2 in at least a portion of
the lung cancer cells is indicative of therapeutic responsiveness
to an FGFR1 ECD and/or an FGFR1 ECD fusion molecule. In some
embodiments, the instructions indicate that a ratio of FGFR1 gene
to chromosome 8 centromere of at least 2.5, at least 3, at least
3.5, or at least 4 in at least a portion of the lung cancer cells
is indicative of therapeutic responsiveness to an FGFR1 ECD and/or
an FGFR1 ECD fusion molecule.
[0126] The term "instructions," as used herein includes, but is not
limited to, labels, package inserts, instructions available in
electronic form such as on a computer readable medium (e.g., a
diskette, compact disk, or DVD), instructions available remotely
such as over the internet, etc. A dosage pack is considered to
include the instructions when the dosage pack provides access to
the instructions, a link to the instructions (such as a uniform
resource locator, or url), or other mechanism for obtaining a copy
of the instructions (such as a return reply card, a physical
address from which instructions may be requested, an e-mail address
from which instructions may be requested, a phone number that may
be called to obtain instructions, etc.).
[0127] FGFR1 ECDs and FGFR1 ECD Fusion Molecules
[0128] Nonlimiting exemplary FGFR1 ECDs include full-length FGFR1
ECDs, FGFR1 ECD fragments, and FGFR1 ECD variants. FGFR1 ECDs may
include or lack a signal peptide. Exemplary FGFR1 ECDs include, but
are not limited to, FGFR1 ECDs having amino acid sequences selected
from SEQ ID NOs.: 1, 2, 3, and 4.
[0129] Non-limiting exemplary FGFR1 ECD fragments include human
FGFR1 ECD ending at amino acid 339 (counting from the first amino
acid of the mature form, without the signal peptide). In some
embodiments, an FGFR1 ECD fragment ends at an amino acid between
amino acid 339 and amino acid 360 (counting from the first amino
acid of the mature form, without the signal peptide). Exemplary
FGFR1 ECD fragments include, but are not limited to, FGFR1 ECD
fragments having amino acid sequences selected from SEQ ID NOs.: 3
and 4.
[0130] In some embodiments, an FGFR1 ECD comprises a sequence
selected from SEQ ID NOs: 1 to 4. In some embodiments, an FGFR1 ECD
consists of a sequence selected from SEQ ID NOs: 1 to 4. When an
FGFR1 ECD "consists of" a sequence selected from SEQ ID NOs: 1 to
4, the FGFR1 ECD may or may not contain various post-translational
modifications, such as glycosylation and sialylation. In other
words, when an FGFR1 ECD consists of a particular amino acid
sequence, it does not contain additional amino acids in the
contiguous amino acid sequence, but may contain modifications to
amino acid side chains, the N-terminal amino group, and/or the
C-terminal carboxy group.
[0131] In some embodiments, an FGFR1 ECD fusion molecule comprises
a signal peptide. In some embodiments, an FGFR1 ECD fusion molecule
lacks a signal peptide. In some embodiments, the FGFR1 ECD portion
of an FGFR1 ECD fusion molecule comprises a sequence selected from
SEQ ID NOs: 1 to 4. In some embodiments, the FGFR1 ECD portion of
an FGFR1 ECD fusion molecule consists of a sequence selected from
SEQ ID NOs: 1 to 4. When an FGFR1 ECD portion of an FGFR1 ECD
fusion molecule "consists of" a sequence selected from SEQ ID NOs:
1 to 4, the FGFR1 ECD portion of an FGFR1 ECD fusion molecule may
or may not contain various post-translational modifications, such
as glycosylation and sialylation. In other words, when an FGFR1 ECD
portion of an FGFR1 ECD fusion molecule consists of a particular
amino acid sequence, it does not contain additional amino acids
from FGFR1 in the contiguous amino acid sequence, but may contain
modifications to amino acid side chains, the N-terminal amino
group, and/or the C-terminal carboxy group. Further, because the
FGFR1 ECD is linked to a fusion molecule, there may be additional
amino acids at the N- and/or C-terminus of the FGFR1 ECD, but those
amino acids are not from the FGFR1 sequence, but may be from, for
example, a linker sequence, or a fusion partner sequence.
[0132] In some embodiments, the fusion partner portion of an FGFR1
ECD fusion molecule is selected from Fc, albumin, and polyethylene
glycol. Nonlimiting exemplary fusion partners are discussed
herein.
[0133] The inventors have found that administration of an FGFR1 ECD
and/or an FGFR1 ECD fusion molecule and at least one additional
therapeutic agent selected from docetaxel, paclitaxel, vincristine,
carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil
(5-FU), leucovorin, pemetrexed, sorafenib, etoposide, topotecan, a
VEGF antagonist, pazopanib, an anti-VEGF antibody, a VEGF trap, and
bevacizumab is useful for treating cancers in which at least a
portion of the cancer cells have FGFR1 gene amplification and/or
overexpress at least one, at least two, at least three, or at least
four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and
ETV4. In some embodiments, FGFR1 is FGFR1IIIc. In some embodiments,
an FGFR1 ECD and/or an FGFR1 ECD fusion molecule is administered
with docetaxel.
[0134] Fusion Partners and Conjugates
[0135] As discussed herein, an FGFR1 ECD may be combined with at
least one fusion partner, resulting in an FGFR1 ECD fusion
molecule. These fusion partners may facilitate purification, and
the FGFR1 ECD fusion molecules may show an increased half-life in
vivo. Suitable fusion partners of an FGFR1 ECD include, for
example, polymers, such as water soluble polymers, the constant
domain of immunoglobulins; all or part of human serum albumin
(HSA); fetuin A; fetuin B; a leucine zipper domain; a tetranectin
trimerization domain; mannose binding protein (also known as
mannose binding lectin), for example, mannose binding protein 1;
and an Fc region, as described herein and further described in U.S.
Pat. No. 6,686,179. Nonlimiting exemplary FGFR1 ECD fusion
molecules are described, e.g., in U.S. Pat. No. 7,678,890.
[0136] An FGFR1 ECD fusion molecule may be prepared by attaching
polyaminoacids or branch point amino acids to the FGFR1 ECD. For
example, the polyaminoacid may be a carrier protein that serves to
increase the circulation half life of the FGFR1 ECD (in addition to
the advantages achieved via a fusion molecule). For the therapeutic
purpose of the present invention, such polyaminoacids should
ideally be those that have or do not create neutralizing antigenic
responses, or other adverse responses. Such polyaminoacids may be
chosen from serum albumin (such as HSA), an additional antibody or
portion thereof, for example the Fc region, fetuin A, fetuin B,
leucine zipper nuclear factor erythroid derivative-2 (NFE2),
neuroretinal leucine zipper, tetranectin, or other polyaminoacids,
for example, lysines. As described herein, the location of
attachment of the polyaminoacid may be at the N terminus or C
terminus, or other places in between, and also may be connected by
a chemical linker moiety to the selected molecule.
[0137] Polymers
[0138] Polymers, for example, water soluble polymers, may be useful
as fusion partners to reduce precipitation of the FGFR1 ECD fusion
molecule in an aqueous environment, such as typically found in a
physiological environment. Polymers employed in the invention will
be pharmaceutically acceptable for the preparation of a therapeutic
product or composition.
[0139] Suitable, clinically acceptable, water soluble polymers
include, but are not limited to, polyethylene glycol (PEG),
polyethylene glycol propionaldehyde, copolymers of ethylene
glycol/propylene glycol, monomethoxy-polyethylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol (PVA), polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, poly (.beta.-amino acids)
(either homopolymers or random copolymers), poly(n-vinyl
pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers
(PPG) and other polyakylene oxides, polypropylene oxide/ethylene
oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol)
and other polyoxyethylated polyols, polyoxyethylated sorbitol, or
polyoxyethylated glucose, colonic acids or other carbohydrate
polymers, Ficoll, or dextran and mixtures thereof.
[0140] As used herein, polyethylene glycol (PEG) is meant to
encompass any of the forms that have been used to derivatize other
proteins, such as mono-(C.sub.1-C.sub.10) alkoxy- or
aryloxy-polyethylene glycol. Polyethylene glycol propionaldehyde
may have advantages in manufacturing due to its stability in
water.
[0141] Polymers used herein, for example water soluble polymers,
may be of any molecular weight and may be branched or unbranched.
In some embodiments, the polymers have an average molecular weight
of between about 2 kDa to about 100 kDa (the term "about"
indicating that in preparations of a polymer, some molecules will
weigh more, some less, than the stated molecular weight). The
average molecular weight of each polymer may be between about 5 kDa
and about 50 kDa, or between about 12 kDa and about 25 kDa.
Generally, the higher the molecular weight or the more branches,
the higher the polymer:protein ratio. Other sizes may also be used,
depending on the desired therapeutic profile; for example, the
duration of sustained release; the effects, if any, on biological
activity; the ease in handling; the degree or lack of antigenicity;
and other known effects of a polymer on an FGFR1 ECD.
[0142] Polymers employed in the present invention are typically
attached to an FGFR1 ECD with consideration of effects on
functional or antigenic domains of the polypeptide. In general,
chemical derivatization may be performed under any suitable
condition used to react a protein with an activated polymer
molecule. Activating groups which can be used to link the polymer
to the active moieties include sulfone, maleimide, sulfhydryl,
thiol, triflate, tresylate, azidirine, oxirane, and 5-pyridyl.
[0143] Polymers of the invention are typically attached to a
heterologous polypeptide at the alpha (.alpha.) or epsilon
(.epsilon.) amino groups of amino acids or a reactive thiol group,
but it is also contemplated that a polymer group could be attached
to any reactive group of the protein that is sufficiently reactive
to become attached to a polymer group under suitable reaction
conditions. Thus, a polymer may be covalently bound to an FGFR1 ECD
via a reactive group, such as a free amino or carboxyl group. The
amino acid residues having a free amino group may include lysine
residues and the N-terminal amino acid residue. Those having a free
carboxyl group may include aspartic acid residues, glutamic acid
residues, and the C-terminal amino acid residue. Those having a
reactive thiol group include cysteine residues.
[0144] Methods for preparing fusion molecules conjugated with
polymers, such as water soluble polymers, will each generally
involve (a) reacting an FGFR1 ECD with a polymer under conditions
whereby the polypeptide becomes attached to one or more polymers
and (b) obtaining the reaction product. Reaction conditions for
each conjugation may be selected from any of those known in the art
or those subsequently developed, but should be selected to avoid or
limit exposure to reaction conditions such as temperatures,
solvents, and pH levels that would inactivate the protein to be
modified. In general, the optimal reaction conditions for the
reactions will be determined case-by-case based on known parameters
and the desired result. For example, the larger the ratio of
polymer:polypeptide conjugate, the greater the percentage of
conjugated product. The optimum ratio (in terms of efficiency of
reaction in that there is no excess unreacted polypeptide or
polymer) may be determined by factors such as the desired degree of
derivatization (e.g., mono-, di-, tri-, etc.), the molecular weight
of the polymer selected, whether the polymer is branched or
unbranched and the reaction conditions used. The ratio of polymer
(for example, PEG) to a polypeptide will generally range from 1:1
to 100:1. One or more purified conjugates may be prepared from each
mixture by standard purification techniques, including among
others, dialysis, salting-out, ultrafiltration, ion-exchange
chromatography, gel filtration chromatography, and
electrophoresis.
[0145] One may specifically desire an N-terminal chemically
modified FGFR1 ECD. One may select a polymer by molecular weight,
branching, etc., the proportion of polymers to FGFR1 ECD molecules
in the reaction mix, the type of reaction to be performed, and the
method of obtaining the selected N-terminal chemically modified
FGFR1 ECD. The method of obtaining the N-terminal chemically
modified FGFR1 ECD preparation (separating this moiety from other
monoderivatized moieties if necessary) may be by purification of
the N-terminal chemically modified FGFR1 ECD material from a
population of chemically modified protein molecules.
[0146] Selective N-terminal chemical modification may be
accomplished by reductive alkylation which exploits differential
reactivity of different types of primary amino groups (lysine
versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, substantially
selective derivatization of the protein at the N terminus with a
carbonyl group-containing polymer is achieved. For example, one may
selectively attach a polymer to the N terminus of the protein by
performing the reaction at a pH that allows one to take advantage
of the pKa differences between the e-amino group of the lysine
residues and that of the .alpha.-amino group of the N-terminal
residue of the protein. By such selective derivatization,
attachment of a polymer to a protein is controlled: the conjugation
with the polymer takes place predominantly at the N terminus of the
protein and no significant modification of other reactive groups,
such as the lysine side chain amino groups, occurs. Using reductive
alkylation, the polymer may be of the type described above and
should have a single reactive aldehyde for coupling to the protein.
Polyethylene glycol propionaldehyde, containing a single reactive
aldehyde, may also be used.
[0147] In one embodiment, the present invention contemplates the
chemically derivatized FGFR1 ECD to include mono- or poly- (e.g.,
2-4) PEG moieties. Pegylation may be carried out by any of the
pegylation reactions available. Methods for preparing a pegylated
protein product will generally include (a) reacting a polypeptide
with polyethylene glycol (such as a reactive ester or aldehyde
derivative of PEG) under conditions whereby the protein becomes
attached to one or more PEG groups; and (b) obtaining the reaction
product(s). In general, the optimal reaction conditions will be
determined case by case based on known parameters and the desired
result.
[0148] There are a number of PEG attachment methods known in the
art. See, for example, EP 0 401 384; Malik et al., Exp. Hematol.,
20:1028-1035 (1992); Francis, Focus on Growth Factors, 3(2):4-10
(1992); EP 0 154 316; EP 0 401 384; WO 92/16221; WO 95/34326; and
the other publications cited herein that relate to pegylation.
[0149] Pegylation may be carried out, e.g., via an acylation
reaction or an alkylation reaction with a reactive polyethylene
glycol molecule. Thus, protein products according to the present
invention include pegylated proteins wherein the PEG group(s) is
(are) attached via acyl or alkyl groups. Such products may be
mono-pegylated or poly-pegylated (for example, those containing 2-6
or 2-5 PEG groups). The PEG groups are generally attached to the
protein at the .alpha.- or .epsilon.-amino groups of amino acids,
but it is also contemplated that the PEG groups could be attached
to any amino group attached to the protein that is sufficiently
reactive to become attached to a PEG group under suitable reaction
conditions.
[0150] Pegylation by acylation generally involves reacting an
active ester derivative of polyethylene glycol (PEG) with an FGFR1
ECD. For acylation reactions, the polymer(s) selected typically
have a single reactive ester group. Any known or subsequently
discovered reactive PEG molecule may be used to carry out the
pegylation reaction. An example of a suitable activated PEG ester
is PEG esterified to N-hydroxysuccinimide (NHS). As used herein,
acylation is contemplated to include, without limitation, the
following types of linkages between the therapeutic protein and a
polymer such as PEG: amide, carbamate, urethane, and the like, see
for example, Chamow, Bioconjugate Chem., 5:133-140 (1994). Reaction
conditions may be selected from any of those currently known or
those subsequently developed, but should avoid conditions such as
temperature, solvent, and pH that would inactivate the polypeptide
to be modified.
[0151] Pegylation by acylation will generally result in a
poly-pegylated protein. The connecting linkage may be an amide. The
resulting product may be substantially only (e.g., >95%) mono-,
di-, or tri-pegylated. However, some species with higher degrees of
pegylation may be formed in amounts depending on the specific
reaction conditions used. If desired, more purified pegylated
species may be separated from the mixture (particularly unreacted
species) by standard purification techniques, including among
others, dialysis, salting-out, ultrafiltration, ion-exchange
chromatography, gel filtration chromatography, and
electrophoresis.
[0152] Pegylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with a polypeptide in the
presence of a reducing agent. For the reductive alkylation
reaction, the polymer(s) selected should have a single reactive
aldehyde group. An exemplary reactive PEG aldehyde is polyethylene
glycol propionaldehyde, which is water stable, or mono
C.sub.1-C.sub.10 alkoxy or aryloxy derivatives thereof, see for
example, U.S. Pat. No. 5,252,714.
[0153] Markers
[0154] Moreover, FGFR1 ECDs of the present invention may be fused
to marker sequences, such as a peptide that facilitates
purification of the fused polypeptide. The marker amino acid
sequence may be a hexa-histidine peptide such as the tag provided
in a pQE vector (Qiagen, Mississauga, Ontario, Canada), among
others, many of which are commercially available. As described in
Gentz et al., Proc. Nall. Acad. Sci. 86:821-824 (1989), for
instance, hexa-histidine provides for convenient purification of
the fusion protein. Another peptide tag useful for purification,
the hemagglutinin (HA) tag, corresponds to an epitope derived from
the influenza HA protein. (Wilson et al., Cell 37:767 (1984)). Any
of these above fusions may be engineered using the FGFR1 ECDs
described herein.
[0155] Oligomerization Domain Fusion Partners
[0156] In various embodiments, oligomerization offers some
functional advantages to a fusion protein, including, but not
limited to, multivalency, increased binding strength, and the
combined function of different domains. Accordingly, in some
embodiments, a fusion partner comprises an oligomerization domain,
for example, a dimerization domain. Exemplary oligomerization
domains include, but are not limited to, coiled-coil domains,
including alpha-helical coiled-coil domains; collagen domains;
collagen-like domains; and certain immunoglobulin domains.
Exemplary coiled-coil polypeptide fusion partners include, but are
not limited to, the tetranectin coiled-coil domain; the coiled-coil
domain of cartilage oligomeric matrix protein; angiopoietin
coiled-coil domains; and leucine zipper domains. Exemplary collagen
or collagen-like oligomerization domains include, but are not
limited to, those found in collagens, mannose binding lectin, lung
surfactant proteins A and D, adiponectin, ficolin, conglutinin,
macrophage scavenger receptor, and emilin.
[0157] Antibody Fc Immunoglobulin Domain Fusion Partners
[0158] Many Fc domains that may be used as fusion partners are
known in the art. In some embodiments, a fusion partner is an Fc
immunoglobulin domain. An Fc fusion partner may be a wild-type Fc
found in a naturally occurring antibody, a variant thereof, or a
fragment thereof. Non-limiting exemplary Fc fusion partners include
Fcs comprising a hinge and the CH2 and CH3 constant domains of a
human IgG, for example, human IgG1, IgG2, IgG3, or IgG4. Additional
exemplary Fc fusion partners include, but are not limited to, human
IgA and IgM. In some embodiments, an Fc fusion partner comprises a
C237S mutation, for example, in an IgG1 (see, for example, SEQ ID
NO: 8). In some embodiments, an Fc fusion partner comprises a
hinge, CH2, and CH3 domains of human IgG2 with a P331S mutation, as
described in U.S. Pat. No. 6,900,292. Certain exemplary Fc domain
fusion partners are shown in SEQ ID NOs: 8 to 10.
[0159] Albumin Fusion Partners and Albumin-binding Molecule Fusion
Partners
[0160] In some embodiments, a fusion partner is an albumin.
Exemplary albumins include, but are not limited to, human serum
album (HSA) and fragments of HSA that are capable of increasing the
serum half-life or bioavailability of the polypeptide to which they
are fused. In some embodiments, a fusion partner is an
albumin-binding molecule, such as, for example, a peptide that
binds albumin or a molecule that conjugates with a lipid or other
molecule that binds albumin. In some embodiments, a fusion molecule
comprising HSA is prepared as described, e.g., in U.S. Pat. No.
6,686,179.
[0161] Exemplary Attachment of Fusion Partners
[0162] The fusion partner may be attached, either covalently or
non-covalently, to the N terminus or the C terminus of the FGFR1
ECD. The attachment may also occur at a location within the FGFR1
ECD other than the N terminus or the C terminus, for example,
through an amino acid side chain (such as, for example, the side
chain of cysteine, lysine, serine, or threonine).
[0163] In either covalent or non-covalent attachment embodiments, a
linker may be included between the fusion partner and the FGFR1
ECD. Such linkers may be comprised of at least one amino acid or
chemical moiety. Exemplary methods of covalently attaching a fusion
partner to an FGFR1 ECD include, but are not limited to,
translation of the fusion partner and the FGFR1 ECD as a single
amino acid sequence and chemical attachment of the fusion partner
to the FGFR1 ECD. When the fusion partner and an FGFR1 ECD are
translated as single amino acid sequence, additional amino acids
may be included between the fusion partner and the FGFR1 ECD as a
linker. In some embodiments, the linker is selected based on the
polynucleotide sequence that encodes it, to facilitate cloning the
fusion partner and/or FGFR1 ECD into a single expression construct
(for example, a polynucleotide containing a particular restriction
site may be placed between the polynucleotide encoding the fusion
partner and the polynucleotide encoding the FGFR1 ECD, wherein the
polynucleotide containing the restriction site encodes a short
amino acid linker sequence). When the fusion partner and the FGFR1
ECD are covalently coupled by chemical means, linkers of various
sizes may typically be included during the coupling reaction.
[0164] Exemplary methods of non-covalently attaching a fusion
partner to an FGFR1 ECD include, but are not limited to, attachment
through a binding pair. Exemplary binding pairs include, but are
not limited to, biotin and avidin or streptavidin, an antibody and
its antigen, etc.
[0165] Co-Translational and Post-Translational Modifications
[0166] The invention encompasses administration of FGFR1 ECDs and
FGFR1 ECD fusion molecules that are differentially modified during
or after translation, for example by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or linkage to an
antibody molecule or other cellular ligand. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to, specific chemical cleavage by
cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease;
NABH.sub.4; acetylation; formylation; oxidation; reduction; and/or
metabolic synthesis in the presence of tunicamycin.
[0167] Additional post-translational modifications encompassed by
the invention include, for example, for example, N-linked or
O-linked carbohydrate chains, processing of N-terminal or
C-terminal ends), attachment of chemical moieties to the amino acid
backbone, chemical modifications of N-linked or O-linked
carbohydrate chains, and addition or deletion of an N-terminal
methionine residue as a result of prokaryotic host cell expression.
A nonlimiting discussion of various post-translational
modifications of FGFR1 ECDs and FGFR1 ECD fusion molecules can be
found, e.g., in U.S. Pat. No. 7,678,890.
[0168] FGFR1 ECD and FGFR1 ECD Fusion Molecule Expression and
Production Vectors
[0169] Vectors comprising polynucleotides that encode FGFR1 ECDs
are provided. Vectors comprising polynucleotides that encode FGFR1
ECD fusion molecules are also provided. Such vectors include, but
are not limited to, DNA vectors, phage vectors, viral vectors,
retroviral vectors, etc.
[0170] In some embodiments, a vector is selected that is optimized
for expression of polypeptides in CHO or CHO-derived cells.
Exemplary such vectors are described, e.g., in Running Deer et al.,
Biotechnol. Frog. 20:880-889 (2004).
[0171] In some embodiments, a vector is chosen for in vivo
expression of FGFR1 ECDs and/or FGFR1 ECD fusion molecules in
animals, including humans. In some such embodiments, expression of
the polypeptide is under the control of a promoter that functions
in a tissue-specific manner. For example, liver-specific promoters
are described, e.g., in PCT Publication No. WO 2006/076288. A
nonlimiting discussion of various expression vectors can be found,
e.g., in U.S. Pat. No. 7,678,890.
[0172] Host Cells
[0173] In various embodiments, FGFR1 ECDs or FGFR1 ECD fusion
molecules may be expressed in prokaryotic cells, such as bacterial
cells; or in eukaryotic cells, such as fungal cells, plant cells,
insect cells, and mammalian cells. Such expression may be carried
out, for example, according to procedures known in the art.
Exemplary eukaryotic cells that may be used to express polypeptides
include, but are not limited to, COS cells, including COS 7 cells;
293 cells, including 293-6E cells; CHO cells, including CHO--S and
DG44 cells; and NSO cells. In some embodiments, a particular
eukaryotic host cell is selected based on its ability to make
certain desired post-translational modifications to the FGFR1 ECDs
or FGFR1 ECD fusion molecules. For example, in some embodiments,
CHO cells produce FGFR1 ECDs and/or FGFR1 ECD fusion molecules that
have a higher level of sialylation than the same polypeptide
produced in 293 cells.
[0174] Introduction of a nucleic acid into a desired host cell may
be accomplished by any method known in the art, including but not
limited to, calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection, etc. Nonlimiting
exemplary methods are described, e.g., in Sambrook et al.,
Molecular Cloning, A Laboratory Manual, 3.sup.rd ed. Cold Spring
Harbor Laboratory Press (2001). Nucleic acids may be transiently or
stably transfected in the desired host cells, according to methods
known in the art. A nonlimiting discussion of host cells and
methods of polypeptides in host cells can be found, e.g., in U.S.
Pat. No. 7,678,890.
[0175] In some embodiments, a polypeptide may be produced in vivo
in an animal that has been engineered or transfected with a nucleic
acid molecule encoding the polypeptide, according to methods known
in the art.
[0176] Purification of FGFR1 ECD Polypeptides
[0177] FGFR1 ECDs or FGFR1 ECD fusion molecules may be purified by
various methods known in the art. Such methods include, but are not
limited to, the use of affinity matrices or hydrophobic interaction
chromatography. Suitable affinity ligands include any ligands of
the FGFR1 ECD or of the fusion partner. Suitable affinity ligands
in the case of an antibody that binds FGFR1 include, but are not
limited to, FGFR1 itself and fragments thereof. Further, a Protein
A, Protein G, Protein A/G, or an antibody affinity column may be
used to bind to an Fc fusion partner to purify an FGFR1 ECD fusion
molecule. Antibodies to FGFR1 ECD may also be used to purify FGFR1
ECD or FGFR1 ECD fusion molecules. Hydrophobic interactive
chromatography, for example, a butyl or phenyl column, may also
suitable for purifying some polypeptides. Many methods of purifying
polypeptides are known in the art. A nonlimiting discussion of
various methods of purifying polypeptides can be found, e.g., in
U.S. Pat. No. 7,678,890.
Methods of Identifying Patients Who Would Benefit from FGFR1 ECDs
and/or FGFR1 ECD Fusion Molecules
[0178] In some embodiments, methods of identifying patients with
cancer who may benefit from administration of an FGFR1 ECD or FGFR1
ECD fusion molecule are provided. In some such embodiments, the
method comprises determining whether at least a portion of the
cancer cells comprise an FGFR1 gene amplification in a sample
obtained from the subject. In some embodiments, FGFR1 gene
amplification is indicative of therapeutic responsiveness by the
cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule. In some
embodiments, a sample is taken from a patient having or suspected
of having cancer. A finding of FGFR1 gene amplification in at least
a portion of the cancer cells indicates that the patient having or
suspected of having cancer may benefit from an FGFR1 ECD or FGFR1
ECD fusion molecule therapy. In some embodiments, the patient has
or is suspected of having lung cancer.
[0179] In some embodiments, the method comprises determining
whether at least a portion of the cancer cells comprise
overexpression of at least one, at least two, at least three, or at
least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4 in a sample obtained from the subject. In some
embodiments, the overexpression is mRNA overexpression. In some
embodiments, the overexpression is protein overexpression. In some
embodiments, FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and/or ETV4
overexpression is indicative of therapeutic responsiveness by the
cancer to an FGFR1 ECD or FGFR1 ECD fusion molecule. In some
embodiments, a sample is taken from a patient having or suspected
of having cancer. A finding of FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18,
and/or ETV4 overexpression in at least a portion of the cancer
cells indicates that the patient having or suspected of having
cancer may benefit from an FGFR1 ECD or FGFR1 ECD fusion molecule
therapy. In some embodiments, FGFR1 is FGFR1IIIc. In some
embodiments, the patient has or is suspected of having lung
cancer.
[0180] In some embodiments, FGFR1 gene amplification and/or
overexpression of at least one, at least two, at least three, or at
least four markers selected from FGFR1, FGFR3IIIc, FGF2, DKK3,
FGF18, and ETV4 is determined by a laboratory. A laboratory may be
a hospital laboratory or a laboratory independent of a hospital. In
some embodiments, following a determination of FGFR1 gene
amplification and/or overexpression of at least one, at least two,
at least three, or at least four markers selected from FGFR1,
FGFR3IIIc, FGF2, DKK3, FGF18, and ETV4, the results of the
determination are communicated to a medical professional. In some
such embodiments, the results are communicated for the purpose of
determining whether a patient should benefit from, or be responsive
to, an FGFR1 ECD or FGFR1 ECD fusion molecule therapy. In some
embodiments, medical professionals include, but are not limited to,
doctors, nurses, hospital administration and staff, etc. In some
embodiments, FGFR1 is FGFR1IIIc.
[0181] Any suitable method of determining FGFR1 gene amplification
may be used. Nonlimiting exemplary such methods include
fluorescence in situ hybridization (FISH; see, e.g., Monni et al.
(2001) PNAS 98: 5711-5716), array comparative genomic hybridization
(aCGH), DNA microarrays (see, e.g., Carter et al. (2007)Nat. Genet.
39: S16-21), spectral karyotyping (SKY; see, e.g. Liyanage et al.
(1996) Nat. Genet. 14: 312-5), real-time quantitative PCR (see,
e.g., Dhaene et al. (2010) Methods 50: 262-270), southern blotting,
and sequencing, including, but not limited to, high-throughput
sequencing (HTS; see, e.g. Medvedev et al. (2010) Genome Res. 20:
1613-22), and next generation sequencing technologies such as
RNA-seq, also called "Whole Transcriptome Shotgun Sequencing"
("WTSS"), Applied Biosystems SOLiD.TM. System, Illumina (Solexa)
sequencing, Ion semiconductor sequencing, DNA nanoball sequencing,
Helioscope.TM. single molecule sequencing, Single Molecule SMRT.TM.
sequencing, Single Molecule real time (RNAP) sequencing, Nanopore
DNA sequencing, VisiGen Biotechnologies approach, and 454
pyrosequencing.
[0182] Fluorescence in situ hybridization (FISH) is a cytogenetic
technique to detect and localize the presence or absence of
specific DNA sequences on chromosomes. In some embodiments, FISH
uses fluorescent probes to detect certain regions of chromosomes in
a sequence-specific manner. Thus, in some embodiments, to detect
gene amplification in cancer using FISH, in some embodiments, a
fluorescent probe is developed that binds specifically to the gene
of interest, such as the FGFR1 gene. In some such embodiments, this
gene specific probe is hybridized to a cancer sample and the copy
number determined by counting the number of fluorescent signals
present per cell using fluorescence microscopy. For a normal
diploid cell, the majority of genes will have a copy number of two
(exceptions exist when the gene is present on one of the sex
chromosomes rather than an autosome or the cell is undergoing
division and the genome replicated). If more than two signals are
detected in a cell, in certain instances, the gene may be
amplified.
[0183] Dual color FISH may also be used for assessing gene
amplification in cancer. In some embodiments, a reference probe
that binds to the centromere region of the chromosome on which the
gene of interest is located can be used as a control. In some
instances, the centromere (CEN) region of a chromosome is
considered to be genomically stable and is therefore assumed to be
representative of the entire chromosome. CEN copy number can
therefore, in some embodiments, assist in distinguishing focal gene
amplification from increased gene copy number resulting from
polysomy (.gtoreq.3 copies of the chromosome centromere) of the
chromosome. Gene amplification can be distinguished from polysomy,
in some embodiments, by calculating the ratio the signal from the
gene-of-interest probe/signal from the centromere probe. For a
normal diploid cell, where the gene of interest in located on an
autosome, this ratio is typically 1. In some embodiments, a ratio
of >1 is indicative of gene amplification. In some embodiments,
a probe to a chromosomal reference gene can be used in place of, or
in addition to, a centromere probe (see, e.g., Tse et al. (2011) J.
Clin. Oncol. 29: 4168-74). In some embodiments, the selected
reference gene is also on chromosome 8. In some embodiments, the
reference gene is located close to the centromere of chromosome 8.
In some embodiments, the reference sequence comprises non-coding
DNA on chromosome 8.
[0184] In some embodiments, FISH allows the determination of
multiple parameters of gene amplification, including, but not
limited to, the fraction of cells with an amplified gene, the
amplification levels within various subpopulations of cells, and
the amplification pattern within a cell (for example, a clustered
signal versus multiple scattered signals). In some embodiments, the
ratio of the copy number of the gene of interest to the centromere
reference for each cancer cell is determined. In some such
embodiments, the mean ratio for a particular sample or subset of
cells in a sample is then calculated. A mean ratio of greater than
two is generally considered to indicate gene amplification, whereas
signals between 1.5 to 2 may indicate low-level amplification. In
some embodiments, cells that have a greater copy number of the gene
of interest than a reference control probe are considered amplified
(see, e.g., Kobayashi et al. (2002) Hum. Pathol. 33: 21-8; and
Kunitomo et al. (2002) Pathol. Int. 52: 451-7). In some
embodiments, single-color FISH is used to determine the copy number
of a gene of interest without a chromosomal reference probe
control. In some such embodiments, four or more copies of the gene
per nucleus is considered to be gene amplification (see, e.g.,
Couturier et al. (2000) Mod. Pathol. 13: 1238-43; Jacobs et al.
(1999) J. Clin. Oncol. 17: 1974-82; Wang et al. (2000) J. Clin.
Pathol. 53: 374-81).
[0185] Any suitable method of determining protein overexpression
(FGFR1, FGFR3IIIc, FGF2, DKK3, FGF18, and/or ETV4 overexpression)
may be used. In certain embodiments, the expression of proteins in
a sample is examined using immunohistochemistry ("IHC") and
staining protocols Immunohistochemical staining of tissue sections
has been shown to be a reliable method of assessing or detecting
presence of proteins in a sample. Immunohistochemistry techniques
utilize an antibody to probe and visualize cellular antigens in
situ, generally by chromogenic or fluorescent methods.
[0186] The tissue sample may be fixed (i.e. preserved) by
conventional methodology (See e.g., "Manual of Histological
Staining Method of the Armed Forces Institute of Pathology,"
3.sup.rd edition (1960) Lee G. Luna, H T (ASCP) Editor, The
Blakston Division McGraw-Hill Book Company, New York; The Armed
Forces Institute of Pathology Advanced Laboratory Methods in
Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed
Forces Institute of Pathology, American Registry of Pathology,
Washington, D.C.). One of skill in the art will appreciate that the
choice of a fixative is determined by the purpose for which the
sample is to be histologically stained or otherwise analyzed. One
of skill in the art will also appreciate that the length of
fixation depends upon the size of the tissue sample and the
fixative used. By way of example, neutral buffered formalin,
Bouin's or paraformaldehyde, may be used to fix a sample.
[0187] Generally, the sample is first fixed and is then dehydrated
through an ascending series of alcohols, infiltrated and embedded
with paraffin or other sectioning media so that the tissue sample
may be sectioned. Alternatively, one may section the tissue and fix
the sections obtained. By way of example, the tissue sample may be
embedded and processed in paraffin by conventional methodology (See
e.g., "Manual of Histological Staining Method of the Armed Forces
Institute of Pathology", supra). Examples of paraffin that may be
used include, but are not limited to, Paraplast, Broloid, and
Tissuemay. Once the tissue sample is embedded, the sample may be
sectioned by a microtome or the like (See e.g., "Manual of
Histological Staining Method of the Armed Forces Institute of
Pathology", supra). By way of example for this procedure, sections
may range from about three microns to about five microns in
thickness. Once sectioned, the sections may be attached to slides
by several standard methods. Examples of slide adhesives include,
but are not limited to, silane, gelatin, poly-L-lysine and the
like. By way of example, the paraffin embedded sections may be
attached to positively charged slides and/or slides coated with
poly-L-lysine.
[0188] If paraffin has been used as the embedding material, the
tissue sections are generally deparaffinized and rehydrated to
water. The tissue sections may be deparaffinized by several
conventional standard methodologies. For example, xylenes and a
gradually descending series of alcohols may be used (See e.g.,
"Manual of Histological Staining Method of the Armed Forces
Institute of Pathology", supra). Alternatively, commercially
available deparaffinizing non-organic agents such as Hemo-De7 (CMS,
Houston, Tex.) may be used.
[0189] In some embodiments, subsequent to the sample preparation, a
tissue section may be analyzed using IHC. IHC may be performed in
combination with additional techniques such as morphological
staining and/or fluorescence in-situ hybridization. Two general
methods of IHC are available; direct and indirect assays. According
to the first assay, binding of antibody to the target antigen is
determined directly. This direct assay uses a labeled reagent, such
as a fluorescent tag or an enzyme-labeled primary antibody, which
can be visualized without further antibody interaction. In a
typical indirect assay, unconjugated primary antibody binds to the
antigen and then a labeled secondary antibody binds to the primary
antibody. Where the secondary antibody is conjugated to an
enzymatic label, a chromogenic or fluorogenic substrate is added to
provide visualization of the antigen. Signal amplification occurs
because several secondary antibodies may react with different
epitopes on the primary antibody.
[0190] The primary and/or secondary antibody used for
immunohistochemistry typically will be labeled with a detectable
moiety. Numerous labels are available which can be generally
grouped into the following categories: (a) Radioisotopes, such as
.sup.35S, .sup.14C, .sup.125I, .sup.3H, and .sup.131I. The antibody
can be labeled with the radioisotope using the techniques described
in Current Protocols in Immunology, Volumes 1 and 2, Coligen et
al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for
example and radioactivity can be measured using scintillation
counting. (b) Colloidal gold particles. (c) Fluorescent labels
including, but are not limited to, rare earth chelates (europium
chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine,
umbelliferone, phycocrytherin, phycocyanin, or commercially
available fluorophores such SPECTRUM ORANGE7 and SPECTRUM GREEN7
and/or derivatives of any one or more of the above. The fluorescent
labels can be conjugated to the antibody using the techniques
disclosed in Current Protocols in Immunology, supra, for example.
fluorescence can be quantified using a fluorimeter. (d) Various
enzyme-substrate labels are available and U.S. Pat. No. 4,275,149
provides a review of some of these. The enzyme generally catalyzes
a chemical alteration of the chromogenic substrate that can be
measured using various techniques. For example, the enzyme may
catalyze a color change in a substrate, which can be measured
spectrophotometrically. Alternatively, the enzyme may alter the
fluorescence or chemiluminescence of the substrate. Techniques for
quantifying a change in fluorescence are described above. The
chemiluminescent substrate becomes electronically excited by a
chemical reaction and may then emit light which can be measured
(using a chemiluminometer, for example) or donates energy to a
fluorescent acceptor. Examples of enzymatic labels include
luciferases (e.g., firefly luciferase and bacterial luciferase;
U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,
malate dehydrogenase, urease, peroxidase such as horseradish
peroxidase (HRPO), alkaline phosphatase, .beta.-galactosidase,
glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase,
galactose oxidase, and glucose-6-phosphate dehydrogenase),
heterocyclic oxidases (such as uricase and xanthine oxidase),
lactoperoxidase, microperoxidase, and the like. Techniques for
conjugating enzymes to antibodies are described in O'Sullivan et
al., Methods for the Preparation of Enzyme-Antibody Conjugates for
use in Enzyme Immunoassay, in Methods in Enzym. (ed. J. Langone
& H. Van Vunakis), Academic press, New York, 73:147-166
(1981).
[0191] Examples of enzyme-substrate combinations include, for
example: (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase
as a substrate, wherein the hydrogen peroxidase oxidizes a dye
precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); (ii) alkaline
phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic
substrate; and (iii).beta.-D-galactosidase (.beta.-D-Gal) with a
chromogenic substrate (e.g., p-nitrophenyl-.beta.-D-galactosidase)
or fluorogenic substrate (e.g.,
4-methylumbelliferyl-.beta.-D-galactosidase).
[0192] Numerous other enzyme-substrate combinations are available
to those skilled in the art. For a general review of these, see
U.S. Pat. Nos. 4,275,149 and 4,318,980. Sometimes, the label is
indirectly conjugated with the antibody. The skilled artisan will
be aware of various techniques for achieving this. For example, the
antibody can be conjugated with biotin and any of the four broad
categories of labels mentioned above can be conjugated with avidin,
or vice versa. Biotin binds selectively to avidin and thus, the
label can be conjugated with the antibody in this indirect manner.
Alternatively, to achieve indirect conjugation of the label with
the antibody, the antibody is conjugated with a small hapten and
one of the different types of labels mentioned above is conjugated
with an anti-hapten antibody. Thus, indirect conjugation of the
label with the antibody can be achieved.
[0193] Aside from the sample preparation procedures discussed
above, further treatment of the tissue section prior to, during or
following IHC may be desired. For example, epitope retrieval
methods, such as heating the tissue sample in citrate buffer may be
carried out (see, e.g., Leong et al. Appl. Immunohistochem.
4(3):201 (1996)).
[0194] Following an optional blocking step, the tissue section is
exposed to primary antibody for a sufficient period of time and
under suitable conditions such that the primary antibody binds to
the target protein antigen in the tissue sample. Appropriate
conditions for achieving this can be determined by routine
experimentation. The extent of binding of antibody to the sample is
determined by using any one of the detectable labels discussed
above. In some embodiments, the label is an enzymatic label (e.g.
HRPO) which catalyzes a chemical alteration of the chromogenic
substrate such as 3,3'-diaminobenzidine chromogen. In one
embodiment, the enzymatic label is conjugated to antibody which
binds specifically to the primary antibody (e.g. the primary
antibody is rabbit polyclonal antibody and secondary antibody is
goat anti-rabbit antibody).
[0195] Specimens thus prepared may be mounted and coverslipped.
Slide evaluation is then determined, e.g., using a microscope, and
staining intensity criteria, routinely used in the art, may be
employed.
[0196] In some embodiments, when IHC is used, a tiered system of
staining is used to determine whether a cell or collection of cells
overexpresses FGFR1 protein. For example, in some embodiments, a
four-tiered system is used in which the tiers are no staining, 1+,
2+, and 3+, where 1+, 2+, and 3+ indicate increasing levels of
staining, respectively. In some such embodiments, greater than 1+,
greater than 2+, or greater than 3+ may be used to indicate FGFR1
protein overexpression. As a nonlimiting example, if a particular
cell type typically shows no staining for FGFR1 in an IHC assay,
then any staining in that IHC assay (i.e., 1+, 2+, or 3+) may be
indicative as protein overexpression. As a further nonlimiting
example, if a particular cell type typically shows little to no
staining for FGFR1 in an IHC assay, then any staining above 1+ in
that IHC assay (i.e., 2+ or 3+) may be indicative as protein
overexpression. One skilled in the art can determine the staining
level that indicates protein overexpression depending on the
particular IHC assay (including the particular antibody), the cell
type, etc.
[0197] Any suitable method of determining mRNA overexpression (such
as FGFR1 overexpression, and/or FGF2 overexpression, and/or DKK3
overexpression, and/or FGF18 overexpression, and/or ETV4
overexpression) may be used. Methods for the evaluation of mRNAs in
cells are well known and include, for example, hybridization assays
using complementary DNA probes (such as in situ hybridization using
labeled riboprobes specific for FGFR1, FGF2, DKK3, FGF18, or ETV4
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for FGFR1, FGFR1IIIc, FGFR3IIIc, FGF2, DKK3, FGF18, or
ETV4 and other amplification type detection methods, such as, for
example, branched DNA, SISBA, TMA and the like).
[0198] Tissue or cell samples from mammals can be conveniently
assayed for mRNAs using Northern, dot blot or PCR analysis. For
example, RT-PCR assays such as quantitative PCR assays are well
known in the art. In some embodiments, mRNA expression levels are
levels quantified using real-time qRT-PCR. In some embodiments of
the invention, a method for detecting a target mRNA in a biological
sample comprises producing cDNA from the sample by reverse
transcription using at least one primer; amplifying the cDNA so
produced using a target polynucleotide as sense and antisense
primers to amplify target cDNAs therein; and detecting the presence
of the amplified target cDNA. In addition, such methods can include
one or more steps that allow one to determine the levels of target
mRNA in a biological sample (e.g., by simultaneously examining the
levels a comparative control mRNA sequence of a "housekeeping" gene
such as an actin family member). Optionally, the sequence of the
amplified target cDNA can be determined.
[0199] Optional methods of the invention include protocols which
examine or detect mRNAs, such as target mRNAs, in a tissue or cell
sample by microarray technologies. Using nucleic acid microarrays,
test and control mRNA samples from test and control tissue samples
are reverse transcribed and labeled to generate cDNA probes. The
probes are then hybridized to an array of nucleic acids immobilized
on a solid support. The array is configured such that the sequence
and position of each member of the array is known. Hybridization of
a labeled probe with a particular array member indicates that the
sample from which the probe was derived expresses that gene.
Differential gene expression analysis of disease tissue can provide
valuable information. Microarray technology utilizes nucleic acid
hybridization techniques and computing technology to evaluate the
mRNA expression profile of thousands of genes within a single
experiment. (see, e.g., WO 01/75166 published Oct. 11, 2001; (see,
for example, U.S. Pat. No. 5,700,637, U.S. Pat. No. 5,445,934, and
U.S. Pat. No. 5,807,522, Lockart, Nature Biotechnology,
14:1675-1680 (1996); Cheung, V. G. et al., Nature Genetics
21(Suppl):15-19 (1999) for a discussion of array fabrication). DNA
microarrays are miniature arrays containing gene fragments that are
either synthesized directly onto or spotted onto glass or other
substrates. Thousands of genes are usually represented in a single
array. A typical microarray experiment involves the following
steps: 1) preparation of fluorescently labeled target from RNA
isolated from the sample, 2) hybridization of the labeled target to
the microarray, 3) washing, staining, and scanning of the array, 4)
analysis of the scanned image and 5) generation of gene expression
profiles. Currently two main types of DNA microarrays are being
used: oligonucleotide (usually 25 to 70 mers) arrays and gene
expression arrays containing PCR products prepared from cDNAs. In
forming an array, oligonucleotides can be either prefabricated and
spotted to the surface or directly synthesized on to the surface
(in situ). In some embodiments, a DNA microarray is a
single-nucleotide polymorphism (SNP) microarrays, e.g.,
Affymetrix.RTM. SNP Array 6.0.
[0200] The Affymetrix GeneChip.RTM. system is a commercially
available microarray system which comprises arrays fabricated by
direct synthesis of oligonucleotides on a glass surface. Probe/Gene
Arrays: Oligonucleotides, usually 25 mers, are directly synthesized
onto a glass wafer by a combination of semiconductor-based
photolithography and solid phase chemical synthesis technologies.
Each array contains up to 400,000 different oligos and each oligo
is present in millions of copies. Since oligonucleotide probes are
synthesized in known locations on the array, the hybridization
patterns and signal intensities can be interpreted in terms of gene
identity and relative expression levels by the Affymetrix
Microarray Suite software. Each gene is represented on the array by
a series of different oligonucleotide probes. Each probe pair
consists of a perfect match oligonucleotide and a mismatch
oligonucleotide. The perfect match probe has a sequence exactly
complimentary to the particular gene and thus measures the
expression of the gene. The mismatch probe differs from the perfect
match probe by a single base substitution at the center base
position, disturbing the binding of the target gene transcript.
This helps to determine the background and nonspecific
hybridization that contributes to the signal measured for the
perfect match oligo. The Microarray Suite software subtracts the
hybridization intensities of the mismatch probes from those of the
perfect match probes to determine the absolute or specific
intensity value for each probe set. Probes are chosen based on
current information from Genbank and other nucleotide repositories.
The sequences are believed to recognize unique regions of the 3'
end of the gene. A GeneChip Hybridization Oven ("rotisserie" oven)
is used to carry out the hybridization of up to 64 arrays at one
time. The fluidics station performs washing and staining of the
probe arrays. It is completely automated and contains four modules,
with each module holding one probe array. Each module is controlled
independently through Microarray Suite software using preprogrammed
fluidics protocols. The scanner is a confocal laser fluorescence
scanner which measures fluorescence intensity emitted by the
labeled cRNA bound to the probe arrays. The computer workstation
with Microarray Suite software controls the fluidics station and
the scanner. Microarray Suite software can control up to eight
fluidics stations using preprogrammed hybridization, wash, and
stain protocols for the probe array. The software also acquires and
converts hybridization intensity data into a presence/absence call
for each gene using appropriate algorithms. Finally, the software
detects changes in gene expression between experiments by
comparison analysis and formats the output into .txt files, which
can be used with other software programs for further data
analysis.
EXAMPLES
[0201] The examples discussed below are intended to be purely
exemplary of the invention and should not be considered to limit
the invention in any way. The examples are not intended to
represent that the experiments below are all or the only
experiments performed. It is understood that various other
embodiments may be practiced, given the general description
provided above. Efforts have been made to ensure accuracy with
respect to numbers used (for example, amounts, temperature, etc.)
but some experimental errors and deviations should be accounted
for. Unless indicated otherwise, parts are parts by weight,
molecular weight is weight average molecular weight, temperature is
in degrees Centigrade, and pressure is at or near atmospheric.
Example 1
FGFR1-ECD.339-Fc Inhibits Proliferation of FGFR1 Amplified Lung
Cancer Cell Lines in Tissue Culture
[0202] A panel of lung cancer cell lines displaying potential
amplification of the FGFR1 gene was identified using CONAN
(http://www.sanger.ac.uk/cgi-bin/genetics/CGP/conan/search.cgi) and
Tumorscape
(http://www.broadinstitute.org/tumorscape/pages/portalHomejsf).
CONAN and Tumorscape represent public data mining tools to extract
gene copy number information for predefined or user defined loci
across the SNP6.0 dataset of cancer. Lung cancer cell lines DMS53,
DMS114, NCI-H1581 and NCI-H520 were identified as having potential
amplification of the FGFR1 gene (>4 copies/cell) and were
selected for further analysis. Human small cell lung cancer (SCLC)
cell lines DMS53 and DMS114 were purchased from ATCC (Manassas,
Va.; Cat. No. CRL-2062; Cat. No. CRL-2066, respectfully). The cells
were cultured in Waymouth's MB 752/1 medium+10% FBS+2 mM
L-glutamine at 37.degree. C. in a humidified atmosphere with 5%
CO.sub.2. Human non-small cell lung cancer (NSCLC) cell line
NCI-H1581 was purchased from ATCC (Manassas, Va.; Cat. No.
CRL-5878) and cultured in ACL-4 medium (serum-free). The base
medium for NCI-H1581 is DMEM: F12 (50/50 mix) with the following
components to the base medium: 0.02 mg/ml insulin, 0.01 mg/ml
transferrin, 25 nM sodium selenite (final conc.), 50 nM
Hydrocortisone (final conc.), 1 ng/ml Epidermal Growth Factor
(final conc.), 0.01 mM ethanolamine (final conc.), 0.01 mM
phosphorylethanolamine (final conc.), 100 pM triiodothyronine
(final conc.), 0.5% (w/v) bovine serum albumin (final conc.), 0.5
mM sodium pyruvate (final conc.) and 4.5 mM L-glutamine. Cells were
grown at 37.degree. C. in a humidified atmosphere with 5% CO.sub.2.
Human non-small cell lung cancer (NSCLC) cell line NCI-H520 was
purchased from ATCC (Manassas, Va.; Cat. No. HTB-182). The cells
were cultured in RPMI-1640 Medium+10% FBS+2 mM L-glutamine at
37.degree. C. in a humidified atmosphere with 5% CO.sub.2.
[0203] Amplification status of the FGFR1 gene in the cell lines was
confirmed by QuantiGene.RTM. Plex DNA Assay (Panomics). The
QuantiGene Plex DNA Assay is a hybridization-based assay using
xMAP.RTM. Luminex.RTM. magnetic beads. Individual, bead-based,
oligonucleotide probe sets (including capture, capture extenders,
blockers, and label probes) specific for FGFR1 (NM.sub.--023110),
ALB (NM.sub.--000477) and DCK (NM.sub.--000788) genes were designed
to prevent cross-reactivity (Panomics, Affymetrix, Santa Clara,
Calif.). ALB and DCK were used as reference genes for normalizing
FGFR1 copy number. Cell samples were lysed to release DNA and
incubated overnight with FGFR1 target specific probe sets. On the
second day a signal amplification tree was built via sequential
hybridization of PreAmplifier (PreAmp), Amplifier (Amp) and
biotinylated Label Probe (LP). The signal was detected by adding
phycoerythrin streptavidin (SAPE) substrate. SAPE fluorescence was
detected at 575 nm for each capture bead using a Luminex 200 flow
cytometer instrument (Luminex, Austin, Tex.). All data were
normalized to the reference genes and expressed as a ratio
(FGFR1/ALB). Data for the four cell lines is shown in Table 2.
TABLE-US-00002 TABLE 2 FGFR1 gene amplification in lung cancer cell
lines FGFR1-ECD.339-Fc Cell Line FGFR1 Gene Status Growth
Inhibition (Lung tumor subtype) (Copy number) In vitro In vivo (%
TGI)* DMS53 (SCLC) Amplified + +(64%) (5 copies/cell) DMS114 (SCLC)
Amplified + +(64%) (10 copies/cell) NCI-H1581 (NSCLC) Amplified +
+(74%) (6 copies/cell) NCI-H520 (NSCLC) Amplified + +(47%) (8
copies/cell) *TGI = tumor growth inhibition.
[0204] To determine the impact of FGFR1-ECD.339-Fc on lung cancer
cell lines in tissue culture, cells were plated in a Microtest.TM.
96-well tissue culture plate (Becton Dickenson, Franklin Lakes,
N.J.) at a density of 5.times.10.sup.3 cells/well in medium
containing 10%, 1% or 0.1% FBS in the presence or absence of 15
.mu.g/ml FGFR1-ECD.339-Fc (SEQ ID NO: 6) or an unrelated ECD-Fc
fusion protein (as a negative control). Plates were incubated at
37.degree. C. at 5% CO.sub.2 for 4 days and then assayed to
determine the impact of FGFR1-ECD.339-Fc on cell number and
proliferation.
[0205] To determine cell number the CellTiter-Glo.RTM. Luminescent
Cell Viability Assay (Promega, Madison, Wis.) was employed.
CellTiter-Glo.RTM. is a homogeneous method of determining the
number of viable cells in culture based on quantitation of the ATP
present, an indicator of metabolically active cells. In brief,
CellTiter-Glo.RTM. Reagent was added to each well of the tissue
culture plate at a volume equal to the volume of cell culture
medium present in each well (100 .mu.l), the contents mixed for 2
minutes on an orbital shaker to induce cell lysis and then the
plate incubated for 10 minutes at room temperature. Luminescence
was then determined on an EnVision.TM. Multilabel Plate Reader
(PerkinElmer, Boston, Mass.) with a 0.2 second integration time.
Results were expressed as relative light units (RLU)/well.
[0206] Results from the CellTiter-Glo.RTM. assay demonstrated that
cell number was significantly (P=>0.01) reduced by
FGFR1-ECD.339-Fc incubation in all four cell lines with FGFR1
amplification (FIG. 1A-D show NCI-H1581, NCI-H520, DMS53, and
DMS114, respectively). P-values were determined using an unpaired
t-test. See Mathematical Statistics and Data Analysis, 1988,
Wadsworth & Brooks, Pacific Grove, Calif.
[0207] To determine the impact of FGFR1-ECD.339-Fc on cell
proliferation the tritiated thymidine ([3H]-TdR) incorporation
assay was employed. Following incubation of lung cancer cell lines
with FGFR1-ECD.339-Fc or an unrelated ECD-Fc negative control,
tritiated thymidine ([3H]-TdR; PerkinElmer, Boston, Mass.) was
added at activity of 1 .mu.Ci/well. After 16-h exposure, tritiated
thymidine incorporation was assessed. Cells were washed with
Dulbecco's phosphate-buffered saline (DPBS; Mediatech, Inc.) and
removed from cell culture surface by incubation with trypsin-EDTA
(Mediatech, Inc.). The cell suspension (200 p. 1) was then removed
from the tissue culture plate using a FilterMate harvester
(PerkinElmer) and filtered through a UniFilter-96 GF/B
(PerkinElmer) plate. Cells were lysed using 95% ethanol and 40 p. 1
of Microscint 40 (PerkinElmer) scintillant fluid added per well.
Thymidine incorporation was determined as counts per minute (cpm)
on a Topcount NXT (PerkinElmer) scintillation counter. Results were
expressed as cpm/well.
[0208] In the tritiated thymidine incorporation assay,
FGFR1-ECD.339-Fc reduced NCI-H1581, NCI-H520, DMS53, and DMS114
cell proliferation by 85, 33, 52 and 81%, respectively (FIG. 2A-D,
respectively). The control ECD-Fc demonstrated no impact on cell
proliferation in any cell line. An additional lung tumor cell line,
NCI-H1703 (NSCLC; FGFR1 gene copy number: 6 copies/cell) was also
tested in the tritiated thymidine incorporation assay following
incubation with FGFR1-ECD.339-Fc, as described above.
FGFR1-ECD.339-Fc reduced NCI-H1703 proliferation by 15%.
[0209] Results from the tritiated thymidine incorporation assay
demonstrate that cell proliferation was significantly (* indicates
P=>0.05) reduced by FGFR1-ECD.339-Fc incubation in all four cell
lines with FGFR1 gene amplification (FIG. 2). P-values were
determined using an unpaired t-test. See Mathematical Statistics
and Data Analysis, 1988, Wadsworth & Brooks, Pacific Grove,
Calif. The control ECD Fc had little no impact on cell
proliferation in any cell line.
[0210] Percent reduction in CellTiterGlo relative light units (RLU)
in the presence of FGFR1-ECD.339-Fc was averaged across all FBS
concentrations examined for each of the four FGFR1 gene-amplified
lung cancer cell lines and was compared to a panel of lung cancer
cell lines without FGFR1 gene amplification (FIG. 3). Lung cancer
cell lines without FGFR1 gene amplification examined in this
experiment included NCI-H838, NCI-H1793, A549, Calu-1, NCI-H226,
NCI-H441, NCI-H460, NCI-H522 and NCI-H2126. Non-amplified cell
lines were purchased from ATTC (Manassas, Va.) and cultured
according to supplier instructions. Lung cancer cell lines with
FGFR1 gene amplification on average had a 46.25% reduction in cell
number, as assessed by CellTiterGlo, with the addition of
FGFR1-ECD.339-Fc compared to addition of control ECD-Fc. In
comparison, lung cancer cell lines without FGFR1 gene amplification
displayed on average a 9.33% decrease in cell number, as assessed
by CellTiterGlo, on addition of FGFR1-ECD.339-Fc compared to
addition of control ECD-Fc. This difference between the impact on
cell number of FGFR1-ECD.339-Fc on FGFR1 gene amplified and
non-amplified lung cancer cell lines was statistically significant
(P=0.0039).
[0211] The impact of FGFR1-ECD.339-Fc on cell proliferation as
assessed by tritiated thymidine incorporation was also compared
between FGFR1 gene amplified and non-amplified lung cancer cell
lines (FIG. 4). An average percent reduction in cell proliferation
with FGFR1-ECD.339-Fc addition was determined across all FBS
concentrations examined for each FGFR1 gene amplified cell line and
the panel of non-FGFR1 gene amplified cell lines indicated above.
Lung cancer cell lines with FGFR1 gene amplification on average had
a 62.75% reduction in CPM with the addition of FGFR1-ECD.339-Fc
compared to addition of control ECD-Fc. In comparison, lung cancer
cell lines without FGFR1 gene amplification displayed on average a
17.0% decrease in CPM, on addition of FGFR1-ECD.339-Fc compared to
addition of control ECD-Fc. This difference between FGFR1 gene
amplified and non-amplified lung cancer cell lines was
statistically significant (P=0.0088).
Example 2
Administration of FGFR1-ECD.339-Fc Inhibits Tumor Growth in the
DMS53 Small Cell Lung Cancer (SCLC) Xenograft Model
[0212] Six week old female SCID mice were purchased from Charles
River Laboratories (Wilmington, Mass.) and were acclimated for 1
week before the start of the study. Human small cell lung cancer
(SCLC) cell line DMS53 was used as the tumor model and was
purchased from ATCC (Manassas, Va.; Cat. No. CRL-2062). The cells
were cultured for three passages in Waymouth's MB 752/1 medium+10%
FBS+2 mM L-glutamine at 37.degree. C. in a humidified atmosphere
with 5% CO.sub.2. When the cultured cells reached 85-90%
confluence, cells were harvested and resuspended in cold Ca.sup.2+
and Mg.sup.2+ free phosphate buffered saline (PBS) containing 50%
Matrigel at 5.times.10.sup.7 cells per milliliter. The cells were
implanted subcutaneously over the right flank of the mice at
5.times.10.sup.6 cells/100 .mu.l/mouse. One day following cell
implantation mice were sorted and randomized (n=10) and treatment
initiated according to Table 3, below.
[0213] FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and
administered intraperitoneally (i.p.) at 15 mg/kg (300 .mu.g/100
.mu.l/mouse) twice a week for four weeks. Human albumin was
purchased from Grifols USA (Los Angeles, Calif.; Cat. No. NDC
61953-0002-1), diluted to a working stock (3 mg/ml) with 0.9%
sodium chloride, and was used as negative control at 300 .mu.g/100
.mu.l/mouse (15 mg/kg) administered twice a week for four
weeks.
TABLE-US-00003 TABLE 3 Dosing groups Test Article and Dose (mg test
article per Dosing Route and Group Number of Animals weight mouse)
Schedule 1 10 Albumin Intraperitoneal, 2X/week 2 10
FGFR1-ECD.339-Fc, Intraperitoneal, 15 mg/kg 2X/week
[0214] Tumor sizes were measured in each mouse on days 7, 14, 21,
28, 35 and 39 following the day of tumor cell inoculation. The
length and width of each tumor was measured using calipers and the
tumor size calculated according to the formula:
Tumor size (mm.sup.3)=(width(mm).times.length (mm)).sup.2/2
Mice were euthanized as a "cancer death" when the subcutaneous
tumor volumes exceeded 2000 mm.sup.3 or when the tumors became
excessively necrotic.
[0215] FIG. 5 shows the results of this experiment. Mice that
received FGFR1-ECD.339-Fc showed a 64% reduction of tumor growth
compared to albumin-treated animals. Comparison of DMS 53 tumor
volume at day 37 in the FGFR1-ECD.339-Fc treatment group and
vehicle treated group indicated that this result was statistically
significant (P=0.003). P-values were calculated using an ANOVA
analysis. See, e.g., Mathematical Statistics and Data Analysis,
1988, Wadsworth & Brooks, Pacific Grove, Calif. This analysis
demonstrated that FGFR1-ECD.339-Fc significantly reduced tumor
growth in the lung cancer cell line DMS53, which has amplification
of the gene encoding the FGFR1 receptor.
Example 3
Administration of FGFR1-ECD.339-Fc Inhibits Tumor Growth in the
DMS114 Small Cell Lung Cancer (SCLC) Xenograft Model
[0216] Six week old female SCID mice were purchased from Charles
River Laboratories (Wilmington, Mass.) and were acclimated for 1
week before the start of the study. Human small cell lung cancer
(SCLC) cell line DMS114 was used as the tumor model and was
purchased from ATCC (Manassas, Va.; Cat. No. CRL-2066). The cells
were cultured for three passages in Waymouth's MB 752/1 medium+10%
FBS+2 mM L-glutamine at 37.degree. C. in a humidified atmosphere
with 5% CO.sub.2. When the cultured cells reached 85-90%
confluence, cells were harvested and resuspended in cold Ca.sup.2+
and Mg.sup.2+ free phosphate buffered saline (PBS) containing 50%
Matrigel at 5.times.10.sup.7 cells per milliliter. The cells were
implanted subcutaneously over the right flank of the mice at
5.times.10.sup.6 cells/100 .mu.l/mouse. One day following cell
implantation mice were sorted and randomized (n=10) and treatment
initiated as described in Example 2, above.
[0217] Tumor sizes were measured in each mouse on days 3, 10, 16,
19, 24, 27, and 31 following the day of tumor cell inoculation. The
length and width of each tumor was measured using calipers and the
tumor size calculated according to the formula:
Tumor size (mm.sup.3)=(width(mm).times.length (mm)).sup.2/2
Mice were euthanized as a "cancer death" when the subcutaneous
tumor volumes exceeded 2000 mm.sup.3 or when the tumors became
excessively necrotic.
[0218] FIG. 6 shows the results of this experiment. Mice that
received FGFR1-ECD.339-Fc showed a 64% reduction of tumor growth
compared to albumin-treated animals. Comparison of DMS114 tumor
volume at day 31 in the FGFR1-ECD.339-Fc treatment group and
vehicle treated group indicated that this result was statistically
significant (P=0.002). P-values were calculated using an ANOVA
analysis. See, e.g., Mathematical Statistics and Data Analysis,
1988, Wadsworth & Brooks, Pacific Grove, Calif. This analysis
demonstrated that FGFR1-ECD.339-Fc significantly reduced tumor
growth in the lung cancer cell line DMS114, which has amplification
of the gene encoding the FGFR1 receptor.
Example 4
Administration of FGFR1-ECD.339-Fc Inhibits Tumor Growth in the
NCI-H1581 Non-Small Cell Lung Cancer (NSCLC) Xenograft Model
[0219] Six week old female SCID mice were purchased from Charles
River Laboratories (Wilmington, Mass.) and were acclimated for 1
week before the start of the study. Human non-small cell lung
cancer (NSCLC) cell line NCI-H1581 was used as the tumor model and
was purchased from ATCC (Manassas, Va.; Cat. No. CRL-5878). The
cells were cultured for three passages in ACL-4 medium
(serum-free). The base medium for this cell line is DMEM: F12
(50/50 mix) with the following components to the base medium: 0.02
mg/ml insulin, 0.01 mg/ml transferrin, 25 nM sodium selenite (final
conc.), 50 nM Hydrocortisone (final conc.), 1 ng/ml Epidermal
Growth Factor (final conc.), 0.01 mM ethanolamine (final conc.),
0.01 mM phosphorylethanolamine (final conc.), 100 pM
triiodothyronine (final conc.), 0.5% (w/v) bovine serum albumin
(final conc.), 0.5 mM sodium pyruvate (final conc.) and 4.5 mM
L-glutamine Cells were grown at 37.degree. C. in a humidified
atmosphere with 5% CO.sub.2. When the cultured cells reached 85-90%
confluence, cells were harvested and resuspended in cold Ca.sup.2+
and Mg.sup.2+ free phosphate buffered saline (PBS) containing 50%
Matrigel at 5.times.10.sup.7 cells per milliliter. The cells were
implanted subcutaneously over the right flank of the mice at
5.times.10.sup.6 cells/100 .mu.l/mouse. One day following cell
implantation mice were sorted and randomized (n=10) and treatment
initiated as described in Example 2, above.
[0220] Tumor sizes were measured in each mouse on days 7, 10, 14,
17, 21, 25 and 31 following the day of tumor cell inoculation. The
length and width of each tumor was measured using calipers and the
tumor size calculated according to the formula:
Tumor size (mm.sup.3)=(width(mm).times.length (mm)).sup.2/2
Mice were euthanized as a "cancer death" when the subcutaneous
tumor volumes exceeded 2000 mm.sup.3 or when the tumors became
excessively necrotic.
[0221] FIG. 7 shows the results of this experiment. Mice that
received FGFR1-ECD.339-Fc showed a 74% reduction of tumor growth
compared to albumin-treated animals. Comparison of NCI-H1581 tumor
volume at day 31 in the FGFR1-ECD.339-Fc treatment group and
vehicle treated group indicated that this result was statistically
significant (P<0.001). P-values were calculated using an ANOVA
analysis. See, e.g., Mathematical Statistics and Data Analysis,
1988, Wadsworth & Brooks, Pacific Grove, Calif. This analysis
demonstrated that FGFR1-ECD.339-Fc significantly reduced tumor
growth in the lung cancer cell line NCI-H1581, which has
amplification of the gene encoding the FGFR1 receptor.
Example 5
Administration of FGFR1-ECD.339-Fc Inhibits Tumor Growth in the
NCI-H520 Non-Small Cell Lung Cancer (NSCLC) Xenograft Model
[0222] Six week old female SCID mice were purchased from Charles
River Laboratories (Wilmington, Mass.) and were acclimated for 1
week before the start of the study. Human non-small cell lung
cancer (NSCLC) cell line NCI-H520 was used as the tumor model and
was purchased from ATCC (Manassas, Va.; Cat. No. HTB-182). The
cells were cultured for three passages in RPMI-1640 Medium+10%
FBS+2 mM L-glutamine at 37.degree. C. in a humidified atmosphere
with 5% CO.sub.2. When the cultured cells reached 85-90%
confluence, cells were harvested and resuspended in cold Ca.sup.2+
and Mg.sup.2+ free phosphate buffered saline (PBS) containing 50%
Matrigel at 5.times.10.sup.7 cells per milliliter. The cells were
implanted subcutaneously over the right flank of the mice at
5.times.10.sup.6 cells/100 .mu.l/mouse. One day following cell
implantation mice were sorted and randomized (n=10) and treatment
initiated as described below.
[0223] FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and
administered intraperitoneally (i.p.) at 20 mg/kg (400 .mu.g/125
.mu.l/mouse) twice a week for four weeks. Human albumin was
purchased from Grifols USA (Los Angeles, Calif.; Cat. No. NDC
61953-0002-1), diluted to a working stock (3 mg/ml) with 0.9%
sodium chloride, and was used as negative control at 400 .mu.g/125
.mu.l/mouse (20 mg/kg) administered twice a week for six weeks.
[0224] Tumor sizes were measured in each mouse on days 11, 18, 25,
32, 39 and 46 following the day of tumor cell inoculation. The
length and width of each tumor was measured using calipers and the
tumor size calculated according to the formula:
Tumor size (mm.sup.3)=(width(mm).times.length (mm)).sup.2/2
Mice were euthanized as a "cancer death" when the subcutaneous
tumor volumes exceeded 2000 mm.sup.3 or when the tumors became
excessively necrotic.
[0225] FIG. 8 shows the results of this experiment. Mice that
received FGFR1-ECD.339-Fc showed a 47% reduction of tumor growth
compared to albumin-treated animals. Comparison of NCI-H520 tumor
volume at day 46 in the FGFR1-ECD.339-Fc treatment group and
vehicle treated group indicated that this result was statistically
significant (P<0.01). P-values were calculated using an ANOVA
analysis. See, e.g., Mathematical Statistics and Data Analysis,
1988, Wadsworth & Brooks, Pacific Grove, Calif. This analysis
demonstrated that FGFR1-ECD.339-Fc significantly reduced tumor
growth in the lung cancer cell line NCI-H520, which has
amplification of the gene encoding the FGFR1 receptor.
[0226] Efficacy of FGFR1-ECD.339-Fc treatment in one additional
xenograft model, using non-small cell lung cancer (NSCLC) cell line
NCI-H1703, was tested in a similar manner as the SCLC and NSCLC
cell lines described above. Mice that received FGFR1-ECD.339-Fc
showed a 31% reduction of tumor growth compared to albumin-treated
animals. It is noted that NCI-H1703 cell line contains a
drug-sensitive PDGFRA/PDGFC genomic amplification in addition to
FGFR1 amplification, which may be responsible for the modest
efficacy observed.
Example 6
Certain Lung Cancer Xenograft Models with FGFR1 Gene Amplification
were More Sensitive to FGFR1-ECD.339-Fc-Mediated Growth Inhibition
than Certain Non-FGFR1 Gene Amplified Lung Cancer Xenograft
Models
[0227] The impact of FGFR1-ECD.339-Fc on tumor growth was compared
between FGFR1 gene amplified and non-amplified lung cancer
xenograft models. Lung cell lines without FGFR1-amplification
examined in this experiment were as follows: A549, NCI-H460,
NCI-H226, NCI-H2126, NCI-H441, NCI-H358, NCI-H522 and Colo699.
Non-amplified cell lines were purchased from ATTC (Manassas, Va.)
and cultured according to supplier instructions. Lung cancer
xenograft models using non-FGFR1 gene amplified cell lines were
carried out substantially as described in Example 2.
[0228] A panel of patient-derived xenograft (PDX) models of lung
cancer without FGFR1-amplification was also examined for
sensitivity to FGFR1-ECD.339-Fc. PDX xenografts have been
transplanted directly from cancer patients into nude mice without
in vitro tissue culture. The tumor xenografts retain most of the
characteristics of the parental patient tumors including histology
and sensitivity to anticancer drugs. Lung PDX models examined were
as follows: PDX D35087, PDX D37638, PDX D35376, LXFL-430, LXFE-937,
LXFE-397, LXFA-737 and LXFA-629. Preliminary pathology and patient
characteristics for the lung PDXs examined are outlined in Table
4.
TABLE-US-00004 TABLE 4 Characteristics of lung cancer
patient-derived xenograph (PDX) models Tissue Patient Tumor No.
type Origin Differentiation age Gender Stage LXFE_937 Squamous Lung
moderately 37 female T3N1M0 differentiated LXFE_397 Squamous Lung
poorly 56 male TIN0Mx differentiated LXFL_430 Large cell Lung
poorly 53 male T2N1M0 differentiated LXFA_629 Adeno Lung poorly 59
male T3N2Mx differentiated LXFA_737 Adeno Lung moderately 56 male
T3N2Mx differentiated PDX D35087 Squamous Lung moderately -- --
T3N0M0 differentiated PDX D37638 Squamous Lung poorly -- -- T3N2M0
differentiated PDX D35376 Squamous Lung moderately -- -- T2N0M0
differentiated
[0229] Six week old female SCID mice were purchased from Charles
River Laboratories (Wilmington, Mass.) and were acclimated for 1
week before the start of the study. PDX tumor fragments were
obtained from xenografts in serial passage in donor SCID mice.
After removal of tumors from donor mice, they were cut into
fragments (1-2 mm diameter, .about.25 mgs) and placed in RPMI 1640
culture medium until subcutaneous implantation. Recipient mice were
anaesthetized by inhalation of isoflurane. A small pocket was
formed with blunt forceps and one chunk of tumor PDX was placed in
the pocket. The wound was sealed using dermabond glue and a drop of
bupivicaine placed on the incision. One day following PDX
implantation mice were sorted and randomized (n=10) and treatment
initiated as described below.
[0230] FGFR1-ECD.339-Fc was formulated in PBS at 3 mg/ml and
administered intraperitoneally (i.p.) at 15 mg/kg (300 .mu.g/100
.mu.l/mouse) twice a week for four to eight weeks depending on the
growth rate of the PDX tumor implanted. Human albumin was purchased
from Grifols USA (Los Angeles, Calif.; Cat. No. NDC 61953-0002-1),
diluted to a working stock (3 mg/ml) with 0.9% sodium chloride, and
was used as negative control at 300 .mu.g/100 .mu.l/mouse (15
mg/kg) administered twice a week for four to eight weeks depending
on the growth rate of the PDX tumor implanted.
[0231] Tumor sizes were measured in each mouse on days 11, 18, 25,
32, 39 and 46 following the day of tumor cell inoculation. The
length and width of each tumor was measured using calipers and the
tumor size calculated according to the formula:
Tumor size (mm.sup.3)=(width(mm).times.length (mm)).sup.2/2
Mice were euthanized as a "cancer death" when the subcutaneous
tumor volumes exceeded 2000 mm.sup.3 or when the tumors became
excessively necrotic.
[0232] Percentage tumor growth inhibition by FGFR1-ECD.339-Fc was
determined by area-under-the-curve (AUC) analysis of xenograft
growth curves treated with FGFR1-ECD.339-Fc compared to albumin
control. FIG. 9 shows a scatterplot of the results of this
analysis. Lung cancer xenografts with FGFR1 gene amplification had
an average a 56% reduction in tumor growth with FGFR1-ECD.339-Fc
treatment. In comparison, lung cancer xenografts without FGFR1 gene
amplification displayed an average 22% decrease in xenograft growth
with FGFR1-ECD.339-Fc treatment compared to control. The difference
in FGFR1-ECD.339-Fc-mediated xenograft inhibition between FGFR1
gene amplified and non-amplified lung cancer xenograft models was
statistically significant (P=0.0333).
[0233] Thus, FGFR1 gene amplified tumor cells were found to be more
sensitive to FGFR1-ECD.339-Fc administration than tumor cells with
a non-amplified FGFR1 gene.
Example 7
FGFR1 Overexpression in FGFR1 Gene-Amplified and Non-Amplified Lung
Cancer Cell Lines and Xenografts
[0234] The expression of the FGFR1 at the RNA level was compared
between FGFR1 gene amplified and non-amplified lung cancer cell
lines, xenograft models, and PDX models. Lung cancer cell lines
without FGFR1 gene amplification examined in this experiment were
as follows: A549, NCI-H460, NCI-H226, NCI-H2126, NCI-H441,
NCI-H358, NCI-H522, MSTO-211H, and Colo699. Non-amplified cell
lines were purchased from ATTC (Manassas, Va.) and cultured
according to supplier instructions. A panel of patient-derived
xenograft (PDX) models of lung cancer without FGFR1 gene
amplification was also examined for FGFR1 mRNA expression. Lung PDX
models examined were as follows: PDX D35087, PDX D37638, PDX
D35376, LXFL-430, LXFE-937, LXFE-397, LXFA-737, and LXFA-629.
Preliminary pathology and patient characteristics for the lung PDXs
examined are outlined above in Table 4.
[0235] RNA was extracted from cell lines grown in vitro or tumor
xenografts grown in vivo using the RNAeasy0 mini kit (cat. No.
74104, Qiagen, Germany). Extracted RNA was treated with DNAse I
prior to creating cDNA with random hexamer priming and reverse
transcriptase using the QuantiTect Reverse Transcription Kit (cat.
No. 205311, Qiagen, Germany). Human FGFR1 RNA expression was
determined using an FGFR1 QuantiTect Primer Assay
(Hs_FGFR1.sub.--1_SG, cat. No. QT00102837, Qiagen, Germany) and a
human GUSB control reference QuantiTect Primer Assay
(Hs_GUSB.sub.--1_SG, cat. No. QT00046046, Qiagen, Germany).
QuantiTect SYBR Green PCR Kits (cat. No. 204145, Qiagen, Germany)
were used to quantify mRNA expression levels using real-time
qRT-PCR and an ABI Prism ViiA.TM. 7 Real-Time PCR System (Applied
Biosystems, Foster City, Calif.). Relative gene expression
quantification was calculated according to the comparative Ct
method using human GUSB as a reference and commercial RNA controls
(Stratagene, La Jolla, Calif.). Relative quantification was
determined according to the formula: 2.sup.-(.DELTA.Ct
sample-.DELTA.t calibrator).
[0236] GUSB-normalized FGFR1 RNA expression was compared between
lung cancer cell lines (FIG. 10) and xenograft models (FIG. 12)
with and without FGFR1 gene amplification.
[0237] FIG. 10 shows a scatterplot of FGFR1 RNA expression in cell
lines with and without FGFR1 gene amplification. Lung cancer cell
lines with FGFR1 gene amplification have a statistically
significant increase (P=0.0114) in FGFR1 mRNA expression compared
to cell lines without FGFR1 gene amplification. FIG. 10 also
demonstrates that a sub-population of lung cancer cell lines have
high FGFR1 mRNA expression in the absence of FGFR1 gene
amplification. NCI-H226, which has a GUSB normalized gene
expression of FGFR1 of 1.48, and NCI-H522, which has a GUSB
normalized gene expression of FGFR1 of 1.26, represent the two
uppermost outlier points in the non-amplified lung cancer cell line
population.
[0238] NCI-H226 and NCI-H522 were also sensitive to
FGFR1-ECD.339-Fc in vitro, having decreased cell proliferation and
number using the tritiated thymidine ([3H]-TdR) incorporation assay
and CellTiter-Glo.RTM. Luminescent Cell Viability Assay (Promega,
Madison, Wis.), respectively. FIG. 11A shows results from the
CellTiter-Glo.RTM. assay for the NCI-H226 cell line, demonstrating
that cell number was significantly (* indicates P=>0.05) reduced
by FGFR1-ECD.339-Fc incubation in the NCI-H226 cell line, which
does not have FGFR/-amplification. P-values were determined using
an unpaired t-test. See, e.g., Mathematical Statistics and Data
Analysis, 1988, Wadsworth & Brooks, Pacific Grove, Calif.
[0239] FIG. 11B shows results from the tritiated thymidine
incorporation assay for the NCI-H226 cell line, demonstrating that
cell proliferation was significantly (* indicates P=>0.05)
reduced by FGFR1-ECD.339-Fc incubation in the NCI-H226 cell line,
which does not have FGFR1 gene amplification. P-values were
determined using an unpaired t-test. The control ECD Fc had little
no impact on NCI-H226 cell proliferation.
[0240] Thus, certain lung cancer cell lines that do not have FGFR1
gene amplification, but which have FGFR1 overexpression, are
sensitive to FGFR1-ECD.339-Fc treatment.
[0241] FIG. 12 shows a scatterplot of FGFR1 mRNA expression
comparing FGFR1 gene amplified to non-amplified lung cancer
xenografts. Xenograft models with FGFR1 gene amplification had a
statistically significant (P=0.0146) increase in FGFR1 RNA levels
compared to non-amplified cell lines. In addition, in agreement
with the in vitro data, a sub-population of lung cancer xenograft
models has high FGFR1 RNA expression in the absence of FGFR1 gene
amplification. Xenograft models NCI-H226, NCI-H522 and PDX D35087
represent the 3 outlier points for FGFR1 RNA expression in the
non-amplified lung models (FIG. 12), with GUSB-normalized gene
expression levels of 3.70, 3.75 and 4.30, respectively.
[0242] NCI-H226, NCI-H522, and PDX D35087 were also sensitive to
FGFR1-ECD.339-Fc in vivo, demonstrating a statistically significant
(P<0.05) reduction in tumor growth of 55, 42 and 57%
respectively with FGFR1-ECD.339-Fc treatment. For PDX D35087, the
experiment was carried out substantially as described in Example
6.
[0243] Tumor sizes were measured in each mouse on days 26, 35, 41
and 45 following the day of PDX D35087 implantation. The length and
width of each tumor was measured using calipers and the tumor size
calculated according to the formula:
Tumor size (mm.sup.3)=(width(mm).times.length (mm)).sup.2/2
[0244] FIG. 13 shows the results of this experiment. Mice that
received FGFR1-ECD.339-Fc showed an inhibition of tumor growth
compared to albumin-treated animals. Comparison of PDX 35087 tumor
volume at day 45 in the FGFR1-ECD.339-Fc treatment group and
vehicle treated group indicated that this result was statistically
significant (P<0.01). P-values were calculated using an ANOVA
analysis. See, e.g., Mathematical Statistics and Data Analysis,
1988, Wadsworth & Brooks, Pacific Grove, Calif. This analysis
demonstrated that FGFR1-ECD.339-Fc significantly reduced tumor
growth in the PDX lung tumor model D35087, which does not have
amplification of the FGFR1 gene, but expresses relatively
high-levels of FGFR1 mRNA.
[0245] Thus, certain lung cancer xenograft models that do not have
FGFR1 gene amplification, but which have FGFR1 overexpression, are
sensitive to FGFR1-ECD.339-Fc treatment.
Example 8
Predictors of FGFR1-ECD.339-Fc Response
[0246] The RNA expression of a panel of FGFR1-related genes
including FGF ligands, FGF receptors, FGF binding proteins, FGF
signaling molecules, and a group of angiogenesis-related targets
was determined in a set of 35 tumor cell lines and xenografts using
qRT-PCR. RNA was extracted from cell lines grown in vitro or tumor
xenografts grown in vivo using the RNAeasy.RTM. mini kit (Qiagen,
Germany). Extracted RNA was treated with DNAse I prior to creating
cDNA with random hexamer priming and reverse transcriptase using
the QuantiTect Reverse Transcription Kit (Qiagen, Germany). Human
and mouse RNA expression was determined using QuantiTect Primer
Assays (Qiagen, Germany) employing a human GUSB control reference
QuantiTect Primer Assay (Qiagen, Germany). QuantiTect SYBR Green
PCR Kits (Qiagen, Germany) were used to quantify mRNA expression
levels using real-time qRT-PCR and an ABI Prism ViiA.TM. 7
Real-Time PCR System (Applied Biosystems, Foster City, Calif.).
Relative gene expression quantification was calculated according to
the comparative Ct method using human GUSB as a reference and
commercial RNA controls (Stratagene, La Jolla, Calif.). Relative
quantification was determined according to the formula:
2.sup.-(.DELTA.Ct sample-.DELTA.Ct calibrator).
[0247] The tumor cell lines and xenografts used in this experiment
are shown in Table 5. Also shown in Table 5 are the dosing schedule
for FGFR1-ECD.339-Fc in a mouse xenograft model, the percent tumor
growth inhibition (TGI (%)) and the statistical significance of the
tumor growth inhibition (P Value), as well as whether the FGFRJ
gene is amplified in the cell line.
TABLE-US-00005 TABLE 5 Anti-tumor activity of FGFR1-ECD.339-Fc in a
panel of xenograft models Xenograft Cell line/ Dosing Dose TGI
FGFR1 Tumor type model PDX route Dose sched. (%) P Value amp.
status Colon HCT116 Cell Line IP 15 mg/kg BIW 0% ns Non-amplified
Colo205 Cell Line IV 5 mg/kg BIW 38% P < 0.001 Non-amplified
Colo201 Cell Line IP 15 mg/kg BIW 0% ns Non-amplified Renal G-401
Cell Line IP 15 mg/kg BIW 36% P < 0.05 Non-amplified A498 Cell
Line IP 15 mg/kg BIW 7% ns Non-amplified Caki-1 Cell Line IV 10
mg/kg BIW 81% P < 0.001 Non-amplified Lung A549 Cell Line IP 10
mg/kg BIW 38% P < 0.05 Non-amplified NCI-H460 Cell Line IP 10
mg/kg BIW 35% P < 0.05 Non-amplified NCI-H226 Cell Line IP 15
mg/kg 3x/w 55% P < 0.001 Non-amplified NCI-H520 Cell Line IP 20
mg/kg BIW 47% P < 0.05 Amplified NCI-H1703 Cell Line IP 15 mg/kg
BIW 31% P < 0.05 Amplified NCI-H2126 Cell Line IP 15 mg/kg BIW
0% ns Non-amplified NCI-H441 Cell Line IP 15 mg/kg BIW 0% ns
Non-amplified NCI-H358 Cell Line IP 15 mg/kg BIW 0% ns
Non-amplified NCI-H522 Cell Line IP 10 mg/kg BIW 42% P < 0.05
Non-amplified NCI-H1581 Cell Line IP 15 mg/kg BIW 74% P = 0.002
Amplified DMS53 Cell Line IP 15 mg/kg BIW 64% 0.003 Amplified
DMS114 Cell Line IP 15 mg/kg BIW 64% P < 0.001 Amplified Calu-1
Cell Line IP 15 mg/kg BIW 0% ns Non-amplified D35087 PDX IP 15
mg/kg BIW 57% P < 0.01 Non-amplified D37638 PDX IP 15 mg/kg BIW
0% ns Non-amplified D35376 PDX IP 15 mg/kg BIW 0% ns Non-amplified
LXFA-737 PDX IP 15 mg/kg BIW 0% ns Non-amplified LXFA-629 PDX IP 15
mg/kg BIW 65% P = 0.007 Non-amplified Mesothelioma MSTO-211H Cell
Line IP 15 mg/kg BIW 64% P < 0.0001 Non-amplified Glioblastoma
U-87 Cell Line IP 15 mg/kg BIW 0% ns Non-amplified U-118 Cell Line
IP 15 mg/kg BIW 36% ns Non-amplified U-251 Cell Line IP 15 mg/kg
BIW 48% P = 0.0078 Non-amplified Retinoblastoma Y79 Cell Line IP 10
mg/kg BIW 0% ns Non-amplified Prostate Du145 Cell Line IP 0.15
mg/kg 3x/w 31% ns Non-amplified Endometrial MFE-280 Cell Line IP 15
mg/kg BIW 96% P < 0.001 Non-amplified HEC-1B Cell Line IP 15
mg/kg BIW 30% P < 0.05 Non-amplified MFE-319 Cell Line IP 15
mg/kg BIW 0% ns Non-amplified Breast MDA-MB-231 Cell Line IP 15
mg/kg BIW 0% ns Non-amplified JIMT1 Cell Line IP 1 mg/kg BIW 28% P
< 0.05 Non-amplified
[0248] An exemplary xenograft experiment is as follows. For Caki-1
and MSTO-211H, five million cells were implanted subcutaneously
over the right flank of SCID mice (N=10 per group).
FGFR1-ECD.339-Fc or albumin was administered i.p. twice a week at
the dose indicated in Table 5. FIG. 16 shows anti-tumor activity of
FGFR1-ECD.339-Fc in selected xenograft models. Representative tumor
growth curves are shown for a renal cancer, Caki-1, (A), and
mesothelioma, MSTO-211H, (B) xenograft cancer model. In the renal
cell carcinoma (RCC) Caki-1 model, administration of
FGFR1-ECD.339-Fc at 10 mg/kg twice a week for 6 weeks resulted in
81% (P<0.001) tumor growth inhibition (TGI; FIG. 16a). In the
MSTO-211H mesothelioma model, FGFR1-ECD.339-Fc administration
reduced tumor growth (FIG. 16b) by 64% (P<0.0001). In responding
tumors, FGFR1-ECD.339-Fc significantly reduced tumor volume as
assessed by area-under-the-curve (AUC) analysis. Responses were
observed in 19/35 (54%) of the models examined, with a range of
25-96% inhibition (see Table 5).
[0249] In order to further understand the potential molecular
determinants that make xenograft models sensitive to treatment with
FGFR1-ECD.339-Fc, the RNA expression of a panel of genes including
FGF ligands, FGF receptors, FGF binding proteins and FGF signaling
molecules was examined using qRT-PCR in certain xenograft models
from Table 5. The results are shown in Table 7, below.
[0250] Gene expression was then correlated to FGFR1-ECD.339-Fc
response to determine RNA expression signatures positively and
negatively correlated with anti-tumor activity. Table 8 shows the
results of that analysis. In addition to FGF2, RNA expression of
FGF18 (P=0.02227) was also positively (6.9-fold) correlated with
FGFR1-ECD.339-Fc anti-tumor activity. The downstream target gene of
FGF signaling, ets variant 4 (ETV4), was the most significant
(P=0.01639) gene for its positive (2.897-fold) association with
FGFR1-ECD.339-Fc activity. Expression of FGFR1 (P=0.01276),
including the FGFR1IIIc splice variant (P=0.01603), was a positive
predictor for FGFR1-ECD.339-Fc response. Expression of the
FGFR1IIIb splice variant was not correlated with FGFR1-ECD.339-Fc
response in that experiment. In addition to FGFR1, expression of
the FGFR3IIIc receptor (P=0.02488) was also positively correlated
with FGFR1-ECD.339-Fc response, reflecting the potential overlap in
FGF-ligand binding affinities between the Inc-splice isoforms of
FGFR1 and FGFR3 receptors. Significant genes with a negative
association with FGFR1-ECD.339-Fc activity were not found in this
analysis.
TABLE-US-00006 TABLE 8 Statistical analysis of FGF-related gene
expression in relation to FGFR1-ECD.339-Fc anti-tumor response in
xenograft models Gene Ratio.sup..sctn. P value.sup..dagger. Gene
Ratio.sup..sctn. P value.sup..dagger. ETV4 2.897 0.01639 SPRY3
1.665 0.4944 FGFR1 2.447 0.01669 SPRY1 1.394 0.5008 FGFR3IIIc 9.863
0.01944 DUSP6 0.6418 0.507 FGF18 6.915 0.02227 FGF19 1.203 0.5338
FGF2 247.7 0.03569 FLRT1 1.158 0.5676 FGFR1IIIc 3.647 0.0431 FGF3
1.431 0.5699 DUSP4 0.09578 0.08166 FGFR4 1.347 0.5755 TNC 0.0345
0.1212 FGF9 0.5356 0.6102 VIM 5.155 0.1448 FGFR3 1.767 0.6165 ETV5
1.447 0.1567 SPRY2 0.3142 0.6313 FGFBP3 1.84 0.1592 SERPINE1 0.333
0.6642 PLAU 0.3842 0.1781 FGF21 1.935 0.6744 PLAUR 0.3805 0.2408
FLRT2 0.2276 0.693 FGF7 1.991 0.243 FGFR2b 0.9266 0.7897 FGF5 24.79
0.2691 FGF6 0 0.8316 KDR 0.5892 0.2742 FGFBP1 0.5 0.8372 FGF11
2.153 0.2944 SOX9 1.181 0.8372 MET 0.4225 0.2962 SPRY4 0.9028
0.8372 FGF2 5.48 0.3015 NCAM1 1.661 0.8731 DUSP5 0.4765 0.3238 FGF8
1.052 0.9552 FGF22 1.604 0.3484 ELK4 1.062 0.9815 FGF10 1.91 0.3518
CDH1 0.1158 0.9818 FGFR2 1.402 0.3587 ELK3 1.157 0.9818 FGF1
0.09845 0.398 FGFBP2 0.7737 0.9818 FGFR2IIIc 5.546 0.4195 FGF16
1.076 1 FGF17 1.334 0.4361 FLRT3 0.7523 1 FGFR3IIIb 1.08 0.451
FGF20 5.967 0.4729 FGFR1IIIb 0.6493 0.486 .sup..sctn.Gene
expression ratio determined by median gene expression in
FGFR1-ECD.339-Fc responders/non-responders .sup..dagger.P-values
are determined by a Mann-Whitney test of PCR gene expression in
responders vs. non-responders for each gene using all models in
Table 5.
[0251] To determine what RNA factors may determine lung xenograft
response in the absence of FGFR1-gene amplification, the
correlation of FGFR1-ECD.339-Fc response in the non-FGFR1 amplified
subset of lung models was examined (N=13). The results of that
analysis are shown in Table 9. FGF2 expression was up-regulated
>3,000 fold in responding vs. non-responding FGFR1 non-amplified
lung models (P=0.029). The expression of FGFR1IIIc and FGFR3IIIc
also displayed a positive trend with FGFR1-ECD.339-Fc response in
the non-FGFR1 amplified lung subset in this experiment.
TABLE-US-00007 TABLE 9 Statistical analysis of FGF-related gene
expression in relation to FGFR1-ECD.339-Fc anti-tumor response in
non-FGFR1 amplified lung xenograft models Gene Ratio.sup..sctn. P
value.sup..dagger. Gene Ratio.sup..sctn. P value.sup..dagger. FGF2
3437 0.02857 FGF8 0.3268 0.5338 SPRY2 0.1395 0.05714 FGF20 0.4803
0.6573 FGFR3IIIc 3.765 0.1375 ELK4 1.019 0.6857 DUSP5 0.3241 0.2
FGFBP2 0.6526 0.6857 FGFR1IIIc 3.688 0.2343 FLRT3 0.2211 0.6857
FGF21 6.868 0.2454 FGF11 2.039 0.7308 FGFR2 8.793 0.2949 FGF5 44.05
0.8294 FGFR1 3.72 0.2949 FGFR2IIIc 2.029 0.8357 FGF19 20.79 0.3094
FGF1 1.45 0.8357 FGFR1IIIb 0.553 0.3429 FGFR3 1.285 0.8357 ELK3
0.5091 0.3429 FGFR4 0.8265 0.8357 SPRY4 0.3532 0.3429 FGF10 0.4615
0.8357 FGFBP1 0.1836 0.3429 FGF17 0.4268 0.8357 DUSP6 0.1254 0.3429
ETV5 0.8563 0.8857 DKK3 46.5 0.366 FLRT2 0.828 0.8857 FGF18 2.455
0.366 FLRT1 0.8212 0.8857 FGF22 1.373 0.3836 PLAUR 0.716 0.8857
FGF2 30.92 0.4452 FGFR3IIIb 0.7137 0.8857 VIM 4.122 0.4452 FGFR2b
0.5752 0.8857 ETV4 1.665 0.4452 FGF16 1.786 0.9452 FGFBP3 4.424
0.4857 SPRY3 1.051 0.9452 SOX9 0.3956 0.4857 FGF9 2.07 1 SERPINE1
0.3155 0.4857 NCAM1 1.391 1 SPRY1 0.1799 0.4857 DUSP4 0.9031 1 FGF3
0.8571 1 FGF7 0.738 1 .sup..sctn.Gene expression ratio determined
by median gene expression in FGFR1-ECD.339-Fc responders/median
gene expression in non-responders .sup..dagger.P-values are
determined by a Mann-Whitney test of PCR gene expression in
responders vs. non-responders for each gene using the non-FGFR1
amplified lung models in table 5.
[0252] It was examined if there was a correlation in gene
expression amongst the significant gene markers identified for
their association with FGFR1-ECD.339-Fc response in all models. The
results of that analysis are shown in Table 10. In this experiment,
there was a significant, positive correlation between the majority
of the individual RNA markers identified as predictive for
FGFR1-ECD.339-Fc xenograft response. For example, xenograft FGF2
RNA expression is positively correlated with FGFR3IIIc, FGFR1IIIc
and FGFR1 expression (P<0.05); FGFR1 RNA expression is
positively correlated with FGFR3IIIc, FGF2 and FGF18. The
expression of ETV4 was not associated with other FGFR1-ECD.339-Fc
responsive genes.
TABLE-US-00008 TABLE 10 Spearman correlation of gene expression
markers predictive of FGFR1-ECD.339-Fc efficacy in xenograft models
Gene 1 Gene 2 Correlation P-value.sup..sctn. FGF18 FGFR1 0.47
0.0083 FGF18 FGFR1IIIc 0.57 0.0008 FGF2 FGFR3IIIc 0.49 0.0139 FGFR1
FGFR3IIIc 0.41 0.0244 FGF2 FGFR1IIIc 0.43 0.0336 FGF2 FGFR1 0.39
0.0447 .sup..sctn.2-sided p-values approximated with a Monte Carlo
simulation
[0253] FIG. 14 shows (A) FGF2 mRNA (normalized to GUSB) and (B)
FGF2 protein expression in FGFR1-ECD.339-Fc responder and
non-responder xenografts. Expression of FGF2 (P=0.03569) was
positively associated with FGFR1-ECD.339-Fc response. FGF2
displayed a high ratio (247.7-fold) of mRNA gene expression between
FGFR1-ECD.339-Fc responder and non-responder xenografts. FGF2
protein levels were also confirmed to correlate with
FGFR1-ECD.339-Fc response.
[0254] FIG. 17 shows (A) FGFR1 mRNA expression (normalized to GUSB)
and (B) FGFR3IIIc mRNA expression (normalized to GUSB) in
FGFR1-ECD.339-Fc responder and non-responder xenografts. Expression
of FGFR1 (P=0.01669; FIG. 17a), and the FGFR1IIIc splice variant
(P=0.0431; Table 8), was positively correlated with
FGFR1-ECD.339-Fc anti-tumor activity. In addition to FGFR1,
expression of the FGFR3IIIc receptor (P=0.01944, Table 8) was also
positively correlated with FGFR1-ECD.339-Fc anti-tumor response
(FIG. 5b), reflecting the overlap in FGF-ligand binding specificity
between the c-splice isoforms of FGFR1 and FGFR3 receptors (see,
e.g., Zhang, et al. J. Biol. Chem. 281, 15694-15700 (2006); Ornitz,
et al. J. Biol. Chem. 271, 15292-15297 (1996)).
Example 9
Predictor of FGFR1-ECD.339-Fc Response
[0255] DKK3 mRNA expression was determined in a set of 25
xenografts using qRT-PCR. RNA was extracted from tumor xenografts
grown in vivo using the RNAeasy.RTM. mini kit (Qiagen, Germany).
Extracted RNA was treated with DNAse I prior to creating cDNA with
random hexamer priming and reverse transcriptase using the
QuantiTect Reverse Transcription Kit (Qiagen, Germany). Human DKK3
RNA expression was determined using QuantiTect Primer Assays
(Qiagen, Germany) employing a human GUSB control reference
QuantiTect Primer Assay (Qiagen, Germany). QuantiTect SYBR Green
PCR Kits (Qiagen, Germany) were used to quantify mRNA expression
levels using real-time qRT-PCR and an ABI Prism ViiA.TM. 7
Real-Time PCR System (Applied Biosystems, Foster City, Calif.).
Relative gene expression quantification was calculated according to
the comparative Ct method using human GUSB as a reference and
commercial RNA controls (Stratagene, La. Jolla, Calif.). Relative
quantification was determined according to the formula:
2.sup.-(.DELTA.Ct sample-.DELTA.Ctcalibrator).
[0256] The tumor xenografts used in this experiment are shown in
Table 11. Also shown in Table 11 are the dosing schedule for
FGFR1-ECD.339-Fc in a mouse xenograft model, the percent tumor
growth inhibition (TGI (%)) and the statistical significance of the
tumor growth inhibition (P Value).
TABLE-US-00009 TABLE 11 Panel of xenograft models with microarray
data. Xenograft Cell line/ Dosing Dose TGI Tumor type model PDX
route Dose schedule (%) P Value Colon HCT116 Cell Line IP 15 mg/kg
BIW 0% ns Colo205 Cell Line IV 5 mg/kg BIW 38% P < 0.001 Colo201
Cell Line IP 15 mg/kg BIW 0% ns Renal A498 Cell Line IP 15 mg/kg
BIW 7% ns Caki-1 Cell Line IV 10 mg/kg BIW 81% P < 0.001 Lung
A549 Cell Line IP 10 mg/kg BIW 38% P < 0.05 NCI-H460 Cell Line
IP 10 mg/kg BIW 35% P < 0.05 NCI-H226 Cell Line IP 15 mg/kg 3x/w
55% P < 0.001 NCI-H520 Cell Line IP 20 mg/kg BIW 47% P < 0.05
NCI-H1703 Cell Line IP 15 mg/kg BIW 31% P < 0.05 NCI-H2126 Cell
Line IP 15 mg/kg BIW 0% ns NCI-H441 Cell Line IP 15 mg/kg BIW 0% ns
NCI-H358 Cell Line IP 15 mg/kg BIW 0% ns NCI-H522 Cell Line IP 10
mg/kg BIW 42% P < 0.05 NCI-H1581 Cell Line IP 15 mg/kg BIW 74% P
= 0.002 Calu-1 Cell Line IP 15 mg/kg BIW 0% ns Methothelioma
MSTO-211H Cell Line IP 15 mg/kg BIW 64% P < 0.0001 Glioblastoma
U-87 Cell Line IP 15 mg/kg BIW 0% ns U-118 Cell Line IP 15 mg/kg
BIW 36% ns U-251 Cell Line IP 15 mg/kg BIW 48% P = 0.0078
Retinoblastoma Y79 Cell Line IP 10 mg/kg BIW 0% ns Prostate Du145
Cell Line IP 0.15 mg/kg 3x/w 31% ns Endometrial HEC-1B Cell Line IP
15 mg/kg BIW 30% P < 0.05 Breast MDA-MB-231 Cell Line IP 15
mg/kg BIW 0% ns JIMT1 Cell Line IP 1 mg/kg BIW 28% P < 0.05
[0257] Gene expression was then correlated to FGFR1-ECD.339-Fc
response to determine RNA expression signatures positively and
negatively correlated with anti-tumor activity. Expression of DKK3
mRNA was higher in tumors that were sensitive to FGFR1-ECD.339-Fc
than in tumors that were not sensitive to FGFR1-ECD.339-Fc
(P=0.0069).
[0258] FIG. 15 shows DKK3 mRNA levels (normalized to GUSB) in
FGFR1-ECD.339-Fc responder and non-responder xenografts. The
horizontal line indicates the median expression level for that
group.
Example 10
FGFR1-ECD.339-Fc does not Increase Serum Phosphate Following High
Dose Administration in Rats
[0259] FGFR1-ECD.339-Fc binds to the mitogenic FGFs with 10 to
100-fold higher affinity than to FGF-23. The binding affinity of
FGFR1-ECD.339-Fc for rodent FGF-23 is comparable to that of human
FGF-23 by SPR analysis (6.0.times.10.sup.-8 vs. 6.7.times.10.sup.-8
M). The potential biological impact of this relatively weak
FGFR1-ECD.339-Fc/FGF-23 binding was investigated in rats following
four weekly doses of FGFR1-ECD.339-Fc at a dose range of 10-200
mg/kg/qwk.
[0260] In the first experiment, Sprague Dawley rats (Charles River
Labs; N=5/group) were dosed with vehicle, 10, 50 or 200 mg/kg/qwk
of FGFR1-ECD.339-Fc for four weekly doses and plasma concentrations
of FGFR1-ECD.339-Fc were determined throughout the study by an
ELISA based detection method.
[0261] FGFR1-ECD.339-Fc concentration in plasma was determined
using a quantitative ELISA. Briefly, recombinant human FGF-2
(R&D Systems) was immobilized on a half-well microtiter ELISA
plate, blocked and incubated with test samples (diluted 1:10 with
blocking buffer/20 .mu.g/mL of heparin). The plate was subsequently
washed and a dilute goat anti-human IgG-Fc HRP antibody solution
(Sigma) was added and incubated. After a final wash step, a
tetramethylbenzidine peroxidase substrate solution was added and
incubated at ambient temperature with gentle shaking. The reaction
was stopped with a phosphoric acid solution. Plates were read on a
plate reader (450 nm). FGFR1-ECD.339-Fc concentrations were
determined on a standard curve obtained by plotting optical density
(OD) versus concentration.
[0262] In the second experiment, Sprague Dawley rats (Charles River
Labs; N=5/group) were administered the FGFR kinase inhibitor
PD173074 (Chemdea, Ridgewood, N.J.; 50 mg/kg/day) or vehicle
control by oral gavage for 7 days; or were administered
FGFR1-ECD.339-Fc (200 mg/kg) or appropriate vehicle weekly by
intravenous administration. Blood samples were collected at the
time points indicated and serum phosphate was determined at 24 and
168 hours post-initiation of dosing (Idexx laboratories, Westbrook,
Mass.).
[0263] The results of those experiments are shown in FIG. 18. At
the 200 mg/kg/qwk dose the maximal plasma concentration of the drug
was 3.6 and 4.2 mg/ml for female and male rats, respectively (FIG.
18A). Despite these sustained high levels of drug, no significant
changes in plasma phosphate were observed for any FGFR1-ECD.339-Fc
dose compared to animals that received vehicle (9.61 vs. 10.19
mg/dL for vehicle and 200 mg/kg/qwk FGFR1-ECD.339-Fc,
respectively). In contrast, daily dosing of rats with the small
molecule FGFR kinase inhibitor PD173043 resulted in significantly
elevated plasma phosphate levels either at 24 hour or 1 week of
daily dosing (FIG. 18B). Additionally, histological analysis of 55
tissues in animnals treated with high-dose FGFR1-ECD.339-Fc failed
to reveal any changes consistent with those reported by Brown et
al. (Toxicol. Pathol. 33, 449-455 (2005)), who observed
hyperphosphatemia and calcium-phosphorus deposition in various
organs following administration of a small molecule inhibitor of
FGFR1 kinase activity.
[0264] In addition, FGFR1-ECD.339-Fc has completed a phase 1
dose-escalation study (N=39) of up to 16 mg/kg/qwk in patients with
solid tumors. No impact of FGFR1-ECD.339-Fc on serum phosphate was
observed at any of the dose-levels examined (See, e.g., Tolcher, et
al. Proceedings of the 22nd EORTC-NCI-AACR Symposium on Molecular
Targets and Cancer Therapeutics (2010)). In summary, these results
support the biophysical data that FGFR1-ECD.339-Fc does not bind to
FGF-23 with high-affinity and does not induce hyperphosphatemia as
was shown for other broad inhibitors of the FGFR pathway.
Example 11
FGFR1-ECD.339-Fc Mediated Inhibition of FGF-2 and VEGF-A Induced
Angiogenesis in a Matrigel Plug Assay
[0265] Recombinant human FGF-2 (final concentration 250 ng/ml;
Peprotech) and/or recombinant human VEGF-A (final concentration 100
ng/ml; Peprotech) were added to matrigel (BD Biosciences, Franklin
Lakes, N.J.) with sodium heparin (2 units/ml; Sigma). FGF-2 and/or
VEGF-A containing matrigel plugs (one per animal) were implanted
subcutaneously in the abdomen region of C57BL/6 mice (Charles
River, Wilmington, Mass.). FGFR1-ECD.339-Fc was administered by
tail vein injection on days 1, 4, and 7 post-matrigel implantation.
On day 9, plugs were excised and processed for hematoxylin and
eosin (H&E) staining. Digital images of the stained matrigel
sections were generated using a Retiga 2000R digital camera
(Qlmaging, Burnaby, BC). Image analysis was performed using
Image-Pro Plus 5.1 (Media Cybernetics Inc., Silver Spring, Md.).
Neovascularization was defined as the cellular response in the
Matrigel plugs, consisting of newly formed blood vessels and
migrated cells.
[0266] The results of that experiment are shown in FIG. 19.
Administration of 5 mg/kg or higher FGFR1-ECD.339-Fc completely
blocked in vivo angiogenesis induced by a matrigel plug impregnated
with FGF-2. Administration of 15 or 45 mg/kg FGFR1-ECD.339-Fc also
completely blocked in vivo angiogenesis in response to a matrigel
plug impregnated with VEGF-A only or FGF-2 plus VEGF-A.
Anti-angiogenic activity against VEGF induced angiogenesis in this
model system may reflect inhibition of the synergistic activity
between VEGF in the plug and murine-derived stromal FGFs since SPR
analysis shows that FGFR1-ECD.339-Fc does not directly interact
with VEGF-A.
[0267] To determine whether FGFR1-ECD.339-Fc blocks VEGF-induced
proliferation of endothelial cells, HUVEC cells (Life Technologies,
Grand Island, N.Y.) were seeded at a density of 4.times.10.sup.3
cells/well in basal media (Medium 200 (Life Technologies) with 2%
heat inactivated FBS) and stimulated with either 10 ng/ml FGF2
(R&D Systems, Minneapolis, Minn.) or 15 ng/ml VEGF-A165
(R&D Systems, Minneapolis, Minn.) either in the presence of
absence of 10 .mu.g/ml FGFR1-ECD.339-Fc. HUVEC cell proliferation
was determined 3 days post-stimulation using CellTiter-Glo.RTM.
Luminescent Cell Viability Assay.
[0268] The results of that experiment are shown in FIG. 20.
FGFR1-ECD.339-Fc did not block VEGF-induced proliferation of
HUVECs, although it is capable of blocking FGF-2 induced HUVEC
proliferation.
Example 12
FGFR1-ECD.339-Fc Inhibits Tumor Angiogenesis in the Caki-1 Renal
Cell Carcinoma Xenograft Model
[0269] Human renal carcinoma Caki-1 cells (1.5.times.10.sup.7
cells/mouse) cells were implanted subcutaneously into the right
flank of CB17-SCID mice. One day after tumor implantation, the mice
were randomized and treated intravenously with either vehicle or
FGFR1-ECD.339-Fc (5 mg/kg) twice a week. At the end of the study
(Day 57), tumors were excised (N=3/gp) and used for histological
analysis. Frozen sections were probed with anti-mouse CD31
monoclonal antibody (BD Biosciences, Franklin Lakes, N.J.) and
visualized using HRP-conjugated conjugated secondary antibody
coupled with diaminobenzidine staining (brown color). Slides were
counter-stained with hematoxylin to identify cell nuclei (blue
color). Representative sections are shown (5.times.
magnification).
[0270] The results of that experiment are shown in FIG. 21.
Following treatment with FGFR1-ECD.339-Fc, reduced CD31 staining is
observed, indicatingthat tumor angiogenesis was inhibited by
FGFR1-ECD.339-Fc administration in this experiment.
Example 13
FGFR1-ECD.339-Fc-Mediated Inhibition of FGFR1 Signaling in the
JIMT-1 Breast Cancer Xenograft Model
[0271] Animals with established (200 mm3) human breast cancer
JIMT-1 tumors were administered either a single (24 and 72 hour
timepoints) or three times per week (multidose) i.p. dose(s) of
FGFR1-ECD.339-Fc at 15 mg/kg. Tumor samples were collected at 24
and 72 hours post-dose for the single dose groups and 48 hours post
the last dose in multi-dose group, snap-frozen in liquid nitrogen
and lyzed in RIPA buffer (Sigma Aldrich, St Luis, Mo.). Tumor
lysates were separated by SDS-PAGE and western blotting was
performed using monoclonal antibodies FGFR1, pFGFR1, FRS2a, pFRS2a,
Akt, pAkt, and PActin (Cell Signaling Technology, Inc).
FGFR1-ECD.339-Fc was detected using anti-human Fc monoclonal
antibody (Jackson Immuno Research).
[0272] The results of that experiment are shown in FIG. 22.
FGFR1-ECD.339-Fc reduced levels of phosphorylated FGFR1 by 24 hours
post-dose and completely abolished FGFR1 phosphorylation by 72
hours post-dose. Phosphorylated FRS and Akt levels were reduced 24
hours post-dose and further reduced two days later. Thus,
FGFR1-ECD.339-Fc inhibited FGFR1 signaling in the JIMT-1 breast
cancer xenograft model.
Table of Sequences
[0273] Table 6 lists certain sequences discussed herein. FGFR1
sequences are shown without the signal peptide, unless otherwise
indicated.
TABLE-US-00010 TABLE 6 Sequences and Descriptions SEQ ID NO
Description Sequence 1 Full-length human MWSWKCLLFW AVLVTATLCT
ARPSPTLPEQ AQPWGAPVEV FGFR1 ECD (with signal ESFLVHPGDL LQLRCRLRDD
VQSINWLRDG VQLAESNRTR peptide); SP-hFGFR1- ITGEEVEVQD SVPADSGLYA
CVTSSPSGSD TTYFSVNVSD ECD.353 ALPSSEDDDD DDDSSSEEKE TDNTKPNPVA
PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHR IGGYKVRYAT
WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLP ANKTVALGSN
VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKE MEVLHLRNVS
FEDAGEYTCL AGNSIGLSHH SAWLIVLEAL EERPAVMTSP LYLE 2 Full-length
human RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD FGFR1 ECD (without
VQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYA signal peptide);
hFGFR1- CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKE ECD.353
TDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHR
IGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLP
ANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKE
MEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EERPAVMTSP LYLE 3
SP-hFGFR1-ECD.339 MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEV
ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYA
CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKE TDNTKPNPVA PYWTSPEKME
KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHR IGGYKVRYAT WSIIMDSVVP
SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLP ANKTVALGSN VEFMCKVYSD
PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKE MEVLHLRNVS FEDAGEYTCL
AGNSIGLSHH SAWLTVLEAL 4 hFGFR1-ECD.339 RPSPTLPEQ AQPWGAPVEV
ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYA
CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKE TDNTKPNPVA PYWTSPEKME
KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHR IGGYKVRYAT WSIIMDSVVP
SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLP ANKTVALGSN VEFMCKVYSD
PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKE MEVLHLRNVS FEDAGEYTCL
AGNSIGLSHH SAWLTVLEAL 5 SP-hFGFR1-ECD.339-Fc MWSWKCLLFW AVLVTATLCT
ARPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTR
ITGEEVEVQD SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKE
TDNTKPNPVA PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHR
IGGYKVRYAT WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLP
ANKTVALGSN VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKE
MEVLHLRNVS FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EPKSSDKTHT CPPCPAPELL
GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ
YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR
DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS
RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 6 hFGFR1-ECD.339-Fc RPSPTLPEQ
AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD VQSINWLRDG VQLAESNRTR ITGEEVEVQD
SVPADSGLYA CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKE TDNTKPNPVA
PYWTSPEKME KKLHAVPAAK TVKFKCPSSG TPNPTLRWLK NGKEFKPDHR IGGYKVRYAT
WSIIMDSVVP SDKGNYTCIV ENEYGSINHT YQLDVVERSP HRPILQAGLP ANKTVALGSN
VEFMCKVYSD PQPHIQWLKH IEVNGSKIGP DNLPYVQILK TAGVNTTDKE MEVLHLRNVS
FEDAGEYTCL AGNSIGLSHH SAWLTVLEAL EPKSSDKTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK 7 hFGFR1 signal peptide
MWSWKCLLFWAVLVTATLCTA 8 Fc C237S EPKSSDKTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV
LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL
TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC
SVMHEALHNH YTQKSLSLSP GK 9 Exemplary Fc #1 ERKCCVECPP CPAPPVAGPS
VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV DGVEVHNAKT KPREEQFNST
FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT KGQPREPQVY TLPPSREEMT
KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD SDGSFFLYSK LTVDKSRWQQ
GNVFSCSVMH EALHNHYTQK SLSLSPGK 10 Exemplary Fc #2 ESKYGPPCPS
CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK
TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK AKGQPREPQV
YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS
RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK
TABLE-US-00011 TABLE 7 Gene expression values for certain xenograft
models from Table 5. Gene A498 A549 Caki-1 Calu-1 Colo201 Colo205
D35087 AREG 0.007391 0.188156 1.24833 0.141611 1.1487 0.5 5.41702
CA12 6.49802 1.46409 1.00696 0.064257 0.003826 0.011924 NA CDH1
0.001253 1.79005 0.269807 0.071298 3.94493 11.7942 NA DKK3 0.128514
0.028557 1.01396 2 0.000157 0 3.50594 DUSP4 0.000171 0.066064
0.007977 0.028956 0.085378 0.171943 NA DUSP5 0.026645 0.102238
0.68302 0.697372 0.125869 0.07966 NA DUSP6 0.083043 0.203063
3.27161 1.18921 3.11666 0.570382 NA EGF 0.004072 0.010525 0.036398
0.065607 0.000162 4.11E-05 NA EGFR 0.646176 0.353553 0.450625
0.97942 0.558644 0.438303 NA ELK3 0.04095 0.307786 0.76313 1.44393
0.085378 0.065607 NA ELK4 0.000015 6.28E-05 0.000265 7.67E-05
0.000513 0.000322 NA ERBB2 0.096723 0.185565 0.712025 0.271684
0.528509 0.566442 NA ERBB3 0.456916 0.50698 0.22688 0.001677
1.23114 0.757858 2.53696 EREG 0 0.080772 2.11404 0.673617 1.03526
0.129408 0.03438 ETV4 0.010672 0.697372 0.346277 0.351111 0.297302
0.624165 3.77951 ETV5 0.034674 0.200267 0.784584 0.528509 0.303549
0.389582 NA FGF1 0.037421 0.00357 0.001186 0.043889 0.04095
0.021051 0.681223 FGF10 0 1.17E-05 3.73E-05 0.000147 2.69E-05
2.19E-05 1.73E-05 FGF11 0.076947 0.003173 0.01937 0.000644 0.003696
0.002182 0.753929 FGF16 0 0.000348 0.000804 0.000649 0.002372
0.00143 0.011209 FGF17 4.72E-05 0.000148 4.14E-05 0.000156 0.001391
0.00093 0.000251 FGF18 0.000735 0.00194 0.004129 0.107321 0.006801
0.015303 0.012216 FGF19 0 0.000207 0 0 0.358489 0.721965 NA FGF2
0.035158 0.166086 0.524858 0.000581 0 0 NA FGF20 0.000159 0.000246
0.018841 0.005799 0.000115 0 NA FGF21 4.29E-05 3.58E-05 0 2.55E-05
0.000918 0.000561 NA FGF22 0.003002 0.004581 0.002879 0.004581
0.003285 0.002244 NA FGF3 1.09E-05 0 0 0 0.021945 0.036147 NA FGF4
0 0 0 0 0 0 NA FGF5 0.020054 1.01E-05 0.033262 0.248273 0 2.83E-05
0.005164 FGF6 0 0 0 0 0 0 NA FGF7 4.23E-05 0 9.3E-06 0.000143 0
3.01E-05 0 FGF8 0.000116 3.12E-05 0.000338 3.63E-05 0.00296
0.000918 0.000517 FGF9 0.000672 0.000735 0.001994 0.003545 0.037682
0.035649 NA FGFBP1 0.001245 0.111878 2.01391 0.002405 0.006434
0.0017 NA FGFBP2 7.46E-05 0.001253 0.005839 0.002137 0.00148
0.000355 NA FGFBP3 0.000203 0.001861 0.003217 0.000868 0.001642
0.002438 NA FGFR1 0.356012 0.535887 1.1487 1.53688 0.664343
0.126745 4.30765 FGFR1IIIb 0.000152 0.000309 0.000288 0.000282
0.000963 0.000456 NA FGFR1IIIc 0.119908 0.131215 0.193446 0.646176
0.114229 0.009753 0.381142 FGFR2 0.166086 0.001186 0.00072 0.001554
0.092142 0.003401 2.3227 FGFR2IIIb 0.009163 0.000334 8.63E-05
0.000169 0.045753 0.001797 NA FGFR2IIIc 0.196146 0.000175 0.000133
0.000804 0.000275 8.51E-05 0.00162 FGFR3 0.327598 0.044811 0.456916
0.033493 0.148651 0.038741 4.50554 FGFR3IIIb 0.006661 0.006003
0.006524 0.00014 0.023036 0.010167 NA FGFR3IIIc 0.039555 0.001576
0.063813 0.005048 0 0 0.001059 FGFR4 0.167241 0.111105 0.558644
0.000399 0.184284 0.107321 0.041146 FLRT1 0.002489 0.02352 0.01209
0.007867 0.040107 0.076415 NA FLRT2 4.03E-05 0.042986 0.003879
1.12506 0 5.24E-05 NA FLRT3 0.001586 0.051474 0.042986 0.000052
0.000186 0.001773 NA HGF 0 0.007977 0.033961 0.000725 0 0 0 IGF1
0.000405 0.002613 0 0.000381 3.25E-05 0 NA IGF1R 0.02977 0.598739
0.071794 0.469761 1.10957 1.01396 NA IGF2 0.004129 0.05954 0.060371
0.043285 0.002438 0.000299 NA KDR 0.000502 8.34E-05 0.000238
0.01418 0.000478 0.000122 0.000281 MET 1.28343 0.503478 7.26015
1.50525 0.790041 0.366021 NA MMP1 2.51E-05 0.018841 0.007599
0.303549 0.000413 0.000899 NA MMP2 1.54E-05 0.030186 0.888843
2.39496 0 0 12.3138 NCAM1 0.05366 5.85E-05 0.000485 0.000394
0.000159 2.44E-05 NA PDGFRa 0.000627 0.00095 0.173139 0.219151 0 0
0.023016 PDGFRb 0.001887 0.000735 0.021793 0.952638 0.002405
0.001114 NA PLAU 0.013888 0.267943 5.20537 0.456916 0.271684
0.289172 NA PLAUR 0.228458 0.97942 0.920188 1.94531 0.582367
0.248273 NA SERPINE1 0.61132 0.230047 1.94531 9.00047 0.077482
0.105843 NA SOX9 0.602904 1.26576 2.82843 1.72907 1.87905 4.85678
NA SPRY1 0.013415 0.022718 0.160428 0.198884 0.119908 0.186856 NA
SPRY2 0.028756 0.136787 0.5 0.301452 0.395021 0.50698 NA SPRY3
0.002668 0.003086 0.014579 0.001491 0.002668 0.003521 0.003134
SPRY4 0.002372 0.001565 0.005336 0.022876 0.009163 0.020905 NA TGFa
0.456916 0.051833 0.258816 0.009552 0.271684 0.127626 NA TNC
0.002542 0.007139 0.222211 1.67018 0.50698 0.123279 NA VIM 27.0958
13.8326 122.786 60.9688 0.336808 0.166086 43.9259 Gene D35376
D37638 DMS114 DMS53 Du145 G-401 HCT116 AREG 0.004051 1.51362
0.000292 0.008144 0.166086 0.0019 2.18859 CA12 NA NA NA NA 0.015303
0.02936 0.026278 CDH1 NA NA NA NA 0.933033 0.003262 1.09429 DKK3
0.000737 3.12315 NA NA 0.010237 0.018073 5.43E-05 DUSP4 NA NA NA NA
0.01468 0.000155 0.052193 DUSP5 NA NA NA NA 0.028956 0.011281
0.316439 DUSP6 NA NA NA NA 0.692555 2.63902 4.08405 EGF NA NA 0
0.000918 0.065607 1.09E-05 0.008609 EGFR NA NA NA NA 0.594604
0.000399 1.42405 ELK3 NA NA NA NA 0.041521 0.156041 0.234881 ELK4
NA NA NA NA 0.000023 6.28E-05 0.000104 ERBB2 NA NA NA NA 0.389582
0.121582 0.217638 ERBB3 0.000903 0.108909 0.001913 0.012691
0.260616 0.031686 0.231647 EREG 0 0.002591 0 9.93E-06 0.034197
0.003853 5.65685 ETV4 0.151082 1.54928 NA NA 0.014579 1.20581
0.15822 ETV5 NA NA NA NA 0.046071 0.426317 0.371131 FGF1 0.000328
0.050036 NA NA 0.001631 0.000176 0.034674 FGF10 0.000157 0.00023 NA
NA 3.39E-05 0.5 0.000192 FGF11 0.012728 0.101173 NA NA 0.008669
0.251739 0.022876 FGF16 0.026669 0.026479 NA NA 0.000585 0.000311
0.000918 FGF17 0.000632 0.006306 NA NA 0.006801 0.000681 0.011359
FGF18 0.000445 0.002484 NA NA 0.00286 0.003826 0.03082 FGF19 NA NA
NA NA 0.000128 0.000937 0.035897 FGF2 NA NA NA NA 0.107321 0.008373
0.10083 FGF20 NA NA NA NA 0.00145 0.30566 0.00613 FGF21 NA NA NA NA
0.000193 4.59E-05 0.000231 FGF22 NA NA NA NA 0.008373 0.002668
0.01937 FGF3 NA NA NA NA 0 6.23E-05 0.000331 FGF4 NA NA NA NA 0 0 0
FGF5 5.91E-05 0.000808 NA NA 0 1.84E-05 0 FGF6 NA NA NA NA 0.000052
0 0.00015 FGF7 0 0 NA NA 7.11E-05 0.000233 0.000045 FGF8 0.000961
0.001714 NA NA 0.000301 0.01541 0.006003 FGF9 NA NA NA NA 0.003065
0.001137 0.009227 FGFBP1 NA NA NA NA 0.050067 0 0.248273 FGFBP2 NA
NA NA NA 0.001211 0.00029 0.005048 FGFBP3 NA NA NA NA 0.000618
0.060371 0.00588 FGFR1 0.581641 0.709808 0.678302 0.078563 0.220676
1.32869 0.517632 FGFR1IIIb NA NA 0 0 0.001665 5.62E-05 0.085378
FGFR1IIIc 0.069464 0.386462 0.027585 0.01698 0.057512 0.473029
0.063373 FGFR2 0.000917 1.05416 0.008974 0.001084 0.033032 1.22264
0.137738 FGFR2IIIb NA NA NA NA 0.023036 0.049721 0.118257 FGFR2IIIc
0.000498 0.012137 NA NA 0.00075 0.972655 0.000294 FGFR3 0.009346
0.580312 0.009163 0.002093 0.033262 0.025559 0.329877 FGFR3IIIb NA
NA NA NA 0.005799 0.000844 0.030607 FGFR3IIIc 9.87E-05 0.00035 NA
NA 0.000135 0.003747 6.36E-05 FGFR4 0.000564 0.009061 0.002879
0.000168 0.004395 0.015953 0.042394 FLRT1 NA NA NA NA 0.01698
0.005839 0.034197 FLRT2 NA NA NA NA 0.009889 0.010027 0 FLRT3 NA NA
NA NA 0.007867 0.000886 0.002372 HGF 0.044508 0.009057 NA NA
6.2E-06 2.23457 0 IGF1 NA NA NA NA 0.002036 0.000294 0 IGF1R NA NA
NA NA 0.297302 0.065154 0.088388 IGF2 NA NA NA NA 0.006754 0.104386
0.20166 KDR 0.000377 0.009784 NA NA 0.00294 0.000142 0.000557 MET
NA NA NA NA 0.119908 0.003747 1.1487 MMP1 NA NA NA NA 0.044502
0.000184 0.002339 MMP2 0.000158 0.138658 NA NA 0 0.325336 0 NCAM1
NA NA NA NA 0.000061 0.562529 0.003401 PDGFRa 0.005323 0.038353 NA
NA 0.000208 0.001748 0 PDGFRb NA NA NA NA 0.001381 0.007443 0.00294
PLAU NA NA NA NA 0.289172 0.00324 0.297302 PLAUR NA NA NA NA
0.194791 0.035403 0.429283 SERPINE1 NA NA NA NA 0.03983 0.001153
0.45376 SOX9 NA NA NA NA 0.063813 0.012174 1.94531 SPRY1 NA NA NA
NA 0.004876 0.088388 0.030396 SPRY2 NA NA NA NA 0.027017 0.721965
0.055553 SPRY3 0.00269 0.006099 7.89E-05 0.000644 0.007599 0.007922
0.020054 SPRY4 NA NA NA NA 0.000162 0.00162 0.003773 TGFa NA NA NA
NA 0.05954 0.000428 0.121582 TNC NA NA NA NA 0.014579 0.000162
0.000118 VIM 16.4293 3.26549 NA NA 2.15846 38.5858 0.051119 LXFA-
LXFA- MDA-MB- MFE- MFE- Gene HEC-1B JIMT1 629 737 231 280 319 AREG
0.000804 0.0625 0.794269 0.941087 1.37554 0.001511 0.001271 CA12
2.8481 0.010672 NA NA 0.119908 0.02683 0.035403 CDH1 0.033493
3.20428 NA NA 0.000139 0.602904 0.895025 DKK3 0.646176 0.118257
0.039949 0.067093 0.000516 0.188156 0.000761 DUSP4 0.000446
0.023683 NA NA 0.070805 0.001511 6.87E-05 DUSP5 0.203063 0.050067
NA NA 0.432269 0.039282 0.02936 DUSP6 2.36199 0.183011 NA NA
3.68075 1.3566 0.084202 EGF 0.00588 0.023196 NA NA 0.011125
0.001061 0.00362 EGFR 0.432269 3.03143 NA NA 1.86607 0.092783
0.307786 ELK3 0.628507 0.154963 NA NA 0.539614 0.03983 0.037163
ELK4 0.000032 0.00143 NA NA 8.28E-05 0 8.3E-06 ERBB2 0.535887
5.06303 NA NA 0.11744 1.31039 0.48971 ERBB3 0.072293 0.271684
0.152936 1.94598 0.046071 0.309927 0.080214 EREG 2.08E-05 0.06164
0.067803 0.041083 0.25349 1.78E-05 0.000119 ETV4 0.528509 0.493116
0.185141 0.889459 0.210224 0.888843 0.011598 ETV5 0.371131 0.179244
NA NA 0.248273 0.05672 0.017824 FGF1 0.003354 0.036398 0.0984
0.004799 0.077482 0.000462 0.001032 FGF10 3.03E-05 0 3.2E-05
2.51E-05 3.23E-05 0.000168 4.9E-06 FGF11 0.009552 0.017948 0.173307
0.554631 0.003086 0.057115 0.009037 FGF16 9.78E-05 0.002137
0.016327 0.025879 0.000341 0.000485 0.000147 FGF17 0.000821
0.024349 0.000633 0.003234 0.000391 0.034197 0.013139 FGF18 1.45397
0.057115 0.00032 0.001085 0.001362 0.049378 0.043586 FGF19 0 0 NA
NA 7.26E-05 0.008432 7.3E-06 FGF2 0.021793 0 NA NA 0 0.009889
0.001598 FGF20 0.006896 0 NA NA 0.001785 0.001004 0.000016 FGF21
2.66E-05 0.000452 NA NA 1.25E-05 0.000084 7.1E-06 FGF22 0.00519
0.019237 NA NA 0.003401 0.012691 0.049037 FGF3 0.000011 0.001289 NA
NA 0 0.000735 0 FGF4 0 0 NA NA 0 0.000437 0 FGF5 0.001011 0.004364
0.006428 5.47E-05 0.181747 2.23E-05 7.8E-06 FGF6 0 0 NA NA 9.5E-06
0 3.09E-05 FGF7 2.14E-05 0 0 0 4.14E-05 9.85E-05 0.003173 FGF8
0.001325 0.00064 7.08E-05 0.000522 8.11E-05 0.000331 0.000368 FGF9
0.001011 0.008549 NA NA 0.000495 0.001245 0.013697 FGFBP1 0.20733
0.664343 NA NA 0.002244 0.002355 0.002065 FGFBP2 0.003195 0.000428
NA NA 0.000127 0.001887 0.003961 FGFBP3 0.000267 0.003065 NA NA
0.00734 0.001047 0.00162 FGFR1 0.479632 5.89708 0.6208 0.448755
0.524858 1.22264 0.554785 FGFR1IIIb 0.000475 0.204476 NA NA 0.00097
0.00734 0.000509 FGFR1IIIc 0.236514 1.86607 0.114633 0.108525
0.204476 1.02101 0.267943 FGFR2 0.050067 1.21419 0.121945 0.001513
0.003065 0.027394 0.211686 FGFR2IIIb 0.012344 0.602904 NA NA
0.001169 0.014279 0.160428 FGFR2IIIc 0.016289 0.005448 2.79E-05
0.000266 0.000137 0.001178 0.009486 FGFR3 0.200267 0.840896 1.05256
1.51215 0.005154 0.094732 0.062935 FGFR3IIIb 0.023196 0.148651 NA
NA 0.00147 0.007391 0.005486 FGFR3IIIc 0.013139 0.000194 0.000669
0.000864 0.000132 0.000144 0.000152 FGFR4 0.225313 0.094732
0.005931 0.111491 0.000523 0.013985 0.004581 FLRT1 0.00362 0.018711
NA NA 0.031034 0.041521 0.040667 FLRT2 0.001677 0 NA NA 0.069348
0.00362 0.089003 FLRT3 0.041521 0 NA NA 2.87E-05 0.002228 0.034197
HGF 2.62E-05 0 4.75E-05 0 0 5.13E-05 2.36E-05 IGF1 0 0.000581 NA NA
0.000045 0.030186 0.000653 IGF1R 0.125869 0.61132 NA NA 0.200267
0.063373 0.004743 IGF2 0.137738 0.196146 NA NA 0.034197 0.0625
0.11744 KDR 0.000375 0.000233 0.000274 0.000304 0.01038 0.000686
0.001532 MET 4.46915 0.920188 NA NA 0.450625 0.019915 0.057115 MMP1
0.021642 0.00162 NA NA 0.45376 0.00093 0.002981 MMP2 0.162668
0.038741 0.67301 0.009119 0.000419 0.001381 0.000509 NCAM1 0.000104
9.58E-05 NA NA 9.7E-06 0.039555 0.010027 PDGFRa 8.51E-05 0.001011
4.55E-06 0.001835 0.004016 0.018581 0.001253 PDGFRb 0.000862
0.002559 NA NA 0.019915 0.003521 0.001025 PLAU 1.34723 1.40444 NA
NA 2.32947 0.007139 0.004581 PLAUR 0.316439 0.632878 NA NA 0.757858
0.080772 0.008201 SERPINE1 0.096723 7.51618 NA NA 2.82843 0.008432
0.001069 SOX9 0.858565 0.000145 NA NA 0.429283 0.149685 0.004395
SPRY1 0.234881 0.00982 NA NA 0.061214 0.039282 0.014989 SPRY2
0.271684 0.035403 NA NA 0.297302 0.017098 0.029157 SPRY3 0.008432
0.012604 0.001365 0.045286 0.004518 0.006087 0.015843 SPRY4
0.020334 0.002981 NA NA 0.018581 0.001861 0.000821 TGFa 0.118257
0.120742 NA NA 0.034915 0.027776 0.087172 TNC 0.01541 0.737135 NA
NA 0.146604 0.020617 0.00613 VIM 69.551 5.54044 0.091157 0.065954
44.3235 2.39496 0.463294 MSTO- NCI- NCI- NCI- NCI- NCI- NCI- Gene
211H H1581 H1703 H2126 H226 H358 H441 AREG 0.0017 0.000868 1.87E-05
0.064704 0.013048 5.73582 2.44528 CA12 0.084788 0.084202 0.000012
0.003645 0.015734 0 0 CDH1 0.009618 0.073302 0.000772 1.81504
0.042986 12.7286 9.84916 DKK3 4.11246 0.127626 0.094732 0.000255
0.161544 0 3.71E-05 DUSP4 0.000309 0.000219 0.0007 0.045123
0.003496 0.040386 0.013508 DUSP5 0.186856 0.02797 0.02977 0.02836
0.174343 0.223756 0.190782 DUSP6 0.255253 1.47427 0.149685 0.062935
0.063813 4.34694 2.86791 EGF 0.003595 0.000997 0.00011 0.000542
0.00982 0.07966 0.049721 EGFR 1.56917 0.108819 0.34151 0.460094
3.05252 0.628507 0.895025 ELK3 0.473029 0.214641 0.376312 0.063813
0.435275 0.463294 0.329877 ELK4 3.97E-05 3.55E-05 0.000788 2.85E-05
4.44E-05 2.64E-05 0.00181 ERBB2 0.189465 0.368567 0.246558 0.20733
0.156041 0.641713 0.482968 ERBB3 0.011125 0.208772 0.00942 0.099442
0.073812 0.721965 0.447513 EREG 0 0.000157 1.41E-05 2.93E-05
0.000145 0.907519 1.18099 ETV4 0.063813 0.408951 0.466516 0.019641
0.166086 0.230047 0.148651 ETV5 0.15932 0.271684 0.907519 0.03125
0.293209 0.183011 0.20733
FGF1 0.007813 0.00564 0.002668 0.000158 0.016289 0.0819 0.006849
FGF10 0.000194 0.000546 9.58E-05 1.35E-05 0.000343 7.31E-05
3.58E-05 FGF11 0.022876 0.301452 0.001543 0.00282 0.005486 0.042394
0.019641 FGF16 0.002079 0.000523 7.46E-05 0.000239 0.002372 0.00029
0.001099 FGF17 4.32E-05 0.00879 0.001887 0.000117 0.001091 0.002307
1.46E-05 FGF18 0.005373 0.119908 0.005154 0.000549 0.619854
0.000686 0.000816 FGF19 9.25E-05 0.01038 2.25E-05 5.7E-06 3.63E-05
0.000804 0 FGF2 3.07375 0.528509 0.069348 7.26E-05 2.12874 0.000273
4.63E-05 FGF20 0.008432 0.121582 0.000174 0 0 0.000478 3.36E-05
FGF21 0 2.01E-05 6.28E-05 0.000003 7.94E-05 0 9.9E-06 FGF22
0.004158 0.009685 0.003173 0.00162 0.014082 0.00292 0.004843 FGF3 0
0.000109 0 1.12E-05 6.28E-05 3.48E-05 0 FGF4 0 0 0 0 0 0 0 FGF5
0.939523 0.00181 0.514057 0 0.148651 0.002595 0 FGF6 0 1.11E-05
2.68E-05 4.1E-06 3.03E-05 0 0 FGF7 0.013322 0.001609 0.000378
2.87E-05 4.11E-05 0.000112 2.08E-05 FGF8 0.000472 0.10083 0.000495
1.39E-05 0.000193 0.000397 0.000695 FGF9 0.002008 0.001253 0.000146
0.000234 0.028164 0.070316 0.010599 FGFBP1 0.111105 0.000782
2.77E-05 0.007239 0.469761 2.88786 0.607097 FGFBP2 0.000109
0.000478 4.03E-05 0.002743 0.000318 0.00128 0.000296 FGFBP3
0.004187 3.20428 0.001797 0.000597 0.00012 0.002405 0.001773 FGFR1
3.75809 2.05623 1.76541 0.146604 3.70635 0.607097 0.397768
FGFR1IIIb 0.000593 5.54E-05 0.000228 0.000589 0.00141 0.000362
0.001654 FGFR1IIIc 1.33793 1.17283 0.521233 0.011842 1.12506
0.045437 0.048027 FGFR2 0.002152 4.85678 0.02936 0.00071 0.023196
0.033726 0.001861 FGFR2IIIb 0.000644 0.303549 0.001773 0.000277
0.009355 0.020054 0.001106 FGFR2IIIc 0.000345 3.78423 0.008974
4.35E-05 0.006302 0.000531 0.000173 FGFR3 0.008315 0.043586
0.277392 0.051119 0.086569 0.156041 0.00367 FGFR3IIIb 7.57E-05
0.001835 0.01278 0.00849 0.005719 0.009889 2.23E-05 FGFR3IIIc
0.00088 0.006615 0.035403 0.000026 0.003377 0.000443 0 FGFR4
0.001343 0.004645 0.010309 0.005048 0.001642 0.004581 0.004334
FLRT1 0.004044 0.029564 0.036906 0.027017 0.002743 0.016863
0.033961 FLRT2 0.028164 0.008729 0.41466 0.013048 0.11908 0.118257
0.077482 FLRT3 2.77E-05 0.002559 0.001114 0.190782 0.001665
0.005226 0.005563 HGF 6.59E-05 0.005524 2.44E-05 0.00013 0 0 0 IGF1
0 0.006801 9.71E-05 0.000005 3.97E-05 0.030186 0.008729 IGF1R
0.275476 0.965936 0.021793 0.179244 0.840896 0.737135 0.211686 IGF2
2.36199 0.047366 0.005448 0.048361 0.023357 0.214641 3.94E-05 KDR
0.001253 0.004044 4.03E-05 8.63E-05 0.036398 0.5 0.271684 MET
1.75321 0.017337 0.128514 0.173139 2.53151 0.558644 4.82323 MMP1
0.035403 0.022718 0.307786 0.002542 0.058315 0.503478 0.001797 MMP2
3.11666 0.004809 0.003906 0.001099 0.078563 0 0 NCAM1 0.002524
0.000174 5.13E-05 0.000413 0.000856 0.000264 0.000169 PDGFRa
0.005962 0.486327 6.45313 0.000142 0.001926 0.001253 6.73E-05
PDGFRb 0.392292 0.178006 0.000627 0 0.267943 0.004518 0.001654 PLAU
1.6358 0.641713 0.00471 0.054788 0.021793 1.43396 3.53081 PLAUR
0.646176 0.11908 0.143587 0.447513 2.23457 0.773782 0.732043
SERPINE1 37.7918 0.275476 1.07923 0.06983 18.1261 0.316439 0.554785
SOX9 0.417544 0.450625 0.006087 0.214641 0.124137 1.45397 0.103665
SPRY1 0.012344 0.50698 0.185565 0.010525 0.00879 0.119908 0.0625
SPRY2 0.044502 0.030186 0.021642 0.062068 0.019641 0.186856
0.161544 SPRY3 0.001522 0.007289 0.01278 0.004016 0.003472 0.00296
0.001797 SPRY4 0.002323 0.009291 0.015093 0.000288 0.001091
0.004843 0.00471 TGFa 0.001161 0.008088 0.000581 0.01937 0.010097
0.320857 0.521233 TNC 0.02352 0.003262 3.76E-05 0.007546 0.100134
2.14355 1.07923 VIM 78.249 21.8566 32.6724 0.110338 19.8353 5.38893
0.479632 NCI- NCI- NCI- Gene H460 H520 H522 U-118 U-251 U-87 Y79
AREG 0.052556 0.05329 0.111878 0.000605 0.000065 4.3E-06 9.9E-06
CA12 0.082469 0.003906 0.010237 0.659754 0.087172 1.02811 0.358489
CDH1 0.004809 0.111105 0.005839 6.02E-05 0.007867 0.000181 0.000121
DKK3 0.017824 0.091505 0.017098 5.20537 1.51572 0.089003 0.00026
DUSP4 0.059129 0.002668 3.73E-05 0.000343 0.005448 0.01468 0.001785
DUSP5 0.032129 0.013697 0.016863 0.021642 0.094732 0.06164 0.046391
DUSP6 0.30566 1.49485 0.946058 0.273573 0.63728 0.476319 0.001491
EGF 0.07966 0.01176 5.35E-05 0.014885 0.15822 0.005083 2.14E-05
EGFR 0.11344 0.017948 0.473029 0.673617 0.993092 0.48971 0 ELK3
0.055169 0.006302 0.096055 0.368567 0.25349 0.084788 0.008669 ELK4
7.62E-05 7.41E-05 0 1.49E-05 4.89E-05 0.000129 4.1E-06 ERBB2
0.04181 0.049378 0.348686 0.169575 0.111878 0.005013 0.005962 ERBB3
0.001773 0.018841 0.011518 0.002275 0.019641 0.000416 0.001913 EREG
0.01698 3.03E-05 0.089622 0.034435 0.004216 0.395021 1.44E-05 ETV4
0.5 0.312083 0.266093 0.003424 0.085971 0.026278 0.00015 ETV5
0.133972 0.858565 0.056328 0.156041 1.09429 0.271684 0.003065 FGF1
0.000192 0.002137 0.001511 0.035649 0.120742 NA 0.000388 FGF10
3.55E-05 0.000233 0.02683 0.000236 0.000482 NA 0 FGF11 0.007289
0.010672 0.072796 0.00176 0.025033 0.003401 0.005759 FGF16 0.001554
0.00176 0.000383 0.000163 0.000225 NA 0.000112 FGF17 0.000176
0.006615 0.000288 4.14E-05 0.002421 NA 0.000681 FGF18 0.001665
0.055939 0.002065 0.039282 0.014378 NA 0.004487 FGF19 8.22E-05
0.447513 2.79E-05 0 0.000167 NA 0.000231 FGF2 0.162668 0.125
1.02101 0.325336 0.456916 NA 0.021493 FGF20 0 0.070805 0.000892
0.000104 0.001362 NA 1.27E-05 FGF21 5.28E-05 0.002022 0 6.28E-05
0.00012 NA 0.00002 FGF22 0.001913 0.028164 0.005719 0.001848
0.006708 NA 0.018073 FGF3 0 3.29436 9.6E-06 0 0 NA 7.2E-06 FGF4 0
0.000147 0 0 0 NA 0 FGF5 0 0.00052 0.000042 0.230047 0.032577 NA
5.7E-06 FGF6 4.5E-06 4.32E-05 0 4.4E-06 1.81E-05 NA 6.9E-06 FGF7
0.00143 0.001106 3.07E-05 0.00294 0.001554 NA 7.5E-06 FGF8 0.000148
0.002197 0.001236 8.94E-05 0 NA 0.006172 FGF9 0.001106 0.217638
0.03983 0.000886 0.001289 NA 0.000341 FGFBP1 0.000943 0.02352
6.32E-05 0.000113 0.000475 7.78E-05 0.000019 FGFBP2 0.002307
0.000943 0.000977 0.001271 0.002079 0.000502 0.000411 FGFBP3
0.017824 0.008549 0.00982 0.00639 0.007239 0.005263 0.00128 FGFR1
0.101531 7.46426 4.16986 1.25701 1.81504 NA 0.10083 FGFR1IIIb
1.47E-05 0.11744 0.000217 0.000104 0.000527 NA 1.02E-05 FGFR1IIIc
0.020054 2.17347 3.83706 0.952638 0.231647 NA 0.032804 FGFR2
0.001631 0.006003 0.004129 0.00088 0.082469 NA 0.044502 FGFR2IIIb
7.57E-05 0.001848 0.000462 0.000103 0.007189 NA 0.004876 FGFR2IIIc
0.000402 0.000109 0.003853 0.000488 0.023036 NA 0.016516 FGFR3
0.004016 0.291183 0.01937 0.002291 0.373712 NA 0.029977 FGFR3IIIb
5.35E-05 0.049378 0.00044 1.12E-05 0.010672 NA 0.000756 FGFR3IIIc
0.000136 0.000299 0.002981 1.55E-05 0.021051 NA 0.000899 FGFR4
0.000715 0.007041 0.001047 7.16E-05 0.001748 NA 0.006003 FLRT1
0.0625 0.020475 0.012517 0.001848 0.016747 0.006434 0.015625 FLRT2
0.395021 0.001381 0.006944 0.329877 0.033262 0.084788 0.007239
FLRT3 0.000618 0.00074 0.00072 0.000108 0 0.002108 0.000223 HGF
2.87E-05 0.007391 0.011679 1.19748 0.000411 1.32869 2.01E-05 IGF1 0
5.02805 0.001689 0.070805 0.015303 0.00471 0.000226 IGF1R 0.368567
0.028956 1.34723 0.041521 0.668964 0.052193 0.142595 IGF2 0.008258
0.00357 0.05872 0.097396 0.000459 0.000197 0.035649 KDR 0.007705
0.001145 0.000196 0.001228 0.003308 0.000108 0.00004 MET 0.262429
0.066064 0.089622 1.3566 0.366021 0.697372 0.00088 MMP1 0.00639
0.000125 0.033493 0.104386 0.003906 0.049378 5.4E-06 MMP2 0.006708
0.139661 0.003545 5.61778 2.37841 10.9283 0.001289 NCAM1 0.022251
0.02836 8.7E-06 0.000446 0.125 0.004016 0.030186 PDGFRa 0 0.001325
0.005759 1.07177 0.650671 0.120742 0.000121 PDGFRb 0.021945 0.00148
0.002152 3.50642 0 1.28343 0.000338 PLAU 0.011598 0.000226 0.021493
1.46409 0.933033 2.56685 8.57E-05 PLAUR 0.098755 0.022718 0.003826
0.190782 0.939523 0.933033 0.041235 SERPINE1 0.044811 0.010027
0.003002 1.54756 3.53081 2.2974 2.46E-05 SOX9 0.535887 0.496546
0.02797 0.119908 3.34035 0.30566 0.000983 SPRY1 0.010097 0.001532
0.334482 0.070316 0.092783 0.003496 0.019505 SPRY2 0.028956
0.115024 0.008851 0.092783 0.432269 0.351111 0.017458 SPRY3
0.046391 0.015517 0.001785 0.001598 0.009291 0.007813 0.004425
SPRY4 0.00181 0.007239 0.002668 0.002065 0.002879 0.002197 0.00012
TGFa 0.001665 0.099442 0.021793 0.002259 0.266093 0.024689 0.000296
TNC 0 0.000531 0.001609 2.62079 2.32947 4.02782 0.000341 VIM
13.0864 2.71321 5.1337 31.3414 48.1679 22.4711 0.790041
Sequence CWU 1
1
101374PRTArtificial sequenceSynthetic 1Met Trp Ser Trp Lys Cys Leu
Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 Thr Leu Cys Thr Ala
Arg Pro Ser Pro Thr Leu Pro Glu Gln Ala Gln 20 25 30 Pro Trp Gly
Ala Pro Val Glu Val Glu Ser Phe Leu Val His Pro Gly 35 40 45 Asp
Leu Leu Gln Leu Arg Cys Arg Leu Arg Asp Asp Val Gln Ser Ile 50 55
60 Asn Trp Leu Arg Asp Gly Val Gln Leu Ala Glu Ser Asn Arg Thr Arg
65 70 75 80 Ile Thr Gly Glu Glu Val Glu Val Gln Asp Ser Val Pro Ala
Asp Ser 85 90 95 Gly Leu Tyr Ala Cys Val Thr Ser Ser Pro Ser Gly
Ser Asp Thr Thr 100 105 110 Tyr Phe Ser Val Asn Val Ser Asp Ala Leu
Pro Ser Ser Glu Asp Asp 115 120 125 Asp Asp Asp Asp Asp Ser Ser Ser
Glu Glu Lys Glu Thr Asp Asn Thr 130 135 140 Lys Pro Asn Pro Val Ala
Pro Tyr Trp Thr Ser Pro Glu Lys Met Glu 145 150 155 160 Lys Lys Leu
His Ala Val Pro Ala Ala Lys Thr Val Lys Phe Lys Cys 165 170 175 Pro
Ser Ser Gly Thr Pro Asn Pro Thr Leu Arg Trp Leu Lys Asn Gly 180 185
190 Lys Glu Phe Lys Pro Asp His Arg Ile Gly Gly Tyr Lys Val Arg Tyr
195 200 205 Ala Thr Trp Ser Ile Ile Met Asp Ser Val Val Pro Ser Asp
Lys Gly 210 215 220 Asn Tyr Thr Cys Ile Val Glu Asn Glu Tyr Gly Ser
Ile Asn His Thr 225 230 235 240 Tyr Gln Leu Asp Val Val Glu Arg Ser
Pro His Arg Pro Ile Leu Gln 245 250 255 Ala Gly Leu Pro Ala Asn Lys
Thr Val Ala Leu Gly Ser Asn Val Glu 260 265 270 Phe Met Cys Lys Val
Tyr Ser Asp Pro Gln Pro His Ile Gln Trp Leu 275 280 285 Lys His Ile
Glu Val Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro 290 295 300 Tyr
Val Gln Ile Leu Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu 305 310
315 320 Met Glu Val Leu His Leu Arg Asn Val Ser Phe Glu Asp Ala Gly
Glu 325 330 335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Leu Ser His
His Ser Ala 340 345 350 Trp Leu Thr Val Leu Glu Ala Leu Glu Glu Arg
Pro Ala Val Met Thr 355 360 365 Ser Pro Leu Tyr Leu Glu 370
2353PRTArtificial sequenceSynthetic 2Arg Pro Ser Pro Thr Leu Pro
Glu Gln Ala Gln Pro Trp Gly Ala Pro 1 5 10 15 Val Glu Val Glu Ser
Phe Leu Val His Pro Gly Asp Leu Leu Gln Leu 20 25 30 Arg Cys Arg
Leu Arg Asp Asp Val Gln Ser Ile Asn Trp Leu Arg Asp 35 40 45 Gly
Val Gln Leu Ala Glu Ser Asn Arg Thr Arg Ile Thr Gly Glu Glu 50 55
60 Val Glu Val Gln Asp Ser Val Pro Ala Asp Ser Gly Leu Tyr Ala Cys
65 70 75 80 Val Thr Ser Ser Pro Ser Gly Ser Asp Thr Thr Tyr Phe Ser
Val Asn 85 90 95 Val Ser Asp Ala Leu Pro Ser Ser Glu Asp Asp Asp
Asp Asp Asp Asp 100 105 110 Ser Ser Ser Glu Glu Lys Glu Thr Asp Asn
Thr Lys Pro Asn Pro Val 115 120 125 Ala Pro Tyr Trp Thr Ser Pro Glu
Lys Met Glu Lys Lys Leu His Ala 130 135 140 Val Pro Ala Ala Lys Thr
Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 145 150 155 160 Pro Asn Pro
Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys Pro 165 170 175 Asp
His Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala Thr Trp Ser Ile 180 185
190 Ile Met Asp Ser Val Val Pro Ser Asp Lys Gly Asn Tyr Thr Cys Ile
195 200 205 Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr Tyr Gln Leu
Asp Val 210 215 220 Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln Ala
Gly Leu Pro Ala 225 230 235 240 Asn Lys Thr Val Ala Leu Gly Ser Asn
Val Glu Phe Met Cys Lys Val 245 250 255 Tyr Ser Asp Pro Gln Pro His
Ile Gln Trp Leu Lys His Ile Glu Val 260 265 270 Asn Gly Ser Lys Ile
Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu 275 280 285 Lys Thr Ala
Gly Val Asn Thr Thr Asp Lys Glu Met Glu Val Leu His 290 295 300 Leu
Arg Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala 305 310
315 320 Gly Asn Ser Ile Gly Leu Ser His His Ser Ala Trp Leu Thr Val
Leu 325 330 335 Glu Ala Leu Glu Glu Arg Pro Ala Val Met Thr Ser Pro
Leu Tyr Leu 340 345 350 Glu 3360PRTArtificial sequenceSynthetic
3Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val Leu Val Thr Ala 1
5 10 15 Thr Leu Cys Thr Ala Arg Pro Ser Pro Thr Leu Pro Glu Gln Ala
Gln 20 25 30 Pro Trp Gly Ala Pro Val Glu Val Glu Ser Phe Leu Val
His Pro Gly 35 40 45 Asp Leu Leu Gln Leu Arg Cys Arg Leu Arg Asp
Asp Val Gln Ser Ile 50 55 60 Asn Trp Leu Arg Asp Gly Val Gln Leu
Ala Glu Ser Asn Arg Thr Arg 65 70 75 80 Ile Thr Gly Glu Glu Val Glu
Val Gln Asp Ser Val Pro Ala Asp Ser 85 90 95 Gly Leu Tyr Ala Cys
Val Thr Ser Ser Pro Ser Gly Ser Asp Thr Thr 100 105 110 Tyr Phe Ser
Val Asn Val Ser Asp Ala Leu Pro Ser Ser Glu Asp Asp 115 120 125 Asp
Asp Asp Asp Asp Ser Ser Ser Glu Glu Lys Glu Thr Asp Asn Thr 130 135
140 Lys Pro Asn Pro Val Ala Pro Tyr Trp Thr Ser Pro Glu Lys Met Glu
145 150 155 160 Lys Lys Leu His Ala Val Pro Ala Ala Lys Thr Val Lys
Phe Lys Cys 165 170 175 Pro Ser Ser Gly Thr Pro Asn Pro Thr Leu Arg
Trp Leu Lys Asn Gly 180 185 190 Lys Glu Phe Lys Pro Asp His Arg Ile
Gly Gly Tyr Lys Val Arg Tyr 195 200 205 Ala Thr Trp Ser Ile Ile Met
Asp Ser Val Val Pro Ser Asp Lys Gly 210 215 220 Asn Tyr Thr Cys Ile
Val Glu Asn Glu Tyr Gly Ser Ile Asn His Thr 225 230 235 240 Tyr Gln
Leu Asp Val Val Glu Arg Ser Pro His Arg Pro Ile Leu Gln 245 250 255
Ala Gly Leu Pro Ala Asn Lys Thr Val Ala Leu Gly Ser Asn Val Glu 260
265 270 Phe Met Cys Lys Val Tyr Ser Asp Pro Gln Pro His Ile Gln Trp
Leu 275 280 285 Lys His Ile Glu Val Asn Gly Ser Lys Ile Gly Pro Asp
Asn Leu Pro 290 295 300 Tyr Val Gln Ile Leu Lys Thr Ala Gly Val Asn
Thr Thr Asp Lys Glu 305 310 315 320 Met Glu Val Leu His Leu Arg Asn
Val Ser Phe Glu Asp Ala Gly Glu 325 330 335 Tyr Thr Cys Leu Ala Gly
Asn Ser Ile Gly Leu Ser His His Ser Ala 340 345 350 Trp Leu Thr Val
Leu Glu Ala Leu 355 360 4339PRTArtificial sequenceSynthetic 4Arg
Pro Ser Pro Thr Leu Pro Glu Gln Ala Gln Pro Trp Gly Ala Pro 1 5 10
15 Val Glu Val Glu Ser Phe Leu Val His Pro Gly Asp Leu Leu Gln Leu
20 25 30 Arg Cys Arg Leu Arg Asp Asp Val Gln Ser Ile Asn Trp Leu
Arg Asp 35 40 45 Gly Val Gln Leu Ala Glu Ser Asn Arg Thr Arg Ile
Thr Gly Glu Glu 50 55 60 Val Glu Val Gln Asp Ser Val Pro Ala Asp
Ser Gly Leu Tyr Ala Cys 65 70 75 80 Val Thr Ser Ser Pro Ser Gly Ser
Asp Thr Thr Tyr Phe Ser Val Asn 85 90 95 Val Ser Asp Ala Leu Pro
Ser Ser Glu Asp Asp Asp Asp Asp Asp Asp 100 105 110 Ser Ser Ser Glu
Glu Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val 115 120 125 Ala Pro
Tyr Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala 130 135 140
Val Pro Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 145
150 155 160 Pro Asn Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe
Lys Pro 165 170 175 Asp His Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala
Thr Trp Ser Ile 180 185 190 Ile Met Asp Ser Val Val Pro Ser Asp Lys
Gly Asn Tyr Thr Cys Ile 195 200 205 Val Glu Asn Glu Tyr Gly Ser Ile
Asn His Thr Tyr Gln Leu Asp Val 210 215 220 Val Glu Arg Ser Pro His
Arg Pro Ile Leu Gln Ala Gly Leu Pro Ala 225 230 235 240 Asn Lys Thr
Val Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val 245 250 255 Tyr
Ser Asp Pro Gln Pro His Ile Gln Trp Leu Lys His Ile Glu Val 260 265
270 Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro Tyr Val Gln Ile Leu
275 280 285 Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu Met Glu Val
Leu His 290 295 300 Leu Arg Asn Val Ser Phe Glu Asp Ala Gly Glu Tyr
Thr Cys Leu Ala 305 310 315 320 Gly Asn Ser Ile Gly Leu Ser His His
Ser Ala Trp Leu Thr Val Leu 325 330 335 Glu Ala Leu
5592PRTArtificial sequenceSynthetic 5Met Trp Ser Trp Lys Cys Leu
Leu Phe Trp Ala Val Leu Val Thr Ala 1 5 10 15 Thr Leu Cys Thr Ala
Arg Pro Ser Pro Thr Leu Pro Glu Gln Ala Gln 20 25 30 Pro Trp Gly
Ala Pro Val Glu Val Glu Ser Phe Leu Val His Pro Gly 35 40 45 Asp
Leu Leu Gln Leu Arg Cys Arg Leu Arg Asp Asp Val Gln Ser Ile 50 55
60 Asn Trp Leu Arg Asp Gly Val Gln Leu Ala Glu Ser Asn Arg Thr Arg
65 70 75 80 Ile Thr Gly Glu Glu Val Glu Val Gln Asp Ser Val Pro Ala
Asp Ser 85 90 95 Gly Leu Tyr Ala Cys Val Thr Ser Ser Pro Ser Gly
Ser Asp Thr Thr 100 105 110 Tyr Phe Ser Val Asn Val Ser Asp Ala Leu
Pro Ser Ser Glu Asp Asp 115 120 125 Asp Asp Asp Asp Asp Ser Ser Ser
Glu Glu Lys Glu Thr Asp Asn Thr 130 135 140 Lys Pro Asn Pro Val Ala
Pro Tyr Trp Thr Ser Pro Glu Lys Met Glu 145 150 155 160 Lys Lys Leu
His Ala Val Pro Ala Ala Lys Thr Val Lys Phe Lys Cys 165 170 175 Pro
Ser Ser Gly Thr Pro Asn Pro Thr Leu Arg Trp Leu Lys Asn Gly 180 185
190 Lys Glu Phe Lys Pro Asp His Arg Ile Gly Gly Tyr Lys Val Arg Tyr
195 200 205 Ala Thr Trp Ser Ile Ile Met Asp Ser Val Val Pro Ser Asp
Lys Gly 210 215 220 Asn Tyr Thr Cys Ile Val Glu Asn Glu Tyr Gly Ser
Ile Asn His Thr 225 230 235 240 Tyr Gln Leu Asp Val Val Glu Arg Ser
Pro His Arg Pro Ile Leu Gln 245 250 255 Ala Gly Leu Pro Ala Asn Lys
Thr Val Ala Leu Gly Ser Asn Val Glu 260 265 270 Phe Met Cys Lys Val
Tyr Ser Asp Pro Gln Pro His Ile Gln Trp Leu 275 280 285 Lys His Ile
Glu Val Asn Gly Ser Lys Ile Gly Pro Asp Asn Leu Pro 290 295 300 Tyr
Val Gln Ile Leu Lys Thr Ala Gly Val Asn Thr Thr Asp Lys Glu 305 310
315 320 Met Glu Val Leu His Leu Arg Asn Val Ser Phe Glu Asp Ala Gly
Glu 325 330 335 Tyr Thr Cys Leu Ala Gly Asn Ser Ile Gly Leu Ser His
His Ser Ala 340 345 350 Trp Leu Thr Val Leu Glu Ala Leu Glu Pro Lys
Ser Ser Asp Lys Thr 355 360 365 His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser 370 375 380 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 385 390 395 400 Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 405 410 415 Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 420 425 430
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 435
440 445 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr 450 455 460 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 465 470 475 480 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 485 490 495 Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys 500 505 510 Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 515 520 525 Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 530 535 540 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 545 550 555
560 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
565 570 575 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 580 585 590 6571PRTArtificial sequenceSynthetic 6Arg Pro
Ser Pro Thr Leu Pro Glu Gln Ala Gln Pro Trp Gly Ala Pro 1 5 10 15
Val Glu Val Glu Ser Phe Leu Val His Pro Gly Asp Leu Leu Gln Leu 20
25 30 Arg Cys Arg Leu Arg Asp Asp Val Gln Ser Ile Asn Trp Leu Arg
Asp 35 40 45 Gly Val Gln Leu Ala Glu Ser Asn Arg Thr Arg Ile Thr
Gly Glu Glu 50 55 60 Val Glu Val Gln Asp Ser Val Pro Ala Asp Ser
Gly Leu Tyr Ala Cys 65 70 75 80 Val Thr Ser Ser Pro Ser Gly Ser Asp
Thr Thr Tyr Phe Ser Val Asn 85 90 95 Val Ser Asp Ala Leu Pro Ser
Ser Glu Asp Asp Asp Asp Asp Asp Asp 100 105 110 Ser Ser Ser Glu Glu
Lys Glu Thr Asp Asn Thr Lys Pro Asn Pro Val 115 120 125 Ala Pro Tyr
Trp Thr Ser Pro Glu Lys Met Glu Lys Lys Leu His Ala 130 135 140 Val
Pro Ala Ala Lys Thr Val Lys Phe Lys Cys Pro Ser Ser Gly Thr 145 150
155 160 Pro Asn Pro Thr Leu Arg Trp Leu Lys Asn Gly Lys Glu Phe Lys
Pro 165 170 175 Asp His Arg Ile Gly Gly Tyr Lys Val Arg Tyr Ala Thr
Trp Ser Ile 180 185 190 Ile Met Asp Ser Val Val Pro Ser Asp Lys Gly
Asn Tyr Thr Cys Ile 195 200 205 Val Glu Asn Glu Tyr Gly Ser Ile Asn
His Thr Tyr Gln Leu Asp Val 210 215 220 Val Glu Arg Ser Pro His Arg
Pro Ile Leu Gln Ala Gly Leu Pro Ala 225 230 235 240 Asn Lys Thr Val
Ala Leu Gly Ser Asn Val Glu Phe Met Cys Lys Val 245 250 255 Tyr Ser
Asp Pro Gln Pro His Ile Gln Trp
Leu Lys His Ile Glu Val 260 265 270 Asn Gly Ser Lys Ile Gly Pro Asp
Asn Leu Pro Tyr Val Gln Ile Leu 275 280 285 Lys Thr Ala Gly Val Asn
Thr Thr Asp Lys Glu Met Glu Val Leu His 290 295 300 Leu Arg Asn Val
Ser Phe Glu Asp Ala Gly Glu Tyr Thr Cys Leu Ala 305 310 315 320 Gly
Asn Ser Ile Gly Leu Ser His His Ser Ala Trp Leu Thr Val Leu 325 330
335 Glu Ala Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
340 345 350 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro 355 360 365 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr 370 375 380 Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn 385 390 395 400 Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg 405 410 415 Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 420 425 430 Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 435 440 445 Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 450 455
460 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
465 470 475 480 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe 485 490 495 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu 500 505 510 Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe 515 520 525 Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly 530 535 540 Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr 545 550 555 560 Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 565 570 721PRTArtificial
sequenceSynthetic 7Met Trp Ser Trp Lys Cys Leu Leu Phe Trp Ala Val
Leu Val Thr Ala 1 5 10 15 Thr Leu Cys Thr Ala 20 8232PRTArtificial
sequenceSynthetic 8Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100
105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys 225 230 9228PRTArtificial
sequenceSynthetic 9Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro
Ala Pro Pro Val 1 5 10 15 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Phe Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 100
105 110 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220
Ser Pro Gly Lys 225 10229PRTArtificial sequenceSynthetic 10Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20
25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150
155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225
* * * * *
References