U.S. patent application number 16/704861 was filed with the patent office on 2020-11-19 for anti-flt-1 antibodies in treating bronchopulmonary dysplasia.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF COLORADO, A BODY CORPORATE, SHIRE HUMAN GENETIC THERAPIES, INC.. Invention is credited to Steven ABMAN, Dennis KEEFE, Gregory SEEDORF.
Application Number | 20200362041 16/704861 |
Document ID | / |
Family ID | 1000004991513 |
Filed Date | 2020-11-19 |
United States Patent
Application |
20200362041 |
Kind Code |
A1 |
KEEFE; Dennis ; et
al. |
November 19, 2020 |
ANTI-FLT-1 ANTIBODIES IN TREATING BRONCHOPULMONARY DYSPLASIA
Abstract
The present invention provides, among other things, methods and
compositions for treating chronic lung disorders, in particular,
bronchopulmonary dysplasia (BPD). In some embodiments, a method
according to the present invention includes administering to an
individual who is suffering from or susceptible to BPD an effective
amount of an anti-Flt-1 antibody, or antigen binding fragment
thereof, such that at least one symptom or feature of BPD is
reduced in intensity, severity, or frequency, or has delayed
onset.
Inventors: |
KEEFE; Dennis; (Lexington,
MA) ; ABMAN; Steven; (Aurora, CO) ; SEEDORF;
Gregory; (Aurora, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHIRE HUMAN GENETIC THERAPIES, INC.
THE REGENTS OF THE UNIVERSITY OF COLORADO, A BODY
CORPORATE |
Lexington
Denver |
MA
CO |
US
US |
|
|
Family ID: |
1000004991513 |
Appl. No.: |
16/704861 |
Filed: |
December 5, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15564969 |
Feb 7, 2018 |
10738123 |
|
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PCT/US16/26420 |
Apr 7, 2016 |
|
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16704861 |
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62144241 |
Apr 7, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/07 20130101;
C07K 2317/94 20130101; A61K 2039/54 20130101; A61K 9/0012 20130101;
A23L 33/10 20160801; A61K 39/3955 20130101; C07K 2317/21 20130101;
C07K 2317/33 20130101; C07K 16/2863 20130101; C07K 2317/55
20130101; C07K 2317/626 20130101; C07K 2317/92 20130101; A23V
2002/00 20130101; A61K 9/0053 20130101; A61K 9/007 20130101; C07K
2317/74 20130101; C07K 2317/54 20130101; A61K 2039/545 20130101;
C07K 2317/622 20130101; A61K 9/0019 20130101; A61K 31/56 20130101;
A61K 2039/505 20130101; C07K 2317/76 20130101; C07K 2317/24
20130101; A61K 33/00 20130101; C07K 2317/52 20130101; A61K 45/06
20130101; A61K 9/0073 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 9/00 20060101 A61K009/00; A61K 31/07 20060101
A61K031/07; A61K 31/56 20060101 A61K031/56; A61K 33/00 20060101
A61K033/00; A61K 39/395 20060101 A61K039/395; A61K 45/06 20060101
A61K045/06; A23L 33/10 20060101 A23L033/10 |
Claims
1.-32. (canceled)
33. A method of treating bronchopulmonary dysplasia (BPD) in an
infant comprising administering to an infant in need of treatment
an effective amount of an anti-Flt-1 antibody or antigen binding
fragment thereof, wherein the administration of the anti-Flt-1
antibody or antigen binding fragment thereof results in improved
lung development relative to a control.
34. The method of claim 33, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof has binding affinity to human
Flt-1 greater than 10.sup.-12 M in a surface plasmon resonance
binding assay.
35. The method of claim 33, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof is characterized with an IC.sub.50
below 1 pM in a competition assay with human Flt-I.
36. The method of claim 33, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof does not bind to VEGFR2 and/or
VEGFR3.
37. The method of claim 33, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof is selected from the group
consisting of IgG, F(ab').sub.2, F(ab).sub.2, Fab', Fab, ScFvs,
diabodies, triabodies and tetrabodies.
38. The method of claim 37, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof is IgG.
39. The method of claim 38, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof is IgG1.
40. The method of claim 38 or 39, wherein the anti-Flt-1 antibody
or antigen binding fragment thereof is a monoclonal antibody,
wherein the monoclonal antibody contains a human Fc region.
41. The method of claim 40, wherein the Fc region contains one or
more mutations that enhance the binding affinity between the Fc
region and the FcRn receptor such that the in vivo half-life of the
antibody is prolonged.
42. The method of claim 41, wherein the Fc region contains one or
more mutations at one or more positions corresponding to Thr 250,
Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433,
and/or Asn 434 of human IgG1.
43. The method of claim 33, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof is administered parenterally.
44. The method of claim 43, wherein the parenteral administration
is selected from intravenous, intradermal, intrathecal, inhalation,
transdermal (topical), intraocular, intramuscular, subcutaneous,
pulmonary delivery, and/or transmucosal administration.
45. The method of claim 33, wherein the effective amount of
anti-Flt-1 antibody or antigen-binding fragment thereof ranges from
0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
46. The method of claim 33, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof is administered bimonthly,
monthly, triweekly, biweekly, weekly, daily, or at variable
intervals.
47. The method of claim 33, wherein the anti-Flt-1 antibody or
antigen binding fragment thereof is delivered to one or more target
tissues selected from lungs and heart.
48. The method of claim 33, wherein the administration of the
anti-Flt-1 antibody or antigen binding fragment thereof results in
growth of healthy lung tissue, decreased lung inflammation,
increased alveologenesis, increased angiogenesis, improved
structure of pulmonary vascular bed, reduced lung scarring,
improved lung growth, reduced respiratory insufficiency, improved
exercise tolerance, reduced adverse neurological outcome, and/or
improved pulmonary function relative to a control.
49. The method of claim 33, further comprising co-administering at
least one additional agent or therapy selected from a surfactant,
oxygen therapy, ventilator therapy, a steroid, vitamin A, inhaled
nitric oxide, high calorie nutritional formulation, a diuretic,
and/or a bronchodilator.
50. The method of claim 33, wherein the infant is an unborn infant
and the anti-Flt-1 antibody or antigen binding fragment thereof is
administered to the infant via intra-amniotic injection.
51. The method of claim 33, wherein the method comprises parental
administration to the infant after birth.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 15/564,969
filed Feb. 7, 2018, which is a 35 U.S.C. .sctn. 371 National Stage
Application of International Application No. PCT/US2016/26420,
filed Apr. 7, 2016, which claims priority to U.S. Provisional
Application Ser. No. 62/144,241, filed Apr. 7, 2015, the disclosure
of which is hereby incorporated by reference.
INCORPORATION-BY-REFERENCE
[0002] The content of the text file named "SHR-1188US2_ST25.txt,"
which was created on May 5, 2020 and is 1.78 KB in size, is hereby
incorporated by reference in its entirety.
BACKGROUND
[0003] Bronchopulmonary dysplasia (BPD) is a severe, chronic lung
disease that primarily affects premature infants. Premature infants
can develop BPD after their lungs have been damaged from the use of
supplemental oxygen and mechanical breathing aids. Infants with BPD
have inflammation and scarring in the lungs and in severe cases,
are at high risk for prolonged need for ventilator or oxygen
support, pulmonary hypertension, recurrent respiratory infections,
abnormal lung function, exercise intolerance, late
neuro-developmental conditions, and even death.
[0004] Many infants with BPD recover and improve with time,
however, these children are at increased risk of developing further
complications, including asthma and viral pneumonia. And while most
infants survive, some infants with very severe BPD will still die
from the disease even after months of care.
SUMMARY OF THE INVENTION
[0005] The present invention provides, among other things, improved
methods and compositions for treating chronic lung disorders, in
particular, bronchopulmonary dysplasia (BPD), based on anti-Flt-1
antibody therapy. As described in the Examples below, the invention
is, in part, based on the discovery that anti-Flt-1 antibodies, or
antigen binding fragments thereof, can inhibit VEGF and other
ligands from binding to the Flt-1 receptor, thereby increasing the
amount VEGF and/or other ligands available to bind to VEGF
receptors. This increased binding can induce a pro-angiogenic
effect that increases capillary density and facilitates reduction
of fibrosis and inflammation, and mitigation of symptoms and
features associated with BPD. Indeed, as shown in the Examples, the
present inventors have demonstrated that administration of an
anti-Flt-1 antibody improves measures of lung pathology in BPD
animal models. Therefore, the present invention provides safe and
effective antibody-based therapeutics for the treatment of BPD.
[0006] In one aspect, the present invention provides methods of
treating bronchopulmonary dysplasia (BPD) comprising administering
to an individual in need of treatment an effective amount of an
anti-Flt-1 antibody or antigen binding fragment thereof.
[0007] In some embodiments, an individual is an infant who is
suffering from or susceptible to BPD. In some embodiments, an
individual is pregnant with a fetus who is suffering from or
susceptible to BPD.
[0008] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is characterized with an ability to bind
human Flt-1 at an affinity greater than 10.sup.-9M, greater than
10.sup.-10M, or greater than 10.sup.-12M in a surface plasmon
resonance binding assay.
[0009] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is characterized with an IC.sub.50 below
100 pM, below 10 pM, or below 1 pM in a competition assay with
human Flt-1.
[0010] In some embodiments, a competition assay is inhibition of
binding of VEGF to human Flt-1. In some embodiments, a competition
assay is inhibition of binding of PLGF to human Flt-1.
[0011] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof does not bind to VEGFR2 and/or VEGFR3.
[0012] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof does not bind to a mouse or monkey
Flt-1.
[0013] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof binds to a mouse and/or monkey Flt-1.
[0014] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is selected from the group consisting of
IgG, F(ab').sub.2, F(ab).sub.2, Fab', Fab, ScFvs, diabodies,
triabodies and tetrabodies. In some embodiments, an anti-Flt-1
antibody or antigen binding fragment thereof is IgG. In some
embodiments, an anti-Flt-1 antibody or antigen binding fragment
thereof is IgG1.
[0015] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is a monoclonal antibody. In some
embodiments, a monoclonal antibody is a humanized monoclonal
antibody. In some embodiments, a humanized monoclonal antibody
contains a human Fc region. In some embodiments, a Fc region
contains one or more mutations that enhance the binding affinity
between the Fc region and the FcRn receptor such that the in vivo
half-life of the antibody is prolonged. In some embodiments, a Fc
region contains one or more mutations at one or more positions
corresponding to Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu
380, Met 428, His 433, and/or Asn 434 of human IgG1.
[0016] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is administered parenterally. In some
embodiments, parenteral administration is selected from
intravenous, intradermal, intrathecal, inhalation, transdermal
(topical), intraocular, intramuscular, subcutaneous, pulmonary
delivery, and/or transmucosal administration. In some embodiments,
parenteral administration is intravenous administration.
[0017] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is administered orally.
[0018] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is administered bimonthly, monthly,
triweekly, biweekly, weekly, daily, or at variable intervals.
[0019] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is delivered to one or more target tissues
selected from lungs and heart. In some embodiments, an anti-Flt-1
antibody or antigen binding fragment thereof is delivered to the
lungs. In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof, is delivered to the heart.
[0020] In some embodiments, administration of an anti-Flt-1
antibody or antigen binding fragment thereof results in growth of
healthy lung tissue, decreased lung inflammation, increased
alveologenesis, increased angiogenesis, improved structure of
pulmonary vascular bed, reduced lung scarring, improved lung
growth, reduced respiratory insufficiency, improved exercise
tolerance, reduced adverse neurological outcome, and/or improved
pulmonary function relative to a control.
[0021] In some embodiments, the present invention provides a method
further comprising co-administering at least one additional agent
or therapy selected from a surfactant, oxygen therapy, ventilator
therapy, a steroid, vitamin A, inhaled nitric oxide, high calorie
nutritional formulation, a diuretic, and/or a bronchodilator.
[0022] As used in this application, the terms "about" and
"approximately" are used as equivalents. Any numerals used in this
application with or without about/approximately are meant to cover
any normal fluctuations appreciated by one of ordinary skill in the
relevant art.
[0023] Other features, objects, and advantages of the present
invention are apparent in the detailed description that follows. It
should be understood, however, that the detailed description, while
indicating embodiments of the present invention, is given by way of
illustration only, not limitation. Various changes and
modifications within the scope of the invention will become
apparent to those skilled in the art from the detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows exemplary results illustrating the anti-soluble
human Flt-1 antiserum titer of mice immunized with soluble human
Flt-1 antigen.
[0025] FIG. 2 shows exemplary results illustrating competitive
binding of monoclonal antibodies with human soluble Flt-1 in an
ELISA.
[0026] FIG. 3 shows exemplary monoclonal antibody binding to
soluble human Flt-1.
[0027] FIG. 4 shows exemplary results illustrating monoclonal
antibody binding to soluble human Flt-1 via surface plasmon
resonance (BIACORE) assay.
[0028] FIG. 5 shows exemplary results illustrating cross-reactivity
of monoclonal antibody binding with cyno (monkey) Flt-1.
[0029] FIG. 6 shows exemplary results illustrating competitive
binding of monoclonal antibodies with human soluble Flt-1 in an
ELISA. VEGF:sFlt-1 IC.sub.50 determination of monoclonal antibody
01A04 (sub-clone 02B10-02G07) versus a commercial benchmark is
depicted.
[0030] FIG. 7 shows exemplary results illustrating anti-Flt-1
monoclonal antibody inhibition of VEGF binding to sFlt-1 in a cell
based assay.
[0031] FIG. 8 shows exemplary results illustrating pulmonary artery
endothelial cell (PAEC) growth 3 days after treatment.
[0032] FIG. 9 shows exemplary results illustrating PAEC growth 3
days after treatment.
[0033] FIG. 10 shows exemplary results illustrating tube formation
24 hours after treatment.
[0034] FIG. 11 shows exemplary results illustrating tube formation
24 hours after treatment.
[0035] FIG. 12 shows exemplary results illustrating the effects of
in utero dosing of Vitamin D in an endotoxin (ETX) induced model of
BPD in rats.
[0036] FIG. 13 shows exemplary results illustrating the effects of
in utero dosing of anti-Flt-1 monoclonal antibody in an endotoxin
(ETX) induced model of BPD in rats.
[0037] FIG. 14 shows exemplary results illustrating the effects of
in utero dosing of anti-Flt-1 monoclonal antibody in a soluble Flt1
(sFLT) induced model of BPD on pulmonary vessel density in
rats.
[0038] FIG. 15 shows exemplary results illustrating the effects of
in utero dosing of anti-Flt-1 monoclonal antibody in a soluble Flt1
(sFLT) induced model of BPD on pulmonary vessel density in
rats.
[0039] FIG. 16 shows exemplary results illustrating the effects of
low and high doses of anti-Flt-1 monoclonal antibody (a-sFLT) in a
soluble Flt1 (sFLT) induced model of BPD in rats.
[0040] FIG. 17 shows exemplary results illustrating the effects of
low and high doses of anti-Flt-1 monoclonal antibody (a-sFLT) in a
soluble Flt1 (sFLT) induced model of BPD in rats.
[0041] FIG. 18 shows exemplary results illustrating the effects of
low and high doses of anti-Flt-1 monoclonal antibody (a-sFLT) in a
soluble Flt1 (sFLT) induced model of BPD in rats.
[0042] FIG. 19 shows exemplary results illustrating the effects of
1 mg/kg and 10 mg/kg postnatal doses of anti-Flt-1 monoclonal
antibody (antisFLT) on body weight in an endotoxin (ETX) induced
model of BPD in rats.
[0043] FIG. 20 shows exemplary results illustrating the effects of
1 mg/kg and 10 mg/kg postnatal doses of anti-Flt-1 monoclonal
antibody (Mab) on radial alveolar count (RAC) in an endotoxin (ETX)
induced model of BPD in rats.
[0044] FIG. 21 shows exemplary results illustrating the effects of
1 mg/kg and 10 mg/kg postnatal doses of anti-Flt-1 monoclonal
antibody (antisFLT) on right ventricular hypertrophy (RVH) in an
endotoxin (ETX) induced model of BPD in rats.
[0045] FIG. 22 shows exemplary results illustrating the effects of
1 mg/kg and 10 mg/kg postnatal doses of anti-Flt-1 monoclonal
antibody (anti-sFLT) on lung structure in an endotoxin (ETX)
induced model of BPD in rats.
DEFINITIONS
[0046] In order for the present invention to be more readily
understood, certain terms are first defined below. Additional
definitions for the following terms and other terms are set forth
throughout the specification.
[0047] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans, at any stage of development. In some embodiments,
"animal" refers to non-human animals, at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate, and/or a pig). In some embodiments, animals
include, but are not limited to, mammals, birds, reptiles,
amphibians, fish, insects, and/or worms. In some embodiments, an
animal may be a transgenic animal, genetically-engineered animal,
and/or a clone.
[0048] Antibody: As used herein, the term "antibody" refers to any
immunoglobulin, whether natural or wholly or partially
synthetically produced. All derivatives thereof which maintain
specific binding ability are also included in the term. The term
also covers any protein having a binding domain which is homologous
or largely homologous to an immunoglobulin-binding domain. Such
proteins may be derived from natural sources, or partly or wholly
synthetically produced. An antibody may be monoclonal or
polyclonal. An antibody may be a member of any immunoglobulin
class, including any of the human classes: IgG, IgM, IgA, IgD, and
IgE. In certain embodiments, an antibody may be a member of the IgG
immunoglobulin class. As used herein, the terms "antibody fragment"
or "characteristic portion of an antibody" are used interchangeably
and refer to any derivative of an antibody that is less than
full-length. In general, an antibody fragment retains at least a
significant portion of the full-length antibody's specific binding
ability. Examples of antibody fragments include, but are not
limited to, Fab, Fab', F(ab').sub.2, scFv, Fv, dsFv diabody, and Fd
fragments. An antibody fragment may be produced by any means. For
example, an antibody fragment may be enzymatically or chemically
produced by fragmentation of an intact antibody and/or it may be
recombinantly produced from a gene encoding the partial antibody
sequence. Alternatively or additionally, an antibody fragment may
be wholly or partially synthetically produced. An antibody fragment
may optionally comprise a single chain antibody fragment.
Alternatively or additionally, an antibody fragment may comprise
multiple chains that are linked together, for example, by disulfide
linkages. An antibody fragment may optionally comprise a
multimolecular complex. A functional antibody fragment typically
comprises at least about 50 amino acids and more typically
comprises at least about 200 amino acids. In some embodiments, an
antibody may be a human antibody. In some embodiments, an antibody
may be a humanized antibody.
[0049] Antigen binding fragment: As used herein, the term "antigen
binding fragment" refers to a portion of an immunoglobulin molecule
that contacts and binds to an antigen (i.e., Flt-1).
[0050] Approximately or about: As used herein, the term
"approximately" or "about," as applied to one or more values of
interest, refers to a value that is similar to a stated reference
value. In certain embodiments, the term "approximately" or "about"
refers to a range of values that fall within 25%, 20%, 19%, 18%,
17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, or less in either direction (greater than or less than) of
the stated reference value unless otherwise stated or otherwise
evident from the context (except where such number would exceed
100% of a possible value).
[0051] Biologically active: As used herein, the phrase
"biologically active" refers to a characteristic of any agent that
has activity in a biological system, and particularly in an
organism. For instance, an agent that, when administered to an
organism, has a biological effect on that organism, is considered
to be biologically active. In particular embodiments, where a
peptide is biologically active, a portion of that peptide that
shares at least one biological activity of the peptide is typically
referred to as a "biologically active" portion. In certain
embodiments, a peptide has no intrinsic biological activity but
that inhibits the binding of one or more VEGF ligands, is
considered to be biologically active.
[0052] Carrier or diluent: As used herein, the terms "carrier" and
"diluent" refer to a pharmaceutically acceptable (e.g., safe and
non-toxic for administration to a human) carrier or diluting
substance useful for the preparation of a pharmaceutical
formulation. Exemplary diluents include sterile water,
bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution.
[0053] Dosage form: As used herein, the terms "dosage form" and
"unit dosage form" refer to a physically discrete unit of a
therapeutic protein (e.g., antibody) for the patient to be treated.
Each unit contains a predetermined quantity of active material
calculated to produce the desired therapeutic effect. It will be
understood, however, that the total dosage of the composition will
be decided by the attending physician within the scope of sound
medical judgment.
[0054] Functional equivalent or derivative: As used herein, the
term "functional equivalent" or "functional derivative" denotes, in
the context of a functional derivative of an amino acid sequence, a
molecule that retains a biological activity (either function or
structural) that is substantially similar to that of the original
sequence. A functional derivative or equivalent may be a natural
derivative or is prepared synthetically. Exemplary functional
derivatives include amino acid sequences having substitutions,
deletions, or additions of one or more amino acids, provided that
the biological activity of the protein is conserved. The
substituting amino acid desirably has chemico-physical properties
that are similar to that of the substituted amino acid. Desirable
similar chemico-physical properties include similarities in charge,
bulkiness, hydrophobicity, hydrophilicity, and the like.
[0055] Fusion protein: As used herein, the term "fusion protein" or
"chimeric protein" refers to a protein created through the joining
of two or more originally separate proteins, or portions thereof.
In some embodiments, a linker or spacer will be present between
each protein.
[0056] Half-life: As used herein, the term "half-life" is the time
required for a quantity such as protein concentration or activity
to fall to half of its value as measured at the beginning of a time
period.
[0057] Hypertrophy: As used herein the term "hypertrophy" refers to
the increase in volume of an organ or tissue due to the enlargement
of its component cells.
[0058] Improve, increase, or reduce: As used herein, the terms
"improve," "increase" or "reduce," or grammatical equivalents,
indicate values that are relative to a baseline measurement, such
as a measurement in the same individual prior to initiation of the
treatment described herein, or a measurement in a control subject
(or multiple control subjects) in the absence of the treatment
described herein. A "control subject" is a subject afflicted with
the same form of disease as the subject being treated, who is about
the same age as the subject being treated.
[0059] In vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, etc., rather than within
a multi-cellular organism.
[0060] In vivo: As used herein, the term "in vivo" refers to events
that occur within a multi-cellular organism, such as a human and a
non-human animal. In the context of cell-based systems, the term
may be used to refer to events that occur within a living cell (as
opposed to, for example, in vitro systems).
[0061] Linker: As used herein, the term "linker" refers to, in a
fusion protein, an amino acid sequence other than that appearing at
a particular position in the natural protein and is generally
designed to be flexible or to interpose a structure, such as an
.alpha.-helix, between two protein moieties. A linker is also
referred to as a spacer. A linker or a spacer typically does not
have biological function on its own.
[0062] Pharmaceutically acceptable: As used herein, the term
"pharmaceutically acceptable" refers to substances that, within the
scope of sound medical judgment, are suitable for use in contact
with the tissues of human beings and animals without excessive
toxicity, irritation, allergic response, or other problem or
complication, commensurate with a reasonable benefit/risk
ratio.
[0063] Polypeptide: As used herein, the term "polypeptide" refers
to a sequential chain of amino acids linked together via peptide
bonds. The term is used to refer to an amino acid chain of any
length, but one of ordinary skill in the art will understand that
the term is not limited to lengthy chains and can refer to a
minimal chain comprising two amino acids linked together via a
peptide bond. As is known to those skilled in the art, polypeptides
may be processed and/or modified.
[0064] Prevent: As used herein, the term "prevent" or "prevention",
when used in connection with the occurrence of a disease, disorder,
and/or condition, refers to reducing the risk of developing the
disease, disorder and/or condition. See the definition of
"risk."
[0065] Protein: As used herein, the term "protein" refers to one or
more polypeptides that function as a discrete unit. If a single
polypeptide is the discrete functioning unit and does not require
permanent or temporary physical association with other polypeptides
in order to form the discrete functioning unit, the terms
"polypeptide" and "protein" may be used interchangeably. If the
discrete functional unit is comprised of more than one polypeptide
that physically associate with one another, the term "protein"
refers to the multiple polypeptides that are physically coupled and
function together as the discrete unit.
[0066] Risk: As will be understood from context, a "risk" of a
disease, disorder, and/or condition comprises a likelihood that a
particular individual will develop a disease, disorder, and/or
condition (e.g., BPD). In some embodiments, risk is expressed as a
percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some
embodiments risk is expressed as a risk relative to a risk
associated with a reference sample or group of reference samples.
In some embodiments, a reference sample or group of reference
samples have a known risk of a disease, disorder, condition and/or
event (e.g., BPD). In some embodiments a reference sample or group
of reference samples are from individuals comparable to a
particular individual. In some embodiments, relative risk is 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
[0067] Subject: As used herein, the term "subject" refers to a
human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat,
cattle, swine, sheep, horse or primate). A human includes pre- and
post-natal forms. In many embodiments, a subject is a human being.
A subject can be a patient, which refers to a human presenting to a
medical provider for diagnosis or treatment of a disease. The term
"subject" is used herein interchangeably with "individual" or
"patient." A subject can be afflicted with or susceptible to a
disease or disorder but may or may not display symptoms of the
disease or disorder.
[0068] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. One of ordinary skill in the biological arts will
understand that biological and chemical phenomena rarely, if ever,
go to completion and/or proceed to completeness or achieve or avoid
an absolute result. The term "substantially" is therefore used
herein to capture the potential lack of completeness inherent in
many biological and chemical phenomena.
[0069] Substantial homology: As used herein, the phrase
"substantial homology refers to a comparison between amino acid or
nucleic acid sequences. As will be appreciated by those of ordinary
skill in the art, two sequences are generally considered to be
"substantially homologous" if they contain homologous residues in
corresponding positions. Homologous residues may be identical
residues. Alternatively, homologous residues may be non-identical
residues will appropriately similar structural and/or functional
characteristics. For example, as is well known by those of ordinary
skill in the art, certain amino acids are typically classified as
"hydrophobic" or "hydrophilic" amino acids, and/or as having
"polar" or "non-polar" side chains. Substitution of one amino acid
for another of the same type may often be considered a "homologous"
substitution.
[0070] As is well known in this art, amino acid or nucleic acid
sequences may be compared using any of a variety of algorithms,
including those available in commercial computer programs such as
BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and
PSI-BLAST for amino acid sequences. Exemplary such programs are
described in Altschul, et al., basic local alignment search tool,
J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al., Methods in
Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs", Nucleic Acids Res.
25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical
Guide to the Analysis of Genes and Proteins, Wiley, 1998; and
Misener, et al., (eds.), Bioinformatics Methods and Protocols
(Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In
addition to identifying homologous sequences, the programs
mentioned above typically provide an indication of the degree of
homology. In some embodiments, two sequences are considered to be
substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
of their corresponding residues are homologous over a relevant
stretch of residues. In some embodiments, the relevant stretch is a
complete sequence. In some embodiments, the relevant stretch is at
least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350,
375, 400, 425, 450, 475, 500 or more residues.
[0071] Substantial identity: As used herein, the phrase
"substantial identity" is used to refer to a comparison between
amino acid or nucleic acid sequences. As will be appreciated by
those of ordinary skill in the art, two sequences are generally
considered to be "substantially identical" if they contain
identical residues in corresponding positions. As is well known in
this art, amino acid or nucleic acid sequences may be compared
using any of a variety of algorithms, including those available in
commercial computer programs such as BLASTN for nucleotide
sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid
sequences. Exemplary such programs are described in Altschul, et
al., Basic local alignment search tool, J. Mol. Biol., 215(3):
403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul et
al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al.,
Bioinformatics: A Practical Guide to the Analysis of Genes and
Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics
Methods and Protocols (Methods in Molecular Biology, Vol. 132),
Humana Press, 1999. In addition to identifying identical sequences,
the programs mentioned above typically provide an indication of the
degree of identity. In some embodiments, two sequences are
considered to be substantially identical if at least 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more of their corresponding residues are identical over
a relevant stretch of residues. In some embodiments, the relevant
stretch is a complete sequence. In some embodiments, the relevant
stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.
[0072] Suffering from: An individual who is "suffering from" a
disease, disorder, and/or condition has been diagnosed with or
displays one or more symptoms of the disease, disorder, and/or
condition.
[0073] Susceptible to: An individual who is "susceptible to" a
disease, disorder, and/or condition has not been diagnosed with the
disease, disorder, and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition may not exhibit symptoms of the disease, disorder, and/or
condition. In some embodiments, an individual who is susceptible to
a disease, disorder, condition, or event (for example, BPD) may be
characterized by one or more of the following: (1) a genetic
mutation associated with development of the disease, disorder,
and/or condition; (2) a genetic polymorphism associated with
development of the disease, disorder, and/or condition; (3)
increased and/or decreased expression and/or activity of a protein
associated with the disease, disorder, and/or condition; (4) habits
and/or lifestyles associated with development of the disease,
disorder, condition, and/or event (5) having undergone, planning to
undergo, or requiring a transplant. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[0074] Target tissues: As used herein, the term "target tissues"
refers to any tissue that is affected by a disease to be treated
such as BPD. In some embodiments, target tissues include those
tissues that display disease-associated pathology, symptom, or
feature, including but not limited to lung inflammation, lung
scarring, impaired lung growth, early lung injury, prolonged
respiratory insufficiency, lung infections, exercise intolerance,
and adverse neurological outcome.
[0075] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" of a therapeutic agent means an
amount that is sufficient, when administered to a subject suffering
from or susceptible to a disease, disorder, and/or condition, to
treat, diagnose, prevent, and/or delay the onset of the symptom(s)
of the disease, disorder, and/or condition. It will be appreciated
by those of ordinary skill in the art that a therapeutically
effective amount is typically administered via a dosing regimen
comprising at least one unit dose.
[0076] Treating: As used herein, the term "treat," "treatment," or
"treating" refers to any method used to partially or completely
alleviate, ameliorate, relieve, inhibit, prevent, delay onset of,
reduce severity of and/or reduce incidence of one or more symptoms
or features of a particular disease, disorder, and/or condition.
Treatment may be administered to a subject who does not exhibit
signs of a disease and/or exhibits only early signs of the disease
for the purpose of decreasing the risk of developing pathology
associated with the disease.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0077] The present invention provides, among other things, methods
and compositions for treating chronic lung disorders, in
particular, bronchopulmonary dysplasia (BPD), based on the use of
anti-Flt-1 antibodies, or antigen binding fragments thereof, as
therapeutics for treating BPD. In some embodiments, the present
invention provides methods of treating BPD including administering
to an individual who is suffering from or susceptible to BPD an
effective amount of an Flt-1 antibody or antigen binding fragment
thereof such that at least one symptom or feature of BPD is reduced
in intensity, severity, or frequency, or has delayed onset.
[0078] Various aspects of the invention are described in detail in
the following sections. The use of sections is not meant to limit
the invention. Each section can apply to any aspect of the
invention. In this application, the use of "or" means "and/or"
unless stated otherwise.
Bronchopulmonary Dysplasia (BPD)
[0079] With the introduction of surfactant therapy, maternal
steroids, new ventilator strategies, aggressive management of the
patent ductus arteriosus, improved nutrition, and other treatments,
the clinical course and outcomes of premature newborns with RDS
have dramatically changed over the past 30 years. It has recently
been demonstrated that about two thirds of infants who develop BPD
have only mild respiratory distress at birth. This suggests that
developmental timing of lung injury is a critical factor in the
etiology of BPD.
[0080] In parallel with this changing epidemiologic and clinical
pattern, key features of lung histology in BPD have also changed.
There is now growing recognition that infants with persistent lung
disease after premature birth have a different clinical course and
pathology than was traditionally observed in infants dying with BPD
during this presurfactant era. The classic progressive stages that
first characterized BPD are often absent owing to changes in
clinical management, and BPD has clearly changed from being
predominantly defined by the severity of acute lung injury to its
current characterization, which is primarily defined by a
disruption of distal lung growth. Thus, the so-called new BPD of
the postsurfactant period represents inhibition of lung development
with altered lung structure, growth, and function of the distal
airspaces and vasculature. Physiologically, this suggests a marked
reduction in alveolo-capillary surface area, potentially
contributing to impaired gas exchange with increased risk for
exercise intolerance, pulmonary hypertension, and poor tolerance of
acute respiratory infections.
[0081] Pathogenesis of BPD
[0082] BPD represents the response of the lung to injury during a
critical period of lung growth, that is, during the canalicular
period (17 to 26 weeks in the human), a time during which airspace
septation and vascular development increase dramatically. In some
embodiments, factors that increase the susceptibility of the
premature newborn to the development of BPD, include surfactant
deficiency, decreased antioxidant defenses, impaired epithelial ion
and water transport function, and lung structural immaturity. In
some embodiments, lung injury after premature birth and the
subsequent arrest of lung growth results from complex interactions
between multiple adverse stimuli, including inflammation,
hyperoxia, mechanical ventilation, and infection, of the poorly
defended developing lung. In some embodiments, prenatal exposure to
proinflammatory cytokines, such as TNF-.alpha., IL-6, IL-8, and
others, due to maternal chorioamnionitis, enhance lung maturation
in utero, but increase the risk for BPD.
[0083] Hyperoxia and oxidant stress are critical factors in the
development of BPD. In some embodiments, the transition of the
premature newborn from the low-oxygen tension environment of the
normal fetus to the relative hyperoxia of extrauterine life
increases the risk for BPD with decreased alveolarization and a
dysmorphic vasculature. In some embodiments, the premature change
in the oxygen environment impedes normal epithelial-mesenchymal
interactions and leads to alterations in endothelial cell survival,
differentiation, and organization in the microvasculature. In some
embodiments, a premature infant is especially susceptible to
reactive oxidant species (ROS)-induced damage owing to the lack of
adequate antioxidants after premature birth. In some embodiments,
antioxidant enzymes [e.g., superoxide dismutase (SOD), catalase,
and glutathione peroxidase] markedly increase during late
gestation. In some additional embodiments, the ability to increase
synthesis of antioxidant enzymes in response to hyperoxia is
decreased in preterm animals, so premature birth may precede the
normal up-regulation of antioxidants, which persists during early
postnatal life. In some embodiments, endothelial and alveolar type
II cells are extremely susceptible to hyperoxia and ROS-induced
injury, leading to increased edema, cellular dysfunction, and
impaired cell survival and growth.
[0084] In some embodiments, even in the absence of overt signs of
baro- or volutrauma, treatment of premature neonates with
mechanical ventilation initiates and promotes lung injury with
inflammation and permeability edema, and contributes to BPD. In
some embodiments, ventilator-associated lung injury (VALI) results
from stretching distal airway epithelium and capillary endothelium,
which increases permeability edema, inhibits surfactant function,
and provokes a complex inflammatory cascade. In some embodiments,
even brief periods of positive-pressure ventilation, such as during
resuscitation in the delivery room, can cause bronchiolar
epithelial and endothelial damage in the lung, setting the stage
for progressive lung inflammation and injury.
[0085] Lung inflammation, whether induced prior to birth (from
chorioamnionitis) or during the early postnatal period (due to
hyperoxia or VALI) plays a prominent role in the development of
BPD. In some embodiments, the risk for BPD is associated with
sustained increases in tracheal fluid neutrophil counts, activated
macrophages, high concentrations of lipid products,
oxidant-inactivated .alpha.-1-antitrypsin activity, and
proinflammatory cytokines, including IL-6 and IL-8, and decreased
IL-10 levels. In some embodiments, release of early response
cytokines, such as TNF-.alpha., IL-1.beta., IL-8, and TGF-.beta.,
by macrophages and the presence of soluble adhesion molecules
(i.e., selectins) may impact other cells to release
chemoattractants that recruit neutrophils and amplify the
inflammatory response. In some embodiments, elevated concentrations
of proinflammatory cytokines in conjunction with reduced
anti-inflammatory products (i.e., IL-10) appear in tracheal
aspirates within a few hours of life in infants subsequently
developing BPD. In some embodiments, increased elastase and
collagenase release from activated neutrophils may directly destroy
the elastin and collagen framework of the lung, and markers of
collagen and elastin degradation can be recovered in the urine of
infants with BPD. In some embodiments, infection from relatively
low virulence organisms, such as airway colonization with
Ureaplasma urealyticum, may augment the inflammatory response,
further increasing to the risk for BPD. In some embodiments, other
factors, such as nutritional deficits and genetic factors, such as
vitamin A and E deficiency or single nucleotide polymorphism
variants of the surfactant proteins, respectively, are likely to
increase risk for BPD in some premature newborns.
[0086] Pulmonary Circulation in BPD
[0087] In addition to adverse effects on the airway and distal
airspace, acute lung injury also impairs growth, structure, and
function of the developing pulmonary circulation after premature
birth. In some embodiments, endothelial cells are particularly
susceptible to oxidant injury through hyperoxia or inflammation. In
some embodiments, the media of small pulmonary arteries undergoes
striking changes, including smooth muscle cell proliferation,
precocious maturation of immature mesenchymal cells into mature
smooth muscle cells, and incorporation of
fibroblasts/myofibroblasts into the vessel wall. In some
embodiments, structural changes in the lung vasculature contribute
to high pulmonary vascular resistance (PVR) through narrowing of
the vessel diameter and decreased vascular compliance. In some
embodiments, in addition to these structural changes, the pulmonary
circulation is further characterized by abnormal vasoreactivity,
which also increases PVR. In some embodiments, decreased
angiogenesis may limit vascular surface area, causing further
elevations of PVR, especially in response to high cardiac output
with exercise or stress.
[0088] Overall, early injury to the lung circulation leads to the
rapid development of pulmonary hypertension, which contributes
significantly to the morbidity and mortality of severe BPD. In some
embodiments, high mortality rates occur in infants with BPD and
pulmonary hypertension who require prolonged ventilator support. In
some embodiments, pulmonary hypertension is a marker of more
advanced BPD, and elevated PVR also causes poor right ventricular
function, impaired cardiac output, limited oxygen delivery,
increased pulmonary edema and, perhaps, a higher risk for sudden
death. In some embodiments, physiologic abnormalities of the
pulmonary circulation in BPD include elevated PVR and abnormal
vasoreactivity, as evidenced by the marked vasoconstrictor response
to acute hypoxia. In some embodiments, even mild hypoxia causes
marked elevations in pulmonary artery pressure in infants with
modest basal levels of pulmonary hypertension. In some embodiments,
treatment levels of oxygen saturations above 92-94% effectively
lower pulmonary artery pressure. In some embodiments, strategies to
lower pulmonary artery pressure or limit injury to the pulmonary
vasculature may limit the subsequent development of pulmonary
hypertension in BPD.
[0089] Finally, pulmonary hypertension and right heart function
remain major clinical concerns in infants with BPD. In some
embodiments, pulmonary vascular disease in BPD also includes
reduced pulmonary artery density owing to impaired growth, which
contributes to physiologic abnormalities of impaired gas exchange,
as well as to the actual pathogenesis of BPD. In some embodiments,
impaired angiogenesis impedes alveolarization and strategies that
preserve and enhance endothelial cell survival, growth, and
function provide therapeutic approaches for the prevention of
BPD.
[0090] Altered Signaling of Angiogenic Factors in BPD
[0091] Multiple growth factors and signaling systems play important
roles in normal lung vascular growth. In some embodiments,
premature delivery and changes in oxygen tension, inflammatory
cytokines, and other signals alter normal growth factor expression
and signaling and thus lung/lung vascular development. In some
embodiments, the growth factor is VEGF. Impaired VEGF signaling has
been associated with the pathogenesis of BPD in the clinical
setting. In some embodiments, VEGF is found to be lower in tracheal
fluid samples from premature neonates who subsequently develop BPD
than those who do not develop chronic lung disease (185). In some
embodiments, hyperoxia down-regulates lung VEGF expression, and
pharmacologic inhibition of VEGF signaling impairs lung vascular
growth and inhibits alveolarization. The biologic basis for
impaired VEGF signaling leading to decreased vascular growth and
impaired alveolarization is well established.
[0092] Vascular Growth and Alveolarization
[0093] As described above, close coordination of growth between
airways and vessels is essential for normal lung development. In
some embodiments, failure of pulmonary vascular growth during a
critical period of lung growth (saccular or alveolar stages of
development) decreases septation and ultimately contributes to the
lung hypoplasia that characterizes BPD. In some embodiments,
angiogenesis is involved in alveolarization during lung development
and mechanisms that injure and inhibit lung vascular growth may
impede alveolar growth after premature birth. In some embodiments,
inhibition of lung vascular growth during a critical period of
postnatal lung growth impairs alveolarization.
[0094] Flt-1 Receptor
[0095] Flt-1 receptor, also known as vascular endothelial growth
factor receptor 1, is a receptor that is encoded by the FLT1 gene.
The vascular endothelial growth factor (VEGF) family of signal
glycoproteins act as potent promoters of angiogenesis during
embryogenesis and postnatal growth. Specifically, the binding of
the VEGF-A ligand with the VEGF receptors has been shown to promote
vascular permeability and also trigger endothelial cell migration,
proliferation, and survival, and the newly formed endothelial cells
provide the basic structure of new vasculatures. The dominant VEGF
signal molecule for angiogenesis, VEGF-A, mediates its signal
through VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF
receptor-2 (VEGFR-2, also known as Flk-1). A soluble form of Flt-1
(sFlt-1) also exists, but lacks an intracellular signaling domain
and thus is believed to only serve in a regulatory capacity by
sequestering VEGF-A or other ligands that bind to it. sFlt-1 and
other molecules containing Flt-1 binding sites that are not linked
to an intracellular signal transduction pathway are referred to as
"decoy receptors". Flt-1 and Flk-1 receptors contain an
extracellular VEGF-A-binding domain and an intracellular tyrosine
kinase domain, and both show expression during the developmental
stage and tissue regeneration in hemangioblasts and endothelial
cell lineages. Flt-1 has about 10 times greater binding affinity
for VEGF-A (Kd .about.2-10 pM) compared to Flk-1, but the weaker
tyrosine kinase domain indicates that angiogenic signal
transduction following VEGF-A binding to Flt-1 is comparably weaker
than the Flk-1 signal. As such, homozygous Flt-1 gene knockout mice
die in the embryonic stage from endothelial cell overproduction and
blood vessel disorganization. Inversely, homozygous Flk-1 gene
knockout mice die from defects in the development of organized
blood vessels due to lack of yolk-sac blood island formation during
embryogenesis. Both the Flt-1 and Flk-1 receptors are needed for
normal development, but selective augmentation in VEGF-A
concentration may allow for greater binding to the Flk-1 receptor
and induce a pro-angiogenic effect that increases capillary density
and facilitates reduction of fibrosis and inflammation, and
mitigation of symptoms and features associated with BPD.
[0096] As used herein, the term "Flt-1 receptor" refers to both
soluble and membrane associated Flt-1 receptors, or functional
fragments thereof.
Anti-Flt-1 Antibodies
[0097] As used herein, the term "anti-Flt-1 antibodies" refers to
any antibodies, or antigen binding fragments thereof, that bind to
an Flt-1 receptor (e.g., soluble or membrane associated Flt-1
receptor). In some embodiments, anti-Flt-1 antibodies are produced
that bind with high affinity to Flt-1 receptors. Without wishing to
be bound by theory, it is believed that anti-Flt-1 antibody binding
to Flt-1 receptors inhibits one or more endogenous ligands from
binding to Flt-1 and thereby allowing a greater amount of available
ligand to associate with other VEGF receptors, such as the Flk-1
receptor. Increased activation of the Flk-1 receptor could
increases capillary density and facilitates reduction of fibrosis
and inflammation, and mitigation of symptoms and features
associated with BPD. In some embodiments, antibody binding to Flt-1
receptors increases the amount of VEGF available to bind to other
VEGF receptors. In some embodiments, antibody binding to Flt-1
receptors increases the amount of placental growth factor (PLGF)
available to bind to other VEGF receptors.
[0098] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof, binds human Flt-1 with an affinity
greater than about 10.sup.-9 M, greater than about 10.sup.-10 M,
greater than about 0.5.times.10.sup.-10 M, greater than about
10.sup.-11 M, greater than about 0.5.times.10.sup.-11 M, greater
than about 10.sup.-12 M, or greater than about 0.5.times.10.sup.-12
M. The affinity of an Flt-1 antibody may be measured, for example,
in a surface plasmon resonance assay, such as a BIACORE assay.
[0099] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof, is characterized by an IC.sub.50 below
100 pM, below 10 pM, or below 1 pM in a competition assay with
human Flt-1.
[0100] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof inhibits the binding and/or activity of
VEGF at the Flt-1 receptor. In some embodiments, an anti-Flt-1
antibody, or an antigen binding fragment thereof, is characterized
by an IC.sub.50 below 100 pM, below 10 pM, or below 1 pM for
inhibition of binding of VEGF to human Flt-1 in a competition
assay.
[0101] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof inhibits the binding and/or activity of
PLGF at the Flt-1 receptor. In some embodiments, an anti-Flt-1
antibody, or an antigen binding fragment thereof, is characterized
by an IC.sub.50 below 100 pM, below 10 pM, or below 1 pM for
inhibition of binding of PLGF to human Flt-1 in a competition
assay.
[0102] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof selectively binds Flt-1 and has minimal or
no appreciable binding to other VEGF receptors. In some
embodiments, an anti-Flt-1 antibody, or an antigen binding fragment
thereof selectively binds Flt-1 and has minimal or no appreciable
binding to VEGFR2 (Flk-1) and/or VEGFR3 (Flt-4).
[0103] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof selectively binds human Flt-1, and has
minimal or no appreciable binding to other mammalian Flt-1
receptors (e.g., with a binding affinity less than 10.sup.-7M or
10.sup.-6M). In some embodiments, an anti-Flt-1 antibody, or an
antigen binding fragment thereof selectively binds human Flt-1 and
does not bind to monkey Flt-1. In some embodiments, an anti-Flt-1
antibody, or an antigen binding fragment thereof selectively binds
human Flt-1 and does not bind to mouse Flt-1.
[0104] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof binds human Flt-1 as well as monkey Flt-1.
In some embodiments an anti-Flt-1 antibody, or an antigen binding
fragment thereof binds human Flt-1 as well as mouse Flt-1.
[0105] In some embodiments, an anti-Flt-1 antibody, or an antigen
binding fragment thereof, is selected from the group consisting of
IgG, F(ab').sub.2, F(ab).sub.2, Fab', Fab, ScFvs, diabodies,
triabodies and tetrabodies.
[0106] In some embodiments an anti-Flt-1 antibody, or an antigen
binding fragment thereof, is IgG. In some embodiments an anti-Flt-1
antibody, or an antigen binding fragment thereof, is IgG1.
[0107] In some embodiments, a suitable anti-Flt-1 antibody contains
an Fc domain or a portion thereof that binds to the FcRn receptor.
As a non-limiting example, a suitable Fc domain may be derived from
an immunoglobulin subclass such as IgG. In some embodiments, a
suitable Fc domain is derived from IgG1, IgG2, IgG3, or IgG4.
Particularly suitable Fc domains include those derived from human
or humanized antibodies.
[0108] It is contemplated that improved binding between Fc domain
and the FcRn receptor results in prolonged serum half-life. Thus,
in some embodiments, a suitable Fc domain comprises one or more
amino acid mutations that lead to improved binding to FcRn. Various
mutations within the Fc domain that effect improved binding to FcRn
are known in the art and can be adapted to practice the present
invention. In some embodiments, a suitable Fc domain comprises one
or more mutations at one or more positions corresponding to Thr
250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433,
and/or Asn 434 of human IgG1.
[0109] In some embodiments, an anti-FLT-1 antibody or antigen
binding fragment contains a spacer and/or is linked to another
entity. In some embodiments, the linker or spacer comprises a
sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to
GAPGGGGGAAAAAGGGGGGAP (SEQ ID NO: 1) (GAG linker). In some
embodiments, the linker or spacer comprises a sequence at least 50%
(e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100%) identical to
GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP (SEQ ID NO: 2) (GAG2
linker). In some embodiments, the linker or spacer comprises a
sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to
TABLE-US-00001 (SEQ ID NO: 3)
GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAG GGGGGAP (GAG3
linker).
Production of Anti-Flt-1 Antibodies and Antigen Binding
Fragments
[0110] A recombinant anti-Flt-1 antibody or antigen binding
fragment suitable for the present invention may be produced by any
available means. For example, a recombinant anti-Flt-1 antibody or
antigen binding fragment may be recombinantly produced by utilizing
a host cell system engineered to express a recombinant anti-Flt-1
antibody or antigen binding fragment-encoding nucleic acid.
[0111] Where antibodies are recombinantly produced, any expression
system can be used. To give but a few examples, known expression
systems include, for example, egg, baculovirus, plant, yeast, or
mammalian cells.
[0112] In some embodiments, recombinant anti-Flt-1 antibody or
antigen binding fragments suitable for the present invention are
produced in mammalian cells. Non-limiting examples of mammalian
cells that may be used in accordance with the present invention
include BALB/c mouse myeloma line (NSO/1, ECACC No: 85110503);
human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); and
monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL
1651).
[0113] In some embodiments, the present invention provides
recombinant anti-Flt-1 antibody or antigen binding fragment
produced from human cells. In some embodiments, the present
invention provides anti-Flt-1 antibody or antigen binding fragment
produced from CHO cells.
Pharmaceutical Composition and Administration
[0114] The present invention further provides a pharmaceutical
composition containing an anti-Flt-1 antibody or antigen binding
fragment described herein and a physiologically acceptable carrier
or excipient.
[0115] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), saline,
buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable
oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, sugars such as mannitol,
sucrose, or others, dextrose, magnesium stearate, talc, silicic
acid, viscous paraffin, perfume oil, fatty acid esters,
hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as
combinations thereof. The pharmaceutical preparations can, if
desired, be mixed with auxiliary agents (e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like) which do not deleteriously react
with the active compounds or interfere with their activity. In a
preferred embodiment, a water-soluble carrier suitable for
intravenous administration is used.
[0116] A suitable pharmaceutical composition or medicament, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. A composition can be a liquid
solution, suspension, emulsion, tablet, pill, capsule, sustained
release formulation, or powder. A composition can also be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulations can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, polyvinyl pyrrolidone, sodium
saccharine, cellulose, magnesium carbonate, etc.
[0117] A pharmaceutical composition or medicament can be formulated
in accordance with the routine procedures as a pharmaceutical
composition adapted for administration to human beings. For
example, in some embodiments, a composition for intravenous
administration typically is a solution in sterile isotonic aqueous
buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic to ease pain at the site
of the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampule or sachette indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water, saline or
dextrose/water. Where the composition is administered by injection,
an ampule of sterile water for injection or saline can be provided
so that the ingredients may be mixed prior to administration.
Routes of Administration
[0118] An anti-Flt-1 antibody or antigen binding fragment described
herein (or a composition or medicament containing an anti-Flt-1
antibody or antigen binding fragment described herein) is
administered by any appropriate route. In some embodiments, an
anti-Flt-1 antibody or antigen binding fragment protein or a
pharmaceutical composition containing the same is administered
parenterally. Parenteral administration may be intravenous,
intradermal, intrathecal, inhalation, transdermal (topical),
intraocular, intramuscular, subcutaneous, intramuscular, and/or
transmucosal administration. In some embodiments, an anti-Flt-1
antibody or antigen binding fragment or a pharmaceutical
composition containing the same is administered subcutaneously. As
used herein, the term "subcutaneous tissue", is defined as a layer
of loose, irregular connective tissue immediately beneath the skin.
For example, the subcutaneous administration may be performed by
injecting a composition into areas including, but not limited to,
the thigh region, abdominal region, gluteal region, or scapular
region. In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof or a pharmaceutical composition containing
the same is administered intravenously. In some embodiments, an
anti-Flt-1 antibody or antigen binding fragment thereof or a
pharmaceutical composition containing the same is administered
intra-arterially. In some embodiments, an anti-Flt-1 antibody or
antigen binding fragment or a pharmaceutical composition containing
the same is administered orally. More than one route can be used
concurrently, if desired.
[0119] In some embodiments, administration results only in a
localized effect in an individual, while in other embodiments,
administration results in effects throughout multiple portions of
an individual, for example, systemic effects. Typically,
administration results in delivery of an anti-Flt-1 antibody or
antigen binding fragment to one or more target tissues including
but not limited lungs and heart.
Dosage Forms and Dosing Regimen
[0120] In some embodiments, a composition is administered in a
therapeutically effective amount and/or according to a dosing
regimen that is correlated with a particular desired outcome (e.g.,
with treating or reducing risk for a chronic lung disorder, such as
bronchopulmonary dysplasia).
[0121] Particular doses or amounts to be administered in accordance
with the present invention may vary, for example, depending on the
nature and/or extent of the desired outcome, on particulars of
route and/or timing of administration, and/or on one or more
characteristics (e.g., weight, age, personal history, genetic
characteristic, lifestyle parameter, severity of cardiac defect
and/or level of risk of cardiac defect, etc., or combinations
thereof). Such doses or amounts can be determined by those of
ordinary skill. In some embodiments, an appropriate dose or amount
is determined in accordance with standard clinical techniques.
Alternatively or additionally, in some embodiments, an appropriate
dose or amount is determined through use of one or more in vitro or
in vivo assays to help identify desirable or optimal dosage ranges
or amounts to be administered.
[0122] In various embodiments, an anti-Flt-1 antibody or antigen
binding fragment thereof is administered at a therapeutically
effective amount. Generally, a therapeutically effective amount is
sufficient to achieve a meaningful benefit to the subject (e.g.,
treating, modulating, curing, preventing and/or ameliorating the
underlying disease or condition). In some particular embodiments,
appropriate doses or amounts to be administered may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0123] In some embodiments, a provided composition is provided as a
pharmaceutical formulation. In some embodiments, a pharmaceutical
formulation is or comprises a unit dose amount for administration
in accordance with a dosing regimen correlated with achievement of
the reduced incidence or risk of a chronic lung disorder, such as
bronchopulmonary dysplasia.
[0124] In some embodiments, a formulation comprising an anti-Flt-1
antibody or antigen binding fragment described herein administered
as a single dose. In some embodiments, a formulation comprising an
anti-Flt-1 antibody or antigen binding fragment described herein is
administered at regular intervals. Administration at an "interval,"
as used herein, indicates that the therapeutically effective amount
is administered periodically (as distinguished from a one-time
dose). The interval can be determined by standard clinical
techniques. In some embodiments, a formulation comprising an
anti-Flt-1 antibody or antigen binding fragment described herein is
administered bimonthly, monthly, twice monthly, triweekly,
biweekly, weekly, twice weekly, thrice weekly, daily, twice daily,
or every six hours. The administration interval for a single
individual need not be a fixed interval, but can be varied over
time, depending on the needs of the individual.
[0125] As used herein, the term "bimonthly" means administration
once per two months (i.e., once every two months); the term
"monthly" means administration once per month; the term "triweekly"
means administration once per three weeks (i.e., once every three
weeks); the term "biweekly" means administration once per two weeks
(i.e., once every two weeks); the term "weekly" means
administration once per week; and the term "daily" means
administration once per day.
[0126] In some embodiments, a formulation comprising an anti-Flt-1
antibody or antigen binding fragment described herein is
administered at regular intervals indefinitely. In some
embodiments, a formulation comprising an anti-Flt-1 antibody or
antigen binding fragment described herein is administered at
regular intervals for a defined period.
[0127] In some embodiments, a formulation comprising an anti-Flt-1
antibody or antigen binding fragment described herein is
administered prenatally. In some embodiments, a formulation
comprising an anti-Flt-1 antibody or antigen binding fragment
described herein is administered postnatally.
[0128] In some embodiments, a formulation comprising an anti-Flt-1
antibody or antigen binding fragment described herein is
administered at a dose of about 0.5 mg/kg body weight, about 1.0
mg/kg body weight, about 10 mg/kg body weight or about 20 mg/kg
body weight.
[0129] In some embodiments, a formulation comprising an anti-Flt-1
antibody or antigen binding fragment described herein is
administered at a dose ranging from about 0.5 mg/kg body weight to
about 20 mg/kg body weight, for example about 1 mg/kg body weight
to about 10 mg/kg body weight.
[0130] In some embodiments, a formulation comprising an anti-Flt-1
antibody or antigen binding fragment described herein is
administered to an adult at a unit dose of about 35 mg, about 70
mg, about 700 mg or about 1400 mg. In some embodiments, a
formulation comprising an anti-Flt-1 antibody or antigen binding
fragment described herein is administered at a dose ranging from
about 35 mg to about 1400 mg, for example about 70 mg to about 700
mg.
[0131] In some embodiments, a formulation comprising an anti-Flt-1
antibody or antigen binding fragment described herein is
administered to an infant at a unit dose of about 2 mg, about 4 mg,
about 40 mg or about 80 mg. In some embodiments, a formulation
comprising an anti-Flt-1 antibody or antigen binding fragment
described herein is administered at a dose ranging from about 2 mg
to about 80 mg, for example about 4 mg to about 40 mg.
[0132] In some embodiments, administration of an anti-Flt-1
antibody, or an antigen binding fragment thereof reduces the
intensity, severity, or frequency, or delays the onset of at least
one BPD sign or symptom. In some embodiments administration of an
anti-Flt-1 antibody, or an antigen binding fragment thereof reduces
the intensity, severity, or frequency, or delays the onset of at
least one BPD sign or symptom selected from the group consisting of
lung inflammation, lung scarring, impaired lung growth, early lung
injury, prolonged respiratory insufficiency, lung infections,
exercise intolerance, and adverse neurological outcome.
Combination Therapy
[0133] In some embodiments, an anti-Flt-1 antibody or antigen
binding fragment is administered in combination with one or more
known therapeutic agents (e.g., corticosteroids) currently used for
treatment of a muscular dystrophy. In some embodiments, the known
therapeutic agent(s) is/are administered according to its standard
or approved dosing regimen and/or schedule. In some embodiments,
the known therapeutic agent(s) is/are administered according to a
regimen that is altered as compared with its standard or approved
dosing regimen and/or schedule. In some embodiments, such an
altered regimen differs from the standard or approved dosing
regimen in that one or more unit doses is altered (e.g., reduced or
increased) in amount, and/or in that dosing is altered in frequency
(e.g., in that one or more intervals between unit doses is
expanded, resulting in lower frequency, or is reduced, resulting in
higher frequency).
EXAMPLES
Example 1
Generation and Characterization of High Affinity Anti-Flt-1
Antibodies
[0134] Antibody 01A04
[0135] An antibody was generated against soluble Flt-1 using
traditional mouse monoclonal antibody methodology. Briefly, Balb/c
mice were immunized with recombinant human soluble Flt-1 (purchased
from ABCAM). On day 20 post-immunization, animals were titered for
anti-sFlt-1 production by ELISA (FIG. 1). One mouse was found to be
a high titer responder; this animal was subsequently boosted with
antigen and sacrificed 5 days later. Spleen and lymph node cells
from this animal were fused to mouse myeloma partners to produce
hybridomas. Hybridoma supernatants were screened versus sFlt-1
antigen, and positive responders were scaled up and re-assayed for
binding to both human and mouse sFlt-1, as well as the ability to
compete with sFlt-1 for VEGF binding. There were no cross reactive
hybridomas that could bind to both human and mouse sFlt-1. However,
among human sFlt-1 reactive hybridomas, several sFlt-1:VEGF
antagonists were identified by competition ELISA (see FIG. 2 for a
representative experiment). The most potent of these, fusion
partner 01A04, was subjected to three rounds of single cell cloning
to achieve monoclonal antibody 01A04. This antibody was further
characterized for binding affinity to sFlt-1 antigen (ELISA,
BIACORE and FACs); IC50 in sFlt-1:VEGF competition ELISA; and
performance in cell based assays.
[0136] Antibody 01A04 Characterization--Binding
[0137] Following cloning and sub-cloning of the fusion partner
parent, multiple sub-clones of the 01A04 parent demonstrated
binding to immobilized soluble Flt-1 (FIG. 3). One of these
subclones, monoclonal 01A04-02B10-02G07 was chosen for scale up and
cell banking based upon antigen binding, clone morphology and
viability. The binding constant of 01A04-02B10-02G07 for sFlt-1
antigen was determined via surface plasmon resonance methodology
(BIACORE, see FIG. 4). Monoclonal antibody 01A04-02B10-02G07 is a
sub-nanomolar binder to human sFlt-1.
[0138] Antibody 01A04 Characterization--Cross-Reactivity
[0139] Binding of monoclonal antibody 01A04 to the Flt-1 receptor
expressed on cells was tested with FACS. Three transfected cell
lines were tested expressing human, mouse or cyno Flt-1. Binding to
all three cell lines was tested by incubating the cells with
antibody for one hour. Binding of the antibody to the cells was
then revealed with an anti-mouse IgG PE antibody. Results are shown
in FIG. 5. Consistent with ELISA and BIACORE data, monoclonal
antibody 01A04 does not bind to mouse Flt-1. However, the antibody
does bind to human and cynomolgus Flt-1 expressed on cells.
[0140] Antibody 01A04 Characterization--Competition
[0141] To estimate the potency of the antibodies, the competition
ELISA (using human sFlt-1 and VEGF) that was set-up for the
screening of the llama Fabs and IgGs was used. A concentration
range from 10 to 0.01 .mu.g/ml of IgG was tested. Monoclonal
antibody 01A04 was assayed versus both negative control (purified
polyclonal mouse IgG) and positive control (commercial anti-sFlt-1
monoclonal antibody Abcam56300) molecules. Half maximal inhibition
(IC50) values were calculated. Results are presented in FIG. 6.
[0142] Antibody 01A04 Characterization--Cell Based Assay
[0143] Human primary umbilical vein endothelial cells (HUVECs) were
stimulated with VEGF in the presence or absence of soluble Flt-1
and monoclonal antibody 01A04. VEGF induced activation of cells was
assayed by determining the phosphorylation status of the VEGF R2
receptor. In the presence of soluble Flt-1, VEGF induced HUVEC
activation is attenuated. Addition of monoclonal antibody 01A04
rescues cell activation by antagonizing soluble Flt-1 (FIG. 7).
Example 2
In Vitro Efficacy of Anti-Flt-1 Antibody
[0144] Fetal Pulmonary Artery Endothelial Cell Isolation
[0145] Pulmonary artery endothelial cells (PAECs) were harvested
from the proximal pulmonary arteries of late gestation control
fetal sheep at day 135 (day 147 term). Immunohistochemistry with
standard endothelial markers confirmed the cell phenotype.
Low-passage PAECs (p4-5) were then exposed to ETX, VEGF, sFlt1 or
anti-Flt1 alone or in combination.
[0146] Growth of PAECs while Exposed to ETX, VEGF, sFlt1 and
Anti-Flt1
[0147] Fetal PAECs were plated in triplicate at 50,000 cells/well
in DMEM with 10% FBS into 12 well plates and allowed to adhere
overnight in 21% oxygen. The following day (day 0) the cells were
washed twice with PBS. DMEM with 2.5% FBS with VEGF, ETX, sFlt1, or
anti-F1t1 (alone or in combination) was then added, and cells
incubated in 21% oxygen. Final concentrations of exogenous factors
were as follows: VEGF 50 ng/mL, ETX 1 ng/mL, sFlt1 114 ng/mL and
anti-Flt1 1800 ng/mL. Experimental media was changed daily and
cells were counted on day 3 after removing cells with 0.25% trypsin
and counted with a cell counter (Beckman Coulter; Fullerton,
Calif.). Growth studies with treatment were performed in DMEM with
2.5% FBS, based on previous studies that determined that this was
the lowest serum concentration that supported fetal PAEC survival
with some proliferation.
[0148] PAEC Tube Formation Assay
[0149] To assay in vitro angiogenesis, we cross-linked rat-tail
collagen using 0.2% Flavin mononucleotide and a UV Stratalinker
1800 (Stratagene). 50,000 cells/well were added in serum free DMEM
media supplemented with ETX, VEGF, sFlt1 and anti-Flt1 (alone or in
combination) and each condition was tested in triplicate for each
animal. PAECs were then incubated for 12-18 hours under 3% oxygen
conditions based on previous studies that determined tube formation
was more robust in 3% compared to 21% oxygen. Branch-point counting
was performed in blinded fashion under .times.10 magnification from
each of three wells with three to four field of view per well.
Wells were imaged using an Olympus IX71 fluorescence microscope
(Olympus).
[0150] Statistical Analysis
[0151] Statistical analysis was performed with the Prism software
package (v. 5.0a, GraphPad). Repeated measures one-way analysis of
variance (ANOVA) with Bonferroni post-test analysis were performed.
P values less than 0.05 were considered significant.
[0152] Administration of Anti-Flt-1 Antibody to PAECs Exposed to
sFLT
[0153] Cells were treated with recombinant human VEGF (50 ng/mL),
recombinant human soluble Flt-1 (sFLT, 114 ng/mL) or antibody for
human soluble Flt-1 (a-sFLT, 1800 ng/mL) either alone or in
combination. PAEC growth was measured 3 days after treatment and
the number of tube branch points was measured 24 hours after
treatment.
[0154] Results
[0155] As shown in FIG. 8, treatment with sFLT and VEGF decreased
the number of PAECs compared to cells treated only with VEGF and
treatment, indicating that sFLT prevents VEGF from promoting cell
growth. When both sFLT and a-sFLT were combined with VEGF, the
number of PAECs was brought up to the levels seen when cells were
treated with only VEGF, demonstrating that a-sFLT inhibits the
sFLT-induced decrease in cell growth.
[0156] As shown in FIG. 10, treatment with VEGF alone increased the
number of tube branch points, as did treatment with VEGF and
a-sFLT. Contrastingly, treatment with VEGF and sFLT decreased the
number of branch points as compared with the cells treated with
only VEGF. When both sFLT and a-sFLT were combined with VEGF, the
number of branch points was comparable to the number seen in the
VEGF only group, demonstrating that a-sFLT inhibits the
sFLT-induced decrease in the number of branch points.
[0157] Administration of Anti-Flt-1 Antibody to PAECs Exposed to
ETX
[0158] Cells were treated with either VEGF (50 ng/mL), endotoxin
(ETX, 1 ng/mL), VEGF+ETX, EXT+a-sLFT (1800 ng/mL) or
EXT+VEGF+a-sFLT. PAEC growth was measured 3 days after treatment
and the number of tube branch points was measured 24 hours after
treatment.
[0159] Results
[0160] As shown in FIG. 9, PAEC growth was increased after
treatment with VEGF compared to control (CTL) and PAECs treated
with only ETX showed decreased growth compared to control. The
combination of either VEGF or a-sFLT with ETX brought cells numbers
up to the level seen in the control group, as did treatment with
ETX, VEGF and a-sFLT, demonstrating that treatment with either VEGF
or a-sFLT can reverse the detrimental effects of ETX on PAEC
growth.
[0161] As shown in FIG. 11, the number of branch points increased
after treatment with VEGF only and cells treated with only ETX
showed a decreased number of branch points compared to both the
control and VEGF treated groups. The combination of either VEGF or
a-sFLT with ETX brought the number of branch points up to the level
seen in the control group, as did treatment with ETX, VEGF and
a-sFLT, demonstrating that treatment with either VEGF or a-sFLT can
reverse the detrimental effects of ETX on the number of branch
points in tubes.
Example 3
In Vivo Efficacy of Anti-Flt-1 Antibody in ETX Model of BPD
[0162] Animals
[0163] All procedures and protocols were approved by the Animal
Care and Use Committee at the University of Colorado Health
Sciences Center. Timed pregnant Sprague-Dawley rats were purchased
from Charles River Laboratories (Wilmington, Mass.) and maintained
in room air at Denver's altitude (1,600 m; barometric pressure, 630
mmHg; inspired oxygen tension, 122 mmHg) for at least 1 week before
giving birth. Animals were fed ad libitum and exposed to day-night
cycles alternatively every 12 hours. Rats were killed with an
intraperitoneal injection of pentobarbital sodium (0.3 mg/g body
weight; Fort Dodge Animal Health, Fort Dodge, Iowa).
[0164] Animal Model and Study Design
[0165] Intra-Amniotic ETX, Vitamin D and Anti-sFLT
Administration
[0166] An animal model of chorioamnionitis was utilized. At 20 days
gestation (term: 22 days), pregnant rats were prepared for
receiving intra-amniotic (IA) injections. The timing of injection
during the late canalicular stage of lung development in the rat
was selected to parallel the similar stage of human lung
development in 24 to 26 week premature newborns who are at the
highest risk for BPD. After premedication with buprenorphine
(0.01-0.05 mg/kg, subcutaneous injection), laparotomy was performed
under general anesthesia with 1-2% isoflurane inhalation via
facemask (anesthesia machine: Matrx by Midmark, model VIP3000).
During anesthesia and laparotomy, pregnant rats were kept on a
heating pad for preventing hypothermia. Pregnant rats were randomly
assigned to saline control (CTR), endotoxin (ETX), or ETX+vitamin D
(vit D) group in one study or to saline control (CTR), endotoxin
(ETX) or ETX+anti-sFLT in the other study. The CTR groups received
50 .mu.l of normal 136 saline per amniotic sac, the ETX groups
received 10 .mu.g of Escherichia coli 055:B55 ETX (Sigma Chemical,
St. Louis, Mo.) diluted to 50 .mu.l with normal saline per sac, the
ETX+vit D group received 10 .mu.g of Escherichia coli 055:B55 ETX
and 50 pg diluted to 50 .mu.l with normal saline and the
ETX+anti-sFLT group received 10 .mu.g of Escherichia coli 055:B55
ETX and low dose (1.times. molar equivalent) or high dose
(10.times. molar equivalent) anti-sFlt1 antibody. Under sterile
preparation, a midline abdominal incision of 3-4 cm in length was
made to expose the amniotic sacs for IA injections. The amniotic
sac closest to the right ovary was first identified and injected,
and then in a counterclockwise sequence each sac was identified and
injected with a maximum of 10 sacs injected per dam. Injections
were limited to 10 sacs to prevent maternal mortality due to
systemic toxicities from accumulating doses of IA ETX. The dose of
ETX was established from previous studies that demonstrated ETX at
lower doses (1-5 .mu.g/sac) failed to induce abnormal lung
structure at 14 days of age. The dose of vit D was established
again from previous studies demonstrating vit D at higher doses
(500 ng/gm) produced subcutaneous calcium deposits noted in rat
pups. The abdominal incision was closed with nylon sutures.
Bupivacaine (1-2 mg/kg, intramuscular injection) was applied over
the incision wound for postoperative pain control. Pregnant rats
were monitored closely to ensure arousal within 10 minutes after
surgery, and rats were placed back to the cages and were monitored
for activity and for signs of bleeding or infection.
[0167] Cesarean Section
[0168] Two days after IA injections, cesarean section was performed
on pregnant rats under general anesthesia with isoflurane
inhalation, as described above. The fetus in the amniotic sac
closest to the right ovary was first delivered, which was followed
by delivery of the rest of the fetuses in a counterclockwise
sequence, to identify fetuses exposed to IA injections. Cesarean
sections were performed instead of allowing vaginal deliveries in
order to identify fetuses exposed to specific IA injections, based
on the order of the amniotic sacs and their anatomic locations
related to the ovaries. All of the rat pups in the injected
amniotic sacs were delivered within 5 minutes after onset of
anesthesia. Mother rats were then euthanized with pentobarbital
sodium. Newborn rats were immediately dried and placed on a heating
pad to avoid hypothermia. Pups received no supplemental oxygen or
artificial ventilation at birth. Within 30 minutes after birth,
pups were weighed and either sacrificed for histology or placed
with foster mother rats to be raised through 14 days. Rat lungs
were harvested at birth and 14 days of age for histological
assessment. Survival of the infant rats was monitored and recorded
daily from birth throughout the study period. Survival rate was
calculated as the number of survived pups divided by the number of
sacs that received intra-amniotic injection in each given
litter.
[0169] Study Measurements
[0170] Tissue for Histological Analysis
[0171] Animals were killed with intra-peritoneal pentobarbital
sodium. A catheter was placed in the trachea and the lungs were
inflated with 4% paraformaldehyde and maintained at 20 cm H.sub.2O
pressure for 60 minutes. A ligature was tightened around the
trachea to maintain pressure and the tracheal cannula was removed.
Lungs were immersed in 4% paraformaldehyde at room temperature
overnight for fixation. A 2-mm thick transverse section was taken
from the mid-plane of right lower lobe and left lobe of the fixed
lungs per animal, respectively. Two sections from each animal were
processed and embedded in paraffin wax for study.
[0172] Bronchoalveolar Lavage (BAL)
[0173] Bronchoalveolar lavage was performed on the day of birth
(Day 0) according to standard techniques and sFLT levels in the
lung were measured.
[0174] Radial Alveolar Counts (RAC)
[0175] Alveolarization was assessed by the RAC method of Emery and
Mithal as described (Cooney T P, Thurlbeck W M. The radial alveolar
count method of Emery and Mithal: a reappraisal 1--postnatal lung
growth. Thorax 37: 572-579, 1982; Cooney T P, Thurlbeck W M. The
radial alveolar count method of Emery and Mithal: a reappraisal
2--intrauterine and early postnatal lung growth. Thorax 37:
580-583, 1982). Respiratory bronchioles were identified as
bronchioles lined by epithelium in one part of the wall. From the
center of the respiratory bronchiole, a perpendicular line was
dropped to the edge of the acinus connective tissues or septum or
pleura, and the number of septae intersected by this line was
counted.
[0176] Statistical Analysis
[0177] Statistical analysis was performed with the Prism software
package (v. 5.0a, GraphPad). Repeated measures one-way analysis of
variance (ANOVA) with Bonferroni post-test analysis were performed.
P values less than 0.05 were considered significant.
[0178] Results
[0179] As shown in FIG. 12, sFLT levels were significantly (*
p<0.05) increased in rats exposed to ETX in utero compared to
the control group and treatment with Vitamin D decreased the levels
of sFLT to the level seen in the control group. This demonstrates
that treatment with Vitamin D could be used as a therapeutic for
treating BPD via the action of Vitamin D on levels of sFLT in the
lungs.
[0180] As shown in FIG. 13, by morphometric analysis, RAC was
decreased in rats exposed to ETX in utero compared to the control
group and in utero dosing with anti-sFLT in rats exposed to ETX
significantly (* p<0.05) increased RAC compared to the group
only exposed to ETX. This demonstrates that treatment with
anti-sFLT could be used as a therapeutic for treating BPD.
Example 4
In Vivo Efficacy of Anti-Flt-1 Antibody in sFLT Model of BPD
[0181] Animals
[0182] All procedures and protocols were approved by the Animal
Care and Use Committee at the University of Colorado Health
Sciences Center. Pregnant Sprague-Dawley rats were purchased from
Charles River Laboratories (Wilmington, Mass.) and maintained in
room air at Denver's altitude (1,600 meters; barometric pressure,
630 mmHg; inspired oxygen tension, 122 mmHg) for at least 1 week
before giving birth. Animals were fed ad libitum and exposed to
day-night cycles alternatively every 12 hours. Rats were killed
with an intraperitoneal injection of pentobarbital sodium (0.3 mg/g
body wt; Fort Dodge Animal Health, Fort Dodge, Iowa).
[0183] Study Design
[0184] Intra-Amniotic sFlt-1 Administration
[0185] At 20 days gestation (term: 22 days), pregnant rats were
prepared for receiving intra-amniotic injections. The timing of
injection during the late canalicular stage of lung development in
the rat was selected to parallel a similar stage of human lung
development in 24- to 26-week premature newborns who are at the
highest risk for BPD. After premedication with buprenorphine
(0.01-0.05 mg/kg, intramuscular injection), laparotomy was
performed on pregnant rats under general anesthesia with 1-2%
isoflurane inhalation via a face mask (Anesthesia machine: Matrx by
Midmark, model VIP3000). During anesthesia and laparotomy, pregnant
rats were kept on a heating pad for preventing hypothermia.
Pregnant rats were randomly assigned to saline control or sFlt-1
group; the saline group received 50 .mu.L of normal saline per
amniotic sac, and the sFlt-1 groups received 50 .mu.g of
recombinant human sFlt-1-Fc (R&D Systems, Minneapolis, Minn.)
diluted to 50 .mu.L with normal saline per sac. One sFLT group
received a low dose (1.times. molar equivalent) of anti-sFLT and
the other received a high dose (10.times. molar equivalent) of
anti-sFLT. Under sterile preparation, a midline abdominal incision
of 3-4 cm in length was made to expose the amniotic sacs for
intra-amniotic injections. The amniotic sac closest to the right
ovary was first identified and injected, and then in a
counterclockwise sequence each sac was identified and injected with
a maximum of 10 sacs injected per dam. Limiting sFlt-1 injections
to 10 sacs per pregnant rat was to achieve a consistent total dose
of sFlt-1 on the individual mother rats, given intra-amniotic
sFlt-1 is absorbed into the maternal circulation through an
intramembranous pathway, which is characterized by a microscopic
network of fetal vasculature on the fetal surface of the placenta
to mediate the transfer of intraamniotic substances into fetal and
maternal circulations. Similarly, considering the communication
between the amniotic cavity and maternal and fetal circulations
through the intramembranous pathway, intra-amniotic saline was
given in separate litters to serve as controls. The total number of
amniotic sacs in each mother rat was examined and recorded during
laparotomy. The abdominal incision was closed with nylon sutures.
Bupivacaine (1-2 mg/kg, subcutaneous injection) was applied over
the incision wound for postoperative pain control. Pregnant rats
were monitored closely to ensure arousal within 10 minutes after
surgery, and rats were placed back to the cages and were monitored
for activity, ability to drink and eat, and for signs of bleeding
or infection.
[0186] Cesarean Section
[0187] Two days after intra-amniotic injections, cesarean section
was performed on pregnant rats under general anesthesia with
isoflurane inhalation, as described above. The fetus in the
amniotic sac closest to the right ovary was first delivered, which
was followed by delivery of the rest of the fetuses in a
counterclockwise sequence, to identify fetuses exposed to
intra-amniotic injections. The total number of amniotic sacs in
each mother rat was further verified at the time of delivery. The
main reason for performing cesarean section instead of allowing
vaginal delivery is to identify the fetuses exposed to
intra-amniotic injections, based on the order of the amniotic sacs
and their anatomic locations related to the ovaries. All of the rat
pups in the injected amniotic sacs were delivered within 5 minutes
after the onset of anesthesia. Maternal rats were then killed with
pentobarbital sodium. Newborn rats were immediately placed on a
heating pad to avoid hypothermia and were dried manually with gauze
sponges. Pups received no supplemental oxygen or artificial
ventilation at birth. The survival rate at birth was recorded.
Within 30 minutes after birth, the pups were weighed and placed
with foster mother rats in regular cages. For the first 24 h of
life, the newborn pups were monitored closely for mortality or
signs of respiratory distress.
[0188] Rat lungs were harvested at birth for Western blot analysis
and at birth and 14 days of age for histological assessment. Hearts
were dissected and weighed at birth and 7 and 14 days of age. Three
to nine rats were studied in each group for each measurement at
each time point. Survival of the infant rats was monitored and
recorded daily from birth throughout the study period. Survival
rate was calculated as the number of survived pups divided by the
number of sacs that received intra-amniotic injection in each given
litter. Body weight was measured at birth and at the time of being
killed for study measurements.
[0189] Study Measurements
[0190] Tissue for Histological Analysis
[0191] Animals were killed with intraperitoneal pentobarbital
sodium. A catheter was placed in the trachea, and the lungs were
inflated with 4% paraformaldehyde and maintained at 20 cm H.sub.2O
pressure for 60 min. A ligature was tightened around the trachea to
maintain pressure, and then the tracheal cannula was removed. Lungs
were then immersed in 4% paraformaldehyde at room temperature for
24 hours for fixation. A 2-mm-thick transverse section was taken
from the midplane of the right lower lobe and left lobe of the
fixed lungs per animal, respectively, to process and embed in
paraffin wax.
[0192] Immunohistochemistry
[0193] Slides with 5 .mu.m paraffin sections were stained with
hematoxylin and eosin for assessing alveolar structures and with
von Willebrand Factor (vWF), an endothelial cell-specific marker,
for quantifying vessel density.
[0194] Pulmonary Vessel Density
[0195] Pulmonary vessel density was determined by counting
vWF-stained vessels with an external diameter at 50 .mu.m or less
per high-power field. The fields containing large airways or
vessels with external diameter >50 .mu.m were avoided.
[0196] Radial Alveolar Counts (RAC)
[0197] Alveolarization was assessed by the RAC method of Emery and
Mithal as described (Cooney T P, Thurlbeck W M. The radial alveolar
count method of Emery and Mithal: a reappraisal 1--postnatal lung
growth. Thorax 37: 572-579, 1982; Cooney T P, Thurlbeck W M. The
radial alveolar count method of Emery and Mithal: a reappraisal
2--intrauterine and early postnatal lung growth. Thorax 37:
580-583, 1982). Respiratory bronchioles were identified as
bronchioles lined by epithelium in one part of the wall. From the
center of the respiratory bronchiole, a perpendicular line was
dropped to the edge of the acinus connective tissues or septum or
pleura, and the number of septae intersected by this line was
counted.
[0198] Indices of Right Ventricular Hypertrophy
[0199] The right ventricle (RV) and left ventricle plus septum
(LV+S) were dissected and weighed. The ratios of RV to LV+S weights
(RV/LV+S %) and RV/body weights (RV/BW %) were determined to
evaluate right ventricular hypertrophy (RVH).
[0200] Statistical Analysis
[0201] Statistical analysis was performed with the InStat 3.0
software package (GraphPad Software, San Diego, Calif.).
Statistical comparisons were made between groups using t-test or
ANOVA with Newman-Keuls post hoc analysis for significance.
P<0.05 was considered significant.
[0202] Results
[0203] As shown in FIGS. 14 and 15, pulmonary vessel density was
increased in animals treated with sFLT+anti-sFLT compared to those
treated only with sFLT.
[0204] Alveolarization was assessed by the radial alveolar count
(RAC) method. As shown in FIG. 16, when analyzed by morphometric
analysis, sFLT rats had significantly (P<0.001) decreased RAC
compared with the control group (CTL). Treatment with the low dose
of a-sFLT significantly (P<0.01) increased RAC compared to the
sFLT group. This indicates that treatment with a-sFLT can reverse
the decrease in alveolarization caused by sFLT.
[0205] Right ventricular hypertrophy was assessed by weighing the
right ventricle (RV) and left ventricle plus septum (LV+S) and
calculating the ratio. As shown in FIG. 17, animals exposed to sFLT
had a significantly increased (P<0.05) RV/(LV+S) ratio compared
to the control group. Treatment with the low dose of a-sFLT
significantly (P<0.05) decreased the RV/(LV+S) ratio compared to
the sFLT group. This indicates that treatment with a-sFLT can
reverse the right ventricular hypertrophy caused by sFLT.
[0206] The ratio of the right ventricle (RV) to body weight was
determined to evaluation right ventricular hypertrophy. As shown in
FIG. 18, animals exposed to sFLT had a significantly (P<0.05)
increased RV/body weight ratio compared to the control group.
Treatment with the low dose of a-sFLT significantly decreased the
RV/body weight ratio (P<0.05) compared to the sFLT group. This
indicates that treatment with a-sFLT can reverse the right
ventricular hypertrophy caused by sFLT.
Example 5
In Vivo Efficacy of Anti-Flt-1 Antibody in an Endotoxin (ETX) Model
of BPD
[0207] Study Design
[0208] Intra-Amniotic sFlt-1 and ETX Administration
[0209] At 20 days gestation (term: 22 days), pregnant rats were
prepared for receiving intra-amniotic injections. Pregnant rats
were randomly assigned to saline control or ETX (endotoxin) group;
the saline group received normal saline injection into the amniotic
sac, and the and the ETX groups received 10 .mu.g endotoxin per
sac. Following intra-amniotic administration, the abdominal
incision was closed and rats were monitored closely to ensure
arousal after surgery.
[0210] Cesarean Section and Treatment
[0211] Two days after intra-amniotic injections, cesarean section
was performed on pregnant rats under general anesthesia, as
described above. Pups were treated twice a week for two weeks with
1 mg/kg anti-sFLT monoclonal, 10 mg/kg anti-sFLT monoclonal or 10
mg/kg IgG control (mouse IgG1 isotype control).
[0212] Study Measurements
[0213] At day 14, rat lungs were harvested for morphometric
analysis and for histological assessment. Body weight of the
animals was measured at birth and at the time of sacrifice. Lungs
were fixed after inflation with 4% paraformaldehyde at 20 cm
H.sub.2O. Distal airspace structure was assessed by Radial Alveolar
Counts (RAC). Hearts were collected to determine right ventricular
hypertrophy (RV/LS+S weights)
[0214] Body Weight
[0215] The body weight of animals was measured to determine if
postnatal anti-Flt-1 monoclonal antibody treatment improved body
weight following antenatal ETX treatment. Animals administered ETX
in utero followed by postnatal treatment with IgG (control) or
anti-Flt-1 mAb (1 mg/kg or 10 mg/kg) were weighed. Animals
receiving only ETX or ETX+IgG weighed significantly less than
control animals (FIG. 19). The weight of animals receiving
ETX+either dose of anti-Flt-1 mAb was not significantly different
from the weight of control animals. These data indicate that
animals given postnatal anti-Flt-1 mAb have a growth advantage in
an endotoxin induced model of BPD.
[0216] Radial Alveolar Counts (RAC)
[0217] Radial alveolar count was measured to determine if postnatal
anti-Flt-1 monoclonal antibody treatment improved alveolar growth
after antenatal ETX treatment. The lungs of animals administered
ETX in utero followed by postnatal treatment with IgG (control
treatment) or anti-Flt-1 monoclonal antibody (1 mg/kg or 10 mg/kg)
were studied. Animals receiving only ETX or ETX+IgG demonstrated
significantly reduced alveolar growth as compared to control
animals (FIG. 20). Alveolar growth in animals receiving ETX+10
mg/kg of anti-Flt-1 monoclonal antibody was significantly better
than alveolar growth in animals receiving ETX alone. These data
indicate that animals given postnatal anti-Flt-1 monoclonal
antibody have improved lung structure in an endotoxin induced model
of BPD.
[0218] Indices of Right Ventricular Hypertrophy
[0219] The right ventricle was measured to determine if postnatal
anti-Flt-1 monoclonal antibody treatment prevented right
ventricular hypertrophy (RVH) after antenatal ETX treatment. The
hearts of animals administered ETX in utero followed by postnatal
treatment with IgG (control treatment) or anti-Flt-1 monoclonal
antibody (1 mg/kg or 10 mg/kg) were studied. Animals receiving only
ETX or ETX+IgG demonstrated a significantly increased right
ventricle ratio as compared to control animals (FIG. 21). Right
ventricle ratio in animals receiving ETX+either dose of anti-Flt-1
monoclonal antibody was not significantly different from the right
ventricle ratio of control animals. Right ventricle ratio in
animals receiving ETX+either dose of anti-Flt-1 monoclonal antibody
was significantly different from the right ventricle ratio of
animals receiving ETX alone. These data indicate that animals given
postnatal anti-Flt-1 monoclonal antibody have diminished pulmonary
hypertension in an endotoxin induced model of BPD.
[0220] Lung Structure
[0221] Lung structure and pulmonary vessel density was assessed to
determine if postnatal anti-Flt-1 monoclonal antibody treatment
restored lung structure after antenatal ETX treatment. Lungs of
animals administered ETX in utero followed by postnatal treatment
with IgG (control treatment) or anti-Flt-1 monoclonal antibody (1
mg/kg or 10 mg/kg) were studied (FIG. 22). These data indicate that
postnatal anti-sFlt-1 monoclonal antibody restores lung structure
in experimental chorioamnionitis.
EQUIVALENTS AND SCOPE
[0222] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. The scope of the present invention is not intended to be
limited to the above Description, but rather is as set forth in the
following claims.
Sequence CWU 1
1
3121PRTArtificial SequenceSynthetic Oligopeptide 1Gly Ala Pro Gly
Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly1 5 10 15Gly Gly Gly
Ala Pro 20239PRTArtificial SequenceSynthetic Oligopeptide 2Gly Ala
Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly1 5 10 15Gly
Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly 20 25
30Gly Gly Gly Gly Gly Ala Pro 35357PRTArtificial SequenceSynthetic
Oligopeptide 3Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala
Gly Gly Gly1 5 10 15Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala
Ala Ala Ala Gly 20 25 30Gly Gly Gly Gly Gly Ala Pro Gly Gly Gly Gly
Gly Ala Ala Ala Ala 35 40 45Ala Gly Gly Gly Gly Gly Gly Ala Pro 50
55
* * * * *