SERPIN FUSION POLYPEPTIDES AND METHODS OF USE THEREOF

POLYPEPTIDES DE FUSION DE SERPINE ET LEURS PROCEDES D'UTILISATION

English Abstract

This invention relates to molecules, particularly polypeptides, more particularly fusion proteins that include a serpin polypeptide or an amino acid sequence that is derived from a serpin and second polypeptide comprising of at least one the following: an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide; a cytokine targeting polypeptide or a sequence derived from a cytokine targeting polypeptide; a WAP domain containing polypeptide or a sequence derived from a WAP containing polypeptide; and an albumin polypeptide or an amino acid sequence that is derived from a serum albumin polypeptide. This invention also relates to methods of using such molecules in a variety of therapeutic and diagnostic indications, as well as methods of producing such molecules.

French Abstract

L'invention concerne des molécules, en particulier des polypeptides, plus particulièrement des protéines de fusion qui comprennent un polypeptide de serpine ou une séquence d'acides aminés qui est dérivée d'une serpine, et un second polypeptide comprenant au moins l'un des éléments suivants : un polypeptide Fc ou une séquence d'acides aminés qui est dérivée d'un polypeptide Fc ; un polypeptide de ciblage de cytokines ou une séquence dérivée d'un polypeptide de ciblage de cytokines ; un polypeptide contenant un domaine WAP ou une séquence dérivée d'un polypeptide contenant un domaine WAP ; et un polypeptide d'albumine ou une séquence d'acides aminés qui est dérivée d'un polypeptide de sérum-albumine. La présente invention concerne également des procédés d'utilisation de telles molécules dans une large gamme d'indications thérapeutiques et diagnostiques, ainsi que des procédés de production de telles molécules.

Claims

What is claimed is:

1. An isolated fusion protein comprising at least one human serpin
polypeptide
operably linked to a human immunoglobulin Fc polypeptide or an amino acid
sequence that
is derived from an immunoglobulin Fc polypeptide comprising an amino acid
sequence
selected from the group consisting of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49,
50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65, wherein the
immunoglobulin Fc
polypeptide comprises one or more mutations at a position selected from S228,
L235,
M252, M258, M428, and combinations thereof.
2. The isolated fusion protein of claim 1, wherein the fusion protein
further comprises
an additional polypeptide selected from the group consisting of:
a cytokine targeting polypeptide or a sequence derived from a cytokine
targeting
polypeptide;
a WAP domain containing polypeptide or a sequence derived from a WAP domain
containing polypeptide; or
an albumin polypeptide or an amino acid sequence that is derived from a serum
albumin polypeptide.
3. The fusion protein of claim 1, wherein the human serpin polypeptide is a
human
alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT
polypeptide.
4. The isolated fusion protein of claim 3, wherein AAT polypeptide
comprises the
amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 80.
5. The isolated fusion protein of claim 3, wherein the AAT polypeptide
comprises the
reactive site loop of AAT comprising the amino acid sequence of SEQ ID NO: 1.
6. The isolated fusion protein of claim 3, wherein the AAT polypeptide
comprises a
mutated reactive site loop of AAT comprising the amino acid sequence of SEQ ID
NO: 32
or 33.

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7. The isolated fusion protein of claim 1, wherein the immunoglobulin Fc
polypeptide
comprises the amino acid sequence of SEQ ID NO: 53.
8. The isolated fusion protein of claim 1, wherein the immunoglobulin Fc
polypeptide
comprises the amino acid sequence of SEQ ID NO: 73.
9. The isolated fusion protein of claim 1 or claim 3, wherein the serpin
polypeptide and
the immunoglobulin Fc polypeptide are operably linked via a hinge region, a
linker region,
or both a hinge region and linker region.
10. The isolated fusion protein of claim 9, wherein the hinge region, the
linker region or
both the hinge region and the linker region comprise a peptide sequence.
11. The isolated fusion protein of claim 1, wherein the fusion protein
comprises the
amino acid sequence of SEQ ID NO: 78 or 79.
12. An isolated fusion protein comprising at least one human serpin
polypeptide
operably linked to a modified human IgG4 Fc polypeptide, wherein the modified
human
IgG4 Fc polypeptide comprises an amino acid sequence selected from the group
consisting
of SEQ ID NOs: 6, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.
13. The isolated fusion protein of claim 12, wherein the modified human
IgG4 Fc
polypeptide comprises the amino acid sequence of SEQ ID NO: 60, SEQ ID NO: 69,
or
SEQ ID NO: 70.
14. The isolated fusion protein of claim 13, wherein the modified human
IgG4 Fc
polypeptide comprises one or more mutations at a position selected from M252
(residue 34
of SEQ ID NO: 60), T246 (residue 38 of SEQ ID NO: 60), M428 (residue 210 of
SEQ ID
NO: 60), and combinations thereof.

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15. The isolated fusion protein of claim 13, wherein the modified human
IgG4 Fc
polypeptide comprises a mutation at position M252 (residue 34 of SEQ ID NO:
60) and at
position M428 (residue 210 of SEQ ID NO: 60).
16. The isolated fusion protein of claim 13, wherein the fusion protein
further comprises
an additional polypeptide selected from the group consisting of:
a cytokine targeting polypeptide or a sequence derived from a cytokine
targeting
polypeptide;
a WAP domain containing polypeptide or a sequence derived from a WAP domain
containing polypeptide; or
an albumin polypeptide or an amino acid sequence that is derived from a serum
albumin polypeptide.
17. The fusion protein of claim 13, wherein the human serpin polypeptide is
a human
alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT
polypeptide.
18. The isolated fusion protein of claim 17, wherein AAT polypeptide
comprises the
amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 80.
19. The isolated fusion protein of claim 17, wherein the AAT polypeptide
comprises the
reactive site loop of AAT comprising the amino acid sequence of SEQ ID NO: 1.
20. The isolated fusion protein of claim 17, wherein the AAT polypeptide
comprises a
mutated reactive site loop of AAT comprising the amino acid sequence of SEQ ID
NO: 32
or 33.
21. The isolated fusion protein of claim 13 or claim 17, wherein the serpin
polypeptide
and the modified human IgG4 Fc polypeptide are operably linked via a hinge
region, a
linker region, or both a hinge region and linker region.
22. The isolated fusion protein of claim 21, wherein the hinge region, the
linker region
or both the hinge region and the linker region comprise a peptide sequence.
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23. A method of treating or alleviating a symptom of a disease or disorder
associated
with aberrant serine protease expression or activity in a subject in need
thereof, the method
comprising administering a fusion protein according to claim 1 or claim 12.
24. A method of treating or alleviating inflammation or a symptom of an
inflammatory
disease or disorder while reducing the risk of infection, in a subject in need
thereof, the
method comprising administering a fusion protein according to claim 1 or claim
12.
25. A method of reducing the risk of infection in a subject in need
thereof, the method
comprising administering a fusion protein according to claim 1 or claim 12.
26. The method of claim 25, wherein the subject is a human.
27. The method of claim 25, wherein the fusion protein comprises the amino
acid
sequence of SEQ ID NO: 78 or 79.
28. The method of claim 24, wherein the inflammatory disease or disorder is
selected
from the following: emphysema, chronic obstructive pulmonary disease (COPD),
acute
respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis,
cancers of the lung,
ischemia-reperfusion injury, ischemia/reperfusion injury following cardiac
transplantation,
myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic
arthritis, ankylosing
spondylitis, Crohn's disease, psoriasis, type I and/or type II diabetes,
pneumonia, sepsis,
graft versus host disease (GVHD), wound healing, Systemic lupus erythematosus,
and
Multiple sclerosis.
29. The method of claim 24, wherein the infection is selected from
bacterial infections,
fungal infections, or viral infections.
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Description

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SERPIN FUSION POLYPEPTIDES AND METHODS OF USE THEREOF
Related Applications
[0001] This application claims the benefit of U.S. Patent Application No.
14/524,832, filed October 27, 2014, the contents of which are hereby
incorporated by
reference in their entirety.
Incorporation of Sequence Listing
[0002] The contents of the text file named "INHI002002WO_SeqList.txt",
which
was created on October 26, 2015 and is 228 KB in size, are hereby incorporated
by
reference in their entirety.
Field of the Invention
[0003] This invention relates to molecules, particularly polypeptides,
more
particularly fusion proteins that include a serpin polypeptide or an amino
acid sequence that
is derived from a serpin polypeptides and a second polypeptide. Additionally,
the invention
relates to fusion proteins that include a serpin polypeptide or an amino acid
sequence that is
derived from serpin polypeptides, a second polypeptide, and a third
polypeptide.
Specifically, this invention relates to fusion proteins that include at least
one serpin
polypeptide and a second polypeptide or fusion proteins that include at least
one serpin
polypeptide, a second polypeptide, and a third polypeptide, where the second
and third
polypeptides of the fusion proteins of the invention can be at least one the
following: an Fc
polypeptide or an amino acid sequence that is derived from an Fc polypeptide;
a cytokine
targeting polypeptide or a sequence derived from a cytokine targeting
polypeptide; a WAP
domain containing polypeptide or a sequence derived from a WAP containing
polypeptide;
or an albumin polypeptide or an amino acid sequence that is derived from a
serum albumin
polypeptide. This invention also relates to methods of using such molecules in
a variety of
therapeutic and diagnostic indications, as well as methods of producing such
molecules.
Background of the Invention
[0004] Aberrant serine protease activity or an imbalance of protease-to-
protease
inhibitor can lead to protease-mediated tissue destruction and inflammatory
responses.
Accordingly, there exists a need for therapeutics and therapies that target
aberrant serine
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protease activity and/or imbalance of protease-to-protease inhibitor.
Furthermore, enhanced
therapeutic effects may be gained through the attenuation of aberrant cytokine
signaling and
serine protease activity. In addition, serpin proteins have demonstrated anti-
infective
activities while targeting inflammatory cytokines has been shown to increase
the risk of
infection. The fusion proteins of this invention have the potential to dampen
inflammatory
cytokine activity and limit the risk of infection.
Summary of the Invention
[0005] The fusion proteins described herein include at least a serpin
polypeptide or
an amino acid sequence that is derived from a serpin polypeptide (Polypeptide
1) and
second polypeptide (Polypeptide 2). Additionally, the fusion proteins
described herein
include a serpin polypeptide or an amino acid sequence that is derived from a
serpin
polypeptide (Polypeptide 1), a second polypeptide (Polypeptide 2), and a third
polypeptide
(Polypeptide 3). As used interchangeably herein, the terms "fusion protein"
and "fusion
polypeptide" refer to a serpin polypeptide or an amino acid sequence derived
from a serpin
polypeptide operably linked to at least a second polypeptide or an amino acid
sequence
derived from at least a second polypeptide. The individualized elements of the
fusion
protein can be linked in any of a variety of ways, including for example,
direct attachment,
the use of an intermediate or a spacer peptide, the use of a linker region,
the use of a hinge
region or the use of both a linker and a hinge region. In some embodiments,
the linker
region may fall within the sequence of the hinge region, or alternatively, the
hinge region
may fall within the sequence of the linker region. Preferably, the linker
region is a peptide
sequence. For example, the linker peptide includes anywhere from zero to 40
amino acids,
e.g., from zero to 35 amino acids, from zero to 30 amino acids, from zero to
25 amino acids,
or from zero to 20 amino acids. Preferably, the hinge region is a peptide
sequence. For
example, the hinge peptide includes anywhere from zero to 75 amino acids,
e.g., from zero
to 70 amino acids, from zero to 65 amino acids or from zero to 62 amino acids.
In
embodiments where the fusion protein includes both a linker region and hinge
region,
preferably each of the linker region and the hinge region is a peptide
sequence. In these
embodiments, the hinge peptide and the linker peptide together include
anywhere from zero
to 90 amino acids, e.g., from zero to 85 amino acids or from zero to 82 amino
acids.
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[0006] In some embodiments, the serpin polypeptide and the second
polypeptide can
be linked through an intermediate binding protein. In some embodiments, the
serpin-based
portion and second polypeptide portion of the fusion protein may be non-
covalently linked.
[0007] In some embodiments, fusion proteins according to the invention
can have
one of the following formulae, in an N-terminus to C-terminus direction or in
a C-terminus
to N-terminus direction:
Polypeptide 1(a) ¨ hingeni ¨ Polypeptide 2(b)
Polypeptide 1(a) ¨ linker, ¨ Polypeptide 2(b)
Polypeptide 1(a) ¨ linker, ¨ hingeni ¨ Polypeptide 2(b)
Polypeptide 1(a) ¨ hingen, ¨ linker, ¨ Polypeptide 2(b)
Polypeptide 1(a) - Polypeptide 2(b)¨ Polypeptide 3()
Polypeptide 1(a) ¨ hingeni ¨ Polypeptide 2(b)¨ hingeni ¨ Polypeptide 3()
Polypeptide 1(a) ¨ linker, ¨ Polypeptide 2(b)¨ linker, ¨ Polypeptide 3()
Polypeptide 1(a) ¨ hingeni ¨ linker, ¨ Polypeptide 2(b)¨hingeni ¨ linker, ¨
Polypeptide
3() Polypeptide 1(a) ¨ linker, ¨ hingen, ¨ Polypeptide 2(b)¨ linker, ¨
hingeni¨
Polypeptide 3()
where n is an integer from zero to 20, where m is an integer from 1 to 62 and
where
a, b, and c integers of at least 1. These embodiments include the above
formulations
and any variation or combination thereof For example, the order of
polypeptides in
the formulae also includes Polypeptide 3() ¨ Polypeptide 1(a)¨ Polypeptide
2(b),
Polypeptide 2(b) ¨ Polypeptide 3()¨ Polypeptide 1(a), or any variation or
combination
thereof
[0008] In some embodiments, the Polypeptide 1 sequence includes a serpin
polypeptide. Serpins are a group of proteins with similar structures that were
first identified
as a set of proteins able to inhibit proteases. Serpin proteins suitable for
use in the fusion
proteins provided herein include, by way of non-limiting example, alpha-1
antitrypsin
(AAT), antitrypsin-related protein (SERPINA2), alpha 1-antichymotrypsin
(SERPINA3),
kallistatin (SERPINA4), monocyte neutrophil elastase inhibitor (SERPINB1), PI-
6
(SERPINB6), antithrombin (SERPINC1), plasminogen activator inhibitor 1
(SERPINE1),
alpha 2-antiplasmin (SERPINF2), complement 1-inhibitor (SERPING1), and
neuroserpin
(SERPINI1).
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[0009] In some embodiments, the Polypeptide 1 sequence includes an alpha-
1
antitrypsin (AAT) polypeptide sequence or an amino acid sequence that is
derived from
AAT. In some embodiments, the Polypeptide 1 sequence includes a portion of the
AAT
protein. In some embodiments, the Polypeptide 1 sequence includes at least the
reactive site
loop portion of the AAT protein. In some embodiments, the reactive site loop
portion of the
AAT protein includes at least the amino acid sequence:
GTEAAGAMFLEAIPMSIPPEVKFNK SEQ ID NO:1).
[00010] In a preferred embodiment, the AAT polypeptide sequence or an
amino acid
sequence that is derived from AAT is or is derived from a human AAT
polypeptide
sequence.
[00011] In some embodiments, the fusion protein includes a full-length
human AAT
polypeptide sequence having the following amino acid sequence:
1 EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA
61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL
121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT
181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL
241 LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK
301 SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA MFLEAIPMSI
361 PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK (SEQ ID NO: 2)
[00012] In some embodiments, the fusion protein includes a human AAT
polypeptide
sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 2.
[00013] In some embodiments, the AAT polypeptide sequence is, or the amino
acid
sequence derived from an AAT polypeptide is derived from, one or more of the
human
AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1,
CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP 001002235.1,
CAA34982.1, NP 001002236.1, NP 000286.3, NP 001121179.1, NP 001121178.1,
NP 001121177.1, NP 001121176.16,NP 001121175.1, NP 001121174.1,
NP 001121172.1, and/or AAA51547.1.
[00014] In some embodiments, the fusion proteins contain one or more
mutations.
For example, the fusion protein contains at least one mutation at a methionine
(Met) residue
in the serpin portion of the fusion protein. In these Met mutations, the Met
residue can be
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substituted with any amino acid. For example, the Met residue can be
substituted with an
amino acid with a hydrophobic side chain, such as, for example, leucine (Leu,
L). Without
wishing to be bound by theory, the Met mutation(s) prevent oxidation and
subsequent
inactivation of the inhibitory activity of the fusion proteins of the
invention. In some
embodiments, the Met residue can be substituted with a charged residue, such
as, for
example, glutamate (Glu, E). In some embodiments, the Met mutation is at
position 358 of
an AAT polypeptide. For example, the Met mutation is Met358Leu (M358L). In
some
embodiments, the Met mutation is at position 351 of an AAT polypeptide. For
example, the
Met mutation is Met351Glu (M351E). In some embodiments, the Met mutation is at

position 351 and at position 358 of an AAT polypeptide, for example, the Met
mutation is
Met351Glu (M351E) and Met358Leu (M358L). For example, the reactive site loop
of this
variant of the fusion AAT polypeptide has the following sequence:
GTEAAGAEFLEAI PLS I PPEVKFNK (SEQ ID NO: 32) . In some embodiments, the Met
mutation is at position 351 and at position 358 of an AAT polypeptide, for
example, the Met
mutation is Met351Leu (M351L) and Met358Leu (M358L). For example, the reactive
site
loop of this variant of the fusion AAT polypeptide has the following sequence:

GTEAAGALFLEAI PLS I PPEVKFNK (SEQ ID NO: 33).
[00015] In some embodiments, the fusion protein includes a full-length
human AAT
polypeptide sequence containing a variant reactive site loop modified at M351
and M358,
having the following amino acid sequence:
1 EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA
61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL
121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT
181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL
241 LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK
301 SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA OFLEAIPOSI
361 PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK (SEQ ID NO: 80)
[00016] In some embodiments, the fusion protein includes a human AAT
polypeptide
sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID
NO: 80.

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[00017] In some embodiments, the second polypeptide (Polypeptide 2) of the
serpin
fusion protein is an Fc polypeptide or derived from an Fc polypeptide. These
embodiments
are referred to collectively herein as "serpin-Fc fusion proteins." The serpin-
Fc fusion
proteins described herein include at least a serpin polypeptide or an amino
acid sequence
that is derived from a serpin and an Fc polypeptide or an amino acid sequence
that is
derived from an Fc polypeptide. In some embodiments, the serpin-Fc fusion
protein
includes a single serpin polypeptide. In other embodiments, the serpin-Fc
fusion proteins
includes more than one serpin polypeptide, and these embodiments are
collectively referred
to herein as "serpin(a,)-Fc fusion protein," wherein (a') is an integer of at
least 2. In some
embodiments, each serpin polypeptides in a serpin(a,)-Fc fusion protein
includes the same
amino acid sequence. In other embodiments, each serpin polypeptide in a
serpin(a,)-Fc
fusion protein includes serpin polypeptides with distinct amino acid
sequences. The serpin
polypeptides of serpin(a,)-Fc fusion proteins can be located at any position
within the fusion
protein.
[00018] In some embodiments, the serpin polypeptide of the serpin-Fc
fusion protein
includes at least the amino acid sequence of the reactive site loop portion of
the AAT
protein. In some embodiments, the reactive site loop portion of the AAT
protein includes at
least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin

polypeptide of the serpin-Fc fusion protein includes at least the amino acid
sequence of a
variant of the reactive site loop portion of the AAT protein. In some
embodiments, the
variant of the reactive site loop portion of the AAT protein includes at least
the amino acid
sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin
polypeptide of the serpin-Fc fusion protein includes at least the full-length
human AAT
polypeptide sequence having amino acid sequence of SEQ ID NO: 2. In some
embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes
at least the
full-length human AAT polypeptide sequence having amino acid sequence of SEQ
ID NO:
80. In some embodiments the serpin polypeptide of the serpin-Fc fusion protein
includes
human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%,
85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid

sequence of SEQ ID NO: 2 or 32 or 33 or 80.
[00019] In some embodiments, the serpin polypeptide of the serpin-Fc
fusion protein
includes the AAT polypeptide sequence is or the amino acid sequence derived
from an AAT
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polypeptide is derived from one or more of the human AAT polypeptide sequences
shown
in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1,
AAA51546.1, CAA25838.1,NP 001002235.1, CAA34982.1,NP 001002236.1,
NP 000286.3, NP 001121179.1, NP 001121178.1,NP 001121177.1, NP 001121176.16,
NP 001121175.1, NP 001121174.1, NP 001121172.1, and/or AAA51547.1.
[00020] In some embodiments, the Fc polypeptide of the fusion protein is a
human Fc
polypeptide, for example, a human IgG Fc polypeptide sequence or an amino acid
sequence
that is derived from a human IgG Fc polypeptide sequence. For example, in some

embodiments, the Fc polypeptide is a human IgG1 Fc polypeptide or an amino
acid
sequence that is derived from a human IgG1 Fc polypeptide sequence. In some
embodiments, the Fc polypeptide is a human IgG2 Fc polypeptide or an amino
acid
sequence that is derived from a human IgG2 Fc polypeptide sequence. In some
embodiments, the Fc polypeptide is a human IgG3 Fc polypeptide or an amino
acid
sequence that is derived from a human IgG3 Fc polypeptide sequence. In some
embodiments, the Fc polypeptide is a human IgG4 Fc polypeptide or an amino
acid
sequence that is derived from a human IgG4 Fc polypeptide sequence. In some
embodiments, the Fc polypeptide is a human IgM Fc polypeptide or an amino acid
sequence
that is derived from a human IgM Fc polypeptide sequence.
[00021] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG1
Fc
polypeptide sequence having the following amino acid sequence:
1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK
61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT
121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL
181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 3)
[00022] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the fusion protein includes a hinge region coupled to the N-
terminus of the
Fc polypeptide of the fusion protein, where the Fc polypeptide includes a
human IgG1 Fc
polypeptide sequence having the following amino acid sequence:
1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 43)
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[00023] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG1
Fc
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 3 or 43.
[00024] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn
binding. In
these embodiments the mutated or modified Fc polypeptide includes the
following
mutations: Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) or Met428Leu
and Asn434Ser (M428L, N4345) or Met428Val and Asn434Ser (M428V, N4345) using
the
Kabat numbering system. In some embodiments, the mutated or modified Fc
polypeptide
includes one or more mutations selected from the group consisting of Met252Tyr
(M252Y),
Ser254Thr (5256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M428V),
Asn434Ser (N4345), and combinations thereof In some embodiments the Fc
polypeptide
portion is mutated or otherwise modified so as to disrupt Fc-mediated
dimerization. In
these embodiments, the fusion protein is monomeric in nature.
[00025] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide is mutated or modified. In some
embodiments, the
mutated or modified Fc polypeptide includes one or more mutations at a
position selected
from M252, T246, M428, and combinations thereof In some embodiments, the
mutated or
modified Fc polypeptide includes the following mutations: Met25211e,
Thr256Asp,
Met428Leu (M252I, T256D, M428L) using the Kabat numbering system.
[00026] In some embodiments where the fusion protein of the invention
includes a
modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion
protein
includes mutations at residues M252, T256, and M428, which correspond to
residues 22,
26, and 198 of SEQ ID NO: 3 or residues 32, 36, and 208 of SEQ ID NO: 43 shown
above,
and has the following amino acid sequence, where the mutated amino acid
residues are
boxed:
1 APELLGGPSV FLFPPKPKDT LOISRT)PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK
61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT
121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL
181 TVDKSRWQQG NVFSCSVEHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 44)
8

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[00027] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG1 Fc polypeptide, the
modified
IgG1 Fc polypeptide of the fusion protein includes mutations at residues M252,
T256, and
M428, which correspond to residues 22, 26, and 198 of SEQ ID NO: 3 or residues
32, 36,
and 208 of SEQ ID NO: 43 shown above, and the fusion protein includes at least
the
following amino acid sequence, where the mutated amino acid residues are
boxed:
1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LgISREPEVT CVVVDVSHED PEVKFNWYVD
61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
181 DGSFFLYSKL TVDKSRWQQG NVESCSVEHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 45)
[00028] In some embodiments where the fusion protein of the invention
includes a
modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion
protein
includes a modified human IgG1 Fc polypeptide sequence where residue G236,
which
corresponds to residue 6 of SEQ ID NO: 3 or residue 16 of SEQ ID NO: 43 shown
above, is
deleted and has the following amino acid sequence:
1 APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP
61 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL
121 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT
181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 46)
[00029] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG1 Fc polypeptide, the
modified
IgG1 Fc polypeptide of the fusion protein includes a modified human IgG1 Fc
polypeptide
sequence where residue G236, which corresponds to residue 6 of SEQ ID NO: 3 or
residue
16 of SEQ ID NO: 43 shown above, is deleted, and the fusion protein includes
at least the
following amino acid sequence:
1 DKTHTCPPCP APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG
61 VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD
181 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 47)
[00030] In some embodiments where the fusion protein of the invention
includes a
modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion
protein
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includes mutations at residues L234 and L235, which correspond to residues 4
and 5 of
SEQ ID NO: 3 or residues 14 and 15 of SEQ ID NO: 43 shown above, and has the
following amino acid sequence, where the mutated amino acid residues are
boxed:
1 APEVAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK
61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT
121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL
181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 48)
[00031] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG1 Fc polypeptide, the
modified
IgG1 Fc polypeptide of the fusion protein includes mutations at residues L234
and L235,
which correspond to residues 4 and 5 of SEQ ID NO: 3 or residues 14 and 15 of
SEQ ID
NO: 43 shown above, and the fusion protein includes at least the following
amino acid
sequence, where the mutated amino acid residues are boxed:
1 DKTHTCPPCP APEVAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD
61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 49)
[00032] In some embodiments where the fusion protein of the invention
includes a
modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion
protein
includes a deletion at residue G236 and mutations at residues L234 and L235,
and the
fusion protein includes at least the following amino acid sequence, where the
mutated
amino acid residues are boxed:
1 APEVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP
61 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL
121 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT
181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 50)
[00033] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG1 Fc polypeptide, the
modified
IgG1 Fc polypeptide of the fusion protein includes a deletion at residue G236
and mutations
at residues L234 and L235, and has the following amino acid sequence, where
the mutated
amino acid residues are boxed:

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1 DKTHTCPPCP APEVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG
61 VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD
181 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 51)
[00034] In some embodiments where the fusion protein of the invention
includes a
modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion
protein
includes a deletion at residue G236 and mutations at residues L234, L235,
M252, T256, and
M428, and the fusion protein includes at least the following amino acid
sequence, where the
mutated amino acid residues are boxed:
1 APEVAGPSVF LFPPKPKDTL EISREPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP
61 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL
121 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT
181 VDKSRWQQGN VFSCSVLHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 52)
_
[00035] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG1 Fc polypeptide, the
modified
IgG1 Fc polypeptide of the fusion protein includes a deletion at residue G236
and mutations
at residues L234, L235, M252, T256, and M428, and has the following amino acid

sequence, where the mutated amino acid residues are boxed:
1 DKTHTCPPCP APEVAGPSVF LFPPKPKDTL EISREIPEVTC VVVDVSHEDP EVKFNWYVDG
61 VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG
121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD
181 GSFFLYSKLT VDKSRWQQGN VFSCSVEIHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 53)
[00036] In some embodiments where the fusion protein of the invention
includes a
modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion
protein
includes a modified human IgG1 Fc polypeptide sequence that is at least 50%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52,
or 53.
[00037] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG2
Fc
polypeptide sequence having the following amino acid sequence:
1 APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP
11

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61 REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL
121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT
181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 4)
[00038] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG2
Fc
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 4.
[00039] In some embodiments where the fusion protein of the invention
includes a
modified IgG2 Fc polypeptide, the modified IgG2 Fc polypeptide of the fusion
protein
includes a modified human IgG2 Fc polypeptide sequence having the following
amino acid
sequence, where the mutated amino acid residues are boxed:
1 APPVAGPSVF LFPPKPKDTL EIStPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP
61 REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL
121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT
181 VDKSRWQQGN VFSCSVEHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 54)
[00040] In some embodiments where the fusion protein of the invention
includes a
modified IgG2 Fc polypeptide, the modified IgG2 Fc polypeptide of the fusion
protein
includes a modified human IgG2 Fc polypeptide sequence that is at least 50%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of SEQ ID NO: 54.
[00041] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG3
Fc
polypeptide sequence having the following amino acid sequence:
1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK
61 PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT
121 LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL
181 TVDKSRWQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 5)
[00042] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG3
Fc
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
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92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 5.
[00043] In some embodiments where the fusion protein of the invention
includes a
modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion
protein
includes a modified human IgG3 Fc polypeptide sequence having the following
amino acid
sequence, where the mutated amino acid residues are boxed:
1 APELLGGPSV FLFPPKPKDT LOISRT)PEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK
_
61 PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT
121 LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL
181 TVDKSRWQQG NIFSCSVEHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 55)
[00044] In some embodiments where the fusion protein of the invention
includes a
modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion
protein
includes a modified human IgG3 Fc polypeptide sequence where residue G236,
which
corresponds to residue 6 of SEQ ID NO: 5 shown above, is deleted and has the
following
amino acid sequence:
1 APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFKWYVDG VEVHNAKTKP
61 REEQYNSTFR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKTKG QPREPQVYTL
121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESSGQPENNY NTTPPMLDSD GSFFLYSKLT
181 VDKSRWQQGN IFSCSVMHEA LHNRFTQKSL SLSPGK (SEQ ID NO: 56)
[00045] In some embodiments where the fusion protein of the invention
includes a
modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion
protein
includes mutations at residues L234 and L235, which correspond to residues 4
and 5 of
SEQ ID NO: 5 shown above, and has the following amino acid sequence, where the
mutated
amino acid residues are boxed:
1 APEVAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK
61 PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT
121 LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL
181 TVDKSRWQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 57)
[00046] In some embodiments where the fusion protein of the invention
includes a
modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion
protein
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includes a deletion at residue G236 and mutations at residues L234 and L235
and has the
following amino acid sequence:
1 APEVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFKWYVDG VEVHNAKTKP
61 REEQYNSTFR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKTKG QPREPQVYTL
121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESSGQPENNY NTTPPMLDSD GSFFLYSKLT
181 VDKSRWQQGN IFSCSVMHEA LHNRFTQKSL SLSPGK (SEQ ID NO: 58)
[00047] In some embodiments where the fusion protein of the invention
includes a
modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion
protein
includes a deletion at residue G236 and mutations at residues L234, L235,
M252, T256, and
M428, and has the following amino acid sequence:
1 APEVAGPSVF LFPPKPKDTL NISRE)PEVTC VVVDVSHEDP EVQFKWYVDG VEVHNAKTKP
61 REEQYNSTFR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKTKG QPREPQVYTL
121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESSGQPENNY NTTPPMLDSD GSFFLYSKLT
181 VDKSRWQQGN IFSCSVEHEA LHNRFTQKSL SLSPGK (SEQ ID NO: 59)
[00048] In some embodiments where the fusion protein of the invention
includes a
modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion
protein
includes a modified human IgG3 Fc polypeptide sequence that is at least 50%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of SEQ ID NO: 55, 56, 57, 58, or 59.
[00049] In some embodiments, the human IgG3 Fc region is modified at amino
acid
Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g.,
Asn297Ala
(N297A). In some embodiments, the human IgG3 Fc region is modified at amino
acid 435
to extend the half-life, e.g., Arg435His (R435H). In some embodiments, the
human IgG3
Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of
Immunological Interest).
[00050] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG4
Fc
polypeptide sequence having the following amino acid sequence:
1 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK
61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT
121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL
181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 6)
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[00051] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a hinge
region coupled to
the N-terminus of the Fc polypeptide of the fusion protein, where the Fc
polypeptide
includes a human IgG4 Fc polypeptide sequence having the following amino acid
sequence:
1 ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 60)
[00052] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG4
Fc
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 6 or 60.
[00053] In some embodiments where the fusion protein of the invention
includes a
modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion
protein
includes mutations at residues M252, T256, and M428, which correspond to
residues 22,
26, and 19 of SEQ ID NO: 6 or residues 34, 38, and 210 of SEQ ID NO: 60 shown
above,
and has the following amino acid sequence, where the mutated amino acid
residues are
boxed:
1 APEFLGGPSV FLFPPKPKDT LOISRT)PEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK
_
61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT
121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL
181 TVDKSRWQEG NVFSCSVEHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 61)
[00054] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide, the
modified
IgG4 Fc polypeptide of the fusion protein includes mutations at residues M252,
T256, and
M428, which correspond to residues 22, 26, and 197 of SEQ ID NO: 6 or residues
34, 38,
and 210 of SEQ ID NO: 60 shown above, and the fusion protein includes at least
the
following amino acid sequence, where the mutated amino acid residues are
boxed:
1 ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLIIISREIPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK

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121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVE HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 62)
[00055] In some embodiments where the fusion protein of the invention
includes a
modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion
protein
includes a modified human IgG4 Fc polypeptide sequence where residue G236,
which
corresponds to residue 6 of SEQ ID NO: 6 or residue 19 of SEQ ID NO: 60 shown
above, is
deleted and has the following amino acid sequence:
1 APEFLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP
61 REEQFNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKGLPSS IEKTISKAKG QPREPQVYTL
121 PPSQEEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT
181 VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK (SEQ ID NO: 63)
[00056] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide, the
modified
IgG4 Fc polypeptide of the fusion protein includes a modified human IgG4 Fc
polypeptide
sequence where residue G236, which corresponds to residue 6 of SEQ ID NO: 6 or
residue
19 of SEQ ID NO: 60 shown above, is deleted, and the fusion protein includes
at least the
following amino acid sequence:
1 ESKYGPPCPS CPAPEFLGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV
61 DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA
121 KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD
181 SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK (SEQ ID NO: 64)
[00057] In some embodiments where the fusion protein of the invention
includes a
modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion
protein
includes a mutation at residue L235, which corresponds to residue 5 of SEQ ID
NO: 6 or
residue 17 of SEQ ID NO: 60 shown above, and has the following amino acid
sequence,
where the mutated amino acid residue is boxed:
1 APEFHGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK
61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT
121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL
181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 65)
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[00058] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide, the
modified
IgG4 Fc polypeptide of the fusion protein includes a mutation at residue L235,
which
corresponds to residue 5 of SEQ ID NO: 6 or residue 17 of SEQ ID NO: 60 shown
above,
and the fusion protein includes at least the following amino acid sequence,
where the
mutated amino acid residue is boxed:
1 ESKYGPPCPS CPAPEFOGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 66)
[00059] In some embodiments where the fusion protein of the invention
includes a
modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion
protein
includes mutations at residues L234 and L235, which correspond to residues 4
and 5 of
SEQ ID NO: 6 or residues 16 and 17 of SEQ ID NO: 60 shown above, and has the
following amino acid sequence, where the mutated amino acid residues are
boxed:
1 APEVAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK
61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT
121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL
181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 67)
[00060] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide, the
modified
IgG4 Fc polypeptide of the fusion protein includes mutations at residues L234
and L235,
which correspond to residues 4 and 5 of SEQ ID NO: 6 or residues 16 and 17 of
SEQ ID
NO: 60 shown above, and the fusion protein includes at least the following
amino acid
sequence, where the mutated amino acid residues are boxed:
1 ESKYGPPCPS CPAPEVAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 68)
[00061] In some embodiments where the fusion protein of the invention
includes a
modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion
protein
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includes a mutation at residue S228, which corresponds to residue 10 of SEQ ID
NO: 60
shown above, and has the following amino acid sequence, where the mutated
amino acid
residue is boxed:
1 ESKYGPPCP11 CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 69)
[00062] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide, the
modified
IgG4 Fc polypeptide of the fusion protein includes mutations at residues S228
and L235,
which correspond to residues 10 and 17 of SEQ ID NO: 60 shown above, and the
fusion
protein includes at least the following amino acid sequence, where the mutated
amino acid
residues are boxed:
1 ESKYGPPCPM CPAPEFOGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 70)
[00063] In some embodiments where the fusion protein of the invention
includes a
modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion
protein
includes mutations at residues L235, M252, T256, and M428, which correspond to
residues
5, 22, 26, and 197 of SEQ ID NO: 6 or residues 17, 34, 38, and 210 of SEQ ID
NO: 60
shown above, and has the following amino acid sequence, where the mutated
amino acid
residues are boxed:
1 APEFEGGPSV FLFPPKPKDT LIIISREIPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK
61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT
121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL
181 TVDKSRWQEG NVFSCSVEHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 71)
[00064] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide, the
modified
IgG4 Fc polypeptide of the fusion protein includes mutations at residues L235,
M252,
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T256, and M428, which correspond to residues 5, 22, 26, and 197 of SEQ ID NO:
6 or
residues 17, 34, 38, and 210 of SEQ ID NO: 60 shown above, and the fusion
protein
includes at least the following amino acid sequence, where the mutated amino
acid residues
are boxed:
1 ESKYGPPCPS CPAPEFOGGP SVFLFPPKPK DTLEIS4TIPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVE HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 72)
[00065] In some embodiments where the fusion protein of the invention
includes a
hinge region coupled to the N-terminus of a modified IgG4 Fc polypeptide, the
modified
IgG4 Fc polypeptide of the fusion protein includes mutations at residues S228,
L235, M252,
T256, and M428, which correspond to residues 10, 17, 34, 38, and 210 of SEQ ID
NO: 60
shown above, and the fusion protein includes at least the following amino acid
sequence,
where the mutated amino acid residues are boxed:
1 ESKYGPPCPM CPAPEFOGGP SVFLFPPKPK DTLEISRPPE VTCVVVDVSQ EDPEVQFNWY
61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVE HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 73)
[00066] In some embodiments where the fusion protein of the invention
includes a
modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion
protein
includes a modified human IgG4 Fc polypeptide sequence that is at least 50%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, or
73.
[00067] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgM
Fc
polypeptide sequence having the following amino acid sequence:
1 IAELPPKVSV FVPPRDGFFG NPRKSKLICQ ATGFSPRQIQ VSWLREGKQV GSGVTTDQVQ
61 AEAKESGPTT YKVTSTLTIK ESDWLGQSMF TCRVDHRGLT FQQNASSMCV PDQDTAIRVF
121 AIPPSFASIF LTKSTKLTCL VTDLTTYDSV TISWTRQNGE AVKTHTNISE SHPNATFSAV
181 GEASICEDDW NSGERFTCTV THTDLPSPLK QTISRPKG (SEQ ID NO: 7)
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[00068] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgM
Fc
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 7.
[00069] In some embodiments, the serpin-Fc fusion protein includes at
least the
amino acid sequence of the reactive site loop portion of the AAT protein
operably linked to
an Fc polypeptide sequence that includes or is derived from the amino acid
sequence of any
one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some
embodiments, the Fc
polypeptide includes an amino acid sequence selected from the group consisting
of 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68,
69, 70, 71, 72, and 73. In some embodiments, the Fc polypeptide includes an
amino acid
sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected
from the
group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the
reactive site
loop portion of the AAT protein includes at least the amino acid sequence of
SEQ ID NO:l.
In some embodiments, the serpin polypeptide and the Fc polypeptide are
operably linked
via a linker region, for example, a glycine-serine linker or glycine-serine
based linker. In
some embodiments, the serpin polypeptide and the Fc polypeptide are operably
linked via a
hinge region. In some embodiments, the serpin polypeptide and the Fc
polypeptide are
operably linked via a linker region and a hinge region. In other embodiments,
the serpin
polypeptide and the Fc polypeptide are directly attached.
[00070] In some embodiments, the serpin-Fc fusion protein includes at
least the
amino acid sequence of a variant of the reactive site loop portion of the AAT
protein
operably linked to an Fc polypeptide sequence that includes or is derived from
the amino
acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, or 73. In some
embodiments, the Fc polypeptide includes an amino acid sequence selected from
the group
consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63,

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64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the Fc
polypeptide
includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%,
85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and
73. In some
embodiments, the variant of the reactive site loop portion of the AAT protein
includes at
least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some
embodiments,
the serpin polypeptide and the Fc polypeptide are operably linked via a linker
region, for
example, a glycine-serine linker or glycine-serine based linker. In some
embodiments, the
serpin polypeptide and the Fc polypeptide are operably linked via a hinge
region. In some
embodiments, the serpin polypeptide and the Fc polypeptide are operably linked
via a linker
region and a hinge region. In other embodiments, the serpin polypeptide and
the Fc
polypeptide are directly attached.
[00071] In some embodiments, the serpin-Fc fusion protein includes at
least the full-
length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO:
2
operably linked to an Fc polypeptide sequence that includes or is derived from
the amino
acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, or 73. In some
embodiments, the Fc polypeptide includes an amino acid sequence selected from
the group
consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the Fc
polypeptide
includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%,
85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid
sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and
73. In some
embodiments the serpin-Fc fusion protein includes human AAT polypeptide
sequence that
is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 operably
linked to
an Fc polypeptide sequence that includes or is derived from the amino acid
sequence of any
one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some
embodiments, the Fc
polypeptide includes an amino acid sequence selected from the group consisting
of 43, 44,
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45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68,
69, 70, 71, 72, and 73. In some embodiments, the Fc polypeptide includes an
amino acid
sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected
from the
group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the
serpin
polypeptide and the Fc polypeptide are operably linked via a linker region,
for example, a
glycine-serine linker or glycine-serine based linker. In some embodiments, the
serpin
polypeptide and the Fc polypeptide are operably linked via a hinge region. In
some
embodiments, the serpin polypeptide and the Fc polypeptide are operably linked
via a linker
region and a hinge region. In other embodiments, the serpin polypeptide and
the Fc
polypeptide are directly attached.
[00072] In some embodiments, the serpin-Fc fusion protein includes at
least the full-
length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO:
80
operably linked to an Fc polypeptide sequence that includes or is derived from
the amino
acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, or 73. In some
embodiments the serpin-Fc fusion protein includes human AAT polypeptide
sequence that
is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 80 operably
linked
to an Fc polypeptide sequence that includes or is derived from the amino acid
sequence of
any one of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some
embodiments,
the serpin polypeptide and the Fc polypeptide are operably linked via a linker
region, for
example, a glycine-serine linker or glycine-serine based linker. In some
embodiments, the
serpin polypeptide and the Fc polypeptide are operably linked via a hinge
region. In some
embodiments, the serpin polypeptide and the Fc polypeptide are operably linked
via a linker
region and a hinge region. In other embodiments, the serpin polypeptide and
the Fc
polypeptide are directly attached.
[00073] In some embodiments of the fusion proteins provided herein, the
second
polypeptide (Polypeptide 2) of the serpin fusion protein is a cytokine
targeting polypeptide
or derived from a cytokine targeting polypeptide. These embodiments are
referred to
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collectively herein as "serpin-cytokine targeting polypeptide fusion
proteins." The serpin-
cytokine targeting polypeptide fusion proteins described herein include at
least a serpin
polypeptide or an amino acid sequence that is derived from a serpin
polypeptide and a
cytokine targeting polypeptide, or derivation thereof In some embodiments, the
serpin-
cytokine targeting polypeptide fusion protein includes a single serpin
polypeptide. In other
embodiments, the serpin-cytokine targeting polypeptide fusion protein includes
more than
one serpin polypeptide, and these embodiments are collectively referred to
herein as
"serpin(a,)-cytokine targeting polypeptide fusion proteins," wherein (a') is
an integer of at
least 2. In some embodiments, each serpin polypeptide in a serpin(a,)-cytokine
targeting
polypeptide fusion protein includes the same amino acid sequence. In other
embodiments,
each serpin polypeptide of a serpin(a)-cytokine targeting polypeptide fusion
protein includes
serpin polypeptides with distinct amino acid sequences.
[00074] In some embodiments, the cytokine targeting polypeptide of the
serpin-
cytokine targeting polypeptide fusion protein is a cytokine receptor or
derived from a
cytokine receptor. In a preferred embodiment, the cytokine targeting
polypeptide or an
amino acid sequence that is derived from the cytokine receptor is or is
derived from a
human cytokine receptor sequence. In other embodiments, the cytokine targeting

polypeptide is an antibody or an antibody fragment, for example an anti-
cytokine antibody
or anti-cytokine antibody fragment. In a preferred embodiment, the cytokine
targeting
polypeptide or an amino acid sequence that is derived from the antibody or
antibody
fragment is derived from a chimeric, humanized, or fully human antibody
sequence. The
term antibody fragment includes single chain, Fab fragment, a F(ab')2
fragment, a scFv, a
scAb, a dAb, a single domain heavy chain antibody, and a single domain light
chain
antibody.
[00075] In other embodiments, the cytokine targeting polypeptide binds a
cytokine
receptor and prevents binding of a cytokine to the receptor. In other
embodiments, the
cytokine targeting polypeptide is an antibody or an antibody fragment, for
example an anti-
cytokine receptor antibody or anti-cytokine receptor antibody fragment.
[00076] In some embodiments, the serpin polypeptide of the serpin-cytokine
targeting polypeptide fusion proteins includes at least the amino acid
sequence of the
reactive site loop portion of the AAT protein. In some embodiments, the
reactive site loop
portion of the AAT protein includes at least the amino acid sequence of SEQ ID
NO: 1. In
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some embodiments, the serpin polypeptide of the serpin-cytokine targeting
fusion proteins
includes at least the amino acid sequence of a variant of the reactive site
loop portion of the
AAT protein. In some embodiments, the variant of the reactive site loop
portion of the
AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ
ID
NO:33. In some embodiments, the serpin polypeptide of the serpin-cytokine
targeting
fusion protein includes or is derived from at least the full-length human AAT
polypeptide
sequence having amino acid sequence of SEQ ID NO: 2. In some embodiments, the
serpin
polypeptide of the serpin-cytokine targeting fusion protein includes or is
derived from at
least the full-length human AAT polypeptide sequence having amino acid
sequence of SEQ
ID NO: 80. In some embodiments the serpin polypeptide of the serpin-cytokine
targeting
fusion protein includes human AAT polypeptide sequence that is at least 50%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.
[00077] In some embodiments, the serpin polypeptide of the serpin-cytokine
targeting fusion protein includes an AAT polypeptide sequence or an amino acid
sequence
derived from an AAT polypeptide that is or is derived from one or more of the
human AAT
polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1,
P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1,
NP 001002236.1, NP 000286.3,NP 001121179.1,NP 001121178.1, NP 001121177.1,
NP 001121176.16,NP 001121175.1,NP 001121174.1, NP 001121172.1, and/or
AAA51547.1.
[00078] The serpin-cytokine targeting polypeptide fusion protein can
incorporate a
portion of the serpin-Fc fusion protein. For example, an antibody contains an
Fc
polypeptide. Therefore, in some embodiments where the cytokine targeting
polypeptide is a
cytokine-targeting antibody, the serpin-cytokine targeting polypeptide fusion
protein will
incorporate a portion of the serpin-Fc fusion protein. Furthermore, most
receptor fusion
proteins that are of therapeutic utility are Fc fusion proteins. Thus, in some
embodiments,
wherein the serpin-cytokine targeting polypeptide fusion protein is a serpin-
cytokine
receptor fusion protein, the serpin-cytokine targeting polypeptide fusion
protein may
incorporate an Fc polypeptide in addition to the serpin portion and the
cytokine receptor
portion.
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[00079] In some embodiments, where the serpin-cytokine targeting
polypeptide
fusion protein includes an Fc polypeptide sequence, the Fc polypeptide
sequence includes or
is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6,
7, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69,
70, 71, 72, or 73. In some embodiments where the serpin-cytokine targeting
fusion protein
includes an Fc polypeptide sequence, the Fc polypeptide sequence has at least
50%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identity to any one of the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7,
43, 44, 45, 46,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70,
71, 72, or 73. In some embodiments, the serpin polypeptide and the cytokine
targeting
polypeptide are operably linked via a linker region, for example, a glycine-
serine linker or
glycine-serine based linker. In some embodiments, the serpin polypeptide and
the cytokine
targeting polypeptide are operably linked via a hinge region. In some
embodiments, the
serpin polypeptide and the cytokine targeting polypeptide are operably linked
via a linker
region and a hinge region. In other embodiments, the serpin polypeptide and
the cytokine
targeting polypeptide are directly attached.
[00080] In some embodiments of the fusion proteins provided herein, the
second
polypeptide (Polypeptide 2) of the serpin fusion protein is a whey acidic
protein (WAP)
domain containing polypeptide, or an amino acid sequence that is derived from
a WAP
domain containing polypeptide. These embodiments are referred to collectively
herein as
"serpin-WAP domain fusion proteins." The serpin-WAP domain fusion proteins
include at
least a serpin polypeptide or at least an amino acid sequence that is derived
from a serpin, a
WAP domain-containing polypeptide or an amino acid sequence that is derived
from a
WAP domain-containing polypeptide. In some embodiments, the serpin-WAP domain
fusion protein includes a single serpin polypeptide. In other embodiments, the
serpin-WAP
targeting polypeptide fusion protein includes more than one serpin
polypeptide. These
embodiments are collectively referred to herein as "serpin(a,)-WAP domain
fusion proteins,"
wherein (a') is an integer of at least 2. In some embodiments, serpin
polypeptides of the
serpin(a,)-WAP domain fusion protein includes the same amino acid sequence. In
other
embodiments, the serpin polypeptides of the serpin(a,)-cytokine targeting
polypeptide fusion
protein, includes serpin polypeptides with distinct amino acid sequences.

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[00081] These serpin-WAP domain fusion proteins include a WAP domain
containing polypeptide or polypeptide sequence that is or is derived from a
WAP domain
containing polypeptide. The WAP domain is an evolutionarily conserved sequence
motif of
50 amino acids containing eight cysteines found in a characteristic 4-
disulfide core
arrangement (also called a four-disulfide core motif). The WAP domain sequence
motif is a
functional motif characterized by serine protease inhibition activity in a
number of proteins.
[00082] WAP domain-containing polypeptides suitable for use in the fusion
proteins
provided herein include, by way of non-limiting example, secretory leukocyte
protease
inhibitor (SLPI), Elafin, and Eppin.
[00083] In some embodiments, the WAP domain-containing polypeptide
sequence of
the fusion protein includes a secretory leukocyte protease inhibitor (SLPI)
polypeptide
sequence or an amino acid sequence that is derived from SLPI. These
embodiments are
referred to herein as "serpin-SLPI-derived fusion proteins." In some
embodiments, the
SLPI polypeptide sequence comprises a portion of the SLPI protein, such as for
example,
the WAP2 domain or a sub-portion thereof In a preferred embodiment, the SLPI
polypeptide sequence or an amino acid sequence that is derived from SLPI is or
is derived
from a human SLPI polypeptide sequence.
[00084] In some embodiments of the serpin-SLPI fusion proteins of the
invention, the
SLPI sequence or a SLPI-derived sequence of the fusion protein includes a full-
length
human SLPI polypeptide sequence having the following amino acid sequence:
1 MKSSGLFPFL VLLALGTLAP WAVEGSGKSF KAGVCPPKKS AQCLRYKKPE CQSDWQCPGK
61 KRCCPDTCGI KCLDPVDTPN PTRRKPGKCP VTYGQCLMLN PPNFCEMDGQ CKRDLKCCMG
121 MCGKSCVSPV KA (SEQ ID NO:8)
[00085] In some embodiments of the serpin¨SLPI fusion protein of the
invention, the
SLPI sequence or a SLPI-derived sequence of the fusion protein includes a
human SLPI
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 8.
[00086] In some embodiments of the serpin¨SLPI fusion protein of the
invention, the
SLPI sequence or a SLPI-derived sequence of the fusion protein includes a
portion of the
full-length human SLPI polypeptide sequence, where the portion has the
following amino
acid sequence:
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1 SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP DTCGIKCLDP VDTPNPTRRK
61 PGKCPVTYGQ CLMLNPPNFC EMDGQCKRDL KCCMGMCGKS CVSPVKA (SEQ ID NO: 9)
[00087] In some embodiments of the serpin¨SLPI fusion protein of the
invention, the
SLPI sequence or a SLPI-derived sequence of the fusion protein includes a
human SLPI
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 9.
[00088] In some embodiments of the serpin¨SLPI fusion protein of the
invention, the
SLPI sequence or a SLPI-derived sequence of the fusion protein includes the
WAP2 domain
of the full-length human SLPI polypeptide sequence, where the WAP2 domain has
the
following amino acid sequence:
1 TRRKPGKCPV TYGQCLMLNP PNFCEMDGQC KRDLKCCMGM CGKSCVSPVK A (SEQ ID
NO: 10)
[00089] In some embodiments of the serpin¨SLPI fusion protein of the
invention, the
SLPI sequence or a SLPI-derived sequence of the fusion protein includes a
human SLPI
polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence
of SEQ
ID NO: 10.
[00090] In some embodiments of the serpin¨SLPI fusion proteins of the
invention,
the SLPI polypeptide sequence or the amino acid sequence derived from an SLPI
polypeptide is or is derived from, one or more of the human SLPI polypeptide
sequences
shown in GenBank Accession Nos. CAA28187.1, NP_003055.1, EAW75869.1, P03973.2,

AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1, and/or BAG35125.1.
[00091] In some embodiments of the serpin¨SLPI fusion proteins of the
invention,
the SLPI polypeptide sequence or a SLPI-derived sequence of the fusion protein
includes a
human SLPI polypeptide sequence that is modified at a Methionine (Met)
residue. In these
Met mutations, the Met residue can be substituted with any amino acid. For
example, the
Met residue can be substituted with an amino acid with a hydrophobic side
chain, such as,
for example, leucine (Leu, L) or valine (Val, V). Without wishing to be bound
by theory,
the Met mutation(s) prevent oxidation and subsequent inactivation of the
inhibitory activity
of the fusion proteins of the invention. In some embodiments, the Met mutation
is at
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position 98 of an SLPI polypeptide. For example, the modified SLPI polypeptide
sequence
of the serpin-SLPI includes mutations M98L or M98V in SEQ ID NO: 8.
[00092] In other embodiments, the WAP domain-containing polypeptide
sequence of
the fusion protein includes an elafin polypeptide sequence or an amino acid
sequence that is
derived from elafin. These embodiments are referred to herein as
"serpin¨elafin fusion
proteins. In some embodiments, the elafin polypeptide sequence includes a
portion of the
elafin protein, such as for example, the WAP domain or a sub-portion thereof
In a
preferred embodiment, the elafin polypeptide sequence or an amino acid
sequence that is
derived from elafin is or is derived from a human elafin polypeptide sequence.
[00093] In some embodiments of the serpin¨elafin fusion proteins, the
fusion protein
includes a full-length human elafin polypeptide sequence having the following
amino acid
sequence:
1 MRASSFLIVV VFLIAGTLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ VSVKGQDKVK
61 AQEPVKGPVS TKPGSCPIIL IRCAMLNPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ
(SEQ ID NO: 11)
[00094] In some embodiments of the serpin¨elafin fusion proteins, the
fusion protein
includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%,
70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequence of SEQ ID NO: 11.
[00095] In some embodiments of the serpin¨elafin fusion proteins, the
fusion protein
includes a portion of the full-length human elafin polypeptide sequence, where
the portion
has the following amino acid sequence:
1 AVTGVPVKGQ DTVKGRVPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK PGSCPIILIR
61 CAMLNPPNRC LKDTDCPGIK KCCEGSCGMA CFVPQ (SEQ ID NO: 12)
[00096] In some embodiments of the serpin¨elafin fusion proteins, the
fusion protein
includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%,
70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequence of SEQ ID NO: 12.
[00097] In some embodiments of the serpin¨elafin fusion proteins, the
fusion protein
includes the WAP domain of the full-length human elafin polypeptide sequence,
where the
WAP domain has the following amino acid sequence:
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1 VSTKPGSCPI ILIRCAMLNP PNRCLKDTDC PGIKKCCEGS CGMACFVPQ (SEQ ID NO: 13)
[00098] In some embodiments of the serpin¨elafin fusion proteins, the
fusion protein
includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%,
70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the
amino acid sequence of SEQ ID NO: 13.
[00099] In some embodiments of the serpin¨elafin fusion proteins, the
elafin
polypeptide sequence or the amino acid sequence derived from an elafin
polypeptide is
derived from one or more of the human elafin polypeptide sequences shown in
GenBank
Accession Nos. P19957.3, NP 002629.1, BAA02441.1, EAW75814.1, EAW75813.1,
Q8IUB2.1, and/or NP 542181.1.
[000100] In other embodiments, the WAP domain-containing polypeptide
sequence of
the fusion protein includes an Eppin polypeptide sequence or an amino acid
sequence that is
derived from Eppin. These embodiments are referred to herein as
"serpinoi¨Eppin fusion
proteins. In some embodiments, the Eppin polypeptide sequence of the
serpin¨Eppin fusion
protein includes a portion of the Eppin protein, such as for example, the WAP
domain or a
sub-portion thereof In a preferred embodiment, the Eppin polypeptide sequence
or an
amino acid sequence that is derived from Eppin is or is derived from a human
Eppin
polypeptide sequence.
[000101] In some embodiments of the serpin¨Eppin fusion proteins, the Eppin
polypeptide sequence or amino acid sequence derived from an Eppin polypeptide
is or is
derived from one or more of the human Eppin polypeptide sequences shown in
GenBank
Accession Nos. 095925.1, NP 065131.1, AAH44829.2, AAH53369.1, AAG00548.1,
AAG00547.1, and/or AAG00546.1.
[000102] In some embodiments, the serpin polypeptide of the serpin-WAP
domain
fusion protein includes at least the amino acid sequence of the reactive site
loop portion of
the AAT protein. In some embodiments, the reactive site loop portion of the
AAT protein
includes at least the amino acid sequence of SEQ ID NO: 1. In some
embodiments, the
serpin polypeptide of the serpin-WAP fusion protein includes at least the
amino acid
sequence of a variant of the reactive site loop portion of the AAT protein. In
some
embodiments, the variant of the reactive site loop portion of the AAT protein
includes at
least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some
embodiments,
the serpin polypeptide of the serpin-WAP domain fusion protein includes at
least the full-
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length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO:
2.
In some embodiments, the serpin polypeptide of the serpin-cytokine targeting
fusion protein
includes or is derived from at least the full-length human AAT polypeptide
sequence having
amino acid sequence of SEQ ID NO: 80. In some embodiments the serpin
polypeptide of
the serpin-WAP domain fusion protein includes human AAT polypeptide sequence
that is at
least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or
33 or 80.
[000103] In some embodiments, the serpin polypeptide of the serpin-WAP
domain
fusion protein includes the AAT polypeptide sequence is, or the amino acid
sequence
derived from an AAT polypeptide is derived from, one or more of the human AAT
polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1,
P01009.3, AAB59375.1, AAA51546.1, CAA25838.1,NP_001002235.1, CAA34982.1,
NP 001002236.1, NP 000286.3,NP 001121179.1,NP 001121178.1, NP 001121177.1,
NP 001121176.16,NP 001121175.1,NP 001121174.1, NP 001121172.1, and/or
AAA51547.1.
[000104] In some embodiments, the serpin-WAP domain fusion protein can also
include an Fc polypeptide or an amino acid sequence that is derived from an Fc
polypeptide.
These embodiments are referred to collectively herein as "serpin-Fc-WAP domain
fusion
proteins." In these embodiments, no particular order is to be construed by
this terminology.
For example, the order of the fusion protein can be serpin-Fc-WAP domain,
serpin-WAP
domain-Fc, or any variation combination thereof The serpin-Fc-WAP domain
fusion
proteins described herein include at least a serpin polypeptide or an amino
acid sequence
that is derived from a serpin, WAP domain-containing polypeptide or an amino
acid
sequence that is derived from a WAP domain-containing polypeptide, and an
Fc polypeptide or an amino acid sequence that is derived from an Fc
polypeptide.
[000105] In some embodiments, where the serpin-WAP domain fusion protein
includes an Fc polypeptide sequence, the Fc polypeptide sequence can have the
amino acid
sequence of SEQ ID NO: 3-7 and 43-73. In other embodiments, where the serpin-
WAP
domain fusion protein includes an Fc polypeptide sequence, the Fc polypeptide
sequence
can have at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NOs. 3-7
and 43-
73. In some embodiments, the serpin-WAP domain fusion protein can also include
an

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albumin polypeptide, or an amino acid sequence that is derived from an albumin

polypeptide. These embodiments are referred to collectively herein as "serpin-
albumin-
WAP domain fusion proteins." In these embodiments, no particular order is to
be construed
by this terminology. For example, the order of the fusion protein can be
serpin-albumin-
WAP domain, serpin-WAP domain-albumin, or any variation combination thereof
The
serpin-albumin-WAP domain fusion proteins described herein include at least a
serpin
polypeptide or an amino acid sequence that is derived from a serpin, WAP
domain-
containing polypeptide, or an amino acid sequence that is derived from a WAP
domain-
containing polypeptide, and an albumin polypeptide, or an amino acid sequence
that is
derived from an albumin polypeptide.
[000106] In some embodiments where the serpin-WAP domain fusion protein
includes
an albumin polypeptide sequence, the albumin polypeptide sequence includes the
amino
acid sequence of SEQ ID NO: 14-15, described herein. In other embodiments,
where the
serpin-WAP domain fusion protein includes an albumin polypeptide sequence, the
albumin
polypeptide sequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the any one of the amino acid
sequences having SEQ ID NO: 14 or 15.
[000107] In some embodiments, the second polypeptide (Polypeptide 2) of the
serpin
fusion protein is an albumin polypeptide or is derived from an albumin
polypeptide. These
embodiments are referred to collectively herein as "serpinoi-albumin fusion
proteins." The
serpin-albumin fusion proteins described herein include at least a serpin
polypeptide or an
amino acid sequence that is derived from a serpin and an albumin polypeptide
or an amino
acid sequence that is derived from an albumin polypeptide. In addition this
invention relates
to serpin albumin binding polypeptide fusion proteins, wherein the albumin is
operably
linked to the serpin via an intermediate binding molecule. Herein, the serpin
is non-
covalently or covalently bound to human serum albumin.
[000108] In embodiments where the fusion protein of the invention includes
an
albumin polypeptide sequence, the albumin polypeptide sequence of the fusion
protein is a
human serum albumin (HSA) polypeptide or an amino acid sequence derived from
HSA. In
some embodiments, the fusion protein includes a HSA polypeptide sequence
having the
following amino acid sequence:
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DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQC PFEDHVKLVNEVTEFAKTCVADESAEN
CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV
MC TAFHDNEET FLKKYLYE I ARRHPY FYAPELLF FAKRYKAAFTECCQAADKAACLLPKLD
ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVT DLTKVHTE
CCHGDLLECADDRADLAKY I CENQDS I SSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL
AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP
HECYAKVFDEFKPLVEEPQNL IKQNCELFEQLGEYKFQNALLVRYTKKVPQVS TPTLVEVS
RNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS DRVTKCCTESLVNRRPCF
SALEVDE TYVPKE FNAET FT FHAD I C TLSEKERQ I KKQTALVE LVKHKPKATKEQLKAVMD
DFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL ( SEQ ID NO: 14)
[000109] In embodiments where the fusion protein of the invention includes
an
albumin polypeptide sequence, the albumin polypeptide sequence of the fusion
protein
includes a human serum albumin polypeptide sequence that is at least 50%, 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of SEQ ID NO: 14.
[000110] In embodiments where the fusion protein of the invention includes
an
albumin polypeptide sequence, the albumin polypeptide sequence of the fusion
protein
fusion protein includes a domain 3 of human serum albumin polypeptide sequence
having
the following amino acid sequence:
EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE
AKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFN
AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK
ETCFAEEGKKLVA (SEQ ID NO: 15)
[000111] In embodiments where the fusion protein of the invention includes
an
albumin polypeptide sequence, the albumin polypeptide sequence of the fusion
protein
includes a human serum albumin polypeptide sequence that is at least 50%, 60%,
65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
identical
to the amino acid sequence of SEQ ID NO: 15.
[000112] In some embodiments where the fusion protein of the invention
includes an
albumin polypeptide sequence, the fusion protein is linked to the human serum
albumin via
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an intermediate albumin binding polypeptide. The albumin binding polypeptide
can be an
antibody or an antibody fragment or derived from an antibody or antibody
fragment. In a
preferred embodiment, the albumin binding polypeptide or an amino acid
sequence that is
derived from the antibody or antibody fragment is derived from a chimeric,
humanized, or
fully human antibody sequence. The term antibody fragment includes single
chain, Fab
fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy
chain antibody,
and a single domain light chain antibody. In addition, the albumin binding
polypeptide can
be an albumin binding peptide. Another embodiment of the invention is a serpin
albumin
binding polypeptide fusion, wherein the albumin binding polypeptide is domain
3 of
Streptococcal protein G or a sequence derived from domain 3 of Streptococcal
protein G.
[000113] In some embodiments, the serpin polypeptide of the serpin(a,)-
albumin fusion
proteins includes at least the amino acid sequence of the reactive site loop
portion of the
AAT protein. In some embodiments, the reactive site loop portion of the AAT
protein
includes at least the amino acid sequence of SEQ ID NO: 1. In some
embodiments, the
serpin polypeptide of the serpin-albumin fusion protein includes at least the
amino acid
sequence of a variant of the reactive site loop portion of the AAT protein. In
some
embodiments, the variant of the reactive site loop portion of the AAT protein
includes at
least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some
embodiments,
the serpin polypeptide of the serpin-albumin fusion proteins includes at least
the full-length
human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2. In
some
embodiments, the serpin polypeptide of the serpin-cytokine targeting fusion
protein includes
or is derived from at least the full-length human AAT polypeptide sequence
having amino
acid sequence of SEQ ID NO: 80. In some embodiments the serpin polypeptide of
the
serpin-albumin fusion proteins includes human AAT polypeptide sequence that is
at least
50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.
[000114] In some embodiments, the serpin polypeptide of the serpin-albumin
fusion
proteins includes the AAT polypeptide sequence or the amino acid sequence
derived from
an AAT polypeptide is or is derived from one or more of the human AAT
polypeptide
sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3,
AAB59375.1, AAA51546.1, CAA25838.1, NP 001002235.1, CAA34982.1,
NP 001002236.1, NP 000286.3,NP 001121179.1,NP 001121178.1, NP 001121177.1,
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NP 001121176.16,NP 001121175.1,NP 001121174.1, NP 001121172.1, and/or
AAA51547.1.
[000115] In some embodiments, the fusion proteins are modified to increase
or
otherwise inhibit proteolytic cleavage, for example, by mutating one or more
proteolytic
cleavage sites. In some embodiments, the fusion proteins are modified to alter
or otherwise
modulate an Fc effector function of the fusion protein, while simultaneously
retaining
binding and inhibitory function as compared to an unaltered fusion protein. Fc
effector
functions include, by way of non-limiting examples, Fc receptor binding,
prevention of
proinflammatory mediator release upon binding to the Fc receptor,
phagocytosis, modified
antibody-dependent cell-mediated cytotoxicity (ADCC), modified complement-
dependent
cytotoxicity (CDC), modified glycosylation at Asn297 residue (EU index of
Kabat
numbering, Kabat et al 1991 Sequences of Proteins of Immunological Interest)
of the Fc
polypeptide. In some embodiments, the fusion proteins are mutated or otherwise
modified
to influence Fc receptor binding. In some embodiments, the Fc polypeptide is
modified to
enhance FcRn binding. Examples of Fc polypeptide mutations that enhance
binding to FcRn
are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) (Kabat numbering,
Dall'Acqua et al 2006, J. Biol Chem Vol 281(33) 23514-23524), or Met428Leu and

Asn434Ser (M428L, N4345) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-
159).
(EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest)
or
Met428Val and Asn434Ser (M428V, N4345) using the Kabat numbering system. In
some
embodiments, the mutated or modified Fc polypeptide includes one or more
mutations
selected from the group consisting of Met252Tyr (M252Y), Ser254Thr (5256T),
Thr256Glu
(T256E), Met428Leu (M428L), Met428Val (M428V), Asn434Ser (N4345), and
combinations thereof In some embodiments the Fc polypeptide portion is mutated
or
otherwise modified so as to disrupt Fc-mediated dimerization (Ying et al 2012
J. Biol Chem
287(23): 19399-19408). In these embodiments, the fusion protein is monomeric
in nature.
[000116] The fusion proteins and variants thereof provided herein exhibit
inhibitory
activity, for example by inhibiting a serine protease such as human neutrophil
elastase (NE),
a chemotrypsin-fold serine protease that is secreted by neutrophils during an
inflammatory
response. The fusion proteins provided herein completely or partially reduce
or otherwise
modulate serine protease expression or activity upon binding to, or otherwise
interacting
with, a serine protease, e.g., a human serine protease. The reduction or
modulation of a
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biological function of a serine protease is complete or partial upon
interaction between the
fusion proteins and the human serine protease protein, polypeptide and/or
peptide. The
fusion proteins are considered to completely inhibit serine protease
expression or activity
when the level of serine protease expression or activity in the presence of
the fusion protein
is decreased by at least 95%, e.g., by 96%, 97%, 9no ,/o ,
76 99% or 100%
as compared to the
level of serine protease expression or activity in the absence of interaction,
e.g., binding,
with a fusion protein described herein. The fusion proteins are considered to
partially
inhibit serine protease expression or activity when the level of serine
protease expression or
activity in the presence of the fusion protein is decreased by less than 95%,
e.g., 10%, 20%,
25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of
serine
protease expression or activity in the absence of interaction, e.g., binding,
with a fusion
protein described herein.
[000117] The fusion
proteins described herein are useful in a variety of therapeutic,
diagnostic and prophylactic indications. For example, the fusion proteins are
useful in
treating a variety of diseases and disorders in a subject. In some
embodiments, the serpin
fusion proteins, including, fusion proteins described herein, are useful in
treating, alleviating
a symptom of, ameliorating and/or delaying the progression of a disease or
disorder in a
subject suffering from or identified as being at risk for a disease or
disorder selected from
alpha-l-antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary
disease
(COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic
fibrosis,
cancers of the lung, ischemia-reperfusion injury, including, e.g.,
ischemia/reperfusion injury
following cardiac transplantation, myocardial infarction, rheumatoid
arthritis, septic
arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,
psoriasis, type I and/or
type II diabetes, bacterial infections, fungal infections, viral infections,
pneumonia, sepsis,
graft versus host disease (GVHD), wound healing, Systemic lupus erythematosus,
and
Multiple sclerosis.
[000118]
Pharmaceutical compositions according to the invention include a fusion
protein of the invention, including modified fusion proteins and other
variants, along with a
suitable carrier. These pharmaceutical compositions can be included in kits,
such as, for
example, diagnostic kits.

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Brief Description of the Drawings
[000119] Figure lA is a schematic representation of some embodiments of
serpin-Fc
fusion proteins according to the invention. The serpin can be located at any
position within
the fusion protein. Serpin-Fc fusion protein incorporating more than one
serpin polypeptide
are also represented. Figure 1B is a photograph of a SDS-PAGE gel showing
serum derived
AAT (lane 1), AAT-Fcl (lane 2, human IgG1 Fc), and AAT-EL-Fcl (lane 3,
Met351G1u,
Met358Leu mutations within AAT, human IgG1 Fc). Figure 1C is a graph showing
the
inhibition of neutrophil elastase activity by AAT-Fc fusion proteins. Figure
1D is a
photograph of a SDS-PAGE gel showing tetravalent AAT-Fc-AAT, having two AAT
polypeptides per Fc polypeptide. Figure lE is a graph showing the inhibition
of neutrophil
elastase activity by a tetravalent AAT-Fc-AAT fusion protein. Figure 1F is a
graphing
demonstrating the effect of low pH elution from protein A resin, wherein the
NE inhibiting
capacity of the AAT-Fc fusion protein eluted at low pH is drastically reduced.
Figure 1G is
a graph showing that the double mutant, AAT-EL-Fc (Met351G1u, Met358Leu
mutations)
is resistant to H202 inactivation (conc.), compared to wild type AAT and the
single mutant
AAT-EM-Fc (Met351G1u). Figure 1H is a graph depicting the serum clearance
rates of
serum derived AAT (sdAAT) compared to AAT-Fc in rats dosed with 10mg/kg
protein (3
rats/test protein). The half-life of AAT-Fc is substantially longer than that
of sdAAT.
[000120] Figure 2A is a schematic representation of some embodiments of the
serpin-
cytokine targeting fusion proteins of the invention. The serpin can be fused
to either the
heavy chain, the light chain, or both of an antibody. Serpin-cytokine receptor
fusion
proteins are also depicted. Figure 2B is a photograph of a SDS-PAGE gel
showing the
D2E7 antibody (lane 1), and the D2E7 antibody with-AAT fused to heavy chain
(lane 2).
Figure 2C is a graph showing the inhibition of neutrophil elastase activity by
a D2E7
antibody fused to AAT. Serum derived AAT is shown as a positive control,
whereas the
D2E7 antibody alone is shown as a negative control for NE inhibition.
[000121] Figure 3A is a schematic representation of some embodiments of the
serpin-
Fc-WAP fusion proteins. Figure 3B is a photograph of a SDS-PAGE gel showing
AAT-Fc-
ELAFIN (lane 1) and AAT-Fc-SLPI (lane 2). Figure 3C is a graph showing the
inhibition
of neutrophil elastase activity by an AAT-Fc-ELAFIN fusion protein and an AAT-
Fc-SLPI
fusion protein. An AAT-Fc fusion protein and serum derived AAT are included
for
comparison.
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[000122] Figure 4A is a schematic representation of some embodiments of the
AAT-
HSA fusion proteins. Figure 4B is a photograph of a SDS-PAGE gel showing an
AAT-
HSA fusion. Figure 4C is a graph showing the inhibition of neutrophil elastase
activity by
an AAT-HSA compared to serum derived AAT.
Detailed Description of the Invention
[000123] Human neutrophil elastase (NE) is a chymotrypsin-fold serine
protease,
secreted by neutrophils during inflammation. Aberrant activity of NE results
in a
progressive degradation of elastin tissues and the slow destruction of the
alveolar structures
of the lungs leading to emphysema and lung fibrosis (Lungarella et al 2008
Int. J. Biochem
Cell Biol 40:1287). Often, misguided NE activity is due to an imbalance of the
protease
with its natural inhibitor, alphal-antitrypsin (AAT). This imbalance can
result from
enhanced neutrophil infiltration into the lungs, as observed in the lungs of
smokers and
patients with Cystic Fibrosis (CF), or Acute Respiratory Distress Syndrome
(ARDS).
Conversely, a deficiency of AAT, usually as a result of a point mutation that
causes ATT to
aggregate and accumulate in the liver, leaves the lungs exposed to unchecked
NE activity.
Individuals with AAT deficiencies are at increased the risk of emphysema,
COPD, liver
disease, and numerous other conditions.
[000124] AAT deficiency affects approximately 100,000 Americans (according
to
estimates from the Alpha-1 Foundation), and many of the afflicted people die
in their 30's
and 40's. There are currently only a few FDA-approved drugs for treatment of
ATT
deficiency (ProlastinO, AralastTM, Zemaira0, GlassiaTm). Each drug is the
natural AAT
derived from pooled human plasma, which appears to be insufficient to meet the
anticipated
clinical demand. Furthermore, these products have short serum half-lives (T112
of
approximately 5 days) and require high dose (60 mg/kg body weight) weekly
infusions. The
current market for these drugs is estimated at approximately $400 million. The
market for
AAT-like drugs is likely substantially larger, based on the estimation that as
many as 95%
of individuals with AAT-deficiencies go undiagnosed, and the fact that these
drugs have the
potential to be effective therapies for pathologies characterized by enhanced
NE activity in
individuals that are not AAT-deficient (e.g., cystic fibrosis (CF), acute
respiratory distress
syndrome (ARDS), smoking-induced emphysema and/or COPD).
[000125] AAT has been suggested to have broad spectrum anti-inflammatory
activity
(Tilg et al 1993 JEpMed 178:1629-1636, Libert et al 1996 Immunoi 157:5126-
5129, Pott
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et al, Journal of Leukocyte Biology 85 2009, Janciauskiene et al 2007 J. Biol
Chem
282(12): 8573-8582, Nita et al 2007 Int J Biochem Cell Biol 39:1165-1176).
Recently,
evidence has mounted that AAT may be useful in treating numerous human
pathologies,
outside of the commonly suggested inflammatory pulmonary conditions. Human AAT
has
shown to protect mice from clinical and histopathological signs of
experimental
autoimmune encephalomyelitis (EAE), suggesting it could be a potential
treatment of
autoimmune diseases, such as multiple sclerosis or systemic lupus
erythematosus (SLE)
(Subramanian et al 2011 Metab Brain Dis 26:107-113). Serum AAT has shown
activity in
rodent models of Graft Versus Host Disease (GVHD) (Tawara et al 2011 Proc.
Natl. Acad.
Sci. USA 109: 564-569, Marcondes et al 2011 Blood Nov 3;118(18):5031-9), which
has led
to a human clinical trial using AAT to treat individuals with Steroid Non-
responsive Acute
GVHD (NCT01523821). Additionally, AAT has been effective in animal models of
type I
and type II diabetes, dampening inflammation, protecting islet cells from
apoptosis and
enabling durable islet cell allograft (Zhang et al 2007 Diabetes 56:1316--
1323, Lewis et al
2005 Proc Nati Acad Sci USA 102:12153-12158, Lewis et al 2008 Proc. Natl Acad
Sci
USA 105:16236-16241, Kalis et al 2010 Islets 2:185-189). Currently, there are
numerous
early human clinical trials of type I diabetes using serum derived AAT
products
(NCT01183468, NCT01319331, NCT01304537).
[000126] The current serum-derived AAT products undergo extensive
purification and
testing to ensure the removal of pathogenic viruses, however, the risk of
transmission of
infectious agents cannot be completely eliminated. Moreover, serum is limited,
which
limits the production capacity of serum derived AAT. Attempts to address the
concerns of
serum derived products and production issues have been aimed at the expression
of
recombinant AAT. However, after 20 years of work, the generation of a
therapeutically
viable recombinant AAT has yet to reach the market (Karnaukhova et al 2006
Amino Acids
30: 317). Like the plasma-derived products, recombinant versions of AAT suffer
from short
serum half-lives, low production yields, and poor lung distribution.
[000127] The fusion proteins of the present invention have enhanced
functionalities
compared to the unmodified AAT molecule. The fusion of an AAT polypeptide with
a
second polypeptide that interacts with the neonatal Fc receptor (FcRn), serves
to increase
the serum half-life, providing a much needed dosing benefit for patients.
These FcRn
interacting polypeptides of the fusion protein include immunoglobulin (Ig) Fc
polypeptides
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derived from human IgGl, IgG2, IgG3, IgG4, or IgM, and derivatives of human
albumin.
In some embodiments, the fusion protein incorporates mutations with the AAT
portion that
render the molecule more resistant to inactivation by oxidation. For example
Met351G1u,
Met358Leu (AAT-EL-Fc), demonstrates resistance inactivation by H202 oxidation
(Figure
1G). While AAT is a natural anti-inflammatory protein, some embodiments of the
invention provide enhanced inflammation dampening capacity through the fusion
of an
AAT polypeptide and a cytokine targeting polypeptide. The coupling of dual
anti-
inflammatory functionalities from AAT and a second polypeptide, will provide
more a
potent therapeutic protein than either polypeptide on their own. Additionally,
the coupling
the anti-infective activity of AAT will mitigate the infection risk of most
cytokine targeting
biologics. Some embodiments provide for more potent anti-inflammatory and anti-
infective
proteins through the fusion an AAT-polypeptide and WAP domain contain
polypeptide.
The fusion proteins of the present invention are expected to be a great
therapeutic utility and
be superior to the current serum derived AAT products.
[000128] To extend the half-life of recombinant AAT, recombinant DNA
technology
was used to create a AAT gene fusion with the Fc domain of human IgGl, IgG2,
IgG3,
IgG4, IgM, or HSA, such that the expected protein product would be AAT
followed by an
Fc domain ((AAT-Fc (IgGl), AAT-Fc (IgG2), AAT-Fc (IgG3), AAT-Fc (IgG4), AAT-Fc

(IgM)) or AAT followed by HSA. While it was known that fusion of Fc domains of
HSA
to some proteins, protein domains or peptides could extend their half-lives
(see e.g.,
Jazayeri et al. BioDrugs 22, 11-26, Huang et al. (2009) Curr Opin Biotechnol
20, 692-699,
Kontermann et al. (2009) BioDrugs 23, 93-109, Schmidt et al. (2009) Curr Opin
Drug
Discov Devel 12, 284-295), it was unknown if an Fc domain or HSA fused to AAT
would
allow for proper folding and maintenance of NE inhibitory activity, or could
extend the
half-life of recombinant AAT. The fusion proteins of the present invention are
shown to be
potent inhibitors of NE, have extended serum half-lives, and in some
embodiments resistant
to oxidation. In other embodiments, the fusion proteins described herein have
distinct
properties by the incorporation of other functional polypeptides, including
cytokine
targeting polypeptides, and WAP domain containing polypeptides.
[000129] Neutrophils, the primary source of neutrophil elastase (NE), often
undergo
an oxidative burst simultaneously with secretion of NE. Therefore, it is of
great therapeutic
utility for the fusion proteins of the present invention to be active in an
oxidizing
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environment. Oxidation of AAT within the reactive site loop at Met351 and or
Met358
dampens the ability of AAT to inhibit neutrophil elastase. As shown in Figure
1G,
oxidative inactivation can be reduced through mutations Met351Glu and
Met358Leu
(M351E/M358L) shown in SEQ ID NO:32. Furthermore, oxidation of an Fc region at

Met252 and Met428 has been shown to reduce FcRn binding and subsequently
reduce the
serum half-life of the Fc containing protein. Mutation of Met252 and Met428
reduces
oxidation of the Fc region. The present invention discloses and optimized AAT-
Fc fusion
protein, wherein it is resistant to oxidation-inactivation within the AAT
portion of the fusion
protein through mutations at M351 and M358; and oxidative disruption of the
FcRn
interaction through mutations at M252 and M428 and has an extended half-life
through the
set mutation of M252I, T256D, and M428L.
[000130] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn
binding. In
these embodiments the mutated or modified Fc polypeptide includes the
following
mutations: Met25211e, Thr256Asp and Met428Leu (M252I, T256D, M428L) using the
Kabat numbering system.
[000131] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide is a modified IgG1 Fc polypeptide, and the
fusion
protein includes at least the amino acid sequence of SEQ ID NO: 53.
[000132] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide is a modified IgG1 Fc polypeptide, and the
fusion
protein includes at least the amino acid sequence of SEQ ID NO: 73.
[000133] In some embodiments where the fusion protein of the invention
includes an
Fc polypeptide, the Fc polypeptide is mutated or modified to reduce binding to
Fc-gamma
receptors (FcgRs). In some embodiments, reduced FcgRs binding can be achieved
by
modification of Fc glycosylation at Asn297. For example, mutation of Asn297Ala
(N297A)
or Asn297Gln (N297Q). In some embodiments, reduced FcgRs binding is achieved
by
modification of the lower hinge region of Fc. In some embodiments, the Fc
polypeptide is
derived from human IgGl. In some of these embodiments, lower hinge region is
modified
to mimic that of IgG2, through mutation of Leu234Val and Leu235Ala
(L235V/L235A) and
deletion of Gly236 (AG236) using the Kabat numbering system:
IgGl-hinge wt: DKTHTCPPCPAP INGPS (SEQ ID NO: 74)

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IgGl-VAAG Hinge: DKTHTCPPCPAP MGPS (SEQ ID NO: 75)
In some of these embodiments, the fusion protein includes at least the amino
acid sequence
of SEQ ID NO: 51.
[000134] In some embodiments, the Fc polypeptide is derived from human
IgG4. In
some of these embodiments the lower hinge region is modified by mutation at
Leu235Glu
(L235E). In addition, embodiments of the present invention wherein the Fc
polypeptide is
derived from IgG4, the hinge region is modified through the stabilizing
mutation Ser228Pro
(S228P) using the Kabat numbering system:
IgG4-hinge wt: ESKYGPPCPECPAPEFLGGPS (SEQ ID NO: 76)
IgG4-hinge PE: ESKYGPPCPPCPAPEFEGGPS (SEQ ID NO: 77)
In some of these embodiments, the fusion protein includes at least the amino
acid sequence
of SEQ ID NO: 70.
[000135] The fusion proteins described herein include at least a serpin
polypeptide or
an amino acid sequence that is derived from a serpin and a second polypeptide.
In some
embodiments, for example, the invention provides a serpin polypeptide fused to
human
IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA derivatives. The serpin-
fusion
described herein are expected to be useful in treating a variety of
indications, including, by
way of non-limiting example, alpha- 1-antitrypsin (AAT) deficiency, emphysema,
chronic
obstructive pulmonary disease (COPD), acute respiratory distress syndrome
(ARDS),
allergic asthma, cystic fibrosis, cancers of the lung, ischemia-reperfusion
injury, including,
e.g., ischemia/reperfusion injury following cardiac transplantation,
myocardial infarction,
rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing
spondylitis, Crohn's
disease, psoriasis, type I and/or type II diabetes, bacterial infections,
fungal infections, viral
infections, pneumonia, sepsis, graft versus host disease (GVHD), wound
healing, Systemic
lupus erythematosus, and Multiple sclerosis.
[000136] In some embodiments, the fusion proteins described herein include
at least
an alpha- 1-antitrypsin (AAT) polypeptide or an amino acid sequence that is
derived from
AAT and second polypeptide. For example, the invention provides alpha- 1-
antitrypsin
(AAT) fused to human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA
derivatives.
[000137] In some embodiments, the fusion proteins described herein include
at least a
serpin polypeptide or an amino acid sequence that is derived from a serpin
polypeptide and
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a cytokine targeting polypeptide or an amino acid sequence that is derived
from a cytokine
targeting polypeptide. For example, the invention provides serpin polypeptide
or a
sequence derived from a serpin polypeptide fused to a human cytokine receptor
or
derivative thereof Another embodiment of the invention provides serpin
polypeptide or a
sequence derived from a serpin polypeptide fused to a cytokine targeting
antibody, e.g., an
anti-cytokine antibody, or a sequence derived from of a cytokine targeting
antibody, e.g., an
anti-cytokine antibody, or sequence derived from a fragment of cytokine
targeting antibody,
e.g., a fragment of an anti-cytokine antibody. For example, the invention
provides a serpin
polypeptide or a sequence derived from a serpin polypeptide fused to a
cytokine targeting
polypeptide in which the cytokine targeting polypeptide binds any of the
following human
cytokines: TNFa, IgE, IL-12, IL-23, IL-6, IL-la, IL-113, IL-17, IL-13, IL-4,
IL-10, IL-2,
IL-18, IL-27, or IL-32.
[000138] For example, in some embodiments, the cytokine targeting
polypeptide
targets TNFa and includes any of the following TNFa-targeting polypeptide or
sequences
derived from the following TNFa-targeting polypeptides: Remicade0, Humira0,
Simponi0, Cimiza0, Enbrel0 or ATN-103 and ATN-192.
[000139] For example, in some embodiments, the cytokine targeting
polypeptide
targets IgE and includes any of the following IgE-targeting polypeptide or
sequences
derived from the following IgE-targeting polypeptides: Xolair or FcERI.
[000140] For example, in some embodiments, the cytokine targeting
polypeptide
targets the shared p40 subunit of IL-12 and IL-23 and includes the Stelara0
polypeptide or
sequences derived from the Stelara0 polypeptide.
[000141] For example Stelara0 the cytokine targeting polypeptide targets IL-
13 and
includes the CDP7766 polypeptide or sequences derived from the CDP7766
polypeptide.
[000142] In some embodiments, the fusion proteins described herein include
at least a
alpha-l-antitrypsin (AAT) polypeptide or an amino acid sequence that is
derived from AAT
and a cytokine targeting polypeptide or an amino acid sequence that is derived
from a
cytokine targeting polypeptide. For example, the invention provides alpha-l-
antitrypsin
inhibitor (AAT) fused a cytokine targeting polypeptide in which the cytokine
targeting
polypeptide binds any of the following human cytokines: TNFa, IgE, IL-6, IL-
la, IL-113,
IL-12, IL-17, IL-13, IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.
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[000143] In some embodiments the cytokine targeting polypeptide binds a
cytokine
receptor and prevents binding of the cytokine. For example, the present
invention includes a
serpin fused to a cytokine receptor targeting antibody. For example, the
invention provides
alpha-l-antitrypsin inhibitor (AAT) fused a cytokine targeting polypeptide in
which the
cytokine targeting polypeptide binds the receptor of any of the following
human cytokines:
TNFa, IgE, IL-6, IL-la, IL-113, IL-12, IL-17, IL-13, IL-23, the p40 subunit of
IL-12 and
IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.
[000144] For example, in some embodiments, the cytokine targeting
polypeptide
targets the IL-6 receptor and includes the Actemra0 polypeptide (as described
in patent
publication EP0628639), or the ALX-0061 polypeptide (as described in
W02010/115998),
or sequences derived from the Actemra0 polypeptide, or ALX-0061 polypeptide.
[000145] For example, Actemra0 the cytokine targeting polypeptide targets
the IL-6
receptor and includes the tocilizumab polypeptide or sequences derived from
the
tocilizumab polypeptide.
[000146] The targeting of inflammatory cytokines and immune-stimulating
agents by
protein therapeutics has demonstrated clinical success in numerous
inflammatory
conditions. The most common proteins used as cytokine targeting agents are the
soluble
cytokine receptors and monoclonal antibodies and fragments thereof A
significant
drawback with targeting cytokines is the increased risk of infection in these
patients, as
evidenced by the TNFa targeting biologics, Remicade0, Humira0, Simponi0,
Cimiza0,
and Enbre10, and the IL-12/23 p40 targeting antibody, Stelara0. This is likely
to be a
common problem of targeting inflammatory cytokines leading to immune
suppression in
patients. AAT and other serpin proteins are interesting in that they
demonstrate both anti-
infective and anti-inflammatory activities. Thus, the serpin-cytokine
targeting polypeptide
fusion proteins of this invention can dampen aberrant cytokine activities
while alleviating
the risk of infections.
[000147] In some embodiments, the fusion proteins described herein include
a serpin
polypeptide or an amino acid sequence that is derived from a serpin, a WAP
domain-
containing polypeptide or an amino acid sequence that is derived from a WAP
domain-
containing polypeptide, and an Fc polypeptide or an amino acid sequence that
is derived
from an Fc polypeptide. For example, the invention provides a serpin
polypeptide, a WAP
domain-containing polypeptide and human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc or
IgM-Fc
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derivatives operably linked together in any functional combination. In some
embodiments,
the WAP domain containing protein is human SLPI or derived from human SLPI. In
other
embodiments, the WAP domain containing protein is human ELAFIN or derived from

human ELAFIN. In some embodiments, the fusion proteins described herein
include at
least an alpha-l-antitrypsin (AAT) polypeptide or an amino acid sequence that
is derived
from AAT and a SLPI polypeptide or an amino acid sequence that is derived from
SLPI. In
some embodiments, the fusion proteins described herein include at least an AAT

polypeptide or an amino acid sequence that is derived from AAT and an ELAFIN
polypeptide or an amino acid sequence that is derived from Elafin.
[000148] SLPI and Elafin are WAP domain containing proteins that display
serine
protease inhibitory activity. Both of these proteins are anti-inflammatory in
function. In
addition these proteins possess broad anti-infective capacities toward
numerous strains of
bacteria, viruses, and fungi.
[000149] In some embodiments, the fusion proteins described herein include
at least a
serpin polypeptide or an amino acid sequence that is derived from a serpin and
a human
serum albumin (HSA) polypeptide or an amino acid sequence that is derived from
a HSA
polypeptide. Further embodiments of invention include serpin-albumin binding
polypeptide
fusion proteins, wherein the albumin binding polypeptide is responsible for
the association
of the serpin and HSA. Thereby the invention includes both covalent and non-
covalent
linkages of the serpin polypeptide and the HSA polypeptide or sequences
derived from the
serpin polypeptide or a HSA polypeptide. For example, the invention provides a
serpin
polypeptide fused to human HSA, or HSA derivatives, or HSA binding peptide or
polypeptides.
[000150] In some embodiments, the fusion proteins described herein include
at least
an alpha-l-antitrypsin (AAT) polypeptide or an amino acid sequence that is
derived from
AAT and a HSA polypeptide or an amino acid sequence that is derived from a HSA

polypeptide. For example, the invention provides alpha-l-antitrypsin (AAT)
fused to HSA
or a fragment derived from HSA, or an albumin binding polypeptide.
[000151] In some embodiments, the fusion proteins described herein include
a serpin
polypeptide or an amino acid sequence that is derived from a serpin, a HSA
polypeptide or
an amino acid sequence that is derived from a HSA polypeptide, and a WAP
domain-
containing polypeptide or an amino acid sequence that is derived from a WAP
domain-
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containing polypeptide. In some embodiments, the fusion proteins described
herein include
at least an alpha- 1-antitrypsin (AAT) polypeptide or an amino acid sequence
that is derived
from AAT and a HSA polypeptide or an amino acid sequence that is derived from
a HSA
polypeptide, and a SLPI polypeptide or amino acid sequence derived from SLPI.
In other
embodiments, the fusion proteins described herein include at least an alpha- 1-
antitrypsin
(AAT) polypeptide or an amino acid sequence that is derived from AAT and a HSA

polypeptide or an amino acid sequence that is derived from a HSA polypeptide,
and an
Elafin polypeptide or amino acid sequence derived from Elafin.
[000152] The fusion proteins of the present invention can be readily
produced in
mammalian cell expression systems. For example Chinese Hamster Ovary (CHO)
cells,
Human Embryonic Kidney (HEK) 293 cells, COS cells, PER.C6C), NSO cells, 5P2/0,

YB2/0 can readily be used for the expression of the serpin fusion proteins
described herein.
Importantly, mammalian cell expression systems produce proteins that are
generally more
optimal for therapeutic use. In contrast to bacterial, insect, or yeast-based
expression
systems, mammalian cell expression systems yield proteins with glycosylation
patterns that
are similar or the same as those found in natural human proteins. Proper
glycosylation of a
protein can greatly influence serum stability, pharmacokinetics,
biodistribution, protein
folding, and functionality. Therefore, the ability to produce therapeutic
proteins in
mammalian expression systems has distinct advantages over other systems.
Furthermore,
most of the mammalian cell expression systems (e.g., CHO, NSO, PER.C6C) cells)
can be
readily scaled in commercial manufacturing facilities to produce therapeutic
proteins to
meet clinical demands. The fusion proteins described herein have enhanced
functionalities
over the natural form of AAT and can be produced in mammalian expression
systems for
clinical and commercial supply. Some embodiments of the invention include a
purification
system that enables the isolation of serpin fusion proteins that retain their
ability to inhibit
NE. Importantly, the purification process of the present invention can be
readily
incorporated into today's commercial mammalian cell-based manufacturing
processes.
[000153] Unless otherwise defined, scientific and technical terms used in
connection
with the present invention shall have the meanings that are commonly
understood by those
of ordinary skill in the art. Further, unless otherwise required by context,
singular terms
shall include pluralities and plural terms shall include the singular.
Generally,
nomenclatures utilized in connection with, and techniques of, cell and tissue
culture,

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molecular biology, and protein and oligo- or polynucleotide chemistry and
hybridization
described herein are those well-known and commonly used in the art. Standard
techniques
are used for recombinant DNA, oligonucleotide synthesis, and tissue culture
and
transformation (e.g., electroporation, lipofection). Enzymatic reactions and
purification
techniques are performed according to manufacturer's specifications or as
commonly
accomplished in the art or as described herein. The foregoing techniques and
procedures
are generally performed according to conventional methods well known in the
art and as
described in various general and more specific references that are cited and
discussed
throughout the present specification. See e.g., Sambrook et al. Molecular
Cloning: A
Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor,
N.Y. (1989)). The nomenclatures utilized in connection with, and the
laboratory procedures
and techniques of, analytical chemistry, synthetic organic chemistry, and
medicinal and
pharmaceutical chemistry described herein are those well-known and commonly
used in the
art. Standard techniques are used for chemical syntheses, chemical analyses,
pharmaceutical preparation, formulation, and delivery, and treatment of
patients. The term
patient includes human and veterinary subjects.
[000154] It will
be appreciated that administration of therapeutic entities in accordance
with the invention will be administered with suitable carriers, buffers,
excipients, and other
agents that are incorporated into formulations to provide improved transfer,
delivery,
tolerance, and the like. A multitude of appropriate formulations can be found
in the
formulary known to all pharmaceutical chemists: Remington's Pharmaceutical
Sciences
(15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87
by Blaug,
Seymour, therein. These formulations include, for example, powders, pastes,
ointments,
jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles
(such as
LipofectinTm), DNA conjugates, anhydrous absorption pastes, oil-in-water and
water-in-oil
emulsions, emulsions carbowax (polyethylene glycols of various molecular
weights), semi-
solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing
mixtures
may be appropriate in treatments and therapies in accordance with the present
invention,
provided that the active ingredient in the formulation is not inactivated by
the formulation
and the formulation is physiologically compatible and tolerable with the route
of
administration. See also Baldrick P. "Pharmaceutical excipient development:
the need for
preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W.
46

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"Lyophilization and development of solid protein pharmaceuticals." Int. J.
Pharm. 203(1-
2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-
some
emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al.
"Compendium of
excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311
(1998) and
the citations therein for additional information related to formulations,
excipients and
carriers well known to pharmaceutical chemists.
[000155] Therapeutic formulations of the invention, which include a fusion
protein of
the invention, are used to treat or alleviate a symptom associated with a
disease or disorder
associated with aberrant serine protease activity in a subject. The present
invention also
provides methods of treating or alleviating a symptom associated with a
disease or disorder
associated with aberrant serine protease activity in a subject. A therapeutic
regimen is
carried out by identifying a subject, e.g., a human patient suffering from (or
at risk of
developing) a disease or disorder associated with aberrant serine protease
activity, using
standard methods, including any of a variety of clinical and/or laboratory
procedures. The
term patient includes human and veterinary subjects. The term subject includes
humans and
other mammals.
[000156] Efficaciousness of treatment is determined in association with any
known
method for diagnosing or treating the particular disease or disorder
associated with aberrant
serine protease activity. Alleviation of one or more symptoms of the disease
or disorder
associated with aberrant serine protease activity indicates that the fusion
protein confers a
clinical benefit.
[000157] Methods for the screening of fusion proteins that possess the
desired
specificity include, but are not limited to, enzyme linked immunosorbent assay
(ELISA),
enzymatic assays, flow cytometry, and other immunologically mediated
techniques known
within the art.
[000158] The fusion proteins described herein may be used in methods known
within
the art relating to the localization and/or quantitation of a target such as a
serine protease,
e.g., for use in measuring levels of these targets within appropriate
physiological samples,
for use in diagnostic methods, for use in imaging the protein, and the like).
The terms
"physiological sample" and "biological sample," used interchangeably, herein
are intended
to include tissues, cells and biological fluids isolated from a subject, as
well as tissues, cells
and fluids present within a subject. Included within the usage of the terms
"physiological
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sample" and "biological sample", therefore, is blood and a fraction or
component of blood
including blood serum, blood plasma, or lymph.
[000159] In a given embodiment, fusion proteins specific for a given
target, or
derivative, fragment, analog or homolog thereof, that contain the target-
binding domain, are
utilized as pharmacologically active compounds (referred to hereinafter as
"Therapeutics").
[000160] A fusion protein of the invention can be used to isolate a
particular target
using standard techniques, such as immunoaffinity, chromatography or
immunoprecipitation. Detection can be facilitated by coupling (i.e.,
physically linking) the
fusion protein to a detectable substance. Examples of detectable substances
include various
enzymes, prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent
materials, and radioactive materials. Examples of suitable enzymes include
horseradish
peroxidase, alkaline phosphatase, 13-galactosidase, or acetylcholinesterase;
examples of
suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples
of suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of
bioluminescent materials include luciferase, luciferin, and aequorin, and
examples of
suitable radioactive material include 1251, 131-,
I 35S or 3H.
[000161] A therapeutically effective amount of a fusion protein of the
invention relates
generally to the amount needed to achieve a therapeutic objective. As noted
above, this
may be a binding interaction between the fusion protein and its target that,
in certain cases,
interferes with the functioning of the target. The amount required to be
administered will
furthermore depend on the binding affinity of the fusion protein for its
specific target, and
will also depend on the rate at which an administered fusion protein is
depleted from the
free volume other subject to which it is administered. Common ranges for
therapeutically
effective dosing of an fusion protein or fragment thereof invention may be, by
way of
nonlimiting example, from about 0.1 mg/kg body weight to about 250 mg/kg body
weight.
Common dosing frequencies may range, for example, from twice daily to once a
month.
[000162] Where fusion protein fragments are used, the smallest inhibitory
fragment
that specifically binds to the target is preferred. For example, peptide
molecules can be
designed that retain the ability to bind the target. Such peptides can be
synthesized
chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco
et al.,
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Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also
contain more
than one active compound as necessary for the particular indication being
treated, preferably
those with complementary activities that do not adversely affect each other.
Alternatively,
or in addition, the composition can comprise an agent that enhances its
function, such as, for
example, a cytotoxic agent, cytokine, chemotherapeutic agent, growth-
inhibitory agent, an
anti-inflammatory agent or anti-infective agent. Such molecules are suitably
present in
combination in amounts that are effective for the purpose intended.
[000163] The active ingredients can also be entrapped in microcapsules
prepared, for
example, by coacervation techniques or by interfacial polymerization, for
example,
hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems (for example,
liposomes,
albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in
macroemulsions.
[000164] The formulations to be used for in vivo administration must be
sterile. This
is readily accomplished by filtration through sterile filtration membranes.
[000165] Sustained-release preparations can be prepared. Suitable examples
of
sustained-release preparations include semipermeable matrices of solid
hydrophobic
polymers containing the fusion protein, which matrices are in the form of
shaped articles,
e.g., films, or microcapsules. Examples of sustained-release matrices include
polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7
ethyl-L-
glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-
glycolic acid
copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of
lactic
acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-
hydroxybutyric acid.
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid
enable release
of molecules for over 100 days, certain hydrogels release proteins for shorter
time periods.
Pharmaceutical compositions
[000166] The fusion proteins of the invention (also referred to herein as
"active
compounds"), and derivatives, fragments, analogs and homologs thereof, can be
incorporated into pharmaceutical compositions suitable for administration.
Such
compositions typically comprise the fusion protein and a pharmaceutically
acceptable
carrier. As used herein, the term "pharmaceutically acceptable carrier" is
intended to
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include any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents,
isotonic and absorption delaying agents, and the like, compatible with
pharmaceutical
administration. Suitable carriers are described in the most recent edition of
Remington's
Pharmaceutical Sciences, a standard reference text in the field, which is
incorporated herein
by reference. Preferred examples of such carriers or diluents include, but are
not limited to,
water, saline, ringer's solutions, dextrose solution, and 5% human serum
albumin.
Liposomes and non-aqueous vehicles such as fixed oils may also be used. The
use of such
media and agents for pharmaceutically active substances is well known in the
art. Except
insofar as any conventional media or agent is incompatible with the active
compound, use
thereof in the compositions is contemplated. Supplementary active compounds
can also be
incorporated into the compositions.
[000167] A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of routes of
administration
include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation),
transdermal (i.e., topical), transmucosal, and rectal administration.
Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can include the
following
components: a sterile diluent such as water for injection, saline solution,
fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial
agents such as benzyl alcohol or methyl parabens; antioxidants such as
ascorbic acid or
sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid
(EDTA); buffers
such as acetates, citrates or phosphates, and agents for the adjustment of
tonicity such as
sodium chloride or dextrose. The pH can be adjusted with acids or bases, such
as
hydrochloric acid or sodium hydroxide. The parenteral preparation can be
enclosed in
ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[000168] Pharmaceutical compositions suitable for injectable use include
sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous
administration, suitable carriers include physiological saline, bacteriostatic
water,
Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In
all cases,
the composition must be sterile and should be fluid to the extent that easy
syringeability
exists. It must be stable under the conditions of manufacture and storage and
must be
preserved against the contaminating action of microorganisms such as bacteria
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The carrier can be a solvent or dispersion medium containing, for example,
water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the
like), and suitable mixtures thereof The proper fluidity can be maintained,
for example, by
the use of a coating such as lecithin, by the maintenance of the required
particle size in the
case of dispersion and by the use of surfactants. Prevention of the action of
microorganisms
can be achieved by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases,
it will be
preferable to include isotonic agents, for example, sugars, polyalcohols such
as manitol,
sorbitol, sodium chloride in the composition. Prolonged absorption of the
injectable
compositions can be brought about by including in the composition an agent
which delays
absorption, for example, aluminum monostearate and gelatin.
[000169] Sterile injectable solutions can be prepared by incorporating the
active
compound 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, 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
[000170] Oral compositions generally include an inert diluent or an edible
carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral
therapeutic administration, the active compound can be incorporated with
excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also
be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is
applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible
binding agents, and/or adjuvant materials can be included as part of the
composition. The
tablets, pills, capsules, troches and the like can contain any of the
following ingredients, or
compounds of a similar nature: a binder such as microcrystalline cellulose,
gum tragacanth
or gelatin; an excipient such as starch or lactose, a disintegrating agent
such as alginic acid,
Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes;
a glidant such
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as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring
agent such as peppermint, methyl salicylate, or orange flavoring.
[000171] For administration by inhalation, the compounds are delivered in
the form of
an aerosol spray from pressured container or dispenser which contains a
suitable propellant,
e.g., a gas such as carbon dioxide, or a nebulizer.
[000172] Systemic administration can also be by transmucosal or transdermal
means.
For transmucosal or transdermal administration, penetrants appropriate to the
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art, and
include, for example, for transmucosal administration, detergents, bile salts,
and fusidic acid
derivatives. Transmucosal administration can be accomplished through the use
of nasal
sprays or suppositories. For transdermal administration, the active compounds
are
formulated into ointments, salves, gels, or creams as generally known in the
art.
[000173] The compounds can also be prepared in the form of suppositories
(e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention
enemas for rectal delivery.
[000174] In one embodiment, the active compounds are prepared with carriers
that
will protect the compound against rapid elimination from the body, such as a
controlled
release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid. Methods
for preparation of such formulations will be apparent to those skilled in the
art. The
materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions can also be used as
pharmaceutically
acceptable carriers. These can be prepared according to methods known to those
skilled in
the art, for example, as described in U.S. Patent No. 4,522,811.
[000175] It is especially advantageous to formulate oral or parenteral
compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form as
used herein refers to physically discrete units suited as unitary dosages for
the subject to be
treated; each unit containing a predetermined quantity of active compound
calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical
carrier. The specification for the dosage unit forms of the invention are
dictated by and
directly dependent on the unique characteristics of the active compound and
the particular
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therapeutic effect to be achieved, and the limitations inherent in the art of
compounding
such an active compound for the treatment of individuals.
[000176] The pharmaceutical compositions can be included in a container,
pack, or
dispenser together with instructions for administration.
[000177] The invention will be further described in the following examples,
which do
not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: AAT-Fc Fusion Proteins and Variants
[000178] Exemplary, but non-limiting examples of AAT-Fc fusion proteins
according
to the invention include the following sequences. While these examples include
a hinge
sequence and/or a linker sequence, fusion proteins of the invention can be
made using any
hinge sequence and/or a linker sequence suitable in length and/or flexibility.
Alternatively
fusion proteins can be made without using a hinge and/or a linker sequence.
For example,
the polypeptide components can be directly attached.
[000179] An exemplary AAT-Fc fusion protein is the AAT-hFcl (human IgG1 Fc)
described herein. As shown below, AAT polypeptide portion of the fusion
protein is
underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion
protein is
italicized (SEQ ID NO: 3).
AAT-hFcl (human IgG1 Fc)
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF
NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
D TLMI SRT PEVTCVVVDVSHE D PEVKFNWY VDGVEVHNAKTKPREEQYNS TYRVVSVL TVL
HQ DWLNGKE YKCKVSNKAL PAPIEKT I SKAKGQ PRE PQVY TL PPSRDEL TKNQVSLTCLVK
GFY PS D I AVEWE SNGQ PENNYKT TPPVL DS DGS FFL YSKL TVDKSRWQQ GNVFS CSVMHEA
LHNHYTQKSLSLSPGK (SEQ ID NO:16)
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[000180] An exemplary AAT-Fc fusion protein is the AAT-hFc2 (human IgG2
Fc),
described herein. As shown below, AAT polypeptide portion of the fusion
protein is
underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion
protein is
italicized (SEQ ID NO: 4).
AAT-hFc2 (human IgG2 Fc)
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF
NKPFVFLMIEQNIKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK (SEQ ID NO: 17)
[000181] An exemplary AAT-Fc fusion protein is the AAT-MM-EL-hFcl (human
IgG1 Fc, Met351G1u/Met358Leu), described herein. As shown below, AAT
polypeptide
portion of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc
polypeptide
portion of the fusion protein is italicized (SEQ ID NO: 3), and the Met351Glu
mutation is
boxed, and the Met358Leu mutation is shaded in grey.
AAT-MM-EL-hFcl(human IgG1 Fc, Met351G1u/Met358Leu)
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVESNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGATFLEAIPLSIPPEVKF
NKPFVFLMIEQNIKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
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HQ DWLNGKE YKCKVSNKAL PAPIEKT I SKAKGQ PRE PQVY TL PPSRDEL TKNQVSL TCLVK
GFY PS D I AVE WE SNGQ PENNY KT T PPVL DS DGS FFL Y SKL TVDKSRWQQ GNVFS
CSVMHEA
LHNHY TQKSLS LS PGK (SEQ ID NO: 18)
[000182] An exemplary AAT-Fc fusion protein is the AAT-MM-EL-hFc2 (human
IgG2 Fc, Met351G1u/Met358Leu), described herein. As shown below, AAT
polypeptide
portion of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc
polypeptide
portion of the fusion protein is italicized (SEQ ID NO: 4), the Met351Glu
mutation is
boxed, and the Met358Leu mutation is shaded in grey.
AAT-MM-EL-hFc2 (human IgG2 Fc, Met351G1u/Met358Leu)
EDPQGDAAQKTDTSHHDQDHPTFNKI TPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEI PEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ INDYVEKGTQGKIVDLVKELDRDTVFA
LVNY I FFKGKWERPFEVKDTEEE DFHVDQVTTVKVPMMKRLGMFNI QHCKKLS SWVLLMKY
LGNATAI FFLPDEGKLQHLENELTHDI I TKFLENEDRRSASLHLPKLS I TGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLT I DEKGTEAAGAEFLEAI ns I PPEVKF
NKPFVFLMIEQNIKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH
YTQKSLSLSPGK (SEQ ID NO: 19)
[000183] An exemplary AAT-Fc fusion protein is the AAT-MM-LL-hFcl(human
IgG1 Fc, Met351Leu/Met358Leu), described herein. As shown below, AAT
polypeptide
portion of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc
polypeptide
portion of the fusion protein is italicized (SEQ ID NO: 3), the Met351Leu
mutation is
shaded in black, and the Met358Leu mutation is shaded in grey.
AAT-MM-LL-hFcl (human IgG1 Fc, Met351Leu/Met358Leu)
EDPQGDAAQKTDTSHHDQDHPTFNKI TPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEI PEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ INDYVEKGTQGKIVDLVKELDRDTVFA

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LVNY I FFKGKWERPFEVKDTEEE DFHVDQVTTVKVPMMKRLGMFN I QHCKKLS SWVLLMKY
LGNATAI FFLP DEGKLQHLENELTHD I I TKFLENEDRRSASLHLPKLS I TGTYDLKSVLGQ
LG I TKVF SNGADLSGVTEEAPLKLSKAVHKAVLT I DEKGTEAAGAIFLEAI PLS I PPEVKF
NKPFVFLMIEQNTKS PLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQ DWLNGKE YKCKVSNKAL PAPIEKT I SKAKGQ PRE PQVY TL PPSRDEL TKNQVSL TCLVK
GFY PS D I AVEWESNGQ PENN YKT TPPVL DS DGS FFL YSKL TVDKSRWQQ GNVFS CSVMHEA
LHNHYTQKSLSLSPGK(SEQ ID NO:36)
[000184] An exemplary AAT-Fc fusion protein is the AAT-MM:LL-hFc2(human
IgG2 Fc, Met351Leu/Met358Leu), described herein. As shown below, AAT
polypeptide
portion of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc
polypeptide
portion of the fusion protein is italicized (SEQ ID NO: 4), the Met351Leu
mutation is
shaded in black, and the Met358Leu mutation is shaded in grey.
AAT-MM:LL-hFc2 (human IgG2 Fc, Met351Leu/Met358Leu)
EDPQGDAAQKT DT SHHDQDHPT FNKI T PNLAEFAFSLYRQLAHQSNS TN I FFS PVS IATAF
AMLSLGTKADT HDE I LEGLNFNLTE I PEAQ I HEGFQELLRTLNQPDSQLQLTT GNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ INDYVEKGTQGKIVDLVKELDRDTVFA
LVNY I FFKGKWERPFEVKDTEEE DFHVDQVTTVKVPMMKRLGMFN I QHCKKLS SWVLLMKY
LGNATAI FFLP DEGKLQHLENELTHD I I TKFLENEDRRSASLHLPKLS I TGTYDLKSVLGQ
LG I TKVF SNGADLSGVTEEAPLKLSKAVHKAVLT I DEKGTEAAGALLEAI PLS I PPEVKF
NKPFVFLMI EQNTKS PLFMGKVVNPTQKERKCCVEC PPC P APPVAGPSVFL FPPKPKDTLM
I SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW
LNGKE Y KCKVSNKGL PAPIEKT I SKTKGQ PRE PQVY TL PPSREEMTKNQVSL TCLVKGFY P
S D I AVEWESNGQ PENN YKT T PPML DS DGS FFL YS KL TVDKSRWQQGNVFS CSVMHEALHNH
YTQKSLSLSPGK (SEQ ID NO:20)
[000185] An exemplary AAT-Fc fusion protein is the AAT-hFc 1-AAT (human
IgG1),
described herein. As shown below, AAT polypeptide portion of the fusion
protein is
underlined (SEQ ID NO: 2), the IgG-Fc polypeptide portion of the fusion
protein is
italicized (SEQ ID NO: 3).
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AAT-hFcl-AAT (human IgG1)
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF
NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVL
HQ DWLNGKE Y KCKVSNKAL PAPIEKT I SKAKGQ PRE PQVY TL PPSRDEL TKNQVSL TCLVK
GFY PS D I AVEWESNGQ PENN YKT T PPVL DS DGS FFL Y SKLTVDKSRWQQ GNVFSCSVMHEA
LHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQ
LAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLR
TLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVE
KGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKR
LGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSA
SLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGT
EAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID
NO:21)
AAT-EL-Fc-IgGl-DV,AG,IDL (AAT: Met351G1u/Met358Leu; Fc IgGl:
Leu234Val/Leu235Ala, Deleted G1y236, Met25211e, Thr256Asp,
Met428Leu)
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVESNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAEFLEAIPESIPPEVKF
NKPFVFLMIEQNTKSPLFMGKVVNPTQKGGGGDKTHTCPPCPAPEVAGPSVFLFPPKPKDT
LMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF
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YPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVOHEALH
NHYTQKSLSLSPGK (SEQ ID NO: 78)
AAT-EL-Fc-IgG4-PE,IDL (AAT: Met351G1u/Met358Leu; Fc IgG4:
Ser228Pro Leu235G1u, Met25211e, Thr256Asp, Met428Leu)
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAEFLEAIPESIPPEVKF
NKPFVFLMIEQNTKSPLFMGKVVNPTQKESKYGPPCPHCPAPEFEGGPSVFLFPPKPKDTL
_
EISRDPEVICVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD
_
WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVOHEALHN
HYTQKSLSLSLGK (SEQ ID NO: 79)
[000186] These exemplary AAT-Fc fusion proteins were made using the
following
techniques.
[000187] The gene encoding human AAT was PCR amplified from human liver
cDNA
(Zyagen). Specific point mutations within the gene encoding AAT or the Fc
region were
generated by overlapping PCR. The AAT encoding gene was cloned in frame with a
gene
encoding the hinge region, followed by a CH2 domain, and a CH3 domain of human
IgGl,
IgG2, IgG3, IgG4, or IgM into a mammalian expression vector, containing a
mammalian
secretion signal sequence up stream of the AAT gene insertion site. These
expression
vectors were transfected into mammalian cells (specifically HEK293 or CHO
cells) and
grown for several days in 8% CO2 at 37 C. The recombinant AAT-Fc fusion
proteins were
purified from the expression cell supernatant by protein A chromatography.
Importantly, a
near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo
Scientific) to elute
the AAT-Fc fusion protein from the protein A resin. The AAT-Fc fusion protein
cannot be
eluted from protein A resin using a standard low pH elution, as the ability of
AAT to inhibit
NE is compromised following low pH treatment, likely due to a low pH mediated
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oligomerization of AAT. Figure 1F shows the effects of low pH elution on the
ability of
AAT to inhibit neutrophil elastase. AAT-Fc fusion protein can be purified
either by protein
A and a near neutral pH elution buffer, by CaptureSelect Alpha-1 Antitrypsin
affinity
matrix (BAC BV).
[000188] The purified AAT-Fc fusion proteins were tested for activity by
determining
their ability to inhibit neutrophil elastase (NE). Figure 1B and 1D show a
reducing SDS-
PAGE gel of purified serum derived AAT (sdAAT) and AAT-Fc fusion proteins (Fig
1B-
lane 1: sdAAT, lane 2: AAT-Fc (SEQ ID NO: 16), lane 3: AAT-EL-Fc (SEQ ID NO:
18),
Fig 1D AAT-Fc-AAT (SEQ ID NO: 20). The proteins were visualized by staining
with
Coomassie blue.
[000189] To monitor human Neutrophil Elastase (NE) activity a fluorescent
microplate
assay was used. Inhibitory activity was measured by a concomitant decrease in
the residual
NE activity using the following assay. This assay buffer is composed of 100 mM
Tris pH
7.4, 500 mM NaCl, and 0.0005% Triton X-100. Human NE is used at a final
concentration
of 5 nM (but can also be used from 1-20 nM). The fluorescent peptide substrate
AAVP-
AMC is used at a final concentration of 100 uM in the assay. The Gemini EM
plate reader
from Molecular Devices is used to read the assay kinetics using excitation and
emission
wavelengths of 370 nm and 440 nm respectively, and a cutoff of 420 nm. The
assay is read
for 10 min at room temperature scanning every 5 to 10 seconds. The Vmax per
second
corresponds to the residual NE activity, which is plotted for each
concentration of inhibitor.
The intercept with the x-axis indicates the concentration of inhibitor needed
to fully
inactivate the starting concentration of NE in the assay. Human serum derived
AAT
(sdAAT) was used as a positive control in these assays. The AAT-Fc fusion
proteins
display potent NE inhibitory activity as shown in Figure 1C. The fusion
wherein there are
two AAT polypeptides fused to single Fc polypeptide (AAT-Fc-AAT) displays
enhanced
potency over both sdAAT and the AAT-Fc fusion protein comprising a single AAT
polypeptide (Figure 1E). These findings presented here demonstrate for the
first time the
AAT can be fused to an Fc region and maintain its ability to inhibit NE. Of
particular
interest, the AAT-Fc-AAT fusion protein was found to be a more potent NE
inhibitor.
[000190] Figure 1F demonstrates the resistance of the AAT-EL-Fc (M351E,
M358L)
fusion protein to inactivation by oxidation. AAT fusion proteins, AAT-Fc (wt),
AAT-EL-
Fc (M351E, M358L), and AAT-EM-Fc (M351E), were treated with 33mM H202 and
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compared to untreated fusion proteins in the NE inhibition assays. The
inhibition of NE by
AAT-EL-Fc was not comprised by oxidation, converse to the other proteins
tested.
[000191] Furthermore, AAT-Fc fusion protein displayed a longer serum half-
life in
rats compared to serum derived AAT (Figure 1H). In these studies 3 rats per
each test
protein were injected I.V. with 10mg/kg of sdAAT or AAT-Fc. Serum sample were
taken
at various time points over a 48 period. The serum ATT concentration was using
an
ELISA. These findings demonstrate that the fusion proteins of the invention
have improved
pharmacokinetic properties and are a superior therapeutic format over serum
derived AAT,
for treating numerous human inflammatory conditions and infectious diseases.
Example 2: AAT-TNFa Targeting Molecule Fusion Proteins
[000192] The studies presented herein describe several, non-limiting
examples of
recombinant AAT derivatives comprising human AAT fused to an anti-TNFa
antibody or a
derivative of a TNFa receptor. These examples are provided below to further
illustrate
different features of the present invention. The examples also illustrate
useful methodology
for practicing the invention. These examples do not and are not intended to
limit the
claimed invention.
[000193] The fusion proteins below include cytokine targeting polypeptide
sequences
that are from or are derived from (i) the anti-TNFa antibody D2E7 (also known
as
Adalimumab or Humira0), or (ii) the extracellular domain of Type 2 TNFa
Receptor
(TNFR2-ECD). The AAT polypeptide portion of the fusion protein is underlined,
the
antibody constant regions (CH1-hinge-CH2-CH3, or CL) are italicized, and D2E7-
VH,
D2E7-VK, and TNFR2-ECD are denoted in bold text. While these examples include
a hinge
sequence and/or a linker sequence, fusion proteins of the invention can be
made using any
hinge sequence and/or a linker sequence suitable in length and/or flexibility.
Alternatively
fusion proteins can be made without using a hinge and/or a linker sequence.
[000194] An exemplary AAT-TNFa fusion protein is D2E7-Light Chain-AAT
(G3S)2
Linker, described herein. As shown below, the AAT polypeptide portion of the
fusion
protein is underlined (SEQ ID NO: 2), D2E7-VK is denoted in bold text (SEQ ID
NO: 37),
and the antibody constant regions are italicized (SEQ ID NO: 38)

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D2E7-Light Chain-AAT (G3S) 2 Linker
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR
FSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKR TVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSEDPQGDAAQKTDTSHHDQDHPT
FNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFN
LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFT
VNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEE
EDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENE
LTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPL
KLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKV
VNPTQK (SEQ ID NO:22)
[000195] An exemplary AAT-TNFa fusion protein is D2E7-Light Chain-AAT
ASTGS Linker, described herein. As shown below, the AAT polypeptide portion of
the
fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is denoted in bold text
(SEQ ID
NO: 37), and the antibody constant regions is italicized (SEQ ID NO: 38)
D2E7-Light Chain-AAT ASTGS Linker
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR
FSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVF/FPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGECASTGSEDPQGDAAQKTDTSHHDQDHPTFNK
ITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTE
IPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNF
GDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDF
HVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTH
DIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLS
KAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNP
TQK (SEQ ID NO:23)
[000196] An exemplary AAT-TNFa fusion protein is D2E7-Heavy Chain-AAT
(G35)2
Linker, described herein. As shown below, the AAT polypeptide portion of the
fusion
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protein is underlined (SEQ ID NO: 2), D2E7-VH is denoted in bold text (SEQ ID
NO: 39),
and the antibody constant regions is italicized (SEQ ID NO: 40)
D2E7-Heavy Chain-AAT (G3S)2 Linker
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA
DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPN
LAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEI PEA
QIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTE
EAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ
VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIIT
KFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVH
KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
(SEQ ID NO:24)
[000197] An exemplary AAT-TNFcc fusion protein is D2E7-Heavy Chain-AAT
ASTGS Linker, described herein. As shown below, the AAT polypeptide portion of
the
fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is denoted in bold text
(SEQ ID
NO: 39), and the antibody constant regions is italicized (SEQ ID NO: 40)
D2E7-Heavy Chain-AAT ASTGS Linker
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA
DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAE
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FAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIH
EGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAK
KQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTT
VKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL
ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAV
LTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
(SEQ ID NO:25)
[00149] An exemplary AAT-TNFa fusion protein is TNFR2-ECD-Fcl-AAT(G3S)2
Linker, described herein. As shown below, the AAT polypeptide portion of the
fusion
protein is underlined (SEQ ID NO: 2), TNFR2-ECD is denoted in bold text (SEQ
ID NO:
41), and the antibody constant regions is italicized (SEQ ID NO: 42)
TNFR2-ECD-Fcl-AAT (G3S) 2 Linker
LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCED STY
TQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCR
PGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSP
TRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH
NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDT
SHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHD
EILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVK
KLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWER
PFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDE
GKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADL
SGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNT
KSPLFMGKVVNPTQK (SEQ ID NO:26)
[00150] An exemplary AAT-TNFa fusion protein is TNFR2-ECD-Fcl-AAT ASTGS
Linker, described herein. As shown below, the AAT polypeptide portion of the
fusion
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protein is underlined (SEQ ID NO: 2), TNFR2-ECD is denoted in bold text (SEQ
ID NO:
41), and the antibody constant regions is italicized (SEQ ID NO: 42)
TNFR2-ECD-Fcl-AAT ASTGS Linker
LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCED STY
TQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCR
PGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSP
TRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH
NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHH
DQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEIL
EGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLY
HSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE
VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKL
QHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGV
TEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSP
LFMGKVVNPTQK (SEQ ID NO:27)
[000198] These exemplary AAT-TNFcc targeting molecule fusion proteins were
made
using the following techniques.
[000199] The genes encoding the variable heavy (VH) and variable kappa (VK)
regions of the anti-TNFcc antibody, D2E7, were generated by gene synthesis.
The D2E7-
VH gene was cloned in frame with a gene encoding a human IgG1 antibody heavy
chain
constant region, consisting of a CH1 domain, a hinge domain, a CH2 domain, and
a CH3
domain, into a mammalian expression vector, containing a mammalian secretion
signal
sequence up stream of the VH domain insertion site (D2E7-HC). The D2E7-VK gene
was
cloned in frame with a human antibody kappa light chain constant (CL) domain,
into a
mammalian expression vector, containing a mammalian secretion signal sequence
up stream
of the VK domain insertion site (D2E7-LC). The AAT encoding gene and the
adjacent 5'
linker sequence were cloned in frame into the 3' end of either, the CH3 domain
of the D2E7
heavy chain gene (D2E7-HC-AAT), or the CL domain of the D2E7 light chain gene
(D2E7-
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LC-AAT) coding sequences in the above described mammalian expression vectors.
The
extracellular domain of the TNFa Receptor 2 (TNFR2-ECD) was generated by gene
synthesis and cloned in frame with a gene encoding the hinge region, followed
by a CH2
domain and a CH3 domain of human IgG1 (hFc1) into a mammalian expression,
containing
a mammalian secretion signal sequence up stream of the TNFR2-ECD insertion
site. The
AAT encoding gene and the adjacent 5' linker sequence were cloned in frame
into the 3'
end of the gene encoding TNFR2-ECD-hFcl into a mammalian expression vector
(TNFR2-
ECD-hFc1-AAT).
[000200] The D2E7-HC-AAT expression vector was co-transfected with either
the
D2E7-LC or the D2E7-LC-AAT expression vector into mammalian cells
(specifically
HEK293 or CHO cells) to generate the D2E7 antibody with AAT fused to the C-
terminus of
the heavy chain or to the C-terminus of both the heavy chain and light chain,
respectively.
The D2E7-LC-AAT was co-transfected with the D2E7-HC expression vector into
mammalian cells to generate the D2E7 antibody with AAT fused to the C-terminus
of the
light chain. The TNFR2-hFc 1-AAT expression vector was transfected into
mammalian
cells. Transfected cells were grown for several days in 8% CO2 at 37 C.
[000201] The recombinant AAT-TNFa targeting fusion proteins were purified
from
the expression cell supernatant by protein A chromatography. A near neutral pH
buffer was
used (Gentle Ag/Ab Elution Buffer, Thermo Scientific) to elute the AAT-TNFa
targeting
fusion proteins from the protein A resin.
[000202] Figure 2B shows an SDS-PAGE gel of the D2E7 antibody alone (lane
1) and
variant wherein AAT is fused to the heavy chain of D2E7 (lane 2). The proteins
were
visualized by staining with Coomassie blue.
[000203] The purified AAT-TNFa targeting molecule fusion proteins were
tested for
activity by determining their ability to inhibit neutrophil elastase. Human
serum derived
AAT (sdAAT) was used as a positive control in these assays. NE inhibitory
assay were
conducted as described above. Figure 2C demonstrates relative to sdAAT, the
AAT-TNFa
targeting molecule fusion protein shows similar inhibition of neutrophil
elastase, indicating
that the inhibitory capacity of AAT has not been compromised by its fusion to
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Example 3 AAT-Fc-SLPI and AAT-Fc-Elafin
[000204] The studies presented herein describe several, non-limiting
examples of
recombinant AAT derivatives comprising human AAT fused a WAP domain containing

protein. These examples are provided below to further illustrate different
features of the
present invention. The examples also illustrate useful methodology for
practicing the
invention. The AAT polypeptide portion of the fusion protein is underlined,
the Fc portion
is italicized, and the WAP domain containing polypeptide is in bold font.
While these
examples include a hinge sequence and/or a linker sequence, fusion proteins of
the
invention can be made using any hinge sequence and/or a linker sequence
suitable in length
and/or flexibility. Alternatively fusion proteins can be made without using a
hinge and/or a
linker sequence. For example, the polypeptide components can be directly
attached.
[000205] An exemplary AAT-Fc-SLPI fusion protein is AAT-hFc 1-SLPI (human
IgG1 Fc), described herein. As shown below, the AAT polypeptide portion of the
fusion
protein is underlined (SEQ ID NO: 2), the Fc portion is italicized (SEQ ID NO:
3), and the
WAP domain containing polypeptide is in bold font (SEQ ID NO: 9)
AAT-hFcl-SLPI (human IgG1 Fc)
EDPQGDAAQKT DT SHHDQDHPT FNKI T PNLAEFAFSLYRQLAHQSNS TN I FES PVS IATAF
AMLSLGTKADT HDE I LEGLNFNLTE I PEAQ I HEGFQELLRTLNQPDSQLQLTT GNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQ INDYVEKGTQGKIVDLVKELDRDTVFA
LVNY I FFKGKWERPFEVKDTEEE DFHVDQVTTVKVPMMKRLGMEN I QHCKKLS SWVLLMKY
LGNATAI FFLP DEGKLQHLENELTHD I I TKFLENEDRRSASLHLPKLS I TGTYDLKSVLGQ
LG I TKVF SNGADLSGVTEEAPLKLSKAVHKAVLT I DEKGTEAAGAMFLEAI PMS I PPEVKF
NKPFVFLMIEQNTKS PLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPK
DTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQ DWLNGKE Y KCKVSNKAL PAPIEKT I SKAKGQ PRE PQVY TL P PSRDEL TKNQVSL TCLVK
GFY PS D I AVEWESNGQ PENN YKT T PPVL DS DGS FFL Y SKL TVDKSRWQQ GNVFSCSVMHEA
LHNHYTQKSLSLSPGKAS TGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCP
D T CG I KC LD PVD T PNP TRRK PGKC PVTYGQCLMLNP PNFCEMD GQCKRD LKCCMGMCGKSC
VSPVKA (SEQ ID NO:28)
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[000206] An exemplary AAT-Fc-SLPI fusion protein is AAT-hFc 1-SLPI (human
IgGl Fc), described herein. As shown below, the AAT polypeptide portion of the
fusion
protein is underlined (SEQ ID NO: 2), the Fc portion is italicized (SEQ ID NO:
3), and the
WAP domain containing polypeptide is in bold font (SEQ ID NO: 12)
AAT-hFcl-Elafin (human IgG1 Fc)
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLT IDEKGTEAAGAMFLEAIPMSIPPEVKF
NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
D T LMI SRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNS T YRVVSVL TVL
HQ DWLNGKE Y KCKVSNKAL PAPIEKT I SKAKGQ PRE PQVYTLPPSRDELTKNQVSLTCLVK
GFY PS D I AVE WE SNGQ PENNY KT T PPVL DS DGS FFL Y SKL TVDKSRWQQ GNVFS
CSVMHEA
LHNHYTQKSLSLSPGKAS TGSAVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQ
EPVKGPVSTKPGSCPI I LIRCAMLNPPNRCLKDTDCPGI KKCCEGSCGMACFVPQ ( SEQ
ID NO:29)
[000207] The genes encoding the SLPI and Elafin were PCR amplified from
human
spleen cDNA (Zyagen). These genes were cloned into the mammalian expression
vectors
of example 1, wherein the SLPI or Elafin gene was inserted in frame with the
AAT-Fc gene.
These expression vectors were transfected into mammalian cells (specifically
HEK293 or
CHO cells) and grown for several days in 8% CO2 at 37 C. The recombinant AAT-
Fc-
WAP domain fusion proteins were purified from the expression cell supernatant
by protein
A chromatography. A near neutral pH buffer was used (Gentle Ag/Ab Elution
Buffer,
Thermo Scientific) to elute the AAT-Fc-WAP domain fusion protein from the
protein A
resin.
[000208] Figure 3B shows an SDS-PAGE gel of the AAT-Fc-WAP fusion proteins
(lane 1 AAT-Fc-Elafin, lane 2 AAT-Fc-SLPI). The proteins were visualized by
staining
with Coomassie blue. The purified AAT-Fc-WAP domain fusion proteins were
tested for
activity by determining their ability to inhibit neutrophil elastase. NE
inhibitory assays
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were conducted as described above. Human serum derived AAT (sdAAT) and the AAT-
Fc
fusion protein were used as a positive control in these assays. Relative to
sdAAT, the AAT-
Fc-WAP targeting molecule fusion proteins display enhanced potency of NE
inhibition of
neutrophil elastase (Figure 3C).
Example 4 AAT-Albumin
[000209] The studies presented herein describe several, non-limiting
examples of
recombinant AAT derivatives comprising human AAT fused an albumin polypeptide.
These
examples are provided below to further illustrate different features of the
present invention.
The examples also illustrate useful methodology for practicing the invention.
These
examples do not and are not intended to limit the claimed invention. The AAT
portion is
underlined and the albumin portion is italicized. For example, the polypeptide
components
can be directly attached.
[000210] An exemplary AAT-Albumin fusion protein is AAT-HSA, described
herein.
As shown below, the AAT polypeptide portion of the fusion protein is
underlined (SEQ ID
NO: 2), and the albumin polypeptide is italicized (SEQ ID NO: 14)
AAT-HSA
EDPQGDAAQKTDTSHHDQDHPTENKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNENLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLELSE
GLKLVDKFLEDVKKLYHSEAFTVNEGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMENIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLT IDEKGTEAAGAMFLEAIPMSIPPEVKF
NKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSDAHKSEVAHRFKDLGEENFKALVLIAFA
QY LQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD
CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKY L YE I ARRHPY FY
APELL FFAKRY KAAFTECCQAADKAACLL PKL DE LRDEGKAS SAKQRLKCASL QKFGERAF
KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKY I CENQDS IS
SKLKECCEKPLLEKSHCIAEVENDEMPADL PSLAADFVE SKDVCKNYAEAKDVFLGMFL YE
YARRHPD Y SVVLLLRLAKT YE T TLEKCCAAAD PHECYAKVFDE FKPLVEE PQNL I KQNCEL
FEQLGEYKFQNALLVRYTKKVPQVST PTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKT PVS DRVTKCC TESLVNRR PCFSALEVDE T YVPKE FNAE TFT FHAD I CTL
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SE KERQ I KKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA
ASQAALGL (SEQ ID NO:30)
[000211] An exemplary AAT-Albumin fusion protein is AAT-HSA Domain 3,
described herein. As shown below, the AAT polypeptide portion of the fusion
protein is
underlined (SEQ ID NO: 2), and the albumin polypeptide is italicized (SEQ ID
NO: 15)
AAT-HSA Domain 3
EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF
AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE
GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA
LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY
LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ
LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF
NKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSEEPQNL/KQNCELFEQLGEYKFQNALLV
RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS
DRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVE
LVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO:31)
[000212] The gene encoding human serum albumin (HSA) was PCR amplified from
human liver cDNA (Zyagen). A mammalian expression vector was generated,
wherein
gene encoding HSA or the domain 3 of HSA, was cloned in frame to the 3' end of
the AAT
encoding gene, containing a mammalian secretion signal sequence up stream of
AAT.
[000213] These expression vectors were transfected into mammalian cells
(specifically
HEK293 or CHO cells) and grown for several days in 8% CO2 at 37 C. The
recombinant
AAT-HSA fusion proteins were purified from the expression cell supernatant
using the
CaptureSelect Alpha-1 Antitrypsin affinity matrix (BAC BV), wherein the
binding buffer
consisted of 20mM Tris, 150mM NaC1, pH 7.4 and the elution buffer consisted of
20mM
Tris, 2M MgC12 pH 7.4.
[000214] Figure 4B shows an SDS-PAGE gel of the AAT-HSA fusion protein The
proteins were visualized by staining with Coomassie blue. The purified AAT-HSA
fusion
proteins were tested for activity by determining their ability to inhibit
neutrophil elastase.
NE inhibitory assays were conducted as described above. Human serum derived
AAT
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(sdAAT) was used as a positive control in these assays. Relative to sdAAT, the
AAT-HS
fusion protein displays similar potency of NE inhibition, demonstrating that
the fusion to
albumin does not dampen the capacity of AAT to inhibit NE (Figure 4C.)
Other Embodiments
[000215] While the invention has been described in conjunction with the
detailed
description thereof, the foregoing description is intended to illustrate and
not limit the scope
of the invention, which is defined by the scope of the appended claims. Other
aspects,
advantages, and modifications are within the scope of the following claims.

Representative Drawing