U.S. patent application number 13/785253 was filed with the patent office on 2013-07-11 for administering anti-placental growth factor antibodies.
The applicant listed for this patent is Peter CARMELIET, Desire Collen, Sandro De Falco, Ruvo Menotti. Invention is credited to Peter CARMELIET, Desire Collen, Sandro De Falco, Ruvo Menotti.
Application Number | 20130177564 13/785253 |
Document ID | / |
Family ID | 8171493 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177564 |
Kind Code |
A1 |
CARMELIET; Peter ; et
al. |
July 11, 2013 |
ADMINISTERING ANTI-PLACENTAL GROWTH FACTOR ANTIBODIES
Abstract
The present invention relates to the field of pathological
angiogenesis and arteriogenesis and, in particular, to a
stress-induced phenotype in a transgenic mouse (PIGF.sup.-/-) that
does not produce Placental Growth Factor (PIGF) and that
demonstrates an impaired vascular endothelial growth factor
(VEGF)-dependent response. PIGF deficiency has a negative influence
on diverse pathological processes of angiogenesis, arteriogenesis
and vascular leakage comprising ischemic retinopathy, tumor
formation, pulmonary hypertension, vascular leakage (edema
formation) and inflammatory disorders. The invention thus relates
to molecules that can inhibit the binding of PIGF to its receptor
(VEGFR-1), such as monocloncal antibodies and tetrameric peptides,
and to the use of these molecules to treat the above-mentioned
pathological processes.
Inventors: |
CARMELIET; Peter; (Landen,
BE) ; Collen; Desire; (Winksele, BE) ; De
Falco; Sandro; (Napoli, IT) ; Menotti; Ruvo;
(Trevico, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CARMELIET; Peter
Collen; Desire
De Falco; Sandro
Menotti; Ruvo |
Landen
Winksele
Napoli
Trevico |
|
BE
BE
IT
IT |
|
|
Family ID: |
8171493 |
Appl. No.: |
13/785253 |
Filed: |
March 5, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13470615 |
May 14, 2012 |
|
|
|
13785253 |
|
|
|
|
12400993 |
Mar 10, 2009 |
|
|
|
13470615 |
|
|
|
|
12341177 |
Dec 22, 2008 |
|
|
|
12400993 |
|
|
|
|
10291979 |
Nov 11, 2002 |
7482004 |
|
|
12341177 |
|
|
|
|
PCT/EP01/05478 |
May 10, 2001 |
|
|
|
10291979 |
|
|
|
|
Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/142.1; 424/145.1; 424/158.1 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
7/00 20180101; A61P 9/10 20180101; A61P 29/00 20180101; C07K 16/22
20130101; A61K 38/07 20130101; A61P 9/12 20180101; A61K 39/3955
20130101; A61P 9/14 20180101; C07K 1/047 20130101; A61P 27/02
20180101; A61K 2039/505 20130101; G01N 2333/71 20130101; A61P 35/00
20180101 |
Class at
Publication: |
424/135.1 ;
424/158.1; 424/145.1; 424/133.1; 424/142.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
May 12, 2000 |
EP |
00201714.3 |
Claims
1. A method of suppressing inflammation in a mammal by
administering to a mammal in need of suppressing inflammation an
antibody or a fragment thereof that specifically binds to placental
growth factor and that neutralizes the activity of placental growth
factor, such that said inflammation is suppressed.
2. The method according to claim 1 wherein said inflammation is an
inflammatory disorder or inflammatory disease.
3. A method of suppressing development of inflammation-associated
edema in a mammal by administering to a mammal likely to develop
inflammation-associated edema an antibody or a fragment thereof
that specifically binds to placental growth factor and that
neutralizes the activity of placental growth factor, such that
development of inflammation-associated edema is suppressed.
4. A method of reducing inflammation in a mammal by administering
to a mammal suffering from inflammation an antibody or a fragment
thereof that specifically binds to placental growth factor and that
neutralizes the activity of placental growth factor, such that said
inflammation is reduced.
5. The method according to claim 4 wherein said inflammation is an
inflammatory disorder or inflammatory disease.
6. A method of reducing development of inflammation-associated
edema in a mammal by administering to a mammal likely to develop
inflammation-associated edema an antibody or a fragment thereof
that specifically binds to placental growth factor and that
neutralizes the activity of placental growth factor, such that
development of inflammation-associated edema is reduced.
7. The method according to claim 1 wherein said antibody is
selected from the group consisting of a monoclonal antibody, a
human antibody, a human monoclonal antibody, a humanized antibody,
and a humanized monoclonal antibody; or wherein said antibody
fragment is selected from the group consisting of a Fab, F(ab)'2 or
scFv fragment.
8. The method according to claim 3 wherein said antibody is
selected from the group consisting of a monoclonal antibody, a
human antibody, a human monoclonal antibody, a humanized antibody,
and a humanized monoclonal antibody; or wherein said antibody
fragment is selected from the group consisting of a Fab, F(ab)'2 or
scFv fragment.
9. The method according to claim 4 wherein said antibody is
selected from the group consisting of a monoclonal antibody, a
human antibody, a human monoclonal antibody, a humanized antibody,
and a humanized monoclonal antibody; or wherein said antibody
fragment is selected from the group consisting of a Fab, F(ab)'2 or
scFv fragment, a human monoclonal antibody, a humanized antibody,
or a humanized monoclonal antibody.
10. The method according to claim 6 wherein said antibody is
selected from the group consisting of a monoclonal antibody, a
human antibody, a human monoclonal antibody, a humanized antibody,
and a humanized monoclonal antibody; or wherein said antibody
fragment is selected from the group consisting of a Fab, F(ab)'2 or
scFv fragment.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 13/470,615, filed May 14, 2012 (published as US-2012-0263710 A1
on Oct. 18, 2012) (pending), which is a continuation of U.S.
application Ser. No. 12/400,993, filed Mar. 10, 2009 (published as
US-2009-0238826 A1 on Sep. 24, 2009) (abandoned), which is a
divisional of U.S. application Ser. No. 12/341,177, filed Dec. 22,
2008 (published as US-2009-0162354 A1 on Jun. 25, 2009)
(abandoned), which is a divisional of U.S. application Ser. No.
10/291,979, filed Nov. 11, 2002 (U.S. Pat. No. 7,482,004, and which
published as US-2003-0180286 A1 on Sep. 25, 2003), which is a
continuation of PCT/EP01/05478, filed May 10, 2001 (which
designated the U.S. and was published in English as WO 01/85796
A2), which claims benefit of EP 00201714.3, filed May 12, 2000, the
entire contents of each of which is hereby incorporated herein by
reference. Related U.S. application Ser. No. 12/265,046, filed Nov.
5, 2008 (published as US-2009-0074765 A1 on Mar. 19, 2009)
(abandoned), is a divisional of U.S. application Ser. No.
10/291,979, filed Nov. 11, 2002 (U.S. Pat. No. 7,482,004, and which
published as US 2003-0180286 A1 on Sep. 25, 2003), which is a
continuation of PCT/EP01/05478, filed May 10, 2001 (which
designated the U.S. and was published in English as WO 01/85796
A2), which claims benefit of EP 00201714.3, filed May 12, 2000, the
entire contents of each of which is hereby incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of pathological
angiogenesis and arteriogenesis. In particular, the invention
describes a stress-induced phenotype in a transgenic mouse (PIGF-)
that does not produce Placental Growth Factor (PIGF) and that
demonstrates an impaired vascular endothelial growth factor
(VEGF)-dependent response. It is revealed that PIGF deficiency has
a negative influence on diverse pathological processes of
angiogenesis, arteriogenesis and vascular leakage comprising
ischemic retinopathy, tumor formation, pulmonary hypertension,
vascular leakage (edema formation) and inflammatory disorders. The
invention thus relates to molecules that can inhibit the binding of
PIGF to its receptor (VEGFR-1), such as monoclonal antibodies and
tetrameric peptides. The invention further relates to the use of
these molecules to treat the above-mentioned pathological
processes.
BACKGROUND OF THE INVENTION
[0003] Abnormal blood vessel formation contributes to the
pathogenesis of numerous diseases with high morbidity and
mortality. Elucidation of the mechanisms underlying vascular growth
might allow the development of therapeutic strategies to stimulate
vascular growth in ischemic tissues or to suppress their formation
in tumors. Recent gene targeting studies in embryos have identified
some of the mechanisms involved in the initial formation of
endothelial channels (angiogenesis) and their subsequent maturation
by coverage with smooth muscle cells (arteriogenesis). Evidence is
emerging that distinct molecular mechanisms may mediate growth of
blood vessels during pathological conditions, but the molecular
players remain largely undetermined.
[0004] VEGF has been implicated in development and pathological
growth of the vasculature (N. Ferrara et al., 1999, Curr. Top.
Microbiol. Immunol. 237, 1-30). Deficiency of a single VEGF allele
causes fatal vascular defects (P. Carmeliet et al., 1996, Nature
380, 435-439; and N. Ferrara et al., 1996, Nature 380, 439-442),
whereas suppression of VEGF in the neonate or expression of a
single VEGF 120 isoform results in impaired vascular growth (H. P.
Gerber et al., 1999, Development 126, 1149-1159; and P. Carmeliet
et al., 1999, Nat. Med. 5, 495-502). In the adult, VEGF affects
vascular growth during reproduction, wound healing, and malignant
and inflammatory disorders (N. Ferrara et al., 1999, Curr. Top.
Microbiol. Immunol. 237, 1-30). VEGF is currently being tested for
therapeutic angiogenesis in the ischemic heart and limb, but
initial clinical trials have resulted in both promising and
disappointing results (J. M. Isner et al., 1999, J. Clin. Invest.
103, 1231-1236). An outstanding question is whether VEGF is able to
stimulate the maturation of vessels with a smooth muscle coat
(arteriogenesis). Naked endothelial channels remain vulnerable to
traumatic insults, regress during changes in oxygen, and lack
vasomotor control to accommodate changes in tissue perfusion (L. E.
Benjamin et al., 1998, Development 125, 1591-1598). In some
diseases such as pulmonary hypertension excess arteriogenesis is an
undesired and poorly controllable phenomenon. In pulmonary
hypertension, remodelling of the pulmonary vasculature occurs
because vascular smooth muscle cells proliferate and migrate
distally around the terminal arterioles, increasing thereby the
pulmonary vascular resistance. Another aspect of VEGF is that this
molecule affects the permeability and growth of adult quiescent
vessels. In normal human serum, no detectable levels of VEGF are
present, but under pathological conditions, such as cancer and
inflammatory disorders, VEGF is highly up-regulated and mediates
the formation of undesired edema. Edema formation is also an
important clinical problem associated with several tumors leading
to ascites in peritoneal tumors, pleuritis in lung cancer and
cerebral edema in brain tumors (possibly leading to fatal
intracranial hypertension) and often facilitates metastasis of
tumors. Vascular congestion and edema are important pathogenic
mechanisms in asthma, brain infarct expansion after stroke,
peritoneal sclerosis after dialysis or abdominal interventions,
etc. Other VEGF homologues have been identified, but their role in
angiogenesis and arteriogenesis remains unclear.
[0005] One interesting homologue of VEGF is Placental Growth Factor
(PIGF) but its role in vascular growth and pathogenesis has been
poorly studied (M. G. Persico et al., 1999, Curr. Top. Microbial.
Immunol. 237, 31-40). U.S. Pat. No. 5,919,899 describes PIGF and
its use in the treatment of inflammatory disorders, wounds and
ulcers. Donnini et al. (J. Pathol. 189, 66, 1999) have observed a
correlation between up-regulation of PIGF and human meningiomas but
it is clear that there is no indication whatsoever that PIGF has a
role in tumor formation. The role of PIGF in edema was studied by
Monsky et al. (Cancer Res. 59, 4129, 1999), but no in viva role for
PIGF in edema formation during pathological processes could be
found in several mouse and human tumors.
[0006] Inhibitors for PIGF are not known in the art except for a
goat polyclonal antibody against human PIGF (R&D
Pharmaceuticals, Abingdon, UK) and a chicken polyclonal antibody
(Gassmann et al., 1990, Faseb J. 4, 2528). Those antibodies are
used for western blotting, histochemistry and immunoprecipitation
studies. The role of the PIGF receptor (=VEGFR-1) for endothelial
cell biology has also remained enigmatic (A. Sawano et al., 1996,
Cell Growth Differ. 7, 213-221 and M. Clauss et al., 1996, J. Biol.
Chem. 271, 17629-17634). Gene-targeting studies yielded conflicting
results on the role of VEGFR-1, either as a possible signaling
receptor (suggested by the vascular defects in VEGFR-1-deficient
embryos (G. H. Fong et al., 1999, Development 126, 3015-3025)) or
as an inert binding site, a "sink," for VEGF, regulating
availability of VEGF for the angiogenic VEGFR-2 (suggested by the
normal vascular development in mice expressing a truncated VEGFR-1,
lacking the tyrosine kinase domain (S. Hiratsuka et al., 1998,
Proc. Natl. Acad. Sci. U.S.A. 95, 9349-9354)).
[0007] The present invention relates to the surprising finding that
PIGF is a specific modulator of VEGF during a variety of
pathological conditions, such as ischemic retinopathy,
tumorigenesis, inflammatory disorders, wound healing, edema and
pulmonary hypertension. This finding has implications for the
inhibition of vascular leakage (edema formation), inflammatory
disorders, tumor formation, pathological angiogenesis and the
prevention of pulmonary hypertension that occurs during
pathological arteriogenesis.
DISCLOSURE OF THE INVENTION
[0008] The present invention aims at providing research tools and
therapeutics for patients suffering from pathological angiogenesis,
pathological arteriogenesis and edema formation. In particular, the
invention aims at providing molecules, such as antibodies, small
molecules, tetrameric peptides, ribozymes, antisense nucleic acids,
receptor antagonists or soluble receptors that can block the
activity and/or synthesis of PIGF or antagonize the VEGFR-1
activity or can inhibit the signal transduction from the VEGFR-1 to
VEGFR-2. The invention further aims at using these molecules for
the treatment and/or the prevention of, but not limited to,
pulmonary hypertension, cancer, edema, ischemic retinopathy and
inflammatory disorders. The present invention also aims at
providing a method to screen for molecules that bind on VEGFR-1 or
PIGF.
[0009] In other words, the present invention aims at providing
therapeutics or a medicament that can be used for the treatment of
pulmonary hypertension, tumor formation, edema, ischemic
retinopathy or inflammatory disorders.
BRIEF DESCRIPTION OF THE DRAWING
[0010] FIG. 1: Role of PIGF in pathological vascular growth.
[0011] Panel A: PIGF--constitutively produced by adult quiescent
endothelial cells (EC)--is not essential for maintenance of the
adult quiescent vasculature, presumably because it is ineffective
in the presence of minimal VEGF expression. When expression of VEGF
is up-regulated during ischemia, inflammation (macrophages: M.phi.)
or malignancy (tumor cells), PIGF amplifies the response of
endothelial and smooth muscle cells (SMC) to VEGF, resulting in
enhanced angiogenesis, vascular permeability and arteriogenesis.
PIGF can act in an autocrine manner on endothelial, smooth muscle
and inflammatory cells, but is also produced by nearby tumor cells,
ischemic cardiomyocytes, etc. Panel B: In the absence of PIGF,
vessels are normally formed during development, but respond less to
VEGF during pathological conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0012] In previous studies, the PIGF gene was inactivated in the
mouse genome via homologous recombination in embryonic stem (ES)
cells (P. Carmeliet, 2000, J. Pathol. 190, 387-405; P. Carmeliet,
1999, Curr. Interv. Cardiol. Reports 1, 322-335; and P. Carmeliet
and D. Collen, 1999, Curr. Top. Microbiol. Immunol. 237, 133-158).
PIGF-deficient (PIGF.sup.-/-) mice are viable and fertile, and did
not exhibit spontaneous vascular defects. In the present invention,
it is shown that growth of endothelial channels (angiogenesis),
vascular maturation by smooth muscle cells (arteriogenesis) and
vascular permeability are significantly impaired in adult
PIGF.sup.-/- mice during a variety of conditions where pathological
angiogenesis and edema formation occurs. The latter conditions
comprise ischemic retinopathy, tumor formation, pulmonary
hypertension, edema and inflammation also known to involve VEGF. In
another aspect of the invention, it is shown that the role of PIGF
is not only restricted to the formation of immature capillaries,
but also includes the maturation/stabilization of newly formed
vessels via stimulating their coverage with smooth muscle cells
(arteriogenesis), a therapeutic prerequisite for functional and
sustainable angiogenesis, but an undesired effect of pathological
arteriogenesis as in the case of pulmonary hypertension.
[0013] Thus, in one embodiment, the present invention relates to
molecules that comprise a region that can specifically bind to
placental growth factor or to vascular endothelial growth factor
receptor-1; these molecules can suppress or prevent placental
growth factor-induced pathological angiogenesis, vascular leakage
(edema), pulmonary hypertension, tumor formation and/or
inflammatory disorders. With "suppression," it is understood that
suppression can occur for at least 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or even 100%. More specifically, the invention relates to
molecules that can be used to neutralize the activity of PIGF by
interfering with its synthesis, translation, dimerization,
receptor-binding and/or receptor-binding-mediated signal
transduction.
[0014] By "molecules," it is meant peptides, tetrameric peptides,
proteins, organic molecules, mutants of the VEGFR-1, soluble
receptors of VEGFR-1 and any fragment or homologue thereof having
the same neutralizing effect as stated above. Also, the invention
is directed to antagonists of PIGF such as anti-PIGF antibodies and
functional fragments derived thereof, antisense RNA and DNA
molecules and ribozymes that function to inhibit the translation of
PIGF, all capable of interfering/or inhibiting the VEGFR-1 signal
transduction.
[0015] By "synthesis," it is meant transcription of PIGF. Small
molecules can bind on the promoter region of PIGF and inhibit
binding of a transcription factor or the molecules can bind the
transcription factor and inhibit binding to the PIGF promoter.
[0016] By "PIGF" is also meant its isoforms, which occur as a
result of alternative splicing, and allelic variants thereof. As a
result of alternative splicing, three PIGF RNAs encoding monomeric
human PIGF-1, PIGF-2 and PIGF-3 isoform precursors containing 149,
179 and 219 amino acid residues, respectively, have been described.
In normal mouse tissues, only one mouse PIGF mRNA encoding the
equivalent of human PIGF-2 has been identified.
[0017] In a specific embodiment, the invention provides a murine
monoclonal antibody against PIGF. In another specific embodiment,
the murine monoclonal antibody is MabPL5D11. This monoclonal
antibody is available in the Department of Transgene Technology and
Gene Therapy, UZ Gasthuisberg, Herestraat 49, B-3000 Leuven.
[0018] The terms "antibody" or "antibodies" relate to an antibody
characterized as being specifically directed against PICT or
VEGFR-1 or any functional derivative thereof, with the antibodies
being preferably monoclonal antibodies, or an antigen-binding
fragment thereof, of the F(ab').sub.2, F(ab) or single chain Fv
type, or any type of recombinant antibody derived thereof. These
antibodies of the invention, including specific polyclonal antisera
prepared against PIGF or VEGFR-1 or any functional derivative
thereof, have no cross-reactivity to other proteins. The monoclonal
antibodies of the invention can, for instance, be produced by any
hybridoma liable to be formed according to classical methods from
splenic cells of an animal, particularly of a mouse or rat
immunized against PIGF or VEGFR-1 or any functional derivative
thereof, and of cells of a myeloma cell line, and to be selected by
the ability of the hybridoma to produce the monoclonal antibodies
recognizing PIGF or VEGFR-1 or any functional derivative thereof
that have been initially used for the immunization of the
animals.
[0019] The monoclonal antibodies according to this embodiment of
the invention may be humanized versions of the mouse monoclonal
antibodies made by means of recombinant DNA technology, departing
from the mouse and/or human genomic DNA sequences coding for H and
L chains or from cDNA clones coding for H and L chains.
[0020] Alternatively, the monoclonal antibodies according to this
embodiment of the invention may be human monoclonal antibodies.
Such human monoclonal antibodies are prepared, for instance, by
means of human peripheral blood lymphocytes (PBL) repopulation of
severe combined immune deficiency (SLID) mice as described in
PCT/EP 99/03605 or by using transgenic non-human animals capable of
producing human antibodies as described in U.S. Pat. No. 5,545,806.
Also, fragments derived from these monoclonal antibodies such as
Fab, F(ab)'2 and ssFv ("single chain variable fragment"), providing
they have retained the original binding properties, form part of
the present invention. Such fragments are commonly generated by,
for instance, enzymatic digestion of the antibodies with papain,
pepsin, or other proteases. It is well known to the person skilled
in the art that monoclonal antibodies, or fragments thereof, can be
modified for various uses. The antibodies involved in the invention
can be labeled by an appropriate label of the enzymatic,
fluorescent, or radioactive type.
[0021] Small molecules, e.g., small organic molecules, and other
drug candidates can be obtained, for example, from combinatorial
and natural product libraries. To screen for the candidate/test
molecules, cell lines that express VEGFR-1 and VEGFR-2 may be used
and the signal transduction is monitored as described in detail in
the examples.
[0022] Monitoring can be measured using standard biochemical
techniques. Other responses, such as activation or suppression of
catalytic activity, phosphorylation (e.g., the tyrosine
phosphorylation of the intracellular domain of VEGFR-2) or
dephosphorylation of other proteins, activation or modulation of
second messenger production, changes in cellular ion levels,
association, dissociation or translocation of signaling molecules,
or transcription or translation of specific genes, may also be
monitored. These assays may be performed using conventional
techniques developed for these purposes in the course of screening.
Inhibition of ligand binding to its cellular receptor may, via
signal transduction pathways, affect a variety of cellular
processes.
[0023] Cellular processes under the control of the VEGFR-1/PIGF
signaling pathway may include, but are not limited to, normal
cellular functions, proliferation, differentiation, maintenance of
cell shape, and adhesion, in addition to abnormal or potentially
deleterious processes such as unregulated cell proliferation, loss
of contact inhibition, blocking of differentiation or cell death.
The qualitative or quantitative observation and measurement of any
of the described cellular processes by techniques known in the art
may be advantageously used as a means of scoring for signal
transduction in the course of screening.
[0024] Random peptide libraries, such as tetrameric peptide
libraries further described herein, consisting of all possible
combinations of amino acids attached to a solid phase support, may
be used to identify peptides that are able to bind to the ligand
binding site of a given receptor or other functional domains of a
receptor such as kinase domains (K. S. Lam et al., 1991, Nature
354, 82). The screening of peptide libraries may have therapeutic
value in the discovery of pharmaceutical agents that act to inhibit
the biological activity of receptors through their interactions
with the given receptor.
[0025] Identification of molecules that are able to bind to the
VEGFR-1 or PIGF may be accomplished by screening a peptide library
with recombinant soluble VEGFR-1 protein or PIGF protein. For
example, the kinase and extracellular ligand binding domains of
VEGFR-1 may be separately expressed and used to screen peptide
libraries. In addition to using soluble VEGFR-1 molecules, in
another embodiment, it is possible to detect peptides that bind to
cell surface receptors using intact cells. The cells used in this
technique may be either alive or fixed cells. The cells will be
incubated with the random peptide library and will bind certain
peptides in the library to form a "rosette" between the target
cells and the relevant solid phase support/peptide. The rosette can
thereafter be isolated by differential centrifugation or removed
physically under a dissecting microscope.
[0026] In another embodiment, transdominant-negative mutant forms
of VEGF receptors (e.g., a transdominant-negative receptor of
VEGFR-1) can be used to inhibit the signal transduction of PIGF.
The use of the transdominant-negative mutant forms of VEGF
receptors is fully described in U.S. Pat. No. 5,851,999.
[0027] Also within the scope of the invention are
oligoribonucleotide sequences that include antisense RNA and DNA
molecules and ribozymes that function to inhibit the translation of
VEGFR-1 mRNA or PIGF mRNA. Antisense RNA and DNA molecules act to
directly block the translation of mRNA by binding to targeted mRNA
and preventing protein translation. In regard to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site, e.g., between -10 and +10 regions of the VEGFR-1 or PIGF
nucleotide sequence, are preferred. Ribozymes are enzymatic RNA
molecules capable of catalyzing the specific cleavage of RNA. The
mechanism of ribozyme action involves sequence-specific
hybridization of the ribozyme molecule to complementary target RNA,
followed by an endonucleolytic cleavage. Within the scope of the
invention are engineered hammerhead motif ribozyme molecules that
specifically and efficiently catalyze endonucleolytic cleavage of
VEGFR-1 or PIGF RNA sequences.
[0028] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites that include the following sequences, GUA,
GUU and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides corresponding to the region of the target gene
containing the cleavage site may be evaluated for predicted
structural features such as secondary structures that may render
the oligonucleotide sequence unsuitable. The suitability of
candidate targets may also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using ribonuclease protection assays.
[0029] Both antisense RNA and DNA molecules and ribozymes of the
invention may be prepared by any method known in the art for the
synthesis of RNA molecules. These include techniques for chemically
synthesizing oligodeoxyribonucleotides well known in the art, such
as, for example, solid phase phosphoramidite chemical
synthesis.
[0030] Alternatively, RNA molecules may be generated by in vitro
and in vivo transcription of DNA sequences encoding the antisense
RNA molecule. Such DNA sequences may be incorporated into a wide
variety of vectors that incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly, depending on the promoter used can
be stably introduced into cell lines.
[0031] In another embodiment of the invention, the above-described
molecules can be used as a medicament to treat pathological
conditions of angiogenesis and/or arteriogenesis and/or edema
formation.
[0032] "Edema" is a condition that is caused by vascular leakage.
Vasodilation and increased permeability during inflammation can be
predominant pathogenetic mechanisms. For instance, edema
contributes to infarct expansion after stroke and may cause
life-threatening intracranial hypertension in cancer patients.
Further, extravasation of plasma proteins favors metastatic spread
of occult tumors, and airway congestion may cause fatal asthmatic
attacks. The increased vascular leakage that occurs during
inflammation can lead to respiratory distress, ascites, peritoneal
sclerosis (in dialysis patients), adhesion formation (abdominal
surgery) and metastatic spreading.
[0033] By "angiogenesis," it is meant a fundamental process by
which new blood vessels are formed. The primary angiogenic period
in humans takes place during the first three months of embryonic
development, but angiogenesis also occurs as a normal physiological
process during periods of tissue growth, such as an increase in
muscle or fat and during the menstrual cycle and pregnancy. The
term "pathological angiogenesis" refers to the formation and growth
of blood vessels during the maintenance and the progression of
several disease states, for example, in blood vessels
(atherosclerosis, hemangioma, hemangioendothelioma), bone and
joints (rheumatoid arthritis, synovitis, bone and cartilage
destruction, osteomyelitis, pannus growth, osteophyte formation,
neoplasms and metastasis), skin (warts, pyogenic granulomas, hair
growth, Kaposi's sarcoma, scar keloids, allergic edema, neoplasms),
liver, kidney, lung, ear and other epithelia (inflammatory and
infectious processes (including hepatitis, glomerulonephritis,
pneumonia), asthma, nasal polyps, otitis, transplantation, liver
regeneration, neoplasms and metastasis), uterus, ovary and placenta
(dysfunctional uterine bleeding (due to intra-uterine contraceptive
devices), follicular cyst formation, ovarian hyperstimulation
syndrome, endometriosis, neoplasms), brain, nerves and eye
(retinopathy of prematurity, diabetic retinopathy, choroidal and
other intraocular disorders, leukomalacia, neoplasms and
metastasis), heart and skeletal muscle due to work overload,
adipose tissue (obesity), endocrine organs (thyroiditis, thyroid
enlargement, pancreas transplantation), hematopoiesis (AIDS
(Kaposi), hematologic malignancies (leukemias, etc.), lymph vessels
(tumor metastasis, lymphoproliferative disorders).
[0034] By "retinal ischemic diseases," it is meant that the
retina's supply of blood and oxygen is decreased, and the
peripheral portions of the retina lose their source of nutrition
and stop functioning properly. Common diseases that lead to
retinopathy are diabetic retinopathy, central retinal vein
occlusion, stenosis of the carotid artery, and sickle cell
retinopathy. Diabetic retinopathy is a major cause of visual loss
in diabetic patients. In the ischemic retina, the growth of new
blood vessels occurs (neovascularization). These vessels often grow
on the surface of the retina, at the optic nerve, or in the front
of the eye on the iris. The new vessels cannot replace the flow of
necessary nutrients and, instead, can cause many problems such as
vitreous hemorrhage, retinal detachment, and uncontrolled glaucoma.
These problems occur because new vessels are fragile and are prone
to bleed. If caught in its early stages, proliferative diabetic
retinopathy can sometimes be arrested with panretinal
photocoagulation. However, in some cases, vitrectomy surgery is the
only option.
[0035] By the term "pulmonary hypertension," it is meant a disorder
in which the blood pressure in the pulmonary arteries is abnormally
high. In the absence of other diseases of the heart or lungs, it is
called primary pulmonary hypertension. Diffuse narrowing of the
pulmonary arterioles occurs as a result of pathological
arteriogenesis followed by pulmonary hypertension as a response to
the increased resistance to blood flow. The incidence is eight out
of 100,000 people. However, pulmonary hypertension can also occur
as a complication of Chronic Obstructive Pulmonary Diseases (COPD)
such as emphysema, chronic bronchitis or diffuse interstitial
fibrosis and in patients with asthmatiform COPD. The incidence of
COPD is approximately five out of 10,000 people.
[0036] In another embodiment of the invention, the above-described
molecules can be used to manufacture a medicament to treat
inflammation and, more specifically, inflammatory disorders.
"Inflammation," as used herein, means the local reaction to injury
of living tissues, especially the local reaction of the small blood
vessels, their contents, and their associated structures. The
passage of blood constituents through the vessel walls into the
tissues is the hallmark of inflammation, and the tissue collection
so formed is termed the "exudates" or "edema." Any noxious process
that damages living tissue (infection with bacteria, excessive
heat, cold, mechanical injury such as crushing, acids, alkalis,
irradiation, or infection with viruses) can cause inflammation
irrespective of the organ or tissue involved. It should be clear
that diseases of animals and man classed as "inflammatory diseases"
and tissue reactions ranging from burns to pneumonia, leprosy,
tuberculosis, and rheumatoid arthritis are all "inflammations."
[0037] In another embodiment of the invention, the above-described
molecules can be used to manufacture a medicament to treat tumor
formation. By "tumor," it is meant a mass of abnormal tissue that
arises without obvious cause from pre-existing body cells, has no
purposeful function, and is characterized by a tendency to
autonomous and unrestrained growth. Tumors are quite different from
inflammatory or other swellings because the cells in tumors are
abnormal in their appearance and other characteristics. Abnormal
cells, the kind that generally make up tumors, differ from normal
cells in that they have undergone one or more of the following
alterations: (1) hypertrophy, or an increase in the size of
individual cells; this feature is occasionally encountered in
tumors but occurs commonly in other conditions; (2) hyperplasia or
an increase in the number of cells within a given zone; in some
instances, it may constitute the only criterion of tumor formation;
(3) anaplasia, or a regression of the physical characteristics of a
cell toward a more primitive or undifferentiated type; this is an
almost constant feature of malignant tumors, though it occurs in
other instances both in health and in disease. In some instances,
the cells of a tumor are normal in appearance, faithful
reproductions of their parent types; the differences between them
and normal body cells are difficult to discern. Such tumors are
also often benign.
[0038] Other tumors are composed of cells that appear different
from normal adult types in size, shape, and structure; they usually
belong to tumors that are malignant. Such cells may be bizarre in
form or be arranged in a distorted manner. In more extreme cases,
the cells of malignant tumors are described as primitive, or
undifferentiated, because they have lost the appearance and
functions of the particular type of (normal) specialized cell that
was their predecessor. As a rule, the less differentiated a
malignant tumor's cells are, the more quickly that tumor may grow.
Malignancy refers to the ability of a tumor to ultimately cause
death. Any tumor, either benign or malignant in type, may produce
death by local effects if it is "appropriately" situated.
[0039] The common and more specific definition of malignancy
implies an inherent tendency of the tumor's cells to metastasize
(invade the body widely and become disseminated by subtle means)
and eventually to kill the patient unless all the malignant cells
can be eradicated. Metastasis is thus the outstanding
characteristic of malignancy. Metastasis is the tendency of tumor
cells to be carried from their site of origin by way of the
circulatory system and other channels, which may eventually
establish these cells in almost every tissue and organ of the body.
In contrast, the cells of a benign tumor invariably remain in
contact with each other in one solid mass centered on the site of
origin. Because of the physical continuity of benign tumor cells,
they may be removed completely by surgery if the location is
suitable. But the dissemination of malignant cells, each one
individually possessing (through cell division) the ability to give
rise to new masses of cells (new tumors) in new and distant sites,
precludes complete eradication by a single surgical procedure in
all but the earliest period of growth. A benign tumor may undergo
malignant transformation, but the cause of such change is unknown.
It is also possible for a malignant tumor to remain quiescent,
mimicking a benign one clinically, for a long time. All benign
tumors tend to remain localized at the site of origin. Many benign
tumors are encapsulated. The capsule consists of connective tissue
derived from the structures immediately surrounding the tumor.
[0040] Well-encapsulated tumors are not anchored to their
surrounding tissues. These benign tumors enlarge by accretion,
pushing aside the adjacent tissues without involving them
intimately. Among the major types of benign tumors are the
following: lipomas, which are composed of fat cells; angiomas,
which are composed of blood or lymphatic vessels; osteomas, which
arise from bone; chondromas, which arise from cartilage; and
adenomas, which arise from glands. For malignant tumors, examples
comprise carcinomas (occur in epithelial tissues, which cover the
body (the skin) and line the inner cavitary structures of organs
(such as the breast, the respiratory and gastrointestinal tracts),
the endocrine glands, and the genitourinary system) and sarcomas
that develop in connective tissues, including fibrous tissues,
adipose (fat) tissues, muscle, blood vessels, bone, and cartilage.
A cancer can also develop in both epithelial and connective tissue
and is called a carcinosarcoma. Cancers of the blood-forming
tissues (such as leukemias and lymphomas), tumors of nerve tissues
(including the brain), and melanoma (a cancer of the pigmented skin
cells) are classified separately.
[0041] In a specific embodiment, it should be clear that the
therapeutic method of the present invention against tumors can also
be used in combination with any other tumor therapy known in the
art such as irradiation, chemotherapy or surgery.
[0042] The term "medicament to treat" relates to a composition
comprising molecules as described above and a pharmaceutically
acceptable carrier or excipient (both terms can be used
interchangeably) to treat diseases as indicated above. Suitable
carriers or excipients known to the skilled man are saline,
Ringer's solution, dextrose solution, Hank's solution, fixed oils,
ethyl oleate, 5% dextrose in saline, substances that enhance
isotonicity and chemical stability, buffers and preservatives.
Other suitable carriers include any carrier that does not itself
induce the production of antibodies harmful to the individual
receiving the composition such as proteins, polysaccharides,
polylactic acids, polyglycolic acids, polymeric amino acids and
amino acid copolymers.
[0043] The "medicament" may be administered by any suitable method
within the knowledge of the skilled man. The preferred route of
administration is parenterally. In parental administration, the
medicament of this invention will be formulated in a unit dosage
injectable form such as a solution, suspension or emulsion, in
association with the pharmaceutically acceptable excipients as
defined above. However, the dosage and mode of administration will
depend on the individual. Generally, the medicament is administered
so that the protein, polypeptide, or peptide of the present
invention is given at a dose between 1 .mu.g/kg and 10 mg/kg, more
preferably between 10 .mu.g/kg and 5 mg/kg, most preferably between
0.1 and 2 mg/kg. Preferably, it is given as a bolus dose.
Continuous infusion may also be used and includes continuous
subcutaneous delivery via an osmotic minipump. If so, the
medicament may be infused at a dose between 5 and 20
.mu.g/kg/minute, more preferably between 7 and 15
.mu.g/kg/minute.
[0044] In another embodiment, antibodies or functional fragments
thereof can be used for the manufacture of a medicament for the
treatment of the above-mentioned disorders. Non-limiting examples
are the commercially available goat polyclonal antibody from
R&D Pharmaceuticals, Abingdon, UK, or the chicken polyclonal
antibody (Gassmann et al., 1990, Faseb J. 4, 2528). Preferentially,
the antibodies are humanized (Rader et al., 2000, J. Biol. Chem.
275, 13668) and, more preferentially, human antibodies are used as
a medicament.
[0045] Another aspect of administration for treatment is the use of
gene therapy to deliver the above-mentioned antisense gene or
functional parts of the PIGF gene or a ribozyme directed against
the PIGF mRNA or a functional part thereof. "Gene therapy" means
the treatment by the delivery of therapeutic nucleic acids to a
patient's cells. This is extensively reviewed in Lever and
Goodfellow 1995; Br. Med. Bull. 51, 1-242; Culver 1995; F. D.
Ledley, 1995, Hum. Gene Ther. 6, 1129. To achieve gene therapy,
there must be a method of delivering genes to the patient's cells
and additional methods to ensure the effective production of any
therapeutic genes. There are two general approaches to achieve gene
delivery; these are non-viral delivery and virus-mediated gene
delivery.
[0046] In another embodiment of the invention, a molecule to
inhibit the activity of PIGF, as described above, can be used in
combination with a molecule to inhibit the activity of VEGF,
according to the same inhibition levels as described above for
PIGF. Indeed, PIGF is found to be angiogenic at sites where VEGF
levels are increased.
[0047] In another embodiment, the invention provides a method to
identify molecules that can interfere with the binding of PIGF to
the VEGF Receptor-1 (VEGFR-1). This method comprises exposing PIGF
or VEGFR-1 to at least one molecule and measuring the ability of at
least one molecule to interfere with the binding of PIGF to VEGFR-1
and monitoring the ability of at least one molecule to prevent or
to inhibit pathological angiogenesis, vascular leakage, pulmonary
hypertension, tumor formation and/or inflammatory disorders.
[0048] In another embodiment, the invention provides a method to
identify molecules comprising: exposing placental growth factor or
vascular endothelial growth factor receptor-1 and/or neuropilin-1
or nucleic acids encoding the growth factor to at least one
molecule whose ability to suppress or prevent placental growth
factor-induced pathological angiogenesis, vascular leakage (edema),
pulmonary hypertension, tumor formation and/or inflammatory
disorders is sought to be determined, and monitoring the
pathological angiogenesis, vascular leakage (edema), pulmonary
hypertension, tumor formation and/or inflammatory disorders.
[0049] The invention also provides methods for identifying
compounds or molecules that bind on the VEGFR-1 or on PIGF and
prevent the interaction between PIGF and VEGFR-1 and consequently
are able to antagonize the signal transduction. The latter methods
are also referred to as "drug screening assays" or "bioassays" and
typically include the step of screening a candidate/test compound
or agent for the ability to interact with VEGFR-1 or PIGF.
Candidate compounds or agents that have this ability can be used as
drugs to combat or prevent pathological conditions of angiogenesis,
arteriogenesis or edema formation. Candidate/test compounds, such
as small molecules, e.g., small organic molecules, and other drug
candidates can be obtained, for example, from combinatorial and
natural product libraries as described above.
[0050] Typically, the assays are cell-free assays, which include
the steps of combining VEGFR-1 or PIGF and a candidate/test
compound, e.g., under conditions that allow for interaction of
(e.g., binding of) the candidate/test compound with VEGFR-1 or PIGF
to form a complex, and detecting the formation of a complex in
which the ability of the candidate compound to interact with
VEGFR-1 or PIGF is indicated by the presence of the candidate
compound in the complex. Formation of complexes between the VEGFR-1
or PIGF and the candidate compound can be quantitated, for example,
using standard immunoassays. The VEGFR-1 or PIGF employed in such a
test may be free in solution, affixed to a solid support, born on a
cell surface, or located intracellularly.
[0051] To perform the above-described drug screening assays, it is
feasible to immobilize VEGFR-1 or PIGF or its (their) target
molecule(s) to facilitate separation of complexes from uncomplexed
forms of one or both of the proteins, as well as to accommodate
automation of the assay. Interaction (e.g., binding of) of VEGFR-1
or PIGF to a target molecule can be accomplished in any vessel
suitable for containing the reactants.
[0052] Examples of such vessels include microtiter plates, test
tubes, and microcentrifuge tubes. In one embodiment, a fusion
protein can be provided that adds a domain that allows the protein
to be bound to a matrix. For example, VEGFR-1-His or PIGF tagged
can be adsorbed onto Ni-NTA microtiter plates, or VEGFR-1-ProtA or
PIGF fusions adsorbed to IgG, which are then combined with the cell
lysates (e.g., .sup.35S-labeled) and the candidate compound, and
the mixture incubated under conditions conducive to complex
formation (e.g., at physiological conditions for salt and pH).
Following incubation, the plates are washed to remove any unbound
label, and the matrix immobilized and radiolabel determined
directly, or in the supernatant after the complexes are
dissociated. Alternatively, the complexes can be dissociated from
the matrix and separated by SDS-PAGE, and the level of VEGFR-1 or
PIGF binding protein found in the bead fraction quantitated from
the gel using standard electrophoretic techniques. Other techniques
for immobilizing protein on matrices can also be used in the drug
screening assays of the invention. For example, either VEGFR-1 or
PIGF and VEGFR-1 or its target molecules can be immobilized
utilizing conjugation of biotin and streptavidin. Biotinylated
VEGFR-1 or PIGF can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art
(e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.) and
immobilized in the wells of streptavidin-coated 96-well plates
(Pierce Chemical). Alternatively, antibodies reactive with VEGFR-1
or PIGF, but which do not interfere with binding of the protein to
its target molecule, can be derivatized to the wells of the plate,
and VEGFR-1 or PIGF trapped in the wells by antibody conjugation.
As described above, preparations of a VEGFR-1-binding protein or
PIGF and a candidate compound are incubated in the VEGFR-1 or
PIGF-presenting wells of the plate, and the amount of complex
trapped in each well can be quantitated.
[0053] Methods for detecting such complexes, in addition to those
described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
VEGFR-1-target molecule or PIGF, or reactive with VEGFR-1 or PIGF
and compete with the target molecule, as well as enzyme-linked
assays that rely on detecting an enzymatic activity associated with
the target molecule. Another technique for drug screening that
provides for high throughput screening of compounds having suitable
binding affinity to VEGFR-1 or PIGF is described in detail in
"Determination of Amino Acid Sequence Antigenicity" by H. N.
Geysen, WO 84/03564, published on Sep. 13, 1984. In summary, large
numbers of different small peptide test compounds are synthesized
on a solid substrate, such as plastic pins or some other surface.
The protein test compounds are reacted with fragments of VEGFR-1 or
PIGF and washed. Bound VEGFR-1 or PIGF is then detected by methods
well known in the art. Purified VEGFR-1 or PIGF can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support. This
invention also contemplates the use of competitive drug screening
assays in which neutralizing antibodies capable of binding VEGFR-1
or PIGF specifically compete with a test compound for binding
VEGFR-1 or PIGF. In this manner, the antibodies can be used to
detect the presence of any protein, which shares one or more
antigenic determinants with VEGFR-1 or PIGF.
[0054] In another embodiment, the invention provides a method for
the production of a pharmaceutical composition comprising the usage
of the method according to claims 6-7 and further mixing the
molecule identified, or a derivative or homologue thereof, with a
pharmaceutically acceptable carrier.
[0055] In another embodiment, PIGF promoter polymorphisms can be
used to identify individuals having a predisposition to acquire
pathological angiogenesis, vascular leakage (edema), pulmonary
hypertension, tumor formation and/or inflammatory disorders.
Indeed, it can be expected that promoter polymorphisms can give
rise to much higher or much lower levels of PIGF. Consequently,
higher levels of PIGF can lead to a predisposition to acquire
pathological angiogenesis, vascular leakage (edema), pulmonary
hypertension, tumor formation and/or inflammatory disorders while
much lower levels of PIGF can lead to a protection to acquire
pathological angiogenesis, vascular leakage (edema), pulmonary
hypertension, tumor formation and/or inflammatory disorders.
[0056] The following examples more fully illustrate preferred
features of the invention, but are not intended to limit the
invention in any way. All of the starting materials and reagents
disclosed below are known to those skilled in the art, and are
available commercially or can be prepared using well-known
techniques.
Examples
1. Impaired Pathological Angiogenesis in PIGF.sup.-/- Mice
[0057] In several pathological conditions, in particular when
associated with increased VEGF expression, formation of new
endothelial-lined channels (angiogenesis) was significantly
impaired in PIGF.sup.-/- mice. Growth and angiogenesis of embryonic
stem (ES) cell-derived tumors, known to be mediated by VEGF (N.
Ferrara et al., 1996, Nature 380, 439-442), was also dependent on
PIGF. Indeed, PIGF.sup.+/+ ES cell-derived tumors obtained within
four weeks after subcutaneous inoculation in nu/nu PIGF.sup.+/+
mice, weighed 4.+-.1 g (n=8) and appeared hemorrhagic and bled
profusely (seven of eight tumors). In contrast, PIGF.sup.-/- tumors
in nu/nu PIGF.sup.-/- hosts only weighed 1.0.+-.0.3 g (n=8) and
were homogeneously white with minimal bleeding (five of seven
tumors). Growth and vascularization in PIGF.sup.-/- tumors were
reduced to the same degree as in VEGF.sup.-/- tumors. PIGF.sup.+/+
and PIGF.sup.-/- tumors contained comparable vascular densities of
endothelial cords and capillaries (diameter <8 .mu.m). However,
compared to PIGF.sup.+/+ tumors PIGF.sup.-/- tumors contained fewer
medium-sized or large vessels. In a previous study, we demonstrated
that formation of medium-sized and large vessels is dependent on
VEGF (P. Carmeliet et al., 1998, Nature 394, 485-490). Angiogenesis
of PIGF.sup.+/+ tumors in nu/nu PIGF.sup.-/- mice or of
PIGF.sup.-/- tumors in nu/nu PIGF.sup.+/+ mice was comparable to
that of PIGF.sup.+/+ tumors in nu/nu PIGF.sup.+/+ mice, indicating
that production of PIGF, either by tumor or by host-derived tissue,
could rescue the phenotype. VEGF transcript levels were comparable
between both genotypes (VEGF/10.sup.3 HPRT mRNA molecules:
280.+-.20 in PIGF.sup.+/+ tumors versus 320.+-.50 in PIGF.sup.-/-
tumors; p=NS) and were expressed in epithelial and mesenchymal
cells throughout PIGF.sup.+/+ tumors, whereas PIGF was expressed in
endothelial cells of small and large vessels (in situ
hybridization). Expression of VEGF-B and VEGF-C was also
comparable.
[0058] Exposure of neonatal mice to 80% oxygen from P7 to P12
causes capillary dropout in the retina due to reduced VEGF
expression (VEGF/10.sup.3 HPRT mRNA molecules in PIGF.sup.+/+
retinas: 270.+-.40 at P7 during normoxia versus 100.+-.10 at P12
during hyperoxia; p<0.05 versus P7; n=5) (T. Alon et al., 1995,
Nat. Med. 1, 1024-1028). Upon reexposure to room air at P12,
retinal ischemia up-regulates VEGF expression (VEGF/10.sup.3 HPRT
mRNA molecules: 390.+-.50 at P13, p<0.05 versus P7; and
165.+-.50 at P17; n=5), thereby inducing venous dilatation,
arterial tortuosity, and capillary outgrowth in the vitreous
chamber by P17 (T. S. Kern et al., 1996, Arch. Opthalmol. 114,
986-990). The role of PIGF in ischemic retinopathy remains unknown.
PLGF transcript levels (PIGF/10.sup.3 HPRT mRNA molecules) were
40.+-.10 at P7 (normoxia), 10.+-.2 at P12 (hyperoxia), 90.+-.10 at
P13 (return to normoxia) and 12.+-.2 at P17. Despite a comparable
retinal vascular development during normoxia and a comparable
capillary dropout during hyperoxia (P12) in both genotypes,
PIGF.sup.-/- mice developed .about.75% fewer and significantly
smaller neovascular vitreous tufts by P17 than PIGF.sup.+/+ mice
(endothelial cells per section: 10.+-.2 in PIGF.sup.-/- mice versus
48.+-.4 in PIGF.sup.+/+ mice; n=6; p<0.05). In addition,
PIGF.sup.-/- mice exhibited reduced venous dilatation
(semiquantitative dilatation score, see methods: 0.9.+-.0.05 in
PIGF.sup.-/- mice versus 1.7.+-.0.03 in PIGF.sup.+/+ mice;
p<0.05) and arterial tortuosity (tortuosity score: 0.8.+-.0.05
in PIGF.sup.-/- mice versus 2.3.+-.0.2 in PIGF.sup.+/+ mice;
p<0.05).
2. Reduced Vascular Permeability in PIGF.sup.-/- Mice
[0059] Vascular permeability, a characteristic feature of VEGF (N.
Ferrara et al., 1999, Curr. Top. Microbial. Immunol. 237, 1-30),
was consistently reduced in PIGF.sup.-/- mice as compared to
PIGF.sup.+/+ mice. VEGF has been previously implicated in vascular
permeability of the skin (L. F. Brown et al., 1995, J. Immunol.
154, 2801-2807), but the role of PIGF remains undetermined. Several
models were used: (i) Intradermal injection of 1, 3 or 10 ng
VEGF.sub.165 induced less extravasation of Evans blue in
PIGF.sup.-/- mice than in PIGF.sup.+/+ mice (Miles assay). (ii)
Skin sensitization with ovalbumin caused less extravasation of
plasma in PIGF.sup.-/- mice than in PIGF.sup.+/+ mice (J.
Casals-Stenzel et al., 1987, Immunopharmacology 13, 177-183)
(Arthus reaction: 12.+-.1 .mu.l extravasated plasma after vehicle
versus 130.+-.5 .mu.l plasma after ovalbumin in PIGF.sup.+/+ mice;
p<0.05; n=12 as compared to 11.+-.1 .mu.l plasma after vehicle
versus 13.+-.1 .mu.l plasma after ovalbumin in PIGF.sup.-/- mice;
p=NS; n=12). (iii) Plasma extravasation in normal skin vessels was
similar in both genotypes (mg plasma.times.10.sup.5/min. mg tissue:
35.+-.5 in PIGF.sup.+/+ mice versus 35.+-.4 in PIGF.sup.-/- mice;
p=NS; n=7) but increased significantly more in PIGF.sup.+/+ than in
PIGF.sup.-/- mice after skin wounding (60.+-.4 in PIGF.sup.+/+ mice
versus 40.+-.4 in PIGF.sup.-/- mice; p<0.05; n=10). Thus, PIGF
specifically increased the vascular permeability in response to
VEGF, but not to histamine.
3. Impaired Pathological Arteriogenesis in PIGF.sup.-/- Mice
[0060] Healing of skin wounds is mediated by ingrowth of vessels,
which initially consist of endothelial cells (angiogenesis) and
subsequently become surrounded by smooth muscle cells
(arteriogenesis). VEGF has been implicated in capillary growth
during skin healing (M. Detmar et al., 1998, J. Invest. Dermatol.
111, 1-6), but the role of PIGF remains unknown. Healing of skin
incisions was slightly retarded in PIGF.sup.-/- as compared to
PIGF.sup.+/+ mice. Both genotypes contained comparable densities of
thrombomodulin-stained vessels in unwounded skin (vessels/mm.sup.2:
240.+-.80 in PIGF.sup.+/+ mice versus 200.+-.80 in PIGF.sup.-/-
mice; p=NS; n=5). Smooth muscle .alpha.-actin staining revealed a
comparable density of vessels (i) that were not covered or
surrounded by a few smooth muscle cells (vessels/mm.sup.2: 58.+-.14
in PLGF.sup.+/+ mice versus 40.+-.12 in PIGF.sup.-/- mice; p=NS;
n=5), and (ii) that were completely covered by at least one smooth
muscle cell layer (vessels/mm.sup.2: 15.+-.5 in PIGF.sup.+/+ mice
versus 19.+-.1 in PIGF.sup.-/- mice; p=NS; n=5). Within four days
after wounding, PIGF was expressed in endothelial cells, and PIGF
and VEGF were up-regulated in PIGF.sup.+/+ keratinocytes in the
hyperplastic epidermis at the wound edge, where new vessels formed
(in situ hybridization; not shown). Both strains contained
comparable vessel ingrowth in the wound region
(thrombomodulin-stained vessels/mm.sup.2: 240.+-.50 in PIGF.sup.+/+
mice versus 180.+-.50 in PIGF.sup.-/- mice; n=5; p=NS). However,
both genotypes differed in the degree the new vessels were covered
by SMA-positive smooth muscle cells. The number of vessels that
were not or incompletely covered by smooth muscle cells was 40.+-.7
in PIGF.sup.+/+ mice versus 84.+-.13 in PIGF.sup.-/- mice
(p<0.05; n=5), whereas the number of vessels that were
completely covered by at least one smooth muscle cell layer was
75.+-.18 in PIGF.sup.+/+ mice versus 30.+-.10 in PIGF.sup.-/- mice
(p<0.05). Thus, lack of PIGF impairs coverage of new endothelial
channels with smooth muscle cells.
[0061] Pulmonary hypertension due to hypoxia-induced remodeling of
the pulmonary vasculature is a life-threatening complication of
chronic obstructive pulmonary disease (COPD). Even though VEGF is
highly up-regulated in lungs of patients with COPD (C. D. Cool et
al., 1999, Am. J. Pathol, 155, 411-419) and of hypoxic animals (H.
Christou et al., 1998, Am. J. Respir. Cell. Mol. Biol. 18,
768-776), its role in this process is not understood. Surprisingly,
no information is available about the expression or role of PIGF.
Therefore, adult mice were exposed to hypoxia (10% O.sub.2) during
four weeks, as this causes pulmonary hypertension due to increased
"muscularization" of the pulmonary vessels (C. A. Hales et al.,
1983, Am. Rev. Respir. Dis. 128, 747-751). The ratio of the right
ventricular (RV) to left ventricular (LV) weight (a measure of RV
hypertrophy) was comparable in both genotypes during normoxia
(32.+-.2% in PIGF.sup.+/+ mice versus 33.+-.2% in PIGF.sup.-/-
mice; n=4; p=NS), significantly increased after hypoxia in
PIGF.sup.+/+ mice (48.+-.4%; n=5; p<0.05 versus normoxia), but
only minimally affected by hypoxia in PIGF.sup.-/- mice (37.+-.2%;
n=6; p<0.05 versus normoxia and versus PIGF.sup.+/+).
Significant genotypic differences in pulmonary vascular remodeling
were observed. Elastin staining of normoxic lungs revealed that
both genotypes had a comparable density of intra-acinar thin-walled
arterioles containing only an internal elastic lamina (IEL), or
thick-walled arterioles containing an intact IEL plus an incomplete
external elastic lamina (EEL) (Table 1). Thick-walled arterioles
containing two intact elastic laminae over their entire
circumference were only occasionally detected in both genotypes
(Table 1). Hypoxia caused significant vascular remodeling in
PIGF.sup.+/+ mice, resulting in a larger fraction of thick-walled
vessels with a partial or complete EEL at the expense of
thin-walled vessels with only a single IEL (Table 1). In contrast,
vascular remodeling was much less significant in PIGF.sup.-/- mice,
resulting in a smaller fraction of thick-walled vessels with a
complete TEL and EEL (Table 1). Immunostaining for smooth muscle
.alpha.-actin (SMA) confirmed that PIGF.sup.+/+ mice contained
significantly more fully muscularized arterioles than PIGF.sup.-/-
mice after hypoxia (Table 1). Protection against pulmonary
hypertension in PIGF.sup.-/- mice was not due to a reduced
vasoconstriction response (RV pressure increased by 31.+-.4% in
PIGF.sup.+/+ mice versus 34.+-.5% in PIGF.sup.-/- mice in response
to 30 minutes 7% O.sub.2; p=NS), nor was it due to lower hematocrit
levels (48.+-.3% in PIGF.sup.+/+ mice versus 53.+-.3% in
PIGF.sup.-/- mice; p=NS). Thus, PIGF significantly modulates
arterial remodeling.
TABLE-US-00001 TABLE 1 Pulmonary vascular remodeling after chronic
hypoxia. Vessels per 10.sup.3 alveoli PLGF.sup.+/+ mice
PLGF.sup.-/- mice 20% O.sub.2 10% O.sub.2 20% O.sub.2 10% O.sub.2
Presence of elastic laminae Single IEL 11 .+-. 2 3.6 .+-. 0.6* 11
.+-. 1 5.6 .+-. 0.6*, # IEL + incomplete 11 .+-. 2 12 .+-. 1 10
.+-. 2 11 .+-. 1 EEL IEL + complete <0.5 6 .+-. 1* <0.5 3
.+-. 1*, # EEL Coverage by SMC Absent 2.4 .+-. 0.9 0.5 .+-. 0.3*
3.1 .+-. 0.7 3.4 .+-. 0.7* Incomplete 11 .+-. 1 12 .+-. 2 8 .+-. 2
11 .+-. 1 Complete 1.2 .+-. 0.5 11 .+-. 2*, # 2.6 .+-. 2 2.8 .+-.
2*, # The data represent the number (average .+-. SEM in five mice)
of vessels per 10.sup.3 alveoli containing a single internal
elastic lamina (IEL), an IEL plus an incomplete external elastic
lamina (EEL), or an IEL plus a complete EEL. In addition, the
density of vessels that were not (absent), incompletely or
completely surrounded by smooth muscle .alpha.-actin stained smooth
muscle cells (SMC) is shown. *p < 0.005 versus normoxia (20%
O.sub.2); #: p < 0.05 versus PLGF.sup.+/+.
4. Synergism Between PIGF and VEGF
[0062] Proliferation and survival of endothelial cells in response
to VEGF were studied. VEGF.sub.165 stimulated proliferation of
PIGF.sup.+/+ endothelial cells (Table 2) and protected them against
apoptosis induced by serum deprivation (0.1% serum) or
supplementation of TNF-.alpha. (10 ng/ml). In contrast,
VEGF.sub.165 failed to stimulate proliferation or to protect
PIGF.sup.-/- endothelial cells against serum deprivation- or
TNF-.alpha.-induced apoptosis (Table 2). PIGF itself was not
mitogenic nor anti-apoptotic for endothelial cells of either
genotype. Also, it did not affect the response of PIGF.sup.+/+
endothelial cells to VEGF (Table 2), likely because PIGF.sup.+/+
endothelial cells already produced sufficient PIGF (the variable
degree of PLGF production by endothelial cells and the relative
amounts of VEGF present in the culture conditions may in fact
explain why some, but not others, have previously observed an
angiogenic response in vitro). However, PIGF rescued the impaired
proliferation and survival response of PIGF.sup.-/- endothelial
cells to VEGF.sub.165 (Table 2). PIGF was also found to modulate
the mitogenic response to VEGF of smooth muscle cells, known to
express VEGFR-1 and VEGFR-2 (C. L. Grosskreutz et al., 1999,
Microvasc. Res. 58, 128-136). Indeed, VEGF stimulated proliferation
of PIGF.sup.+/+ but not of PIGF.sup.-/- smooth muscle cells. PIGF,
ineffective by itself, restored the responsiveness of PIGF.sup.-/-
cells to VEGF. PIGF specifically modulated the activity of VEGF,
since PIGF.sup.-/- and PIGF.sup.+/+ cells displayed comparable
responses to bFGF. Thus, PIGF affected endothelial and smooth
muscle cells only when they were stimulated with VEGF.
[0063] The mechanism found to play a role in the synergism between
VEGF and PIGF: (i) PIGF up-regulated the expression of VEGF, as
previously suggested (M. J. Bottomley et al., 2000, Clin. Exp.
Immunol. 119, 182-188). Expression of VEGF by PIGF.sup.-/-
fibroblasts was increased by PIGF (VEGF production per 10.sup.6
cells/ml/24 hours: 180.+-.10 pg after treatment with vehicle versus
440.+-.10 pg after treatment with 100 ng/ml PIGF for 48 hours;
p<0.05 versus vehicle). Similar results were obtained for
PIGF.sup.+/+ fibroblasts (VEGF production per 10.sup.6 cells/ml/24
hours: 200.+-.8 pg after treatment with vehicle versus 430.+-.5 pg
after treatment with 100 ng/ml PIGF for 48 hours; p<0.05 versus
vehicle).
[0064] Induction of VEGF production by PIGF was, however, smaller
than that induced by hypoxia (1% O.sub.2) (expressed per 10.sup.6
cells/ml/24 hours: 3200.+-.150 pg for PIGF.sup.+/+ cells;
2400.+-.150 pg for PIGF.sup.-/- cells; p<0.05 versus normoxia).
VEGF immunoreactivity was also increased in PIGF.sup.+/+ mice after
treatment with 1.5 .mu.g PIGF.sub.132/24 hours.
TABLE-US-00002 TABLE 2 Role of PLGF and VEGF in endothelial
proliferation. Endothelial proliferation PLGF.sup.+/+ PLGF.sup.-/-
Vehicle 11 .+-. 2 11 .+-. 1 VEGF.sup.120 (100 ng/ml) 22 .+-. 1* 12
.+-. 1 VEGF.sup.165 (100 ng/ml) 36 .+-. 4* 12 .+-. 2 VEGF.sup.165
(300 ng/ml) 42 .+-. 3* 13 .+-. 3 VEGF-E (100 ng/ml) 32 .+-. 1* 13
.+-. 1 PLGF (100 ng/ml) 11 .+-. 1 11 .+-. 1 VEGF.sup.165 (100
ng/ml) + PLGF (100 ng/ml) 33 .+-. 3* 33 .+-. 2* VEGF.sup.165 (100
ng/ml) +anti-NP1 MoAb 27 .+-. 3* N.D. +anti-NP2 MoAb 34 .+-. 3 N.D.
+anti-VEGFR-1 Ab 23 .+-. 5* 12 .+-. 2 +anti-VEGFR-2 MoAb 15 .+-. 2
N.D. VEGF.sup.165 (100 ng/ml) + PLGF (100 ng/ml) +anti-VEGFR-1 Ab
22 .+-. 4 21 .+-. 4 +anti-VEGFR-2 MoAb 13 .+-. 3 17 .+-. 2 bFGF (50
ng/ml) 35 .+-. 2* 35 .+-. 5* bFGF (50 ng/ml) + PLGF (100 ng/ml) 32
.+-. 3* 33 .+-. 1* The data represent the mean .+-.SD of nine to
twelve experiments. *p < 0.05 versus control (vehicle). None of
the antibodies affected baseline endothelial proliferation in the
absence of VEGF (not shown). Ab: polyclonal antiserum; MoAb:
monoclonal antibodies; bFGF: basic fibroblast growth factor.
5. PIGF Specifically Modulates the Responsiveness to VEGF
[0065] Since VEGF plays a role in the above-mentioned phenotypes of
angiogenesis, arteriogenesis and permeability, we investigated
whether PIGF determined the responsiveness to VEGF. Subcutaneous
implantation of matrigel (A. Passaniti et al., 1992, Lab. Invest.
67, 519-528) supplemented with VEGF.sub.165 (VEGF.sub.165) induced
a strong angiogenic response in PIGF.sup.+/+ but not in
PIGF.sup.-/- mice (hemoglobin content: 0.28.+-.0.02 g/dl in
PIGF.sup.+/+ mice versus 0.02.+-.0.02 g/dl in PIGF.sup.-/- mice;
n=15; p<0.05).
[0066] In contrast, basic fibroblast growth factor (bFGF) induced a
similar angiogenic response in both genotypes (hemoglobin content:
0.28.+-.0.02 g/dl in PIGF.sup.+/+ mice versus 0.25.+-.0.02 g/dl in
PIGF.sup.-/- mice; n=15; p=NS). These observations were confirmed
by histological analysis and immunostaining for endothelial factor
VIDE-related antigen.
[0067] The reduced response of PIGF.sup.-/- endothelial cells to
VEGF was confirmed using cultured primary PIGF.sup.-/- endothelial
cells. VEGF.sub.165 (100 ng/ml) was chemotactic for PIGF.sup.+/+
but not for PIGF.sup.-/- endothelial cells, whereas both genotypes
responded comparably to bFGF. PIGF (100 ng/ml) itself was not
chemotactic for endothelial cells of either genotype and did not
affect the response of PIGF.sup.+/+ endothelial cells to VEGF,
likely because endothelial cells already produce abundant PIGF.
However, PIGF completely restored the impaired migration of
PIGF.sup.-/- endothelial cells in response to VEGF.sub.165. PIGF
also enhanced the chemo-attractive activity of VEGF on smooth
muscle cells, known to express VEGFR-1 and VEGFR-2 Grosskreutz et
al., 1999, Microvasc. Res. 58, 128-136). VEGF stimulated the
migration of PIGF.sup.+/+ but not of PIGF.sup.-/- smooth muscle
cells, whereas bFGF stimulated smooth muscle cells of both
genotypes. Similar effects were observed for SMC proliferation.
Even though PIGF alone did not stimulate the cells, it rescued the
impaired smooth muscle cell response to VEGF, further underscoring
that PIGF is essential for the biological activity of VEGF. Thus,
PIGF determines and synergistically amplifies the response to
VEGF.
6. Inhibition of PIGF Impairs Pulmonary Hypertension
[0068] Wild-type mice are injected with different concentrations of
a murine anti-PIGF antibody (0 .mu.g, 1 .mu.g, 5 .mu.g, 10 .mu.g
and 50 .mu.g). Murine anti-PIGF antibody is generated in the
PIGF.sup.-/- mouse. After 72 hours, the mice are placed for four
weeks in a tightly sealed chamber under normobaric hypoxia
(FiO.sub.2 10%). After 28 days, the mice are sacrificed and used
for histological analysis as described in the materials and methods
section. The control mouse with 0 .mu.g anti-PIGF antibody
developed a serious hypoxia-induced pulmonary vascular remodeling.
The murine anti-PIGF antibody prevents this pulmonary vascular
remodeling at very low concentrations.
7. Inhibition of PIGF Impairs Inflammation
[0069] Occlusion of a supply artery is a frequent complication of
atherosclerosis or arterial restenosis and deprives downstream
tissues of oxygen and nutrients. However, coincident enlargement of
preexisting collaterals due to endothelial activation and smooth
muscle growth (adaptive arteriogenesis) may allow residual
perfusion to the ischemic regions and prevent tissue necrosis in
the territory of the occluded artery.
[0070] Even though administration of VEGF protein or VEGF gene
transfer has been shown to improve collateral growth, the role of
endogenous VEGF remains controversial. PLGF has not been previously
implicated in this process. Macrophages play a role in adaptive
arteriogenesis, but the role of PIGF remains unknown. Therefore,
the role of macrophages in adaptive arteriogenesis of collateral
arterioles was studied after ligation of the femoral artery.
Mac3-positive macrophages, known to play an essential role in
collateral growth, were found to adhere to the endothelium and to
infiltrate in and through the wall of the collaterals three days
after ligation. However, more collaterals were infiltrated by
Mac3-positive macrophages in PLGF.sup.+/+ than in PLGF.sup.-/- mice
(45 of 66 PLGF.sup.+/+ collaterals versus 29 of 67 PLGF.sup.-/-
collaterals; p<0.05 by Chi-square analysis; n=5 mice). This may
relate to the known monocyte chemo-attractant activity of PLGF.
Indeed, using another model of leukocyte attraction (local
endotoxin injection in the footpad), three-fold more leukocytes
infiltrated in PLGF.sup.+/+ than in PLGF.sup.-/- vessels
(CD45-positive cells/vessel: 5.2.+-.1 in PLGF.sup.+/+ mice versus
1.5.+-.0.2 .mu.m in PLGF.sup.-/- mice; n=5; p<0.05). Macrophages
may also modulate collateral growth via production of PLGF (8.+-.2
PLGF/10.sup.3 HPRT mRNA molecules; n=5). Another characteristic
feature of adaptive arteriogenesis is the extravasation of
fibronectin, providing a scaffold for migrating smooth muscle
cells. Extravasation of fibronectin was greater in PLGF than in
PLGF.sup.-/- collaterals, as revealed by the more numerous
collateral vessels, surrounded by fibronectin-immunoreactive
deposits (57 of 80 PLGF.sup.+/+ collaterals versus 21 of 83
PLGF.sup.-/- collaterals; n=5 mice; p<0.05 by Chi-Square). The
increased permeability in PLGF.sup.+/+ collaterals may be caused by
the synergism between PLGF and VEGF, known to be released by
activated macrophages. VEGF levels in thioglycolate-stimulated
peritoneal macrophages were 200.+-.11 VEGF/10.sup.3 HPRT mRNA
molecules (n=5). Thus, PLGF is essential for collateral growth.
8. Preparation of Monoclonal Antibodies Against PIGF
[0071] Since PIGF deficiency reduces the phenotype of diverse
pathological processes of angiogensis, arteriogenesis and vascular
leakage comprising ischemic retinopathy, tumor formation, pulmonary
hypertension, vascular leakage (edema formation) and inflammatory
disorders, molecules that can inhibit the formation of PIGF, or
binding PIGF to its receptor (VEGFR-1), or signal transduction
initiated by PIGF, can be useful to treat the above-mentioned
pathological processes. Monoclonal antibodies against murine PIGF-2
were produced essentially as previously described (P. J. Declerck
et al. (1995) J. of Biol. Chem. 270, 8397), however, using mice
with inactivated PIGF genes. The mice were immunized by
subcutaneous injection of murine PIGF-2 (R&D Systems). In
total, 120 hybridomas were produced, of which 15 showed a 50%
inhibition, 38 showed 70% inhibition and five gave complete
inhibition of binding of rmPIGF-2 to its receptor (Flt-1). This was
measured in an immunofunctional ELISA in which 96-well plates were
coated with 100 .mu.l of 1 .mu.g/ml of rmFlt-1/Fc chimera overnight
at room temperature in PBS. After blocking for one hour with 1% BSA
in PBS, 100 .mu.l of a mixture of 70 .mu.l of hybridoma medium
pre-incubated with 70 .mu.l of recombinant rmPIGF-2 at 10 ng/ml for
two hours at room temperature was applied to the plate. A standard
of rmPIGF-2 ranging from 20 ng/ml to 156 pg/ml was included
(diluted in PBS-Tween.BSA-EDTA). Plates were incubated one hour at
37.degree. C. and one hour at room temperature, washed five times
with PBS-Tween and 100 .mu.l of biotinylated goat anti-murine
PIGF-2 at 200 ng/ml was applied for two hours at room temperature.
After washing five times with PBS-Tween, 100 .mu.l of avidin-HRP
conjugate (Vectastorin ABC kit) was applied for one hour at room
temperature. After washing five times with PBS-Tween, the plate was
developed with 90 .mu.l of o-phenylene diamine in citrate phosphate
buffer, pH 5.0, for 30 minutes and measured at 490 nm.
[0072] The five positive clones (PL1H5, PL5D11, PL9F7, PL13F11,
PL17A10) were subcloned, grown and injected in mice (PIGF knockouts
in Balb/c background) to produce ascites. The monoclonal antibodies
were purified on protein-A Sepharose and again tested for
inhibition of binding of m-PIGF-2 to Flt-1/Fc. The results (Table
3) indicate that Mab-PL5D11 markedly inhibits binding of m-PIGF-2
to its receptor. This Mab was selected for evaluation in the edema
model (mustard oil skin application).
TABLE-US-00003 TABLE 3 Inhibition by anti-murine PIGF-2 Mab of
murine PIGF-2 binding to murine Flt-1/Fc. The data represent
residual m-PIGF-2 in percent. Molar excess versus m-PIGF-2 Nr 10 X
5 X 2.5 X 1.25 X No antibody 1 PL1H5G5 66 64 63 89 100 2 PL5D11D4
10 15 22 43 100 PL5D11F10 14 19 22 35 100 3 PL13F11C8 57 70 83 100
100 4 PL17A10E12 40 46 60 89 100 PL17A10F12 41 41 53 90 100
Negative Irrelevant 100 100 100 100 100 control antibody 1 C 8
Concentration of m-PIGF-2 5 5 5 5 5 in ng/ml Concentration of
antibody 200 100 50 25 0 in ng/ml
9. Validation of the PIGF Monoclonals in a Mustard Oil Skin
Application Model
[0073] Mustard oil was painted on the ears of Swiss mice, and
extravasation of Evans blue was determined. Antibodies were
injected intravenously at 300 .mu.g/kg 30 minutes before injection
of Evans blue and application of mustard oil. Briefly, 100 .mu.l of
a test agent was injected via a jugular vein catheter, followed 30
minutes later by an intravenous injection of 300 .mu.l of 0.5%
Evans blue. One ear was painted with 0.1% mustard oil and repainted
again 15 minutes later. After 30 minutes, the mouse was perfused
via the left ventricle with saline containing 100 U/ml heparin,
followed by 3% paraformaldehyde in citrate buffer. The ears were
amputated, dissected in small segments and extracted overnight in
formamide at 55.degree. C. The absorbance of the extraction fluid
was measured at 610 nm.
[0074] Anti-PIGF antibodies, which blocked the PIGF response of
endothelial cells (Table 4), reduced vascular leakage in wild-type
mice application of mustard oil on their ears (relative absorbance
units/ear: 13.+-.3 after mineral oil; 53.+-.7 after mustard oil
plus control IgGs; 26.+-.5 after mustard oil plus anti-PIGF; n=5;
p<0.05).
TABLE-US-00004 TABLE 4 Effect of mustard oil on Evans blue
extravasation. Group A.sup.610 nm p No antibody 0.76 .+-. 0.09 --
PL5D11D4 10 .mu.g 0.50 .+-. 0.05 0.12 PL17A10E12 10 .mu.g 0.37 .+-.
0.04 0.03
10. Isolation of PIGF Inhibitors by Screening of a Tetrameric
Library
[0075] A tetrameric library has been assayed for its ability to
inhibit in ELISA assays the binding between the recombinant mouse
Placental Growth Factor 2 (mPIGF-2) (R&D Systems Cat. No.
645-PL) and the recombinant mouse Vascular Endothelial Growth
Factor Receptor 1 (VEGF R1) Flt-1/Fc chimera (R&D Systems, Cat.
No. 471-F1). The ELISA assay was performed following this
procedure. The receptor was coated on a microtiter plate (Costar,
Cat. No. 3590) at 1 .mu.g/ml in NaH.sub.2PO.sub.4 50 mM, NaCl 150
mM pH 7.5 (PBS), 100 .mu.l/well, for 16 hours at room temperature.
The wells were washed five times with PBS containing 0.004% Tween
(PBS-T) and blocked with Bovine Serum Albumin (BSA) 1% in PBS, 150
.mu.l/well, for two hours at room temperature. The plate was washed
with PBS-T five times, and 100 .mu.l of mPIGF diluted at 8 ng/ml in
PBS pH 7.5, BSA 0.1%, EDTA 5 mM, Tween 0.004% (PBET), was added to
each well. After the incubation for one hour at 37.degree. C. and
one hour at room temperature, the plate was washed again, and
biotinylated anti-mPIGF antibodies (R&D Systems, Cat. No. BAF
465) were added at 200 ng/ml in PBET, 100 .mu.l/well, and incubated
for two hours at room temperature. The plate was washed five times
with PBS-T, 100 .mu.l/well of a solution containing an
avidin-biotin system (Vectastatin Elite ABC kit, Vector
Laboratories, Cat. No. PK 6100). This solution was prepared by
mixing one drop of solution A and one drop of solution B in 2.5 ml
of Tris-HCl 50 mM and diluting this mix 1/100 in PBET. After one
hour of incubation, the plate was washed as above, and 90
.mu.l/well of a solution containing 1 mg/ml of O-Phenylenediamine
(OPD) in citrate buffer pH 5.0 was added to each well. After 40
minutes, the developing reaction was stopped by adding 30
.mu.l/well of sulfuric acid 3M. The plate was read at 490 nm with
an ELISA reader. To test the inhibitory activity of the library,
each pool of the library was added in competition with mPIGF using
a molar excess of 1000 times (1:1000, 1:2000 means a molar excess
of 2000; 1:1500 means a molar excess of 1500, etc.). The results of
the screening are shown in Table 5.
TABLE-US-00005 TABLE 5 plate 1 OD 490 nm mPIGF 8 ng/ml 100 0.993
mPIGF 5 ng/ml 67.673716 0.672 R&D mAb (1:5) 51.0574018 0.507 1
D-Ala 80.5639476 0.8 2 D-Asp 75.5287009 0.75 3 D-Val 80.5639476 0.8
4 D-Glu 57.4018127 0.57 5 L-Cha 87.6132931 0.87 6 D-Phe 79.0533736
0.785 7 D-Thr 83.5850957 0.83 8 D-Met 84.592145 0.84 9 D-Cys(Acm)
91.6414904 0.91 10 D-Lys 88.8217523 0.882 11 D-Tyr 90.6344411 0.9
12 D-Pro 93.6555891 0.93 13 D-Leu 95.367573 0.947 14 D-His
107.75428 1.07 15 D-Gln 101.711984 1.01 16 D-Trp 101.711984 1.01 17
D-Arg 105.740181 1.05 18 D-Asn 106.747231 1.06 19 D-Ile 103.222558
1.025 20 D-Arg(Tos) 103.323263 1.026 plate 2 OD 490 nm mPIGF 8
ng/ml 100 0.991 mPIGF 5 ng/ml 71.6448032 0.71 21 D-Ser 83.6528759
0.829 22 L-Cys(Acm) 83.8546922 0.831 23 L-Cys(Bzl) 84.7628658 0.84
24 L-Cys(p-MeBzl) 89.3037336 0.885 25 L-Cys(tBu) 92.0282543 0.912
26 L-Mt(O) 83.9556004 0.832 27 L-Met(O2) 78.6074672 0.779 28
L-Glu(a-OalI) 92.244898 0.904 29 b-Ala 100 0.98 30 Gly 92.1428571
0.903 4 D-Glu 1:500 86.1884368 0.805 4 D-Glu 1:1000 59.1006424
0.552 4 D-Glu 1:1500 48.2869379 0.451 4 D-Glu 1:2000 39.9357602
0.373 2 D-Asp 1:500 89.9357602 0.84 2 D-Asp 1:1000 78.9079229 0.737
2 D-Asp 1:1500 78.0513919 0.729 2 D-Asp 1:2000 74.9464668 0.7 14
D-His 1:500 96.6809422 0.903 14 D-His 1:1000 101.391863 0.947 14
D-His 1:1500 100.535332 0.939 14 D-His 1:2000 96.8950749 0.905
[0076] Thirty pools of 900 different tetrameric peptides were
screened for the possibility to interfere with the binding of
mPIGF-2 to mVEGFR-1. Clearly, pool 4 on Table 5 (with D-Glu on the
terminal position) has inhibitory activity when compared with two
anti-PIGF antibodies to interfere with binding (R&D mAb and a
homemade mAb) of mPIGF-2 to mVEGER-1.
Materials and Methods
Models of Angiogenesis
[0077] Morphometric analysis of myocardial, renal, and retinal
angiogenesis in neonatal mice was performed as described (P.
Carmeliet et al., 1999, Nat. Med. 5, 495-502).
[0078] Matrigel Assay:
[0079] Ingrowth of capillaries in matrigel was performed as
described (A. Passaniti et al., 1992, Lab. Invest. 67, 519-528).
Briefly, 500 .mu.l ice-cold matrigel containing heparin (300
.mu.g/ml) and VEGF (100 ng/ml), or basic fibroblast growth factor
(bFGF; 100 ng/ml) was injected subcutaneously. After seven days,
the matrigel pellet with the neovessels was dissected for analysis
of neovascularization: one part was homogenized to determine the
hemoglobin content determined using Drabkin's reagent (Sigma, St.
Quentin Fallavier, France), whereas the other part was fixed in 1%
paraformaldehyde for histological analysis.
[0080] ES-Tumor Model:
[0081] For ES cell-derived tumor formation, 5.times.10.sup.6 ES
cells were subcutaneously injected into PIGF.sup.+/+ Nu/Nu or
PIGF.sup.-/- Nu/Nu mice, obtained by intercrossing PIGF.sup.+/-
Nu/Nu mice, as described (P. Carmeliet et al., 1998, Nature 394,
485-490). Vascular densities were quantitated by counting the
number of endothelial cords and capillaries (diameter <8 .mu.m),
medium-sized vessels (diameter 10-25 .mu.m), or large vessels
(diameter >30 .mu.m) per field (1.2 mm.sup.2) in six to eight
randomly chosen optical fields on three to five adjacent sections
(320 .mu.m apart) per tumor using the Quantimet Q600 imaging
system.
[0082] Ischemic Retina Model:
[0083] Retinal ischemia was induced by placing P7 neonatal mice in
a cage of hyperbaric (80%) oxygen for five days, after which they
were returned for another five days in normal room air, as
described (L. E. Smith et al., 1994, Invest. Ophthalmol. Vis. Sci.
35, 101-111). Fluorescent retinal angiography and endothelial cell
counts on retinal cross-sections were determined as reported (H. P.
Hammes et al., 1996, Nat. Med. 2, 529-533). Venous dilatation and
arterial tortuosity were semi-quantitatively scored on a scale from
0-3.
[0084] Wound Models:
[0085] Vascular remodeling during skin wound healing was analyzed
within four days after occurrence of a 10 mm full-thickness skin
wound on the back of the mouse, as described (S. Frank et al.,
1995, J. Biol. Chem. 270, 12607-12613). Wound healing was scored by
daily measurements of the width of the wound.
[0086] Pulmonary Hypertension:
[0087] Adult mice were placed for four weeks in a tightly sealed
chamber under normobaric hypoxia (FiO.sub.2 10%), as described (C.
A. Hales et al., 1983, Am. Rev. Respir. Dis. 128, 747-751). After
28 days, mice were used for measurements of hematocrit, using an
automated cell counter (Abbott Cell-Dyn 1330 system, Abbott Park,
Ill.) and for histological analysis. Right ventricular pressures
(RVP) were measured in anesthetized ventilated mice (sodium
pentobarbital, 60 mg/kg, i.p.) by transthoracic puncture using
high-fidelity pressure micromanometers (Millar) after inhalation of
a gas mixture containing 20% O.sub.2 or 7% O.sub.2. For histology,
mice were perfused with 1% phosphate-buffered paraformaldehyde at
100 cm H.sub.2O pressure via the heart and at 30 cm H.sub.2O
pressure through the trachea. Visualization of the internal elastic
lamina (IEL) and external elastic lamina (EEL) was achieved using
Hart's elastin stain. Hypoxia-induced pulmonary vascular remodeling
was assessed by counting the number of non-muscularized (only IEL)
and partially (IEL plus incomplete EEL) or fully (IEL plus complete
EEL) muscularized peripheral vessels (landmarked to airway
structures distal to the terminal bronchioli) per 100 alveoli in
fields containing 5.times.500 alveoli (C. A. Hales et al., 1983,
Am. Rev. Respir. Dis. 128, 747-751).
Vascular Permeability
[0088] Arthus reaction (allergen-induced edema formation in the
skin (J. Casals-Stenzel et al., 1987, Immunopharmacology 13,
177-183)): mice were sensitized by intraperitoneal (i.p.) injection
of saline (1 ml/kg) containing ovalbumin (40 .mu.g/kg; Sigma, St.
Louis, Mo.) and Al(OH).sub.3 (0.2 mg/ml added to the antigen
solution one hour prior to injection) on days 0 and 2.
[0089] Vascular leakage was quantified 14 days after
presensitization by determining the amount of intravenously
injected 125I-bovine serum albumin (BSA) and Evans blue dye
accumulating in the skin injection site. Therefore, the fur on the
dorsal skin of anaesthetized mice was shaved, and 1.5 .mu.Ci/kg of
125I-BSA (2.8 .mu.Ci/.mu.g; NEN-Dupont, France) mixed with a
solution of Evans blue dye in sterile saline (15 mg/kg) was
injected i.v. Ten minutes later, ovalbumin (100 ng/site) was
injected at four intradermal sites. After 60 minutes, the degree of
vascular leakage was quantified: (i) by measuring the diameter of
the edematous spot (visualized by its coloration) using a
micrometer; and (ii) by determining the amount of extravasated
plasma protein at each skin site (expressed as .mu.l extravasated
plasma) after normalizing the 125I-cpm in the skin (10 mm punch)
for the 125I-cpm in 1 .mu.l of plasma.
[0090] Miles assay: Vascular permeability was assayed using the
Miles assay (N. McClure, 1994, J. Pharmacol. Toxicol. Methods).
Briefly, mice were shaved and injected with 50 .mu.l of a solution
containing 0.5% Evans blue in saline 45 minutes prior to
intradermal injection of 20 .mu.l phosphate-buffered saline (PBS)
containing 1, 3 or 10 ng recombinant human VEGF.sub.165; pictures
were taken 45 minutes later.
[0091] Skin healing: After shaving, a standardized 15 mm
full-thickness skin incision was made on the back of anesthetized
mice, taking care not to damage the underlying muscle.
Extravasation of 125I-BSA (expressed as g plasma/g tissue/min) was
measured as described (R. G. Tilton et al., 1999, Invest.
Ophthalmol. Vis. Sci. 40, 689-696).
In Vitro Angiogenesis Assays
[0092] Endothelial and Smooth Muscle Cell Culture:
[0093] In order to obtain mouse capillary endothelial cells,
anesthetized mice were injected s.c. with 500 .mu.l of ice-cold
matrigel containing bFGF (100 ng/ml) and heparin (100 .mu.g/ml).
After seven days, the matrigel pellet was dissected and
enzymatically dispersed using 0.1% type II collagenase (Sigma, St.
Louis, Mo.). Mouse endothelial cells were routinely cultured in T75
flasks coated with 0.1% gelatin in M131 medium supplemented with 5%
MVGS (Gibco-BRL). Smooth muscle cells from mouse aorta were
harvested and cultured as described (J. M. Herbert et al., 1997,
FEBS Lett. 413, 401-404). Before stimulation, cells were starved in
medium with 0.5% serum for 24 hours, after which they were
stimulated with human VEGF.sub.165 and/or murine PIGF, or bFGF (all
from R&D, Abingdon, UK) for 24 hours before analysis of the
total cell number (proliferation) or the number of cells migrated
after scrape-wounding (migration).
Synthesis of a Tetrameric Library
[0094] A tetrameric library has been synthesized using commercially
available 9-Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acids
(purity >99%). All derivatives are listed in Table 6 along with
catalog numbers and company names (provider) from which they have
been purchased.
TABLE-US-00006 TABLE 6 List of building blocks used throughout the
peptide library preparation. Building block Number 3-letter code
Building block Protected derivative Provider Catalog number 1 D-Ala
D-Alanine N.alpha.-Fmoc-D-Alanine Chem-Impex Intl 02372 2 D-Asp
D-Aspartic acid N.alpha.-Fmoc-D-Aspartic acid (t-butyl Chem-Impex
Intl 02478 ester) 3 D-Val D-Valine N.alpha.-FmocD-Valine Chem-Impex
Intl 02471 4 D-Glu D-Glutamic acid N.alpha.-Fmoc-D-Glutamic acid
(t-butyl Chem-Impex Intl 02479 ester) 5 L-Cha L-Cyclohexylalanine
N.alpha.-Fmoc-L-Cyclohexylalanine Sygena FC-01-003-117 6 D-Phe
D-Phenylalanine N.alpha.-Fmoc-D-Phenylalanine Novabiochem
04-13-1030 7 D-Thr D-Threonine N.alpha.-Fmoc-D-Threonine Chem-Impex
Intl 02483 (O-t-butyl-ether) 8 D-Met D-Methionine
N.alpha.-Fmoc-D-Methionine Novabiochem 04-13-1003 9 D-Cys(Acm)
D-Cysteine (S- N.alpha.-Fmoc-D-Cysteine (S- Novabiochem 04-13-1054
acetamydomethyl) acetamydomethyl) 10 D-Lys D-Lysine
N.alpha.-Fmoc-D-Lysine (N.sup..epsilon.-t- Alexis 104-041-G005
butyloxycarbonyl) 11 D-Tyr D-Tyrosine N.alpha.-Fmoc-D-Tyrosine
(O-t-butyl-ether) Chem-Impex Intl 02465 12 D-Pro D-Proline
N.alpha.-Fmoc-D-Proline Novabiochem 04-13-1031 13 D-Leu D-Leucine
N.alpha.-Fmoc-D-Leucine Chem-Impex Intl 02427 14 D-His D-Histidine
N.alpha.-Fmoc-D-Histidine (N.sup.r-trytil) Alexis 104-034-G005 15
D-Gln D-Glutamine N.alpha.-Fmoc-D-Glutamine (N-trytil) Novabiochem
04-13-1056 16 D-Trp D-Triptophan N.alpha.-Fmoc-D-Triptophan
(N.sup.in-t- Chem-Impex Intl 02484 butyloxycarbonyl) 17 D-Arg
D-Arginine N.alpha.-Fmoc-D-Arginine (N.sup.r- Alexis 104-113-G005
pentamethylchroman) 18 D-Asn D-Asparagine
N.alpha.-Fmoc-D-Asparaqine (N-trytil) Chem-Impex Intl 02477 19
D-Ile D-Isoleucine N.alpha.-Fmoc-D-Isoleucine Chem-Impex Intl 03448
20 D-Arg(Tos) D-Arginine (N.sup.r-Tosyl) N.alpha.-Fmoc-D-Arginine
(N.sup.r-Tosyl) Chem-Impex Intl 02382 21 D-Ser D-serine
N.alpha.-Fmoc-D-serine (O-t-butyl-ether) Alexis 104-066-G005 22
L-Cys(Acm) L-Cysteine (S- N.alpha.-Fmoc-L-Cysteine (S- Chem-Impex
Intl 02396 acetamydomethyl) acetamydomethyl) 23 L-Cys(Bzl)
L-Cysteine (S-benzyl) N.alpha.-Fmoc-L-Cysteine (S-benzyl)
Novabiochem 04-12-1015 24 L-Cys(p- L-Cysteine (S-p-methyl-
N.alpha.-Fmoc-L-Cysteine (S-p-methoxy- Chem-Impex Intl 02399 MeBzl)
benzyl) benzyl) 25 L-Cys(tBu) L-Cysteine (S-tert-butyl)
N.alpha.-Fmoc-L-Cysteine (S-tert-butyl) Novabiochem 04-12-1016 26
L-Met(O) L-Methionine-sulphone N.alpha.-Fmoc-L-Methionine-sulphone
Novabiochem 04-12-1112 27 L-Met(O).sub.2 L-Methionine-sulphoxide
N.alpha.-Fmoc-L-Methionine-sulphoxide Novabiochem 04-12-1113 28
L-Glu(.beta.-OAll) L-Glutamic acid-(.beta.-allyl)
N.alpha.-Fmoc-L-Glutamic acid-(.beta.-allyl) Novabiochem 04-12-1158
29 .beta.-Ala .beta.-Alanine N.alpha.-Fmoc-.beta.-Alanine
Chem-Impex Intl 02374 30 Gly Glycine N.alpha.-Fmoc-Glycine
Chem-Impex Intl 02416
[0095] The library has been built onto the following scaffold (G.
Fassina et al. (1996) J. Mol. Recogn. 9, 564; M. Marino et al. Nat.
Biotechn. (1997) 18, 735):
##STR00001##
where G represents the amino acid Glycine and K represents the
amino acid L-Lysine onto which three levels of randomization have
been achieved by applying the Portioning-Mixing method (A. Furka et
al. (1991) Int. J. Pept. Protein. Res. 37, 487; K. S. Lam (1991)
Nature 354, 82). The total number of peptides generated (N.sub.t)
can be calculated by the following formula:
N.sub.tB.sup.x
Where B is the number of building blocks used (30) and x is the
number of randomization (3).
a) Synthesis of the Scaffold
[0096] The initial scaffold has been prepared by manual solid phase
synthesis starting from 419 mg of Fmoc-Gly-derivatized
4-hydroxymethylphenoxyacetic polystyrene resins (PS-HMP)
(substitution 0.75 mmol/g, Novabiochem Cat. No. 04-12-2053),
corresponding to 314 .mu.mol of Glycine onto which two subsequent
couplings with Fmoc-L-Lys(Fmoc)-OH (Chem-Impex Intl. Cat. No.
01578) have been carried out. The resin has been placed in a 35 ml
polypropylene cartridge endowed with a polypropylene septum
(AllTech, Cat. No. 210425) and washed three times with 4.0 ml of
N,N-dimethylformamide (DMF, Peptide synthesis grade, LabScan, Cat.
No. H6533). To remove the Fmoc protection, the resin has been
treated for 15 minutes with 5.0 ml of 20% Piperidine (BIOSOLVE LTD,
Cat. No. 16183301) in DMF and then washed three times with 4.0 ml
of DMF. For the coupling of the first Lysine, 1.5 mmol of
Fmoc-L-Lys(Fmoc)-OH (0.87 g) have been dissolved in 6.0 ml of DMF
and then activated by adding 3.0 ml of a 0.5 M solution of
2-(1H-Benzotriazol-yl)-1,1,3,3-tetramethyl-uranium
tetrafluoroborate (TBTU, >99%, Chem-Impex Intl, Cat. No. 02056)
and 1-Hydroxybenzotriazole (HOBt, SIGMA-ALDRICH, Cat. No. H2006) in
DMF and 3.0 ml of a 1 M solution of Di-isopropyl-ethylamine (DMA
SIGMA-ALDRICH, Cat. No, D-3387) in DMF. After four minutes stirring
at room temperature, the solution has been transferred onto the
resin and stirred for 30 minutes. To remove the excess of amino
acid, the resin has been washed three times with 4.0 ml of DMF. The
deprotection of the Lysine-Fmoc groups has been achieved by
treating the resin for 15 minutes with 5.0 ml of 20% Piperidine in
DMF and washing three times with 4.0 ml of DMF to remove the excess
of reagent. For the coupling of the second Lysine, 3.0 mmol of
Fmoc-L-Lys(Fmoc)-OH (1.77 g) have been dissolved in 6.0 ml of DMF
and then activated adding 6.0 ml of a 0.5 M solution of TBTU/HOBt
in DMF and 6.0 ml of DIEA in DMF. After four minutes stirring at
room temperature, the solution has been transferred onto the resin
and stirred for 30 minutes. The resin has then been washed three
times with 4.0 ml of DMF. The final Fmoc groups have been removed
by treatment with 10.0 ml of 20% Piperidine in DMF for 15 minutes.
The resin has then been submitted to the following washings:
TABLE-US-00007 Number of Solvent washes Volume (ml) DMF 3 4.0 MeOH*
3 4.0 Et.sub.2O** 3 4.0 *Methanol (MeOH, LabScan, Cat. No. A3513)
**Ethyl Ether (Et.sub.2O, LabScan, Cat. No. A3509E)
[0097] The resin has been dried by applying a Nitrogen stream and
then weighed. The final weight of the resin was 442 mg.
b) Assembly of the Library
[0098] The library has been generated by applying the following
procedure:
[0099] b.1 Resin Splitting into 30 Equal Aliquots.
[0100] The resin has been transferred into a 50 ml polypropylene
graduated tube and 35 ml of DMF:DCM (2:3) (DCM, Dichloromethane,
LabScan, Cat. No. H6508L) have been added. The suspension has been
thoroughly mixed and fractions of 1.0 ml have been dispensed in 30
polypropylene syringes (3 ml) endowed with filtration septa at the
bottom (Shimadzu Corp. Cat. No. 292-05250-02). The remaining volume
of suspension has been diluted up to 35 ml with DMF:DCM (2:3) and
1.0 ml fractions have been again dispensed into the syringes. The
graduated tube has been once again filled up to 30 ml and 1.0 ml
aliquots distributed into the syringes. The syringes have been
vacuum drained from the bottom and the resins washed once with 1.0
ml of DMF. The syringes, labeled with numbers from 1 to 30,
contained an equal fraction of resin corresponding to around 10
.mu.moles of scaffold (40 .mu.moles of NH.sub.2 groups).
[0101] b.2 Coupling of the First Random Residue (Position 3).
[0102] 0.75 M stock solutions in DMF of the 30 amino acids (listed
in Table 5) have been prepared and stored at 4.degree. C. until
use. To carry out couplings and deprotection on sub-libraries, from
this step forward, a PSSM8 eight-channel peptide synthesizer
(Shimadzu Corp.) has been used. The syringes have been placed into
the synthesizer and 267 .mu.l (200 .mu.moles) of amino acids have
been dispensed into 2 ml polypropylene tubes (Eppendorf, Cat. No.
24299). The machine automatically performs activations, acylations,
deprotections and washings. Activations were carried out using 400
.mu.l of a 0.5 M solution in DMF of TBTU/HOBt plus 400 .mu.l of a 1
M solution in DMF of DIEA. Acylations have been carried out for 30
minutes, mixing the suspensions by bubbling nitrogen from the
bottom of the syringes, Deprotection have been carried out for 15
minutes with 0.9 ml of 20% piperidine/DMF, while washings have been
performed with 0.9 ml of DMF (three times, two minutes).
[0103] In the first round, amino acids 1 to 8 have been coupled, in
the second round, amino acids 9 to 16 were coupled, in the third
round, amino acids 17 to 24 were coupled, and in the final round,
amino acids 25 to 30 were coupled.
[0104] b.3 Mixing and Re-Splitting of Resins.
[0105] To each syringe, 500 .mu.l of DMF:DCM (2:3) have been added
and the resins suspended by gentle swirling. The suspensions have
been removed from the syringes, collected in a 50 ml polypropylene
tube (graduated) and thoroughly mixed by vigorous shaking.
[0106] After addition of DMF:DCM (2:3) up to a final volume of
around 35 ml, 1.0 ml aliquots of suspension have been re-dispensed
into the 30 syringes, repeating the operations described in step 1
("Resin splitting into 30 equal aliquots"). At the end, the resins
have been washed once with 1.0 ml of DMF.
[0107] b.4 Coupling of the Second Random Residue (Position 2).
[0108] All operations described in step 2 ("Coupling of the first
random residue") were repeated.
[0109] b.5 Mixing and Re-Splitting of Resins.
[0110] All operations described in step 3 ("Mixing and re-splitting
of resins") were repeated.
[0111] b.6 Coupling of the Third Known Residue (Position 1).
[0112] All operations described in step 2 ("Coupling of the first
random residue") have been repeated.
[0113] b.7 Final Washes and Drying of the Resins.
[0114] The resins were washed three times with 1 ml of DCM, three
times with 1 ml of MeOH, and two times with 1 ml of Et.sub.2O. The
resins have then been dried under vacuum.
[0115] b.8 Cleavage.
[0116] Thirty ml of a TFA-H.sub.2O-TIS (100:5:5, v/v/v) mixture
(TIS, tri-iso-propylsilane, SIGMA-ALDRICH Cat. No. 23, 378-1) have
been freshly prepared and 800 .mu.l added to each syringe. After
vortexing for three hours, the resins have been filtered off,
collecting the acidic solution directly in 15 ml polypropylene
tubes (labeled with numbers from 1 to 30) containing 5 ml of cold
Et.sub.2O. The white precipitates have been separated by
centrifugation at 3000 rpm for ten minutes and the organic solvents
discarded. The precipitates have been washed once with 5 ml of cold
Et.sub.2O and, after centrifugation, were dissolved in 2.0 ml of
H.sub.2O/CH.sub.3CN/TFA 50:50:01 and lyophilized. Ten mg/ml stock
solutions of the peptide libraries in DMSO have been prepared and
stored in sealed vials at -80.degree. C.
* * * * *