U.S. patent application number 15/688740 was filed with the patent office on 2018-04-26 for treatment of fibrodysplasia ossificans progressiva.
The applicant listed for this patent is REGENERON PHARMACEUTICALS, INC.. Invention is credited to Aris N. Economides, Sarah J. Hatsell, Vincent J. Idone.
Application Number | 20180111983 15/688740 |
Document ID | / |
Family ID | 54207748 |
Filed Date | 2018-04-26 |
United States Patent
Application |
20180111983 |
Kind Code |
A1 |
Hatsell; Sarah J. ; et
al. |
April 26, 2018 |
Treatment of Fibrodysplasia Ossificans Progressiva
Abstract
Methods for treating Fibrodysplasia Ossificans Progressiva (FOP)
are provided. Such methods involve administering to a subject
having FOP an effective regime of an activin receptor type 2A
(ACVR2A) and/or an activin receptor type 2B (ACVR2B) antagonist or
an activin receptor type 1 (ACVR1) antagonist. Antagonists include
fusion proteins of one or more extracellular domains (ECDs) of
ACVR2A, ACVR2B and/or ACVR1 and the Fc domain of an immunoglobulin
heavy chain, and antibodies against ACVR2A, ACVR2B, ACVR1 or
Activin A.
Inventors: |
Hatsell; Sarah J.; (Nyack,
NY) ; Economides; Aris N.; (Tarrytown, NY) ;
Idone; Vincent J.; (Ridgefield, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
REGENERON PHARMACEUTICALS, INC. |
Tarrytown |
NY |
US |
|
|
Family ID: |
54207748 |
Appl. No.: |
15/688740 |
Filed: |
August 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14850844 |
Sep 10, 2015 |
|
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15688740 |
|
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62049869 |
Sep 12, 2014 |
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62141775 |
Apr 1, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/179 20130101;
C07K 16/18 20130101; C07K 16/28 20130101; A61P 19/08 20180101; C07K
2317/24 20130101; A61K 2039/505 20130101; A61K 39/39533 20130101;
A61P 19/04 20180101; A61P 21/00 20180101; A61P 19/00 20180101; C07K
16/22 20130101; C07K 16/2863 20130101; C07K 2317/33 20130101; C07K
2317/21 20130101; C07K 2319/30 20130101; A61P 43/00 20180101; C07K
14/705 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07K 16/28 20060101 C07K016/28; A61K 39/395 20060101
A61K039/395; C07K 14/705 20060101 C07K014/705; C07K 16/22 20060101
C07K016/22; A61K 38/17 20060101 A61K038/17 |
Claims
1. A method of treating Fibrodysplasia Ossificans Progressiva
(FOP), comprising administering to a subject having FOP an
effective regime of an activin receptor type 2A (ACVR2A) and/or an
activin receptor type 2B (ACVR2B) antagonist.
2-13. (canceled)
14. A method of treating Fibrodysplasia Ossificans Progressiva
(FOP), comprising administering to a subject having FOP an
effective regime of an activin receptor type 1 (ACVR1)
antagonist.
15-18. (canceled)
19. A method of treating Fibrodysplasia Ossificans Progressiva
(FOP), comprising administering to a subject having FOP an
effective regime of an activin receptor type 2A (ACVR2A) and/or an
activin receptor type 2B (ACVR2B) antagonist in combination with an
activin receptor type 1 (ACVR1) antagonist.
20-23. (canceled)
24. A method of treating Fibrodysplasia Ossificans Progressiva
(FOP), comprising administering to a subject having FOP an
effective regime of an antibody against Activin A.
25. The method of claim 24, wherein the antibody competes for
binding with antibody comprising the heavy and light chain variable
regions of the antibody designated H4H10446P, H4H10430P or A1.
26. The method of claim 24, wherein the antibody comprises the
heavy and light chain variable regions of the antibody designated
H4H10446P, H4H10430P or A1.
27. The method of claim 24, wherein the antibody is chimeric,
veneered, humanized or human antibody.
28. The method of claim 24 wherein the antibody is an intact
antibody.
29. The method of claim 24, wherein the antibody is a human kappa
IgG1 antibody.
30. The method of claim 25, wherein the antibody is a human kappa
IgG1 antibody.
31. The method of claim 26, wherein the antibody is a human kappa
IgG1 antibody.
32. The method of claim 27, wherein the antibody is a human kappa
IgG1 antibody.
33. The method of claim 28, wherein the antibody is a human kappa
IgG1 antibody.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 14/850,844 filed Sep. 10, 2015, which claims the benefit under
35 USC 119(e) of US Provisional Application Nos. 62/049,869 filed
Sep. 12, 2014, and 62/141,775 filed Apr. 1, 2015, the disclosures
each of which are herein incorporated by reference in their
entireties
REFERENCE TO A SEQUENCE LISTING
[0002] The application refers to sequences written in the file
T0037US02_SEQLST.txt, created on, Aug. 28, 2017, which is 10,051
bytes. The information contained in this file is hereby
incorporated by reference.
BACKGROUND
[0003] Fibrodysplasia Ossificans Progressiva (FOP) is an autosomal
dominant disorder characterized by early onset, episodic and
progressive ossification of skeletal muscle and associated
connective tissue. FOP is driven by mutations in the intracellular
domain of ACVR1 (ALK2), with the great majority altering Arginine
206 to Histidine (R206H) (Pignolo, R. J. et al. 2011, Orphanet J.
Rare Dis. 6:80). ACVR1 is a type I receptor for bone morphogenic
proteins (BMPs). The R206H mutation, among others, is believed to
increase the sensitivity of the receptor to activation and render
it more resistant to silencing. No effective medical therapy is
known for FOP.
SUMMARY OF THE CLAIMED INVENTION
[0004] The invention provides methods of treating Fibrodysplasia
Ossificans Progressiva (FOP), comprising administering to a subject
having FOP an effective regime of an activin receptor type 2A
(ACVR2A) and/or an activin receptor type 2B (ACVR2B) antagonist. In
some methods, the antagonist comprises an ACVR2A or ACVR2B
extracellular domain. In some methods, the antagonist comprises an
ACVR2A or ACVR2B Fc fusion protein. In some methods, the isotype of
the Fc fusion protein is human IgG1. In some methods, the
antagonist comprises an ACVR2A extracellular domain linked to an
ACVR2B extracellular domain. In some methods, the antagonist
further comprises an Fc domain. In some methods, the antagonist
comprises an ACVR2A extracellular domain fused to a first Fc domain
and an ACVR2B extracellular domain fused to a second Fc domain
wherein the first and second Fc domains are complexed with one
another. In some methods, the antagonist comprises a linker between
the ACVR2A and ACVR2B extracellular domains, each fused to an Fc
domain. In some methods, the antagonist is a fusion protein
comprising an ACVR2A extracellular domain, an ACVR2B extracellular
domain and an Fc domain. In some methods, an effective regime of an
ACVR2A antagonist and an ACVR2B antagonist is administered. In some
methods, the ACVR2A antagonist is an ACVR2A Fc fusion protein and
the ACVR2B antagonist is an ACVR2B Fc fusion protein. In some
methods, the antagonist is an antibody to ACVR2A or ACVR2B. In some
methods, the subject does not have and is not at risk of type II
diabetes, muscular dystrophy, amyotrophic lateral sclerosis (ALS)
or osteoporosis.
[0005] The invention further provides methods of treating FOP,
comprising administering to a subject having FOP an effective
regime of an activin receptor type 1 (ACVR1) antagonist. In some
methods, the antagonist comprises an ACVR1 extracellular domain. In
some methods, the antagonist comprises an ACVR1 fusion protein. In
some methods, the isotype of the Fc fusion protein is human IgG1.
In some methods, the antagonist is an antibody to ACVR1.
[0006] The invention further provides methods of treating
Fibrodysplasia Ossificans Progressiva (FOP), comprising
administering to a subject having FOP an effective regime of an
activin receptor type 2A (ACVR2A) and/or an activin receptor type
2B (ACVR2B) antagonist in combination with an activin receptor type
1 (ACVR1) antagonist. In some methods, the antagonist comprises an
ACVR1, ACVR2A and/or ACVR2B extracellular domain. In some methods,
the antagonist comprises an ACVR1, ACVR2A and/or ACVR2B Fc fusion
protein. In some methods, the isotype of the Fc fusion protein is
human IgG1. In some methods, the antagonist is an antibody to
ACVR1, ACVR2A and/or ACVR2B.
[0007] The invention further provides a method of treating
Fibrodysplasia Ossificans Progressiva (FOP), comprising
administering to a subject having FOP an effective regime of an
antibody against Activin A. Optionally, the antibody competes for
binding with antibody comprising the heavy and light chain variable
regions of the antibody designated H4H10446P, H4H10430P or A1.
Optionally, the antibody comprises the heavy and light chain
variable regions of the antibody designated H4H10446P, H4H10430P or
A1. Optionally, the antibody is a chimeric, veneered, humanized or
human antibody. Optionally, the antibody is an intact antibody.
Optionally, the antibody is a human kappa IgG1 antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1 shows microCT data from mice showing ectopic bone
formation at 6 weeks post initiation of tamoxifen administration
with and without ACVR2A-Fc/ACVR2B-Fc treatment. Nine out of ten
control mFc treated mice (numbers 1 to 9) showed ectopic bone
formation at 6 weeks post tamoxifen administration. Most common
locations are hind limb, neck region and sternum. Two out of eleven
ACVR2A-Fc/ACVR2B-Fc treated mice (numbers 12 and 14) showed ectopic
bone formation at 6 weeks post tamoxifen administration. The
ectopic bone lesions in these two mice were of small size compared
to those seen in the mFc treated group and both located at the
sternum.
[0009] FIG. 2 shows microCT data from mice showing ectopic bone
formation 4 weeks post initiation of tamoxifen administration with
or without LDN212854 treatment. Numbers 1 to 8 correspond to
tamoxifen+vehicle treated mice. Large ectopic bone nodules have
formed in mice numbered 1, 2, 3, 4, 5 and 7, and small ectopic bone
nodules have formed in mice numbered 6 and 8. Numbers 9-16
correspond to tamoxifen+LDN212854 treated mice. Small ectopic bone
nodules have formed in mice numbered 9, 12 and 13. No ectopic bone
nodules could be detected in mice numbered 10, 11, 14, 15 or
16.
[0010] FIG. 3 shows microCT data for mice disposed to ectopic bone
formation treated with an antibody against Activin A, an isotype
matched irrelevant control antibody, and ACVR2A-Fc. The antibody
against Activin A inhibited formation of ectopic bone nodules most
effectively.
[0011] FIG. 4 shows microCT data for mice disposed to ectopic bone
formation treated with an antibody against Activin A, an isotype
matched irrelevant control antibody, and an antibody against
Acvr2a/Acvr2b. The antibody against Activin A and the antibody
against Acvr2a/Acvr2b inhibited formation of ectopic bone
nodules.
[0012] FIG. 5 shows microCT data for mice disposed to ectopic bone
formation treated with varying doses of an antibody against Activin
A compared with an isotype matched irrelevant control antibody.
Dosages between 1 mg/kg and 25 mg/kg were shown to be effective
with 10 mg/kg being the most effective dose tested.
DEFINITIONS
[0013] Antagonists are typically provided in isolated form. This
means that an antagonist is typically at least 50% w/w pure of
interfering proteins and other contaminants arising from its
production or purification, but does not exclude the possibility
that the antagonist is combined with an excess of pharmaceutical
acceptable carrier(s) or other vehicle intended to facilitate its
use. Sometimes antagonists are at least 60, 70, 80, 90, 95 or 99%
w/w pure of interfering proteins and contaminants from production
or purification.
[0014] For purposes of classifying amino acids substitutions as
conservative or nonconservative, amino acids are grouped as
follows: Group I (hydrophobic side chains): met, ala, val, leu,
ile; Group II (neutral hydrophilic side chains): cys, ser, thr;
Group III (acidic side chains): asp, glu; Group IV (basic side
chains): asn, gln, his, lys, arg; Group V (residues influencing
chain orientation): gly, pro; and Group VI (aromatic side chains):
trp, tyr, phe. Conservative substitutions involve substitutions
between amino acids in the same class. Non-conservative
substitutions constitute exchanging a member of one of these
classes for a member of another.
[0015] Percentage sequence identities are determined with antibody
sequences maximally aligned by the Kabat numbering convention for a
variable region or EU numbering for a constant region. For other
proteins, sequence identity can be determined by aligning sequences
using algorithms, such as BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package Release 7.0, Genetics Computer
Group, 575 Science Dr., Madison, Wis.), using default gap
parameters, or by inspection, and the best alignment. After
alignment, if a subject antibody region (e.g., the entire mature
variable region of a heavy or light chain) is being compared with
the same region of a reference antibody, the percentage sequence
identity between the subject and reference antibody regions is the
number of positions occupied by the same amino acid in both the
subject and reference antibody region divided by the total number
of aligned positions of the two regions, with gaps not counted,
multiplied by 100 to convert to percentage.
[0016] Compositions or methods "comprising" one or more recited
elements can include other elements not specifically recited. For
example, a composition that comprises antibody can contain the
antibody alone or in combination with other ingredients.
[0017] A humanized antibody is a genetically engineered antibody in
which the CDRs from a non-human "donor" antibody are grafted into
human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat.
Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539;
Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. Nos. 5,859,205
and 6,881,557; Foote, U.S. Pat. No. 6,881,557). The acceptor
antibody sequences can be, for example, a mature human antibody
sequence, a composite of such sequences, a consensus sequence of
human antibody sequences, or a germline region sequence. Thus, a
humanized antibody is an antibody having some or all CDRs entirely
or substantially from a donor antibody and variable region
framework sequences and constant regions, if present, entirely or
substantially from human antibody sequences. Similarly, a humanized
heavy chain has at least one, two and usually all three CDRs
entirely or substantially from a donor antibody heavy chain, and a
heavy chain variable region framework sequence and heavy chain
constant region, if present, substantially from human heavy chain
variable region framework and constant region sequences. Similarly,
a humanized light chain has at least one, two and usually all three
CDRs entirely or substantially from a donor antibody light chain,
and a light chain variable region framework sequence and light
chain constant region, if present, substantially from human light
chain variable region framework and constant region sequences.
Other than nanobodies and dAbs, a humanized antibody comprises a
humanized heavy chain and a humanized light chain. A CDR in a
humanized antibody is substantially from a corresponding CDR in a
non-human antibody when at least 85%, 90%, 95% or 100% of
corresponding residues (as defined by Kabat) are identical between
the respective CDRs. The variable region framework sequences of an
antibody chain or the constant region of an antibody chain are
substantially from a human variable region framework sequence or
human constant region, respectively, when at least 85, 90, 95 or
100% of corresponding residues defined by Kabat are identical.
[0018] Although humanized antibodies often incorporate all six CDRs
(preferably as defined by Kabat) from a mouse antibody, they can
also be made with less than all CDRs (e.g., at least 3, 4, or 5
CDRs from a mouse antibody) (e.g., Pascalis et al., J. Immunol.
169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:
415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999;
Tamura et al., Journal of Immunology, 164:1432-1441, 2000).
[0019] A chimeric antibody is an antibody in which the mature
variable regions of light and heavy chains of a non-human antibody
(e.g., a mouse) are combined with human light and heavy chain
constant regions. Such antibodies substantially or entirely retain
the binding specificity of the mouse antibody, and are about
two-thirds human sequence.
[0020] A veneered antibody is a type of humanized antibody that
retains some and usually all of the CDRs and some of the non-human
variable region framework residues of a non-human antibody, but
replaces other variable region framework residues that can
contribute to B- or T-cell epitopes, for example exposed residues
(Padlan, Mol. Immunol. 28:489, 1991) with residues from the
corresponding positions of a human antibody sequence. The result is
an antibody in which the CDRs are entirely or substantially from a
non-human antibody and the variable region frameworks of the
non-human antibody are made more human-like by the
substitutions.
[0021] A human antibody can be isolated from a human, or otherwise
result from expression of human immunoglobulin genes (e.g., in a
transgenic mouse, in vitro or by phage display). Methods for
producing human antibodies include the trioma method of Oestberg et
al., Cys muoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664;
and Engleman et al., U.S. Pat. No. 4,634,666. The monoclonal
antibodies can also be produced by transgenic mice bearing human
immune system genes, such as the Veloclmmune.RTM. mouse from
Regeneron Pharmaceuticals, Inc. (Murphy, PNAS 111 no. 14, 5153-5158
(2014), Xenomouse, Jakobovits, Nature Biotechnology 25, 1134-1143
(2007) or HuMAb mouse from Medarex, Inc. (Lonberg, Handbook Exp.
Pharmacol. 181, 69-97 (2008); Lonberg et al., WO93/12227 (1993);
U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No.
5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S.
Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat. No.
5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature
148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996),
Kucherlapati, WO 91/10741 (1991). Human antibodies can also be
produced by phage display methods (see, e.g., Dower et al., WO
91/17271 and McCafferty et al., WO 92/01047, U.S. Pat. No.
5,877,218, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,858,657, U.S.
Pat. No. 5,837,242, U.S. Pat. No. 5,733,743 and U.S. Pat. No.
5,565,332).
[0022] When an antagonist is said to retain a property of a
parental antibody from which it was derived, the retention can be
complete or partial. Complete retention of an activity means the
activity of the antagonist is the same within experimental error or
greater than that of the molecule from which it was derived.
Partial retention of activity means activity significantly above
background level of a negative control (i.e., beyond experimental
error) and preferably at least 50% of the corresponding activity of
the molecule from which it was derived.
[0023] Two antibodies have the same epitope if all amino acid
mutations in the antigen that reduce or eliminate binding of one
antibody reduce or eliminate binding of the other. Two antibodies
have overlapping epitopes if some amino acid mutations that reduce
or eliminate binding of one antibody reduce or eliminate binding of
the other.
[0024] Competition between antibodies is determined by an assay in
which an antibody under test inhibits specific binding of a
reference antibody to a common antigen (see, e.g., Junghans et al.,
Cancer Res. 50:1495, 1990). A test antibody competes with a
reference antibody if an excess of a test antibody (e.g., at least
2.times., 5.times., 10.times., 20.times. or 100.times.) inhibits
binding of the reference antibody by at least 50%, but preferably
75%, 90% or 99%, as measured in a competitive binding assay.
Antibodies identified by competition assay (competing antibodies)
include antibodies binding to the same epitope as the reference
antibody and antibodies binding to an adjacent epitope sufficiently
proximal to the epitope bound by the reference antibody for steric
hindrance to occur.
DETAILED DESCRIPTION
I. Overview
[0025] Methods for treating Fibrodysplasia Ossificans Progressiva
(FOP) are provided. Such methods involve administering to a subject
having FOP an effective regime of an activin receptor type 2A
(ACVR2A) and/or an activin receptor type 2B (ACVR2B) antagonist
and/or an activin receptor type 1 (ACVR1) antagonist, and/or an
Activin A antagonist. Such antagonists include fusion proteins
comprising one or more extracellular domains (ECDs) of ACVR2A,
ACVR2B and/or ACVR1 and the Fc domain of an immunoglobulin heavy
chain. Antibody antagonists of ACVR2A, ACVR2B, ACVR1 or Activin A
are also provided.
II. ACVR1, ACVR2A, ACVR2B and Activin A
[0026] The transforming growth factor .beta. (TGF.beta.)
superfamily of ligands includes, for example, bone morphogenetic
proteins (BMPs) and growth and differentiation factors (GDFs). The
receptors for these ligands are heteromeric receptor complexes made
up of type I and type II transmembrane serine/threonine kinase
receptors. Examples of type I receptors include activin receptor
type IA (ACTRIA, ACVR1, or ALK2), BMP receptor type IA and BMP
receptor type IB. Examples of type II receptors include activin
receptors type IIA and IIB (ACTRIIA or ACVR2A and ACTRIIB or
ACVR2B) and BMP receptor type II. The ligands of the TGF.beta.
superfamily each have differing affinities for the different type I
and type II receptors.
[0027] Both the type I and type II receptors have an extracellular
ligand binding domain (ECD) and an intracellular serine/threonine
kinase domain. In addition, the type I receptors have a
glycine/serine-rich region (GS-box) preceding the kinase domain and
a L45 loop within the kinase domain. Both receptors work together
for ligands to activate downstream signaling pathways, such as Smad
and non-Smad signaling pathways. Activation involves ligand
binding, ligand-receptor oligomerization and transphosphorylation
of the GS box of the type I receptor by the type II receptor
kinase. The type II receptor kinase is constitutively active and
has a role in ligand binding and activation of the type I
receptor.
[0028] ACVR1, also known as activin a receptor type I, ACVR1A,
ACVRLK2, or ALK2, is a type I receptor for the TGF.beta.
superfamily of ligands. ACVR1 has serine/threonine kinase activity
and phosphorylates Smad proteins and activates downstream signaling
pathways. ACVR1 is found in many tissues of the body including
skeletal muscle and cartilage and helps to control the growth and
development of the bones and muscles. As described elsewhere
herein, certain mutations in the ACVR1 gene cause FOP. Examples of
ACVR1 activity include the ability to bind to ligands, the ability
to form a complex with a type II receptor, or the ability to
activate downstream signaling pathways, such as the Smad
pathway.
[0029] ACVR2, also known as activin receptor type II, is a type II
receptor for the TGF.beta. superfamily of ligands. There are at
least two ACVR2 receptors, for example, activin receptor type IIA
(ACVR2A or ACTRIIA) and activin receptor type IIB (ACVR2B or
ACTRIIB). Reference to ACVR2 includes either or both of ACVR2A and
ACVR2B. ACVR2A and ACVR2B can be expressed in multiple tissues,
including skeletal muscle, stomach, heart, endometrium, testes,
prostate, ovary, and neural tissues.
[0030] On ligand binding, an ACVR2 receptor forms a complex with a
type I receptor, such as ACVR1, and phosphorylates the GS box of
the type I receptor, thus enhancing the kinase activity of the type
I receptor. Examples of ACVR2A and ACVR2B activity include the
ability to bind to ligands, the ability to form a complex with a
type I receptor, or the ability to phosphorylate a type I
receptor.
[0031] An exemplary form of human ACVR2A has Swiss Prot accession
number P27037. Residues 1-19 are a signal peptide, residues 20-135
are an extracellular domain, residues 59-116 are an activin types I
and II receptor domain, residues 136-161 are a transmembrane domain
and residues 162-513 are a cytoplasmic domain. An exemplary form of
human ACVR2B is assigned Swiss Prot Number Q13705. Residues 1-18
are a signal sequence, residues 19-137 are an extracellular domain,
residues 27-117 are an activin types I and II receptor domain,
residues 138-158 are a transmembrane domain and residues 159-512
are a cytoplasmic domain. An exemplary form of human ACVR1 has
Swiss Prot accession number Q04771. Residues 1-20 are a signal
sequence, residues 21-123 are extracellular domain, residues 33-104
are an activin types I and II receptor domain, residues 124-146 are
a transmembrane domain and residues 147-509 are a cytoplasmic
domain. Reference to any of ACVR1, ACVR2A and ACVR2B includes these
exemplary forms, known isoforms and polymorphisms thereof, such as
those listed in the Swiss Prot database, cognate forms from other
species, and other variants having at least 90, 95, 96, 97, 98 or
99% sequence identity with an exemplified form.
[0032] Residues of forms of ACVR2A, ACVR2B and ACVR1 other than the
exemplified sequences defined above are numbered by maximum
alignment with the corresponding exemplified sequences so aligned
residues are allocated the same number. Substitutions from
exemplified sequences can be conservative or non-conservative
substitutions. Reference to ACVR1, ACVR2A or ACVR2B also includes
intact extracellular domains (e.g., residues 20-135, 19-137 or
21-123 of ACVR2A, ACVR2B and ACVR1, respectively) or a portion
thereof free or substantially free of transmembrane and cytoplasmic
portion. Portions of an extracellular domain retain sufficient
residues of the intact extracellular domain to bind at least one
ligand or counter receptor that binds to the intact extracellular
domain and thereby antagonize the relevant receptor (e.g., residues
59-116, 27-117 or 33-104 of ACVR2A, ACVR2B and ACVR1,
respectively).
[0033] Activin A in humans can exist as a homo or heterodimeric
protein. The homodimeric protein contains a homodimeric beta A
subunit pair. The heterodimeric protein contains a beta subunit and
a beta B, beta C or beta E subunit (i.e., beta A beta B, beta A
beta C, or beta A beta E. The subunits are each expressed as
precursor polypeptides including a signal peptide, propeptide and
mature polypeptide. An exemplary form of human beta A subunit
precursor is a polypeptide of length 426 amino acids designated
Swiss Prot P08476 of which residues 1-20 are a signal peptide,
residues 21-310 are a propeptide and residues 311-426 are the
mature polypeptide. An exemplary form of a beta B subunit precursor
polypeptide is designated Swiss Prot P09529 of which residues 1-28
are a signal peptide, residues 29-292 a propeptide and residues
293-407 a mature polypeptide. An exemplary form of a beta C subunit
is designated Swiss Prot P55103, of which residues 1-18 are a
signal peptide, residues 19-236 are a propeptide and residues
237-352 are a mature polypeptide. An exemplary form of a beta E
subunit precursor is designated Swiss Prot P58166 of which residues
1-19 are a signal peptide, residues 20-236 are a propeptide and
residues 237-350 are a mature polypeptide. Several variants of
these sequences are known as described in the Swiss Prot Data base.
Reference to Activin A includes any of the beta A homodimer, beta A
beta B, beta A beta C and beta A beta E heterodimer forms, as well
as their subunits, as well as their precursors in which subunits
are attached to the propeptide and/or signal peptide defined by the
exemplary Swiss Prot sequences provided or other natural occurring
human forms of these sequences. Activin A signals through binding
to ACVR2A or ACVR2B, but is not known to be a ligand for ACVR1.
III. Antagonists of ACVR1, ACVR2A, ACVR2B
[0034] Antagonists of the type I receptor ACVR1 and of the type II
receptor ACVR2 proteins (e.g., ACVR2A and/or ACVR2B) are provided
for treating FOP. Such antagonists can antagonize receptors
directly by binding to the receptor (as for an antibody to ACVR1,
ACVR2A or ACVR2B) or indirectly by binding to a ligand or counter
receptor and inhibiting the ligand or counter receptor from binding
to ACVR1, ACVR2A or ACVR2B (as for a fusion protein of ACVR1,
ACVR2A or ACVR2B) among other mechanisms. Antagonists of ACVR2A and
ACVR2B can also bind to Activin A.
[0035] An ACVR1, ACVR2A or ACVR2B antagonist provided herein can
inhibit or reduce the activity of ACVR1, ACVR2A and/or ACVR2B by at
least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%
or more relative to a control cell or animal model that did not
receive the antagonist.
[0036] Any antagonist of ACVR1, ACVR2A or ACVR2B can be used in the
methods for treating FOP. The antagonist can comprise, for example,
an ACVR1, ACVR2A or ACVR2B polypeptide, such as an extracellular
domain, an antagonist antibody, or a small molecule inhibitor.
[0037] A. Extracellular Domains of ACVR1, ACVR2A and ACVR2B
Polypeptides
[0038] Antagonists include ACVR1, ACVR2A and ACVR2B proteins and
fragments thereof effective to inhibit at least one activity of
ACVR1, ACVR2A and ACVR2B, respectively. Such antagonists typically
include the extracellular domain of ACVR1, ACVR2A or ACVR2B or a
portion thereof. Preferably, such extracellular domains are
entirely or substantially free of the transmembrane and cytoplasmic
regions (i.e., any remaining residues from these regions have no
significant effect on function of the extracellular domain). In
other words, the ACVR2A, ACVR2B or ACVR1 component of such
antagonists consists of or consists essentially of the entire
extracellular domain of ACVR2A, ACVR2B or ACVR1 ora portion thereof
as defined above Such antagonists may or may not include other
component(s) distinct from ACVR2A, ACVR2B or ACVR1 as further
described below. Such extracellular domains free or substantially
free of transmembrane and cytoplasmic domains are soluble. Such
extracellular domains can function as an antagonist by binding to a
soluble ligand or counter receptor, effectively competing with the
ligand or counter receptor binding to the ACVR1, ACVR2A or ACVR2B
cell surface receptor, thereby modulating (reducing) the
availability of the ligand or counter receptor in vivo.
[0039] Soluble extracellular domains can be initially expressed
with a signal sequence, which is cleaved in the course of
expression. The signal sequence can be a native signal sequence of
an ACVR1, ACVR2A or ACVR2B, such as those described in U.S. Pat.
No. 7,709,605, which is incorporated by reference herein in its
entirety, or can be a signal sequence from a different protein such
honey bee melittin (HBM) or tissue plasminogen activator (TPA).
Alternatively, soluble extracellular ACVR1, ACVR2A or ACVR2B
polypeptides can be synthesized or expressed without a signal
sequence.
[0040] The ECDs or ligand binding domains of ACVR1, ACVR2A and
ACVR2B are highly conserved among species including mouse and
human. The ECDs contain a cysteine rich region and a C-terminal
tail region. The ECDs of ACVR1, ACVR2A and ACVR2B bind to a diverse
group of TGF.beta. family ligands, including, for example, Activin
A, myostatin (GDF-8), GDF-11 and BMPs. See, e.g., Souza et al.
(2008) Molecular Endocrinology 22(12):2689-2702.
[0041] Examples of ACVR2A and ACVR2B polypeptides and soluble
ACVR2A and ACVR2B polypeptides include those disclosed in U.S. Pat.
No. 7,842,633; U.S. Pat. No. 7,960,343; and U.S. Pat. No.
7,709,605, each of which is incorporated by reference herein in
their entirety.
[0042] The ECD of an ACVR1, ACVR2A or ACVR2B polypeptide can be
mutated such that the variant polypeptide has altered ligand
binding properties (e.g., binding specificity or affinity). Some
variant ACVR1, ACVR2A or ACVR2B polypeptides have altered binding
affinity (e.g., elevated or reduced) for a specific ligand.
Variants have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% sequence identity to the naturally occurring ACVR1, ACVR2A or
ACVR2B sequences, and retain biological activity and hence have an
ACVR1, ACVR2A or ACVR2B activity as described elsewhere herein.
Active variants and fragments of ACVR2A and ACVR2B are described,
for example, in U.S. Pat. No. 7,842,633; U.S. Pat. No. 7,960,343;
and U.S. Pat. No. 7,709,605, each of which is incorporated by
reference herein in its entirety.
[0043] Assays to measure ACVR1, ACVR2A or ACVR2B activity are
disclosed in e.g., U.S. Pat. No. 7,842,633; U.S. Pat. No.
7,960,343; and U.S. Pat. No. 7,709,605. For example, an ACVR1,
ACVR2A or ACVR2B polypeptide variant can be screened for the
ability to bind a ligand or for the ability to prevent binding of a
ligand to an ACVR1, ACVR2A or ACVR2B receptor protein.
[0044] B. Fusion Proteins
[0045] The ACVR1, ACVR2A and ACVR2B polypeptides described above
can be expressed as fusion proteins having at least a portion of an
ACVR1, ACVR2A and/or ACVR2B polypeptide and one or more fusion
domains.
[0046] Fusion domains include an immunoglobulin heavy chain
constant region (Fc), human serum albumin (HSA), glutathione S
transferase (GST), protein A, protein G, or any fusion domain which
can be useful in stabilizing, solubilizing, isolating or
multimerizing a fusion protein.
[0047] An Fc domain of an immunoglobulin heavy chain is a preferred
domain for fusion proteins. Fusions with the Fc portion of an
immunoglobulin confer desirable pharmacokinetic properties on a
wide range of proteins (e.g., increases stability and/or serum
half-life of the protein). Thus, the invention provides fusion
proteins comprising at least one ECD of an ACVR1, ACVR2A and/or
ACVR2B fused to an Fc domain of an immunoglobulin.
[0048] The Fc domain for use in the present methods can be from any
immunoglobulin. Any of the various classes of immunoglobulin can be
used, including IgG, IgA, IgM, IgD and IgE. Within the IgG class
there are different subclasses or isotypes, including, for example,
IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4. In one embodiment,
the Fc fusion protein comprises the Fc domain of an IgG molecule.
In a further embodiment, the Fc domain is from an IgG.sub.1
molecule. The immunoglobulin molecule can be of any animal type,
including, for example, a mammal, a rodent, a human, a mouse, a
rat, a hamster or a rabbit. In one embodiment, the immunoglobulin
Fc domain is from a mammal. In another embodiment, the Fc domain is
from a human. In yet another embodiment, the Fc domain is from a
rodent, such as a mouse or rat. In a specific embodiment, the Fc
domain of the fusion protein is from human IgG1.
[0049] The Fc-fusion proteins provided herein can be made by any
method known in the art. The Fc-fusion proteins can include at
least CH2 and CH3 regions, and typically at least a portion of a
hinge region. Although the CH1 region can be present, it is
typically omitted in fusion proteins.
[0050] The fusion can be made at any site within the Fc portion of
an immunoglobulin constant domain. Fusions can be made to the
C-terminus of the Fc portion of a constant domain, or immediately
N-terminal to the CH1 region of the heavy chain. Particular sites
can be selected to optimize the biological activity, secretion or
binding characteristics of the Fc-fusion protein.
[0051] In some cases, a nucleic acid encoding the ECD of ACVR1,
ACVR2A and/or ACVR2B is fused C-terminally to a nucleic acid
encoding the N-terminus of an immunoglobulin constant domain
sequence. In other cases, N-terminal fusions are also possible. It
is also possible to fuse an ECD of ACVR1, ACVR2A and/or ACVR2B to
both the N-terminus and the C-terminus of an immunoglobulin
constant domain sequence.
[0052] For the production of immunoglobulin fusions, see also U.S.
Pat. No. 5,428,130, U.S. Pat. No. 5,843,725, U.S. Pat. No.
6,018,026 and WO2005/070966, each of which is incorporated by
reference herein in their entirety.
[0053] A fusion protein can be produced, for example, by
recombinant expression of a nucleic acid encoding the fusion
protein. For example, the fusion protein can be made by fusing a
nucleic acid encoding an ECD of ACVR1, ACVR2A and/or ACVR2B to a
nucleic acid encoding an Fc domain. The ACVR1, ACVR2A and/or ACVR2B
ECD nucleic acid can be fused to the N-terminus of a nucleic acid
encoding an Fc domain or can be fused to the C-terminus of a gene
encoding an Fc domain. Alternatively, the ECD can be fused at any
position in the Fc domain.
[0054] The ECD fusion proteins can also include a linker. In the
case of an Fc fusion protein, the linker can be positioned between
the ACVR1, ACVR2A or ACVR2B ECD and the Fc domain, optionally
replacing part or all of the hinge region. The linker can be 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 or more amino acids that
are relatively free of secondary structure. A linker can be rich in
glycine and proline residues and can, for example, contain
repeating sequences of threonine/serine and glycines (e.g.,
TG.sub.4 or SG.sub.4 repeats).
[0055] Two or more ECD-Fc fusion proteins can be joined together by
a linker. In such cases, the linker can be positioned between the
ECDs or the linker can be positioned between the Fc domains to join
the fusion proteins together. For example, 1, 2, 3, 4 or more
ACVR1, ACVR2A and/or ACVR2B Fc fusion proteins can be linked
together.
[0056] Examples of ACVR2A and/or ACVR2B ECD fusion proteins have
been described, such as those disclosed in U.S. Pat. No. 7,842,633;
U.S. Pat. No. 7,960,343; and U.S. Pat. No. 7,709,605, each of which
is incorporated by reference herein in their entirety.
[0057] One example of an ACVR2A antagonist is known as Sotatercept
(also called ACE-011). Sotatercept contains the ECD of ACVR2A fused
to a human IgG1 Fc domain and is described in detail in Carrancio
et al., (2014) British J Haematology. 165(6):870-872, which is
incorporated by reference herein in its entirety.
[0058] One example of an ACVR2B antagonist is known as ACE-031.
ACE-031 contains the ECD of ACVR2B fused to a human IgG1 Fc domain
and is described in detail in Sako et al., (2010) J. Biol. Chem.
285(27):21037-21048, which is incorporated by reference herein in
its entirety.
[0059] Examples of ACVR1 ECD fusion proteins are known, such as
those disclosed in Berasi, et al., (2011) Growth Factors,
29(4):128-139; which is incorporated by reference herein in its
entirety.
[0060] C. Hybrid ECD Fusion Proteins
[0061] Hybrid or multispecific ECD fusion protein antagonists are
also provided. Hybrid ECD fusion proteins can comprise a
combination of two or more ACVR1, ACVR2A and/or ACVR2B ECDs. For
example, the fusion proteins can comprise 1, 2, 3, 4 or more
molecules of an ACVR1, ACVR2A and/or ACVR2B ECD. In one embodiment,
the antagonist comprises an ACVR2A ECD linked to an ACVR2B ECD. In
a further embodiment, the antagonist further comprises an Fc
domain.
[0062] In one embodiment, a fusion protein can comprise one or more
molecules of an ACVR2A ECD and one or more molecules of an ACVR2B
ECD. In another embodiment, a fusion protein can comprise one or
more molecules of an ACVR1 ECD and one or more molecules of an
ACVR2A ECD. In another embodiment, a fusion protein can comprise
one or more molecules of an ACVR1 ECD and one or more molecules of
an ACVR2B ECD.
[0063] In one embodiment, a fusion protein comprises one or more
ACVR2A ECD-Fc fusion proteins and one or more ACVR2B ECD-Fc fusion
proteins which are complexed together. In another embodiment, a
fusion protein comprises one or more ACVR1 ECD-Fc fusion proteins
and one or more ACVR2A ECD-Fc fusion proteins which are complexed
together. In another embodiment, a fusion protein comprises one or
more ACVR1 ECD-Fc fusion proteins and one or more ACVR2B ECD-Fc
fusion proteins which are complexed together. In such cases, the
fusion proteins can be joined together via their Fc domains, for
example, by at least one disulfide linkage or by a linker sequence.
Alternatively, the ECD portions of the fusion protein can be joined
together by a linker sequence.
[0064] In one embodiment, the antagonist comprises an ACVR2A ECD
fused to a first Fc domain and an ACVR2B ECD fused to a second Fc
domain. In such cases, the Fc domains can be complexed with one
another. In another embodiment, the antagonist comprises a linker
between the ACVR2A and ACVR2B ECDs, each fused to an Fc domain.
[0065] The fusion proteins can be constructed to generate ACVR1,
ACVR2A, and/or ACVR2B antagonists in a tandem format. In one
embodiment, a fusion protein comprises two or more ECDs from ACVR1,
ACVR2A and/or ACVR2B in tandem followed by an Fc domain. In some
cases the ECDs arranged in tandem are separated by a linker
sequence. Such a tandem fusion protein can comprise 1, 2, 3, 4 or
more ACVR1, ACVR2A and/or ACVR2B ECDs.
[0066] D. Antibody Antagonists
[0067] An ACVR1, ACVR2A or ACVR2B antagonist includes antibodies
against (in other words specifically binding to) any of these
receptors, preferably antibodies having an epitope within the
extracellular domain. Specific binding of an antibody or fusion
protein to its target antigen means an affinity of at least
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, or 10.sup.10 M.sup.-1.
Specific binding is detectably higher in magnitude and
distinguishable from non-specific binding occurring to at least one
unrelated target. Methods for preparing antibodies are known to the
art. See, for example, Kohler & Milstein (1975) Nature
256:495-497; and Harlow & Lane (1988) Antibodies: a Laboratory
Manual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.
[0068] Any antibody that inhibits or reduces the activity of ACVR1,
ACVR2A and/or ACVR2B (e.g., an antagonist antibody) can be used.
Such ACVR2A and ACVR2B antibodies include, for example, those
antibodies disclosed in U.S. Pat. No. 8,486,403, U.S. Pat. No.
8,128,933, WO2009/137075, and Lach-Trifilieff, et al. (2014) Mol.
Cell Biol. 34(4):606-618, each of which is incorporated by
reference herein in their entirety. Humanized, chimeric and
veneered forms of any of these antibodies are included as are
antibodies competing for binding therewith.
[0069] In one embodiment, the antibody is an anti-ACVR2A antibody.
In another embodiment, the antibody is an anti-ACVR2B antibody. In
other embodiments, the antibody can be a bispecific antibody
against both ACVR2A and ACVR2B. In another embodiment, the antibody
is an anti-ACVR1 antibody. In other embodiments, the antibody can
be a bispecific antibody against both ACVR1 and ACVR2A or against
both ACVR1 and ACVR2B.
[0070] The term "antibody" covers intact antibodies with two pairs
of heavy and light chains, antibody fragments that can bind antigen
(e.g., Fab, F(ab').sub.2, Fv, single chain antibodies, diabodies,
antibody chimeras, hybrid antibodies, bispecific antibodies,
humanized antibodies, and the like), and recombinant peptides
comprising the forgoing.
[0071] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen-binding or variable region of the
intact antibody. Examples of antibody fragments include Fab,
F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et
al. (1995) Protein Eng. 10:1057-1062); single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments.
[0072] The antibody can be monoclonal or polyclonal. A "monoclonal
antibody" is an antibody obtained from a population of
substantially homogeneous antibodies, that is, the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that can be present in minor
amounts. Monoclonal antibodies are often highly specific, being
directed against a single antigenic site. Furthermore, in contrast
to conventional (polyclonal) antibody preparations that typically
include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is typically
directed against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
such as those produced by a clonal population of B-cells, and does
not require production of the antibody by any particular
method.
[0073] Monoclonal antibodies to be used in accordance with the
methods provided herein can be made by the hybridoma method first
described by Kohler et al. (1975) Nature 256:495, or a modification
thereof. Typically, an animal, such as a mouse, is immunized with a
solution containing an antigen (e.g., an ACVR1, ACVR2A and/or
ACVR2B polypeptide, or Activin A particularly the extracellular
domain (in receptors) or a portion thereof).
[0074] Immunization can be performed by mixing or emulsifying the
antigen-containing solution in saline, preferably in an adjuvant
such as Freund's complete adjuvant, and injecting the mixture or
emulsion parenterally. After immunization of the animal, the spleen
(and optionally, several large lymph nodes) are removed and
dissociated into single cells. The spleen cells can be screened by
applying a cell suspension to a plate or well coated with the
antigen of interest. The B-cells expressing membrane bound
immunoglobulin specific for the antigen bind to the plate and are
not rinsed away. Resulting B-cells, or all dissociated spleen
cells, are then induced to fuse with myeloma cells to form
hybridomas, and are cultured in a selective medium. The resulting
cells are plated by serial dilution and are assayed for the
production of antibodies that specifically bind the antigen of
interest (and that do not bind to unrelated antigens). The selected
monoclonal antibody (mAb)-secreting hybridomas are then cultured
either in vitro (e.g., in tissue culture bottles or hollow fiber
reactors), or in vivo (as ascites in mice).
[0075] Alternatively, the monoclonal antibodies can be made by
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The
monoclonal antibodies can also be isolated from phage antibody
libraries using the techniques described in, for example, Clackson
et al. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol.
222:581-597; and U.S. Pat. No. 5,514,548.
[0076] "Antibodies" include chimeric, veneered, humanized and human
monoclonal antibodies against any of ACVR1, ACVR2A, ACVR2B and
Activin A as defined above.
[0077] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these can be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and
IgA2. The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called alpha, delta,
epsilon, gamma, and mu, respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0078] The present monoclonal antibodies or Fc fusion proteins can
be any of the various antibody classes. In one embodiment, the
monoclonal antibody is an IgG class antibody. In other embodiments,
the monoclonal antibody can be of the IgM, IgE, IgD, or IgA class.
In specific embodiments, the antibody is an isotype of IgG, such
as, IgG1, IgG2, IgG3 or IgG4, particularly human IgG1, IgG2, IgG3
or IgG4.
[0079] One or several amino acids at the amino or carboxy terminus
of the light and/or heavy chain, such as a C-terminal lysine of the
heavy chain, can be missing or derivatized in a proportion or all
of the molecules. Substitutions can be made in the constant regions
to reduce or increase effector function such as complement-mediated
cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Pat. No.
5,624,821; Tso et al., U.S. Pat. No. 5,834,597; and Lazar et al.,
Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life
in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213,
2004). Exemplary substitutions include a Gln at position 250 and/or
a Leu at position 428 (EU numbering) for increasing the half-life
of an antibody. Substitution at any of positions 234, 235, 236
and/or 237 reduces affinity for Fc.gamma. receptors, particularly
Fc.gamma.RI receptor (see, e.g., U.S. Pat. No. 6,624,821).
Optionally, positions 234, 236 and/or 237 in human IgG2 are
substituted with alanine and position 235 with glutamine. (See,
e.g., U.S. Pat. No. 5,624,821). Effector functions can also be
reduced by substitution of EFLG at positions 232-236 with PVA (see
WO14/121087). Optionally, S at position 428 can be replaced by P,
particularly in human IgG4 to reduce exchange between endogenous
and exogenous immunoglobulins. Other variations can add or remove
sites of post-translational modification, such as N-linked
glycosylation at N-X-S/T motifs. Variations can also include
introduction of knobs (i.e., replacement of one or more amino acids
with larger amino acids) or holes (i.e., replacement of one or more
amino acids with smaller amino acids) to promote formation of
heterodimers between different heavy chains for production of
bispecific antibodies. Exemplary substitutions to form a knob and
hole pair are T336Y and Y407T, respectively (Ridgeway et al.,
Protein Engineering vol. 9 no. 7 pp. 617-621, 1996). Variations can
also include mutations that reduce protein A interaction (e.g.,
H435R and Y436F) in the EU numbering system. Bispecific antibodies
in which one heavy chain has such a variation, and another does
not, can be separated from their parental antibodies by protein-A
affinity chromatography.
[0080] Antibodies can also include antibodies specifically binding
to Activin A. Such antibodies can specifically bind to any or all
of the beta A beta A, beta A beta B, beta A beta C and beta A beta
E forms of Activin A. Some antibodies specifically bind to only one
of these forms (i.e., beta A beta A, beta A beta B, beta A beta C
or beta A beta E). Specificity for the beta A beta B, beta A beta C
and beta A beta E forms can be conferred by an epitope within the
beta B, beta C or beta E subunit, respectively, or for an epitope
to which both components of the heterodimer contribute. Specificity
for beta A beta can be conferred by an epitope contributed by both
molecules within the homodimer (e.g., at the interface of
subunits). Some antibodies specifically bind to all of these forms
of Activin A, in which case the epitope is typically on the beta A
subunit. Antibodies typically have epitopes within the mature
polypeptide component of the precursor proteins. Some antibodies
specifically bind to any or all forms of Activin A without binding
to human inhibin, which exists in the form of alpha (Swiss Prot
P05111) beta A or alpha beta B heterodimers. Some antibodies
specifically bind to any or all forms of Activin A and bind to
either or both forms of human inhibin. Although it is believed that
such antibodies inhibit signal transduction of Activin A through
one or more of its counterreceptors, ACVR2A and/or ACVR2B and/or
BMPR2, an understanding of mechanism is not required for use of
such antibodies in methods of treating FOP.
[0081] A substantial number of antibodies against Activin A have
been reported. For example, US2015/00373339 discloses human
antibodies designated H4H10423P, H4H10424P, H4H10426P, H4H10429P,
H4H10430P, H4H10432P2, H4H10433P2, H4H10436P2, H4H10437P2,
H4H10438P2, H4H10440P2, H4H10442P2, H4H10445P2, H4H10446P2,
H4H10447P2, H4H10447P2, H4H10448P2, H4H10452P2. U.S. Pat. No.
8,309,082 discloses human antibodies A1-A14. Mouse antibodies
against Activin A are available from several commercial suppliers,
such as MAB3381 from R&D Systems or 9H16 from Novus Biologicals
or MM0074-7L18 (a b89307) AbCam.
[0082] Preferred antibodies have an affinity for Activin A
(measured at 25.degree. C. as in Example 3 of US2015/00373339) of
at least 10.sup.8 M.sup.-1, 10.sup.9 M.sup.-1, 10.sup.10M.sup.-1,
10.sup.11 M.sup.-1, 10.sup.12 M.sup.-1, or 10.sup.13 M.sup.-1. Some
antibodies have an affinity within a range of 10.sup.9-10.sup.12
M.sup.-1. Preferred antibodies inhibit signal transduction of
Activin A with an IC50 of less than 4 nM, and preferably less than
400 pM or 40 pM. Some antibodies inhibit signal transduction with
and IC50 in a range of 4 nM to 10 pM or 3.5 nM to 35 pM.
[0083] Signal transduction inhibition can be measured as in Example
6 of US20150037339, which is summarized as follows. A human A204
rhabdomyosarcoma cell line is transfected with a Smad
2/3-luciferase reporter plasmid to produce the A204/CAGAx12-Luc
cell line. A204/CAGAx12-Luc cells were maintained in McCoy's 5A
supplemented with 10% fetal bovine serum,
penicillin/streptomycin/glutamine and 250 .mu.g/mL of G418. For the
bioassay, A204/CAGAx12-Luc cells were seeded onto 96-well assay
plates at 10,000 cells/well in low serum media, 0.5% FBS and
OPTIMEM, and incubated at 37.degree. C. and 5% CO.sub.2 overnight.
Activin A is serially diluted at 1:3 from 100 to 0.002 nM and added
to cells starting along with a control containing no Activin.
Antibodies are serially diluted at 1:3 starting from 100 to 0.002
nM, 1000 to 0.02 nM, or 300 to 0.005 nM including control samples
containing either an appropriate isotype control antibody or no
antibody and added to cells with a constant concentration of 100 pM
Activin A.
[0084] Some antibodies inhibit binding of Activin A to ACVR2A
and/or ACVR2B and/or BMPR2 by at least 1%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 99%, as measured when the receptor is
expressed from a cell or the extracellular domain is fused with an
Fc domain as a fusion protein, and the fusion protein is
immobilized to support (e.g., a Biacore sensor chip). In such
measurements, the antibody and Activin A should be present in
equimolar amounts and the receptor or extracellular domain in
excess.
[0085] Some antibodies bind to an epitope within residues 321-343
or 391-421 of full-length Activin A, which correspond to C11-S33
and C81-E111 of the mature protein.
[0086] An exemplary antibody used in the present examples is
designated H4H10446P in US2015037339. Its heavy chain variable
region and heavy chain CDR1, CDR2 and CDR3 having the amino acid
sequences of SEQ ID NOs:162, 164, 166 and 168, respectively, of
US2015/00373339 (present SEQ ID NOs:1-4, respectively). Its light
chain variable region and light chain CDRs, CDRL1, CDRL2 and CDRL3
having the amino acid sequences of SEQ ID NO:146, 148, 150 and 152,
respectively, of US2015/0037339 (present SEQ ID NOs:5-8,
respectively). H4H10446P inhibits Activin A mediated signaling
through ACVR2A and/or ACVRIIB, but does not inhibit strongly, if at
all, Activin A binding to ACRIIA or ACVR2B. Other antibodies
competing with H4H10446P for binding to human Activin A or binding
to the same epitope on human Activin A as H4H10446P are included
and sharing its inhibition of signaling are also included.
[0087] Another exemplary antibody for use in the present methods is
designated H4H10430P in US2015037339. Its heavy chain variable
region and heavy chain CDRs CDRH1, CDRH2 and CDRH3 having the amino
acid sequences of SEQ ID NOs:66, 68, 70 and 72, respectively, in
US2015/00373339 (present SEQ ID NOs:9-12, respectively). Its light
chain variable region and light chain CDRs, CDRL1, CDRL2 and CDRL3
having the amino acid sequences of SEQ ID NOs:74, 76, 78 and 80,
respectively, in US2015/0037339 (present SEQ ID NOs:13-16,
respectively). This antibody inhibits binding of Activin A to
ACRV2A and/or ACVR2B and inhibits signal transduction through one
or both of these receptors. Other antibodies competing with
H4H10430P for binding to Activin A or binding to the same epitope
on Activin A as H4H10430P and sharing its property of inhibiting
Activin A binding to and signal transduction through ACVR2A and
ACVR2B are also included.
[0088] Another exemplary antibody for use in the present methods is
the antibodies designated A1 in U.S. Pat. No. 8,309,082, which is
characterized by light and heavy chain variable regions having the
sequences SEQ ID NOs:9 and 10 in U.S. Pat. No. 8,309,082 (present
SEQ ID NOs:17 and 18, respectively). Its light chain CDRs, CDRL1,
CDRL2 and CDRL3 having the sequences SEQ ID NO:11, 12, and 13,
respectively, in U.S. Pat. No. 8,309,082 (present SEQ ID NOs:19-21,
respectively), and its heavy chain CDRs, CDRH1, CDRH2 and CDRH3
having the sequences SEQ ID NOs: 62, 63 and 64, respectively, in
U.S. Pat. No. 8,309,082 (present SEQ ID NOs:22-24, respectively).
Other antibodies competing with H4H10430P for binding to Activin A
or binding to the same epitope on Activin A as H4H10430P and
sharing its property of inhibiting Activin A binding to and
transducing a signal through ACVR2A and/or ACVR2B are also
included.
[0089] Other antibodies can be obtained by mutagenesis of cDNA
encoding the heavy and light chains of any of the above-mentioned
antibodies. Monoclonal antibodies that are at least 90%, 95% or 99%
identical to any of the above-mentioned antibodies in amino acid
sequence of the mature heavy and/or light chain variable regions
and maintain its functional properties, and/or which differ from
the respective antibody by a small number of functionally
inconsequential amino acid substitutions (e.g., conservative
substitutions), deletions, or insertions are also included in the
invention. Monoclonal antibodies having at least 1, 2, 3, 4, 5 and
preferably all six CDR(s) that are 90%, 95%, 99% or 100% identical
to corresponding CDRs of any of the exemplified antibodies are also
included. CDRs are preferably as defined by Kabat, but can be
defined by any conventional alternative definition, such as
Chothia, composite Kabat-Chothia, the contact definition or AbM
definition (see world wide web bioinf.org.uk/abs).
[0090] E. Small Molecule Antagonists
[0091] Antagonists of ACVR1, ACVR2A and ACVR2B can also be small
molecule antagonists. Such small molecule antagonists can inhibit
an activity of ACVR1, ACVR2A, ACVR2B or Activin A. Small molecule
antagonists of ACVR1 include, for example, LDN-212854 described in
Mohedas et al., (2013) ACS Chem. Biol. 8:1291-1302, which is
incorporated by reference herein in its entirety.
IV. Screening Assays
[0092] The activity of the various ACVR1, ACVR2A and/or ACVR2B
antagonists and variants or fragments thereof provided herein can
be screened in a variety of assays. For example, ACVR1, ACVR2A
and/or ACVR2B antagonists and variants thereof can be screened for
their ability to bind to ligands or bind to ACVR1, ACVR2A or ACVR2B
receptors, for their ability to inhibit binding of a ligand to an
ACVR1 and/or ACVR2 polypeptide, and/or for their ability to inhibit
activity of the ACVR1 or ACVR2 receptors.
[0093] The activity of an ACVR1 or an ACVR2 antagonist or variants
or fragments thereof can be tested in vitro or in cell based
assays. In vitro binding assays and assays to measure inhibition of
receptor activity are well known. Various assays to measure the
activity of an ACVR1, ACVR2A or ACVR2B antagonist are described in
detail, for example, in U.S. Pat. No. 7,842,663 which is
incorporated by reference herein in its entirety.
[0094] The ability of the antagonist to modulate complex formation
between the ACVR1 or ACVR2 polypeptide and its binding protein can
be detected by a variety of techniques. For instance, modulation of
the formation of complexes can be quantitated using, for example,
detectably labeled proteins such as radiolabeled .sup.32P,
.sup.35S, .sup.14C or .sup.3H), fluorescently labeled (e.g., FITC),
or enzymatically labeled ACVR1 or ACVR2 polypeptide or its binding
protein, by immunoassay, or by chromatographic detection.
[0095] The ability of the ACVR1 or ACVR2 antagonist to inhibit
ACVR1 or ACVR2 receptor-mediated signaling can be monitored. For
example, the effects of downstream signaling such as Smad
activation can be monitored using a Smad-responsive reporter
gene.
[0096] ACVR1 and/or ACVR2 antagonists and variants or fragments
thereof can also be screened for activity in an in vivo assay. For
example, ACVR1 or ACVR2 antagonists or variants thereof can be
screened for their ability to treat FOP in a mouse model of FOP
(e.g., ability to decrease ectopic bone formation). Transgenic
knock-in mice have been developed that carry a conditional allele
encoding Acvr1[R206H]. These Acvr1.sup.[R206H]COIN/+ mice are
described in U.S. Ser. No. 14/207,320 and PCT/US2014/026582, which
are incorporated by reference herein in its entirety. This allele
expresses the R206H variant only after activation by Cre
recombinase. This allows Cre-dependent activation of Acvr1[R206H]
expression at specific tissues and at specific time by using
different types of Cre driver lines. In this manner the resulting
mice also bypass the perinatal lethality that has been observed
with a non-regulated knock-in allele of Acvr1[R206H]. Activation of
Acvr1[R206H] expression in young or in adult mice results in
ectopic bone formation. For example,
Acvr1.sup.[R206H]COIN/+;Gt(ROSA26)Sor.sup.CreERt2/+ mice (wherein
CreERt2 is a tamoxifen-regulatable recombinase (see Feil et al.
(1997) Biochem Biophys Res Commun. 237(3):752-7) that has been
introduced into the Gt(ROSA26)Sor locus, and hence it is
constitutively and globally expressed) develop FOP after exposure
to tamoxifen. Briefly, in the absence of tamoxifen, CreERt2 is
inactive. Tamoxifen activates expression of Cre which then acts
upon the Acvr1.sup.[R206H]COIN/+ to convert it to
Acvr1.sup.[R206H]/+, thereby converting the genotype of the mice to
mirror the genotype of the FOP patients that are ACVR1[R206H]. The
Acvr1.sup.[R206H] allele expresses Acvr1[R206H], and that is
adequate to drive the development of FOP in the
Acvr1.sup.[R206H]/+;Gt(ROSA26)Sor.sup.CreERt2/+ mice. This bypasses
the embryonic lethality experienced with conventional
Acvr1.sup.[R206H] knock-in mice, Acvr1.sup.tm1Emsh
(http://www.informatics.jax.org/allele/key/828153). After tamoxifen
treatment, the ACVR1, ACVR2A and/or ACVR2B antagonists or a control
can be administered to the
Acvr1.sup.[R206H]COIN/+;Gt(ROSA26)Sor.sup.CreERt2/+ mice and the
animals monitored for ectopic bone formation. See Chakkalakal S A,
et al. (20120 An Acvr1 R206H knock-in mouse has fibrodysplasia
ossificans progressiva. J Bone Miner Res. 27(8):1746-56. This assay
is described in detail in the Examples below.
V. Fibrodysplasia Ossificans Progressiva (FOP)
[0097] FOP is a rare heritable disorder in which heterotopic
ossification forms histologically and biomechanically `normal` bone
at extraskeletal sites, such as connective tissue. This disorder,
although episodic, is cumulative, and results in permanent
disability of increasing severity.
[0098] FOP's worldwide prevalence is approximately 1/2,000,000.
There is no ethnic, racial, gender, or geographic predilection to
FOP. It is not only an extremely disabling disease but also a
condition of considerably shortened lifespan.
[0099] Characteristics of FOP include, for example, congenital
malformations of the great toe, flare-ups characterized by painful
soft tissue swellings on the head, neck, and/or back with
inflammation and progressive formation of heterotopic bone via
endochondral ossification.
[0100] FOP can be suspected clinically based on the presence of
malformations of the great toe. Diagnostic tests, such as x-rays or
bone scan can substantiate great toe abnormalities and confirm the
presence of heterotopic ossification. A FOP diagnosis can also be
confirmed by genetic testing, for example, by detecting the 617
G-to-A (R206H) mutation in the ACVR1 gene.
[0101] It is common for FOP to be misdiagnosed as several other
disorders, including other conditions of heterotopic ossification.
FOP should be distinguished by a differential diagnosis from
disorders including, for example, isolated congenital
malformations, lymphedema, soft tissue sarcoma, desmoid tumors,
aggressive juvenile fibromatosis, juvenile bunions, isolated
brachydactyly, progressive osseous heteroplasia and heterotopic
ossification. The presence of great toe congenital malformations
and the painful soft-tissue flare-ups can be used to differentiate
FOP from other disorders.
[0102] Patients with FOP have congenital malformations of the great
toe but otherwise appear normal at birth. The flare-ups associated
with FOP start during the first decade of life. Flare-ups can be
triggered by, for example, soft tissue injury, falls, fatigue,
viral infections or intramuscular injections. The result of the
flare-ups is a transformation of soft tissue, such as ligaments,
skeletal muscle or tendons into heterotopic bone.
[0103] There was no previous therapeutic treatment for FOP. FOP was
managed by preventative measures, such as improved safety and
strategies to minimize injury, avoiding intramuscular injections
and taking care when receiving dental care. High dose
corticosteroid treatments started within the first 24 hours of a
flare-up can help reduce the inflammation and edema associated with
flare-ups. Surgical strategies to remove the heterotopic bone are
not recommended as it is counterproductive and causes new
trauma-induced heterotopic ossification.
[0104] FOP is caused by mutations in ACVR1 (also known as ALK2)
that appear to destabilize the interaction of the GS domain with an
inhibitory molecule, FKBP12 (Groppe, J., et al. 2011, Cells Tissues
Organs, 194:291-295). FKBP12 is a negative modulator of ACVR1 and
functions to stabilize the receptor in an inactive conformation
(Huse, M., et al. 1999, Cell, 96:425-436). See Kaplan, F. S., et
al. 2012, Disease Models & Mechanisms, 5:756-762).
[0105] An example of a mutation in ACVR1 that is associated with
FOP is an Arginine 206 to Histidine (R206H) mutation in the
intracellular domain.
[0106] A subject at risk of developing FOP includes any subject
with the ACVR1 R206H mutation or other mutation associated with
FOP, a subject born with malformations of the great toe, or a
subject that has a family history of FOP, who has not yet developed
symptoms of FOP sufficient for a diagnosis of FOP to be made by
art-recognized criteria.
VI. Methods of Treatment
[0107] Methods of treating FOP, comprising administering to a
subject having FOP an effective regime of an ACVR1, ACVR2A and/or
an ACVR2B antagonist are provided herein. In one embodiment, an
effective regime of an ACVR2A antagonist and an ACVR2B antagonist
is administered. In a further embodiment, the ACVR2A antagonist is
an Fc fusion protein and the ACVR2B antagonist is an Fc fusion
protein. In another embodiment, FOP is treated by administering an
effective regime of an antibody against Activin A.
[0108] "Treating" a subject with FOP means administration of an
effective regime of an ACVR1, an ACVR2A and/or an ACVR2B
antagonist, or an antibody against Activin A, to a subject that has
FOP, where the purpose is to cure, heal, alleviate, relieve, alter,
remedy, ameliorate, improve, or affect the condition of one or more
symptoms of FOP.
[0109] A "subject" is any animal (i.e., mammals) such as, humans,
primates, rodents, such as mice and rats, agricultural and
domesticated animals such as, dogs, cats, cattle, horses, pigs,
sheep, and the like, in which one desires to treat FOP. In any of
the present methods, the subject can be mammal and preferably
human.
[0110] An effective regime of an Activin A, ACVR1, ACVR2A and/or an
ACVR2B antagonist, or an antibody against Activin A, means a
combination of dose, frequency and route of administration of an
antagonist which brings a positive response in at least one sign or
symptom of FOP. A positive response can include reducing,
eliminating, ameliorating, inhibiting worsening of, or delaying at
least one sign or symptom of FOP. Signs or symptoms of FOP that can
be subject of a positive response include for example, ectopic or
heterotopic bone formation, FOP flare-ups, or pain and swelling
associated with flare-ups. The regime can be assessed in a single
patient by comparing signs and symptoms before and after treatment.
A regime is considered effective if at least one sign or symptom
gives a positive response following treatment. A regime can
alternatively or additionally be assessed by comparing signs and
symptoms of population of subjects treated with an antagonist or
antagonists of the present invention with a control population of
subjects not receiving treatment. The subjects for such comparison
can be an animal model, or human subjects in a clinical trial
(e.g., phase I, phase II, IIa, IIb, or III). A regime is considered
effective if there is a statistically significant positive response
between the populations in at least one sign or symptom.
[0111] In some methods for treating FOP, the subject does not have
and is not at risk of other conditions treatable with antagonists
against ACVR1, ACVR2A, and/or ACVR2B, or an antibody against
Activin A. For example, the subject can be free of any or all of
type II diabetes, muscular dystrophy, amyotrophic lateral sclerosis
(ALS) and osteoporosis.
[0112] A. Methods of Administration
[0113] ACVR1, ACVR2A and/or ACVR2B antagonists, or an antibody
against Activin A, are usually administered directly as proteins or
small molecules, but in the case of proteins can also be
administered as nucleic acid encoding such proteins. Such
antagonists can be administered by various methods, such as
cellular transfection, gene therapy, direct administration with a
delivery vehicle or pharmaceutically acceptable carrier, indirect
delivery by providing recombinant cells comprising a nucleic acid
encoding an ACVR1, ACVR2A and/or ACVR2B antagonist, or an antibody
against Activin A, provided herein.
[0114] Various delivery systems can be used to administer the
ACVR1, ACVR2A and/or ACVR2B antagonists, or an antibody against
Activin A, provided herein, e.g., encapsulation in liposomes,
microparticles, microcapsules, recombinant cells capable of
expressing the compound, receptor-mediated endocytosis (see, e.g.,
Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a
nucleic acid as part of a retroviral or other vector, etc.
[0115] Methods of administration can be enteral or parenteral and
include intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, pulmonary, intranasal, intraocular, epidural, and
oral routes. The compounds can be administered by any convenient
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and can be administered
together with other biologically active agents. Administration can
be systemic or local. In addition, it can be desirable to introduce
the pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection; intraventricular injection can be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Omcana reservoir. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent.
[0116] The pharmaceutical compositions of the invention can be
administered locally to the area in need of treatment; this can be
achieved, for example, by local infusion during surgery, topical
application, e.g., by injection, by means of a catheter, or by
means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, fibers, or commercial skin substitutes.
[0117] In another embodiment, the active agent can be delivered in
a vesicle, in particular a liposome (see Langer (1990) Science
249:1527-1533). In another embodiment, the active agent can be
delivered in a controlled release system. In one embodiment, a pump
can be used (see Langer (1990) supra). In another embodiment,
polymeric materials can be used (see Howard et al. (1989) J.
Neurosurg. 71:105). In another embodiment where the active agent of
the invention is a nucleic acid encoding a protein, the nucleic
acid can be administered in vivo to promote expression of its
encoded protein, by constructing it as part of an appropriate
nucleic acid expression vector and administering it so that it
becomes intracellular, e.g., by use of a retroviral vector (see,
for example, U.S. Pat. No. 4,980,286), or by direct injection, or
by use of microparticle bombardment, or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.
USA 88:1864-1868), etc. Alternatively, a nucleic acid can be
introduced intracellularly and incorporated within host cell DNA
for expression, by homologous recombination.
[0118] B. Combination Therapies
[0119] The ACVR1, ACVR2A and ACVR2B antagonists, or an antibody
against Activin A, provided herein can be administered in
combination with one another or other treatments. In one
embodiment, the method of treating FOP involves co-administration
of an ACVR2A antagonist and an ACVR2B antagonist. In another
embodiment, the method of treating FOP involves co-administration
of an ACVR1, an ACVR2A and an ACVR2B antagonist. In other
embodiments, an ACVR1 antagonist can be co-administered with an
ACVR2A and/or an ACVR2B antagonist. The ACVR1, ACVR2A and ACVR2B
antagonists can be administered as separate pharmaceutical
compositions or can be administered as a single pharmaceutical
composition comprising a combination of these agents. The ACVR1,
ACVR2A and/or ACVR2B antagonists, or an antibody against Activin A,
either alone or in combination, can be administered in conjunction
with one or more additional therapeutic compounds. The combination
therapy can encompass simultaneous or alternating administration.
In addition, the combination can encompass acute or chronic
administration.
[0120] C. Pharmaceutical Compositions
[0121] The present invention also provides pharmaceutical
compositions comprising an Activin A, ACVR1, ACVR2A and/or an
ACVR2B antagonist, or an antibody against Activin A, provided
herein and a pharmaceutically acceptable carrier. The term
"pharmaceutically acceptable" means approved by a regulatory agency
of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. The term "carrier" refers
to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is administered. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents.
[0122] These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W.
Martin.
[0123] In one embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. When
necessary, the composition can also include a solubilizing agent
and a local anesthetic such as lidocaine to ease pain at the site
of the injection. When the composition is to be administered by
infusion, it can be dispensed with an infusion bottle containing
sterile pharmaceutical grade water or saline. When the composition
is administered by injection, an ampoule of sterile water for
injection or saline can be provided so that the ingredients can be
mixed prior to administration.
[0124] The Activin A, ACVR1, ACVR2A and/or an ACVR2B antagonists,
or an antibody against Activin A, provided herein can be formulated
as neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the
like, and those formed with free carboxyl groups such as those
derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, and the like.
[0125] The amount and frequency of the Activin A, ACVR1, ACVR2A
and/or ACVR2B antagonist, or an antibody against Activin A,
administered by a specified route effective in the treatment of FOP
(e.g., effective regime) can be determined by standard clinical
techniques based on the present description. In addition, in vitro
assays can be employed to help identify optimal dosage ranges. The
precise dose to be employed in the formulation also depends on the
route of administration, and the seriousness of the condition, and
should be decided according to the judgment of the practitioner and
each subject's circumstances. However, suitable dosage ranges for
parenteral administration, preferably intravenous or subcutaneous,
are generally about 20-50000 micrograms of active compound per
kilogram body weight. For antibodies to Activin A suitable dosage
ranges include 1-25 mg/kg, 2-20 mg/kg 5-15 mg/kg, 8-12 mg/kg and 10
mg/kg.
[0126] Suitable dosage ranges for intranasal administration are
generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
Effective doses can be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0127] Frequencies of administration also vary depending on the
severity of the condition and half-life of the agent among other
factors, but are typically between daily and quarterly, including
for example, twice a week, weekly, fortnightly, monthly, bimonthly.
Agents can also be administered at irregular intervals responsive
to the patient's condition or reduction in serum level of the agent
below a threshold among other factors.
[0128] All patent filings, websites, other publications, accession
numbers and the like cited above or below are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual item were specifically and individually
indicated to be so incorporated by reference. If different versions
of a sequence are associated with an accession number at different
times, the version associated with the accession number at the
effective filing date of this application is meant. The effective
filing date means the earlier of the actual filing date or filing
date of a priority application referring to the accession number if
applicable. Likewise if different versions of a publication,
website or the like are published at different times, the version
most recently published at the effective filing date of the
application is meant unless otherwise indicated. Any feature, step,
element, embodiment, or aspect of the invention can be used in
combination with any other unless specifically indicated
otherwise.
EXAMPLES
Example 1: Use of ACVR2A-Fc/ACVR2B-Fc to Suppress Ectopic Bone
Formation in a Mouse Model of FOP
[0129] Acvr1.sup.[R206H]COIN/+; Gt(ROSA26)Sor.sup.CreERt2/+ were
protected from ectopic bone formation after tamoxifen treatment by
ACVR2A-Fc/ACVR2B-Fc treatment.
A mouse model of FOP, referred to as Acvr1.sup.[R206H]COIN/+;
Gt(ROSA26)Sor.sup.CreERt2/+ were given tamoxifen at 1 mg/mouse dose
i.p. for eight days. Eleven mice were treated with 10 mg/kg of
ACVR2A-Fc and 10 mg/kg of ACVR2B-Fc twice weekly and ten mice were
treated with 10 mg/kg control mFc twice weekly for 6 weeks. Mice
were monitored using in vivo .mu.CT at baseline, 2, 4 and 6 weeks
post initiation of tamoxifen administration. After 6 weeks, 9 out
of 10 mice in the mFc group had developed ectopic bone in at least
one location, in contrast only 2 out of 11 mice in the ACVR2A-Fc
and ACVR2B-Fc group showed development of ectopic bone and this
bone was small in size. These results are shown in FIG. 1.
Example 2: Use of an ACVR1 Kinase Small Molecule Inhibitor to
Suppress Ectopic Bone Formation in a Mouse Model of FOP
[0130] Acvr1.sup.[R206H]COIN/+; Gt(ROSA26)Sor.sup.CreERt2/+ were
protected from ectopic bone formation after tamoxifen treatment by
ACVR1 kinase inhibitor LDN-212854 treatment.
[0131] 16 Acvr1.sup.[R206H]COIN/+; Gt(ROSA26)Sor.sup.CreERt2/+ mice
were given tamoxifen at 1 mg/mouse dose i.p. for eight days. Eight
mice were treated with 3 mg/kg of the ACVR1 kinase inhibitor
LDN-212854 (Mohedas et al. (2013) ACS Chem. Biol. 8:1291-1302)
twice daily for 4 weeks. Eight mice were treated with vehicle
control twice daily for 4 weeks. Mice were monitored using in vivo
.mu.CT at baseline, 2 and 4 weeks post initiation of tamoxifen
administration. After 4 weeks 8 out of 8 mice in the vehicle
control group showed ectopic bone formation, in 6 of these mice the
ectopic bone lesions were large in size. In contrast, in the
LDN-212854 treated group, 3 out of 8 mice showed ectopic bone
formation, the size of the ectopic bone lesions formed in the 3
mice were small compared to the vehicle control group. These
results are shown in FIG. 2.
Example 3: Use of an Antibody Against Activin a to Suppress Ectopic
Bone Formation in a Mouse Model of FOP
[0132] 23 Acvr1.sup.[R206H]COIN/+; Gt(ROSA26).sup.SorCreERt2/+ mice
were treated with tamoxifen at 1 mg/mouse dose i.p. for eight days.
Seven mice were treated with 25 mg/kg isotype control antibody
twice weekly, eight mice were treated with 25 mg/kg of Activin A
antibody (H4H10446P) twice weekly, and eight mice were treated with
10 mg/kg of ACVR2a-Fc twice weekly for 3 weeks. Treatments with
these agents were started concurrent with initiating tamoxifen
treatment. Mice were monitored using in vivo micro computer
tomography (.mu.CT) at baseline, 2 and 3 weeks post initiation of
tamoxifen administration. FIG. 3 shows that after 3 weeks, all mice
in the isotype control antibody group had developed ectopic bone in
at least one location, in contrast none of the mice in the Activin
A antibody group showed development of ectopic bone at this time.
Two mice in the ACVR2a-Fc group developed ectopic bone at 3
weeks.
Example 4
[0133] Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ were protected
from ectopic bone formation after tamoxifen treatment by both an
Activin A and an Acvr2a and b blocking antibody.
[0134] 26 Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ mice were
given with tamoxifen at a 40 mg/kg dose i.p. for eight days. Eight
mice were treated with 10 mg/kg isotype control antibody
(REGN1945), nine mice were treated with 10 mg/kg of Activin A
antibody (H4H10446P) (REGN2477) and nine mice were treated with 10
mg/kg of an Acvr2a/Acvr2b antibody twice weekly for 6 weeks. Mice
were monitored using in vivo .mu.CT at baseline, 2, 3 and 4 weeks
post initiation of tamoxifen administration. FIG. 4 shows that
after 4 weeks, 7 out of 8 mice in the isotype control antibody
group had developed ectopic bone in at least one location, in
contrast only one of the mice in the Activin A antibody treated
group and three of the mice in the Acvr2a/Acvr2b antibody treated
group developed ectopic bone at 4 weeks. The size of the ectopic
bone that formed in the antibody treated group was smaller than the
isotype control treated group.
Example 5
[0135] Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ were protected
from ectopic bone formation after tamoxifen treatment by an Activin
A blocking antibody.
[0136] 35 Acvr1[R206H]COIN/+; Gt(ROSA26)SorCreERt2/+ mice were
given with tamoxifen at 40 mg/kg i.p. for eight days. Eight mice
were treated with 25 mg/kg isotype control antibody (REGN1945),
nine mice were treated with 25 mg/kg of Activin A antibody
(H4H10446P) (REGN2477), nine mice were treated with 10 mg/kg of
Activin A antibody (REGN2477) and nine mice were treated with 1
mg/kg of Activin A antibody (REGN2477) weekly for 6 weeks. Mice
were monitored using in vivo .mu.CT at baseline, 2, 3, 4 and 6.5
weeks post initiation of tamoxifen administration. The volume of
ectopic bone in each mouse was calculated from .mu.CT images. FIG.
5 shows after 4 weeks, all mice in the isotype control antibody
group had developed ectopic bone in at least one location, whereas
only 2 mice each of the Activin A antibody treated groups. At 6.5
weeks the average total volume of ectopic bone in the isotype
treated group was 65.4 mm3 compared to 1.87 mm.sup.3 in the 25
mg/kg, 0.3 mm.sup.3 in the 10 mg/kg and 7.3 mm.sup.3 in the 1 mg/kg
Activin antibody treated groups.
Sequence CWU 1
1
241126PRTArtificial SequenceSynthesized 1Gln Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Phe Ser Ser His 20 25 30 Phe Trp
Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Leu Tyr Thr Gly Gly Thr Ser Phe Asn Pro Ser Leu Lys 50
55 60 Ser Arg Val Ser Met Ser Val Gly Thr Ser Lys Asn Gln Phe Ser
Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95 Arg Ala Arg Ser Gly Ile Thr Phe Thr Gly Ile
Ile Val Pro Gly Ser 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met
Val Thr Val Ser Ser 115 120 125 28PRTArtificial SequenceSynthesized
2Gly Gly Ser Phe Ser Ser His Phe 1 5 37PRTArtificial
SequenceSynthesized 3Ile Leu Tyr Thr Gly Gly Thr 1 5
420PRTArtificial SequenceSynthesized 4Ala Arg Ala Arg Ser Gly Ile
Thr Phe Thr Gly Ile Ile Val Pro Gly 1 5 10 15 Ser Phe Asp Ile 20
5108PRTArtificial SequenceSynthesized 5Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser
Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 67PRTArtificial SequenceSynthesized 6Gln Ser Val Ser Ser
Ser Tyr 1 5 73PRTArtificial SequenceSynthesized 7Gly Ala Ser 1
89PRTArtificial SequenceSynthesized 8Gln Gln Tyr Gly Ser Ser Pro
Trp Thr 1 5 9125PRTArtificial SequenceSynthesized 9Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Lys Ala Ser Gly Phe Ala Phe Asp Asp Phe 20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Gly Ile Val Trp Asn Ser Gly Asp Ile Gly Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr 65 70 75 80 Leu Gln Leu Asn Ser Leu Arg Thr Glu Asp Thr
Ala Leu Tyr Phe Cys 85 90 95 Val Lys Asp Met Val Arg Gly Leu Met
Gly Phe Asn Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 125 108PRTArtificial
SequenceSynthesized 10Gly Phe Ala Phe Asp Asp Phe Ala 1 5
118PRTArtificial SequenceSynthesized 11Ile Val Trp Asn Ser Gly Asp
Ile 1 5 1218PRTArtificial SequenceSynthesized 12Val Lys Asp Met Val
Arg Gly Leu Met Gly Phe Asn Tyr Tyr Gly Met 1 5 10 15 Asp Val
13107PRTArtificial SequenceSynthesized 13Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Thr Ile Ser Thr Tyr 20 25 30 Leu Val
Trp Tyr Arg Gln Arg Pro Gly Gln Ala Pro Ser Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Asp Ile Pro Ala Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn
Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
100 105 146PRTArtificial SequenceSynthesized 14Gln Thr Ile Ser Thr
Tyr 1 5 153PRTArtificial SequenceSynthesized 15Asp Ala Ser 1
169PRTArtificial SequenceSynthesized 16Gln Gln Arg Ser Asn Trp Pro
Ile Thr 1 5 17106PRTHomo sapiens 17Ser Tyr Glu Val Thr Gln Ala Pro
Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys
Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala 20 25 30 Cys Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp
Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65
70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Ser Thr
Ala Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105
18122PRTHomo sapiens 18Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Leu Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ile Pro
Tyr Asn Gly Asn Thr Asn Ser Ala Gln Lys Leu 50 55 60 Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90
95 Ala Arg Asp Arg Asp Tyr Gly Val Asn Tyr Asp Ala Phe Asp Ile Trp
100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120
1911PRTHomo sapiens 19Ser Gly Asp Lys Leu Gly Asp Lys Tyr Ala Cys 1
5 10 207PRTHomo sapiens 20Gln Asp Ser Lys Arg Pro Ser 1 5
219PRTHomo sapiens 21Gln Ala Trp Asp Ser Ser Thr Ala Val 1 5
2210PRTHomo sapiens 22Gly Tyr Thr Phe Thr Ser Tyr Gly Leu Ser 1 5
10 2317PRTHomo sapiens 23Trp Ile Ile Pro Tyr Asn Gly Asn Thr Asn
Ser Ala Gln Lys Leu Gln 1 5 10 15 Gly 2413PRTHomo sapiens 24Asp Arg
Asp Tyr Gly Val Asn Tyr Asp Ala Phe Asp Ile 1 5 10
* * * * *
References