U.S. patent application number 15/631180 was filed with the patent office on 2018-01-04 for fgfr1 extracellular domain combination therapies.
This patent application is currently assigned to Five Prime Therapeutics, Inc.. The applicant listed for this patent is Five Prime Therapeutics, Inc.. Invention is credited to Thomas Brennan, Li Long.
Application Number | 20180002399 15/631180 |
Document ID | / |
Family ID | 46828642 |
Filed Date | 2018-01-04 |
United States Patent
Application |
20180002399 |
Kind Code |
A1 |
Long; Li ; et al. |
January 4, 2018 |
FGFR1 EXTRACELLULAR DOMAIN COMBINATION THERAPIES
Abstract
Methods of treating cancer comprising 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 selected from docetaxel, paclitaxel,
vincristine, carboplatin, cisplatin, oxaliplatin, doxorubicin,
5-fluorouracil (5-FU), leucovorin, pemetrexed, and bevacizumab are
provided. Dosage packs comprising an FGFR1 ECD and/or an FGFR1 ECD
fusion molecule and/or at least one additional therapeutic agent
selected from docetaxel, paclitaxel, vincristine, carboplatin,
cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),
leucovorin, pemetrexed, and bevacizumab are also provided. In some
embodiments, a dosage pack comprises instructions for administering
FGFR1 ECD and/or FGFR1 ECD fusion molecule with at least one
additional therapeutic agent.
Inventors: |
Long; Li; (Lafayette,
CA) ; Brennan; Thomas; (San Jose, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Five Prime Therapeutics, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Five Prime Therapeutics,
Inc.
South San Francisco
CA
|
Family ID: |
46828642 |
Appl. No.: |
15/631180 |
Filed: |
June 23, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14577330 |
Dec 19, 2014 |
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15631180 |
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13296168 |
Nov 14, 2011 |
8951972 |
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14577330 |
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61421462 |
Dec 9, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/519 20130101;
A61K 38/179 20130101; A61K 31/555 20130101; A61K 31/704 20130101;
A61K 31/7048 20130101; A61K 31/704 20130101; A61K 39/3955 20130101;
C07K 2319/30 20130101; A61K 31/282 20130101; A61K 33/24 20130101;
A61K 31/337 20130101; A61K 33/24 20130101; A61K 2039/507 20130101;
A61K 38/179 20130101; A61K 39/3955 20130101; A61K 38/38 20130101;
A61P 35/00 20180101; A61K 2300/00 20130101; A61K 31/555 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 38/38 20130101; A61K 31/513 20130101; A61K 2300/00 20130101;
A61K 31/337 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 31/4745 20130101; A61K 31/475 20130101; A61K 31/513 20130101;
A61K 31/365 20130101; C07K 14/71 20130101; A61K 31/4745 20130101;
A61K 31/7048 20130101; A61K 31/475 20130101; A61K 45/06 20130101;
A61K 31/519 20130101 |
International
Class: |
C07K 14/71 20060101
C07K014/71; A61K 31/337 20060101 A61K031/337; A61K 45/06 20060101
A61K045/06; A61K 39/395 20060101 A61K039/395; A61K 38/38 20060101
A61K038/38; A61K 38/17 20060101 A61K038/17; A61K 33/24 20060101
A61K033/24; A61K 31/7048 20060101 A61K031/7048; A61K 31/282
20060101 A61K031/282; A61K 31/555 20060101 A61K031/555; A61K 31/519
20060101 A61K031/519; A61K 31/513 20060101 A61K031/513; A61K 31/475
20060101 A61K031/475; A61K 31/4745 20060101 A61K031/4745; A61K
31/365 20060101 A61K031/365; A61K 31/704 20060101 A61K031/704 |
Claims
1. A method of treating lung cancer in a human subject comprising
administering to the subject an effective amount of a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) fusion
molecule and at least one additional therapeutic agent selected
from docetaxel, paclitaxel, vincristine, carboplatin, and
topotecan, wherein the FGFR1 ECD fusion molecule comprises an FGFR1
ECD comprising an amino acid sequence selected from SEQ ID NOs: 1
to 4 and a fusion partner.
2. The method of claim 1, wherein the at least one additional
therapeutic agent is docetaxel.
3. The method of claim 1, wherein the at least one additional
therapeutic agent is paclitaxel.
4. (canceled)
5. (canceled)
6. The method of claim 1, wherein the at least one additional
therapeutic agent is vincristine.
7. (canceled)
8. The method of claim 1, wherein the at least one additional
therapeutic agent is topotecan.
9. The method of claim 1, wherein the method comprises
administering to the subject at least two additional therapeutic
agents selected from docetaxel, paclitaxel, vincristine,
carboplatin, and topotecan.
10. (canceled)
11. The method of claim 9, wherein the at least two additional
therapeutic agents are paclitaxel and carboplatin.
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. The method of claim 1, wherein the fusion partner is an Fc.
19. The method of claim 1, wherein the FGFR1 ECD fusion molecule
comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
6.
20. (canceled)
21. The method of claim 1, wherein the lung cancer is non-small
cell lung cancer.
22. The method of claim 2, wherein the fusion partner is an Fc.
23. The method of claim 2, wherein the FGFR1 ECD fusion molecule
comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
6.
24. The method of claim 2, wherein the cancer is non-small cell
lung cancer.
25. The method of claim 9, wherein the fusion partner is an Fc.
26. The method of claim 9, wherein the FGFR1 ECD fusion molecule
comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
6.
27. The method of claim 9, wherein the lung cancer is non-small
cell lung cancer.
28. The method of claim 11, wherein the fusion partner is an
Fc.
29. The method of claim 11, wherein the FGFR1 ECD fusion molecule
comprises a sequence selected from SEQ ID NO: 5 and SEQ ID NO:
6.
30. The method of claim 11, wherein the lung cancer is non-small
cell lung cancer.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/577,330, filed Dec. 19, 2014, which is a
continuation of U.S. patent application Ser. No. 13/296,168, filed
Nov. 14, 2011, now U.S. Pat. No. 8,951,972, issued Feb. 10, 2015,
which claims priority to U.S. Provisional Application No.
61/421,462 filed Dec. 9, 2010, the contents of each of which is
incorporated herein by reference in its entirety for any
purpose.
BACKGROUND AND SUMMARY OF THE INVENTION
[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. Combining an anti-cancer
therapeutic molecule, such as a soluble form of FGFR1, with another
anti-cancer therapeutic molecule, can result in antagonistic,
additive, or synergistic effects on the efficacy of each of the
anti-cancer therapeutics.
[0003] The inventors have discovered that administration of an
FGFR1 ECD and docetaxel in a mouse xenograft model of non-small
cell lung cancer shows synergistic anti-tumor activity of the
therapeutic agents. In addition, the inventors have discovered that
administration of an FGFR1 ECD and at least one additional
therapeutic molecule selected from paclitaxel, vincristine,
carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil
(5-FU), leucovorin, pemetrexed, and bevacizumab, has at least
additive activity relative to each molecule administered alone in
certain mouse xenograft models.
[0004] In some embodiments, methods of treating cancer comprising
administering to a subject a fibroblast growth factor receptor 1
(FGFR1) extracellular domain (ECD) 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
vascular endothelial growth factor (VEGF) antagonist, a VEGF trap,
an anti-VEGF antibody, and bevacizumab are provided. In some
embodiments, the at least one additional therapeutic agent is
docetaxel. In some embodiments, the at least one additional
therapeutic agent is pemetrexed. In some embodiments, the at least
one additional therapeutic agent is cisplatin. In some embodiments,
the at least one additional therapeutic agent is paclitaxel. In
some embodiments, the at least one additional therapeutic agent is
5-FU. In some embodiments, the at least one additional therapeutic
agent is topotecan. In some embodiments, the at least one
additional therapeutic agent is viscristine. In some embodiments,
the at least one additional therapeutic agents is a VEGF
antagonist, such as an anti-VEGF antibody or a VEGF trap. In some
embodiments, the at least one additional therapeutic agent is
bevacizumab. In some embodiments, the at least one additional
therapeutic agent is sorafenib. In some embodiments, the FGFR1 ECD
comprises a sequence selected from SEQ ID NOs: 1 to 4.
[0005] In some embodiments, methods of treating cancer comprising
administering to a subject a fibroblast growth factor receptor 1
(FGFR1) extracellular domain (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 vascular epithelial growth factor (VEGF)
antagonist, a VEGF trap, an anti-VEGF antibody, and bevacizumab are
provided, wherein the FGFR1 ECD fusion molecule comprises an FGFR1
ECD and a fusion partner. In some embodiments, the at least one
additional therapeutic agent is docetaxel. In some embodiments, the
at least one additional therapeutic agent is pemetrexed. In some
embodiments, the at least one additional therapeutic agent is
cisplatin. In some embodiments, the at least one additional
therapeutic agent is paclitaxel. In some embodiments, the at least
one additional therapeutic agent is 5-FU. In some embodiments, the
at least one additional therapeutic agent is viscristine. In some
embodiments, the at least one additional therapeutic agent is
topotecan. In some embodiments, the at least one additional
therapeutic agent is a VEGF antagonist, such as an anti-VEGF
antibody or a VEGF trap. In some embodiments, the at least one
additional therapeutic agent is bevacizumab. In some embodiments,
the at least one additional therapeutic agent is sorafenib.
[0006] In some embodiments, methods of treating cancer comprising
administering to a subject an FGFR1 ECD or FGFR1 ECD fusion
molecule and at least two additional therapeutic agents selected
from docetaxel, paclitaxel, vincristine, carboplatin, cisplatin,
oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,
pemetrexed, etoposide, sorafenib, etoposide, topotecan, a vascular
epithelial growth factor (VEGF) antagonist, a VEGF trap, an
anti-VEGF antibody, and bevacizumab are provided, wherein the FGFR1
ECD fusion molecule comprises an FGFR1 ECD and a fusion partner. In
some embodiments, at least one of the two additional therapeutic
agents is paclitaxel. In some embodiments, at least one of the two
additional therapeutic agents is cisplatin. In some embodiments, at
least one of the two additional therapeutic agents is carboplatin.
In some embodiments, at least one of the two additional therapeutic
agents is oxaliplatin. In some embodiments, at least one of the two
additional therapeutic agents is 5-FU. In some embodiments, at
least one of the two additional therapeutic agents is doxorubicin.
In some embodiments, at least one of the two additional therapeutic
agents is etoposide. In some embodiments, at least one of the two
additional therapeutic agents is topotecan. In some embodiments, at
least one of the two additional therapeutic agents is a VEGF
antagonist, such as an anti-VEGF antibody or a VEGF trap. In some
embodiments, at least one of the two additional therapeutic agents
is 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 two additional therapeutic agents are cisplatin
and etoposide.
[0007] In some embodiments, methods of treating cancer comprising
administering to a subject an FGFR1 ECD or FGFR1 ECD fusion
molecule and at least three additional therapeutic agents selected
from docetaxel, paclitaxel, vincristine, carboplatin, cisplatin,
oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,
pemetrexed, etoposide, sorafenib, a VEGF antagonist, an anti-VEGF
antibody, a VEGF trap, and bevacizumab are provided, wherein the
FGFR1 ECD fusion molecule comprises an FGFR1 ECD and a fusion
partner. In some embodiments, at least one of the three additional
therapeutic agents is oxaliplatin. In some embodiments, at least
one of the three additional therapeutic agents is 5-FU. In some
embodiments, at least one of the three additional therapeutic
agents is leucovorin. In some embodiments, at least one of the
three additional therapeutic agents is carboplatin. In some
embodiments, at least one of the three additional therapeutic
agents is paclitaxel. In some embodiments, at least one of the
three additional therapeutic agents is 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.
In some embodiments, at least two of the three additional
therapeutic agents are cisplatin and etoposide.
[0008] The invention also relates, in some embodiments, to a
combination of an FGFR1 ECD or 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 vascular endothelial growth
factor (VEGF) antagonist, a VEGF trap, an anti-VEGF antibody, and
bevacizumab, for treatment of cancer. The invention further
relates, in some embodiments to a combination of an FGFR1 ECD or
FGFR1 ECD fusion molecule and at least two additional therapeutic
agents selected from docetaxel, paclitaxel, vincristine,
carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil
(5-FU), leucovorin, pemetrexed, etoposide, sorafenib, etoposide,
topotecan, a vascular epithelial growth factor (VEGF) antagonist, a
VEGF trap, an anti-VEGF antibody, and bevacizumab, for treatment of
cancer. The invention also relates, in some embodiments, to a
combination of an FGFR1 ECD or FGFR1 ECD fusion molecule and at
least three additional therapeutic agents selected from docetaxel,
paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin,
doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed,
etoposide, sorafenib, a VEGF antagonist, an anti-VEGF antibody, a
VEGF trap, and bevacizumab, for treatment of cancer.
[0009] In some embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion
molecule is packaged separately from the at least one, at least
two, or at least three additional therapeutic agents. In some
embodiments, the FGFR1 ECD and/or FGFR1 ECD fusion molecule is not
mixed with the at least one, at least two, or at least three
additional therapeutic agents prior to administration.
[0010] In some embodiments, a method of treating cancer comprising
administering to a subject an FGFR1-ECD.339-Fc and docetaxel is
provided, wherein the FGFR1-ECD.339-Fc comprises the amino acid
sequence of SEQ ID NO: 6. In some embodiments, a method of treating
cancer comprising administering to a subject an FGFR1-ECD.339-Fc
and docetaxel is provided, wherein the FGFR1-ECD.339-Fc consists of
the amino acid sequence of SEQ ID NO: 6.
[0011] 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.
[0012] In some embodiments, at least one dose of an FGFR1 ECD
and/or an FGFR1 ECD fusion molecule and at least one dose of at
least one additional therapeutic agent are administered
concurrently. In some embodiments, at least one dose of an FGFR1
ECD and/or an FGFR1 ECD fusion molecule and at least one dose of at
least one additional therapeutic agent are administered at the same
time.
[0013] In some embodiments, the FGFR1 ECD comprises an amino acid
sequence selected from SEQ ID NOs: 1 to 4. 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 comprises a signal
peptide. In some embodiments, the signal peptide comprises the
amino acid sequence of SEQ ID NO: 7.
[0014] In some embodiments, the FGFR1 ECD fusion molecule is an
amount in the range of about 0.5 mg/kg body weight to about 20
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 fusion molecule
is a dose of about 8 mg/kg body weight, while in some embodiments,
the therapeutically effective amount of the FGFR1 ECD fusion
molecule is a dose of about 16 mg/kg body weight (or at about 10
mg/kg body weight or about 20 mg/kg body weight, respectively, when
calculated using an extinction coefficient of 1.11 mL/mg*cm). In
some embodiments, the therapeutically effective amount of 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.
[0015] The invention also relates, in some embodiments, to a dosage
pack. In some embodiments, a dosage pack comprising at least one
component selected from: (i) a fibroblast growth factor receptor 1
(FGFR1) extracellular domain (ECD), (ii) a fibroblast growth factor
receptor 1 (FGFR1) extracellular domain (ECD) fusion molecule, and
(iii) at least one additional therapeutic agent selected from
docetaxel, paclitaxel, vincristine, carboplatin, cisplatin,
oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,
pemetrexed, etoposide, topotecan, a VEGF antagonist, an anti-VEGF
antibody, a VEGF trap, bevacizumab, and sorafenib; and instructions
for administering an FGFR1 ECD or FGFR1 ECD fusion molecule and the
at least one additional therapeutic to a patient is provided. In
some embodiments, the instructions included with the dosage pack
comprise instructions for administering a therapeutically effective
amount of FGFR1 ECD fusion molecule. In some embodiments, the
therapeutically effective amount of the FGFR1 ECD fusion molecule
is an amount in the range of about 0.5 mg/kg body weight to about
20 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 fusion molecule
is a dose of about 8 mg/kg body weight. In some embodiments, the
therapeutically effective amount of the FGFR1 ECD fusion molecule
is a dose of about 16 mg/kg body weight. In some embodiments, the
therapeutically effective amount of 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.
[0016] In some embodiments, the dosage pack comprises an FGFR1 ECD
and does not comprise the at least one additional therapeutic
agent, e.g., docetaxel, paclitaxel, vincristine, carboplatin,
cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),
leucovorin, pemetrexed, etoposide, topotecan, VEGF antagonist,
anti-VEGF antibody, VEGF trap, bevacizumab, or sorafenib. In some
embodiments, the dosage pack comprises an FGFR1 ECD fusion molecule
and does not comprise the at least one additional therapeutic
agent, e.g., docetaxel, paclitaxel, vincristine, carboplatin,
cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil (5-FU),
leucovorin, pemetrexed, etoposide, topotecan, VEGF antagonist,
anti-VEGF antibody, VEGF trap, bevacizumab, or sorafenib. In some
embodiments, the dosage pack comprises at least one additional
therapeutic agent, but does not comprise an FGFR1 ECD or an FGFR1
ECD fusion molecule. In some embodiments, the dosage pack
comprises: (i) an FGFR1 ECD or an FGFR1 ECD fusion molecule, and
(ii) at least one additional therapeutic agent. In some
embodiments, the at least one additional therapeutic agent is
docetaxel, paclitaxel, vincristine, carboplatin, cisplatin,
oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,
pemetrexed, etoposide, topotecan, a VEGF antagonist, an anti-VEGF
antibody, a VEGF trap, bevacizumab, or sorafenib. In some
embodiments, the at least one additional therapeutic agent is
docetaxel. In some embodiments, the at least one additional
therapeutic agent is pemetrexed. In some embodiments, the at least
one additional therapeutic agent is cisplatin. In some embodiments,
the at least one additional therapeutic agent is paclitaxel. In
some embodiments, the at least one additional therapeutic agent is
5-FU. In some embodiments, the at least one additional therapeutic
agent is viscristine. In some embodiments, the at least one
additional therapeutic agent is bevacizumab. In some embodiments,
the at least one additional therapeutic agent is sorafenib. In some
embodiments, the dosage pack comprises at least two additional
therapeutic agents, but does not comprise an FGFR1 ECD or an FGFR1
ECD fusion molecule. In some embodiments, the at least two
additional therapeutic agents are chosen from docetaxel,
paclitaxel, vincristine, carboplatin, cisplatin, oxaliplatin,
doxorubicin, 5-fluorouracil (5-FU), leucovorin, pemetrexed,
etoposide, topotecan, a VEGF antagonist, an anti-VEGF antibody, a
VEGF trap, bevacizumab, and sorafenib.
[0017] In some embodiments described above, the FGFR1 ECD or the
FGFR1 ECD portion of the FGFR1 ECD fusion molecule comprises a
sequence selected from SEQ ID NOs: 1 to 4. 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 FGFR1 ECD fusion molecule consists of 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.
[0018] In certain embodiments, the cancer is prostate cancer,
breast cancer, colorectal cancer, lung cancer, endometrial cancer,
head and neck cancer, laryngeal cancer, liver cancer, renal cancer
glioblastoma or pancreatic cancer. In certain embodiments, the
cancer is lung cancer. In certain embodiments, the cancer is renal
cancer. In certain embodiments, the cancer is colon cancer. In
certain embodiments, the cancer is breast cancer. In certain
embodiments, the cancer is endometrial cancer. In certain
embodiments, the cancer is prostate cancer.
[0019] Any embodiment described herein or any combination of
additional therapeutic agents thereof applies to any and all
methods of the invention described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, docetaxel alone, FGFR1-ECD.339-Fc and
docetaxel sequentially, and FGFR1-ECD.339-Fc and docetaxel
concurrently, as described in Example 1.
[0021] FIGS. 2A-B show the mean tumor volume (A) and body weight
(B) of mice administered FGFR1-ECD.339-Fc alone, pemetrexed alone
(62.5 mg/kg dose), or FGFR1-ECD.339-Fc and pemetrexed (62.5 mg/kg
dose), as described in Example 2.
[0022] FIGS. 3A-B show the mean tumor volume (A) and body weight
(B) of mice administered FGFR1-ECD.339-Fc alone, pemetrexed alone
(125 mg/kg dose), or FGFR1-ECD.339-Fc and pemetrexed (125 mg/kg
dose), as described in Example 2.
[0023] FIGS. 4A-B show the mean tumor volume (A) and body weight
(B) of mice administered FGFR1-ECD.339-Fc alone, pemetrexed alone
(250 mg/kg dose), or FGFR1-ECD.339-Fc and pemetrexed (250 mg/kg
dose), as described in Example 2.
[0024] FIG. 5 shows the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, cisplatin alone, or the combination of
FGFR1-ECD.339-Fc and cisplatin, as described in Example 3A.
[0025] FIG. 6 shows the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, paclitaxel alone, or the combination of
FGFR1-ECD.339-Fc and paclitaxel, as described in Example 3B.
[0026] FIG. 7 shows the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, 5-FU alone, or the combination of
FGFR1-ECD.339-Fc and 5-FU, as described in Example 3C.
[0027] FIGS. 8A-B show the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, docetaxel alone, or the combination of
FGFR1-ECD.339-Fc and docetaxel, at two different dosages of
docetaxel, 3 mg/kg (A) and 10 mg/kg (B), as described in Example
3D.
[0028] FIGS. 9A-C show the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, vincristine alone, or the combination of
FGFR1-ECD.339-Fc and vincristine, at two different dosages of
vincristine, 1 mg/kg beginning on day 1 (A) and 1.5 mg/kg beginning
on day 19 (B), as described in Example 3E. The mean body weight of
mice administered 1.5 mg/kg beginning on day 19 is also shown
(C).
[0029] FIG. 10 shows the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, carboplatin alone, paclitaxel alone, the
combination of carboplatin and paclitaxel, and the combination of
FGFR1-ECD.339-Fc, carboplatin, and paclitaxel, as described in
Example 3F.
[0030] FIGS. 11A-E show the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, the combination of 5-FU (10 mg/kg) and
leucovorin (10 mg/kg), and the combination of FGFR1-ECD.339-Fc,
5-FU (10 mg/kg), and leucovorin (10 mg/kg) (A); FGFR1-ECD.339-Fc
alone, the combination of 5-FU (20 mg/kg) and leucovorin (20
mg/kg), and the combination of FGFR1-ECD.339-Fc, 5-FU (20 mg/kg),
and leucovorin (20 mg/kg) (B); FGFR1-ECD.339-Fc alone, the
combination of 5-FU (30 mg/kg) and leucovorin (30 mg/kg), and the
combination of FGFR1-ECD.339-Fc, 5-FU (30 mg/kg), and leucovorin
(30 mg/kg) (C); FGFR1-ECD.339-Fc alone, bevacizumab alone, and the
combination of FGFR1-ECD.339-Fc and bevacizumab (D); and
FGFR1-ECD.339-Fc alone, the combination of bevacizumab, 5-FU, and
leucovorin, and the combination of FGFR1-ECD.339-Fc, bevacizumab,
5-FU, and leucovorin (E), as described in Example 4A.
[0031] FIGS. 12A-C show the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, oxaliplatin plus 5-FU and leucovorin (LV)
alone, and the combination of FGFR1-ECD.339-Fc and oxaliplatin plus
5-FU/LV, at different dosages of oxaliplatin, 5 mg/kg (A), 10 mg/kg
(B), and 15 mg/kg (C), as described in Example 4B.
[0032] FIG. 13 shows the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, the combination of doxorubicin and
paclitaxel, and the combination of FGFR1-ECD.339-Fc, doxorubicin,
and paclitaxel, as described in Example 5.
[0033] FIG. 14 shows the mean tumor volume in mice administered
FGFR1-ECD.339-Fc alone, a Kinase Insert Domain Receptor (KDR)-ECD
fusion molecule alone, and the combination of FGFR1-ECD.339-Fc and
the KDR-ECD fusion molecule, as described in Example 6.
DETAILED DESCRIPTION
[0034] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
Definitions
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0040] 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.
[0041] 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.
[0042] 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).
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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).
[0047] 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).
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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, non-small cell 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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 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.
[0058] 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
cyclosphosphamide (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
gamma1I and calicheamicin omegaI1 (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, carminomycin, 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 folinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; 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; sizofiran; 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); 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.TM.) combined with 5-FU and
leucovorin.
[0059] 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.
[0060] 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.
[0061] 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. Oncol.
8:200-206 (e.g., Table 1 listing anti-angiogenic agents used in
clinical trials).
[0062] "Docetaxel" refers to
1,7.beta.,10.beta.-trihydroxy-9-oxo-5.beta.,20-epoxytax-11-ene-2.alpha.,4-
,13.alpha.-triyl 4-acetate 2-benzoate
13-{(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoate},
which, in some embodiments, may be sold under the brand name
Taxotere.RTM.. In some embodiments, docetaxel is effective for
treating at least one cancer selected from breast cancer and non
small-cell lung cancer. Nonlimiting exemplary cancers that may be
treated with docetaxel include breast cancer, colorectal cancer,
lung cancer, ovarian cancer, prostate cancer, liver cancer, renal
cancer, gastric cancer, head and neck cancers, and melanoma.
[0063] "Paclitaxel" refers to
(2.alpha.,4.alpha.,5.beta.,7.beta.,10.beta.,13.alpha.)-4,10-bis(acetyloxy-
)-13-{[(2R,3S)-3-(benzoylamino)-2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihyd-
roxy-9-oxo-5,20-epoxytax-11-en-2-yl benzoate, which, in some
embodiments, may be sold under the brand name TAXOL.RTM..
Nonlimiting exemplary cancers that may be treated with paclitaxel
include breast cancer, lung cancer, and Kaposi's sarcoma.
[0064] "Carboplatin" refers to
cis-diammine(cyclobutane-1,1-dicarboxylate-O,O')platinum(II),
which, in some embodiments, may be sold under the brand name
Paraplatin.RTM.. Nonlimiting exemplary cancers that may be treated
with paclitaxel include, ovarian cancer, non-small cell lung
cancer, testicular cancer, stomach cancer, and bladder cancer.
[0065] "Oxaliplatin" refers to
[(1R,2R)-cyclohexane-1,2-diamine](ethanedioato-O,O')platinum(II),
which, in some embodiments, may be sold under the brand name
Eloxatin.RTM.. Nonlimiting exemplary cancers that may be treated
with oxaliplatin include colorectal cancer, gastric cancer, and
ovarian cancer.
[0066] "Cisplatin" refers to (SP-4-2)-diamminedichloridoplatinum.
Nonlimiting exemplary cancers that may be treated with cisplatin
include sarcomas, small cell lung cancer, ovarian cancer, bladder
cancer, testicular cancer, lymphomas, and germ cell tumors.
[0067] "Vincristine" refers to methyl
(1R,9R,10S,11R,12R,19R)-11-(acetyloxy)-12-ethyl-4-[(13S,15S,17S)-17-ethyl-
-17-hydroxy-13-(methoxycarbonyl)-1,11-diazatetracyclo[13.3.1.0.sup.4,12.0.-
sup.5,10]nonadeca-4(12),5,7,9-tetraen-13-yl]-8-formyl-10-hydroxy-5-methoxy-
-8,16-diazapentacyclo[10.6.1.0.sup.1,9.0.sup.2,7.0.sup.16,19]nonadeca-2,4,-
6,13-tetraene-10-carboxylate, which, in some embodiments, may be
sold under the brand name Vincasar.RTM.. Nonlimiting exemplary
cancers that may be treated with vincristine include Hodgkin's
disease, leukemia, non-Hodgkin's lymphoma, neuroblastoma,
rhabdomyosarcoma, acute lymphoblastic leukemia, and Wilms'
tumor.
[0068] "Pemetrexed" refers to
(S)-2-[4-[2-(4-amino-2-oxo-3,5,7-triazabicyclo[4.3.0]nona-3,8,10-trien-9--
yl)ethyl]benzoyl]aminopentanedioic acid, which, in some
embodiments, may be sold under the brand name Alimta.RTM..
Nonlimiting exemplary cancers that may be treated with pemetrexed
include non small cell lung cancer, mesothelioma, and esophageal
cancer.
[0069] "Doxorubicin" refers to
(8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,-
11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5-
,12-dione, which, in some embodiments, may be sold under the brand
name Adriamycin.RTM.. Nonlimiting exemplary cancers that may be
treated with doxorubicin include bladder cancer, breast cancer,
lung cancer, ovarian cancer, stomach cancer, thyroid cancer, soft
tissue sarcoma, multiple myeloma, Hodgkin's disease, leukemia,
non-Hodgkin's lymphoma, neuroblastoma, sarcoma, and Wilms'
tumor.
[0070] "5-FU" and "5-fluorouracil" refer to
5-fluoro-1H-pyrimidine-2,4-dione, which, in some embodiments, may
be sold under the brand name Adrucil.RTM.. Nonlimiting exemplary
cancers that may be treated with 5-FU include colorectal cancer,
pancreatic cancer, breast cancer, esophageal cancer, gastric
cancer, head and neck cancer, hepatoma, and ovarian cancer.
[0071] "Leucovorin" is also known as folinic acid, and refers to
(S)-2-[4-[(2-amino-5-formyl-4-oxo-5,6,7,8-tetrahydro-1H-pteridin-6-yl)met-
hylamino]benzoyl]aminopentanedioic acid. In some embodiments,
leucovorin is administered with 5-fluorouracil.
[0072] 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-A109" 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.
[0073] 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, 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.
[0074] 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.
[0075] 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
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.d 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.
[0076] 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).
[0077] 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.
[0078] "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.
[0079] "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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] Herein, "concurrent" dosing refers to the administration of
two therapeutic molecules within an eight hour time period. In some
embodiments, two therapeutic molecules are administered at the same
time. Two therapeutic molecules are considered to be administered
at the same time (i.e. simultaneously) if at least a portion of a
dose of each therapeutic molecule is administered within 1 hour.
Two therapeutic molecules are administered concurrently if at least
one dose is administered concurrently, even if one or more other
doses are not administered concurrently. In some embodiments,
concurrent administration includes a dosing regimen when the
administration of one or more therapeutic molecule(s) continues
after discontinuing the administration of one or more other
therapeutic molecules(s).
[0084] Administration "in combination with" one or more further
therapeutic agents includes concurrent (including simultaneous) and
consecutive (i.e., sequential) administration in any order.
[0085] 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
[0086] Methods of Treating Cancer Using FGFR1 ECDs and/or FGFR1 ECD
Fusion Molecules in Combination with Other Therapeutic Agents
[0087] The invention features the combination of a fibroblast
growth factor receptor 1 (FGFR1) extracellular domain (ECD) or
FGFR1 ECD fusion molecule with one or more additional anti-cancer
therapies and the use of such combinations in cancer treatment.
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, by
administering therapeutically effective amounts of an FGFR1 ECD
and/or FGFR1 ECD fusion molecule and one or more chemotherapeutic
agents to a subject diagnosed with or suffering from a previously
untreated cancer. 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 another aspect, the invention
provides 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 diagnosed
with a previously untreated cancer. In another aspect, 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
diagnosed with a previously untreated cancer. In yet another
aspect, 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 diagnosed
with a previously untreated cancer. In some embodiments, the one or
more VEGF antagonists are anti-VEGF antibodies and/or VEGF
traps.
[0088] In one example, 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 some embodiments, methods of treating
cancer comprising administering to a subject an FGFR1 ECD and/or
FGFR1 ECD fusion molecule and docetaxel are provided. In some
embodiments, methods of treating cancer comprising administering to
a subject an FGFR1 ECD and/or FGFR1 ECD fusion molecule and
pemetrexed are provided. In some embodiments, methods of treating
cancer comprising administering to a subject an FGFR1 ECD and/or
FGFR1 ECD fusion molecule and paclitaxel are provided. In some
embodiments, methods of treating cancer comprising administering to
a subject an FGFR1 ECD and/or FGFR1 ECD fusion molecule and
cisplatin are provided. In some embodiments, methods of treating
cancer comprising administering to a subject an FGFR1 ECD and/or
FGFR1 ECD fusion molecule and vincristine are provided. In some
embodiments, methods of treating cancer comprising administering to
a subject an FGFR1 ECD and/or FGFR1 ECD fusion molecule and 5-FU
are provided. In some embodiments, methods of treating cancer
comprising administering to a subject an FGFR1 ECD and/or FGFR1 ECD
fusion molecule and etoposide are provided. In some embodiments,
methods of treating cancer comprising administering to a subject an
FGFR1 ECD and/or FGFR1 ECD fusion molecule and topotecan are
provided. In some embodiments, methods of treating cancer
comprising administering to a subject an FGFR1 ECD and/or FGFR1 ECD
fusion molecule and a VEGF antagonist are provided. In some
embodiments, methods of treating cancer comprising administering to
a subject an FGFR1 ECD and/or FGFR1 ECD fusion molecule and an
anti-VEGF antibody are provided. In some embodiments, methods of
treating cancer comprising administering to a subject an FGFR1 ECD
and/or FGFR1 ECD fusion molecule and a VEGF trap are provided. In
some embodiments, methods of treating cancer comprising
administering to a subject an FGFR1 ECD and/or FGFR1 ECD fusion
molecule and bevacizumab are provided. In some embodiments, at
least one dose of the FGFR1 ECD and/or FGFR1 ECD fusion molecule
and at least one dose of at least one additional therapeutic agent
are administered concurrently. In some embodiments, at least one
dose of the FGFR1 ECD and/or FGFR1 ECD fusion molecule and at least
one dose of at least one additional therapeutic agent are
administered at the same time (i.e., simultaneously). In some
embodiments, at least one dose of the FGFR1 ECD and/or FGFR1 ECD
fusion molecule and at least one dose of at least two additional
therapeutic agents are administered concurrently or simultaneously.
In some embodiments, at least one dose of the FGFR1 ECD and/or
FGFR1 ECD fusion molecule and at least one dose of at least three
additional therapeutic agents are administered concurrently or
simultaneously. 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, and etoposide, topotecan, a VEGF antagonist, an
anti-VEGF antibody, a VEGF trap, sorafenib, 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. In some embodiments, at least one dose of
an FGFR1-ECD.339-Fc and at least one dose of at least one
additional therapeutic agent are administered concurrently. In some
embodiments, at least one dose of an FGFR1-ECD.339-Fc and at least
one dose of at least one additional therapeutic agent are
administered at the same time.
[0089] 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, 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. 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.
[0090] 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 protein 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.
[0091] 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.
[0092] 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.
[0093] 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 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.
[0094] 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.
[0095] Routes of Administration and Carriers
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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 some embodiments, the
dosage packs contain an FGFR1 ECD and/or FGFR1 ECD fusion molecule
but do not contain any additional therapeutic agent such as
docetaxel, paclitaxel, vincristine, carboplatin, cisplatin,
oxaliplatin, doxorubicin, 5-fluorouracil (5-FU), leucovorin,
pemetrexed, etoposide, topotecan, sorafenib, a VEGF antagonist, an
anti-VEGF antibody, a VEGF trap, or bevacizumab. In other
embodiments, the dosage packs contain at least one additional
therapeutic agent but do not contain the FGFR1 ECD or FGFR1 ECD
fusion molecule. In other embodiments, the dosage packs contain an
FGFR1 ECD and/or FGFR1 ECD fusion molecule and at least one
additional therapeutic agent, wherein the FGFR1 ECD and/or FGFR1
ECD fusion molecule is in a separate container from the at least
one additional therapeutic agent. In other embodiments, the FGFR1
ECD and/or FGFR1 ECD fusion molecule is in the same container as
the at least one additional therapeutic agent. In certain
embodiments where two or more additional therapeutic agents are
supplied, the two or more additional therapeutic agents may be in
separate or in the same containers. 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. In some
embodiments, a dosage pack comprises an FGFR1 ECD and/or an FGFR1
ECD fusion molecule and/or at least one additional therapeutic
agent selected from docetaxel, paclitaxel, vincristine,
carboplatin, cisplatin, oxaliplatin, doxorubicin, 5-fluorouracil
(5-FU), leucovorin, pemetrexed, etoposide, topotecan, sorafenib, a
VEGF antagonist, an anti-VEGF antibody, a VEGF trap, and
bevacizumab.
[0100] In some embodiments, a dosage pack further comprises
instructions for administering an FGFR1 ECD and/or an FGFR1 ECD
fusion molecule with at least one additional therapeutic agent to a
patient. In some embodiments, the instructions indicate that at
least one dose of the FGFR1 ECD and/or the FGFR1 ECD fusion
molecule should be administered concurrently with the at least one
additional therapeutic agent. In some embodiments, the instructions
indicate that at least one dose of the FGFR1 ECD and/or the FGFR1
ECD fusion molecule should be administered at the same time as the
at least one additional therapeutic agent.
[0101] 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.).
[0102] FGFR1 ECDs and FGFR1 ECD Fusion Molecules
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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 a 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.
[0107] 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.
[0108] 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
vascular epithelial growth factor (VEGF) agonist, a VEGF trap, an
anti-VEGF antibody, and bevacizumab is useful for treating cancer.
In some embodiments, an FGFR1 ECD and/or an FGFR1 ECD fusion
molecule is administered with docetaxel.
[0109] Fusion Partners and Conjugates
[0110] 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.
[0111] 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 album (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.
[0112] Polymers
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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 .epsilon.-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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] Markers
[0129] 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. Natl. 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.
[0130] Oligomerization Domain Fusion Partners
[0131] 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.
[0132] Antibody Fc Immunoglobulin Domain Fusion Partners
[0133] 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.
[0134] Albumin Fusion Partners and Albumin-Binding Molecule Fusion
Partners
[0135] 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.
[0136] Exemplary Attachment of Fusion Partners
[0137] 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).
[0138] 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.
[0139] 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.
[0140] Co-Translational and Post-Translational Modifications
[0141] 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.
[0142] 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.
[0143] FGFR1 ECD and FGFR1 ECD Fusion Molecule Expression and
Production Vectors
[0144] 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.
[0145] 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. Prog. 20:880-889 (2004).
[0146] 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.
[0147] Host Cells
[0148] 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.
[0149] 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.
[0150] 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.
[0151] Purification of FGFR1 ECD Polypeptides
[0152] 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.
EXAMPLES
[0153] 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. 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: Administration of FGFR1-ECD.339-Fc and Docetaxel in the
111703 Non-Small Cell Lung Cancer (NSCLC) Xenograft Model
[0154] 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 H1703 was used as the tumor model and was
purchased from ATCC (Manassas, Va.; Cat. No. CRL-5889). The cells
were cultured for three passages in RPMI+10% FBS+1% 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 2.5.times.10.sup.7 cells
per milliliter. The cells were implanted subcutaneously over the
right flank of the mice at 2.5.times.10.sup.6 cells/100
.mu.l/mouse. One day after tumor implantation, mice were randomized
according to body weight at 10 mice per group.
[0155] 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. Docetaxel was purchased
from Toronto Research Chemicals (North York, Ontario, Canada; cat.
No. D494420) and formulated in H.sub.2O containing 5% Tween 80 and
5% glucose. Docetaxel was administered i.p. at 25 mg/kg (equivalent
to about 75 mg/m.sup.2 in a human) once every three weeks for two
doses. In combination groups, FGFR1-ECD.339-Fc and docetaxel were
either given concurrently or sequentially with FGFR1-ECD.339-Fc
given one day before docetaxel or vice versa. Human albumin was
purchased from Grifols USA (Los Angeles, Calif.; Cat. No. NDC
61953-0002-1), formulated in PBS at 3 mg/mL and was used as
negative control at 300 .mu.g/100 .mu.L/mouse (15 mg/kg). The
dosing schedule for each set of mice is shown in Table 2.
TABLE-US-00002 TABLE 2 Dosing Schedule Group n Route Treatment
Schedule 1 10 i.p. Albumin 2.times./wk .times. 6 wks 2 10 i.p.
FGFR1-ECD.339-Fc 2.times./wk .times. 6 wks 3 10 i.p. Docetaxel
1.times./3 wk .times. 2 doses 4 10 i.p. FGFR1-ECD.339-Fc
2.times./wk .times. 6 wks i.p. Docetaxel 1.times./3 wk .times. 2
doses 5 10 i.p. FGFR1-ECD.339-Fc 2.times./wk .times. 6 wks i.p.
Docetaxel (1 day after) 1.times./3 wk .times. 2 doses 6 10 i.p.
FGFR1-ECD.339-Fc 2.times./wk .times. 6 wks i.p. Docetaxel (1 day
before) 1.times./3 wk .times. 2 doses
[0156] The tumor volume and body weight of the mice were monitored
twice a week throughout the study. The tumor volume was measured by
external caliper to determine the greatest longitudinal diameter
(length) and the greatest transverse diameter (width). The tumor
volume was then calculated using the following formula:
Tumor volume (mm.sup.3)=(length.times.width.sup.2)/2
[0157] On day 38, when the average tumor volume in the Albumin
group reached 850 mm.sup.3, the mice were euthanized by isoflurane
inhalation and cervical dislocation.
[0158] The mean tumor volume throughout the study for each set of
mice is shown in FIG. 1. In that experiment, sequential
administration of FGFR1-ECD.339-Fc and docetaxel inhibited tumor
growth more than either drug alone. Furthermore, in that
experiment, concurrent administration of FGFR1-ECD.339-Fc and
docetaxel was more effective than sequential administration at
inhibiting tumor growth. No weight loss was observed over the
course of the study. (Data not shown.)
[0159] The tumor volume of each group of mice on day 38 was
analyzed by one-way ANOVA followed by Tukey's test. The results of
that analysis are shown in Table 3.
TABLE-US-00003 TABLE 3 Tumor volume analysis on day 38 Mean Tumor
Tumor p Value compare volume growth to control mm.sup.3(.+-.SD)
inhibition (%) (Tukey's test) Albumin 835 (.+-.151) -- --
FGFR1-ECD.339-Fc 557 (.+-.151) 33 <0.01 Docetaxel 209 (.+-.303)
74 <0.001 FGFR1-ECD.339- 27 (.+-.55) 96 <0.001 Fc/Docetaxel
concurrent FGFR1-ECD.339- 99 (.+-.117) 88 <0.001 Fc/Docetaxel
Sequential Docetaxel/FGFR1- 72 (.+-.109) 91 <0.001 ECD.339-Fc
Sequential
[0160] Administration of FGFR1-ECD.339-Fc alone resulted in 33%
(p<0.01) tumor growth inhibition, and administration of
docetaxel alone resulted in 74% (p<0.001) tumor growth
inhibition. Sequential administration with FGFR1-ECD.339-Fc first
resulted in 88% (p<0.001) tumor growth inhibition, and
sequential administration with docetaxel first resulted in 91%
(p<0.001) tumor growth inhibition. Finally, concurrent dosing of
FGFR1-ECD.339-Fc and docetaxel resulted in 96% (p<0.001) tumor
growth inhibition.
[0161] Fractional tumor volume analysis was used to assess the
degree of enhanced (additive or synergistic) or decreased
(antagonistic) tumor growth inhibition in the sequential and
concurrent combinations of FGFR1-ECD.339-Fc and docetaxel. The
results of that analysis are shown in Table 4.
TABLE-US-00004 TABLE 4 Analysis of fractional tumor volume.sup.a on
day 38 FGFR1- Expected/ ECD.339-Fc Docetaxel Expected.sup.b
Observed Observed.sup.c Concurrent 0.68 0.25 0.17 0.03 5.67 FGFR1-
0.68 0.25 0.17 0.11 1.55 ECD.339-Fc/ Docetaxel sequential
Docetaxel/ 0.68 0.25 0.17 0.09 1.89 FGFR1- ECD.339-Fc sequential
.sup.aFractional tumor volume (FTV) = (Mean tumor volume (TV)
treated)/(Mean TV control) .sup.bExpected = (FTV drug 1) .times.
(FTV drug 2) .sup.cRatio of expected over observed, >2 =
synergistic; ~1 = additive; <0.5 = antagonistic.
[0162] Those results demonstrate that concurrent administration of
FGFR1-ECD.339-Fc and docetaxel results in synergistic inhibition of
tumor growth, while sequential administration of FGFR1-ECD.339-Fc
and docetaxel results in additive inhibition.
Example 2: Administration of FGFR1-ECD.339-Fc and Pemetrexed in the
11520 Non-Small Cell Lung Cancer (NSCLC) Xenograft Model
[0163] Six to eight 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. Squamous cell lung cancer
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 to four passages in RPMI+10% FBS+1% 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 PBS containing
50% Matrigel at 3.5.times.10.sup.7 cells/ml. The cells were
implanted subcutaneously over the right flank of the mice at
3.5.times.10.sup.6 cells/100 .mu.l/mouse. One day after tumor
implantation, mice were randomized according to body weight at 10
mice per group. Dosing for all groups began one day post tumor
implantation.
[0164] 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 six weeks. Pemetrexed disodium was
purchased from Fisher Scientific (Pittsburgh, Pa.; Cat. No.
NC9564691) and formulated in Saline USP at 12.5 mg/mL, 25 mg/mL,
and 50 mg/mL. Pemetrexed was administered i.p. at three different
dosage levels: 62.6 mg/kg (1.25 mg/100 .mu.l/mouse); 125 mg/kg (2.5
mg/100 .mu.l/mouse); and 250 mg/kg (5 mg/100 .mu.l/mouse) daily for
five days in week one, and daily for five days in week 2. In
combination groups, FGFR1-ECD.339-Fc and pemetrexed were
administered on the same schedule as when given as single agents,
and were administered concurrently on days 1, 4, 8, and 11. Human
albumin was purchased from Grifols USA (Los Angeles, Calif.; Cat.
No. NDC 61953-0002-1), formulated in PBS at 3 mg/mL and was used as
negative control at 300 .mu.g/100 .mu.L/mouse (15 mg/kg). The
dosing schedule for each group of mice is shown in Table 5.
TABLE-US-00005 TABLE 5 Dosing Schedule Group n Route Treatment Dose
Schedule 1 10 i.p. Albumin 15 mg/kg 2.times./wk .times. 6 wks 2 10
i.p. FGFR1- 15 mg/kg 2.times./wk .times. 6 wks ECD.339-Fc 3 10 i.p.
Pemetrexed 62.5 mg/kg QD .times. 5 .times. 2 cycles 4 10 i.p.
Pemetrexed 125 mg/kg QD .times. 5 .times. 2 cycles 5 10 i.p.
Pemetrexed 250 mg/kg QD .times. 5 .times. 2 cycles 6 10 i.p. FGFR1-
15 mg/kg 2.times./wk .times. 6 wks ECD.339-Fc 62.5 mg/kg QD .times.
5 .times. 2 i.p. Pemetrexed cycles 7 10 i.p. FGFR1- 15 mg/kg
2.times./wk .times. 6 wks ECD.339-Fc 125 mg/kg QD .times. 5 .times.
2 i.p. Pemetrexed cycles 8 10 i.p. FGFR1- 15 mg/kg 2.times./wk
.times. 6 wks ECD.339-Fc 250 mg/kg QD .times. 5 .times. 2 i.p.
Pemetrexed cycles
[0165] The tumor volume and body weight of the mice were monitored
twice a week throughout study. The tumor volume was measured and
calculated using the method and formula described in Example 1.
[0166] Animals were euthanized when any of the following signs were
observed before the end of the study: body weight loss of
.gtoreq.15% of initial body weight; tumor ulceration of .gtoreq.30%
of tumor surface area; mice were moribund; or the individual tumor
volume was .gtoreq.2000 mm.sup.3. The mice were euthanized by
isoflurane inhalation and cervical dislocation.
[0167] The mean tumor volume throughout the study for low
pemetrexed dosage groups, medium pemetrexed dosage groups, and high
pemetrexed dosage groups are shown in FIGS. 2A, 3A, and 4A,
respectively. FIGS. 2B, 3B, and 4B show the body weight of the mice
in each group over the course of the study. It appears that 250
mg/kg pemetrexed is approaching the maximum tolerated dose in the
mice, based on the loss of body weight following administration of
that dose. See FIG. 4B.
[0168] Administration of either FGFR1-ECD.339-Fc or pemetrexed at
the low or medium dose alone resulted in tumor inhibition over the
course of the study. The highest dose of pemetrexed alone did not
appear to inhibit tumor growth. Concurrent dosing of
FGFR1-ECD.339-Fc and pemetrexed resulted in greater inhibition of
tumor growth, although the tumor inhibition observed with the
combination of FGFR1-ECD.339-Fc and the highest dose of pemetrexed
was not statistically significant.
[0169] The body weight graphs show that the mice tolerated the low
and medium doses of pemetrexed well. At the high dose of
pemetrexed, the mice initially lost significant body weight. The
dosing was therefore stopped after the first 5-day dosing. Thus, in
the high dose group, the animals only received the first five doses
and missed the second five doses.
[0170] The tumor volume of each group of mice on day 49 was
analyzed by one-way ANOVA followed by Tukey's test. The results of
that analysis are shown in Table 6.
TABLE-US-00006 TABLE 6 Tumor volume analysis on day 49 Tumor p
Value Mean Tumor growth compare volume inhibition to control
mm.sup.3 (.+-.SD) (%) (Tukey's test) Albumin 917.3 (.+-.313.9) --
-- FGFR1-ECD.339-Fc 630.4 (.+-.461.5) 31 >0.05 Pemetrexed 62.5
mg/kg 544.7 (.+-.341.5) 40 >0.05 FGFR1-ECD.339-Fc/ 292.5
(.+-.198.5) 68 <0.01 Pemetrexed 62.5 mg/kg Pemetrexed 125 mg/kg
689.5 (.+-.328.7) 24 >0.05 FGFR1-ECD.339-Fc/ 389.2 (.+-.271.3)
57 <0.05 Pemetrexed 125 mg/kg Pemetrexed 250 mg/kg 926.6
(.+-.339.9) 1 >0.05 FGFR1-ECD.339-Fc/ 489.3 (.+-.231.0) 46
>0.05 Pemetrexed 250 mg/kg
[0171] Administration of FGFR1-ECD.339-Fc alone resulted in 31%
(p>0.05) tumor growth inhibition, and administration of
pemetrexed alone at 62.5, 125 or 250 mg/kg resulted in 40, 24 or 1%
(p>0.05) tumor growth inhibition respectively. Concurrent dosing
of FGFR1-ECD.339-Fc (15 mg/kg) and pemetrexed at 62.5, 125, or 250
mg/kg resulted in 68% (p<0.01), 57% (p<0.05) or 46%
(p>0.05) tumor growth inhibition respectively.
[0172] Fractional tumor volume analysis was used to assess the
degree of enhanced (additive or synergistic) or decreased
(antagonistic) tumor growth inhibition following administration of
FGFR1-ECD.339-Fc and pemetrexed. The results of that analysis are
shown in Table 7.
TABLE-US-00007 TABLE 7 Analysis of fractional tumor volume.sup.a on
day 49 FGFR1- Ex- Ob- Expected/ ECD.339-Fc Pemetrexed pected.sup.b
served Observed.sup.c Pemetrexed 0.68 0.59 0.40 0.31 1.29 62.5
Pemetrexed 0.68 0.75 0.51 0.42 1.21 125 Pemetrexed 0.68 1.00 0.68
0.53 1.28 250 .sup.aFractional tumor volume (FTV) = (Mean tumor
volume (TV) treated)/(Mean TV control) .sup.bExpected = (FTV drug
1) .times. (FTV drug 2) .sup.cRatio of expected over observed,
>2 = synergistic; ~1 = additive; <0.5 = antagonistic.
[0173] Those results demonstrate that administration of
FGFR1-ECD.339-Fc and pemetrexed results in additive inhibition of
tumor growth.
Example 3: Administration of FGFR1-ECD.339-Fc in Combination with
Various Chemotherapeutics in the A549 Non-Small Cell Lung Cancer
(NSCLC) Xenograft Model
[0174] Six weeks old female SCID mice were purchased from Charles
River Laboratories (Wilmington, Mass.) and were acclimated for 1
week before the start of the study. A549 cells purchased from ATCC
(Manassas, Va.; Cat. No. CCL-185) were cultured for three passages
in RPMI+10% FBS+1% 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' and
Mg' 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 after tumor implantation, mice were
randomized according to body weight at 10 mice per group.
[0175] The tumor volume and body weight of the mice were monitored
twice a week throughout each study. The tumor volume was measured
and calculated using the method and formula described in Example
1.
[0176] The tumor volume of each group of mice at the end of each
study was analyzed by one-way ANOVA followed by Tukey's test.
Fractional tumor volume analysis was then used to assess the degree
of enhanced (additive or synergistic) or decreased (antagonistic)
tumor growth inhibition achieved following administration of
FGFR1-ECD.339-Fc with one or more additional chemotherapeutic
molecules.
[0177] Certain study details, and the results for each combination,
are discussed below.
[0178] A. FGFR1-ECD.339-Fc and Cisplatin
[0179] FGFR1-ECD.339-Fc was formulated in 0.9% Sodium Chloride
Injection USP (Henry Schein, Inc., Melville, N.Y.; Cat. No.
1533826) at 4 mg/ml and administered intraperitoneally (i.p.) at 20
mg/kg (400 .mu.g/100 .mu.l/mouse) twice per week for six weeks.
Cisplatin was purchased from Sigma-Aldrich (St. Louis, Mo.; Cat.
No. P4394), formulated in 0.9% saline, and administered i.p. at 3.5
mg/kg (17 .mu.g/100 .mu.l per mouse) once per week for six weeks.
Saline was used as negative control and was administered i.p. at
100 .mu.l per mouse twice a week for six weeks.
[0180] On day 42, when the average tumor volume in the vehicle
group reached 1300 mm.sup.3, the mice were euthanized by isoflurane
inhalation and cervical dislocation.
[0181] The mean tumor volume throughout the study for each set of
mice is shown in FIG. 5. In that experiment, administration of
FGFR1-ECD.339-Fc and cisplatin inhibited tumor growth more than
either drug alone. Further, the mice did not lose weight over the
course of that study. (Data not shown.)
[0182] The tumor volume of each group of mice on day 42 was
analyzed by one-way ANOVA followed by Tukey's test. The results of
that analysis are shown in Table 8.
TABLE-US-00008 TABLE 8 Tumor volume analysis on day 42 Tumor p
Value Mean Tumor growth compare volume inhibition to control
mm.sup.3 (.+-.SD) (%) (Tukey's test) Saline vehicle 1339 (.+-.411)
-- -- FGFR1-ECD.339-Fc 807 (.+-.431) 39 <0.01 Cisplatin 682
(.+-.195) 49 <0.001 FGFR1-ECD.339-Fc + 382 (.+-.210) 71
<0.001 Cisplatin
[0183] In order to determine whether combination of
FGFR1-ECD.339-Fc and cisplatin resulted in enhanced (additive or
synergistic) or decreased (antagonistic) antitumor activity,
fractional tumor volume was analyzed as described in Example 1. The
results of that analysis are shown in Table 9.
TABLE-US-00009 TABLE 9 Analysis of fractional tumor volume.sup.a on
day 42 relative to control FGFR1- Ex- Ob- Expected/ ECD.339-Fc
Cisplatin pected.sup.b served Observed.sup.c FGFR1- 0.60 0.5 0.30
0.28 1.07 ECD.339-Fc + cisplatin .sup.aFractional tumor volume
(FTV) = (Mean tumor volume (TV) treated)/(Mean TV control)
.sup.bExpected = (FTV drug 1) .times. (FTV drug 2) .sup.cRatio of
expected over observed, >2 = synergistic; ~1 = additive; <0.5
= antagonistic.
[0184] Those results show that the combination of FGFR1-ECD.339-Fc
and cisplatin resulted in additive inhibition of tumor growth in
that experiment.
[0185] B. FGFR1-ECD.339-Fc and Paclitaxel
[0186] The combination of FGFR1-ECD.339-Fc and paclitaxel was
tested in the A549 human non-small cell lung cancer xenograft
model, described above. FGFR1-ECD.339-Fc was formulated in 0.9%
Saline for Injection USP at 3 mg/ml. Paclitaxel was purchased from
Bedford Laboratories (Bedford, Ohio; Cat. No. 1075029) and was
formulated in 0.9% saline for injection containing 5% dextrose at
3.6 mg/ml for a dose of 18 mg/kg. FGFR1-ECD.339-Fc was administered
intraperitoneally (i.p.) at 15 mg/kg twice per week for 5 weeks.
Paclitaxel was administered i.p. at 18 mg/kg on days 8, 12, and
15.
[0187] When the average tumor volume in vehicle control group
reached .about.500 mm.sup.3, the mice were euthanized by isoflurane
inhalation and cervical dislocation.
[0188] The mean tumor volume throughout the study for each set of
mice is shown in FIG. 6. In that experiment, the combination of
FGFR1-ECD.339-Fc and paclitaxel inhibited tumor growth more than
either drug alone. Further, the mice did not lose weight over the
course of that study. (Data not shown.)
[0189] In order to determine whether the combination of
FGFR1-ECD.339-Fc and paclitaxel resulted in additive, synergistic,
or antagonistic activity, fractional tumor volume on day 31 and day
38 was analyzed as described in Example 1. The results of that
analysis are shown in Table 10.
TABLE-US-00010 TABLE 10 Analysis of fractional tumor volume.sup.a
on day 31 and day 38 FGFR1- Expected/ Day ECD.339-Fc Paclitaxel
Expected.sup.b Observed Observed.sup.c 31 0.84 0.57 0.48 0.26 1.81
38 0.89 0.50 0.45 0.39 1.13 .sup.aFractional tumor volume (FTV) =
(Mean tumor volume (TV) treated)/(Mean TV control) .sup.bExpected =
(FTV drug 1) .times. (FTV drug 2) .sup.cRatio of expected over
observed, >2 = synergistic; ~1 = additive; <0.5 =
antagonistic.
[0190] Those results show that administration of FGFR1-ECD.339-Fc
and paclitaxel resulted in additive inhibition of tumor growth.
[0191] C. FGFR1-ECD.339-Fc and 5-FU
[0192] The combination of FGFR1-ECD.339-Fc and 5-fluorouracil
(5-FU) was tested in the A549 human non-small cell lung cancer
xenograft model, described above. FGFR1-ECD.339-Fc was formulated
in 0.9% Saline for Injection USP at 3 mg/ml. 5-FU was purchased
from Sigma-Aldrich (St. Louis, Mo.; Cat. No. F6627) and was
initially dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St.
Louis, Mo.; Cat. No. D8418-50) at the concentration of 50 mg/ml as
a stock solution. The stock solution was further diluted in 0.9%
Sodium Chloride Injection USP to 6.6 mg/ml (for 33 mg/kg dosing).
FGFR1-ECD.339-Fc was administered intraperitoneally (i.p.) at 15
mg/kg twice per week for four weeks. 5-FU was administered i.p. at
33 mg/kg twice a week for three weeks.
[0193] Mice were euthanized on day 31 by isoflurane inhalation and
cervical dislocation.
[0194] The mean tumor volume throughout the study for each set of
mice is shown in FIG. 7. In that experiment, the combination of
FGFR1-ECD.339-Fc and 5-FU inhibited tumor growth more than either
drug alone. Further, the mice did not lose weight over the course
of that study. (Data not shown.)
[0195] In order to determine whether the combination of
FGFR1-ECD.339-Fc and 5-FU resulted in additive, synergistic, or
antagonistic activity, fractional tumor volume on day 31 was
analyzed as described in Example 1. The results of that analysis
are shown in Table 11.
TABLE-US-00011 TABLE 11 Analysis of fractional tumor volume.sup.a
on day 31 FGFR1- Expected/ Day ECD.339-Fc 5-FU Expected.sup.b
Observed Observed.sup.c 31 0.84 1.04 0.89 0.38 2.33
.sup.aFractional tumor volume (FTV) = (Mean tumor volume (TV)
treated)/(Mean TV control) .sup.bExpected = (FTV drug 1) .times.
(FTV drug 2) .sup.cRatio of expected over observed, >2 =
synergistic; ~1 = additive; <0.5 = antagonistic.
[0196] Those results show that administration of FGFR1-ECD.339-Fc
and 5-FU resulted in synergistic inhibition of tumor growth in that
experiment. The inventors note that similar experiments using 20
mg/kg or 50 mg/kg 5-FU did not result in synergy. (Data not
shown.)
[0197] D. FGFR1-ECD.339-Fc and Docetaxel
[0198] The combination of FGFR1-ECD.339-Fc and docetaxel was tested
in the A549 human non-small cell lung cancer xenograft model,
described above. 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. Docetaxel was
administered i.p. at 3 mg/kg or 10 mg/kg twice per week for four
weeks.
[0199] Mice in the albumin control group were euthanized on day 32
by isoflurane inhalation and cervical dislocation when the average
tumor volume in the group reached 1000 mm.sup.3.
[0200] The mean tumor volume throughout the study for each set of
mice is shown in FIGS. 8A (3 mg/kg docetaxel) and 8B (10 mg/kg
docetaxel). In that experiment, the combination of FGFR1-ECD.339-Fc
and docetaxel inhibited tumor growth more than either drug alone,
at either dosage of docetaxel. Further, the mice did not lose
weight over the course of that study, at either dosage of
docetaxel. (Data not shown.)
[0201] In order to determine whether the combination of
FGFR1-ECD.339-Fc and docetaxel resulted in additive, synergistic,
or antagonistic activity, fractional tumor volume on day 32 was
analyzed as described in Example 1. The results of that analysis
are shown in Table 12.
TABLE-US-00012 TABLE 12 Analysis of fractional tumor volume.sup.a
on day 32 FGFR1- ECD.339- Ob- Expected/ Day Fc Docetaxel
Expected.sup.b served Observed.sup.c 32 (3 mg/kg 0.54 0.79 0.43
0.44 0.95 docetaxel) 32 (10 mg/kg 0.54 0.58 0.31 0.42 0.73
docetaxel) .sup.aFractional tumor volume (FTV) = (Mean tumor volume
(TV) treated)/(Mean TV control) .sup.bExpected = (FTV drug 1)
.times. (FTV drug 2) .sup.cRatio of expected over observed, >2 =
synergistic; ~1 = additive; <0.5 = antagonistic.
[0202] Those results show that administration of FGFR1-ECD.339-Fc
and docetaxel resulted in additive inhibition of tumor growth in
that experiment.
[0203] E. FGFR1-ECD.339-Fc and Vincristine
[0204] The combination of FGFR1-ECD.339-Fc and vincristine was
tested in the A549 human non-small cell lung cancer xenograft
model, described above. FGFR1-ECD.339-Fc was formulated in 0.9%
Saline for Injection USP at 1.5 mg/ml for administration at 15
mg/kg (300 .mu.g/200 .mu.l per mouse), or 2 mg/ml for
administration at 20 mg/kg (400 .mu.g/200 .mu.l per mouse).
Vincristine was obtained from Fluka-Sigma (St. Louis, Mo. 63103,
Catalog #V8879) and was formulated in 0.9% Saline for Injection USP
at 0.1 mg/ml or 0.15 mg/mL for administration at 1 mg/kg (0.02
.mu.g/100 .mu.l per mouse) or 1.5 mg/kg (0.03 .mu.g/200 .mu.l per
mouse), respectively.
[0205] In the first experiment, FGFR1-ECD.339-Fc was administered
intraperitoneally (i.p.) at 15 mg/kg twice per week starting on day
1 for six weeks, and vincristine was administered i.p. at 1 mg/kg
on days 8, 15, and 22. In the second experiment, FGFR1-ECD.339-Fc
was administered intraperitoneally (i.p.) at 20 mg/kg twice per
week starting on day 19 for seven weeks, and vincristine was
administered i.p. at 1.5 mg/kg on days 27, 34, and 41.
[0206] Mice from the first experiment were euthanized 46 days post
tumor implantation. In the second study, mice in the albumin
control group and mice in the FGFR1-ECD.339-Fc group were
euthanized 70 days post tumor implantation, while mice in the
vincristine-treated groups were euthanized 77 days post tumor
implantation. All of the mice were euthanized by isoflurane
inhalation and cervical dislocation.
[0207] The mean tumor volume throughout the study for each set of
mice is shown in FIGS. 9A (1 mg/kg vincristine; dosing begun at day
1) and 9B (1.5 mg/kg vincristine; dosing begun at day 19). In that
experiment, the combination of FGFR1-ECD.339-Fc and vincristine
inhibited tumor growth more than either drug alone, at either
dosage of vincristine, and at either dosing schedule. Further, at
the lower dose of vincristine, the mice did not lose weight. (Data
not shown.) At the higher dose of vincristine, the mice lost
weight, indicating that the higher dose is closer to the maximum
tolerated dose. See FIG. 9C.
[0208] In order to determine whether the combination of
FGFR1-ECD.339-Fc and vincristine resulted in additive, synergistic,
or antagonistic activity, fractional tumor volume on days 39 and 46
for the first experiment, and on days 70 for the second experiment,
was analyzed as described in Example 1. The results of that
analysis are shown in Table 13.
TABLE-US-00013 TABLE 13 Analysis of fractional tumor volume.sup.a
Dose of FGFR1- FGFR1- Dose of ECD.339- Expected/ ECD.339-Fc
Vincristine Day Fc Vincristine Exp..sup.b Obs. Observed.sup.c 15
mg/kg 1 mg/kg 39 0.50 0.53 0.27 0.23 1.16 (start day 1) 15 mg/kg 1
mg/kg 46 0.40 0.55 0.22 0.24 0.91 (start day 1) 20 mg/kg 1.5 mg/kg
70 0.71 0.41 0.29 0.14 2.07 (start day 19) .sup.aFractional tumor
volume (FTV) = (Mean tumor volume (TV) treated)/(Mean TV control)
.sup.bExpected = (FTV drug 1) .times. (FTV drug 2) .sup.cRatio of
expected over observed, >2 = synergistic; ~1 = additive; <0.5
= antagonistic.
[0209] Those results show that administration of FGFR1-ECD.339-Fc
and vincristine resulted in additive inhibition of tumor growth at
the lower dose of vincristine, and synergistic inhibition of tumor
growth at the higher dose of vincristine.
[0210] F. FGFR1-ECD.339-Fc, Carboplatin, and Paclitaxel
[0211] The combination of FGFR1-ECD.339-Fc, carboplatin, and
paclitaxel was tested in the A549 human non-small cell lung cancer
xenograft model, described above. FGFR1-ECD.339-Fc was formulated
in 0.9% Saline for Injection USP at 3 mg/ml (for administration at
15 mg/kg). Carboplatin was obtained from Sigma-Aldrich (St. Luis,
Mo. 63103, Catalog # C2538) and was formulated in 0.9% Saline for
Injection USP at 2.5 mg/mL for administration at 25 mg/kg (500
.mu.g/200 .mu.L per mouse). Paclitaxel was obtained from LC
Laboratories (Woburn, Mass. 01801; Catalog # P-9600) and was
formulated in a solution of 50.3% Cremophor.RTM. and 49.7%
dehydrated alcohol at 20 mg/mL as stock solution. The stock
solution was further diluted in 5% dextrose in 0.9% Saline for
Injection USP at 3 mg/mL for administration at 30 mg/kg (600
.mu.g/200 .mu.L per mouse).
[0212] FGFR1-ECD.339-Fc was administered intraperitoneally (i.p.)
at 15 mg/kg twice per week starting on day 7 for three weeks.
Carboplatin was administered i.p. at 25 mg/kg twice per week
starting on day 7 for three weeks. Paclitaxel was administered i.p.
at 30 mg/kg twice per week starting on day 8 for three weeks.
[0213] Mice were euthanized on day 34 in single agent groups and on
day 41 in combination groups. Mice were euthanized by isoflurane
inhalation and cervical dislocation.
[0214] The mean tumor volume throughout the study for each set of
mice is shown in FIG. 10. In that experiment, the combination of
FGFR1-ECD.339-Fc, carboplatin, and paclitaxel inhibited tumor
growth more than any of the drugs alone, and also more than the
combination of carboplatin and paclitaxel. No weight loss was
observed during the course of that study. (Data not shown.)
[0215] In order to determine whether the combination of
FGFR1-ECD.339-Fc, carboplatin, and paclitaxel resulted in additive,
synergistic, or antagonistic activity, fractional tumor volume on
day 28 was analyzed as described in Example 1. The results of that
analysis are shown in Table 14.
TABLE-US-00014 TABLE 14 Analysis of fractional tumor volume.sup.a
FGFR1- ECD.339- Paclitaxel + Expected/ Day Fc carboplatin
Expected.sup.b Observed Observed.sup.c 28 0.58 0.27 0.16 0.14 1.15
.sup.aFractional tumor volume (FTV) = (Mean tumor volume (TV)
treated)/(Mean TV control) .sup.bExpected = (FTV drug 1) .times.
(FTV drug 2) .sup.cRatio of expected over observed, >2 =
synergistic; ~1 = additive; <0.5 = antagonistic.
[0216] Those results show that administration of FGFR1-ECD.339-Fc,
carboplatin, and paclitaxel resulted in additive inhibition of
tumor growth in that experiment.
Example 4: Administration of FGFR1-ECD.339-Fc in Combination with
Various Chemotherapeutics in the Colo205 Colon Cancer Xenograft
Model
[0217] Colo205 cells were purchased from ATCC (Manassas, Va.; Cat.
No. CCL-222) and were cultured for 3 passages in RPMI1640 media,
10% FBS, and 1% L-Glutamine at 37.degree. C. in a humidified
atmosphere with 5% CO.sub.2. Cells were resuspended in a solution
of 50%/v PBS and 50%/v Matrigel at a concentration of 25 million
cells/ml. Resuspended cells were kept on ice until implantation.
Six weeks old female SCID mice were purchased from Charles River
Laboratories (Wilmington, Mass.) and were acclimated for 1 week
before the start of the study. On day 0, 2.5 million cells/100
.mu.l were implanted over the right flank of the each mouse using a
27G1/2 needle. One day after tumor implantation, the mice were
randomized according to the body weight.
[0218] The tumor volume and body weight of the mice were monitored
twice a week throughout each study. The tumor volume was measured
and calculated using the method and formula described in Example
1.
[0219] The tumor volume of each group of mice at the end of each
study was analyzed by one-way ANOVA followed by Tukey's test.
Fractional tumor volume analysis was then used to assess the degree
of enhanced (additive or synergistic) or decreased (antagonistic)
tumor growth inhibition achieved following administration of
FGFR1-ECD.339-Fc with one or more additional chemotherapeutic
molecules.
[0220] Certain study details, and the results for each combination,
are discussed below.
[0221] A. FGFR1-ECD.339-Fc, 5-FU, Leucovorin, and Bevacizumab
[0222] FGFR1-ECD.339-Fc was formulated in 0.9% Sodium Chloride
Injection USP (Henry Schein, Inc, Melville, N.Y.; Cat. No. 1533826)
at 2 mg/ml and stored in screw cap microcentrifuge tubes at
-80.degree. C. The negative control reagent, human albumin, was
purchased from Grifols USA (Los Angeles, Calif.; Cat. No. NDC
61953-0002-1) and was formulated in PBS at 3 mg/ml. 5-FU was
purchased from Sigma-Aldrich (St. Louis, Mo.; Cat. No. F6627) and
was initially dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich,
St. Louis, Mo.; Cat. No. D8418-50) at the concentration of 50 mg/ml
as a stock solution. The stock solution was further diluted in 0.9%
Sodium Chloride Injection USP to 2 mg/ml (for 10 mg/kg dosing), to
4 mg/ml (for 20 mg/kg dosing), and to 6 mg/ml (for 30 mg/kg
dosing). Leucovorin (LV) was also purchased from Sigma-Aldrich
(Cat. No. F8259) and was formulated in 0.9% Sodium Chloride
Injection USP at 2 mg/ml (for 10 mg/kg dosing), to 4 mg/ml (for 20
mg/kg dosing), and to 6 mg/ml (for 30 mg/kg dosing). Bevacizumab
was purchased from Genentech, Inc (South San Francisco, Calif.;
Cat. No. 15734) and was diluted in 0.9% Sodium Chloride Injection
USP to 0.2 mg/ml (for 1 mg/kg dosing).
[0223] In the first experiment, FGFR1-ECD.339-Fc was combined with
three different concentrations of 5-FU/leucovorin. In the second
experiment, FGFR1-ECD.339-Fc was combined with either bevacizumab
or 5-FU/leucovorin, or both. The grouping and dosing schedule for
the experiments are shown in Table 15. The double line separates
the groups from the two experiments.
TABLE-US-00015 TABLE 15 Dosing Schedule (Dosing started one day
post tumor implantation) Drug 1 Drug 2 Drug 3 (Dose, route, (Dose,
route, (Dose, route, N Treatment Schedule) Schedule) Schedule) 10
Albumin + Albumin (10 mg/kg, Saline (100 .mu.l, -- Vehicle i.p.,
3x/wk .times. q.d. .times. 5 days) 4 wks) 10 FGFR1- FGFR1- -- --
ECD.339-Fc ECD.339-Fc (15 mg/kg, i.p., 3x/wk .times. 4 wks) 10
5-Fu/LV 5-FU (10 mg/kg, LV (10 mg/kg, i.p., q.d. .times. 5 days)
i.p., q.d. .times. 5 days) FGFR1- FGFR1- 5-FU (10 mg/kg, LV (10
mg/kg, ECD.339-Fc + ECD.339-Fc (15 mg/kg, i.p., q.d. .times. 5
days) i.p., q.d. .times. 5 5-Fu/LV i.p., 3x/wk .times. days) (10
mg/kg) 4 wks) 10 5-Fu/LV 5-FU (20 mg/kg, LV (20 mg/kg, i.p., q.d.
.times. 5 days) i.p., q.d. .times. 5 days) FGFR1- FGFR1- 5-FU (20
mg/kg, LV (20 mg/kg, ECD.339-Fc + ECD.339-Fc (15 mg/kg, i.p., q.d.
.times. 5 days) i.p., q.d. .times. 5 5-Fu/LV i.p., 3x/wk .times.
days) (20 mg/kg) 4 wks) 10 5-Fu/LV 5-FU (30 mg/kg, LV (30 mg/kg,
i.p., q.d. .times. 5 days) i.p., q.d. .times. 5 days) FGFR1- FGFR1-
5-FU (30 mg/kg, LV (30 mg/kg, ECD.339-Fc + ECD.339-Fc (15 mg/kg,
i.p., q.d. .times. 5 days) i.p., q.d. .times. 5 5-Fu/LV i.p., 3x/wk
.times. days) (30 mg/kg) 4 wks) 10 Bevacizumab Bev (1 mg/kg, --
i.p., 2x/wk .times. 4 wks) 10 Bevacizumab + Bev (1 mg/kg, 5-Fu/LV
(10 mg/kg -- 5-Fu/LV i.p., 2x/wk .times. 4 wks) each, i.p., q.d.
.times. 5 days) 10 FGFR1- FGFR1- -- -- ECD.339-Fc ECD.339-Fc (15
mg/kg, i.p., 3x/wk .times. 4 wks) 10 FGFR1- FGFR1- Bev (1 mg/kg, --
ECD.339-Fc + ECD.339-Fc (15 mg/kg, i.p., 2x/wk .times. 4 wks)
Bevacizumab i.p., 3x/wk .times. 4 wks) 10 FGFR1- FGFR1- Bev (1
mg/kg, 5-Fu/LV (10 mg/kg ECD.339-Fc + ECD.339-Fc (15 mg/kg, i.p.,
2x/wk .times. 4 wks) each, Bevacizumab + i.p., 3x/wk .times. i.p.,
q.d. .times. 5 5-Fu/LV 4 wks) days)
[0224] When the average tumor volume in a group was near 600
mm.sup.3, the mice in that group were euthanized by isoflurane
inhalation and cervical dislocation. The mean tumor volume
throughout the study for each set of mice is shown in FIG. 11. At
20 mg/kg 5-FU/leucovorin and 30 mg/kg 5-FU/leucovorin, the mice
lost about 3 grams and about 4 grams of body weight, respectively
(about 14% and 19%, respectively). (Data not shown.) No weight loss
in the mice was observed in any of the remaining groups. (Data not
shown.)
[0225] The order of effectiveness of the various treatments was
evaluated two ways: 1) the average tumor volume in each group on a
single time point (day); and 2) time for the average tumor volume
in each group to reach 500 mm.sup.3 (see the dotted line in FIG.
11E).
[0226] The results of that experiment according to both methods
showed that the antitumor effect of various treatment groups was in
the following order: FGFR1-ECD.339-Fc, bevacizumab, and
5-Fu/leucovorin>FGFR1-ECD.339-Fc and bevacizumab>bevacizumab
and 5-Fu/leucovorin=bevacizumab>FGFR1-ECD.339-Fc>vehicle.
[0227] The average tumor volume of each group of mice in the second
experiment on day 24 was analyzed by one-way ANOVA followed by
Tukey's test. The results of that analysis are shown in Table
16.
TABLE-US-00016 TABLE 16 Tumor volume analysis on day 24 Mean Tumor
p Value Tumor growth compare volume inhibition to control mm.sup.3
(.+-.SD) (%) (Tukey's test) Albumin + vehicle 693 (.+-.159) -- --
FGFR1-ECD.339-Fc 509 (.+-.144) 26 <0.05 5-FU/LV 511 (.+-.196) 26
>0.05 Bevacizumab 405 (.+-.56) 41 <0.001 Bevacizumab + 399
(.+-.131) 42 <0.001 5-Fu/LV FGFR1-ECD.339-Fc + 322 (.+-.116) 53
<0.001 Bevacizumab FGFR1-ECD.339-Fc + 269 (.+-.83) 61 <0.001
Bevacizumab + 5-Fu/LV
[0228] In order to determine whether administration of
FGFR1-ECD.339-Fc and 5-FU/leucovorin at various concentrations;
FGFR1-ECD.339-Fc and bevacizumab; or FGFR1-ECD.339-Fc, bevacizumab,
and 5-Fu/leucovorin resulted in additive, synergistic, or
antagonistic activity, fractional tumor volume was analyzed as
described in Example 1. The results of that analysis are shown in
Table 17.
TABLE-US-00017 TABLE 17 Analysis of fractional tumor volume.sup.a
FGFR1- ECD. Ex- Ob- Expected/ 339-Fc pected.sup.b served
Observed.sup.c 5-FU/LV (10 mg/kg) Day 16 0.76 0.86 0.67 0.62 1.08
5-FU/LV (20 mg/kg) Day 16 0.76 0.56 0.42 0.42 1.00 5-FU/LV (30
mg/kg) Day 16 0.76 0.36 0.27 0.28 0.96 Bevacizumab Day 24 0.73 0.58
0.42 0.46 0.91 Bevacizumab/ 5-Fu/LV Day 24 0.73 0.57 0.42 0.38 1.10
.sup.aFractional tumor volume (FTV) = (Mean tumor volume (TV)
treated)/(Mean TV control) .sup.bExpected = (FTV drug 1) .times.
(FTV drug 2) .sup.cRatio of expected over observed, >2 =
synergistic; ~1 = additive; <0.5 = antagonistic.
[0229] Those results show that administration of FGFR1-ECD.339-Fc
and 5-FU/leucovorin at various concentrations; FGFR1-ECD.339-Fc and
bevacizumab; or FGFR1-ECD.339-Fc, bevacizumab, and 5-Fu/leucovorin,
resulted in additive inhibition of tumor growth.
[0230] B. FGFR1-ECD.339-Fc, 5-FU, Leucovorin, and Oxaliplatin
[0231] The combination of FGFR1-ECD.339-Fc, 5-FU, leucovorin, and
oxaliplatin was tested in the Colo205 human colon cancer xenograft
model, described above. FGFR1-ECD.339-Fc was formulated in PBS at 3
mg/ml (for administration at 15 mg/kg). 5-FU was purchased from
Sigma-Aldrich (St. Louis, Mo.; Cat. No. F6627) and was initially
dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis,
Mo.; Cat. No. D8418-50) at the concentration of 50 mg/ml as a stock
solution. The stock solution was further diluted in 0.9% Sodium
Chloride Injection USP to 2 mg/ml (for 10 mg/kg dosing). Leucovorin
was also purchased from Sigma-Aldrich (Cat. No. F8259) and was
formulated in 0.9% Sodium Chloride Injection USP at 2 mg/ml (for 10
mg/kg dosing). Oxaliplatin was obtained from LC laboratories
(Woburn, Mass. 01801; Catalog #0-7111) and was formulated in 5%
Dextrose Injection (Baxter, Deerfield, Ill. 60015; Catalog #2B0082)
at 1 mg/mL, 2 mg/mL, and 3 mg/ml for administration at 5 mg/kg (100
.mu.g/100 .mu.L per mouse), 10 mg/kg (200 .mu.g/100 .mu.L per
mouse), and 15 mg/kg (300 .mu.g/100 .mu.l per mouse).
[0232] FGFR1-ECD.339-Fc was administered intraperitoneally (i.p.)
at 15 mg/kg twice per week starting on day 1 for four weeks. 5-FU
and leucovorin were each administered i.p. at 10 mg/kg daily for
five days. Oxaliplatin was administered i.p. at 5 mg/kg, 10 mg/kg,
or 15 mg/kg in one dose on day 1.
[0233] Mice were euthanized on day 28 by isoflurane inhalation and
cervical dislocation.
[0234] The mean tumor volume throughout the study for each set of
mice is shown in FIG. 12. In that experiment, the combination of
FGFR1-ECD.339-Fc, 5-FU, leucovorin, and oxaliplatin inhibited tumor
growth more than FGFR1-ECD.339-Fc alone or the combination 5-FU,
leucovorin, and oxaliplatin. Minimal weight loss in the mice was
observed at 5 mg/kg oxaliplatin (0.78 grams, or 4% body weight);
moderate weight loss was observed at 10 mg/kg (2.6 grams, or 13%
body weight); and more severe weight loss was observed at 15 mg/kg
oxaliplatin twice per week (3.7 grams, or 19% body weight). (Data
not shown.)
[0235] In order to determine whether the combination of
FGFR1-ECD.339-Fc, 5-FU, leucovorin, and oxaliplatin resulted in
additive, synergistic, or antagonistic activity, fractional tumor
volume on day 17 was analyzed as described in Example 1. The
results of that analysis are shown in Table 18.
TABLE-US-00018 TABLE 18 Analysis of fractional tumor volume.sup.a
on day 17 FGFR1- Oxaliplatin ECD.339- Oxaliplatin/ Ob- Expected/
dose Fc 5-FU/LV Expected.sup.b served Observed.sup.c 5 mg/kg 0.77
0.78 0.60 0.47 1.27 10 mg/kg 0.77 0.63 0.48 0.5 0.96 15 mg/kg 0.77
0.46 0.35 0.36 0.97 twice per week .sup.aFractional tumor volume
(FTV) = (Mean tumor volume (TV) treated)/(Mean TV control)
.sup.bExpected = (FTV drug 1) .times. (FTV drug 2) .sup.cRatio of
expected over observed, >2 = synergistic; ~1 = additive; <0.5
= antagonistic.
[0236] Those results show that administration of FGFR1-ECD.339-Fc,
5-FU, leucovorin, and oxaliplatin resulted in additive inhibition
of tumor growth at each dosage of oxaliplatin tested in that
experiment.
Example 5: Administration of FGFR1-ECD.339-Fc, Doxorubicin, and
Paclitaxel in the JIMT-1 Breast Cancer Xenograft Model
[0237] 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 breast cancer cell line
JIMT-1 was used as the tumor model and was purchased from Deutsche
Sammlung von Mikroorganismen and Zellkulturen GmbH (DMSZ,
Braunschweig, Germany; Cat. No. ACC 589). The cells were cultured
for ten passages in DMEM+10% FBS, 1% L-glutamine, and 1%
penicillin/streptomycin 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 after tumor
implantation, mice were randomized according to body weight at 10
mice per group.
[0238] FGFR1-ECD.339-Fc was formulated in 0.9% Saline for Injection
USP at 3 mg/ml and administered intraperitoneally (i.p.) at 15
mg/kg (300 .mu.g/100 .mu.l/mouse) twice per week for five weeks
beginning on day 7. Doxorubicin was obtained from Sigma-Aldrich
(St. Luis, Mo.; Catalog #44583) and was formulated in 0.9% Saline
for Injection USP at 0.05 mg/mL for administration at 0.5 mg/kg (10
.mu.g/200 .mu.L per mouse) once per week for five weeks, beginning
on day 7. Paclitaxel was obtained from Bedford Laboratories
(Bedford, Ohio; Cat. No. 1075029) and was formulated in a solution
of 50.3% Cremophor.RTM. and 49.7% dehydrated alcohol. The stock
solution was diluted in 5% dextrose/0.9% Saline for Injection USP
to a final concentration of 3 mg/mL paclitaxel in 16.8%
Cremophor.RTM., 16.6% dehydrated alcohol, 3.3% dextrose, 0.6%
Saline for Injection USP. Paclitaxel was administered at 30 mg/kg
(60 .mu.g/200 .mu.L per mouse) twice per week for five weeks
beginning on day 7.
[0239] The tumor volume and body weight of the mice were monitored
twice a week throughout the study. The tumor volume was measured
and calculated using the method and formula described in Example
1.
[0240] Mice were euthanized on day 42 by isoflurane inhalation and
cervical dislocation.
[0241] The mean tumor volume throughout the study for each set of
mice is shown in FIG. 13. In that experiment, the combination of
FGFR1-ECD.339-Fc, doxorubicin, and paclitaxel inhibited tumor
growth more than FGFR1-ECD.339-Fc alone or the combination of
doxorubicin and paclitaxel without FGFR1-ECD.339-Fc. No weight loss
was observed over the course of the study. (Data not shown.)
[0242] Fractional tumor volume analysis was used to assess the
degree of enhanced (additive or synergistic) or decreased
(antagonistic) tumor growth inhibition by the combination of
FGFR1-ECD.339-Fc, doxorubicin, and paclitaxel. The results of that
analysis are shown in Table 19.
TABLE-US-00019 TABLE 19 Analysis of fractional tumor volume.sup.a
on day 21 FGFR1- Doxorubicin ECD.339- and Expected/ Fc paclitaxel
Expected.sup.b Observed Observed.sup.c Day 21 0.93 0.59 0.55 0.50
1.10 .sup.aFractional tumor volume (FTV) = (Mean tumor volume (TV)
treated)/(Mean TV control) .sup.bExpected = (FTV drug 1) .times.
(FTV drug 2) .sup.cRatio of expected over observed, >2 =
synergistic; ~1 = additive; <0.5 = antagonistic.
[0243] Those results demonstrate that the combination of
FGFR1-ECD.339-Fc, doxorubicin, and paclitaxel resulted in additive
inhibition of tumor growth in that experiment.
Example 6: Treatment with FGFR1-ECD.339-Fc and KDR ECD-Fc in Mice
Having 11520 Lung Xenograft Tumor Cells Showed Tumor Inhibition
[0244] SCID CB17 mice at 8 weeks of age were administered either
vehicle, kinase insert domain receptor (KDR) cDNA (Five Prime
Therapeutics, South San Francisco, Calif.) (which expresses a
protein that acts as a VEGF antagonist and VEGF trap), FGFR1 cDNA
(Five Prime Therapeutics, South San Francisco, Calif.), or a
combination of KDR and FGFR1, by hydrodynamic tail vein
transfection (TVT), substantially as described in Chen et al.,
Human Gene Therapy 16(1): 126-131 (2005), and then inoculated 4
days later with 5.times.10.sup.6 H520 lung xenograft tumor cells
s.c. on the flank, with the cells in a 1:1 mixture of Matrigel in
100.mu.1 total volume. KDR and FGFR1 cDNA constructs each contain
their respective extracellular domain fused to an IgG1 Fc domain.
Tumor volume measurements were made at approximately 10 day
intervals. On day 29, the tumor volume was reduced in groups
treated with single agents relative to vehicle (Mann Whitney test
P<0.05). On day 29, the tumor volume was reduced in the
combination group relative to groups treated with single agents
(Mann Whitney test P<0.001).
Example 7: Administration of FGFR1-ECD.339-Fc and
Cisplatin/Etoposide in the DMS 53 Small Cell Lung Cancer (SCLC)
Xenograft Model
[0245] 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 DMS 53 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. After tumors reached a size
of 100-125 mm.sup.3, mice were sorted and randomized so each group
(n=10) has the approximately the same average tumor volume, and
treatment initiated according to Table 20 below.
[0246] 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. Cisplatin was purchased
from Amatheon, Inc., Miami, Fla. (Cat No. 5539-0112-50)
pre-formulated at concentration of 1 mg/ml. 0.45 ml of cisplating
stock solution (1 mg/ml) was added to 1.05 ml of 5% dextrose
solution, for a 1.5 ml working solution with a concentration of 0.3
mg/ml. Each mouse received 100 .mu.l of working solution (0.3
mg/ml) to provide a dose of 3 mg/kg every 7 or 21 days depending on
group. Etoposide was purchased from Amatheon, Inc (Cat. No.
5539-0291-01) pre-formulated at concentration of 20 mg/ml. 0.140 ml
of stock solution (20 mg/ml) was added to 3.36 ml of 5% dextrose
solution, for a 3.5 ml working solution with a concentration of 0.8
mg/ml. Each mouse received 50 .mu.l of working solution (0.8 mg/ml)
to provide a dose of 4 mg/kg for 3 days in a row. Etoposide dosing
was repeated every 7 or 21 days depending on group. In combination
groups, FGFR1-ECD.339-Fc and cisplatin/etoposide was administered
concurrently. 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). The dosing
schedule for each set of mice is shown in Table 20.
TABLE-US-00020 TABLE 20 FGFR1- Cisplatin Etoposide ECD.339- (mg/kg,
(mg/kg, Fc (mg/kg, Group Drugs schedule) schedule) schedule) Route
1 Albumin 15 mg/kg 0 0 0 IP 2 FGFR1-ECD.339-Fc 0 0 15, IP 2x/week 3
Cisplatin/Etoposide 3, once every 4, qd .times. 3 0 IP 21 days
every 21 days 4 Cisplatin/Etoposide/ 3, once every 4, qd .times. 3
15, IP FGFR1-ECD.339-Fc 21 days every 21 2x/week days 5
Cisplatin/Etoposide 3, once every 4, qd .times. 3 0 IP 7 days every
7 days 6 Cisplatin/Etoposide/ 3, once every 4, qd .times. 3 15, IP
FGFR1-ECD.339-Fc 7 days every 7 days 2x/week
Example 8: Administration of FGFR1-ECD.339-Fc and Topotecan in the
DMS 53 Small Cell Lung Cancer (SCLC) Xenograft Model
[0247] 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 DMS 53 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. After tumors reached a size
of 100-125 mm.sup.3, mice were sorted and randomized so each group
(n=10) has the approximately the same average tumor volume, and
treatment initiated according to Table 21 below.
[0248] 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. Topotecan powder was
purchased from Sigma-Aldrich, Inc. (Cat No. T2705-50MG) and a 5
mg/ml stock solution made in a 5% dextrose solution. Dilutions are
also made with 5% dextrose solution. 0.6 ml of stock solution (5
mg/ml) was added to 5.4 ml of 5% dextrose solution, for a 6 ml
working solution with a concentration of 0.5 mg/ml. Each mouse
received 100 .mu.l of working solution (0.5 mg/ml) to give a dose
of 2.5 mg/kg. Topotecan dosing was repeated every 7 or 21 days
depending on group. In combination groups, FGFR1-ECD.339-Fc and
topotecan was administered concurrently. 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). The dosing schedule for each set of mice is
shown in Table 21.
TABLE-US-00021 TABLE 21 FGFR1- ECD.339- Topotecan Fc (mg/kg,
(mg/kg, Group Drugs schedule) schedule) Route 1 Albumin 15 mg/kg 0
0 IP 2 FGFR1-ECD.339-Fc 0 15, IP 2x/week 3 Topotecan 2.5, qd
.times. 5 0 IP every 21 days 4 Topotecan/FGFR1- 2.5, qd .times. 5
15, IP ECD.339-Fc every 21 2x/week days 5 Topotecan 2.5, qd .times.
5 0 IP every 7 days 6 Topotecan/FGFR1- 2.5, qd .times. 5 15, IP
ECD.339-Fc every 7 days 2x/week
TABLE OF SEQUENCES
[0249] Table 22 lists certain sequences discussed herein. FGFR1
sequences are shown without the signal peptide, unless otherwise
indicated.
TABLE-US-00022 TABLE 22 Sequences and Descriptions SEQ ID NO
Description Sequence 1 Full-length human MWSWKCLLFW AVLVTATLCT
ARPSPTLPEQ AQPWGAPVEV FGFR1 ECD (with ESFLVHPGDL LQLRCRLRDD
VQSINWLRDG VQLAESNRTR signal peptide); ITGEEVEVQD SVPADSGLYA
CVTSSPSGSD TTYFSVNVSD SP-hFGFR1-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 SAWLTVLEAL EERPAVMTSP LYLE 2
Full-length human RPSPTLPEQ AQPWGAPVEV ESFLVHPGDL LQLRCRLRDD FGFR1
ECD (without VQSINWLRDG VQLAESNRTR ITGEEVEVQD SVPADSGLYA signal
peptide); CVTSSPSGSD TTYFSVNVSD ALPSSEDDDD DDDSSSEEKE
hFGFR1-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-
MWSWKCLLFW AVLVTATLCT ARPSPTLPEQ AQPWGAPVEV Fc 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 F Cc237S 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
Sequence CWU 1
1
101374PRTHomo sapiens 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 2353PRTHomo sapiens
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 3360PRTHomo
sapiens 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 4339PRTHomo sapiens 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 5592PRTHomo sapiens
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 6571PRTHomo sapiens
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 721PRTHomo sapiens 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 8232PRTHomo sapiens 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
9228PRTHomo sapiens 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 10229PRTHomo sapiens 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
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