Note: Descriptions are shown in the official language in which they were submitted.
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TRIMERIC IL-1Ra
Cross-Reference to Related Application
[0001] This application claims the benefit of U.S. Provisional Patent
Application Serial
No. 60/978,254, filed October 8, 2008, which is incorporated by reference
herein in its
entirety.
Field of the Invention
[0002] The invention relates to treatment of diseases that are mediated by
interleukin 1.
More particularly, the invention relates to interleukin 1 receptor antagonists
(IL-1Ra) that are
useful for treating such diseases.
Background of the Invention
[0003] The IL-1 family is an important part of the innate immune system, which
is a
regulator of the adaptive immune system. The balance between IL-1 and IL-1Ra
in local
tissues influences the possible development of inflammatory diseases and
resulting structural
damage. In the presence of an excess amount of IL-1, inflammatory and
autoimmune
diseases may develop in the joints, lungs, gastrointestinal tract, central
nervous system
(CNS), or blood vessels. Treatment of human disease with IL-1Ra has been
carried out
through injection of recombinant IL-IRa or through gene therapy approaches.
Treatment with
recombinant IL-IRa has been approved for rheumatoid arthritis (RA) and phase 2
studies are
ongoing for osteoarthritis (OA).
[0004] An important pro-inflammatory role for IL-1 in many human diseases has
been
described over the past 10 years. The balance between IL-1 and IL-IRa has been
extensively
studied in a variety of animal disease models including rheumatoid arthritis
(RA),
osteoarthritis (OA), inflammatory bowel disease (IBD), granulomatous and
fibrotic lung
disorders, kidney diseases, diseases of the liver and pancreas, graft-versus-
host disease
(GVHD), leukemia, cancer, osteoporosis, diabetes, central nervous system
diseases,
infectious diseases, and arterial diseases. In each of these diseases, local
overproduction of
IL-1 and/or underproduction of IL-lRa pre-disposes subjects to disease
development. The
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therapeutic administration of IL-lRa has been shown to be efficacious in
preventing tissue
damage (See W.P. Arend, Cytokine & Growth Factor Reviews, 13 (2002) pp. 323-
240).
[0005] The IL-1 family consists of two agonists, IL-la and IL-1(3; the
specific receptor
antagonist IL-1Ra; and three different receptors, IL-1R type I (IL-1RI), IL-1R
type II (IL-
lRII) and IL-1 receptor accessory protein (IL-1R AcP). IL-1RI is an 80 kDa
protein with a
long cytoplasmic domain of 215 residues. The biologically inert IL-1RII is a
60 kDa protein
with a short cytoplasmic domain of 29 residues. IL-1R AcP is recruited to the
complex after
binding of IL-la or IL-1(3 to the single chain IL-iRI. Signal transduction
pathways activated
by the approximated cytoplasmic domains of IL-1RI and IL-1R AcP include the NF-
xB,
JNK/AP-1, and p38 MAP kinase pathways. IL-1RII functions as a decoy receptor,
binding
IL-1 both on the plasma membrane and as a soluble receptor in the fluid phase,
thereby
preventing IL-1 from interacting with the functional IL-IRI.
[0006] The third ligand in the IL-1 family, IL-1Ra, is a structural variant of
IL-1 that
binds to both IL-IR but fails to activate cells. IL-IRa is a 17 kDa protein
with 18% amino
acid homology to IL-la and 26% homology to IL-1 R. The originally described
isoform of IL-
1Ra is secreted from monocytes, macrophages, neutrophils, and other cells and
is now termed
sIL-1Ra. Three additional intracellular isoforms of IL-1Ra have been described
to date. An
18 kDa form of IL-lRa, created by an alternative transcriptional splice
mechanism from an
upstream exon is called icIL-1Ral and is found inside keratinocytes and other
epithelial cells,
monocytes, tissue macrophages, fibroblasts, and endothelial cells. IL-lRa cDNA
cloned from
human leukocytes contains an additional 63 bp sequence as an insert in the 5'
region of the
cDNA. A 15 kDa isoform of IL-1Ra, termed icIL-1Ra3, is found in monocytes,
macrophages, neutrophils, and hepatocytes, and may be created both by an
alternative
transcriptional splice as well as by alternative translational initiation.
[0007] Both soluble IL-lRa and icIL-1Ral bind equally well to IL-1R, but icIL-
lRa3
exhibits weak receptor binding. IL-lRa functions as a specific receptor
antagonist by binding
to IL-1RI but preventing IL-1R AcP from associating with the IL-1RI, which
results failure
of initiation of signal transduction pathways.
[0008] The decoy receptor IL-1RII binds IL-1 both on the plasma membrane and
as a
soluble receptor in the fluid phase, preventing IL-1 from interacting with the
functional IL-
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IRI. Therefore, soluble IL-1RII and IL-IRa can inhibit IL-1 in co-operation.
Soluble IL-RI
can bind to IL-1 as well as IL-IRa, but due to the balance between IL-1 and IL-
1Ra, soluble
IL-1RI seems to act as a pro-inflammatory agent.
[0009] KINERET is an E. coli produced IL-1Ra from Amgen, which has been shown
to
benefit patients with active rheumatoid arthritis. KINERET has to be injected
subcutaneously once per day. With subcutaneous administration, KINERET has a
half-life
ranged from 4 to 6 hours; for i.v. administration the half life is
approximately 2%z hours. IL-
IRa is cleared by renal clearance. KINERET is a specific receptor antagonist
of IL-1 that
differs from naturally occurring IL-1 receptor antagonist by the presence of a
methionine
group. When given alone or in combination with methotrexate, KINERET has been
shown
to benefit patients as assessed by improvement in clinical signs and symptoms,
decreased
radiographic progression and improvement in patient function, pain and
fatigue. KINERET
has a favorable safety profile as demonstrated in clinical trials.
[0010] Several attempts have been made to improve the poor pharmacokinetics of
IL-
1 Ra. Antibodies targeting just IL-I beta have been developed. However, in
contrast to IL-
Ira, these only block IL-1 beta, but not IL-la action.
[0011] Accordingly, the inventors have identified a need in the art for an
improved
delivery method for IL1-Ra, which provides for a longer half-life of the
molecule and
provides a favorable safety profile.
Brief Description of the Drawings
[0012] Figure 1 shows alignment of the amino acid sequences of the trimerising
structural element of the tetranectin protein family. Amino acid sequences
(one letter code)
corresponding to residue V17 to K52 comprising exon 2 and the first three
residues of exon 3
of human tetranectin (SEQ ID NO: 59); murine tetranectin (SEQ ID NO: 60);
tetranectin
homologous protein isolated from reefshark cartilage (SEQ ID NO: 61) and
tetranectin
homologous protein isolated from bovine cartilage (SEQ ID NO: 62. Residues at
a and d
positions in the heptad repeats are listed in boldface. The listed consensus
sequence of the
tetranectin protein family trimerising structural element comprise the
residues present at a
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and d positions in the heptad repeats shown in the figure in addition to the
other conserved
residues of the region. "hy" denotes an aliphatic hydrophobic residue.
[0013] Figure 2 shows the results of CII-H6-GrB-TripK-IL-IRa refolding by
dialysis.
[0014] Figure 3 displays the capturing CII-H6-GrB-TripK-IL- 1 Ra on NiNTA.
[0015] Figure 4 is a graph showing the ability of GG-TripV-IL- 1 Ra (trip V-IL-
1Ra), GG-
TripK-IL-lRa (trip K-IL-1Ra), GG-TripT-IL-lRa (trip T-IL-lRa) and GG-TripT-IL-
lRa
(trip T-IL-1Ra) to inhibit IL-1 induction of IL-8 in U937 cells.
[0016] Figure 5 is a graph showing the ability of pegylated TripT and TripV to
inhibit IL-
1 induction of IL-8 in U937 cells as compared to non-pegylated forms and
KINERET .
[0017] Figure 6 is a graph showing the ability of TripT-IL-lRa, I10-TripT-IL-
1Ra, V17-
TripT-IL-lRa used in the PK study to inhibit IL-1 induction of IL-8 in U937
cells
[0018] Figure 7 is a graph showing the blood concentrations of TripT-IL- I Ra,
I10-TripT-
IL-1Ra, and V17-TripT-IL-lRa after 100mg/kg i.v. injection in rats.
[0019] Figure 8 shows an SDS-PAGE analysis of multiple batches of Met-I l0-
TrpT-IL-
1 Ra (LM022 and LM023) and GG-V 17-TrpT-IL-1 Ra (CFO 19 and CF020) protein
yields.
[0020] Figure 9 shows analytical SEC results of Met-I I O-TrpT-IL- 1 Ra and GG-
V17-
TrpT-IL-lRa protein yields.
[0021] Figure 10 shows the results of the rat CIA study. Ankle diameters of
female
Lewis rats with type II collagen arthritis were measured following treatment
with Vehicle (10
mM phosphate buffer pH 7.4), or equimolar amounts of IL-Ira administering
either
monomeric IL-Ira (100 mg/kg KINERET ), or trimerized ILIra (120 mg/kg Met-IlO-
TripT-
ILlra, or 120 mg/kg GG-V17-TripT-ILlra).
[0022] Figure 11 shows study reduction of final paw weight when rats treated
with
KINERET , Met-IlO-TripT-ILlra QD, or GG-V17-TripT-ILlra QD, as compared to
vehicle
treated disease control animals.
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[0023] Figure 12 shows reduction of blood glucose levels observed after daily
i.p. dosing
of either I10-TripT-IL1-Ra or KINERET .
Summary of the Invention
[0024] The present invention provides a fusion protein comprising a
trimerizing domain
and an IL-IRa polypeptide sequence that inhibits IL-1 activity. In one
embodiment, the
fusion protein comprises an IL-iRa sequence that comprises a variant or
fragment of SEQ ID
NO: 38 that inhibits IL-1 activity. In an additional embodiment, the fusion
protein comprises
an IL-iRa polypeptide sequence that is at least 85% identical to SEQ ID NO:
38. The fusion
proteins may include polyethylene glycol. The trimerizing domain of the fusion
protein may
be derived from tetranectin.
[0025] The present invention also provides a trimeric complex comprising three
fusion
proteins of the invention. In one embodiment, the trimeric complex comprises a
trimerizing
domain that is a tetranectin trimerizing structural element (TTSE). In one
embodiment, the
trimeric complex comprises a trimerizing domain is at least 66% identical to
SEQ ID NO: 1.
In s further embodiment, the trimeric complex comprises at least one of the
fusion proteins
selected from the group consisting of TripK-IL-ira (SEQ ID NO: 39); TripV-IL-
ira (SEQ ID
NO: 40); TripT-IL-ira (SEQ ID NO: 41); TripQ-IL-lra (SEQ ID NO: 42); I10-TripK-
IL-lra
(SEQ ID NO: 43); I10-TripV-IL-Ira (SEQ ID NO: 44); I10-TripT-IL-Ira (SEQ ID
NO: 45);
I10-TripQ-IL-Ira (SEQ ID NO: 46); V17-TripT-ILIRa (SEQ ID NO: 55); V17-TripK-
IL-
IRa (SEQ ID NO: 56); V17-TripV-IL-IRA (SEQ ID NO: 57); and V17-TripQ-ILIRA
(SEQ
ID NO: 58).
[0026] In a further embodiment, the present invention provides a
pharmaceutical
composition comprising a trimeric and at least one pharmaceutically acceptable
excipient.
[0027] Even further, the invention is directed to a method for treating a
disease mediated
by interleukin 1. The method includes administering to a patient in need
thereof of the
pharmaceutical composition of the invention. The disease may be an
inflammatory disease
such as rheumatoid arthritis or diabetes. The method also includes
administering to the
patient, either simultaneously or sequentially, an anti-inflammatory agent.
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[0028] The invention also provides a fusion protein further comprising an anti-
inflammatory agent covalently linked to the fusion protein.
[0029] These and other aspects of the invention are described in further
detail below.
Detailed Description of the Invention
[0030] The invention is directed to compounds and methods for treating
diseases
mediated by IL-1. In one aspect, the invention is directed to a fusion protein
of an IL-iRa
polypeptide sequence fused to a trimerizing or multimerizing domain. Three or
more of
fusion proteins may trimerize or multimerize to provide compositions providing
for greater
stability and improved pharmacokinetic properties than IL-1Ra alone, and
provide a
favorable safety profile.
[0031] In an additional aspect the invention provides a nucleic acid which
encodes any
one of the polypeptides defined above, as well as methods of preparing these
polypeptides
under conditions that allow for specific expression and recovery.
[0032] The polypeptides of the invention may be used for the preparation of
pharmaceutical compositions for use in the treatment of a subject having a
pathology
mediated by IL-1, such as a method of treatment of inflammatory diseases, by
administering
to the subject an effective amount of pharmaceutical composition.
[0033] As used herein, a disease or medical condition is considered to be an
"interleukin-
1 mediated disease" or "a disease mediated by interleukin-1" if the
spontaneous or
experimental disease or medical condition is associated with elevated levels
of IL-1 in bodily
fluids or tissue or if cells or tissues taken from the body produce elevated
levels of IL-1 in
culture. In many cases, such interleukin-1 mediated diseases are also
recognized by the
following additional two conditions: (1) pathological findings associated with
the disease or
medical condition can be mimicked experimentally in animals by the
administration of IL-1;
and (2) the pathology induced in experimental animal models of the disease or
medical
condition can be inhibited or abolished by treatment with agents which inhibit
the action of
IL-1. In most IL-1 mediated diseases at least two of the three conditions are
met, and in many
IL-1 mediated diseases all three conditions are met. A non-exclusive list of
acute and chronic
IL-1-mediated inflammatory diseases includes but is not limited to the
following: gout, acute
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pancreatitis; ALS; Alzheimer's disease; cachexia/anorexia; asthma;
atherosclerosis; chronic
fatigue syndrome, fever; diabetes (e.g., insulin diabetes);
glomerulonephritis; graft versus
host rejection; hemohorragic shock; hyperalgesia, inflammatory bowel disease;
inflammatory
conditions of a joint, including osteoarthritis, psoriatic arthritis, juvenile
arthritis, and
rheumatoid arthritis; ischemic injury, including cerebral ischemia (e.g.,
brain injury as a
result of trauma, epilepsy, hemorrhage or stroke, each of which may lead to
neurodegeneration); lung diseases (e.g., ARDS); multiple myeloma; multiple
sclerosis;
myelogenous (e.g., AML and CML) and other leukemias; myopathies (e.g., muscle
protein
metabolism, esp. in sepsis); osteoporosis; Parkinson's disease; pain; pre-term
labor; psoriasis;
reperfusion injury; septic shock; side effects from radiation therapy,
temporal mandibular
joint disease, tumor metastasis; or an inflammatory condition resulting from
strain, sprain,
cartilage damage, trauma, orthopedic surgery, infection or other disease
processes, and
Cryopyrin-associated periodic syndromes, including Muckle Wells syndrome,
familial cold
autoinflammatory syndrome and neonatal-onset multisystem inflammatory disease.
[0034] As used herein, the term "multimerizing domain" means an amino acid
sequence
that comprises the functionality that can associate with two or more other
amino acid
sequences to form trimers or other multimerized complexes. In one example, the
fusion
protein contains an amino acid sequence -- a trimerizing domain -- which forms
a trimeric
complex with two other trimerizing domains. A trimerizing domain can associate
with other
trimerizing domains of identical amino acid sequence (a homotrimer), or with
trimerizing
domains of different amino acid sequence (a heterotrimer). Such an interaction
may be
caused by covalent bonds between the components of the trimerizing domains as
well as by
hydrogen bond forces, hydrophobic forces, van der Waals forces and salt
bridges. In various
embodiment so of the invention, the multimerizing domain is a dimerizing,
domain, a
trimerizing domain, a tetramerizing domain, a pentamerizing domain, etc. These
domains are
capaple of forming polypeptide complexes of two, three, four, five or more
fusion proteins of
the invention.
[0035] The trimerizing domain of a fusion protein of the invention may be
derived from
tetranectin as described in U.S. Patent Application Publication No.
2007/0154901 (`901
Application), which is incorporated by reference in its entirety. The full
length human
tetranectin polypeptide sequence is provided herein as SEQ ID NO: 63. Examples
of a
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tetranectin trimerizing domain includes the amino acids 17 to 49, 17 to 50, 17
to 51 and 17-
52 of SEQ ID NO: 63, which represent the amino acids encoded by exon 2 of the
human
tetranectin gene, and optionally the first one, two or three amino acids
encoded by exon 3 of
the gene. Other examples include amino acids I to 49, 1 to 50, 1 to 51 and 1
to 52, which
represents all of exons 1 and 2, and optionally the first one, two or three
amino acids encoded
by exon 3 of the gene. Alternatively, only a part of the amino acid sequence
encoded by
exon 1 is included in the trimerizing domain. In particular, the N-terminus of
the trimerizing
domain may begin at any of residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16 and 17
of SEQ ID NO: 63. In particular embodiments, the N terminus is 110 or V17 and
the C-
terminus is Q47, T48, V49, C(S)50, L51 or K52 (numbering according to SEQ ID
NO: 63).
[0036] In one aspect of the invention, the trimerizing domain is a tetranectin
trimerizing
structural element ("TTSE") having a amino acid sequence of SEQ ID NO: 1 which
a
consensus sequence of the tetranectin family trimerizing structural element as
more fully
described in US 2007/00154901. As shown in Figure 1, the TTSE embraces
variants of a
naturally occurring member of the tetranectin family of proteins, and in
particular variants
that have been modified in the amino acid sequence without adversely
affecting, to any
substantial degree, the ability of the TTSE to form alpha helical coiled coil
Crimes. In
various aspects of the invention, the trimeric polypeptide according to the
invention includes
a TTSE as a trimerizing domain having at least 66% amino acid sequence
identity to the
consensus sequence of SEQ ID NO: 1; for example at least 73%, at least 80%, at
least 86% or
at least 92% sequence identity to the consensus sequence of SEQ ID NO: 1
(counting only
the defined (not Xaa) residues). In other words, at least one, at least two,
at least three, at
least four, or at least five of the defined amino acids in SEQ ID NO: 1 may be
substituted.
[0037] In one particular embodiment, the cysteine at position 50 (C50) of SEQ
ID NO:
63 can be advantageously be mutagenized to serine, threonine, methionine or to
any other
amino acid residue in order to avoid formation of an unwanted inter-chain
disulphide bridge,
which can lead to unwanted multimerization. Other known variants include at
least one
amino acid residue selected from amino acid residue nos. 6, 21, 22, 24, 25,
27, 28, 31, 32, 35,
39, 41, and 42 (numbering according to SEQ ID NO:63), which may be substituted
by any
non-helix breaking amino acid residue. These residues have been shown not to
be directly
involved in the intermolecular interactions that stabilize the trimeric
complex between three
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TTSEs of native tetranectin monomers. In one aspect shown in FIG. 1, the TTSE
has a
repeated heptad having the formula a-b-c-d-e-f-g (N to C), wherein residues a
and d (i.e.,
positions 26, 33, 37, 40, 44, 47, and 51 may be any hydrophobic amino acid
(numbering
according to claim 63).
[0038] In further embodiments, the TTSE trimerization domain may be modified
by the
incorporation of polyhistidine sequence and/or a protease cleavage site, e.g,
Blood
Coagulating Factor Xa or Granzyme B (see US 2005/0199251, which is
incorporated herein
by reference), and by including a C-terminal KG or KGS sequence. Also, to
assist in
purification, Proline at position 2 may be substituted with Glycine to assist
in purification.
[0039] Particular non-limiting examples of TTSE truncations and variants are
shown in
Table 1 below.
Table 1
TTSE variants
SEQ ID NO: 2 EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLK
SEQ ID NO: 3 EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSL
SEQ ID NO: 4 EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVS
SEQ ID NO:5 EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTV
SEQ ID NO:6 PPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 7 PTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 8 TQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 9 QKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 10 KPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 11 PKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 12 KKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 13 KIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 14 IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 15 VNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 16 NAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 17 AKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 18 KKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 19 KDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
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SEQ ID NO: 20 VVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 21 VVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 22 VVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLK
SEQ ID NO: 23 VVNTKMFEELKSRLDTLAQEVALLKEQQALQTV
SEQ ID NO: 24 VVNTKMFEELKSRLDTLAQEVALLKEQQALQT
SEQ ID NO: 25 VNTKMFEELKSRLDTLAQEVALLKEQQALQ
SEQ ID NO: 26 NTKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 27 TKMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 28 KMFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 29 MFEELKSRLDTLAQEVALLKEQQALQTVSLKG
SEQ ID NO: 30 EGPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLK
SEQ ID NO: 31 EGPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTV
SEQ ID NO: 32 EGPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQT
SEQ ID NO: 33 EGPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQ
SEQ ID NO: 34 IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVSLK
SEQ ID NO: 35 IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTV
SEQ ID NO: 36 IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQT
SEQ ID NO: 37 IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQ
[0040] Another example of a trimerizing domain is disclosed in US 6,190,886
(incorporated herein in its entirety), which describes polypeptides comprising
a collectin neck
region. Trimers can then be made under appropriate conditions with three
polypeptides
comprising the collectin neck region amino acid sequence.
[0041] Another example of a trimerizing domain is an MBP trimerizing domain,
as
described in U.S. Provisional Patent Application Serial No. 60/996,288, filed
by the assignee
of the present application on November 9, 2007, which is incorporated by
reference in its
entirety. This trimerizing domain can oligomerize even further and create
higher order
multimeric complexes.
[0042] The IL-1 Ra polypeptide of the invention may either be linked to the N-
or the C-
terminal amino acid residue of the trimerization domain. A flexible molecular
linker
optionally may be interposed between, and covalently join, the polypeptide
representing the
IL-1 Ra and the trimerization domain. Preferably, the linker is a polypeptide
sequence of
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about 1 to 20, 2 to 10, or 3 to 7 amino acid residues. In further embodiments,
the linker is
non-immunogenic, not prone to proteolytic cleavage, and does not comprise
amino acid
residues which are known to interact with other residues (e.g. cystein
residues).
[0043] As used herein "IL-1 Ra" refers to a polypeptide having the amino acid
sequence
shown below:
RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK]ID
VVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQD
KRFAFIRSD SGPTTSFESAACPGWFLCTAMEADQPV SLTNMPDEGVMV
TKFYFQEDE (SEQ ID NO: 38)
[0044] Also included in the "IL-IRa" definition are variants and fragments of
SEQ ID
NO: 38 that provide for IL-iRa binding to IL-1R, and preferably IL-1R
inhibitory activity.
Such fragments may be truncated at the N-terminus or C-terminus of the IL-1Ra,
or may lack
internal residues, when compared with the full length native IL-lRa protein.
Certain
fragments may lack amino acid residues that are not essential for a desired
biological activity
of the trimeric IL-1 Ra protein according to the invention. For example,
Evans, et al. (J.
Biol. Chem. 1995, 19:11477-11483) demonstrated by site directed mutagenesis
that only
Trpl6, G1n20, Tyr34, G1n36 and Tyr147 are critical for binding to the IL-1R
and that other
amino acid positions can be altered while still maintaining a functional
molecule.
Furthermore, affinity of IL-1Ra to its receptor can be improved by mutating
amino acids
outside the binding region to increase loop interactions of IL-lRa with its
receptor as shown
by Dahlen, et al, (J. Immunotoxicology 5:189-199 (2008)). This is can be
accomplished
through mutations of amino acids outside the IL-1Ra receptor binding region,
and
particularly, for example: D47N, E52R, E90Y, P38Y, H54R, Q129L and M136N. Id.
Furthermore, natural IL-iRa variants exist, any of which may be used. An 18
kDa form of
IL-1Ra, created by an alternative transcriptional splice mechanism from an
upstream exon is
called icIL-1Ral and is found inside keratinocytes and other epithelial cells,
monocytes,
tissue macrophages, fibroblasts, and endothelial cells. IL-lRa cDNA cloned
from human
leukocytes contains an additional 63 bp sequence as an insert in the 5' region
of the cDNA. A
15 kDa isoform of IL-1Ra, termed icIL-1Ra3, is found in monocytes,
macrophages,
neutrophils, and hepatocytes, and may be created both by an alternative
transcriptional splice
as well as by alternative translational initiation.
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[0045] IL-1Ra peptides that are useful for fusion proteins of the invention
include
polypeptides that are at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at
least 90%, or at least 95% identical to SEQ ID NO: 38. In particular
embodiments, the fusion
proteins include an IL-1Ra peptide sequence that is 85% identical to SEQ ID
NO: 38 and has
IL-1R binding activity, and preferably IL-1Ra inhibitory activity. In another
particular
embodiment, the fusion proteins include an IL-1Ra peptide sequence that is 95%
identical to
SEQ ID NO: 38 and has IL-1R binding activity, and preferably IL-1Ra inhibitory
activity. In
these embodiment, the polypeptides comprise Trpl6, Gln2O, Tyr34, G1n36 and
Tyr147
according to the numbering of SEQ ID NO: 38 These polypeptides may further
include one
or more amino acids substitutions D47N, E52R, E90Y, P38Y, H54R, Q129L and
M136N
(numbering according to SEQ ID NO: 38). Furthermore, variations of the IL-lRa
polypeptides can be accomplished by replacing one or more amino acids with
another amino
acid having similar structural or chemical properties, for example,
conservative amino acid
substitutions.
[0046] In a further embodiment, the fusion protein according to the invention
is selected
from an IL-1 receptor antagonist selected from the following:
TripK-IL- l ra
EGPTOKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEOQALO
TV SLKRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPN
VNLEEKID V VPIEPHALFL GIHGGKMCLS C V KSGDETRLQLEA VNI
TDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPV
SLTNMPDEGVMVTKFYFQEDE (SEQ ID NO: 39);
TripV-IL-Ira
EGPTOKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEOQALQ
TVRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL
EEK W V VPEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLS
ENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTN
MPDEGVMVTKFYFQEDE (SEQ ID NO: 40);
TripT-IL- l ra
EGPTQKPKKIVNAKKDV VNTKMFEELKSRLDTLAOEVALLKEQOALQ
TRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE
EKED V VPIEPHALFLGIHGGKMCLSCVKS GDETRLQLEAVNITDLS
ENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTN
MPDEGVMVTKFYFQEDE (SEQ ID NO: 41)
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TripQ-IL-Ira
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHHIH-IHGSIEPDG
GEGPTOKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQAL
QRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE
EKIDV VPIEPHALFLGIHGGKMCLSCVKS GDETRLQLEAVNITDLS
ENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTN
MPDEGVMVTKFYFQEDE (SEQ ID NO: 42)
I10-TripK-ILlra
IVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQOALQTVSLKRPSG
RKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV
VPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQ
DKRFAFIRSD SGPTTSFESAACPGWFLCTAMEAD QPV SLTNMPDE
GVMVTKFYFQEDE (SEQ ID NO: 43);
I10-TripV-IL-lra
IVNAKKDVVNTKMFEELKSRLDTLAOEVALLKEQQALQTVRPSGRKS
SKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIE
PHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKR
FAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVM
VTKFYFQEDE (SEQ ID NO: 44);
I10-TripT-IL-lra
IVNAKKDVVNTKMFEELKSRLDTLAOEVALLKEOQALQTRPSGRKSS
KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEP
HALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRF
AFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMV
TKFYFQEDE (SEQ ID NO: 45);
I10-TripQ-IL-1 ra
IVNAKKDV VNTKMFEELKSRLDTLAQEVALLKEQQALQRPSGRKS SK
MQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDV VPIEPH
ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA
FIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVT
KFYFQEDE (SEQ ID NO: 46);
wherein the underlined part denotes the trimerization unit, and the bold part
denotes the IL-
1 Ra part.
Production of Fusion Proteins
[0047] The trimeric IL-1 Ra protein of the invention may be chemically
synthesized or
expressed in any suitable standard protein expression system. Preferably, the
protein
expression systems are systems from which the desired protein may readily be
isolated and
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refolded in vitro. Prokaryotic expression systems are preferred since high
yields of protein
can be obtained and efficient purification and refolding strategies are
available. Eukaryotic
expression systems may also be used. Thus, it is well within the abilities and
discretion of
the skilled artisan to choose an appropriate expression system. Similarly,
once the primary
amino acid sequence for the fusion proteins of the present invention is
chosen, one of
ordinary skill in the art can easily design appropriate recombinant DNA
constructs which will
encode the desired proteins, taking into consideration such factors as codon
biases in the
chosen host, the need for secretion signal sequences in the host, the
introduction of proteinase
cleavage sites within the signal sequence, and the like. These recombinant DNA
constructs
may be inserted in-frame into any of a number of expression vectors
appropriate to the
chosen host. Preferably, the expression vector will include a strong promoter
to drive
expression of the recombinant constructs.
[0048] The fusion protein of the invention can be expressed in any suitable
standard
protein expression system by culturing a host transformed with a vector
encoding the fusion
protein under such conditions that the fusion protein is expressed.
Preferably, the expression
system is a system from which the desired protein may readily be isolated and
refolded in
vitro. As a general matter, prokaryotic expression systems are preferred since
high yields of
protein can be obtained and efficient purification and refolding strategies
are available. Thus,
selection of appropriate expression systems (including vectors and cell types)
is within the
knowledge of one skilled in the art. Similarly, once the primary amino acid
sequence for the
fusion protein of the present invention is chosen, one of ordinary skill in
the art can easily
design appropriate recombinant DNA constructs which will encode the desired
amino acid
sequence, taking into consideration such factors as codon biases in the chosen
host, the need
for secretion signal sequences in the host, the introduction of proteinase
cleavage sites within
the signal sequence, and the like.
[0049] In one embodiment the isolated polynucleotide encodes a fusion protein
of the
invention. In other embodiments, an IL-lRa polypeptide and the trimerizing
domain are
encoded by non-contiguous polynucleotide sequences. Accordingly, in some
embodiments
an IL-1Ra polypeptide and the trimerizing domain are expressed, isolated, and
purified as
separate polypeptides and fused together to form the fusion protein of the
invention.
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[0050] These recombinant DNA constructs may be inserted in-frame into any of a
number of expression vectors appropriate to the chosen host, In certain
embodiments, the
expression vector comprises a strong promoter that controls expression of the
recombinant
fusion protein constructs. When recombinant expression strategies are used to
generate the
fusion protein of the invention, the resulting fusion protein can be isolated
and purified using
suitable standard procedures well known in the art, and optionally subjected
to further
processing such as e.g. lyophilization.
[0051] Standard techniques may be used for recombinant DNA molecule, protein,
and
fusion protein production, as well as for tissue culture and cell
transformation. See, e.g.,
Sambrook, et al. (below) or Current Protocols in Molecular Biology (Ausubel et
al., eds.,
Green Publishers Inc. and Wiley and Sons 1994). Purification techniques are
typically
performed according to the manufacturer's specifications or as commonly
accomplished in
the art using conventional procedures such as those set forth in Sambrook et
al. (Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
NY (1989), or as described herein. Unless specific definitions are provided,
the
nomenclature utilized in connection with the laboratory procedures, and
techniques relating
to molecular biology, biochemistry, analytical chemistry, and
pharmaceutical/formulation
chemistry described herein are those well known and commonly used in the art.
Standard
techniques can be used for biochemical syntheses, biochemical analyses,
pharmaceutical
preparation, formulation, and delivery, and treatment of patients.
[0052] It will be appreciated that a flexible molecular linker optionally may
be interposed
between, and covalently join, the IL-1Ra polypeptide and the trimerizing
domain. In certain
embodiments, the linker is a polypeptide sequence of about 1 to 20 amino acid
residues. The
linker may be less than 10 amino acids, most preferably, five, four, three,
two, or one amino
acid. It may be in certain cases that nine, eight, seven, or six amino acids
are suitable. In
useful embodiments the linker is essentially non-immunogenic, not prone to
proteolytic
cleavage and does not comprise amino acid residues which are known to interact
with other
residues (e.g. cysteine residues).
[0053] The description below also relates to methods of producing fusion
proteins and
trimeric complexes that are covalently attached (hereinafter "conjugated") to
one or more
chemical groups. Chemical groups suitable for use in such conjugates are
preferably not
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significantly toxic or immunogenic. The chemical group is optionally selected
to produce a
conjugate that can be stored and used under conditions suitable for storage. A
variety of
exemplary chemical groups that can be conjugated to polypeptides are known in
the art and
include for example carbohydrates, such as those carbohydrates that occur
naturally on
glycoproteins, polyglutamate, and non-proteinaceous polymers, such as polyols
(see, e.g.,
U.S. Pat. No. 6,245,901).
[0054] A polyol, for example, can be conjugated to fusion proteins of the
invention at one
or more amino acid residues, including lysine residues, as is disclosed in WO
93/00109,
supra. The polyol employed can be any water-soluble poly(alkylene oxide)
polymer and can
have a linear or branched chain. Suitable polyols include those substituted at
one or more
hydroxyl positions with a chemical group, such as an alkyl group having
between one and
four carbons. Typically, the polyol is a poly(alkylene glycol), such as
poly(ethylene glycol)
(PEG), and thus, for ease of description, the remainder of the discussion
relates to an
exemplary embodiment wherein the polyol employed is PEG and the process of
conjugating
the polyol to a polypeptide is termed "pegylation." However, those skilled in
the art
recognize that other polyols, such as, for example, poly(propylene glycol) and
polyethylene-
polypropylene glycol copolymers, can be employed using the techniques for
conjugation
described herein for PEG.
[0055] The average molecular weight of the PEG employed in the pegylation of
IL-lRa
can vary, and typically may range from about 500 to about 30,000 daltons (D).
Preferably,
the average molecular weight of the PEG is from about 1,000 to about 25,000 D,
and more
preferably from about 1,000 to about 5,000 D. In one embodiment, pegylation is
carried out
with PEG having an average molecular weight of about 1,000 D. Optionally, the
PEG
homopolymer is unsubstituted, but it may also be substituted at one end with
an alkyl group.
Preferably, the alkyl group is a C 1-C4 alkyl group, and most preferably a
methyl group. PEG
preparations are commercially available, and typically, those PEG preparations
suitable for
use in the present invention are non-homogeneous preparations sold according
to average
molecular weight. For example, commercially available PEG(5000) preparations
typically
contain molecules that vary slightly in molecular weight, usually 500 D. The
fusion protein
of the invention can be further modified using techniques known in the art,
such as,
conjugated to a small molecule compounds (e.g., a chemotherapeutic);
conjugated to a signal
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molecule (e.g., a fluorophore); conjugated to a molecule of a specific binding
pair (e.g,.
biotin/streptavidin, antibody/antigen); or stabilized by glycosylation,
PEGylation, or further
fusions to a stabilizing domain (e.g., Fc domains).
[0056] A variety of methods for pegylating proteins are known in the art.
Specific
methods of producing proteins conjugated to PEG include the methods described
in U.S. Pat.
Nos. 4,179,337, 4,935,465 and 5,849,535. Typically the protein is covalently
bonded via one
or more of the amino acid residues of the protein to a terminal reactive group
on the polymer,
depending mainly on the reaction conditions, the molecular weight of the
polymer, etc. The
polymer with the reactive group(s) is designated herein as activated polymer.
The reactive
group selectively reacts with free amino or other reactive groups on the
protein. The PEG
polymer can be coupled to the amino or other reactive group on the protein in
either a
random or a site specific manner. It will be understood, however, that the
type and amount of
the reactive group chosen, as well as the type of polymer employed, to obtain
optimum
results, will depend on the particular protein or protein variant employed to
avoid having the
reactive group react with too many particularly active groups on the protein.
As this may not
be possible to avoid completely, it is recommended that generally from about
0.1 to 1000
moles, preferably 2 to 200 moles, of activated polymer per mole of protein,
depending on
protein concentration, is employed. The final amount of activated polymer per
mole of
protein is a balance to maintain optimum activity, while at the same time
optimizing, if
possible, the circulatory half-life of the protein.
[0057] The term "polyoi" when used herein refers broadly to polyhydric alcohol
compounds. Polyols can be any water-soluble poly(alkylene oxide) polymer for
example, and
can have a linear or branched chain. Preferred polyols include those
substituted at one or
more hydroxyl positions with a chemical group, such as an alkyl group having
between one
and four carbons. Typically, the polyol is a poly(alkylene glycol), preferably
poly(ethylene
glycol) (PEG). However, those skilled in the art recognize that other polyols,
such as, for
example, poly(propylene glycol) and polyethylene-polypropylene glycol
copolymers, can be
employed using the techniques for conjugation described herein for PEG. The
polyols of the
invention include those well known in the art and those publicly available,
such as from
commercially available sources.
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[0058] Furthermore, other half-life extending molecules can be attached to the
N-or C-
terminus of the trimerization domain including serum albumin-binding peptides,
FcRn-
binding peptides or IgG-binding peptides.
[0059] In one embodiment, the trimeric IL-1 Ra protein of the invention is
expressed in a
prokaryotic host cell such as E. coli and is additionally linked to a third
polypeptide, i.e. a
third fusion partner. Thus, it may be that by adding such third fusion partner
to the trimeric
IL-1 Ra protein of the invention, high yields of the trimeric IL-1 Ra protein
may be obtained.
The third fusion partner may be any suitable peptide, oligopeptide,
polypeptide or protein,
including a di-peptide, a tri-peptide, tetra-peptide, penta-peptide or hexa-
peptide. The fusion
partner may in certain instances be a single amino acid. It may be selected
such that it renders
the fusion protein more resistant to proteolytic degradation, facilitates
enhanced expression
and secretion of the fusion protein, improves solubility, and/or allows for
subsequent affinity
purification of the fusion protein.
[0060] In one embodiment, the junction region between the fusion protein of
the
invention (i.e. the IL-lRa portion and the trimerization domain) and the third
fusion partner
such as ubiquitin, comprises a Granzyme B protease cleavage site such as human
Granzyme
B (E.C. 3.4.21.79) as described in US 2005/0199251.
[0061] The third fusion partner may in further embodiments be coupled to an
affinity-tag.
Such an affinity-tag may be an affinity domain which allows for the
purification of the fusion
protein on an affinity resin. The affinity-tag may be a polyhistidine-tag such
as a hexahis-tag,
polyarginine-tag, FLAG-tag, Strep-tag, c-myc-tag, S-tag, calmodulin-binding
peptide,
cellulose-binding peptide, chitin-binding domain, glutathione S-transferase-
tag, or maltose
binding protein.
[0062] The method of the invention may be in an isolation step for isolating
the trimeric
IL-1 Ra protein that is formed by the enzymatic cleavage of the fusion protein
that has been
immobilized by the use of the above mentioned affinity-tag systems. This
isolation step can
be performed by any suitable means known in the art for protein isolation,
including the use
of ion exchange and fractionation by size, the choice of which depends on the
character of
the fusion protein. In one embodiment, the region between the third fusion
partner and the
region comprising the trimerization domain and IL-1 Ra is contacted with the
human serine
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protease Granzyme B to cleave off the fusion protein at a Granzyme B protease
cleavage site
which yields the fusion protein of the invention.
[0063] The present invention also provides plasmids, vectors, transcription or
expression
cassettes which comprise at least one nucleic acid as described above.
Suitable vectors can be
chosen or constructed containing appropriate regulatory sequences, including
promoter
sequences, terminator sequences, polyadenylation sequences, enhancer
sequences, marker
genes and other sequences as appropriate. Vectors may be plasmids, viral,
phage, or
phagemid, as appropriate. (Molecular Cloning: a Laboratory Manual: 2nd
edition, Sambrook
et al., 1989, Cold Spring Harbor Laboratory Press).
[0064] The present invention also provides a recombinant host cell which
comprises one
or more constructs of the invention. Suitable host cells include bacteria,
mammalian cells,
yeast and baculovirus systems. Mammalian cell lines available for expression
of a
heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby
hamster
kidney cells, NSO mouse melanoma cells and many others. A preferred bacterial
host is E.
coli.
[0065] Pharmaceutical Compositions
[0066] In yet another aspect, the invention relates to a pharmaceutical
composition
comprising a therapeutically effective amount of the fusion protein of the
invention along
with a pharmaceutically acceptable carrier or excipient. As used herein,
"pharmaceutically
acceptable carrier" or "pharmaceutically acceptable excipient" includes any
and all solvents,
dispersion media, coating, antibacterial and antifungal agents, isotonic and
absorption
delaying agents, and the like that are physiologically compatible. Examples of
pharmaceutically acceptable carriers or excipients include one or more of
water, saline,
phosphate buffered saline, dextrose, glycerol, ethanol and the like as well as
combinations
thereof. In many cases, it will be preferable to include isotonic agents, for
example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition.
Pharmaceutically acceptable substances such as wetting or minor amounts of
auxiliary
substances such as wetting or emulsifying agents, preservatives or buffers,
which enhance the
shelf life or effectiveness of the of the antibody or antibody portion also
may be included.
Optionally, disintegrating agents can be included, such as cross-linked
polyvinyl pyrrolidone,
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agar, alginic acid or a salt thereof, such as sodium alginate and the like. In
addition to the
excipients, the pharmaceutical composition can include one or more of the
following, carrier
proteins such as serum albumin, buffers, binding agents, sweeteners and other
flavoring
agents; coloring agents and polyethylene glycol.
[0067] The compositions can be in a variety of forms including, for example,
liquid,
semi-solid and solid dosage forms, such as liquid solutions (e.g. injectable
and infusible
solutions), dispersions or suspensions, tablets, pills, powders, liposomes and
suppositories.
The preferred form will depend on the intended route of administration and
therapeutic
application. In an embodiment the compositions are in the form of injectable
or infusible
solutions, such as compositions similar to those used for passive immunization
of humans
with antibodies. In an embodiment the mode of administration is parenteral
(e.g.,
intravenous, subcutaneous, intraperitoneal, intramuscular). In an embodiment,
the fusion
protein (or trimeric complex) is administered by intravenous infusion or
injection. In another
embodiment, the fusion protein or trimeric complex is administered by
intramuscular or
subcutaneous injection.
[0068] Other suitable routes of administration for the pharmaceutical
composition
include, but are not limited to, oral, rectal, transdermal, vaginal,
transmucosal or intestinal
administration.
[0069] Therapeutic compositions are typically sterile and stable under the
conditions of
manufacture and storage. The composition can be formulated as a solution,
microemulsion,
dispersion, liposome, or other ordered structure suitable to high drug
concentration. Sterile
injectable solutions can be prepared by incorporating the active compound
(i.e. fusion protein
or trimeric complex) in the required amount in an appropriate solvent with one
or a
combination of ingredients enumerated above, as required, followed by filtered
sterilization.
Generally, dispersions are prepared by incorporating the active compound into
a sterile
vehicle that contains a basic dispersion medium and the required other
ingredients from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, the preferred methods of preparation are vacuum drying and freeze-
drying that
yields a powder of the active ingredient plus any additional desired
ingredient from a
previously sterile-filtered solution thereof. The proper fluidity of a
solution can be
maintained, for example, by the use of a coating such as lecithin, by the
maintenance of the
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required particle size in the case of dispersion and by the use of
surfactants. Prolonged
absorption of injectable compositions can be brought about by including in the
composition
an agent that delays absorption, for example, monostearate salts and gelatin.
[0070] An article of manufacture such as a kit containing therapeutic agents
useful in the
treatment of the disorders described herein comprises at least a container and
a label. Suitable
containers include, for example, bottles, vials, syringes, and test tubes. The
containers may be
formed from a variety of materials such as glass or plastic. The label on or
associated with
the container indicates that the formulation is used for treating the
condition of choice. The
article of manufacture may further comprise a container comprising a
pharmaceutically-
acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and
dextrose solution.
It may further include other materials desirable from a commercial and user
standpoint,
including other buffers, diluents, filters, needles, syringes, and package
inserts with
instructions for use. The article of manufacture may also comprise a container
with another
active agent as described above.
[0071] Typically, an appropriate amount of a pharmaceutically-acceptable salt
is used in
the formulation to render the formulation isotonic. Examples of
pharmaceutically-acceptable
carriers include saline, Ringer's solution and dextrose solution. The pH of
the formulation is
preferably from about 6 to about 9, and more preferably from about 7 to about
7.5. It will be
apparent to those persons skilled in the art that certain carriers may be more
preferable
depending upon, for instance, the route of administration and concentrations
of therapeutic
agent.
[0072] Therapeutic compositions can be prepared by mixing the desired
molecules
having the appropriate degree of purity with optional pharmaceutically
acceptable carriers,
excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition,
Osol, A. ed.
(1980)), in the form of lyophilized formulations, aqueous solutions or aqueous
suspensions.
Acceptable carriers, excipients, or stabilizers are preferably nontoxic to
recipients at the
dosages and concentrations employed, and include buffers such as Tris, HEPES,
PIPES,
phosphate, citrate, and other organic acids; antioxidants including ascorbic
acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol;
resorcinol;
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cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about
10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates
including glucose, mannose, or dextrins; sugars such as sucrose, mannitol,
trehalose or
sorbitol; salt-forming counter-ions such as sodium; and/or non-ionic
surfactants such as
TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
[0073] Additional examples of such carriers include ion exchangers, alumina,
aluminum
stearate, lecithin, serum proteins, such as human serum albumin, buffer
substances such as
glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of
saturated vegetable fatty
acids, water, salts, or electrolytes such as protamine sulfate, disodium
hydrogen phosphate,
potassium hydrogen phosphate, sodium chloride, colloidal silica, magnesium
trisilicate,
polyvinyl pyrrolidone, and cellulose-based substances. Carriers for topical or
gel-based forms
include polysaccharides such as sodium carboxymethylcellulose or
methylcellulose,
polyvinylpyrrolidone, polyacrylates, polyoxyethylene-polyoxypropylene-block
polymers,
polyethylene glycol, and wood wax alcohols. For all administrations,
conventional depot
forms are suitably used. Such forms include, for example, microcapsules, nano-
capsules,
liposomes, plasters, inhalation forms, nose sprays, sublingual tablets, and
sustained-release
preparations.
[0074] Formulations to be used for in vivo administration should be sterile.
This is
readily accomplished by filtration through sterile filtration membranes, prior
to or following
lyophilization and reconstitution. The formulation may be stored in
lyophilized form or in
solution if administered systemically. If in lyophilized form, it is typically
formulated in
combination with other ingredients for reconstitution with an appropriate
diluent at the time
for use. An example of a liquid formulation is a sterile, clear, colorless
unpreserved solution
filled in a single-dose vial for subcutaneous injection.
[0075] Therapeutic formulations generally are placed into a container having a
sterile
access port, for example, an intravenous solution bag or vial having a stopper
pierceable by a
hypodermic injection needle. The formulations are preferably administered as
repeated
intravenous (i.v.), subcutaneous (s.c.), intramuscular (i.m.) injections or
infusions, or as
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aerosol formulations suitable for intranasal or intrapulmonary delivery (for
intrapulmonary
delivery see, e.g., EP 257,956).
[0076] The molecules disclosed herein can also be administered in the form of
sustained-
release preparations. Suitable examples of sustained-release preparations
include
semipermeable matrices of solid hydrophobic polymers containing the protein,
which
matrices are in the form of shaped articles, e.g., films, or microcapsules.
Examples of
sustained-release matrices include polyesters, hydrogels (e.g., poly(2-
hydroxyethyl-
methacrylate) as described by Langer et al., J. Biomed. Mater. Res., 15: 167-
277 (1981) and
Langer, Chem. Tech., 12: 98-105 (1982) or poly(vinylalcohol)), polylactides
(U.S. Pat. No.
3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-
glutamate
(Sidman et al., Biopolymers, 22: 547-556 (1983)), non-degradable ethylene-
vinyl acetate
(Langer et al., supra), degradable lactic acid-glycolic acid copolymers such
as the Lupron
Depot (injectable microspheres composed of lactic acid-glycolic acid copolymer
and
leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0077] Methods of Treatment
[0078] Another aspect the invention relates to a method of treating diseases
that are
mediated by IL-1Ra. The method includes treating a subject suffering from such
as disease
with a therapeutically effective amount of the pharmaceutical compositions of
the invention.
[0079] Another aspect of the invention is directed to a combination therapy.
Formulations comprising therapeutic agents are also provided by the present
invention. It is
believed that such formulations will be particularly suitable for storage as
well as for
therapeutic administration. The formulations may be prepared by known
techniques. For
instance, the formulations may be prepared by buffer exchange on a gel
filtration column.
[0080] The pharmaceutical compositions can be administered in accord with
known
methods, such as intravenous administration as a bolus or by continuous
infusion over a
period of time, by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-
articular, intrasynovial, intrathecal, oral, topical, or inhalation routes.
Optionally,
administration may be performed through mini-pump infusion using various
commercially
available devices.
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[0081] Effective dosages and schedules for administering the trimeric IL-lRa
may be
determined empirically, and making such determinations is within the skill in
the art. Single
or multiple dosages may be employed. It is presently believed that an
effective dosage or
amount of the trimeric IL-lRa used alone may range from about 1 mg/kg to about
100 mg/kg
of body weight or more per day. Interspecies scaling of dosages can be
performed in a
manner known in the art, e.g., as disclosed in Mordenti, et al., Pharmaceut.
Res., 8:1351
(1991).
[0082] When in vivo administration of the IL-lRa fusion protein is employed,
normal
dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammal body
weight
or more per day, preferably about 1 g/kg/day to 50 mg/kg/day, depending upon
the route of
administration. Guidance as to particular dosages and methods of delivery is
provided in the
literature (see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344; or
5,225,212). One of skill
will appreciate that different formulations will be effective for different
treatment compounds
and different disorders, that administration targeting one organ or tissue,
for example, may
necessitate delivery in a manner different from that to another organ or
tissue. Those skilled
in the art will understand that the dosage of the trimeric IL-1Ra that must be
administered
will vary depending on, for example, the mammal which will receive trimeric IL-
1Ra, the
route of administration, and other drugs or therapies being administered to
the mammal.
[0083] The trimeric complexes and other therapeutic agents (and one or more
other
therapies) may be administered concurrently (simultaneously) or sequentially.
In particular
embodiments, a fusion protein or trimeric complex and a therapeutic agent are
administered
concurrently. In another embodiment, a fusion protein or trimeric complex is
administered
prior to administration of a therapeutic agent. In another embodiment, a
therapeutic agent is
administered prior to a fusion protein or trimeric complex. Following
administration, treated
cells in vitro can be analyzed. Where there has been in vivo treatment, a
treated mammal can
be monitored in various ways well known to the skilled practitioner. For
instance, serum
cytokine responses can be analyzed.
[0084] The IL-lRa fusion proteins described herein may be used in combination
(pre-
treatment, post-treatment, or concurrent treatment) with any of one or more
TNF inhibitors
for the treatment or prevention of the diseases and disorders recited herein,
such as but not
limited to, all forms of soluble TNF receptors including Etanercept (such as
ENBREL ), as
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WO 2009/048961 PCT/US2008/079216
well as all forms of monomeric or multimeric p75 and/or p55 TNF receptor
molecules and
fragments thereof; anti-human TNF antibodies, such as but not limited to,
Infliximab (such as
REMICADE ), and D2E7 (such as HUMIRA ), and the like. Such TNF inhibitors
include
compounds and proteins which block in vivo synthesis or extracellular release
of TNF. In a
specific embodiment, the present invention is directed to the use of an IL-
17RA IL-IRa
fusion proteins in combination (pre-treatment, post-treatment, or concurrent
treatment) with
any of one or more of the following TNF inhibitors: TNF binding proteins
(soluble TNF
receptor type-I and soluble TNF receptor type-II ("sTNFRs"), as defined
herein), anti-TNF
antibodies, granulocyte colony stimulating factor; thalidomide; BN 50730;
tenidap; E 5531;
tiapafant PCA 4248; nimesulide; panavir; rolipram; RP 73401; peptide T; MDL
201,449A;
(1R,3S)-Cis-1-[9-(2,6-diaminopurinyl)]-3-hydroxy-4-cyclopentene hydrochloride;
(1R,3R)-
trans-1-(9-(2,6-diamino)purine]-3-acetoxycyclopentane; (1R,3R)-trans-1-[9-
adenyl)-3-
azidocyclopentane hydrochloride and (1R,3R)-trans-1-(6-hydroxy-purin-9-yl)-3-
azidocyclo-
pentane. TNF binding proteins are disclosed in the art (EP 308 378, EP 422
339, GB 2 218
101, EP 393 438, WO 90/13575, EP 398 327, EP 412 486, WO 91/03553, EP 418 014,
JP
127,800/1991, EP 433 900, U.S. Pat. No. 5,136,021, GB 2 246 569, EP 464 533,
WO
92/01002, WO 92/13095, WO 92/16221, EP 512 528, EP 526 905, WO 93/07863, EP
568
928, WO 93/21946, WO 93/19777, EP 417 563, WO 94/06476, and PCT International
Application No. PCT/US97/12244).
[0085] For example, EP 393 438 and EP 422 339 teach the amino acid and nucleic
acid
sequences of a soluble TNF receptor type I (also known as "sTNFR-I" or "30 kDa
TNF
inhibitor") and a soluble TNF receptor type II (also known as "sTNFR-II" or
"40 kDa TNF
inhibitor"), collectively termed "sTNFRs", as well as modified forms thereof
(e.g., fragments,
functional derivatives and variants). EP 393 438 and EP 422 339 also disclose
methods for
isolating the genes responsible for coding the inhibitors, cloning the gene in
suitable vectors
and cell types and expressing the gene to produce the inhibitors.
Additionally, polyvalent
forms (i.e., molecules comprising more than one active moiety) of sTNFR-1 and
sTNFR-II
have also been disclosed. In one embodiment, the polyvalent form may be
constructed by
chemically coupling at least one TNF inhibitor and another moiety with any
clinically
acceptable linker, for example polyethylene glycol (WO 92/16221 and WO
95/34326), by a
peptide linker (Neve et al. (1996), Cytokine, 8(5):365-370, by chemically
coupling to biotin
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and then binding to avidin (WO 91/03553) and, finally, by combining chimeric
antibody
molecules (U.S. Pat. No. 5,116,964, WO 89/09622, WO 91/16437 and EP 315062.
[0086] Anti-TNF antibodies include the MAK 195F Fab antibody (Holler et al.
(1993),
1st International Symposium on Cytokines in Bone Marrow Transplantation, 147);
CDP 571
anti-TNF monoclonal antibody (Rankin et al. (1995), British Journal of
Rheumatology,
34:334-342); BAY X 1351 murine anti-tumor necrosis factor monoclonal antibody
(Kieft et
al. (1995), 7th European Congress of Clinical Microbiology and Infectious
Diseases, page 9);
CenTNF cA2 anti-TNF monoclonal antibody (Elliott et al. (1994), Lancet,
344:1125-1127
and Elliott et al. (1994), Lancet, 344:1105-1110).
[0087] The IL-1Ra fusion proteins described herein may be used in combination
with all
forms of IL- 17 inhibitors (e.g. anti-IL 17 receptor antibody, Amgen; anti-IL-
17A, anti-
IL17F), RORc inhibitors.
[0088] The IL-1Ra fusion proteins described herein may be used in combination
with all
forms of CD28 inhibitors, such as but not limited to, abatacept (for example
ORENCIA ).
[0089] The IL-1Ra fusion proteins described herein may be used in combination
with all
forms of IL-6 and/or IL-6 receptor inhibitors, such as but not limited to,
Tocilizumab (for
example ACTEMRA ).
[0090] The IL-lRa fusion proteins described herein may be used in combination
with all
forms of anti-IL- 18 compounds, such as IL-18BP or a derivative, an IL- 18
trap, anti-IL-18,
anti-IL-18R1, or anti-IL- 1 8RAcP.
[0091] The IL-1Ra fusion proteins described herein may be used in combination
with all
forms of anti-11,22, such as anti-IL22 or anti-IL22R.
[0092] The IL-1Ra fusion proteins described herein may be used in combination
with all
forms of anti-IL-23 and or IL-12 such as anti-p19, anti-p40 (Ustekinumab),
anti-IL-23R.
[0093] The IL-1Ra fusion proteins described herein may be used in combination
with all
forms of anti-IL21, such as anti-1L21 or anti-1L21R.
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WO 2009/048961 PCTIUS2008/079216
[0094] The IL-1Ra fusion proteins described herein may be used in combination
with all
forms of anti-TGF-beta.
[0095] The IL-1Ra fusion proteins may be used in combination with one or more
cytokines, lymphokines, hematopoietic factor(s), and/or an anti-inflammatory
agent.
[0096] Treatment of the diseases and disorders recited herein can include the
use of first
line drugs for control of pain and inflammation in combination (pretreatment,
post-treatment,
or concurrent treatment) with treatment with one or more of the IL-1Ra fusion
proteins
provided herein. These drugs are classified as non-steroidal, anti-
inflammatory drugs
(NSAIDs). Secondary treatments include corticosteroids, slow acting
antirheumatic drugs
(SAARDs), or disease modifying (DM) drugs. Information regarding the following
compounds can be found in The Merck Manual of Diagnosis and Therapy, Sixteenth
Edition,
Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, N.J. (1992)
and in
Pharmaprojects, PJB Publications Ltd.
[0097] The IL-1Ra fusion proteins described herein may be used in combination
with any
of one or more NSAIDs for the treatment of the diseases and disorders recited
herein.
NSAIDs owe their anti-inflammatory action, at least in part, to the inhibition
of prostaglandin
synthesis (Goodman and Gilman in "The Pharmacological Basis of Therapeutics,"
MacMillan 7th Edition (1985)). NSAIDs can be characterized into at least nine
groups: (1)
salicylic acid derivatives; (2) propionic acid derivatives; (3) acetic acid
derivatives; (4)
fenamic acid derivatives; (5) carboxylic acid derivatives; (6) butyric acid
derivatives; (7)
oxicams; (8) pyrazoles and (9) pyrazolones.
[0098] The IL-1Ra fusion proteins described herein may be used in combination
(pretreatment, post-treatment, or concurrent treatment) with any of one or
more salicylic acid
derivatives, prodrug esters or pharmaceutically acceptable salts thereof. Such
salicylic acid
derivatives, prodrug esters and pharmaceutically acceptable salts thereof
comprise:
acetaminosalol, aloxiprin, aspirin, benorylate, bromosaligenin, calcium
acetylsalicylate,
choline magnesium trisalicylate, magnesium salicylate, choline salicylate,
diflusinal,
etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate,
lysine
acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate,
olsalazine,
parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide,
salicylamide O-acetic
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WO 2009/048961 PCT/US2008/079216
acid, salsalate, sodium salicylate and sulfasalazine. Structurally related
salicylic acid
derivatives having similar analgesic and anti-inflammatory properties are also
intended to be
encompassed by this group.
[0099] In an additional specific embodiment, the present invention is directed
to the use
of an IL-1Ra fusion proteins in combination (pretreatment, post-treatment, or
concurrent
treatment) with any of one or more propionic acid derivatives, prodrug esters
or
pharmaceutically acceptable salts thereof. The propionic acid derivatives,
prodrug esters, and
pharmaceutically acceptable salts thereof comprise: alminoprofen,
benoxaprofen, bucloxic
acid, carprofen, dexindoprofen, fenoprofen, flunoxaprofen, fluprofen,
flurbiprofen,
furcloprofen, ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen, isoprofen,
ketoprofen,
loxoprofen, miroprofen, naproxen, naproxen sodium, oxaprozin, piketoprofen,
pimeprofen,
pirprofen, pranoprofen, protizinic acid, pyridoxiprofen, suprofen, tiaprofenic
acid and
tioxaprofen. Structurally related propionic acid derivatives having similar
analgesic and anti-
inflammatory properties are also intended to be encompassed by this group.
[00100] In yet another specific embodiment, the present invention is directed
to the use of
an IL-1Ra fusion proteins in combination (pretreatment, post-treatment, or
concurrent
treatment) with any of one or more acetic acid derivatives, prodrug esters or
pharmaceutically
acceptable salts thereof. The acetic acid derivatives, prodrug esters, and
pharmaceutically
acceptable salts thereof comprise: acemetacin, alclofenac, amfenac, bufexamac,
cinmetacin,
clopirac, delmetacin, diclofenac potassium, diclofenac sodium, etodolac,
felbinac,
fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac, glucametacin,
ibufenac,
indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, oxametacin,
oxpinac,
pimetacin, proglumetacin, sulindac, talmetacin, tiaramide, tiopinac, tolmetin,
tolmetin
sodium, zidometacin and zomepirac. Structurally related acetic acid
derivatives having
similar analgesic and anti-inflammatory properties are also intended to be
encompassed by
this group.
[00101] In another specific embodiment, the present invention is directed to
the use of an
IL-1Ra fusion proteins in combination (pretreatment, post-treatment, or
concurrent treatment)
with any of one or more fenamic acid derivatives, prodrug esters or
pharmaceutically
acceptable salts thereof. The fenamic acid derivatives, prodrug esters and
pharmaceutically
acceptable salts thereof comprise: enfenamic acid, etofenamate, flufenamic
acid, isonixin,
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WO 2009/048961 PCT/US2008/079216
meclofenamic acid, meclofenamate sodium, medofenamic acid, mefenamic acid,
niflumic
acid, talniflumate, terofenamate, tolfenamic acid and ufenamate. Structurally
related fenamic
acid derivatives having similar analgesic and anti-inflammatory properties are
also intended
to be encompassed by this group.
[00102] In an additional specific embodiment, the present invention is
directed to the use
of an IL-1Ra fusion proteins in combination (pretreatment, post-treatment, or
concurrent
treatment) with any of one or more carboxylic acid derivatives, prodrug esters
or
pharmaceutically acceptable salts thereof. The carboxylic acid derivatives,
prodrug esters,
and pharmaceutically acceptable salts thereof which can be used comprise:
clidanac,
diflunisal, flufenisal, inoridine, ketorolac and tinoridine. Structurally
related carboxylic acid
derivatives having similar analgesic and anti-inflammatory properties are also
intended to be
encompassed by this group.
[00103] In yet another specific embodiment, the present invention is directed
to the use of
an IL-1Ra fusion proteins in combination (pretreatment, post-treatment, or
concurrent
treatment) with any of one or more butyric acid derivatives, prodrug esters or
pharmaceutically acceptable salts thereof. The butyric acid derivatives,
prodrug esters, and
pharmaceutically acceptable salts thereof comprise: bumadizon, butibufen,
fenbufen and
xenbucin. Structurally related butyric acid derivatives having similar
analgesic and anti-
inflammatory properties are also intended to be encompassed by this group.
[00104] In another specific embodiment, the present invention is directed to
the use of an
IL-lRa fusion proteins in combination (pretreatment, post-treatment, or
concurrent treatment)
with any of one or more oxicams, prodrug esters, or pharmaceutically
acceptable salts
thereof. The oxicams, prodrug esters, and pharmaceutically acceptable salts
thereof comprise:
droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicam and 4-hydroxyl-
1,2-
benzothiazine 1,1-dioxide 4-(N-phenyl)-carboxamide. Structurally related
oxicams having
similar analgesic and anti-inflammatory properties are also intended to be
encompassed by
this group.
[00105] In still another specific embodiment, the present invention is
directed to the use of
an IL-1Ra fusion proteins in combination (pretreatment, post-treatment, or
concurrent
treatment) with any of one or more pyrazoles, prodrug esters, or
pharmaceutically acceptable
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WO 2009/048961 PCT/US2008/079216
salts thereof. The pyrazoles, prodrug esters, and pharmaceutically acceptable
salts thereof
which may be used comprise: difenamizole and epirizole. Structurally related
pyrazoles
having similar analgesic and anti-inflammatory properties are also intended to
be
encompassed by this group.
[00106] In an additional specific embodiment, the present invention is
directed to the use
of an IL-1Ra fusion proteins in combination (pretreatment, post-treatment or,
concurrent
treatment) with any of one or more pyrazolones, prodrug esters, or
pharmaceutically
acceptable salts thereof. The pyrazolones, prodrug esters and pharmaceutically
acceptable
salts thereof which may be used comprise: apazone, azapropazone,
benzpiperylon, feprazone,
mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone,
propylphenazone,
ramifenazone, suxibuzone and thiazolinobutazone. Structurally related
pyrazalones having
similar analgesic and anti-inflammatory properties are also intended to be
encompassed by
this group.
[00107] In another specific embodiment, the present invention is directed to
the use of an
IL-lRa fusion proteins in combination (pretreatment, post-treatment, or
concurrent treatment)
with any of one or more of the following NSAIDs:.epsilon.-acetamidocaproic
acid, S-
adenosyl-methionine, 3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen,
antrafenine,
bendazac, bendazac lysinate, benzydamine, beprozin, broperamole, bucolome,
bufezolac,
ciproquazone, cloximate, dazidamine, deboxamet, detomidine, difenpiramide,
difenpyramide,
difisalamine, ditazol, emorfazone, fanetizole mesylate, fenflumizole,
floctafenine, flumizole,
flunixin, fluproquazone, fopirtoline, fosfosal, guaimesal, guaiazolene,
isonixirn, lefetamine
HCI, leflunomide, lofemizole, lotifazole, lysin clonixinate, meseclazone,
nabumetone,
nictindole, nimesulide, orgotein, orpanoxin, oxaceprol, oxapadol, paranyline,
perisoxal,
perisoxal citrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone,
proxazole, thielavin
B, tiflamizole, timegadine, tolectin, tolpadol, tryptamid and those designated
by company
code number such as 480156S, AA861, AD1590, AFP802, AFP860, A177B, AP504,
AU8001, BPPC, BW540C, CHINOIN 121, CN100, EB382, EL508, F1044, FK-506,
GV3658, ITF182, KCNTE16090, KME4, LA2851, MR714, MR897, MY309, 0N03144,
PR823, PV102, PV108, R830, RS2131, SCR152, SH440, S1R133, SPAS510, SQ27239,
ST281, SY6001, TA60, TAI-901 (4-benzoyl-i-indancarboxylic acid), TVX2706,
U60257,
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WO 2009/048961 PCT/US2008/079216
UR2301 and WY41770. Structurally related NSAIDs having similar analgesic and
anti-
inflammatory properties to the NSAIDs are also intended to be encompassed by
this group.
[00108] In still another specific embodiment, the present invention is
directed to the use of
an IL-1Ra fusion proteins in combination (pretreatment, post-treatment or
concurrent
treatment) with any of one or more corticosteroids, prodrug esters or
pharmaceutically
acceptable salts thereof for the treatment of the diseases and disorders
recited herein,
including acute and chronic inflammation such as rheumatic diseases, graft
versus host
disease and multiple sclerosis. Corticosteroids, prodrug esters and
pharmaceutically
acceptable salts thereof include hydrocortisone and compounds which are
derived from
hydrocortisone, such as 21-acetoxypregnenolone, alclomerasone, algestone,
amcinonide,
beclomethasone, betamethasone, betamethasone valerate, budesonide,
chloroprednisone,
clobetasol, clobetasol propionate, clobetasone, clobetasone butyrate,
clocortolone,
cloprednol, corticosterone, cortisone, cortivazol, deflazacon, desonide,
desoximerasone,
dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone,
fluazacort,
flucloronide, flumethasone, flumethasone pivalate, flucinolone acetonide,
flunisolide,
fluocinonide, fluorocinolone acetonide, fluocortin butyl, fluocortolone,
fluocortolone
hexanoate, diflucortolone valerate, fluorometholone, fluperolone acetate,
fluprednidene
acetate, fluprednisolone, flurandenolide, formocortal, halcinonide,
halometasone,
halopredone acetate, hydro-cortamate, hydrocortisone, hydrocortisone acetate,
hydro-
cortisone butyrate, hydrocortisone phosphate, hydrocortisone 21-sodium
succinate,
hydrocortisone tebutate, mazipredone, medrysone, meprednisone,
methylprednisolone,
mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone
21-
diedryaminoacetate, prednisolone sodium phosphate, prednisolone sodium
succinate,
prednisolone sodium 2 1 -m-sulfobenzoate, prednisolone sodium 2 1 -
stearoglycolate,
prednisolone tebutate, prednisolone 21-trimethylacetate, prednisone,
prednival, prednylidene,
prednylidene 21-diethylaminoacetate, tixocortol, triamcinolone, triamcinolone
acetonide,
triamcinolone benetonide and triamcinolone hexacetonide. Structurally related
corticosteroids
having similar analgesic and anti-inflammatory properties are also intended to
be
encompassed by this group.
[00109] In another specific embodiment, the present invention is directed to
the use of an
IL-1Ra fusion proteins in combination (pretreatment, post-treatment, or
concurrent treatment)
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with any of one or more slow-acting antirheumatic drugs (SAARDs) or disease
modifying
antirheumatic drugs (DMARDS), prodrug esters, or pharmaceutically acceptable
salts thereof
for the treatment of the diseases and disorders recited herein, including
acute and chronic
inflammation such as rheumatic diseases, graft versus host disease and
multiple sclerosis.
SAARDs or DMARDS, prodrug esters and pharmaceutically acceptable salts thereof
comprise: allocupreide sodium, auranofm, aurothioglucose, aurothioglycanide,
azathioprine,
brequinar sodium, bucillamine, calcium 3-aurothio-2-propanol-l-sulfonate,
chlorambucil,
chloroquine, clobuzarit, cuproxoline, cyclo-phosphamide, cyclosporin, dapsone,
15-
deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquine gold
salt, gold sodium
thiomalate, gold sodium thiosulfate), hydroxychloroquine, hydroxychloroquine
sulfate,
hydroxyurea, kebuzone, levamisole, lobenzarit, melittin, 6-mercaptopurine,
methotrexate,
mizoribine, mycophenolate mofetil, myoral, nitrogen mustard, D-penicillamine,
pyridinol
imidazoles such as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin and
vincristine. Structurally related SAARDs or DMARDs having similar analgesic
and anti-
inflammatory properties are also intended to be encompassed by this group.
[00110] In another specific embodiment, the present invention is directed to
the use of an
IL-lRa fusion proteins in combination (pretreatment, post-treatment, or
concurrent treatment)
with any of one or more COX2 inhibitors, prodrug esters or pharmaceutically
acceptable salts
thereof for the treatment of the diseases and disorders recited herein,
including acute and
chronic inflammation. Examples of COX2 inhibitors, prodrug esters or
pharmaceutically
acceptable salts thereof include, for example, celecoxib. Structurally related
COX2 inhibitors
having similar analgesic and anti-inflammatory properties are also intended to
be
encompassed by this group. Examples of COX-2 selective inhibitors include but
not limited
to etoricoxib, valdecoxib, celecoxib, licofelone, lumiracoxib, rofecoxib, and
the like.
[00111] In still another specific embodiment, the present invention is
directed to the use of
an IL-lRa fusion proteins in combination (pretreatment, post-treatment, or
concurrent
treatment) with any of one or more antimicrobials, prodrug esters or
pharmaceutically
acceptable salts thereof for the treatment of the diseases and disorders
recited herein,
including acute and chronic inflammation. Antimicrobials include, for example,
the broad
classes of penicillin, cephalosporins and other beta-lactams, aminoglycosides,
azoles,
quinolones, macrolides, rifamycins, tetracyclines, sulfonamides, lincosamides
and
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polymyxins. The penicillins include, but are not limited to penicillin G,
penicillin V,
methicillin, nxfcillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,
ampicillin,
ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate, hetacillin,
cyclacillin,
bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin,
ticarcillin/clavulanate,
azlocillin, meziocillin, peperacillin, and mecillinam. The cephalosporins and
other beta-
lactams include, but are not limited to cephalothin, cephapirin, cephalexin,
cephradine,
cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan, cefoxitin,
ceruroxime, cefonicid,
ceforadine, cefixime, cefotaxime, moxalactam, ceftizoxime, cetriaxone,
cephoperazone,
ceftazidime, imipenem and aztreonam. The aminoglycosides include, but are not
limited to
streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin and
neomycin. The
azoles include, but are not limited to fluconazole. The quinolones include,
but are not limited
to nalidixic acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin,
sparfloxacin and
temafloxacin. The macrolides include, but are not limited to erythomycin,
spiramycin and
azithromycin. The rifamycins include, but are not limited to rifampin. The
tetracyclines
include, but are not limited to spicycline, chlortetracycline, clomocycline,
demeclocycline,
deoxycycline, guamecycline, lymecycline, meclocycline, methacycline,
minocycline,
oxytetracycline, penimepicycline, pipacycline, rolitetracycline, sancycline,
senociclin and
tetracycline. The sulfonamides include, but are not limited to sulfanilamide,
sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole and co-
trimoxazole
(trimethoprim/sulfamethoxazole). The lincosamides include, but are not limited
to
clindamycin and lincomycin. The polymyxins (polypeptides) include, but are not
limited to
polymyxin B and colistin.
[00112] It should be noted that the section headings are used herein for
organizational
purposes only, and are not to be construed as in any way limiting the subject
matter
described. All references cited herein are incorporated by reference in their
entirety for all
purposes.
[00113] The Examples that follow are merely illustrative of certain
embodiments of the
invention, and are not to be taken as limiting the invention, which is defined
by the appended
claims.
[00114] Examples
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[00115] Example 1: Format, production and purification of trimeric IL-1Ra
[00116] It has been previously been shown that IL-lRa can be produced as
recombinant
protein in E. coli. (Steinkasserer et al 1992. FEBS 310:63-65). The protein is
very stable and
refolds efficiently. Isoforms of IL-lRa with additional amino acids in the N-
terminal have
been also described (Haskill et al 1991, PNAS 88:3681-3685; Muzio et al 1995,
JEM 182,
623-628)). These molecules bind IL-1R as well as the mature secreted form
indicating that it
is possible to fuse extra peptide to the N-terminal of the antagonist without
compromising the
binding to the receptor. Crystal structure analysis of IL-lRa interaction with
IL-1R also
supports that N-terminal alterations do not affect interactions with IL1R
(Schreuder et al
1997, Nature 386: 190-194). IL-lRa was cloned from a human cDNA library
derived from
bone marrow and/or human placenta.
[00117] Trimeric IL-lRa was designed as a C-terminal fusion to the Trip-
trimerization
unit. Eight different fusion proteins were designed, four with full length
trimerization units
(Trip) and four with a nine amino acid truncation of the trimerization unit
(IlOTrip). IL-lra
was than fused with either trimerization unit using four different C-terminal
fusions. C-
terminal variations termed Trip V, Trip T, Trip Q and Trip K allow for unique
presentation of
the CTLD domains on the trimerization domain. The Trip K variant is the
longest construct
and contains the longest and most flexible linker between the CTLD and the
trimerization
domain. Trip V, Trip T, Trip Q represent fusions of the CTLD molecule directly
onto the
trimerization module without any structural flexibility but are turning the
CTLD molecule
1/3`a going from Trip V to Trip T and from Trip T to Trip Q. This is due to
the fact that each
of these amino acids is in an a-helical turn and 3.2 as are needed for a full
turn
[00118] The following proteins were produced as the following Granzyme B
cleavable
fusion proteins in BL21 Al bacteria. The underlined portions denotes the
trimerization unit,
and the bold part denotes the IL-1Ra part:
C11-H6-GrB-GG-TripK-IL- l ra:
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHHHHHHGSIEPDG
GEGPTOKPKKIVNAKKDV VNTKMFEELKSRLDTLAOEVALLKEOOAL
TQ VSLKRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGP
NVNLEEKID V VPIEPHALFLGIHGGKMCLS CVKSGDETRLQLEAV
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NITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQ
PVSLTNMPDEGVMVTKFYFQEDE (SEQ ID NO: 47);
CH-H6-GrB-GG-TripV-IL-1 ra:
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHHIl IllHGSIEPDG
GEGPTOKPKKIVNAKKDV VNTKMFEELKSRLDTLAOEVALLKEQQAL
QTVRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVN
LEEKIDV VPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITD
LSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSL
TNMPDEGVMVTKFYFQEDE (SEQ ID NO: 48);
CH-H6-GrB-GG-TripT-IL-1 ra:
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHHHIiHHGSIEPDG
GEGPTOKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQAL
QTRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNL
EEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDL
SENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLT
NMPDEGVMVTKFYFQEDE (SEQ ID NO: 49);
CII-H6-GrB-GG-TripQ-IL-1 ra:
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHIMH HGSIEPDG
GEGPTOKPKKIVNAKKDV VNTKMFEELKSRLDTLAOEVALLKEQOAL
QRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLE
EKIDV VPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLS
ENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTN
MPDEGVMVTKFYFQEDE (SEQ ID NO: 50);
CH-H6-GrB-I10-TripK-IL-1 ra
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHHIII HHGSIEPDIV
NAKKDVVNTKMFEELKSRLDTLAOEVALLKEOOALQTVSLKRPSGR
KSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV V
PIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQD
KRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEG
VMVTICFYFQEDE (SEQ ID NO: 51);
CII-H6-GrB-I10-TripV-IL-l ra
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHIIHHHHGSIEPDIV
NAKKDVVNTKMFEELKSRLDTLAOEVALLKEQQALQTVRPSGRKSS
KMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKID V VPIEP
HALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRF
AFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMV
TKFYFQEDE (SEQ ID NO: 52);
CII-H6-GrB-I1 O-TripT-IL-1 ra:
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WO 2009/048961 PCT/US2008/079216
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHHHHHHGSIEPDIV
NAKKDVVNTKMFEELKSRLDTLAOEVALLKEOOALOTRPSGRKSSK
MQAFRIWDVNQKTFYLRNNQLVAGYLQ GPNVNLEEKIDV VPIEPH
ALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFA
FIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVT
KFYFQEDE (SEQ ID NO: 53); and
CH-H6-GrB-I10-TripQ-IL-1 ra:
MVRANKRNEALRIESALLNKIAMLGTEKTAEGGSHHH HHGSIEPDIV
NAKKDV VNTKMFEELKSRLDTLAOEVALLKEQQALQRPS GRKSSKM
QAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDV VPIEPHA
LFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFI
RSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKF
YFQEDE (SEQ ID NO: 54)
[00119] All constructs were captured on NiNTA Superflow (Qiagen), refolded and
further
purified on SP-Sepharose FF (GE Heathcare). From expression in shake flask or
from a
fermentation of the trimeric IL1-Ra, inclusion bodies were purified. Packed
cell pellet was
homogenized in lysis-buffer (50 mM Tris-HCI, pH 8.0, 25 w/v % Sucrose, 1 mM
EDTA ) by
sonication (50 g wet cell pellet per 100 mL lysis buffer). Then 100 mg
lysozyme per 100 mL
lysis-buffer was added and mixed before the sample was left for 15 min at R.T.
The sample
was then sonicated for 2-5 min with mixing in between. Detergent buffer (0.2 M
NaCl, 1 w/v
% Deoxycholate, Na salt, 1 w/v % Nonidet P40, 20 mM Tris-HCI, pH 7.5, 2 mM
EDTA) was
added and the sample is mixed and sonified again. The inclusion bodies were
recovered by
centrifugation for 25 min at 8.000 rpm, 4 C. The supernatant was stored at 4 C
and the pellet
resuspended in 100 mL TRITON X-100 buffer (0,5 w/v % TRITON X-100, 1 mm EDTA,
pH 8) per 50 g original cell pellet. Inclusion bodies were recovered by
centrifugation for 25
min at 8.000 rpm, 4 C and the supernatant was stored at 4 C. The TRITON X-100
buffer
wash is repeated once more and the inclusion bodies were recovered by
centrifugation for 5
min at 12.000 rpm, 4 C.
[00120] The inclusion bodies were re-suspended in 30 mL denaturing buffer/gram
original
cell paste (6 M urea, 10 mM EDTA, 20 mM Tris/HCl and 20 mM (3-Mercaptoethanol,
pH
8.0) at 28 C for 2 h. The suspension was centrifuged at 7500 g for 15 min to
remove
insoluble material. Following this CaC12 was added to 20 mM final
concentration and the
solution was applied to a 100 mL Ni-NTA Superflow column equillibrated in NTA
buffer (8
M Urea; 1000 mM NaCl; 50 mM Tris HCl pH 8.0; 5 mM (3-Mercaptoethanol) and
washed
36
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WO 2009/048961 PCTIUS2008/079216
until a stable baseline was obtained. A further wash with 250 mL guanidine-
HCI, 50 mM
Tris-HCI pH 8.0, 5 mM (3-Mercaptoethanol followed by wash with 100 mL buffer
NTA.
[00121] Two refolding methods have been used, dialysis refolding and on-column
refolding and both have yielded pure and soluble protein. For dialysis
refolding the
resuspended inclusion bodies was used directly for dialysis over into lx PBS
containing 3 M
urea, 1 mM EDTA, pH 7.2 over night. The day after the dialysis was continued
into lx PBS
containing 0 M urea, 1 mM EDTA, pH 7.2.
[00122] For on-column refolding the washed Ni-NTA Superflow column with
protein
bound, the resin was washed with 4 CV ml 1 x PBS containing 3 M urea, pH 7.2
before a
linear gradient of 10 CV 1 x PBS containing 3 M urea, pH 7.2 and 10 CV 1 x PBS
containing
0 M urea, pH 7.2 was run. To recover the refolded trimeric IL-Ira, the column
eluted with 1 x
PBS, 10 mM EDTA, pH 6.0 and fraction were collected.
[00123] Following refolding cleavage with recombinant human Granzyme B was
performed by adjusting the pH in the eluate to 7.5 with NaOH before Granzyme B
was added
at a 1:500 ratio (granzyme/protein) and incubated at 25 C over night. The
progress was
followed by SDS-PAGE.
[00124] Finally, the cleaved protein was purified using SP-Sepharose FF (GE
Healthcare)
cation exchange step. A -50 mL SP-Sepharose FF was packed and equilibrated in
buffer A (1
x PBS, 1 mM EDTA pH 5,5) until stabile basis line was obtained. The cleavage
reaction was
diluted 1:3 with buffer A and loaded on the column followed by a wash in
buffer A until
stabile basis line was monitored. A gradient from 10 CV buffer A to 10 CV
Buffer B (1 x
PBS, 1 mM EDTA + 0,5 M NaCl pH 5,5) was setup and fractions collected in 5 mL.
Protein
containing fractions were analysed on SDS-PAGE before pooling the protein
product.
[00125] Alternatively, the supernatant from the above inclusion body
preparation were
used to purify the protein. The soluble Trimeric IL1-ra in the supernatant was
purified on Ni-
NTA Superflow (Qiagen) column equilibrated in Buffer A (20mM TrisHCL, 50mM
NaCl pH
8Ø A pool was made of the washes from the inclusion body purification and it
was
centrifuged at 10000 rpm for 10min before CaC12 was added to 5 mM and Tris-HCl
to 20
mM and the pH adjusted to 6.0 with HCI/NaOH. The pool was loaded on the column
and
37
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WO 2009/048961 PCT/US2008/079216
washed in buffer A until stabile basis line. Following a wash in buffer A + 1
M NaCl until
stabile basis line, the bound protein was eluted with buffer A + 20mM EDTA and
fractions
were collected. Hereafter the protein pool was cleaved with Granzyme B and
polished on a
SP-Sepharose FF column as described above. The soluble fraction of CII-H6-GrB-
GG-
TripK-IL-lra from 3L expression culture gave a final yield of 95 mg of TripK-
IL-lRa
following (250 mg CII-H6-GrB-TripK-IL-lRa after capture Ni-NTA Superflow
(Qiagen).
Since the yield and purity of the protein from the soluble fraction was
significantly better
than doing refolding, this path was chosen following the intial construct
testing.
[00126] The ability of the refolded protein to bind to IL-1 Receptor 1 was
analysed on a
Biacore 3000 (Biacore, Uppsala, Sweden) where mouse ILl-RUFc was coupled to
CM5
sensor chips and binding of soluble TripK-IL-Ira to IL-1 RI protein was
measured. Results of
uncleaved C11-H6-GrB-TripK-IL-lra refolding by dialysis are shown in Figure 2
and
uncleaved CH-H6-GrB-TripK-IL-lra on-column NiNTA refolding is shown in Figure
3. The
cleavage and purification assays produced the trimeric IL-1Ra compounds of SEQ
ID NOs:
47-54.
[00127] Example 2: Trimeric IL-lRa compounds ability to inhibit IL-1 induction
of IL-8
in U937 cells
[00128] GG-TripV-IL-lra (trip V-IL1Ra), GG-TripK-IL-lra (trip K-IL1Ra), GG-
TripT-
IL-Ira (trip T-ILlRa) and CII-H6-GrB-GG-TripT-IL-Ira (trip Q-IL1Ra) were
further
analysed for their ability to inhibit IL-1 induction of IL-8 in U937 cells.
Results are shown in
Figure 4.
[00129] The compounds are essentially equally effective in blocking the
response and they
appear all to be as effective as KINERET (when compared on w/w). Due to
buffer effects in
the assay, at the highest protein concentration used (100 .tg/mL) IL-8
production increases
instead of further decreasing. Based on several in vitro efficacy assays as
well as Biacore
assays, it was determined that TripT IL1Ra was the best compound based on
blocking and
binding efficacy as well as production yields.
[00130] Example 3: Pegylated trimeric IL-lRa compounds
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[00131] Since the in vivo half life is a crucial parameter in the efficacy of
KINERET
(KINERET has only a half life in humans of 4-6 hours and has therefore, to be
applied once
daily) the ability to pegylate the TripT IL1Ra by N-terminal pegylation was
tested. The
trimeric IL1-Ra is pegylated at the N terminus. Trimeric ILl-Ra antagonist
proteins after the
final step of the purification procedure described above were used as starting
point for
pegylations. The proteins were buffer changed into PBS buffer pH 6.0 for the
pegylation
reaction. The protein concentration in the reaction was between 0.5 and 3.5
mg/mL and a 5-
molar excess of mPeg5K-Aldehyde or mPeg20K-Aldehyde (Nektar) supplemented with
mM cyanoborohydride (NaCNBH3) was used. The reaction was carried out at 20 C
for 16
hours. Following the reaction mixture was applied to Source 15S column (GE
Healtcare) to
purify the monopegylated form. As shown in Figure 5, antagonistic activity of
the pegylated
version was reduced compared to the unpegylated protein. However, the
pegylated protein
still has good ILl blocking efficacy.
[00132] Example 4: Pharmacokinetic analysis of trimeric IL1Ra proteins in male
Lewis
rats after i.v. injection
[00133] Three of the trimeric ILIRa polypeptides described in the previous
examples were
chosen for pharmacokinetic analysis. The differences in the constructs were in
the N-
terminus of the trimerization domain: full length (FL), first nine amino acids
truncated (110)
and the first 16 amino acids truncated (V17). The 110 construct represents a
naturally
occurring deletion variant of the trimerization domain and lacks the 0-
glycosylation site at
Thr 4. The V17 derivative represents a deletion of the first exon encoding the
trimerization
domain and lacks a characterized heparin binding site. This site is also
partially removed in
the I10 construct. In vitro efficacy of the IL-1Ra molecules was verified in a
U937 cell assay
as shown in Figure 6.
[00134] The pharmacokinetic profile of these three constructs polypeptides
were analysed
in Lewis rats after intravenous (i.v.) injections. The profiles obtained were
compared to the
pharmacokinetic profile of KINERET in the same experiment. The
pharmacokinetic study
was conducted using four male Lewis rats per group, and the constructs that
were used were
FL IL-1Ra, 110 IL-1Ra, V17 IL-1Ra and KINERET . Single i.v. doses of 100 mg/kg
were
given to the animals. The test compound was dissolved in vehicle (4.4 mM
NaCitrate, pH
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WO 2009/048961 PCT/US2008/079216
6.5, 93.8 mM NaCl, 0.33 mM EDTA, 0.7 g TWEEN -80) and administered through the
tail
vein (vena sacralis media) or the hind paw vein (vena saphena).
[00135] Blood was then collected from four animals per time-point at baseline
(zero
hours) and 0.5, 1, 2, 4, 8, 12, 24, 48, 72 h post dosing. Blood samples of
approximately 100
gl were collected from the tip of the tails in MicrotainersTM. Plasma was
collected and
transferred into polypropylene tubes. Plasma samples were then stored at <-70
C until
measurements were performed. Animals were then sacrificed by CO2 inhalation
and the
carcasses were discarded without pathological examination. The IL-1Ra compound
levels
and KINERET levels in plasma were then determined by ELISA.
[00136] The average body weight of each rat was 250 grams. Assuming that the
rat
average blood volume was 16.5 mL a theoretical maximum initial concentration
of the
compounds of 1,500,000 ng/mL was calculated after i.v. injection. These
concentrations are
shown in Figure 7. This starting level was used as starting value for the
analysis. No
observations of side effects or changes in animal well being were observed.
[00137] Following blood sampling at the above indicated time points, an ELISA
assay was
used to measure the injected protein in the blood samples. Based on these
ELISA results, area
under the curve (AUC) was used as a measure of drug exposure and the plasma
half life were
calculated using standard software. The areas under the curve are shown in
Table 2 and the
plasma half lives of the proteins are shown in Table 3.
Table 2
AUC protein/
Protein AUC (ng/mL*h) AUC KINERET
FL IL1Ra 809292 1.89
110IL1Ra 1637866 3.82
V171L1Ra 2177781 5.08
KINERET 428414 1
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Table 3
Half life protein/
Protein Half life (min.) Half life HINERET
FL IL1Ra 20 17
I10 IL1Ra 54 45
V17 IL1Ra 69 58
KINERET 1.2 1
[00138] These i.v. data indicate that the trimeric compounds have superior
plasma half
lives in comparison to KINERET . The half life of KINERET is about 1.2
minutes, whereas
the half life of the V17 IL1Ra trimeric protein after i.v. injection is about
69 minutes.
Dependent on the criteria used in the analysis the relative increase in AUC is
between two-
fold for FL IL1Ra trimer and five-fold for V17 IL1Ra trimer, indicating
substantially
improved drug exposure using the trimerized variants compared to KINERET .
[00139] Example 5: Production of Met-IlO-TripT-ILlra and GG-Vl7-TripT-ILlra
and Rat
CIA model
[00140] Both molecules were produced by BL21 AI bacteria in 10 L fermentor
runs using
either 2 x TY medium (Met-Il0-TripT-IL-Ira) or chemically defined minimal
medium (GG-
V17-TripT-IL-lra). Cell pellets were obtained by centrifugation at 5887 x g
for 20 min, then
resuspended in 10 mM Na2HPO4 pH 6. For Met-10-TripT-IL-lra, the soluble cell
fraction
containing the protein of interest was obtained by high pressure
homogenization (2 x 17.000
psi) followed by 10 min centrifugation at 10.000 x g. The supernatant was
diluted with 10
mM Na2HPO4 pH 7.4 and run over a SP-Sepharose FF column (cation exchange, GE
Healthcare) followed by Q-Sepharose FF (anion exchange. GE Healthcare) using
an AKTA
fPLC. In a last step, proteins were run through a Mustang E filter (Pall) to
remove endotoxin,
followed by buffer exchange into PBS pH 7.4 and concentration to 50 mg/mL. The
GG-V 17
-TripT-IL- 1 Ra protein was expressed as a fusion protein comprising an N-
terminal booster
domain, phage CII protein, followed by a human Granzyme B cleavage site. The
GG-V1 7 -
TripT-IL-iRa was purified from fermentation cell pellets by homogenization in
lysis buffer
containing lysozyme followed by centrifugation for 25 min at 8000 rpm. The
supernatant
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was then run through a Fractogel EMD Chelate (M) column (EMD Chemicals Inc.),
and the
eluate was buffer exchanged into 20 mM Tris pH 7.5, 150 mM NaCl. The protein
fraction
was then digested with recombinant human Granzyme B (made in house, ref to
patent). After
dilution with PBS pH 6, the proteins were purified using SP Sepharose FF
followed by
Mustang E filtration and Fractogel EMD Chelate (M) column in flow through
mode to
remove the fusion tag and human Granzyme B. Final, the protein was buffer
exchanged into
PBS pH 7.4 and concentrated to 50 mg/mL. Yields for both Met-I10-TripT-IL-Ira
and GG-
V17-TripT-IL-Ira proteins were 3-5 g/L, purity >95% as determined by SDS-PAGE
(Figure
8), RP-HPLC and MS. Endotoxin levels were <3EU/mg as determined using a LAL
assay
(Lonza). Aggregates were <0.5% as determined by analytical SEC (Figure 9) and
host cell
protein < 6 ng/mL. Two batches (LM022, LM023) of Met-IIO-TripT-IL-Ira and two
batches
(CFO 19, CF020) of GG-V 17-TripT-IL-1 ra were tested in above assays.
[00141] Female Lewis rats with 4-day established type II collagen arthritis
were treated
subcutaneously (SC), daily (QD) on arthritis days 1-3 with Vehicle (10 mM
phosphate buffer
pH 7.4), or equimolar amounts of IL-lra administering either monomeric IL-lra
(100 mg/kg
KINERET ), or trimerized ILlra (120 mg/kg Met-Il0-TripT-ILlra, or 120 mg/kg GG-
V17-
TripT-ILlra). In order to have only one set of controls, all rats in the QD
groups were dosed
with the respective vehicle (10 mM phosphate buffer pH 7.4, or sodium citrate
buffer pH 6.5
for KINERET ) at the 2nd and 3rd dosings to keep manipulations constant.
Animals were
terminated on arthritis day 4. Efficacy evaluation was based on ankle caliper
measurements,
expressed as area under the curve (AUC), terminal hind paw weights and body
weights
(Bendele et al 2000, Arthritis + Rheumatism 43:2648-2659). All animals
survived to study
termination. Rats injected with KINERET or its vehicle (CSEP) vocalized
during the
injection process thus suggesting that subcutaneous irritation was occurring.
No vocalization
occurred with any other injections.
[00142] Animals (8/group for arthritis, 4/group for normal), housed 4/cage,
were
anesthetized with Isoflurane and received subcutaneous/intradermal (SC/ID)
injections with
300 l of Freund's Incomplete Adjuvant (Difco, Detroit, MI) containing 2 mg/ml
bovine type
II collagen (Elastin Products, Owensville, Missouri) at the base of the tail
and 2 sites on the
back on days 0 and 6. Dosing by subcutaneous route (QD at 24 hour intervals)
was initiated
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on arthritis day 1 and continued through day 3. Experimental groups were as
shown in Table
4
Table 4
QD SC Treatment 2.3 ml/kg, days 1-3,
Group N Dose volumes are based on equivalent IL-lra molecules
1 4 Normal controls, vehicle (10 mM phosphate buffer pH 7.4) TID
2 8 Arthritis+ KINERET QD (100 mg/kg), vehicle (sodium citrate buffer
pH 6.5) at other times
3 8 Arthritis+ Met-I10-TripT-ILira QD (120 mg/kg), vehicle (10 mM
phosphate buffer pH 7.4) at other times
4 8 Arthritis+ V 17-TripT-ILira QD (120 mg/kg), vehicle (10 mM
phosphate buffer pH 7.4) at other times
[00143] Rats were weighed on days 0-4 of arthritis, and caliper measurements
of ankles
were taken every day beginning on day 0 of arthritis (study day 9). After
final body weight
measurement, animals were euthanized, and hind paws were transected at the
level of the
medial and lateral malleolus and weighed (paired).
[00144] Significant reduction of ankle diameter was seen in rats treated with
100 mg/kg
KINERET QD (d3-4), 120 mg/kg Met-110-TripT-ILira QD (d2-4), or 120 mg/kg GG-
V17-
TripT-ILira QD (d3-4), as compared to vehicle treated disease control animals.
Reduction of
ankle diameter AUC was significant for rats treated with 100 mg/kg KINERET QD
(34%),
120 mg/kg Met-I10-TripT-ILira QD (54%), or 120 mg/kg GG-V17-TripT-ILlra QD
(49%),
as compared to vehicle treated disease control animals. Met-I10-TripT-ILira QD
treatment
resulted in significantly reduced ankle diameter AUC compared to KINERET QD
treatment
(p<0.035 at the end of the study). Also, GG-V17-TripT-ILlra QD treatment
resulted in
significantly reduced ankle diameter AUC compared to KINERET QD treatment at
the end
of the study (p<0.001). (Figure 10)
[00145] Reduction of final paw weight was significant for rats treated with
100 mg/kg
KINERET QD (61%), 120 mg/kg Met-I10-TripT-ILira QD (79%), or 120 mg/kg GG-V17-
TripT-ILira QD (91%), as compared to vehicle treated disease control animals.
GG-V17-
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TripT-IL lra QD treatment resulted in significantly reduced final paw weights
compared to
KINERET QD treatment (p<0.006). (Figure 11)
[00146] Change in body weight was significantly increased toward normal for
rats treated
with 100 mg/kg KINERET QD (54%), 120 mg/kg Met-I10-TripT-ILlra QD (49%), or
120
mg/kg GG-V17-TripT-ILlra QD (65%), as compared to vehicle treated disease
control
animals.
[00147] Example 6: Streptozocin (STZ)-induced diabetes model
[00148] STZ (Sigma Aldrich) was administered once daily for five successive
days at 50
mg/kg i.p. to fasted C57BL/6J male mice. The mice gradually developed higher
levels of
blood glucose from Day 1 to Day 4. The levels rose from 6.9 nmol/L to 13.1
nmol/L during
the STZ induction period. Five days (Day 4) after the last STZ dosing, the
mice were
randomly distributed into 10 treatment groups each containing 10 mice in good
condition.
Treatment started on this day, before onset of diabetes and continued beyond
the onset. The
treatment groups were as shown in Table 5.
Table 5
Group Induction of Dose
No. Diabetes Test Article mg/kg Administration
1 + Vehicle 0 i.p. once daily
(QD)
2 + KINERET 100 i.p. once daily
(QD)
3 + KINERET 30 i.p. once daily
(QD)
4 + 110-TripT-IL1-RA 100 i.p. once daily
(QD)
+ I10-TripT-IL1-RA 30 i.p. once daily
(QD)
6 + I10-TripT-ILI-RA 100 i.p. twice
weekly (QD)
[00149] The study period was 28 days and the mice were weighed once weekly
during the
treatment period. Blood glucose levels were measured every other day during
the study
period in order to monitor development of diabetes. A droplet of whole blood
was collected
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by tail vein bleeding and placed on an Ascensia ELITE blood glucose test
strip and
analyzed with an Ascensia ELITE blood glucose meter (Bayer). The values were
recorded,
and x-fold increase in any given group compared to levels at treatment
initiation was
calculated. Clinical symptoms were observed daily or as appropriate in groups
where adverse
symptoms occurred.
[00150] As shown in Figure 12, a marked reduction of blood glucose levels was
observed
after daily i.p. dosing of either I10-TripT-IL1-Ra or KINERET at both 100 and
30 mg/kg.
Furthermore, twice weekly dosing of 100 mg/kg I10-TripT-IL1Ra was equally
effective as
daily dosing of 100 mg/kg KINERET . These data demonstrate that trimerized IL-
1Ra is an
effective treatment of experimentally induced diabetes.
[00151] The examples given above are merely illustrative and are not meant to
be an
exhaustive list of all possible embodiments, applications or modifications of
the invention.
Thus, various modifications and variations of the described methods and
systems of the
invention will be apparent to those skilled in the art without departing from
the scope and
spirit of the invention. Although the invention has been described in
connection with specific
embodiments, it should be understood that the invention as claimed should not
be unduly
limited to such specific embodiments. Indeed, various modifications of the
described modes
for carrying out the invention which are obvious to those skilled in molecular
biology,
immunology, chemistry, biochemistry or in the relevant fields are intended to
be within the
scope of the appended claims.
[00152] It is understood that the invention is not limited to the particular
methodology,
protocols, and reagents, etc., described herein, as these may vary as the
skilled artisan will
recognize. It is also to be understood that the terminology used herein is
used for the purpose
of describing particular embodiments only, and is not intended to limit the
scope of the
invention. It also is to be noted that, as used herein and in the appended
claims, the singular
forms "a," "an," and "the" include the plural reference unless the context
clearly dictates
otherwise. Thus, for example, a reference to "a linker" is a reference to one
or more linkers
and equivalents thereof known to those skilled in the art.
[00153] Unless defined otherwise, all technical and scientific terms used
herein have the
same meanings as commonly understood by one of ordinary skill in the art to
which the
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invention pertains. The embodiments of the invention and the various features
and
advantageous details thereof are explained more fully with reference to the
non-limiting
embodiments and/or illustrated in the accompanying drawings and detailed in
the following
description. It should be noted that the features illustrated in the drawings
are not necessarily
drawn to scale, and features of one embodiment may be employed with other
embodiments
as the skilled artisan would recognize, even if not explicitly stated herein.
[00154] Any numerical values recited herein include all values from the lower
value to the
upper value in increments of one unit provided that there is a separation of
at least two units
between any lower value and any higher value. As an example, if it is stated
that the
concentration of a component or value of a process variable such as, for
example, size, angle
size, pressure, time and the like, is, for example, from 1 to 90, specifically
from 20 to 80,
more specifically from 30 to 70, it is intended that values such as 15 to 85,
22 to 68, 43 to 51,
30 to 32, etc. are expressly enumerated in this specification. For values
which are less than
one, one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate.
These are only
examples of what is specifically intended and all possible combinations of
numerical values
between the lowest value and the highest value enumerated are to be considered
to be
expressly stated in this application in a similar manner.
[00155] Particular methods, devices, and materials are described, although any
methods
and materials similar or equivalent to those described herein can be used in
the practice or
testing of the invention. The disclosures of all references and publications
cited above are
expressly incorporated by reference in their entireties to the same extent as
if each were
incorporated by reference individually.
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