U.S. patent application number 10/575977 was filed with the patent office on 2007-11-29 for fibroblast growth factor receptor-1 inhibitors and methods of treatment thereof.
This patent application is currently assigned to IMCLONE SYSTEMS INCORPORATION. Invention is credited to Juqun Shen, Haijun Sun, James R. Tonra.
Application Number | 20070274981 10/575977 |
Document ID | / |
Family ID | 34465334 |
Filed Date | 2007-11-29 |
United States Patent
Application |
20070274981 |
Kind Code |
A1 |
Sun; Haijun ; et
al. |
November 29, 2007 |
Fibroblast Growth Factor Receptor-1 Inhibitors and Methods of
Treatment Thereof
Abstract
The present invention is directed to an antibody or fragments
thereof that are specific for a fibroblast growth factor receptor
(FGFR)-1(IIIb), FGFR-1(IIIc), and/or FGFR-4. Also, provided herein,
are vectors and host cells comprising the nucleic acids encoding
those antibodies. The present invention further provides methods of
antagonizing FGFR-1 or FGFR-4 as a treatment for obesity, diabetes,
or a condition related thereto, and methods of reducing food
intake.
Inventors: |
Sun; Haijun; (New York,
NY) ; Shen; Juqun; (Flushing, NY) ; Tonra;
James R.; (Scarsdale, NY) |
Correspondence
Address: |
KENYON & KENYON LLP
ONE BROADWAY
NEW YORK
NY
10004
US
|
Assignee: |
IMCLONE SYSTEMS
INCORPORATION
180 VARICK STREET
NEW YORK
NY
10014
|
Family ID: |
34465334 |
Appl. No.: |
10/575977 |
Filed: |
October 18, 2004 |
PCT Filed: |
October 18, 2004 |
PCT NO: |
PCT/US04/34970 |
371 Date: |
May 2, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60512255 |
Oct 16, 2003 |
|
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|
Current U.S.
Class: |
424/130.1 ;
424/143.1; 435/252.31; 435/252.33; 435/252.34; 435/252.35;
435/254.11; 435/254.2; 435/325; 435/348; 435/410; 435/70.1;
436/501; 530/387.1; 536/23.53 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 31/00 20130101; A61P 9/12 20180101; C07K 16/2863 20130101;
A61P 25/02 20180101; A61P 9/00 20180101; A61P 7/02 20180101; A61P
9/10 20180101; A61P 3/00 20180101; A61P 35/00 20180101; A61K
2039/505 20130101; A61P 3/06 20180101; A61P 1/16 20180101; A61P
17/00 20180101; A61P 25/00 20180101; A61P 27/02 20180101; C07K
2317/76 20130101; C07K 2317/56 20130101; A61P 13/12 20180101; A61P
17/02 20180101; A61P 37/04 20180101; C07K 2317/565 20130101; C07K
2317/73 20130101; A61K 45/06 20130101; A61P 13/10 20180101; A61P
43/00 20180101; A61P 1/18 20180101; C07K 2317/55 20130101; A61P
9/14 20180101; A61P 15/08 20180101; A61P 3/04 20180101; A61P 19/02
20180101; A61P 19/06 20180101; A61P 31/00 20180101; A61P 25/26
20180101; A61P 3/10 20180101; A61P 11/00 20180101 |
Class at
Publication: |
424/130.1 ;
435/252.31; 435/252.33; 435/252.34; 435/252.35; 435/254.11;
435/254.2; 435/325; 435/348; 435/410; 435/070.1; 436/501;
530/387.1; 536/023.53; 424/143.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 3/04 20060101 A61P003/04; A61P 3/10 20060101
A61P003/10; A61P 9/12 20060101 A61P009/12; C07H 21/04 20060101
C07H021/04; C07K 16/28 20060101 C07K016/28 |
Claims
1. A purified antibody, or fragment thereof, which specifically
binds to a fibroblast growth factor receptor (FGFR)-1(IIIb),
FGFR-1(IIIc), or FGFR-4(IIIc).
2. The antibody of claim 1, wherein the antibody, or fragment
thereof, binds to an extracellular domain of fibroblast growth
factor receptor FGFR-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc) and
neutralizes activation of the receptor.
3. The antibody of claim 1, wherein the antibody, or fragment
thereof, inhibits binding of a ligand of FGFR-1(IIIb),
FGFR-1(IIIc), or FGFR-4(IIIc) to its receptor.
4. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:5, and SEQ ID NO:6.
5. The antibody of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least 70% homology
to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,
and SEQ ID NO:6.
6. The antibody of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least about 80%
homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, and SEQ ID NO:6.
7. The antibody of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least about 90%
homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ
ID NO:5, and SEQ ID NO:6.
8. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody comprises
a sequence selected from the group consisting of SEQ ID NO:9, SEQ
ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID
NO:14.
9. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least 70% homology
to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, and SEQ ID NO:14.
10. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least about 80%
homology to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, and SEQ ID NO:14.
11. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least about 90%
homology to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, and SEQ ID NO:14.
12. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody comprises
a sequence selected from the group consisting of SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID
NO:22.
13. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least 70% homology
to SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, and SEQ ID NO:22.
14. The antibody of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least about 80%
homology to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID
NO:20, SEQ ID NO:21, and SEQ ID NO:22.
15. The antibody of any of claims 1-3, wherein at least one
complementarity-determining region (CDR) of the antibody, or
fragment thereof, comprises a sequence with at least about 90%
homology to SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, and SEQ ID NO:22.
16. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence of SEQ ID
NO:7 or a light chain variable region sequence of SEQ ID NO:8.
17. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 70% homology to SEQ ID NO:7 or a light chain variable
region sequence with at least about 70% homology to SEQ ID
NO:8.
18. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 80% homology to SEQ ID NO:7 or a light chain variable
region sequence with at least about 80% homology to SEQ ID
NO:8.
19. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 90% homology to SEQ ID NO:7 or a light chain variable
region sequence with at least about 90% homology to SEQ ID
NO:8.
20. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence of SEQ ID
NO:15 or a light chain variable region sequence of SEQ ID
NO:16.
21. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 70% homology to SEQ ID NO:15 or a light chain variable
region sequence with at least about 70% homology to SEQ ID
NO:16.
22. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 80% homology to SEQ ID NO:15 or a light chain variable
region sequence with at least about 80% homology to SEQ ID
NO:16.
23. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 90% homology to SEQ ID NO:15 or a light chain variable
region sequence with at least about 90% homology to SEQ ID
NO:16.
24. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence of SEQ ID
NO:23 or a light chain variable region sequence of SEQ ID
NO:24.
25. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 70% homology to SEQ ID NO:23 or a light chain variable
region sequence with at least about 70% homology to SEQ ID
NO:24.
26. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 80% homology to SEQ ID NO:23 or a light chain variable
region sequence with at least about 80% homology to SEQ ID
NO:24.
27. The antibody of any of claims 1-3, wherein the antibody
comprises at least a heavy chain variable region sequence with at
least about 90% homology to SEQ ID NO:23 or a light chain variable
region sequence with at least about 90% homology to SEQ ID
NO:24.
28. An isolated nucleic acid encoding the antibody of any of claims
4-27
29. An expression vector comprising the nucleic acid of claim 28
operably linked to a control sequence.
30. A host cell comprising the expression vector of claim 29.
31. A method of producing an antibody comprising culturing the host
cell of claim 30 under conditions permitting expression of the
antibody.
32. The method of claim 31, wherein the method further comprises
purifying the antibody.
33. A pharmaceutical composition comprising the antibody of any of
claims 1-27 and a pharmaceutically acceptable carrier.
34. A method of identifying a fibroblast growth factor receptor
(FGFR)-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc) specific antibody, or
fragment thereof, comprising: providing a library of antibody
fragments, screening the library for the antibody that is specific
for FGFR-1(IIIb) and/or FGFR-1(IIIc) or the antibody specific for
FGFR-1(IIIc) and/or FGFR-4, and purifying the antibody that is
specific for FGFR-1(IIIb) and/or FGFR-1(IIIc) or the antibody
specific for FGFR-1(IIIc) and/or FGFR-4.
35. The method of claim 34, wherein the screening of the library
comprises providing an affinity matrix having the FGFR-1(IIIb),
FGFR-1(IIIc), and/or FGFR-4 bound to a solid support, contacting
the affinity matrix with the library of antibody fragments, and
separating the antibody fragments that bind to the affinity matrix
from the antibody fragments that do not bind the affinity
matrix.
36. Am antibody identified using the method of claim 34 or 35.
37. A method of treating obesity or an obesity related condition
comprising administering to a mammal in need thereof a
therapeutically effective amount of a FGFR-1(IIIb), FGFR-1(IIIc),
and/or FGFR-4 antagonist.
38. A method of treating diabetes or a diabetes related condition
comprising administering to a mammal in need thereof a
therapeutically effective amount of a FGFR-1(IIIb), FGFR-1(IIIc),
and/or FGFR-4 antagonist.
39. A method of reducing food intake or a condition affected by
reducing food intake comprising administering to a mammal in need
thereof a therapeutically effective amount of a FGFR-1(IIIb),
FGFR-1(IIIc), and/or FGFR-4 antagonist.
40. The method of any of claims 37-39, wherein the condition is
hypertension.
41. The method of any one of claims 37-39, wherein the condition is
cardiovascular disease.
42. The method of any of claims 37-39, wherein said method reduces
body mass index.
43. The method of any of claims 37-39, wherein said method reduces
serum triglycerides.
44. The method of any of claims 37-39, wherein said method alters
leptin levels.
45. The method of any of claims 37-39, wherein said method inhibits
angiogenesis.
46. The method of any of claims 37-39, wherein said method alters
energy metabolism.
47. The method of any of claims 37-39, wherein said method alters
the Respiratory Exchange Ratio.
48. The method of any of claims 37-39, wherein said method has an
FGFR-1 or FGFR-4 pathway related anorexic effect.
49. The method of any of claims 37-39, wherein the antagonist is a
biological molecule.
50. The method of claim 49, wherein the biological molecule is an
antibody or fragment thereof.
51. The method of claim 50, wherein the antibody is an antibody of
any of claims 1-7.
52. The method of any of claims 37-39, wherein the antagonist is a
small molecule that blocks FGFR-1(IIIb), FGFR-1(IIIc), and/or
FGFR-4 signaling.
53. The method of claim 37-39, wherein the FGFR-1 antagonist binds
FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4 internally.
54. The method of claim 37-39, wherein the FGFR-1 antagonist
inhibits FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4
phosphorylation.
55. The method of claim 37-39, wherein the FGFR-1 antagonist
inhibits binding of ATP to FGFR-1(IIIb), FGFR-1(IIIc), and/or
FGFR-4.
56. The method of claim 37-39, wherein the FGFR-1 antagonist
competes with ATP for FGFR-1(IIIb), FGFR-1(IIIc), and/or
FGFR-4.
57. The method of claim 37-39, wherein the FGFR-1 antagonist
inhibits FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4 tyrosine kinase
activity.
58. The method of any of claims 52-57, wherein the small molecule
comprises pyrimido-pyridine derivative A or B, SU-6668, PD-173074,
SU-5402, CHIR-258, or PD-166285.
59. The method of any of claims 37-58, wherein the mammal is a
human.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an antibody, or fragment
thereof, that is specific for fibroblast growth factor receptor
(FGFR)-1(IIIb), FGFR-1(IIIc), and/or FGFR-4(IIIc). The present
invention further provides methods of antagonizing and neutralizing
FGFR-1 and/or FGFR-4 as a treatment for obesity, diabetes, and/or a
condition related thereto, including reducing food intake or total
body mass.
BACKGROUND OF THE INVENTION
[0002] Fibroblast growth factor (FGF) pathways in general are
implicated in many physiological processes, such as morphogenesis
during development and angiogensis which is the process of
developing new blood vessels that involves the proliferation,
migration, and tissue infiltration of capillary endothelial cells
from pre-existing blood vessels. FGFs are some of the factors that
have been implicated as possible regulators of angiogenesis along
with transforming growth factor (TGF), vascular endothelial growth
factor (VEGF), and platelet deried growth factor (PDGF). FGF
pathways are also implicated in neuronal survival and wound
healing. They are also thought to be important in a number of
pathological processes.
[0003] In particular, FGFR-1 has been implied to be involved in
diseases such as cancers and arthritis. Although the involvement of
FGF pathways in metabolism, such as feeding behavior and adipose
tissue development has been suggested, it is not clear whether
these findings entail fundamental mechanisms through which
metabolism is regulated. For example, a recent study performed in
mice has shown that injections of FGF-2, in combination with
basement membrane proteins, can induce development of new adipose
tissue at the site of the injection. This suggests that locally
produced FGFs may act in a paracrine manner to affect adipogenesis
and thereby influence the regional distribution of adipose tissue
in the body and the relationship between adipose tissue and insulin
resistance is well-established, both of which are strongly
implicated in type 2 diabetes and cardiovascular disease.
[0004] Fibroblast growth factor receptors (FGFRs) have common
structural features and consist of an extracellular ligand-binding
domain containing 2 or 3 Ig-like loops and a unique acid region, a
trans-membrane domain, and the cytoplasmic region, which contains
the tyrosine kinase catalytic domain and kinase insert. The FGFRs
belong to Subclass IV of the receptor tyrosine kinase family of
proteins. These receptors bind in an overlapping pattern to FGFs.
It has been established that 22 FGFs act on 5 FGFRs in FGF ligand
paracrine interaction.
[0005] FGFR-1 has two alternative splicing forms that differ from
each other by the amino acid substitutions in the third IgG-like
domain of the extracellular structure of the receptor designated
IIIb and IIIc, FGFR-4 has only one. These substitutions constitute
what is believed to be part of the binding domain of the receptor,
and therefore are most likely to cause the two splicing forms to
have distinct ligand specificities. The two forms have also been
shown to be differentially expressed, which may be part of an
exquisite control mechanism of complex functions mediated by
FGFR-1.
[0006] Ligand binding, which is strengthened by the presence of
heparin sulfate, causes the FGFRs to dimerize and activate specific
intracellular signaling pathways (Bellot et al. 1991). The receptor
becomes auto-phosphorylated and thus capable of activating
downstream cellular pathways. Among different cellular responses,
stimulation of proliferation or induction of differentiation is
most commonly observed for FGFR-1 mediated signaling.
SUMMARY OF THE INVENTION
[0007] The present invention provides antibodies, or fragments
thereof, specific for fibroblast growth factor receptor
(FGFR)-1(IIIb), FGFR-1(IIIc), and/or FGFR-4(IIIc) as well as
nucleic acids encoding these antibodies or fragments thereof.
Vectors comprising such nucleic acids and host cells are also
provided for production of these antibodies.
[0008] The present invention also provides a method of treating
obesity (or an obesity related condition), diabetes (or a diabetes
related condition) and/or a method to reduce food intake by
administering to a mammal in need thereof a therapeutically
effective amount of an FGFR-1 and/or FGFR-4 antagonist.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 shows the amino acid and nucleic acid sequences of
the Variable Regions and CDRs of FR1-H7. FIG. 1A shows the amino
acid and DNA sequences of the Variable Region of the Heavy Chain of
FR1-H7 (SEQ ID NOS:7 and 31). FIG. 1B shows the amino acid and DNA
sequences of the Variable Region of the Light Chain of FR1-H7 (SEQ
ID NOS:8 and 32). FIG. 1C shows the nucleic acid sequences of the
CDRs of the variable region of the heavy chain and the variable
region of the light chain of FR1-H7 (SEQ ID NOS:25-30). FIG. 1D
shows the amino acid sequences of the CDRs of the variable region
of the heavy chain and the variable region of the light chain of
FR1-H7 (SEQ ID NOS:1-6).
[0010] FIG. 2 shows the amino acid and nucleic acid sequences of
the Variable Regions of FR1-A1. FIG. 2A shows the amino acid and
DNA sequences of the Variable Region of the Heavy Chain of FR1-A1
(SEQ ID NOS: 15 and 39). FIG. 2B shows the amino acid and DNA
sequences of the Variable Region of the Light Chain of FR1-A1 (SEQ
ID NOS:16 and 40). FIG. 1C shows the nucleic acid sequences of the
CDRs of the variable region of the heavy chain and the variable
region of the light chain of FR1-A1 (SEQ ID NOS:33-38). FIG. 1D
shows the amino acid sequences of the CDRs of the variable region
of the heavy chain and the variable region of the light chain of
FR1-A1 (SEQ ID NOS:9-14).
[0011] FIG. 3 shows binding of FR1-H7 antibody to FGFRs as
determined using the ELISA binding assay. Each data point is an
average of duplicates and error bars are standard deviations.
[0012] FIG. 4 shows binding of recombinant FGFR-1 to FGF ligand as
determined using the ELISA blocking assay. FIG. 4A shows binding to
immobilized FGF-1 and FIG. 4B shows binding to immobilized FGF-2.
Each data point is an average of duplicates and error bars are
standard deviation.
[0013] FIG. 5 shows binding of .sup.125I-FGF-2 to cell surface
FGFR-1. In FIG. 5A, the binding of .sup.125I-FGF-2 to the cells had
two distinct components: non-specific and specific bindings. In
FIG. 5B, .sup.125I-FGF-2 binding to the cells was inhibited by the
presence of FR1-H7 antibody. Each data point is an average of
triplicates. Error bars are standard deviations.
[0014] FIG. 6 shows a Western blot of FGFR-1 phosphorylation. The
upper blot was probed with anti-phospho-Tyrosine antibody and the
lower blot shows control protein bands in the cell lysates for
estimation of relative gel loading.
[0015] FIG. 7 shows proliferation of Human Umbilical Vascular
Endothelial Cells (HUVECs) in vitro in presence of antibodies. Each
data point is an average of triplicates and error bars are standard
deviation.
[0016] FIG. 8 shows proliferation of adipocytes in vitro. FIG. 8A
shows the effects of FGF-2 on adipocyte proliferation and FIG. 8B
shows the effects of FR1-H7 on FGF-2-stimulated adipocyte
proliferation. Each data point is an average of triplicates. Error
bars are standard deviations.
[0017] FIG. 9 shows the effect of FR1-H7 on body weight in nu/nu
female mice (Mean.+-.SEM).
[0018] FIG. 10 shows the effect of FR1-H7 on food intake in nu/nu
female mice.
[0019] FIG. 11 shows the effect of FR1-H7 on rearing behavior in a
novel environment (Mean.+-.SEM).
[0020] FIG. 12 shows the effect of FR1-H7 on blood glucose. FIG.
12A shows the effect of FR1-H7 on non-fasted blood glucose
(Mean.+-.SEM). FIG. 12B shows the effect of FR1-H7 on non-fasted
blood glucose after weights are fully recovered (Mean.+-.SEM).
[0021] FIG. 13 shows body weight loss in nu/nu mice after a single
does of FR1-H7 treatment (Mean.+-.SEM, n=4).
[0022] FIG. 14 shows the effects on tissue weights after a single
does of FR1-H7 treatment. Each bar represents the value calculated
as 100.times. the ratio of tissue weight over total body weight.
Error bars are standard deviation.
[0023] FIG. 15 shows the effects on serum chemistry after a single
dosing of FR1-H7 treatment. In FIG. 15A, serum glucose levels
(mean.+-.SEM) are determined; in FIG. 15B, serum triglycerides
levels (mean.+-.SEM) are determined; in FIG. 15C, serum insulin
levels are determined; and in FIG. 15D, serum leptin levels are
determined. All individual measurements are shown. Data taken at
the same time points were separated according to treatment groups
for viewing purpose.
[0024] FIG. 16 shows the effect of FR1-H7 on body weight in C57
black mice (Mean.+-.SEM).
[0025] FIG. 17 shows the effect of FR1-H7 on body weight in db/db
mice (Mean.+-.SEM).
[0026] FIG. 18 shows the effects of FR1-H7 on food intakes in db/db
mice (Mean.+-.SEM).
[0027] FIG. 19 shows the effects of FR1-H7 on the sizes of adipose
tissue (Mean.+-.SEM).
[0028] FIG. 20 shows the binding of FR1-A1 antibody to FGFRs as
determined using the ELISA binding assay.
[0029] FIG. 21 shows binding of recombinant FGFR-1 to FGF ligand as
determined using the ELISA blocking assay.
[0030] FIG. 22 shows a Western blot of FGFR-1 phosphorylation.
[0031] FIG. 23 shows mitogenesis of G18 cells in vitro in presence
of antibodies. Each data point is an average of triplicates. Error
bars are standard deviations.
[0032] FIG. 24 shows the effect of FR1-A1 on body weights in nu/nu
female mice (Mean.+-.SEM).
[0033] FIG. 25 shows the effect of FR1-A1 on food intakes in nu/nu
female mice.
[0034] FIG. 26 shows treatment of C57 black mice with FR1-H7, which
caused decreases in body weights, food intake, muscle and fat mass,
energy expenditure, ambulatory activities and Respiratory Exchange
Ratio (RER) as compared to the control. FIG. 26A shows decrease in
daily body weight with FR1-H7 treatment as compared to control.
FIG. 26B shows decrease in daily food intake with FR1-H7 treatment
as compared to control. FIG. 26C shows decrease in fat and muscle
weights with FR1-H7 treatment as compared to control. FIG. 26D
shows decrease in energy expenditure and ambulatory activities with
FR1-H7 treatment as compared to control. FIG. 26E shows decrease in
oxygen consumption and RER with FR1-H7 treatment as compared to
control.
[0035] FIG. 27 shows a paired-feeding of FR1-H7 and control treated
animals resulting in identical decreases in body weights, muscle
and fat mass, and energy expenditure between the two groups. Both
groups also exhibited similar decreases in ambulatory activities
and RER. FIG. 27A shows decrease in daily body weight of both
FR1-H7 treated and control animals. FIG. 27B shows a decrease in
fat and muscle weights of both FR1-H7 treated and control animals.
FIG. 27C shows a decrease in energy expenditure and ambulatory
activities of both FR1-H7 treated and control animals. FIG. 27D
shows decrease in oxygen consumption of both FR-1-H7 treated and
control animals.
[0036] FIG. 28 shows the amino acid and nucleic acid sequences of
the Variable Regions and the Variable Region CDRs of FR1-4H. FIG.
28A shows the amino acid and nucleic acid sequences of the Variable
Region of the Heavy Chain of FR1-4H and the Variable Region of the
Light Chain of FR1-4H (SEQ ID NOS:23-24 and 47-48). FIG. 28B shows
the amino acid and nucleic acid sequences of the CDRs of the
Variable Region of the Heavy and Light Chains of FR1-4H (SEQ ID
NOS:17-22 and 41-46).
[0037] FIG. 29 shows that FR1-4H inhibited the binding of FGFR-1
(IIIb) to FGF ligand. Percent binding was determined using the
ELISA blocking assay. Each data point is an average of duplicates.
Error bars are standard deviations.
[0038] FIG. 30 shows examples of FGFR small molecule inhibitors
including indolinone derivatives, quinolinone derivatives and
pyrimido-pyridine derivatives.
[0039] FIG. 31 shows that FGFR small molecule inhibitors inhibited
the auto-phosphorylation of FGFR-1(IIIc) in a cell-based
phosphorylation assay. Equal amounts of cell lysate were applied to
each sample lane. Receptor auto-phosphorylation was probed using
anti-phospho-tyrosine antibody as described in Example 20.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention provides purified antibodies, or
fragments thereof, specific for fibroblast growth factor receptor
(FGFR)-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc). An example of an
antibody that is specific for FGFR-1(IIIb) and/or FGFR-1(IIIc),
and/or FGFR-4(IIIc) is FR1-H7 (FIG. 1). It should be appreciated
that the description herein with respect to FR1-H7 applies to all
antibodies specific for FGFR-1(IIIb) and/or FGFR-1(IIIc). FR1-A1 is
an example of an antibody specific for FGFR-1(IIIc) and/or
FGFR-4(IIIc) (FIG. 2). Similarly to FR1-H7, any description herein
with respect to FR1-A1 applies to all antibodies specific for
FGFR-1(IIIc) and/or FGFR-4(IIIc). FR1-4H is an example of an
antibody of the invention specific for at least FGFR(IIIb) (FIG.
28). Preferably, the antibodies, or fragments thereof, of the
present invention bind to an extracellular domain of FGFR-1(IIIb),
FGFR-1(IIIc), or FGFR-4(IIIc) and neutralize activation of the
receptor. More preferably, the antibodies, or fragments thereof, of
the present invention inhibit binding of a ligand of FGFR-1(IIIb),
FGFR-1(IIIc), or FGFR-4(IIIc) to its receptor.
[0041] Naturally occurring antibodies typically have two identical
heavy chains and two identical light chains, with each light chain
covalently linked to a heavy chain by an interchain disulfide bond
and multiple disulfide bonds further link the two heavy chains to
one another. Individual chains can fold into domains having similar
sizes (110-125 amino acids) and structures, but different
functions. The light chain can comprise one variable domain
(V.sub.L) and/or one constant domain (C.sub.L). The heavy chain can
also comprise one variable domain (V.sub.H) and/or, depending on
the class or isotype of antibody, three or four constant domains
(C.sub.H1, C.sub.H2, C.sub.H3 and C.sub.H4). In humans, the
isotypes are IgA, IgD, IgE, IgG, and IgM, with IgA and IgG further
subdivided into subclasses or subtypes (IgA.sub.1-2 and
IgG.sub.1-4).
[0042] Generally, the variable domains show considerable amino acid
sequence variability from one antibody to the next, particularly at
the location of the antigen-binding site. Three regions, called
hypervariable or complementarity-determining regions (CDRs), are
found in each of V.sub.L and V.sub.H, which are supported by less
variable regions called framework variable regions
[0043] The portion of an antibody consisting of V.sub.L and V.sub.H
domains is designated Fv (Fragment variable) and constitutes the
antigen-binding site. Single chain Fv (scFv) is an antibody
fragment containing a V.sub.L domain and a V.sub.H domain on one
polypeptide chain, wherein the N terminus of one domain and the C
terminus of the other domain are joined by a flexible linker (see,
e.g., U.S. Pat. No. 4,946,778 (Ladner et al.); WO 88/09344, (Huston
et al.). WO 92/01047 (McCafferty et al.) describes the display of
scFv fragments on the surface of soluble recombinant genetic
display packages, such as bacteriophage.
[0044] The peptide linkers used to produce the single chain
antibodies can be flexible peptides selected to assure that the
proper three-dimensional folding of the V.sub.L and V.sub.H domains
occurs. The linker is generally 10 to 50 amino acid residues.
Preferably, the linker is 10 to 30 amino acid residues. More
preferably the linker is 12 to 30 amino acid residues. Most
preferably is a linker of 15 to 25 amino acid residues. An example
of such linker peptides includes repeats of four Glycines followed
by Serine.
[0045] Single chain antibodies lack some or all of the constant
domains of the whole antibodies from which they are derived.
Therefore, they can overcome some of the problems associated with
the use of whole antibodies. For example, single-chain antibodies
tend to be free of certain undesired interactions between
heavy-chain constant regions and other biological molecules.
Additionally, single-chain antibodies are considerably smaller than
whole antibodies and can have greater permeability than whole
antibodies, allowing single-chain antibodies to localize and bind
to target antigen-binding sites more efficiently. Furthermore, the
relatively small size of single-chain antibodies makes them less
likely to provoke an unwanted immune response in a recipient than
whole antibodies.
[0046] Multiple single chain antibodies, each single chain having
one V.sub.H and one V.sub.L domain covalently linked by a first
peptide linker, can be covalently linked by at least one or more
peptide linker to form multivalent single chain antibodies, which
can be monospecific or multispecific. Each chain of a multivalent
single chain antibody includes a variable light chain fragment and
a variable heavy chain fragment, and is linked by a peptide linker
to at least one other chain. The peptide linker is composed of at
least fifteen amino acid residues. The maximum number of amino acid
residues is about one hundred.
[0047] Two single chain antibodies can be combined to form a
diabody, also known as a bivalent dimer. Diabodies have two chains
and two binding sites, and can be monospecific or bispecific. Each
chain of the diabody includes a V.sub.H domain connected to a
V.sub.L domain. The domains are connected with linkers that are
short enough to prevent pairing between domains on the same chain,
thus driving the pairing between complementary domains on different
chains to recreate the two antigen-binding sites.
[0048] Three single chain antibodies can be combined to form
triabodies, also known as trivalent trimers. Triabodies are
constructed with the amino acid terminus of a V.sub.L or V.sub.H
domain directly fused to the carboxyl terminus of a V.sub.L or
V.sub.H domain, i.e., without any linker sequence. The triabody has
three Fv heads with the polypeptides arranged in a cyclic,
head-to-tail fashion. A possible conformation of the triabody is
planar with the three binding sites located in a plane at an angle
of 120 degrees from one another. Triabodies can be monospecific,
bispecific or trispecific.
[0049] Fab (Fragment, antigen binding) refers to the fragments of
the antibody consisting of V.sub.L C.sub.L V.sub.H C.sub.H1
domains. Those generated following pepsin digestion simply are
referred to as Fab and do not retain the heavy chain hinge region.
Following pepsin digestion, various Fabs retaining the heavy chain
hinge are generated. Those fragments with the interchain disulfide
bonds intact are referred to as F(ab').sub.2, while a single Fab'
results when the disulfide bonds are not retained. F(ab').sub.2
fragments have higher avidity for antigen than the monovalent Fab
fragments.
[0050] Fc (Fragment crystallization) is the designation for the
portion or fragment of an antibody that comprises paired heavy
chain constant domains. In an IgG antibody, for example, the Fc
comprises C.sub.H2 and C.sub.H3 domains. The Fc of an IgA or an IgM
antibody further comprises a C.sub.H4 domain. The Fc is associated
with Fc receptor binding, activation of complement-mediated
cytotoxicity and antibody-dependent cellular-cytoxicity (ADCC). For
antibodies such as IgA and IgM, which are complexes of multiple IgG
like proteins, complex formation requires Fc constant domains.
[0051] Finally, the hinge region separates the Fab and Fc portions
of the antibody, providing for mobility of Fabs relative to each
other and relative to Fc, as well as including multiple disulfide
bonds for covalent linkage of the two heavy chains.
[0052] Thus, antibodies of the invention include, but are not
limited to, naturally occurring antibodies, bivalent fragments such
as (Fab').sub.2, monovalent fragments such as Fab, single chain
antibodies, single chain Fv (scFv), single domain antibodies,
multivalent single chain antibodies, diabodies, triabodies, and the
like that bind specifically with antigens. Antibody fragments also
include polypeptides with amino acid sequences substantially
similar to the amino acid sequence of the variable or hypervariable
regions of the antibodies of the invention. Substantially the same
amino acid sequence is defined herein as a sequence with at least
70%, preferably at least about 80%, and more preferably at least
about 90% homology to a compared amino acid sequence, as determined
by the FASTA search method in accordance with Pearson and Lipman,
Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988) and which binds to
FGFR-1 and/or FGFR-2.
[0053] The antibodies, or fragments thereof, of the present
invention are specific for FGFR-1(IIIb), FGFR-1(IIIc), and/or
FGFR-4(IIIc). Antibody specificity refers to selective recognition
of the antibody for a particular epitope of an antigen. Antibodies,
or fragments thereof, of the present invention, for example, can be
monospecific or bispecific. Bispecific antibodies (BsAbs) are
antibodies that have two different antigen-binding specificities or
sites. Where an antibody has more than one specificity, the
recognized epitopes can be associated with a single antigen or with
more than one antigen. Thus, the present invention provides
bispecific antibodies, or fragments thereof, that bind to two
different antigens, with at least one specificity for FGFR-1(IIIb),
FGFR-1(IIIc), and/or FGFR-4(IIIc).
[0054] Specificity of the present antibodies, or fragments thereof,
for FGFR can be determined based on affinity and/or avidity.
Affinity, represented by the equilibrium constant for the
dissociation of an antigen with an antibody (K.sub.d), measures the
binding strength between an antigenic determinant and an
antibody-binding site. Avidity is the measure of the strength of
binding between an antibody with its antigen. Avidity is related to
both the affinity between an epitope with its antigen binding site
on the antibody, and the valence of the antibody, which refers to
the number of antigen binding sites of a particular epitope.
Antibodies typically bind with a dissociation constant (K.sub.d) of
10.sup.-5 to 10.sup.-11 liters/mol (e.g., K.sub.D<100 nM). Any
K.sub.d less than 10.sup.-4 liters/mol is generally considered to
indicate nonspecific binding. The lesser the value of the K.sub.d,
the stronger the binding strength between an antigenic determinant
and the antibody binding site.
[0055] The present invention provides a purified antibody, or
fragment thereof, specific for FGFR-1(IIIb) and/or FGFR-1(IIIc),
and/or FGFR-4(IIIc), wherein the antibody binds to an extracellular
domain of an FGFR1-(IIIb) and/or FGFR1-(IIIc) and/or FGFR-4(IIIc)
and neutralizes activation of the receptor. The present invention
also provides a purified antibody or fragment thereof, specific for
FGFR-1(IIIc) and/or FGFR-4(IIIc) (FR1-A1), wherein the antibody
binds to an extracellular domain of an FGFR-1(IIIc) and/or
FGFR-4(IIIc) and neutralizes activation of the receptor. The
present invention also provides a purified antibody or fragment
thereof, specific for at least FGFR-1(IIIb) (FR1-4H), wherein the
antibody binds to an extracellular domain of at least an
FGFR-1(IIIb) and neutralizes activation of the receptor. In this
specification, neutralizing a receptor means inactivating the
intrinsic kinase activity of the receptor to transduce a signal. A
reliable assay for FGFR-1 or FGFR-4 neutralization is the
inhibition of receptor phosphorylation. The present invention is
not limited by any particular mechanism of FGFR neutralization.
Some possible mechanisms include preventing binding of the FGF
ligand to the extracellular binding domain of the FGF receptor,
inducing the internalization of the receptors, and preventing
dimerization or oligomerization of receptors.
[0056] Neutralization of FGF activation of an FGFR-1 or FGFR-4 can
be determined by any suitable method. For example, neutralization
of FGF activation of an FGFR in a sample of endothelial or
non-endothelial cells, such as in adipose tissue or tumor cells,
may be performed in vitro or in vivo. Such neutralizing in a sample
of FGFR-1 or FGFR-4 expressing cells involves contacting the cells
with an antibody of the invention. In vitro, the cells are
contacted with the antibody before, simultaneously with, or after,
adding FGF to the cell sample.
[0057] Further, the invention provides the antibody of the
invention inhibits binding of a ligand of FGFR1-(IIIb) and/or
FGFR1-(IIIc) or FGFR1-(IIIc) and/or FGFR4 to its receptor. The
antibody may be of an alternative splicing form containing the
ligand binding function. Some examples of the ligands of
FGFR1-(IIIb) include the protein fibroblast growth factor (FGF)-1,
-2, -3 and -10. Some examples of the ligands of FGFR1-(IIIc)
include FGF-1, -2, -4, -5, and -6. Some examples of the ligands of
FGFR4-(IIIa) include FGF-1, -2, -4, -6, -8b, -8e, -8f, -9, -16,
-17b, and -19. (See Endocrine-Related Cancer (2000) 7 165-197 at
165-169, "Fibroblast growth factors, their receptors and signaling"
C. J. Powers, S. W. McLeskey and A. Wellstein).
[0058] In a preferred embodiment, one, two, three, four, five, or
all six complementarity-determining regions (CDR) of the present
antibodies have sequences selected from the group consisting of SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and
SEQ ID NO:6 (i.e., any one of the CDRs of FR1-H7). In an
alternatively preferred embodiment, one, two, three, four, five, or
all six complementarity-determining regions (CDR) of the present
antibodies have sequences selected from the group consisting of SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13,
and SEQ ID NO:14 (i.e., any one of the CDRs of FR1-A1). In an
alternatively preferred embodiment, one, two, three, four, five, or
all six complementarity-determining regions (CDR) of the present
antibodies have sequences selected from the group consisting of SEQ
ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
and SEQ ID NO:22 (i.e., any one of the CDRs of FR1-4H).
[0059] The antibodies of the present invention, in another
preferred embodiment, have a heavy chain variable region sequence
of SEQ ID NO:7 (i.e., the heavy chain variable region of FR1-H7)
and/or a light chain variable region sequence of SEQ ID NO:8 (i.e.,
the light chain variable region of FR1-H7). Alternatively, the
antibodies of the present invention preferably have a heavy chain
variable region sequence of SEQ ID NO:15 (i.e., the heavy chain
variable region of FR1-A1 ) or a light chain variable region
sequence of SEQ ID NO:16 (i.e., the light chain variable region of
FR1-A1). Alternatively, the antibodies of the present invention
preferably have a heavy chain variable region sequence of SEQ ID
NO:23 (i.e., the heavy chain variable region of FR1-4H) or a light
chain variable region sequence of SEQ ID NO:24 (i.e., the light
chain variable region of FR1-4H).
[0060] The nucleic acid and amino acid sequences of the CDRs and
variable heavy and light chains of the antibodies are described in
sequences listed in SEQ ID NO:1 to 48. Also, the invention provides
an isolated nucleic acid encoding the antibody of the invention,
antibody equivalents or fragments thereof (SEQ ID NO:25-48). The
nucleic acids that encode for the heavy and light chains of the
antibodies of the invention or their equivalents are obtained by
standard molecular biology techniques. Nucleic acid molecules of
the invention include those that bind under stringent conditions to
SEQ ID NOS:25-48 and which encode functionally equivalent
polypeptide antibody subunits capable of binding to FGFR-1(IIIb),
FGFR-(IIIc) and/or FGFR-4(IIIc). Stringent conditions denotes
conditions for hybridization such as, hybridization to filter-bound
DNA in 0.5M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA
at 65.degree. C., and washing, in 0.1.times.SSC/0.1% SDS at
68.degree. C. (Ausubel F. M. et al., eds., 1989, Current Protocols
in Molecular Biology, Vol. I, Green Publishing Associates, Inc.,
and John Wiley & sons, Inc., New York, at p. 2.20.3) or for
moderately stringent conditions, washing in 0.2SSC/0.1% SDS at
42.degree. C. (Ausubel et al., 1989 supra).
[0061] The monoclonal antibodies of the invention, e.g., FR1-H7,
FR1-A1 and FR1-4H, may be produced by methods known in the art.
These methods include immunological methods described by Kohleer
and Milstein in Nature 256, 495-497 (1975) and Campbell in
"Monoclonal Antibody Technology, The Production and
Characterization of Rodent and Human Hybridomas" in Burdon et al.,
Eds., Laboratory Techniques in Biochemistry and Molecular Biology,
Volume 13, Elsevier Science Publishers, Amsterdam (1985); as well
as by the recombinant DNA method described by Huse et al. in
Science 246, 1275-1281 (1989).
[0062] The antibodies of the invention may be prepared by
immunizing a mammal with a soluble FGFR-1(IIIb), FGFR-1(IIIc), or
FGFR-4(IIIc). The soluble receptors may be used by themselves as
immunogens, or attached to a carrier protein or other objects, such
as beads, i.e. sepharose beads. After the mammal has produced
antibodies, a mixture of antibody producing cells, such as
splenocytes, are isolated. Monoclonal antibodies may be produced by
isolating individual antibody-producing cells from the mixture and
immortalizing them by, for example, fusing them with tumor cells,
such as myeloma cells. The resulting hybridomas are preserved in
culture, and express monoclonal antibodies, which are harvested
from the culture medium.
[0063] The antibodies may also be prepared from FGFR-1(IIIb),
FGFR-1(IIIc), or FGFR-4(IIIc) bound to the surface of cells that
express the FGFR-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc). The cell
to which these receptors may be bound may be a cell known to
preferentially express individual receptors, for example, the human
FGFR-1(IIIb) is expressed on cells such as fibroblast cells,
endothelial cells, certain epithelial cells, flg-1, cek-1, vascular
smooth muscle cells, lymphocytes, the human FGFR-1(IIIc) is
expressed on cells such as macrophage cells, hematopoietic
progenitor cells, and numerous tumor cells, and the human
FGFR-4(IIIc) is expressed on cells such as embryonic and
multipotential stem cells. (See R & D Systems, Cytokine
Mini-Review, 2001 "FGFR expression") Alternatively, the cell to
which the receptor is bound may be a cell into which the DNA
encoding the FGFR-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc) has been
transfected.
[0064] The FGFR-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc) may be used
as an immunogen to raise an antibody of the invention. The receptor
peptide may be obtained from natural sources, such as from cells
that express the above receptors. Also, a synthetic receptor
peptide may be obtained using commercially available machines and
the corresponding amino acid sequence. (See Endocrine--Related
Cancer (2000)7 165-197, at 174.) A further alternative still, is
that a nucleic acid encoding a FGFR-1 (IIIb), FGFR-1(IIIc), or
FGFR-4(IIIc) such as a cDNA or a fragment thereof, may be cloned
and expressed and the resulting polypeptide recovered and used as
an immunogen to raise an antibody of the invention. In order to
prepare the above receptors against which the antibodies are made,
nucleic acid molecules that encode the FGFR-1(IIIb), FGFR-1(IIIc),
or FGFR-4(IIIc), or portions thereof, especially the extracellular
portions thereof, may be inserted into known vectors for expression
in host cells using standard recombinant DNA techniques. Suitable
sources of such nucleic acid molecules include cells that express
FGFR-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc).
[0065] The present invention also provides an expression vector
containing a nucleic acid encoding an antibody, or fragment
thereof, of the present invention operably linked to a control
sequence, as well as a host cell containing such an expression
vector. These host cells can be cultured under specific conditions
permitting expression of antibodies, or fragments thereof, of the
present invention and the antibodies then can be purified from the
host cells.
[0066] Again, standard recombinant techniques and known expression
vectors are used to express the antibodies of the invention.
Vectors for expressing proteins in bacteria, especially E. coli,
are known. Such vectors include the PATH vectors described by
Dieckmann and Tzagoloff in J. Biol. Chem. 260, 1513-1520 (1985).
These vectors contain DNA sequences that encode anthranilate
synthetase (TrpE) followed by a polylinker at the carboxy terminus.
Other expression vector systems are based on beta-galactosidase
(pEX); lambda P.sub.L; maltose binding protein (pMAL); and
glutathione S-transferase (pGST)-see Gene 67, 31 (1988) and Peptide
Research 3, 167 (1990).
[0067] Vectors useful in yeast are available. A suitable example is
the 2.mu. plasmid. Suitable vectors for expression in mammalian
cells are also known. Such vectors include well-known derivatives
of SV-40, adenovirus, retrovirus-derived DNA sequences and shuttle
vectors derived from combination of functional mammalian vectors,
such as those described above, and functional plasmids and phage
DNA.
[0068] Further eukaryotic expression vectors are known in the art
(e.g., P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341
(1982); S. Subramani et al., Mol. Cell. Biol. 1, 854-864 (1981); R.
J. Kaufmann and P. A. Sharp, "Amplification And Expression Of
Sequences Cotransfected with A Modular Dihydrofolate Reductase
Complementary DNA Gene," J. Mol. Biol. 159, 601-621 (1982); R. J.
Kaufmann and P. A. Sharp, "Amplification And Expression Of
Sequences Cotransfected with A Modular Dihydrofolate Reductase
Complementary DNA Gene," J. Mol. Biol. 159, 601-664 (1982); S. I.
Scahill et al., "Expression And Characterization Of the Product Of
A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells,"
Proc. Natl. Acad. Sci. USA 80, 4654-4659 (1983); G. Urlaub and L.
A. Chasin, Proc. Natl. Acad. Sci. USA 77, 4216-4220, (1980)).
[0069] The expression vectors useful in the present invention
contain at least one expression control sequence that is
operatively linked to the DNA sequence or fragment to be expressed.
The control sequence is inserted in the vector in order to control
and to regulate the expression of the cloned DNA sequence. Examples
of useful expression control sequences are the lac system, the trp
system, the tac system, the trc system, major operator and promoter
regions of phage lambda, the control region of fd coat protein, the
glycolytic promoters of yeast, e.g., the promoter for
3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,
e.g., Pho5, the promoters of the yeast alphamating factors, and
promoters derived from polyoma, adenovirus, retrovirus, and simian
virus, e.g., the early and late promoters or SV40, and other
sequences known to control the expression of genes of prokaryotic
or eukaryotic cells and their viruses or combination thereof.
[0070] Vectors containing the control signals and DNA to be
expressed, such as that encoding antibodies of the invention,
antibody equivalents thereof, or FGFR-1(IIIb), FGFR-1(IIIc), or
FGFR-4(IIIc), are inserted into a host cell for expression. Some
useful expression host cells include well-known prokaryotic and
eukaryotic cells. Some suitable prokaryotic hosts include, for
example, E. coli, such as E. coli SG-936, E. coli HB 101, E. coli
W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRC1,
Pseudomonas, Bacillus, such as Bacillus subtilis, and Streptomyces.
Suitable eukaryotic cells include yeast and other fungi, insect,
animal cells, such as COS cells and CHO cells, human cells and
plant cells in tissue culture.
[0071] A method of producing an antibody comprising culturing the
host cell comprising the vector comprising the nucleic acid
sequence encoding for the antibodies of the invention under
conditions permitting expression of the antibody. Following
expression in a host cell maintained in a suitable medium, the
polypeptide or peptide to be expressed, such as that encoding the
antibodies of the invention, antibody equivalents thereof, or
FGFR-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc), maybe isolated from
the medium, and purified by methods known in the art. If the
polypeptide or peptide is not secreted into the culture medium, the
host cells are lysed prior to isolation and purification.
[0072] This invention further provides a pharmaceutical composition
comprising the antibody of this invention or fragment thereof and a
pharmaceutically acceptable carrier.
[0073] Carrier as used herein includes pharmaceutically acceptable
carriers, excipients, or stabilizers which are nontoxic to the cell
or mammal being exposed thereto at the dosages and concentrations
employed. Often the physiologically acceptable carrier is an
aqueous pH buffered solution. Examples of physiologically
acceptable carriers include buffers such as phosphate, citrate and
other organic acids; antioxidants including ascorbic acid; low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; salt forming counterions
such as sodium; and/or nonionic surfactants such as TWEEN.RTM.,
polyethylene glycol (PEG), and PLURONICS.RTM..
[0074] The active ingredients may also be entrapped in
microcapsules prepared, for example, by interfacial polymerization,
for example, hydroxymethylcellulose or gelatin-microcapsules and
poly(methylmethacylate) microcapsules, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles, and nanocapsules) or
in macroemulsions. The formulations to be used for in vivo
administration must be sterile. This is readily accomplished by
filtration through sterile filtration membranes. Sustained-release
preparations may be prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of
solid hydrophobic polymers containing the antibody, which matrices
are in the form of shaped articles, e.g., films, or microcapsules.
Examples of sustained-release matrices include polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),
copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate,
non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers such as the LUPRON DEPOT.RTM.
(injectable microspheres composed of lactic acid-glycolic acid
copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric
acid. While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods.
[0075] When encapsulated antibodies remain in the body for a long
time, they may denature or aggregate as a result of exposure to
moisture at 37.degree. C., resulting in a loss of biological
activity and possible changes in immunogenicity. Rational
strategies can be devised for stabilization depending on the
mechanism involved. For example, if the aggregation mechanism is
discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization maybe achieved by
modifying sulffiydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0076] A method of identifying FR1-H7, FR1-A1 or FR1-4H or
fragments thereof can involve providing a library of antibody
fragments, screening the library for an antibody that is specific
for FGFR1-IIIb and/or FGFR1-IIIc or FGFR1-(IIIc) and/or
FGFR-4(IIIc), and purifying the antibody that is specific for
FGFR1-IIIb and/or FGFR1-IIIc or FGFR1-(IIIc) and/or FGFR-4(IIIc).
Thus, the present invention also provides a method of identifying a
fibroblast growth factor receptor (FGFR)-1(IIIb), FGFR-1(IIIc), or
FGFR-4(IIIc) specific antibody, or fragment thereof, which is as
follows: (i) providing a library of antibody fragments, (ii)
screening the library for the antibody that is specific for
FGFR-1(IIIb) and/or FGFR-1(IIIc) or the antibody specific for
FGFR-1(IIIc) and/or FGFR-4, and (iii) purifying the antibody that
is specific for FGFR-1(IIIb) and/or FGFR-1(IIIc) or the antibody
specific for FGFR-1(IIIc) and/or FGFR-4. Screening of the library
can involve (i) providing an affinity matrix having the
FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4 bound to a solid support,
(ii) contacting the affinity matrix with the library of antibody
fragments, and (iii) separating the antibody fragments that bind to
the affinity matrix from the antibody fragments that do not bind
the affinity matrix. These methods can be used to identify an
antibody.
[0077] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boemer et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p.77 (1985) and Boemer et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by the introduction of human immunoglobulin loci into
transgenic animals, e.g., mice in which the endogenous
immunoglobulin genes have been partially or completely inactivated.
Upon challenge, human antibody production is observed, which
closely resembles that seen in humans in all respects, including
gene rearrangement, assembly, and antibody repertoire. This
approach is described, for example, in U.S. Pat. Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the
following scientific publications: Marks et al., Bio/Technology
10,779-783(1992); Lonberg et al., Nature 368856-859 (1994);
Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature
Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology
14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93
(1995).
[0078] A purified antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment,
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous solutes, generally have been
removed.
[0079] The monoclonal antibodies of the invention secreted by the
subclones may be isolated or purified from the culture medium or
ascites fluid by conventional immunoglobulin purification
procedures such as, for example protein A-Sepharose, hydrolyapatite
chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
[0080] The present invention further provides the method of
identifying and isolating FR1-H7 and/or FR1-A1 and/or FR1-4H,
wherein the screening of the library comprises providing an
affinity matrix having the FGFR1-(IIIb), FGFR1-(IIIc),
FGFR-4(IIIc), and/or alternative splicing form containing ligand
binding function bound to a solid support, contacting the affinity
matrix with the library of antibody fragments, and separating the
antibody fragments that bind to the affinity matrix from the
antibody fragments that do not bind the affinity matrix.
[0081] By solid support is meant a non-aqueous matrix to which the
FGFR-1(IIIb), FGFR-1(IIIc), or FGFR-4(IIIc) can adhere. Examples of
solid phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0082] Methods of treatment involving administration to a mammal in
need thereof of a therapeutically effective amount of a
FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4 antagonist are also
provided by the present invention. Conditions for which these
methods are useful include obesity or an obesity related condition,
and diabetes or a diabetes related condition. Specific conditions
include, but are not limited to hypertension, cardiovascular
disease, and angiogenesis. The methods are also useful for reducing
food intake, body mass index, or altering energy metabolism.
Moreover, these methods can be useful in modulating serum
triglycerides and leptin levels.
[0083] An obesity related condition refers to a condition, which is
the result of or which is exasperated by obesity, such as, but not
limited to dermatological disorders such as infections, varicose
veins, Acanthosis nigricans, and eczema, exercise intolerance,
diabetes (Type 2), insulin resistance, hypertension,
hypercholesterolemia, cholelithiasis, orthopedic injury,
thromboembolic disease, cancer, and coronary (or cardiovascular)
heart disease, particularly those cardiovascular conditions
associated with high triglycerides and free fatty acids in an
individual, Arthritis, daytime sleepiness, sleep apnea, end stage
renal disease, gallbladder disease, gout, heat disorders, impaired
immune response, impaired respiratory function, infections
following wounds, infertility, liver disease, lower back pain,
obstetric and gynecologic complications, pain, pancreatitis,
stroke, surgical complications, urinary stress incontinence,
gastro-intestinal disorders.
[0084] Treatment means any treatment of a disease in an animal and
includes: (1) preventing the disease from occurring in a mammal
which may be predisposed to the disease but does not yet experience
or display symptoms of the disease; e.g. prevention of the outbreak
of the clinical symptoms; (2) inhibiting the disease, e.g.,
arresting its development; or (3) relieving the disease, e.g.,
causing regression of the symptoms of the disease.
[0085] Effective amount for the treatment of a disease means that
amount which, when administered to a mammal in need thereof, is
sufficient to effect treatment, as defined above, for that
disease.
[0086] Also, the present invention provides a method of treating
diabetes (type 2) or a diabetes (type 2) related condition
comprising administering to a mammal in need thereof a
therapeutically effective amount of an FGFR-1(IIIb), FGFR-1(IIIc),
FGFR-4(IIIc) and/or alternative splicing form containing ligand
binding function antagonist.
[0087] A diabetes related condition refers to a condition, which is
the result of or which is exasperated by diabetes, such as, but not
limited to heart and blood vessel disease, heart attack, stroke,
poor blood circulation in legs and feet, high blood pressure,
hypertension, blindness or vision problems, kidney failure or
infection, urinary bladder infection, nerve damage, slow healing
wounds, foot infections, or gum infections.
[0088] Also, the present invention provides a method of reducing
food intake comprising administering to a mammal in need thereof a
therapeutically effective amount of an antagonist of FGFR-1(IIIb),
FGFR-1(IIIc), FGFR-4(IIIc) and/or an alternative splicing form
having ligand binding function antagonist.
[0089] The identification of mammals in need of treatment is well
within the ability and knowledge of one skilled in the art. For
example, human individuals who are either suffering from clinically
significant obesity and diabetes such as hypertension,
cardiovascular disease, blood glucose levels, body mass, serum
triglyseride levels, angiogenesis, and/or energy metabolism (or
other related disease) or who are at risk of developing clinically
such significant disease are suitable for administration of the
present antagonist. A clinician skilled in the art can readily
determine, for example, by the use of clinical tests, physical
examination and medical/family history, if an individual is a
patient has such disease.
[0090] Further, the present invention provides a method of
treatment to affect conditions related to obesity and diabetes such
as hypertension, cardiovascular disease, blood glucose levels, body
mass, serum triglyseride levels, angiogenesis, and/or energy
metabolism.
[0091] Angiogenesis is the process of developing new blood vessels
that involves the proliferation, migration and tissue infiltration
of capillary endothelial cells from pre-existing blood vessels.
[0092] In the context of the present inventive methods, the
antagonist can be a biological molecule or a small molecule that
blocks FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4 signaling.
Preferred biological molecules are antibodies or fragment thereof,
including the antibodies and fragments thereof described herein.
The small molecules suitable in the present methods binds
FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4 internally, inhibits
FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4 phosphorylation. In
addition, the small molecule FGFR-1 antagonists inhibit binding of
ATP to FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4, which can involve
competing with ATP for FGFR-1(IIIb), FGFR-1(IIIc), and/or FGFR-4.
Ultimately, these small molecule antagonists of FGFR-1(IIIb),
FGFR-1(IIIc), and/or FGFR-4 inhibit tyrosine kinase activity.
Preferred small molecules include SU-6668, PD-173074, SU-5402,
CHIR-258, PD-166285 or derivatives A or B of pyrimido-pyridine as
described in FIG. 30.
[0093] The present invention also provides a method of treatment,
wherein the antagonist is a small molecule that blocks FGFR-1 or
FGFR-4 signaling. The FGFR-1 or FGFR-4 signaling is blocked by a
method wherein the FGFR-1 and/or FGFR-4 antagonist binds the FGFR-1
and/or FGFR-4 internally, inhibits the FGFR-1 and/or FGFR-4
phosphorylation, inhibits binding of ATP to FGFR-1 and/ or FGFR-4,
competes with ATP for the FGFR-1 and/or FGFR-4, and/or inhibits the
FGFR-1 and/or FGFR-4 tyrosine kinase activity.
[0094] Further, the present invention provides the method of
treatment wherein the small molecule is selected from the group
consisting of pyrimido-pyridines, quinolinones, indolinones such as
those shown in FIG. 30, as well as, SU-6668
(3-[2,4-Dimethyl-5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3--
yl]-propionic acid), PD-173074
(1-tert-Butyl-3-{6-(3,5-dimethoxy-phenyl)-2-[4-(ethyl-methyl-amino)-butyl-
amino]-pyrido[2,3-d]pyrimidin-7-yl}-urea), SU-5402
(3-[4-Methyl-2-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]--
propionic acid), CHIR-258
(4-Amino-5-fluoro-3-[6-(4-methyl-piperazin-1-yl)-1H-benzoimidazol-2-yl]-1-
H-quinolin-2-one), PD-166285
(3-(2,6-Dichloro-phenyl)-7-[4-(2-diethylamino-ethoxy)-phenylamino]-1-meth-
yl-1H-quinolin-2-one), Sugen Inc. U.S. Pat. No. 6,569,868,
6,599,902, 6,486,185, 6,514,981, 6,573,293; Institut Pasteur U.S.
Pat. No. 6,559,126, Agouron Pharmaceuticals, Inc. U.S. Pat. No.
6,534,524, 6,462,060, 6,620,828, 6,531,491; Pharmacia & Upjohn
Company U.S. Pat. No. 6,451,838; Ariad Pharmaceuticals, Inc. U.S.
Pat. No. 6,576,766, 6,482,852, 6,573,295; Bridges et al. U.S. Pat.
No. 6,602,863; Warner-Lambert Company U.S. Pat. No. 6,602,863;
Pharmacia & Upjohn Co. U.S. Pat. No. 6,451,838.
[0095] The method of treatment described herein can be used to
treat any suitable mammal, preferably the mammal is a human.
[0096] All patents and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0097] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. The examples do not include detailed
descriptions of conventional methods, such as those employed in the
construction of vectors and plasmids, the insertion of genes
encoding polypeptides into such vectors and plasmids, or the
introduction of plasmids into host cells. Such methods are well
known to those of ordinary skill in the art and are described in
numerous publications including Sambrook, J., Fritsch, E. F. and
Maniatis, T. (1989) Molecular Cloning: A laboratory Manual,
2.sup.nd Edition, Cold Spring Harbor Laboratory Press.
Example 1
Assays
[0098] ELISA Screening Assay
[0099] Recombinant FGFs (R&D systems, Minneapolis, Minn.) were
coated on Immulon.RTM. 2B microtiter plates (ThermoLab Systems,
Franklin, Mass.) at concentrations of 0.5-2 .mu.g/ml for 2 hrs. The
plates were then washed with 0.2% Tween20/PBS, and blocked with 5%
milk/PBS for 2 hrs before use. Phage or antibody was serially
diluted with 5 .mu.g/ml heparin (Sigma, St Louis, Mo.), 5% milk,
PBS. Recombinant FGFR-1 (R&D Systems) was then added to a final
concentration of 1 .mu.g/ml. The mixture was incubated at room
temperature for 1 hr before transfer to the FGF-2 coated plates,
and incubated at room temperature for an additional 2 hrs. Plates
were then washed 3 times with 0.2% Tween20/PBS. The bound receptors
were detected using an anti-human Fc monoclonal antibody coupled
with HRP (Pierce Biotechnology Inc., Rockford, Ill.) solution
prepared according to supplier's instructions. Data were presented
as % inhibition of ligand-receptor binding of the controls.
[0100] Binding Assay
[0101] Recombinant FGFRs (R&D systems) at a concentration of 1
.mu.g/ml in PBS were coated on 96-well plates at room temperature
for 2 hrs. The plates were washed 3 times with 0.2% Tween20/PBS,
and blocked with 5% milk/PBS for 2 hrs before use. FR1-H7 or FR1-A1
was added to the plate and serially diluted in 0.2% Tween20/PBS.
The plate was incubated at room temperature for 2 more hrs. The
captured antibodies were detected using an anti-human light chain
monoclonal antibody conjugated with HRP (Pierce Biotechnology,
Rockford, Ill.) solution prepared according to supplier's
instructions. The binding kinetics of the antibody to FGFR-1 was
determined using a BiaCore.RTM. 3000 biosensor (BiaCore, Inc.,
Piscataway, N.J.) following the standard protocols suggested by the
manufacturer. The Binding Kinetic (Kd) of FR1-H7 determined using
BiaCore biosensor are as follows: TABLE-US-00001 FGFR-1b: 60 pM
FGFR-1c: 40 pM FGFR-2b: >1 .mu.M FGFR-2c: >1 .mu.M FGFR-3b:
>1 .mu.M FGFR-3c: >1 .mu.M FGFR-4: 0.2 .mu.M
[0102] Cell-Based Blocking Assay
[0103] An FGFR-1(IIIc) expressing cell line was constructed by
retro-viral transfecting L6 (ATCC, Manassas, Va.) using a pBABE
vector that has a puromycin resistant gene (Invitrogen).
Transfected cells were cultured in 10% FBS, DMEM (Invitrogen) in
6-well tissue culture plates until reaching confluency, Before the
blocking experiment, cells were serum starved for 8-24 hrs at
37.degree. C. in DMEM medium that contains 0.1% FBS and 5 .mu.g/ml
heparin. Before the addition of any reagents, plates were put on
ice for 1 hr to minimize receptor internalization. For binding
experiment, iodinated recombinant FGF-2 was added at various
concentrations to the wells of the plates. For blocking
experiments, serially diluted FR1-H7 antibody was added and
incubated with the cells on ice for 1 hr before iodinated
recombinant FGF-2 was added to each well to a final concentration
of 15 ng/ml. Binding was for 1 hr at 0-4.degree. C., after which
time the solutions were aspirated and the plates were washed 5
times with ice-cold PBS. Cells were lyzed with 0.5 ml ice-cold cell
lysis buffer for 30 minutes. Radioactivity of the cell lysates was
detected using a Wizard.TM. 1470 automatic gamma counter (Turku,
Finland). Non-specific interaction was determined in a binding
experiment in which iodinated FGF-2 was incubated with the cells in
presence of 200 fold excess of cold ligand.
[0104] Phosphorylation Assay
[0105] FGFR-1 expressing L6 cells were cultured in 10% FBS, DMEM,
in a 24-well tissue culture plate till confluency. Cells were serum
starved in 5 .mu.g/ml heparin, 0.1% FBS, DMEM, for 8-24 hrs. Either
FR1-H7 or FR1-A1 was added to the media and allowed to bind with
the cell surface receptors at 37.degree. C. for 1 hr. Cells were
then stimulated with 20 ng/ml FGFs at 37.degree. C. for 10 min
before they were lyzed using ice-cold lysis buffer for 30 min. Cell
lysate was subjected to SDS-PAGE followed by western blot.
Membranes were probed with anti-phospho-tyrosine antibody (Cell
Signaling Technology, Inc., Beverly, Mass.) for detection of
phosphorylated FGFR-1 receptors according to supplier's
instructions.
[0106] Proliferation Assay
[0107] Human umbilical vascular endothelial cells (HUVECs) were
seeded in 96-well tissue culture plates at a concentration of
5.times.10.sup.4 cells/ml. After attachment, cells were quiesced in
EMG-2 medium lacking EGF, VEGF and FGF-2 (Cambrex, East Rutherford,
N.J.) for 24 hrs. Quiescent media in wells were aspirated and
replaced with EMG-2 that contained all the growth factors mentioned
above. Antibodies were serially diluted and added to the wells.
Cells were incubated at 37.degree. C. with 5% CO.sub.2 in a
Muaire.TM. DH autoflow incubator (Cryostar Industries, Inc., White
Hall, Pa.) for 48-72 hrs. Cell growth was determined by monitoring
.sup.3H-thymidine incorporation using a 1450 Micorbeta liquid
scintillation counter (Perkin Elmer, Gaithersburg, Md.).
[0108] Two-week-old (after induced differentiation) grade 2 human
adipocytes were obtained from Zen-Bio Inc. (Research Triangle,
N.C.), and were maintained in the shipping media in a 96-well
tissue culture plate. To evaluate the effects of FGF-2 on the
proliferation of adipocytes, recombinant FGF-2 (R&D systems)
was serially diluted and added to the wells of the plate. The cells
were allowed to grow in the incubator for 60 hrs, before the level
of .sup.3H-thymidine incorporation was determined. To evaluate the
effects of FR1-H7 on adipocytes proliferation, the antibody was
serially diluted and added to the cells before addition of
recombinant FGF-2 to a final concentration of 15 ng/ml.
.sup.3H-thymidine incorporation was determined after 60 hrs of
incubation.
[0109] Mitogenesis Assay
[0110] Human astrocytoma G18 cells were seeded in 96-well tissue
culture plates at a concentration of 5.times.10.sup.4 cells/ml.
After attachment, cells were quiesced for 24 hrs in RPMI media
(Invitrogen) containing 0.1% FBS and 5 .mu.g/ml heparin. Antibodies
were serially diluted, added to the wells, and incubated with the
cells at 37.degree. C. for 1 hr. Recombinant FGF-2 was then added
to a final concentration of 5 ng/ml. Cells were cultured in an
incubator at 37.degree. C. with 5% CO.sub.2. Cell growth was
determined 24 hrs later by monitoring .sup.3H-thymidine
incorporation as described previously.
[0111] Animal Studies--Food Consumption, Behavior, and Serum
Glucose
[0112] Female athymic nude mice (Crl:NU/NU-nuBR, Charles River
Laboratories, Wilmington, Mass.) were housed 4-5 per cage. Mice
were given autoclaved food (Lab Diet #5001, PMI Feeds Inc., St.
Louis, Mo.) and water ad libitum. All animal use in this study was
conducted in compliance with approved institutional animal care and
use protocols, and according to NIH guidelines (Guide for the Care
and Use of Laboratory Animals, NIH publication no. 86-23,
1985).
[0113] In one assay, mice were injected intraperitoneally (i.p.)
with FR1-H7 at 0.19, 1.9, and 19 mg/kg body weight (n=5 per group).
In another assay, mice were injected subcutaneously (s.c.) with the
antibody at 0.4, 4, and 40 mg/kg body weight. In control arms, mice
were treated with saline i.p. or s.c. In addition, one group of
mice was left untreated (n=4). All treatments were given at a
volume of 10 .mu.l/g body weight in Saline. Treatments were started
on a Wednesday (Day 0) with additional treatments on the following
Friday, Monday, Wednesday, and Friday. After this 9-day treatment
period mice received no additional treatments.
[0114] Alternatively, female athymic nude mice were injected (i.p.)
with saline, 2 mg/kg, or 20 mg/kg FR1-A1 on a Monday (Day 0) and
Wednesday. Body weights were measured before the first injection,
and on Day 2 and Day 5. Food intake per group was measured 2 days
after the first injection.
[0115] The body weights of all animals were measured 3 times/week.
Food consumption, behavior, and serum glucose were evaluated as
follows. Food Intake Measurement: Prior to the treatment on Day 2
mice were placed in a new cage and the weight of the food container
in the cage top was weighed. Prior to the treatment on Day 5 the
weight of the food container was measured again. The difference in
the Day 2 to Day 5 food container weight was divided by the number
of mice per cage and the number of days between the measurements to
give a food intake per mouse per 24 hrs. Activity Measurement: On
Day 12, 3 days after the final treatment, mice were placed
individually into a new cage and the number of rearings was counted
over a 1 min period. Blood Glucose Measurement: On Day 16, 7 days
after the final treatment, non-fasted blood glucose was measured in
blood collected from the tip of the tail using an Ascensia Elite XL
blood glucose meter (glucose oxidase method, Bayer, Pittsburg,
Pa.).
Animal Studies--Energy Expenditure and Body Composition
[0116] The concept of energy homeostasis suggests that weight loss
is the result of decreased food consumption, increased energy
expenditure, or the combination of both. To elucidate the mechanism
of the weight-loss induced by FGFR antagonistic antibodies, the
effect of FR1-H7 on energy expenditure and body composition of mice
was studied. Mice treated with FR1-H7 exhibited decreases in body
weight, food intake, muscle and fat mass, energy expenditure and
Respiratory Exchange Ratio (RER) compared to the controls (FIG.
26). A paired-feeding experiment was also conducted which food
consumption of control antibody treated animals was restrained to
exact that of FR1-H7 treated animals (FIG. 27). Animals from the
two treatment groups exhibited identical weight-loss trajectories
in a 5-day period. Moreover, body composition (Day 1 and 5) and
energy consumption determined for the two groups were mostly
indistinguishable. These results suggest that the weight-loss
induced by FR1-H7 treatment can be mostly attributed to reduced
food intake. Corroboratively, the more prominent decrease in RER
values in these animals on later days of the experiment suggests
the greater dependence on internal energy source (i.e. lipid
metabolism), as to compensate the deficit in food intake.
[0117] This study consisted of two stages. In the first stage,
female C57 black mice (Charles River Laboratories, Wilmington,
Mass.) were randomized into two groups (n=16) and individually
housed in a metabolic cage. Both groups of Mice were given
hi-carbohydrate content chow and water ad libitum. The two groups
of mice were treated with FR1-H7 and control human IgG (10 mg/kg
body weight, intraperitoneally), respectively, on Day 2 and Day 4.
Body composition was determined using NMR on Day 1 and Day 5 Body
weight and food consumption were determined daily. Energy
expenditure, ambulatory activities, and oxygen consumption and were
monitored continuously. RER were calculated based on energy
expenditure and oxygen consumption.
[0118] In the second stage, both groups of Mice were given water ad
libitum, but only 2 g of hi-carbohydrate content chow each animal
per day. The amount of food given was determined in the first stage
experiment to be the averaged daily food assumption of those
animals one day after being treated with FR1-H7 (10 mg/kg body
weight, intraperitonaeally).The two groups of mice were treated and
monitored the same way as described in the first stage
experiment.
Example 2
Isolation of Monoclonal Antibodies (FR1-H7)
[0119] A phage-displayed human Fab library from Dyax Corp.
(Cambridge, Mass.) was panned for anti-FGFR-1(IIIb) monoclonal
antibody (Fab) clones. Polystyrene Maxi-soap tubes (75.times.12 mm,
Nalge Nunc International, Rochester, N.Y.) were coated with 50
.mu.g of FGFR-(IIIb) (R&D Systems, Minneapolis, Minn.)
overnight, and blocked with 3% milk/PBS at 37.degree. C. for 2 hrs.
A mixture of 0.8 ml of phage library (>10.sup.13 cfu/ml), 200
.mu.l of 2 mg/ml ICMC-1C11 (ImClone Systems Incorporated, New York,
N.Y.), and 200 .mu.l of 18% milk/PBS were added and incubated in
the tube at room temperature for 2 hrs. IMC-1C11 antibody served to
block the retention of Fab clones that are reactive only to the
Fc-tag of the recombinant receptor. The tube was washed 15 times
with 0.1% tween-20/PBS (PBST), followed by 15 times with PBS. Bound
Fab-phage was eluted by incubating the tube with 1 ml of freshly
made 100 mM triethylamine (SIGMA, St Louis, Mo.) at room
temperature for 10 min. The eluent was transferred to a 50 ml
Falcon tube containing 0.5 ml 1 M Tris-HCl buffer, pH 7.5. Phage
was rescued by adding 12.5 ml and 1 ml of fresh E. coli TG1 cells
(OD.sub.600 nm: 0.5-0.8) to the eluent and panning tube,
respectively. E. coli cells were incubated at 37.degree. C. without
shaking for 30 min, and then with shaking at 100 rpm for an
additional 30 min. Infected TG1 cells were combined and grown in
2.times.YT(Bio 101.RTM. systems, Carlsbad, Calif.)/Amp/glucose
(2.times.YTAG) at 30.degree. C. overnight, then harvested and
stored at -80.degree. C. for future use.
[0120] Phage was grown by culturing 25 ml infected cells in
presence of 1 ml M13KO7 helper phage (Invitrogen, Carlsbad,
Calif.). The culture was incubated at 37.degree. C. without shaking
for 30 min, and with shaking (225 rpm) for an additional 30 min.
The cell culture was transferred into a 50 ml Falcon.RTM. tube
(Becton Dickinson, Franklin lakes, N.J.), and centrifuged at
1,500.times.g for 10 min. The cell pellet was then re-suspended in
25 ml of 2.times.YT/Amp/Kan (2.times.YTAK) medium, transferred into
a fresh 250 ml flask, and grown at 30.degree. C. overnight with
shaking (225 rpm). The culture was then transferred into a
centrifuge tube and centrifuged at 7,000.times.g for 10 min.
Supernatant was carefully removed to a fresh centrifuge tube, and
mixed with PEG/NaCl solution (6:1, v:v). The mixture was incubated
on ice for 1 hr, and centrifuged at 20,000.times.g for 30 min.
Phage pellet was re-suspended in 1 ml PBS.
[0121] The panning was repeated one more time with tubes coated
with 10 .mu.g FGFR-1(IIIb). Single colonies of infected cells were
inoculated into 96-well plates containing 100 .mu.l/well of
2.times.YTAG, and phage was grown in presence of 10 .mu.l M13KO7
helper phage (5.times.10.sup.10 pfu/ml). Plates were incubated at
37.degree. C. for 30 min without shaking followed by 30 min with
shaking (100 rpm). Cell pellets were prepared by centrifugation at
2,500 rpm for 10 min, re-suspended in 200 .mu.l of 2.times.YTAK,
and incubated at 30.degree. C. with shaking (100 rpm) for
overnight. The plates were then centrifuged at 2,500 rpm for 10
min. Supernatants were transferred in fresh plates and mixed with
6.times. blocking buffer (18% milk/PBS) for 1 hr. Phage clones were
screened in the ELISA blocking assay as described below. Blocking
clones was selected and soluble phage was prepared for another
round of screening using the ELISA blocking assay. Confirmed
blockers were engineered into full size antibodies.
Example 3
Modulation of Serum Levels
[0122] Short-term effects of the FR1-H7 antibody on serum levels of
insulin, leptin, glucose, and triglycerides were examined by
injecting 4 mg/kg antibody or control vehicle into female athymic
nude mice. Samples were collected and evaluated before treatment
and 6, 24, and 48 hours after treatment. Alanine aminotransferase
(ALT), creatine (CPK), blood urea nitrogen (BUN), total serum
proteins, and serum albumin levels were measured using serum
samples taken 48 hours after treatment. The mice were sacrificed
and weights of their left parametrial fat pad, left tibialis
anterior muscle, left posterior liver lobe, and spleen were also
taken.
Example 4
Modulation of Food Intake
[0123] The antibodies weight-reducing effects on other strains of
mice, C57 black mice and db/db mice were tested (The Jackson
Laboratory, Bar Harbor, Me.). The C57 mice were injected with 0.8
mg/kg, 4 mg/kg, and 40 mg/kg antibody and saline on a Monday,
Wednesday, Friday, and following Monday rotation schedule with body
weights being measured before each injection and on day 9.
[0124] The db/db 7 week old mice were injected subcutaneously with
4 mg/kg antibody on a Monday, Wednesday, and Friday schedule with
body weights being measured before each injection and on day 7.
Food intake was monitored as well on days 1, 2, 3, 4, and 7. The
db/db animals were sacrificed on day 7 and their intrascapular
brown fat, epididymal white adipose tissue, and inguinal
subcutaneous white adipose tissue were sampled and weighed.
Example 5
In Vitro Activity (FR1-H7)
[0125] ELISA and immunoprecipitation was performed using antibody
FR1-H7. The Fabs were screened according to their ability to
prevent FGF from binding to FGFR-1. The binding specificity of the
antibody was examined using an ELISA binding assay, and the results
are shown in FIG. 3. FR1-H7 binds FGFR-1(IIIb) and -1(IIIc) with
strong affinities, but does not recognize any of the other FGFRs.
Results from kinetic analysis indicated the antibody binds to
FGFR-1b and -1c equally well. The KDS of the interactions were
determined to be approximately 50 pM for both receptors.
Example 6
Inhibition of Ligand-Receptor Binding (FR1-H7)
[0126] FIGS. 4A & 4B shows the inhibitory effect of FR1-H7 on
ligand-receptor binding as determined in an ELISA blocking assay.
Two FGFR1-binding ligands, FGF-1 (FIG. 4A) and FGF-2 (FIG. 4B) were
tested in the blocking assay. FR1-H7 was found to block recombinant
FGFR-1(IIIb) and -1(IIIc) equally well in their binding to FGF-1,
with IC.sub.50s of approximately 5 nM. The antibody also blocks the
receptors from binding to FGF-2, with IC.sub.50s in the range of
2-5 nM, although it is slightly more potent in inhibiting
FGFR-1(IIIb) than -1(IIIc). The blocking activities to fully
active, native FGFR-1 receptor were determined in the cell-based
blocking assay. As shown in FIG. 5A, the total binding of FGF-2 to
FGFR-1-expressing cells has two components: non-specific binding,
and specific binding. The latter accounts for the majority of the
total binding. FIG. 5B shows that FR1-H7 inhibits the specific
binding of .sup.125I-FGF-2 to the cells, and the blocking is near
completion at the concentration of 200 nM. The IC.sub.50 is lower
than 5 nM by estimation.
Example 7
Ligand Mediated FGFR-1 Signaling, Cell Growth In Vitro (FR1-H7)
[0127] Under normal condition, the activation of FGFRs is
ligand-dependent. The blocking of ligand binding by FR1-H7
consequently leads to inhibition of auto-phosphorylation of the
receptor. This is demonstrated in FIG. 6, in which the western blot
was probed with anti-phospho-tyrosine antibody for activated
receptor--the upper blot was probed with anti-phospho-tyrosine
antibody and the lower blot showed control protein bands in the
cell lysates for estimation of relative gel loading. The results
show that the FR1-H7 by itself does not activate the receptor, and
significantly inhibits FGF-2 mediated receptor phosphorylation.
[0128] HUVEC is known to express FGFR-1(IIIb) on the cell surface
(Ferning and Gallagher, 1994). FGFs have been shown to stimulate,
the growth of endothelial cells strongly, and thus are considered
major pro-angiogenic factors. FIG. 7 shows that FR1-H7 inhibited
the proliferation of HUVECs in vitro in a dose-dependant manner.
These results demonstrate that FR1-H7 may be used as an
anti-angiogenic therapeutic in certain diseases.
[0129] The effects of the FGF pathway on the proliferation of
Adipocytes in vitro are shown in FIG. 8. FGF-2 had a profound
stimulating effect on the growth of the adipocytes. The level of
thymidine incorporation was capable of a 10-fold increase in
presence of FGF-2 (FIG. 8A). FR1-H7 inhibited the FGF-2-stimulated
proliferation of adipocytes in a dose dependant manner (FIG.
8B).
Example 8
Antibody Activity (FR1-H7)
[0130] FR1-H7 induces reversible weight loss in a dose-dependent
manner. Both s.c. and i.p. administration of FR1-H7 caused
dose-dependent weight loss of the animals (FIG. 9). At the two
lowest dosages, 0.19 and 0.4 mg/kg, mice had slight weight gains
that are identical as the untreated or vehicle treated animals. At
1.9 mg/kg dosage, steep weight loss of .about.20% of the total body
weight occurred within the first 3 treatments, and then the weights
of the animals showed a trend of stabilization. Moderate weight
loss (.about.0.5 g) continued one week after the treatment was
stopped. This was followed by rapid recovery of the weights.
Complete recovery was reached 25 days after treatment was stopped,
and the averaged weight of this group was identical to that of the
controls. At dosage higher than 4 mg/kg, the antibody inflicted the
same degree of weight loss independent of the doses. Rapid weight
loss occurred within the first 3 treatments, followed by a gradual
stabilization of the weights. The maximal weight loss amounts to
.about.1/3 of the averaged body weight of the controls. Weight
recovery after the stop of the treatments seemed to be
dose-dependent. Lagging times of the first significant weight gains
after the stop of the treatments were 14 days, 19 days, and 24
days, for antibody treatment of 4 mg/kg s.c., 40 mg/kg s.c., and 19
mg/kg i.p., respectively. Except that one animal in the 19 mg/kg
group was euthanized due to unusual weight loss of .about.50% of
the total body weight, all mice in the antibody treated groups
eventually recovered their weights completely.
Example 9
Food Intake and Exploratory Behavior
[0131] Non-fasting food intake, measured over the period between
the second and third treatment, was reduced by approximately 35% in
groups of mice that lost weight, but not in the low dosage groups
in which weight loss did not occur (FIG. 10--Effect of FR1-H7 on
food intake in nu/nu female mice). The exploratory behavior of
mice, measured as the number of rearings per min in a novel
environment, was not significantly altered by the antibody
treatments (FIG. 11--Effect of FR1-H7 on rearing behavior in a
novel environment; Mean.+-.SEM). This is in agreement with the
general observation that mice were not moribund, in spite of
dramatic weight loss.
Example 10
Modulation of Glucose Levels
[0132] Non-fasting blood glucose level was determined one week
after the end of the antibody treatment It appeared that the
antibody reduced the blood glucose in a dose-dependent manner (FIG.
12A--Effect of FR1-H7 on non-fasted blood glucose;Mean .+-.SEM).
The greatest reduction occurred in 19 mg/kg i.p. and 40 mg/kg s.c.
groups, which amounted to .about.1/3 of the normal glucose level.
On day 64, 52 days after the final treatment, non-fasted blood
glucose was again measured. However, serum glucose levels were
restored to normal range by day 64 after the animals fully
recovered their body weights (FIG. 12B--Effect of FR1-H7 on
non-fasted blood glucose after weights are fully recovered;
Mean.+-.SEM).
Example 11
Reduction in Adipose Tissue, Serum Triglycerides, Insulin, and
Leptin
[0133] To study the short-term effect of FR1-H7, nude mice were
treated with a single injection of 4 mg/kg FR1-H7 s.c., and
monitored the effects 48 hours after. FIG. 13, which is a graph of
body weight loss in nu/nu mice after a single does of FR1-H7
treatment (Mean.+-.SEM, n=4), showed that the antibody treatment
caused significant body weight reduction within 48 hours. The
averaged weight in the antibody-treated group was reduced by
approximately 6% compared to that of the control groups. To
investigate the source of this reduction, the weight measurement of
representative tissue samples was taken. As shown in FIG. 14
(effects on tissue weights after a single does of FR1-H7 treatment;
each bar represents the value calculated as 100.times. the ratio of
tissue weight over total body weight; error bars are standard
deviation) weight reduction of approximately 60% was observed in
parametrial fat pad, while the weights of muscle, liver, and spleen
appear to be normal.
[0134] FIGS. 15A-D show that FR1-H7 did not affect the serum
glucose level 48 hours after the treatment. However, serum
triglycerides, insulin, and leptin levels were significantly
reduced, indicating that FR1-H7 systematically affected the energy
metabolism of the animals. There were no significant differences in
the levels of ALT, CPK, BUN, total serum proteins, and serum
albumin between the antibody-treated and control groups, suggesting
normal liver, muscle and kidney function in the treated
animals.
Example 12
Induction of Weight Loss
[0135] FIG. 16 shows that FR1-H7 treatment caused dose-dependent
weight loss of C57 black mice, though the effect is less dramatic
than in the nude mice. After 4 treatments, the weight reduction
compared to the starting weights were approximately 5%, 16% and 16%
for 0.8 mg/kg, 4 mg/kg and 40 mg/kg treated groups, respectively.
In db/db mice, weight reduction is even more moderate compared to
that in the nude mice (FIG. 17). In the 7-day period, weight
reduction occurred gradually, and reached approximately 10% of the
total body weight on Day 7. This was accompanied by a gradual
reduction in food intakes (FIG. 18). Analysis of different adipose
tissues showed that significant reduction occurred in intrascapular
brown fat tissue, while the weights of epididymal white adipose
tissue and inguinal subcutaneous white adipose tissue changed
little (FIG. 19). The more moderate weight loss caused by FR1-H7
db/db mice may be attributed to the leptin pathway deficiency, or
simply due to the abundance of fat tissues in these animals.
Example 13
Isolation of Antibody (FR1-A1)
[0136] Using the method described in Example 2, with the exception
that the Phage-displayed human Fab library from Dyax was panned for
anti-FGFR-1(IIIc) Fab clones, an antibody specific for
anti-FGFR-1(IIIc) was identified. The identified antibody was
designated FR1-A1.
Example 14
In Vitro Activity (A1-A1)
[0137] FR1-A1 exhibits strong binding affinity to FGFR-1(IIIc), and
moderate affinity to FGFR-4. It shows little binding to all the
other receptors (FIG. 20). The K.sub.Ds towards these receptors as
determined from kinetic analyses are as follows: TABLE-US-00002
FGFR-1b: >10 .mu.M FGFR-1c: 0.7 nM FGFR-2b: >10 .mu.M
FGFR-2c: >10 .mu.M FGFR-3b: >10 .mu.M FGFR-3c: >10 .mu.M
FGFR-4: 90 nM
[0138] FIG. 21 shows the inhibitory effects of FR1-A1 on
ligand-receptor binding as determined in an ELISA blocking assay.
Two FGFR-1(IIIc) binding ligands, FGF-1 and FGF-2 were tested in
the blocking assay. FR1-A1 blocked the binding of FGFR-1(IIIc) to
FGF-1 and FGF-2 with IC.sub.50s of approximately 5 and 10 nM,
respectively.
Example 15
Inhibition of Ligand-Mediated FGFR-1(IIIc) Signaling, and Cell
Growth in Vitro (FR1-A1)
[0139] The blocking of ligand binding by FR1-A1 consequently leads
to inhibition of auto-phosphrylation of the receptor. This is
demonstrated in FIG. 22, in which the western blot of the cell
lysates was probed with anti-phospho-tyrosine for activated
FGFR-1(IIIc) receptor. The results show that the FR1-A1 by itself
does not activated the receptor, but significantly inhibits ligand
induced receptor phosphorylation.
[0140] Auto-phosphorylation of the FGFR-1 receptors frequently
leads to the mitogenic response of the cells through mitogen
activated protein kinase (MAPK) cascade in cytoplasma. Therefore,
inhibition of receptor phosphorylation by FR1-A1 leads to
inhibition of cell mitogenesis. Using flow cytometry analysis, it
was found that G18 cells express FGFR-1(IIIc) receptors on the cell
surface. FIG. 23 shows that the addition of 20 ng/ml FGF-2
increased the level of DNA synthesis by approximately 5 folds
compared to the control, and the antibody decreased this
stimulation in a dose-dependant manner.
Example 16
Induction of Weight Loss (FR1-A1)
[0141] Similar to what was observed with FR1-H7 antibody, FR1-A1
caused rapid weight loss to female athymic nude mice. As shown in
FIG. 24, on day 5, after two treatments, mice in 2 mg/kg dosage
group lost an average of .about.9% of the body weight compared to
the control. Mice in 20 mg/kg group lost .about.23%. Non-fast food
intake, measured over the period between the first and second
treatments, was reduced by .about.14% and .about.44% in 2 mg/kg and
20 mg/kg groups, respectively (FIG. 25).
Example 17
Isolation of Monoclonal Antibody(FR1-4H)
[0142] In addition to FR1-A1 and FR1-H7, yet another FGFR-1
specific antibody FR1-4H (FIG. 28) was clones. Through BiaCore.RTM.
analyses, the KDa of this antibody to FGFR-1(IIIb)-was determined
to be 1.1 nM. In comparison, the KDa to FGFR-1(IIIc) was greater
than 10 .mu.M. Therefore, FR1-4H is considered to be specific to
only the b-splicing form of FGFR-1. Like FR1-A1 and FR1-H7, FR1-4H
is a neutralizing antibody, which blocks the receptor from binding
to the ligand (FIG. 29). It was therefore deduced that FR1-4H would
afford many similar effects displayed by the other two FGFR-1
antibodies as described previously.
[0143] FR1-A1 and FR1-H7 exhibited high affinity binding towards
FGFR-1, and low affinity binding towards FGFR-4. Both antibodies
could inhibit the ligand-induced activation of FGFR-1 potently. In
animals, the two antibodies produced almost identical weight-loss
phenomena, accompanied by significant food intake reduction. From
the commonalities of these two antibodies it may be concluded that
other FGFR antagonists are capable of inducing weight reduction
through a FGFR-1 or FGFR-4 pathway related anorexic effect.
[0144] The procedures to isolate and clone FR1-4H is identical to
those of FR1-A1 and FR1-H7 as described in Examples 2 and 12,
except that phage candidates during the screening process were
selected for specific binding to the FGFR-1(IIIb) instead of
FGFR-1(IIIc).
Example 18
ELISA blocking assays for FR1-4H
[0145] This experiment was thoroughly described in Examples 6 and
14, except that FGFR-1(IIIb) recombinant protein was used instead
of FGFR-1(IIIc) in all occasions. FR1-4H inhibited the binding of
FGFR-(IIIb) to FGF Ligand (FIG. 29). Percent binding was determined
using the ELISA blocking assay described in Examples 6 and 14.
Percent binding was statistically reduced by treatment with FR1-4H
over control above 1 nm.
Example 19
FGFR Small Molecule Inhibitors
[0146] Many small molecules inhibit FGFR kinase activity; a few
examples are given in FIG. 30. The two pyrimido-pyridine
derivatives were tested.
[0147] PolyE-Y peptide (Sigma, St. Louis, Mo.) was coated to a
96-well Immulon.RTM. 2B microtiter plates (ThermoLab Systems,
Franklin, Mass.) by incubating 50 .mu.l/well of the solution (5
.mu.g/ml in PBS) for 12 hours at 4.degree. C. The solution was then
disposed, and the plate was washed twice with 200 .mu.l washing
buffer (PBS, 0.1% Tween-20). The following solutions were then
mixed in each well: 25 .mu.l Reaction buffer (100 mM Hepes, 10 mM
MgCl.sub.2, 10 mM MnCl.sub.2 and 1 mM DTT, pH7.5), 2 .mu.l compound
solution (various concentrations in DMSO) and 20 .mu.l FGFR kinase
domain recombinant protein solution (100 ng/.mu.l). After 5 min
incubation, 4.5 .mu.l/well ATP solution (40 .mu.M) was added and
the reaction was allowed to proceed for 20 min at 28.degree. C. The
reaction was stopped by washing the plate 3 times with the washing
buffer. The plate was then blocked with 3% of BSA in PBS for 1 hr.
Phosphorylation of polyE-Y peptides was detected using PY-20-HRP
antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) as
described by the manufacturer of the antibody. Raw data were
processed using a custom-made arithmetic to generate an IC.sub.50
value for each compound.
[0148] Both pyrimido-pyridines derivatives inhibited the kinase
activities of all FGFRs in an enzymatic assay (Table 1). In
cell-based assay, these two compounds were found to potently
inhibit the ligand-induced phosphorylation of FGFR-1c receptor
(FIG. 31). Because these compounds showed antagonistic activity
toward FGFR-1, and to a lesser extend, FGFR-4, they should share
the same weight-loss inducing properties in animals with FR1-A1 and
FR1-H7 antibodies. TABLE-US-00003 TABLE 1 FGFR small molecule
inhibitors inhibited the kinase enzymatic activities in vitro.
Values are IC50s determined using the enzymatic assay described in
Methods. Pryimido-pyridines Pryimido-pyridines derivative A
derivative B FGFR-1 18 nM 20 nM FGFR-2 60 nM N/A FGFR-3 20 nM 3 nM
FGFR-4 440 nM 360 nM
Example 20
Phosphorylation Assay for Small Molecule Inhibitors
[0149] The phosphorylation assay for small molecules is essentially
the same as the phosphorylation assay described in Examples 7 and
15 and Phosphorylation Assay, except that small molecule compounds
was used instead of FGFR-1 antibodies in all occasions. FGFR small
molecule inhibitors inhibited the auto-phosphorylation of
FGFR-1(IIIc) in a cell-based phosphorylation assay (FIG. 31). Equal
amount of cell lysates were applied to each sample lane. Receptor
auto-phosphorylation was probed using anti-phospho-tyrosine
antibody as described in the original patent.
Sequence CWU 1
1
48 1 5 PRT Homo sapiens 1 Asp Tyr Tyr Met His 1 5 2 17 PRT Homo
sapiens 2 Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu Lys
Phe Gln 1 5 10 15 Gly 3 6 PRT Homo sapiens 3 Asp Asp Tyr Met Asp
Val 1 5 4 12 PRT Homo sapiens 4 Arg Ala Ser Gln Ser Val Ser Gly Ser
Ala Leu Ala 1 5 10 5 7 PRT Homo sapiens 5 Asp Ala Ser Ser Arg Ala
Thr 1 5 6 9 PRT Homo sapiens 6 Gln Gln Tyr Gly Ser Ser Pro Leu Thr
1 5 7 123 PRT Homo sapiens 7 Met Ala Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Val Ser
Cys Lys Val Ser Gly Tyr Thr Phe Thr 20 25 30 Asp Tyr Tyr Met His
Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Met Gly
Leu Val Asp Pro Glu Asp Gly Glu Thr Ile Tyr Ala Glu 50 55 60 Lys
Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr 65 70
75 80 Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Ala Arg Asp Asp Tyr Met Asp Val Trp Gly Lys
Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 8 153 PRT Homo sapiens 8 Leu Glu Thr Thr Leu Thr Gln Ser
Pro Asp Thr Leu Ser Leu Ser Pro 1 5 10 15 Gly Glu Gly Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Gly 20 25 30 Ser Ala Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile
Tyr Asp Ala Ser Ser Arg Ala Thr Gly Val Pro Asp Arg Phe 50 55 60
Ser Gly Ser Gly Ser Gly Ala Asp Phe Ser Leu Thr Ile Ser Arg Leu 65
70 75 80 Glu Pro Glu Asp Phe Ala Val Tyr Ser Cys Gln Gln Tyr Gly
Ser Ser 85 90 95 Pro Leu Thr Phe Gly Pro Gly Thr Lys Val Asp Val
Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp
Lys Val Asp 145 150 9 5 PRT Homo sapiens 9 Gly Tyr Tyr Met His 1 5
10 17 PRT Homo sapiens 10 Arg Ile Ile Pro Ile Leu Gly Ile Ala Asn
Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 11 9 PRT Homo sapiens 11 Gly
Gly Asp Leu Gly Gly Met Asp Val 1 5 12 16 PRT Homo sapiens 12 Arg
Ser Ser Gln Ser Leu Arg His Ser Asn Gly Tyr Asn Tyr Leu Asp 1 5 10
15 13 7 PRT Homo sapiens 13 Leu Ala Ser Asn Arg Ala Ser 1 5 14 9
PRT Homo sapiens 14 Met Gln Ala Leu Gln Ile Pro Pro Thr 1 5 15 113
PRT Homo sapiens 15 Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro 1 5 10 15 Gly Ser Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Gln Thr Phe Thr 20 25 30 Gly Tyr Tyr Met His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu 35 40 45 Trp Met Gly Arg Ile Ile
Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln 50 55 60 Lys Phe Gln Gly
Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr 65 70 75 80 Ala Tyr
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 85 90 95
Tyr Cys Ala Arg Gly Gly Asp Leu Gly Gly Met Asp Val Trp Gly Gln 100
105 110 Gly 16 118 PRT Homo sapiens 16 Leu Glu Ile Val Leu Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro 1 5 10 15 Gly Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Arg His 20 25 30 Ser Asn Gly
Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser
Pro Gln Leu Leu Ile Tyr Leu Ala Ser Asn Arg Ala Ser Gly Val 50 55
60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln 85 90 95 Ala Leu Gln Ile Pro Pro Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile 100 105 110 Lys Arg Thr Val Ala Ala 115 17 5 PRT
Homo sapiens 17 Ser Tyr Tyr Trp Ser 1 5 18 16 PRT Homo sapiens 18
Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5
10 15 19 16 PRT Homo sapiens 19 Glu Tyr Tyr Tyr Asp Ser Ser Gly Tyr
Tyr Phe Tyr Ala Phe Asp Ile 1 5 10 15 20 13 PRT Homo sapiens 20 Ser
Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr 1 5 10 21 7 PRT
Homo sapiens 21 Arg Asn Asn Gln Arg Pro Ser 1 5 22 11 PRT Homo
sapiens 22 Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val 1 5 10 23
124 PRT Homo sapiens 23 Gln Val Gln Leu Val Glu Phe Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Ser Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val
Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Glu Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Phe Tyr Ala Phe Asp
100 105 110 Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
24 111 PRT Homo sapiens 24 Leu Pro Val Leu Thr Gln Pro Pro Ser Ala
Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Ser Ile Ser Cys Ser Gly
Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln
Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Phe Arg Asn
Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85
90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 25 15 DNA Homo sapiens 25 gactactaca tgcac 15 26 51 DNA
Homo sapiens 26 cttgttgatc ctgaagatgg tgaaacaatc tacgcagaga
agttccaggg c 51 27 18 DNA Homo sapiens 27 gatgactaca tggacgtc 18 28
36 DNA Homo sapiens 28 agggccagtc agagtgttag cggcagtgcg ttggcc 36
29 21 DNA Homo sapiens 29 gatgcatcca gtagggccac t 21 30 27 DNA Homo
sapiens 30 cagcaatatg gtagctcacc tctcact 27 31 369 DNA Homo sapiens
31 atggccgagg tgcagctggt gcagtctggg gctgaggtga agaagcctgg
ggcctcagtg 60 aaggtttcct gcaaggtttc tggatacacc ttcaccgact
actacatgca ctgggtgcaa 120 caggcccctg gaaaagggct tgagtggatg
ggacttgttg atcctgaaga tggtgaaaca 180 atctacgcag agaagttcca
gggcagagtc accataaccg cggacacgtc tacagacaca 240 gcctacatgg
agctgagcag cctgagatct gaggacacgg ccgtgtatta ctgtgcgaga 300
gatgactaca tggacgtctg gggcaaaggc accctggtca ccgtctcaag cgcctccacc
360 aagggccca 369 32 460 DNA Homo sapiens 32 cttgaaacga cactcacgca
gtctccagac accctgtctt tgtctccagg agaaggagcc 60 accctctcct
gtagggccag tcagagtgtt agcggcagtg cgttggcctg gtaccagcag 120
aaacctggcc aggctcccag actcctcatc tatgatgcat ccagtagggc cactggcgtc
180 ccagacaggt tcagtggcag tgggtctggg gcagacttca gtctcaccat
cagcagactg 240 gagcctgaag attttgcagt gtattcctgt cagcaatatg
gtagctcacc tctcactttc 300 ggccctggga ccaaagtgga tgtcaaacga
actgtggctg caccatctgt cttcatcttc 360 ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420 ttctatccca
gagaggccaa agtacagtgg aaggtggatt 460 33 15 DNA Homo sapiens 33
ggctactata tgcac 15 34 51 DNA Homo sapiens 34 aggatcatcc ctatccttgg
tatagcaaac tacgcacaga agttccaggg c 51 35 27 DNA Homo sapiens 35
ggaggagatc tgggcggtat ggacgtc 27 36 30 DNA Homo sapiens 36
aggtctagtc agagcctccg gcatagtaat 30 37 21 DNA Homo sapiens 37
ttggcttcta atcgggcctc c 21 38 27 DNA Homo sapiens 38 atgcaagctc
tacaaattcc tccgact 27 39 340 DNA Homo sapiens 39 atggcccagg
tccagctggt gcagtctggg gctgaggtga agaagcctgg gtcctcggtg 60
aaggtctcct gcaaggcttc tggatcgacc ttcaccggct actatatgca ctgggtgcga
120 caggcccctg gacaagggct tgagtggatg ggaaggatca tccctatcct
tggtatagca 180 aactacgcac agaagttcca gggcagagtc acgattaccg
cggacaaatc cacgagcaca 240 gcctacatgg agctgagcag cctgagatct
gaggacacgg ccgtgtacta ctgtgcgaga 300 ggaggagatc tgggcggtat
ggacgtctgg ggccaaggga 340 40 354 DNA Homo sapiens 40 cttgaaattg
tgctgactca gtctccactc tccctgcccg tcacccctgg agagccggcc 60
tccatctcct gcaggtctag tcagagcctc cggcatagta atggatacaa ctatttggat
120 tggtacctgc agaagccagg gcagtctcca cagctcctga tctatttggc
ttctaatcgg 180 gcctccgggg tccctgacag gttcagtggc agtggatcag
gcacagattt tacactgaaa 240 atcagcagag tggaggctga ggatgttggg
gtttattact gcatgcaagc tctacaaatt 300 cctccgactt tcggccctgg
gaccaaagtg gatatcaaac gaactgtggc tgca 354 41 15 DNA Homo sapiens 41
agttactact ggagc 15 42 48 DNA Homo sapiens 42 tatatctatt acagtgggag
caccaactac aacccctccc tcaagagt 48 43 48 DNA Homo sapiens 43
gagtattact atgatagtag tggttattac ttttatgctt ttgatatc 48 44 39 DNA
Homo sapiens 44 tctggaagca gctccaacat cggaagtaat tatgtatac 39 45 21
DNA Homo sapiens 45 aggaataatc agcggccctc a 21 46 33 DNA Homo
sapiens 46 gcagcatggg atgacagcct gagtggttgg gtg 33 47 372 DNA Homo
sapiens 47 caggtgcagc tggtggagtt tggcccagga ctggtgaagc cttcggagac
cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt agttactact
ggagctggat ccggcagccc 120 ccagggaagg gactggagtg gattgggtat
atctattaca gtgggagcac caactacaac 180 ccctccctca agagtcgagt
cgccatatca gtagacacgt ccaagaacca gttctccctg 240 aagctgagct
ctgtgaccgc cgcggacacg gccgtgtatt actgtgcgag agagtattac 300
tatgatagta gtggttatta cttttatgct tttgatatct ggggccaagg gaccacggtc
360 accgtctcaa gc 372 48 333 DNA Homo sapiens 48 ctgcctgtgc
tgactcagcc cccctcagcg tctgggaccc ccgggcagag ggtctccatc 60
tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccagcagctc
120 ccaggaacgg cccccaaact cctcatcttt aggaataatc agcggccctc
aggggtccct 180 gaccgattct ctggctccaa gtctggcact tcagcctccc
tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgca
gcatgggatg acagcctgag tggttgggtg 300 ttcggcggag ggaccaagct
gaccgtccta ggt 333
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