U.S. patent application number 12/756078 was filed with the patent office on 2011-01-06 for tweak receptor agonists as anti-angiogenic agents.
This patent application is currently assigned to BIOGEN IDEC MA INC.. Invention is credited to Jeffrey Browning, Linda Burkly, Aniela Jakubowski, Timothy Zheng.
Application Number | 20110002924 12/756078 |
Document ID | / |
Family ID | 31715635 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110002924 |
Kind Code |
A1 |
Browning; Jeffrey ; et
al. |
January 6, 2011 |
TWEAK RECEPTOR AGONISTS AS ANTI-ANGIOGENIC AGENTS
Abstract
The present invention relates to methods of modulating
angiogenesis and inhibiting tumor progression by using TWEAK
receptor (Fn14) agonists. In particular, methods for inhibiting
angiogenesis are disclosed.
Inventors: |
Browning; Jeffrey;
(Brookline, MA) ; Burkly; Linda; (West Newton,
MA) ; Jakubowski; Aniela; (Arlington, MA) ;
Zheng; Timothy; (Boston, MA) |
Correspondence
Address: |
BIOGEN IDEC / FINNEGAN HENDERSON, LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Assignee: |
BIOGEN IDEC MA INC.
Cambridge
MA
|
Family ID: |
31715635 |
Appl. No.: |
12/756078 |
Filed: |
April 7, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11725870 |
Mar 20, 2007 |
7731963 |
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12756078 |
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10380611 |
Mar 14, 2003 |
7208151 |
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PCT/US01/28451 |
Sep 12, 2001 |
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11725870 |
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60232355 |
Sep 14, 2000 |
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Current U.S.
Class: |
424/134.1 ;
424/152.1; 424/172.1 |
Current CPC
Class: |
C07K 16/2878 20130101;
A61P 35/00 20180101; A61P 43/00 20180101; A61K 38/00 20130101; C07K
2317/73 20130101; A61P 9/00 20180101 |
Class at
Publication: |
424/134.1 ;
424/172.1; 424/152.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; A61P 9/00 20060101
A61P009/00 |
Claims
1. A method of modulating angiogenesis comprising the step of
administering to a subject a therapeutically effective amount of a
TWEAK receptor agonist.
2. The method of claim 1 wherein said TWEAK receptor is Fn14.
3. The method of claim 1 wherein said TWEAK receptor agonist is an
Fn14 agonist.
4. The method of claim 3 wherein said Fn14 agonist is an anti-Fn14
antibody.
5. The method of claim 4 wherein said anti-Fn14 antibody is a
monoclonal antibody.
6. The method of claim 3 wherein said Fn14 agonist is administered
in the presence of a therapeutically effective amount of at least
one endothelial cell modulator.
7. A method of inhibiting tumor progression comprising the step of
administering to a subject a therapeutically effective amount of an
agonist to a TWEAK receptor.
8. The method of claim 7 wherein said TWEAK receptor is Fn14.
9. The method of claim 7 wherein said TWEAK receptor agonist is an
Fn14 agonist.
10. The method of claim 9 wherein said Fn14 agonist is an anti-Fn14
antibody.
11. The method of claim 10 wherein said anti-Fn14 antibody is a
monoclonal antibody.
12. The method of claim 9 wherein said Fn14 agonist is administered
in the presence of a therapeutically effective amount of at least
one endothelial cell modulator.
13. A method of inhibiting tumor progression comprising the step of
administering a therapeutically effective amount of a Fn14-Fc
fusion protein.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 10/380,611, filed Mar. 14, 2003, which is a
National Stage entry of PCT/US01/28451, filed Sep. 12, 2001, which
claims priority to U.S. Application Ser. No. 60/232,355, filed Sep.
14, 2000. The contents of each of the foregoing applications are
incorporated in their entireties by reference herein.
BACKGROUND
[0002] Angiogenesis is an integral component of tissue remodeling
during a variety of normal and pathological events, such as the
female reproductive cycle, fetal development, wound healing,
inflammation and tumor progression (Han, Z. C., and Y. Liu, Int. J.
Hematol, 70(2):68 (1999); Folkman, J., Nat. Med., 1(1):27 (1995)).
The growth of microvessels involves the coordinated migration,
proliferation, differentiation and morphogenetic organization of
endothelial cells ("EC") into new capillary structures. Further
stabilization and maturation of neovessels occurs through the
recruitment of adjacent mesenchymal cells to the vessel walls
(Bussolino, F. A., Trands Biochem Sci., 22(7):251 (1997)). The
maintenance of developing and quiescent vessels critically depends
on the availability of appropriate survival signals. These
angiogenic events are orchestrated by a network of extracellular
factors, including several classes of cytokines, extracellular
matrix and integrins, and by their cognate receptors.
[0003] A number of known angiogenic regulators belong to the Tumor
Necrosis Factor (TNF) family. Ligands of this family are expressed
as type II membrane proteins which may be proteolytically cleaved
to produce soluble cytokines (Smith, C. A. et al., Cell, 76:959
(1994)). These ligands trigger biological activities by binding and
signalling through their corresponding receptors in the TNF
receptor family. The majority of the TNF family members mediate
host defense, inflammation and immunological regulation (Vassalli,
P., Annu Rev Immunol, 10:411 (1992); De Togni, P. J. et al.,
Science, 264(5159):703 (1994); Nagata, S., and Goldstein, P.,
Science, 267:1449 (1995); Foy, T. M. et al., Annu Rev Immunol,
14:591 (1996); Mackay, F. et al., J Exp Med, 190(11)1697 (1999)).
In addition, some of these ligands, including TNF-.alpha., Fas
ligand (FasL), Vascular Endothelian Growth Inhibitor (VEGI) or TL1,
and TWEAK have been shown to regulate EC functions
(Frater-Schroder, M. W. et al., Proc. Natl. Acad. Sci. USA, 84:5277
(1987); Yoshida, S. et al., Mol Cell Biol, 17(7):4015 (1997);
Ruegg, C. et al., Nat Med, 4(4):408 (1998); Fajardo, L. F. et al.,
Am J Pathol, 140(3):539 (1992); Leibovich, S. J., Nature,
329(6140):630 (1987); Biancone, L., J Exp Med, 186(1):147 (1997);
Yue, T. et al., J. Biol. Chem., 274:1479 (1999); Zhai, Y., et al.,
Faseb J, 13(1):181 (1999)). The effects of TNF-.alpha. on EC
behavior are complex. TNF-.alpha. inhibits EC growth yet induces
capillary tube formation in vitro (Frater-Schroder, M. et al.,
Proc. Natl. Acad. Sci. USA, 84:5277 (1987); Yoshida, S. et al., Mol
Cell Biol, 17(7):4015 (1997)). It also can be antiangiogenic in the
context of solid tumors (Ruegg, C. A., Nat Med, 4(4):408 (1998)) or
angiogenic in corneal settings in vivo (Frater-Schroder, M. et al.,
Proc. Natl. Acad. Sci. USA, 84:5277 (1987); Yoshida, S. et al., Mol
Cell Biol, 17(7):4015 (1997); Fajardo, L. F. et al., Am J Pathol,
140(3):539 (1992); Leibovich, S. J. et al., Nature, 329(6140):630
(1987)). Fas/FasL interaction can induce endothelial capillary tube
formation in vivo, probably by triggering the production of
heparin-binding growth factors (Biancone, L. et al., J Exp Med,
186(1):147 (1997)), while VEGI inhibits EC survival and
proliferation (That, Y. et al., Faseb J, 13(1):181 (1999); Yue, T.
et al., J. Biol. Chem., 274:1479 (1999)). TWEAK, a novel member of
the TNF ligand family (Chicheportiche, Y. et al., J Biol Chem,
272(51):32401 (1997)), induces the expression of the angiogenic
chemokine Interleukin-8 (IL-8) in some epithelial tumor cell lines.
Recently, TWEAK was reported to induce both proliferation of
cultured human ECs under reduced growth factor conditions and
corneal neovascularization (Lynch, C. N. et al., J. Biol. Chem.,
271(13):8455 (1999)).
[0004] Through the use of expression cloning, a receptor protein
for TWEAK has recently been identified as "Fn14." This protein was
originally characterized as an FGF-1 induced immediate early
response gene (J. Biol. Chem. 274:33166-33176 (1999) and WO
98/55508). Fn14 has a more restricted expression pattern in adult
tissues (as compared to TWEAK) and is the smallest TNF receptor
described to date, with only one cysteine-rich repeat.
SUMMARY OF THE INVENTION
[0005] In addition to the roles played by TWEAK and Fn14 in
inducing angiogenesis (see WO 01/45730), the present inventors have
discovered that Fn14 agonists or activating agents (e.g., TWEAK and
agonist anti-Fn14 monoclonal antibodies) actually (1) inhibit
angiogenesis and (2) slow tumor progression, either indirectly
through inhibition of angiogenesis or by direct anti-tumor
activity.
[0006] Accordingly, the present invention provides methods of
modulating angiogenesis by administering a
therapeutically-effective amount of an agonist to a TWEAK receptor.
In preferred embodiments, the TWEAK receptor is Fn14. In other
preferred embodiments, the Fn14 receptor agonist is TWEAK or an
anti-Fn14 monoclonal antibody. The present invention preferably
provides methods of inhibiting angiogenesis by administering a
therapeutically effective amount of an agonist to a TWEAK
receptor.
[0007] The invention also includes methods of inhibiting tumor
progression by administering a therapeutically-effective amount of
an agonist to a TWEAK receptor. In preferred embodiments, the TWEAK
receptor is Fn14. In other preferred embodiments, the Fn14 agonist
is TWEAK or an anti-Fn14 monoclonal antibody.
[0008] The invention also contemplates pharmaceutical compositions
comprising a therapeutically-effective amount of an agonist to a
TWEAK receptor and a pharmaceutically-acceptable carrier.
[0009] To the extent that methods of the present invention
contemplate modulating angiogenesis, they are useful as a treatment
in diseases where enhanced angiogenic activity is desirable to
promote neovascularization. Such diseases and conditions include:
myocardial ischemic conditions (e.g., myocardinal infarction,
improve blood flow in patents with coronary artery disease
suffering from myocardial ischemia or inadequate blood flow to
areas other than the heart such as in peripheral vascular disease,
where decreased blood flow is a problem, revascularization of
necrotic tissue, for example of the myocardium after an infarction
or an angioplasty, angina, heart transplants, vascular grafts, and
reopening vessels to improve vascularization, perfusion,
collagenization and organization of said lesions), wound healing,
and tissue and organ transplantations (e.g., enhancement of
autologous or heterologous microvascular transplantation).
Promotion of wound healing includes healing of incisions, bone
repair, burn healing, post-infarction repair in myocardial injury,
healing of gastric ulcers and other ulcers of the gastrointestinal
tract and generally in promoting the formation, maintenance and
repair of tissue. Neovascularization of grafted or transplated
tissue is also contemplated, especially in subjects suffering from
vascular insufficiency, such as diabetic patients.
[0010] To the extent that the methods of the present invention
contemplate inhibiting angiogenesis, they are useful as treatment
in diseases where diminished angiogenic activity is desirable to
inhibit neovascularization. Such conditions include cancer,
inflammatory macular degeneration, and diabetic retinopathy.
[0011] To the extent that the methods of the present invention
contemplate inhibiting tumor progression, they are useful to treat
conditions associated with undesired cell proliferation, such as
cancers, including but not limited to, prostate cancers, lymphomas,
adenocarcinomas, neuronal neoplasms, colon cancers, pancreatic
tumors, paraganglioma, breast cancer, renal cell cancers, lung
cancers, ovarian cancers, leiomyomas, lung cancers, memingiomas,
pheochromacytomas, osteosarcomas and thyroid cancers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A and FIG. 1B depict two plots in which Mean
Fluorescence Intensity (MFI) vs. TWEAK concentration is shown
(left), with the dotted line indicating the background MFI with the
indirect detection step alone. Histograms (right) correspond to (1)
background fluorescence with indirect detection step alone, (2)
TWEAK binding, and (3) inhibition of TWEAK binding by the AB.D3
mAb.
[0013] FIG. 2 depicts two bar graphs showing percentage of (A)
HUVEC and (B) HDMEC cells that were viable apoptotic or dead after
treatment with TWEAK. EC were cultured for 48 hours in CM (CS-C
Complete Media containing 10% FBS and growth supplements), 0% FBS
(CS-C media without FBS and growth supplements and supplemented
with 0.1% BSA and 10 .mu.g/ml heparin), and 0% FBS with TWEAK or
VEGF. Anti-TWEAK mAb AB.G11 and control Ig were added as specified.
Cells were stained with FITC-Annexin-V and PI and the percentage of
viable, apoptotic and dead cells indicated. Each of these results
is representative of two independent experiments.
[0014] FIG. 3 depicts two bar graphs showing bFGF-dependent
proliferation. HUVEC were cultured for 3 days in complete media or
in basal media. TWEAK (100 ng/ml), bFGF (1/500 dilution) VEGF (10
ng/ml) or combinations of these factors were added to basal media
as indicated and proliferation measured by .sup.3H-thymidine
incorporation. (A) Data shown are the mean value+/-SD of triplicate
wells. These results are representative of 4 independent
experiments. In addition to growth factors, blocking anti-TWEAK mAb
AB.D3, nonblocking anti-TWEAK mAb BE.B3, and an irrelevant hamster
control Ig Ha4/8 (10 ug/ml) were added where indicated, with
results representative of two independent experiments. (B) Results
shown are the mean value+/-SD of triplicate wells and are
representative of 4 independent experiments.
[0015] FIG. 4 depicts a bar graph showing the effect of TWEAK on
bFGF-dependent HUVEC migration. Confluent HUVEC monolayers treated
with TWEAK, bFGF, VEGF, and combinations of these factors were
wounded and repair measured after 18 hours of culture. Results
shown are the average of 4 experiments+/-SEM.
[0016] FIG. 5(A) depicts phase contrast images of HUVECs on the
surface of fibrin gel matrices after 3 days of culture, untreated
or treated with bFGF, TWEAK or bFGF+TWEAK. All images are 4.times.
magnification. These results are representative of 8 independent
experiments. FIG. 5(B) depicts hematoxylin and eosin-stained
cross-sections of the fibrin gel cultures. EC cells in untreated
and TWEAK-treated cultures remain on the fibrin gel surface.
Invading EC cord-like structures are shown in bFGF-treated cultures
and structural organization of endothelial lumens in
TWEAK+bFGF-treated cultures. Similar results were obtained with
four independent EC types.
[0017] FIG. 6 depicts a bar graph showing the member of capillary
sprouts after treatment with TWEAK and bFGF. HUVECS were plated on
fibrin gel matrices and cultured for 48 hours without treatment, or
with factors and anti-TNF and anti-IL-8 mAbs as indicated.
Capillary sprouts were counted visually using five fields per well
and the total number per culture shown. These results are
representative of four independent experiments.
[0018] FIG. 7 depicts phase contrast images of HUVECs on the
surface of fibrin gel matrices after 3 days of culture, untreated
or treated with VEGF, TWEAK or TWEAK+VEGF. All images are 4.times.
magnification.
[0019] FIG. 8 is a gel electrophoresis image showing VEGF receptor
FLT1 mRNA levels are decreased in cultures treated with TWEAK+VEGF
as compared to cultures treated with VEGF alone.
[0020] FIG. 9A-1, FIG. 9A-2, FIG. 9B-1 and FIG. 9B-2 depict growth
of Lewis Lung Carcinoma in TWEAK-transgenic mice plotted as tumor
weight (mg) at days post implantation.
[0021] FIG. 10A. Amino acid sequence (SEQ ID NO:1) of Type I
transmembrane protein Fn14 [Homo Sapiens].
[0022] FIG. 10B. Amino acid sequence (SEQ ID NO:2) of Type I
transmembrane protein Fn14 [Mus Musculus].
DETAILED DESCRIPTION OF THE INVENTION
[0023] In order that the invention herein described may be fully
understood, the following detailed description is set forth.
[0024] The term "anti-tumor activity" refers to the ability of a
substance or composition to block the proliferation of, or to
induce the death of tumor cells which interact with that substance
or composition.
[0025] The term "inhibition of tumor progression" refers to the
ability of a substance or compound to block the proliferation of,
or to decrease growth and development of tumor cells which interact
with that substance or compound.
[0026] The term "apoptosis" refers to a process of programmed cell
death.
[0027] The term "cytotoxic activity" refers to the ability of a
substance or composition to induce the death of cells which
interact with that substance or composition.
[0028] The term "epitope" (or antigenic determinant) is defined as
the part of a molecule that combines with a single antigen binding
site on an antibody molecule. A single epitope is recognized by a
monoclonal antibody (mAb). Multiple epitopes are normally
recognized by polyclonal antibodies (Ab).
[0029] The term "angiogenic factor" refers to factors which promote
the angiogenic process, including but not limited to the following
phases of the process, ie, the degradation of the extracellular
matrix, cell proliferation, cell migration and structural
organization (Kumar et al., Int. J. Oncology 12:749-757 (1998);
Bussolino et al., Trends in Biochem, 22:251-256 (1997)). Angiogenic
factors include but are not limited to fibroblast growth factor
(bFGF), acidic FGF (aFGF), FGF-5, vascular endothelial growth
factor isoforms (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2
(Ang-2), Platelet-derived endothelial cell growth factor (PD-ECGF),
hepatocyte growth factor, proliferin, B61, soluble vascular cell
adhesion molecular-1, soluble E-selection,
12-hydrozyeicosatetraenoic acid, Tat protein of HIV-1, angiogenin,
TNF .alpha., FasL, Transforming growth factor-.beta..
[0030] The "Fc domain" of an antibody refers to a part of the
molecule comprising the CH2, CH3 and hinge regions but lacking the
antigen binding sites.
[0031] The term "Fn14" refers to the TWEAK receptor protein as
characterized in J. Biol. Chem. 274:33166-33176 (1999), the entire
disclosure of which is incorporated herein by reference. The human
and mouse amino acid sequences for this type I transmembrane
protein are provided in FIGS. 10A (SEQ ID NO:1) and 10B (SEQ ID
NO:2).
[0032] The terms "Fn14 agonist" or "Fn14 activating agent" refers
to any agent which can augment ligand binding to Fn14, Fn14
signaling, or which can influence how the Fn14 signal is
interpreted within the cell. Examples of Fn14 agonists include
TWEAK, soluble anti-Fn14 Abs, cross-linked anti-Fn14 Abs and
multivalent anti-Fn14 Abs.
[0033] The term "Fn14 signaling" refers to all molecular reactions
associated with the Fn14 pathway and subsequent molecular reactions
which result therefrom.
[0034] The term "anti-Fn14 antibody" ("anti-Fn14 Ab") refers to any
antibody that recognizes and binds to at least one epitope of the
Fn14 receptor.
[0035] The term "anti-Fn14 monoclonal antibody" ("anti-Fn14 mAb")
refers to any monoclonal antibody that recognizes and binds to a
single epitope of the Fn14.
[0036] The term "cross-linked anti-Fn14 (m)Abs" refer to antibodies
directed against the Fn14 which have either been cross-linked to
each other to form antibody agglomerates in solution using an
anti-Fn14 antibody (Ab) or (mAb) cross-linking agent, or which have
been immobilized in close proximity to one another on a surface or
matrix.
[0037] The term "anti-Fn14 Ab (or mAb) cross-linking agent" refers
to any agent which can covalently or non-covalently aggregate
anti-Fn14 Abs in solution so that the Abs can bind to and
potentiate target cell surface Fn14 clustering. Such cross-linking
agents include but are not limited to chemical cross-linking
agents, secondary antibodies which react with portions of the
anti-Fn14 Abs or mAbs, and soluble or surface-bound Fc
receptors--either endogenous or added exogenously--which can bind
to anti-Fn14 Abs.
[0038] The term "multivalent ligand" refers to a molecule or
complex which has more than one receptor binding site and which is
capable of simultaneously binding and bringing into close proximity
at least two receptor molecules.
[0039] A "type I leader sequence" is an amino-terminal portion of a
eukaryotic protein which serves as a signal to direct the protein
to the endoplasmic reticular (ER) membrane and often through the
entire secretion pathway. The leader sequence is usually cleaved
off by a signal peptidase in the ER membrane.
[0040] A "signal sequence" is the functional equivalent of a
eukaryotic type I leader sequence in prokaryotic hosts, and directs
the translocation of proteins into or across lipid bilayer
membranes of a bacterium.
Source of Anti-Fn14 Antibodies
[0041] Anti-protein (anti-peptide antisera or monoclonal antibodies
can be made by standard protocols (see, for example, Antibodies: A
Laboratory Manual, ed. by Harlow and Lane, Cold Spring Harbor
press: 1988).
[0042] Polyclonal antibody sera directed against the human Fn14 are
prepared using conventional techniques by injecting animals such as
goats, rabbits or mice subcutaneously with a human Fn14 Fc fusion
protein in complete Freund's adjuvant, followed by booster
intraperitoneal or subcutaneous injection in complete Freunds.
Polyclonal antisera containing the desired antibodies which are
directed against Fn14 are screened by conventional procedures.
[0043] Mouse monoclonal antibodies (mAbs) directed against a human
Fn14 Fc fusion protein are prepared by intraperitoneal immunization
of RBF mice repetitively with a CHO cell-derived recombinant Fn14
Fc fusion protein (Fn14 Fc) attached to protein A sepharose beads
in the absence of adjuvant. Animals are finally boosted with
soluble Fn14 Fc (both i.p. and i.v.), spleen cells are fused using
classical protocols, and hybridomas are screened by ELISA (Ling et
al., J. Interferon and Cytokine Res., 15, pp. 53-59 (1995)). Pure
mAbs are prepared by protein A sepharose purification of IgG from
hybridoma culture supernatants.
[0044] Various forms of anti-Fn14 antibodies can also be made using
standard recombinant DNA techniques (Winter and Milstein, Nature,
349, pp. 293-99 (1991)). For example, "chimeric" antibodies can be
constructed in which the antigen binding domain from an animal
antibody is linked to a human constant domain (e.g. Cabilly et al.,
U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.
U.S.A., 81, pp. 6851-55 (1984)). Chimeric antibodies reduce the
observed immunogenic responses elicited by animal antibodies when
used in human clinical treatments.
[0045] In addition, recombinant "humanized antibodies" which
recognize Fn14 can be synthesized. Humanized antibodies are
chimeras comprising mostly human IgG sequences into which the
regions responsible for specific antigen-binding have been inserted
(e.g. WO 94/04679). Animals are immunized with the desired antigen,
the corresponding antibodies are isolated, and the portion of the
variable region sequences responsible for specific antigen binding
are removed. The animal-derived antigen binding regions are then
cloned into the appropriate position of human antibody genes in
which the antigen binding regions have been deleted. Humanized
antibodies minimize the use of heterologous (inter-species)
sequences in human antibodies, and are less likely to elicit immune
responses in the treated subject. Also, primatized and/or fully
human mAbs can be generated using techniques well-known in the art,
and can be utilized in the present invention.
[0046] Construction of different classes of recombinant anti-Fn14
antibodies can also be accomplished by making chimeric or humanized
antibodies comprising the anti-Fn14 variable domains and human
constant domains (CH1, CH2, CH3) isolated from different classes of
immunoglobulins. For example, anti-Fn14 IgM antibodies with
increased antigen binding site valencies can be recombinantly
produced by cloning the antigen binding site into vectors carrying
the human .mu. chain constant regions (Arulanandam et al., J. Exp.
Med., 177, pp. 1439-50 (1993); Lane et al., Eur. J. Immunol., 22,
pp. 2573-78 (1993); Traunecker et al., Nature, 339, pp. 68-70
(1989)).
[0047] In addition, standard recombinant DNA techniques can be used
to alter the binding affinities of recombinant antibodies with
their antigens by altering amino acid residues in the vicinity of
the antigen binding sites. The antigen binding affinity of a
humanized antibody can be increased by mutagenesis based on
molecular modeling (Queen et al., Proc. Natl. Acad. Sci. U.S.A.,
86, pp. 10029-33 (1989); WO 94/04679).
[0048] It may be desirable to increase or to decrease the affinity
of anti-Fn14 Abs for Fn14 depending on the targeted tissue type or
the particular treatment schedule envisioned. For example, it may
be advantageous to treat a patient with constant levels of
anti-Fn14 Abs with reduced ability to signal through the Fn14
pathway for semi-prophylactic treatments. Likewise, anti-Fn14 Abs
with increased affinity for the Fn14 may be advantageous for
short-term, tumor-targeted treatments.
Multiple Anti-Fn14 Abs in Solution Act as Fn14 Agonists
[0049] Compositions comprising multiple anti-Fn14 Abs in solution
which act as Fn14 agonists are provided in this invention.
Polyclonal anti-Fn14 Abs directed against different epitopes of the
Fn14 can be used. Preferably, the anti-Fn14 Abs are monoclonal Abs
directed against different and non-overlapping epitopes of the
Fn14.
[0050] The combined anti-Fn14 mAb approach to Fn14 activation
requires combining two non-overlapping epitopes. Additional
epitopes (as defined by new mAbs) may be identified by continuing
to fuse immunized mouse spleen cells, by immunizing different
species of animals, and by using different routes of
immunization.
[0051] Epitopes can also be directly mapped by assessing the
ability of different mAbs to compete with each other for binding to
the Fn14 using BIAcore chromatographic techniques (Pharmacia
BIAtechnology Handbook, "Epitope Mapping", Section 6.3.2, (May
1994); see also Johne et al., J. Immunol. Methods, 160, pp. 20
191-8 (1993)).
Anti-Fn14 IgM Monoclonal Antibodies Function as Fn14 Agonist
[0052] Anti-Fn14 mAbs which comprise more than the usual two IgG
antigen binding sites will also function in solution as cell
surface Fn14 cross-linking agents, and will accordingly fall within
the definition of a Fn14 agonist according to this invention. The
antigen binding sites of an anti-Fn14 mAb can be built into IgM
molecules--which have ten antigen binding sites--using standard
recombinant DNA and hybridoma techniques.
[0053] Alternatively, one may collect and enrich for complete mouse
(or other animal) IgM molecules isolated by hybridoma fusion
techniques after a single immunization with antigen. One way to
enrich for IgM molecules would be to immunize CD4O
signaling-deficient mice (Kawabe et al., Immunity, 1, pp. 167-78
(1994); Xu et al., Immunity, 1, pp. 423-31 (1994)). These mice
cannot effectively produce IgGs and therefore their response to
challenge by antigen is enriched for IgM isotypes.
[0054] Anti-Fn14 IgM antibodies, by virtue of their increased
valency, can effectively aggregate Fn14 molecules within the plane
of the membrane, thereby enhancing Fn14 signaling as compared to
their IgG counterparts having two antigen binding sites. A dramatic
example of the increased efficiency of multivalent antibodies in
receptor clustering is seen with antibodies to the Fas receptor,
where the IgM form is very potent and normal bivalent IgGs are not
effective in solution (Yonihara and Yonihara, J. Exp. Med., 169,
pp. 1747-56 (1989); Alderson et al., Int. Immunol., 6, pp.
1799-1806 (1994)).
[0055] Likewise, the apo-1 mAb to the Fas receptor is an IgG3 mAb.
This mAb is a potent cytotoxic agent which relies on Fc
interactions unique to IgG3 subtypes to aggregate into larger
polyvalent forms. Removal of the Fc region creates a F(ab).sub.2
form that cannot associate into larger aggregates and which is
inactive (Dhein et al., J. Immunol., 149, pp. 3166-73 (1992)). Thus
by analogy, it is predicted that IgM versions of anti-Fn14 mAbs
will be potent anti-tumor agents.
Treatments Using Fn14 Agonist
[0056] The compositions of this invention will be administered at
an effective dose to treat the particular clinical condition
addressed. Determination of a preferred pharmaceutical formulation
and a therapeutically efficient dose regiment for a given
application is well within the skill of the art taking into
consideration, for example, the condition and weight of the
patient, the extent of desired treatment and the tolerance of the
patient for the treatment.
[0057] The therapeutic agents of the invention may be administered
by any route of administration which is compatible with the
selected agent, and may be formulated with any pharmaceutically
acceptable carrier appropriate to the route of administration.
Preferred routes of administration are parenteral and, in
particular, intravenous, intraperitoneal, and intracapsular.
[0058] Treatments are also preferably conducted over an extended
period on an outpatient basis. Daily dosages of the therapeutic
agents are expected to be in the range of about 0.01-1000 .mu.g/kg
body weight, and more preferably about 10-300 .mu.g/kg body weight,
although precise dosages will vary depending upon the particular
therapeutic agent employed and the particular subject's medical
condition and history.
[0059] Administration of the anti-Fn14 Abs of this invention,
including isolated and purified forms of the antibodies or
complexes, their salts or pharmaceutically acceptable derivatives
thereof, may be accomplished using any of the conventionally
accepted modes of administration of agents which exhibit anti-tumor
activity.
[0060] The pharmaceutical compositions used in these therapies may
also be in a variety of forms. These include, for example, solid,
semi-solid and liquid dosage forms such as tablets, pills, powders,
liquid solutions or suspensions, suppositories, and injectable and
infusible solutions. The preferred form depends on the intended
mode of administration and therapeutic application. Modes of
administration may include oral, parenteral, subcutaneous,
intravenous, intralesional or topical administration.
[0061] The anti-Fn14 Abs may, for example, be placed into sterile,
isotonic formulations with or without cofactors which stimulate
uptake or stability. The formulation is preferably liquid, or may
be lyophilized powder. For example, the anti-Fn14 Abs may be
diluted with a formulation beffer comprising 5.0 mg/ml citric acid
monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/ml mannitol, 1
mg/ml glycine and 1 mg/ml polysorbate 20. This solution can by
lyophilized, stored under refrigeration and reconstituted prior to
administration with sterile Water-For-Injection (USP).
[0062] The compositions also will preferably include conventional
pharmaceutically acceptable carriers well known in the art (see for
example Remington's Pharmaceutical Sciences, 16.sup.th Edition,
1980, Mac Publishing Company). Such pharmaceutically acceptable
carriers may include other medicinal agents, carriers, genetic
carriers, adjuvants, excipients, etc., such as human serum albumin
or plasma preparations. The compositions are preferably in the form
of a unit dose and will usually be administered one or more times a
day.
[0063] The pharmaceutical compositions of this invention may also
be administered using microspheres, liposomes, other
microparticulate delivery systems or sustained release formulations
placed in, near, or otherwise in communication with affected
tissues or the bloodstream. Suitable examples of sustained release
carriers include semipermeably polymer matrices in the form of
shaped articles such as suppositories or microcapsules. Implantable
or microcapsular sustained release matrices include polylactides
(U.S. Pat. No. 3,773,319; EP 58,481), copolymers of L-glutamic acid
and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22, pp.
547-56 (1985)); poly(2-hydroxyethyl-methacrylate) or ethylene vinyl
acetate (Langer et al., J. Biomed. Mater. Res., 15, pp. 167-277
(1981); Langer, Chem. Tech., 12, pp. 98-105 (1982)).
[0064] Liposomes containing anti-Fn14 Abs can be prepared by
well-known methods (See, e.g. DE 3,218,121; Epstein et al., Proc.
Natl. Acad. Sci. U.S.A., 82, pp. 3688-92 (1985); Hwang et al.,
Proc. Natl. Acad. Sci. U.S.A., 77, pp. 4030-34 (1980); U.S. Pat.
Nos. 4,485,045 and 4,544,545). Ordinarily the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid
content is greater than about 30 mol. % cholesterol. The proportion
of cholesterol is selected to control the optimal rate of anti-Fn14
Abs release.
[0065] The anti-Fn14 Abs of this invention may also be attached to
liposomes containing other Fn14 activating agents, chemotherapeutic
agents to supplement the IFN-.gamma. typically found in the region
of tumors. Attachment of TWEAK complexes and anti-Fn14 Abs to
liposomes may be accomplished by any known cross-linking agent such
as heterobifunctional cross-linking agents that have been widely
used to couple toxins or chemotherapeutic agents to antibodies for
targeted delivery. Conjugation to liposomes can also be
accomplished using the carbohydrate-directed cross-linking reagent
4-(4-maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes et
al., J. Cell. Biochem. Abst. Suppl. 16E 77 (1992)).
[0066] It is envisioned that the mAbs would be administered to
tumor-bearing people in conjunction with a conventional anti-tumor
therapy (i.e. radiation and chemotherapy). A combined treatment of
Fn14 activation with conventional chemotherapies may provide an
extra factor of tumor killing activity that would be more likely to
clear a patient of tumorigenic cells than when conventional
anti-tumor therapy is used alone.
[0067] It is further possible that this approach may have
relatively few side effects and therefore could be given in a
semi-prophylactic sense in cases of carcinomas that may not have
metastasized, or in patients from families who show a genetic
pre-disposition for a certain type of cancer.
[0068] The following are examples which illustrate the methods of
this invention. These examples should not be construed as limiting:
the examples are included for purposes of illustration and the
present invention is limited only by the claims.
EXAMPLES
Example 1
TWEAK Modulates Endothelial Cell Responses to Basic Fibroblast
Growth Factor and Vascular Endothelial Growth Factor
[0069] Cells and Culture Conditions--Human Umbilical Vein
Endothelial Cells (HUVEC) and Human Dermal Microvascular
Endothelial Cells (HDMEC) were obtained from Cell System
Corporation (CS-C) (Kirkland, Wash.) or Clonetics (San Diego,
Calif.). Aortic EC, aortic smooth muscle cells, lung fibroblasts,
embryonic myoblasts and human peripheral blood dendritic cells were
purchased from Clonetics. HUVEC and HDMEC were routinely passaged
in CS-C Complete Medium which contains 10% fetal bovine serum (FBS)
and supplier growth supplement and were used in experiments until
passage seven. Survival studies were performed in CS-C Complete
Media and CS-C Media (without FBS and without growth supplement) as
specified. For the proliferation, migration and immunofluorescent
staining experiments, EC Basal Medium (EBM) containing 2% FBS and
supplier growth supplements, defined herein as "complete media" and
EBM containing 2% FBS, defined herein as "basal media", were used
(Clonetics). For the capillary tube formation assay, EC Basal
Medium 2 (EBM-2) with 5% FBS and supplier growth supplements were
used (Clonetics). The MS-1 and M210B4 cell lines were purchased
from American Type Culture Collection (Manassas, Va.). Mouse
lymphoid cells were obtained 24 to 48 hours after in vivo
activation with 200 .mu.g/ml of anti-CD3 monoclonal antibody (mAb)
(clone 2C11) (Pharmingen, San Diego, Calif.).
Thioglycollate-induced peritoneal macrophages were stimulated in
vitro for 48 hours with interferon gamma (IFN.gamma.) (100
Units/ml), TNF-.alpha. (10 ug/ml) or Lipopolysaccharide (LPS) (1
ug/ml). Reagents and Antibodies--Recombinant human bFGF was
obtained as a growth supplier supplement (Clonetics), and bFGF and
VEGF also were purchased from R&D Systems (Minneapolis, Minn.)
and Sigma (St. Louis, Mo.). Annexin V-FITC and propidium iodide
(PI) were from Pharmingen. Mouse anti-human TNF-.alpha. and mouse
anti-human IL-8 mAbs from R&D (Minneapolis, Minn.) and
isotype-matched control Ig MOPC21 (ICN Biomedicals Inc., Irvine,
Calif.) were used for blocking studies. Biotin-conjugated anti-FLAG
from Eastman Kodak Company (New Haven, Conn.), and Phycoerythrin
(PE)-Streptavidin from Southern Biotechnology Associates, Inc.
(Birmingham, Ala.). TWEAK-specific mAbs--BE.B3, AB.G11 and AB.D3
were generated in Armenian hamsters by immunizing with soluble
human TWEAK protein and standard hybridoma generation procedures.
The ability of AB.D3 and AB.G11 to bind to human and murine TWEAK
and BE.B3 to bind to human TWEAK was demonstrated in an ELISA assay
using recombinant soluble TWEAK proteins immobilized on 96 well
microtiter plates. The blocking activity of AB.D3 and AB.G11 were
demonstrated by these mAbs but not BE.B3 to inhibit soluble
flag-tagged human TWEAK binding to HT29 cells in a FACS analysis.
BE.B3 was biotinylated using ImmunoPure Biotinylation kits
following the manufacturer's protocol (Pierce, Rockford, Ill.). A
hamster control Ig (clone Ha4/8-3.1) was obtained from the American
Type Culture Collection and mAb was purified from culture
supernatant by Protein A Fast Flow column (Pharmacia, Piscataway,
N.J.). Recombinant Soluble Human TWEAK protein--Recombinant soluble
human TWEAK (GenBank Accession #AF030099) containing amino acid
residues A106-H249 was expressed either with or without an
N-terminal flag epitope in the yeast Pichia pastoris (Invitrogen,
location). The fermentation medium from pichia expressing soluble
human TWEAK was concentrated and dia-filtrated in 20 mM Tris-HCl,
pH 8.0 and ion-exchanged on a Q Sepharose column prior to loading
onto a Zn-chelating column. The soluble TWEAK was eluted by an
imidazole gradient in 20 mM sodium phosphate, 0.5 M sodium
chloride, pH 7.5 prior to final fractionation by a size-excluded
using a Sephacryl 300 column. Analysis of Apoptosis--For serum
starvation experiments, HUVEC or HDMEC were seeded in 6-well plates
at a density of 1.2.times.10.sup.5 cells per well and were
incubated overnight in CS-C Complete Medium. Immediately before
each experiment, cells were washed with phosphate-buffered saline
(PBS) and cultured for 48 hours in CS-C Complete Media, or CS-C
Media supplemented with 0.1% bovine serum albumin (BSA) and heparin
(10 ug/ml), with or without VEGF (10 ng/ml) or TWEAK (200 ng/ml).
Where indicated, 2 ug/ml of anti-TWEAK mAb AB.G11 or control Ig
also were added. After 48 hours, cells were washed with PBS and
detached by incubation with dispase (CS-C) for 15 minutes at
37.degree. C. followed by replacement with PBS containing 5 mM EDTA
and 0.1% BSA for 15 minutes at 37.degree. C. After an additional
wash in PBS, cells were stained with FITC-Annexin-V and 5 ug/ml PI
according to the supplier. Fluorescence was analyzed within the
hour using FACStar.sup.PLUS (Becton Dickinson, San Jose, Calif.).
Proliferation Assays--HUVEC were plated in 96-well microtiter
plates at subconfluence (4000 cells per well) and cultured
overnight in CS-C Medium without addition of supplier growth
supplements. Media was replaced with complete media or with basal
media as defined above. Cells were cultured in basal media with or
without TWEAK (100 ng/ml), bFGF using a 1/500-1/1000 dilution of
the bFGF supplement (Clonetics) or 1 ng/ml (R&D Systems), VEGF
(10 ng/ml) or combinations of these factors. Where indicated, 10
ug/ml anti-TWEAK mAbs AB.D3, BE.B3 or hamster control Ig Ha4/8 also
were added. Cells were incubated at 37.degree. C. with 5% CO.sub.2
for three days and proliferation was measured by pulsing with
.sup.3H-Thymidine for the last 10 hours of culture. Cell-bound
radioactivity was measured with a Betaplate.TM. (EG&G Wallac,
Gaithersburg, Md.). Endothelial Wound Repair Assay--A standard
wound repair assay was employed as previously described (Morales,
D. E. et al., Circulation, 91(3):755 (1995)). In brief, a confluent
monolayer of HUVEC was grown in CS-C Complete Medium in 35.times.10
mm cell culture dishes with 2 mm grids (Nalge Nunc International,
Naperville, Ill.). The monolayer was wounded by two perpendicular
strokes across the diameter of the dish with a 1 mm tip. Dislodged
cells were aspirated and plates were rinsed with PBS. Cells were
cultured for 18 hours in complete media or in basal media as
defined above with or without TWEAK (200 ng/ml), bFGF (1/1000 or 1
ng/ml), VEGF (10 ng/ml) or combinations of these before fixing with
1% paraformaldehyde followed by staining with Harris Hematoxylin
(Sigma, St. Louis, Mo.). Wound repair was quantified by visually
counting the number of grids in which the gap was obscured by
migrating cells. This number was divided by the total number of
grids that aligned the wound and results were expressed as mean
percentage wound repair+/-SEM. Immunofluorescent staining--Cells
were analyzed for TWEAK binding by incubation with flag-TWEAK and
binding was detected with either biotinylated mouse anti-flag mAb
or biotinylated BE.B3 and streptavidin-PE. Cold competition was
performed with flag-TWEAK at 100 ng/ml and increasing
concentrations of nontagged TWEAK, with binding detected with
biotinylated mouse anti-flag mAb. Blocking of TWEAK binding by the
AB.D3 Ab was performed by preincubation of flag-TWEAK with 10
.mu.g/ml of mAb. Capillary tube formation assay--Capillary tube
formation by ECs was analyzed using a fibrin matrix gel assay based
on a method previously described (Mach et al., Am J Pathol 154,
(1):229 (1999)). Briefly, 4 mg/ml plasminogen free human fibrinogen
(Calbiochem, San Diego, Calif.) was dissolved in serum free EBM-2
media with heparin and polymixin B both at 1 ug/ml (Sigma) as well
as all of the supplier supplements except for VEGF and bFGF. The
fibrin solution was filtered-sterilized and fibrin matrices were
prepared by adding thrombin (20-50 milliunits/ml) (Sigma) and
distributing 300 ul per well in 24-well plates. HUVEC
(4.times.10.sup.4 cells/cm.sup.2) were seeded onto the gel surfaces
and overlayered with EBM-2 media as above and 5% FBS in the
presence or absence of TWEAK, bFGF, VEGF or combinations of these
factors. TWEAK was used at 1 ug/ml or 100 ng/ml with similar
results obtained, bFGF at 100 ng/ml, and VEGF at 50 ng/ml. In some
experiments, neutralizing mAbs specific for TNF (1 ug/ml) and IL-8
(10 ug/ml) or isotype control Ig were used. After 48-72 hours of
culture, phase-contrast photomicrographs of the gel surface were
taken. Gels were fixed with 10% ethanol for 10 minutes, transferred
from the original wells to new wells, fixed with 4%
paraformaldehyde, embedded in paraffin, cross sectioned (5 microns)
and stained with Hematoxylin and eosin. RNAse Protection Assay
(RPA)--RPA was performed using the hAngio multiprobe Template Set
(Pharmingen). HUVEC were cultured as adherant monolayers in EBM-2
media with 5% FBS and supplier supplements except for bFGF and
VEGF. TWEAK, VEGF or VEGF+TWEAK were added and RNA was isolated
after 16 hours of culture.
Results:
[0070] TWEAK binding is cell type-restricted--In order to determine
which cell types might be targets for TWEAK activity, we surveyed
various primary cells for their ability to bind to recombinant
soluble human TWEAK by immunofluorescent staining. As shown in
Table I, TWEAK bound to human venous and aortic ECs, aortic smooth
muscle cells, embryonic myoblasts and to a lower degree to lung
fibroblasts. Flag-TWEAK binding was dose-dependent and specific as
evidenced by blocking with the AB.D3 mAb and by cold competition
with an independent TWEAK preparation (FIG. 1 and data not shown).
Similarly, TWEAK bound to murine EC and fibroblast cell lines.
However, TWEAK did not bind detectably to any human leukocytes,
either freshly isolated or activated under a variety of conditions.
TWEAK promotes EC survival--The ability of TWEAK to promote EC
survival was tested by culturing HUVEC in serum-free media in the
absence or presence of TWEAK. HUVEC viability under these
conditions was compared with that in serum-free media supplemented
with VEGF (Nor, J. E. et al., Am J Pathol, 154(2):375 (1999)) or in
CS-C Complete Media. Cell viability was measured by standard double
staining with Annexin-V, an early marker for poptosis, and PI dye
exclusion. As shown in FIG. 2A, cells cultured in CS-C Complete
Media remained viable at 48 hours (97%), whereas in serum-free
cultures without additional factors the viable cells decreased to
25% and a significant fraction of apoptotic (48%) and dead cells
(26%) appeared. However, in the presence of TWEAK, HUVECs remained
largely protected from apoptosis (73% viable). The degree of EC
survival achieved with TWEAK was comparable to that with VEGF. The
survival of HDMEC in serum-free media also was enhanced by TWEAK
but to a lesser degree than HUVECs, and this activity was
specifically inhibited by anti-TWEAK mAb AB.G11 (FIG. 2B). TWEAK
cooperates with bFGF to increase EC proliferation--We further
examined the effect of TWEAK on proliferation by measuring
.sup.3H-thymidine incorporation of HUVEC cultured in basal media
with TWEAK, alone or in combination with two key angiogenic
factors. TWEAK induced a small but not significant increase in
HUVEC proliferation, on average a 1.6 fold increase relative to
basal medium (n=7 independent experiments). By contrast, the cells
cultured with TWEAK and bFGF displayed a significantly enhanced
proliferative response as compared to cells cultured in the
presence of bFGF alone (FIG. 3A). The level of .sup.3H-thymidine
incorporation achieved was comparable to or greater than that of
ECs cultured in complete media. Similar results were obtained using
bFGF at 1 ng/ml. The synergistic activity of TWEAK with bFGF was
completely inhibited by anti-TWEAK mAb AB.D3 indicating that the
effect of TWEAK was specific, whereas there was no inhibition by a
anti-TWEAK mAb BE.B3 or a control Ig. By contrast, TWEAK did not
affect the proliferative response to VEGF (FIG. 3B). TWEAK enhances
bFGF-dependent and inhibits VEGF-dependent EC migration--The
ability of TWEAK to effect EC migration also was evaluated in the
presence and absence of other angiogenic factors. Confluent HUVEC
monolayers were wounded and EC migration was monitored within the
first 18 hours by determining the degree of wound repair. Addition
of TWEAK or bFGF to the basal media induced a low level of wound
repair. By contrast, cultures treated with both TWEAK and bFGF were
repaired more efficiently than cultures kept in basal media or
stimulated with either agent alone (FIG. 4). By contrast, wound
repair was decreased in cultures treated with TWEAK+VEGF as
compared to those with VEGF alone. Thus TWEAK cooperates with bFGF
and antagonizes the effect of VEGF on wound repair. TWEAK
differentially regulates EC morphogenesis induced by bFGF and
VEGF--The growth of microvessels involves the coordinated
proliferation, migration and morphogenetic organization of ECs into
capillary tubes. The effect of TWEAK on morphogenic activity was
assessed using cultures of EC seeded onto the surface of fibrin
gels in the presence or absence of bFGF or VEGF. We found that bFGF
but not TWEAK induced morphological changes in the EC monolayer on
the fibrin gel surface as evidenced by phase-contrast microscopy
(FIG. 5A) and that the addition of TWEAK to bFGF significantly
enhanced these morphogenic changes, inducing a two-fold increase in
the number of capillary sprouts (FIG. 6). Furthermore, histological
analysis of cross-sections perpendicular to the matrix surface
revealed that bFGF but not TWEAK promoted EC invasion into the
fibrin matrix and that addition of TWEAK to bFGF induced the
formation of lumen-containing structures (FIG. 5B). Similar results
were obtained with several different EC types, including HUVECs,
HDMEC, Human Pulmonary Artery EC, and Human Lung Microvascular EC,
and no stimulation of lumen morphogenesis was observed when TWEAK
was substituted for by another TNF family member, CD40L (data not
shown). The cooperation between TWEAK and bFGF could be due to the
ability of TWEAK to induce IL-8 (Chicheportiche, Y. et al., J Biol
Chem, 272(51):32401 (1997)) and/or TNF (Schneider, P. R. et al.,
Eur J Immunol, 29(6):1785 (1999)), cytokines previously shown to
cooperatively promote EC morphogenesis in vitro (Yoshida, S. et
al., Mol Cell Biol, 17(7):4015 (1997); Koolwijk, P. et al., The J.
of Biol. Chem., 132:1177 (1996)). However, as shown in FIG. 6, the
number of capillary sprouts formed by TWEAK+bFGF was not reduced in
the presence of neutralizing mAbs for these factors. The inhibitory
activity of these mAbs was independently confirmed, anti-TNF mAb
inhibiting tumor killing and anti-IL-8 inhibiting IL-8-induced EC
proliferation (data not shown).
[0071] VEGF also induced EC morphogenesis, as evidenced by the
reorganization of the HUVEC monolayer on the fibrin gel surface
(FIG. 7). The appearance of the structures induced by VEGF were
qualitatively different from those induced by bFGF and there was no
EC invasion into the fibrin matrix. Interestingly, confluent EC
monolayers treated with TWEAK+VEGF did not reorganize, and were
similar in appearance to untreated cultures and cultures treated
with TWEAK alone. Thus TWEAK cooperates with bFGF to induce the
formation of capillary-like structures but antagonizes the
morphogenic response of HUVECs to VEGF.
TWEAK inhibits expression of VEGF receptor FLT1--TWEAK may regulate
EC responses to VEGF by modulating VEGF signaling through its
receptors. RPA expression analysis on a panel of angiogenic
cytokines and receptors was performed as a means to address this
possibility. Our results (FIG. 8) show an increase in the
expression of FLT1 in cultures treated with VEGF as compared to
control cultures. Cultures treated with TWEAK alone were similar to
controls (data not shown). Interestingly, this response was
inhibited in cultures treated with TWEAK+VEGF. Thus, TWEAK
decreased mRNA levels of FLT1 in the presence of VEGF. Our RPA
analysis also reveals inhibition by TWEAK of the expression of TIE,
another EC receptor required for angiogenesis (Sato, T. N., et al.,
Nature, 376(6535):70-74 (1995)). These data suggest a mechanism
whereby TWEAK may modulate signal transduction by angiogenic
factors.
[0072] Accordingly, these results show that TWEAK antagonizes the
morphogenic response of ECs to VEGF and inhibits VEGF-induced EC
migration, but has no measurable effect on EC proliferation in the
presence of VEGF, and the action of TWEAK may be pro-angiogenic or
anti-angiogenic, depending upon the particular angiogenic
context.
[0073] In contrast with the pro-angiogenic activity of TWEAK in the
context of bFGF, these results show that TWEAK can inhibit the
angiogenic behavior of ECs in other settings. TWEAK inhibits EC
morphogenesis induced by VEGF, as evidenced by little if any change
in the appearance of confluent EC monolayers plated onto fibrin gel
surfaces in the presence of both factors. This anti-angiogenic
effect of TWEAK may reflect TWEAK inhibition of VEGF-dependent EC
migration which was observed independently in the wound repair
assay. There was no changes in expression of the integrins
.alpha..sub.v, .alpha..sub.1, .alpha..sub.2, .alpha..sub.5,
.beta..sub.3 and .beta..sub.1, in cultures treated with TWEAK and
VEGF as compared to VEGF alone (A. Jakubowski, unpublished
observations). However, we found that the inducible expression of
the VEGF receptor Flt-1 was inhibited by addition of TWEAK. Thus
TWEAK may limit EC responses to VEGF by modulating VEGF signaling
through its receptor. This is especially important in understanding
and effecting tumor biology, where the role of endogenous VEGF is
critical to tumor progression (Ferrars, N. and Davis-Smyth, T.,
Endocrine Reviews, 18:4-25 (1997)).
[0074] This data together with the TNF paradigm support the
hypothesis that TWEAK can differentially modulate angiogenesis
depending upon the particular angiogenic setting. These results
indicate that it plays a role in the regulation of microvascular
growth, remodeling, and/or maintenance in vivo, being
pro-angiogenic or anti-angiogenic depending on the angiogenic
context. Agonists (or antagonists) of the TWEAK pathway can provide
useful therapeutic approaches to treatment in settings of ischemic
injury, cancer, angioproliferative and inflammatory disorders.
Example 2
Growth of Lewis Lung Carcinoma is Inhibited in Tweak-Transgenic
(Tg) Mice
[0075] The Lewis Lung Carcinoma in vitro cell line was obtained
from the Tumor Repository, NCI-Frederick Cancer Research and
Development Center. The cell line was passed in vitro four passages
in RPMI-1640/10% FBS without antibiotics prior to implantation into
animals. The tumor cells (of C57BL/6 mouse strain origin) were
injected at an innoculum of 1.times.106 cells per mouse
subcutaneously in the right flank area of TWEAK-Tg mice and NonTg
littermates which had been successively bred onto a C57BL/6
background. In each experiment, tumor cells were injected into 8
TWEAK-Tg mice and 8 NonTg littermates. Tumor measurements were
recorded twice weekly and the average tumor weight at each time
point calculated for each experimental group. Plots of tumor weight
vs. time for each of two individual experiments are shown in FIG.
9, indicating that tumor growth was slower in mice expressing TWEAK
as compared to their normal littermates.
TABLE-US-00001 TABLE I TWEAK Binding to Primary Cell Types TWEAK
Binding.sup.a Human Cell Types HUVEC +++ Aortic EC ++ Aortic Smooth
Muscle Cells +++ Lung Fibroblasts + Embryonic Myoblasts ++
Peripheral Blood Lymphocytes - Peripheral Blood Dendritic Cells -
Murine Cell Types EC cell line (MS-1) ++ Bone marrow fibroblast
line (M210B4) + Spleen cells with and without anti-CD3 mAb
activation - Lymph node cells with and without anti-CD3 - mAb
activation Thymocytes with and without anti-CD3 mAbactivation -
Resident Peritoneal Macrophages - Thioglycollate-induced
Macrophages - (with and without LPS, TNF or IFN.alpha. stimulation)
.sup.aRelative Binding compared to HUVECs as measured by
immunofluorescent staining
Sequence CWU 1
1
21129PRTHomo sapiens 1Met Ala Arg Gly Ser Leu Arg Arg Leu Leu Arg
Leu Leu Val Leu Gly1 5 10 15Leu Trp Leu Ala Leu Leu Arg Ser Val Ala
Gly Glu Gln Ala Pro Gly 20 25 30Thr Ala Pro Cys Ser Arg Gly Ser Ser
Trp Ser Ala Asp Leu Asp Lys 35 40 45Cys Met Asp Cys Ala Ser Cys Arg
Ala Arg Pro His Ser Asp Phe Cys 50 55 60Leu Gly Cys Ala Ala Ala Pro
Pro Ala Pro Phe Arg Leu Leu Trp Pro65 70 75 80Ile Leu Gly Gly Ala
Leu Ser Leu Thr Phe Val Leu Gly Leu Leu Ser 85 90 95Gly Phe Leu Val
Trp Arg Arg Cys Arg Arg Arg Glu Lys Phe Thr Thr 100 105 110Pro Ile
Glu Glu Thr Gly Gly Glu Gly Cys Pro Ala Val Ala Leu Ile 115 120
125Gln2129PRTMus musculus 2Met Ala Pro Gly Trp Pro Arg Ser Leu Pro
Gln Ile Leu Val Leu Gly1 5 10 15Phe Gly Leu Val Leu Met Arg Ala Ala
Ala Gly Glu Gln Ala Pro Gly 20 25 30Thr Ser Pro Cys Ser Ser Gly Ser
Ser Trp Ser Ala Asp Leu Asp Lys 35 40 45Cys Met Asp Cys Ala Ser Cys
Pro Ala Arg Pro His Ser Asp Phe Cys 50 55 60Leu Gly Cys Ala Ala Ala
Pro Pro Ala His Phe Arg Leu Leu Trp Pro65 70 75 80Ile Leu Gly Gly
Ala Leu Ser Leu Val Leu Val Leu Ala Leu Val Ser 85 90 95Ser Phe Leu
Val Trp Arg Arg Cys Arg Arg Arg Glu Lys Phe Thr Thr 100 105 110Pro
Ile Glu Glu Thr Gly Gly Glu Gly Cys Pro Gly Val Ala Leu Ile 115 120
125Gln
* * * * *