U.S. patent application number 17/453960 was filed with the patent office on 2022-06-16 for peptides and compositions for treatment of joint damage.
The applicant listed for this patent is Novartis AG. Invention is credited to Kristen Johnson, Jian Shi.
Application Number | 20220184182 17/453960 |
Document ID | / |
Family ID | 1000006172023 |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220184182 |
Kind Code |
A1 |
Johnson; Kristen ; et
al. |
June 16, 2022 |
PEPTIDES AND COMPOSITIONS FOR TREATMENT OF JOINT DAMAGE
Abstract
The present invention provides new protease-resistant
polypeptides, as well as compositions and methods for treating,
ameliorating or preventing conditions related to joint damage,
including acute joint injury and arthritis.
Inventors: |
Johnson; Kristen; (Santee,
CA) ; Shi; Jian; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Family ID: |
1000006172023 |
Appl. No.: |
17/453960 |
Filed: |
November 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16403677 |
May 6, 2019 |
11179442 |
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17453960 |
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15457656 |
Mar 13, 2017 |
10328126 |
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16403677 |
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14772209 |
Sep 2, 2015 |
9649359 |
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PCT/US14/22102 |
Mar 7, 2014 |
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15457656 |
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61938123 |
Feb 10, 2014 |
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61775400 |
Mar 8, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/1891 20130101;
C07K 14/515 20130101; A61K 38/1825 20130101; A61K 45/06 20130101;
C12N 2506/1346 20130101; A61K 35/32 20130101; C12N 2501/17
20130101; A61K 38/225 20130101; A61K 31/192 20130101; A61K 38/014
20130101; A61K 38/1875 20130101; A61K 31/728 20130101; C12N 5/0655
20130101; A61K 31/593 20130101; A61K 31/198 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/515 20060101 C07K014/515; A61K 38/01 20060101
A61K038/01; A61K 38/22 20060101 A61K038/22; A61K 45/06 20060101
A61K045/06; A61K 35/32 20060101 A61K035/32; A61K 31/192 20060101
A61K031/192; A61K 31/198 20060101 A61K031/198; A61K 31/593 20060101
A61K031/593; A61K 31/728 20060101 A61K031/728; C12N 5/077 20060101
C12N005/077 |
Claims
1. A method of ameliorating arthritis or joint injury in a human
patient, comprising: administering to a joint of the patient an
isolated polynucleotide encoding a polypeptide comprising an amino
acid sequence that has at least 95% amino acid sequence identity to
an amino acid sequence selected from any one of SEQ ID NOs: 58-70,
wherein the amino acid at position 423, as determined with
reference to SEQ ID NO:1, is deleted, whereupon expression of the
encoded polypeptide ameliorates or prevents arthritis or joint
injury in the patient.
2. The method of claim 1, wherein the isolated polynucleotide is
complementary DNA (cDNA) or messenger RNA (mRNA).
3. The method of claim 1, wherein the encoded polypeptide further
comprises a secretion signal sequence.
4. The method of claim 1, wherein the encoded polypeptide further
comprises a fusion domain selected from the group consisting of a
polyhistidine, Glu-Glu, glutathione S transferase (GST),
thioredoxin, protein A, protein G, an immunoglobulin heavy chain
constant region (Fc), maltose binding protein (MBP), a human serum
albumin (HSA), a FLAG tag, influenza virus haemagglutinin (HA), and
a c-myc tag.
5. The method of claim 1, wherein the isolated polynucleotide is
codon-optimized for expression in a host cell.
6. The method of claim 1, wherein the isolated polynucleotide is
operably linked to a promoter or other regulatory sequence.
7. A method of ameliorating arthritis or joint injury in a human
patient, comprising: administering to a joint of the patient an
expression vector comprising a polynucleotide encoding a
polypeptide comprising an amino acid sequence that has at least 95%
amino acid sequence identity to an amino acid sequence selected
from any one of SEQ ID NOs: 58-70, wherein the amino acid at
position 423, as determined with reference to SEQ ID NO:1, is
deleted, whereupon expression of the encoded polypeptide
ameliorates or prevents arthritis or joint injury in the
patient.
8. A kit comprising an isolated polynucleotide encoding a
polypeptide comprising an amino acid sequence that has at least 95%
amino acid sequence identity to an amino acid sequence selected
from any one of SEQ ID NOs: 58-70, wherein the amino acid at
position 423, as determined with reference to SEQ ID NO:1, is
deleted, wherein the kit comprises a first container comprising the
encoded polypeptide, and a second container having an aqueous
reconstitution formula.
9. The kit according to claim 8, wherein the encoded polypeptide in
the first container is in dry form.
10. The kit according to claim 8, wherein one container comprises a
single chambered pre-filled syringe.
11. The kit according to claim 8, wherein the containers are
encompassed as a multi-chambered pre-filled syringe.
Description
BACKGROUND OF THE INVENTION
[0001] Osteoarthritis (OA) represents the most common
musculoskeletal disorder. Approximately 40 million Americans are
currently affected; a number predicted to increase to 60 million
within the next twenty years as a result of aging population and an
increase in life expectancy, making it the fourth leading cause of
disability. OA is characterized by a slow degenerative breakdown of
a joint including both articular cartilage (containing the cells
and matrix which produce lubrication and cushioning for the joint)
and subchondral bone underlying the articular cartilage. OA can be
considered a consequence of various etiologic factors. For example,
it can be caused by abnormal biomechanical stress or genetic or
acquired abnormalities of articular cartilage or bone. Current OA
therapies include pain relief with oral NSAIDs or selective
cyclooxygenase 2 (COX-2) inhibitors, intra-articular (IA) injection
with agents such as corticosteroids and hyaluronan, and surgical
approaches.
[0002] Joint damage, e.g., acute joint injury, such as a meniscal
or ligament tear, or an intra-articular fracture can also lead to
arthritis, e.g., posttraumatic arthritis. Because articular
cartilage has a limited ability to repair, even small undetectable
damage can often get worse over time and lead to OA. Current
treatments for joint injury can include surgery and other invasive
procedures focused on regeneration of damaged joints as well as
treatment with agents to reduce pain and inflammation.
[0003] Mesenchymal stem cells (MSCs) are present in adult articular
cartilage and upon isolation can be programmed in vitro to undergo
differentiation to chondrocytes and other mesenchymal cell
lineages, and may be used for cartilage regeneration. In part, the
process is regulated by growth factors (TGF.beta.s, BMPs), serum
conditions and cell-cell contact. WO2011/008773 describes peptide
compositions and use of those compositions for treating or
preventing arthritis and joint injury and for inducing
differentiation of mesenchymal cells into chondrocytes.
Additionally, WO2012/129562 describes small molecule compounds,
compositions and use of those compositions for amelioration of
arthritis and joint injury and for inducing differentiation of
mesenchymal cells into chondrocytes.
[0004] Though surgical techniques, and regenerative technology have
made some progress in restoration of cartilage, slowing
degeneration, and improved repair of joint damage, a continued need
exists for improvement of compositions and methods for effective
cartilage regeneration, treatment of joint damage and amelioration
or prevention of OA.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention relates to the identification of new
polypeptide and protein variants of angiopoietin-like 3 (ANGPTL3)
that have improved pharmaceutical properties, e.g., are more
stable, less susceptible to proteolysis and enzymatic degradation
than wild-type ANGPTL3. Also provided are pharmaceutical
compositions and methods for treatment of joint damage or joint
injury, and methods of ameliorating or preventing arthritis, joint
damage or joint injury in a mammal.
[0006] Thus, provided are protease-resistant polypeptides
comprising an amino acid sequence that has at least 95% amino acid
sequence identity, or at least 96%, 97%, 98%, 99% or 100% amino
acid sequence identity to an amino acid sequence selected from any
one or more of the sequences of TABLE 1, and as further described
herein. The modified polypeptides of TABLE 1 include an amino acid
that is a polar amino acid other than K or R at position 423, as
determined with reference to the full length ANGPTL3 polypeptide
sequence, SEQ ID NO:1. In some embodiments the amino acid at
position 423 as determined with reference to SEQ ID NO:1 is Q or S.
In certain embodiments the amino acid at position 423 as determined
with reference to SEQ ID NO:1 is Q. In certain embodiments the
amino acid at position 423 as determined with reference to SEQ ID
NO:1 is S. In certain embodiments the amino acid at position 423 as
determined with reference to SEQ ID NO:1 is deleted. In addition,
provided polypeptides have chondrogenic activity.
[0007] In some embodiments, the polypeptide comprises a sequence
having at least 95% identity or at least 96%, 97%, 98%, 99% or 100%
to any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65,
SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID
NO:70. In some embodiments the polypeptide comprises a sequence
having at least 95% identity or at least 96%, 97%, 98%, 99% or 100%
to any one of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ ID
NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, and
SEQ ID NO:64. In some embodiments, the polypeptide comprises any
one of the sequences of TABLE 1. In some embodiments, the
polypeptide comprises any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID
NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, and SEQ ID NO:70. In some embodiments the polypeptide
comprises any one of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ
ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ
ID NO:61, SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:64. In some
embodiments, the polypeptide is any one of the sequences of TABLE
1. In some embodiments, the polypeptide is any one of SEQ ID NO:30,
SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID
NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70. In some embodiments
the polypeptide is any one of SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,
SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ ID NO:59, SEQ ID
NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, and SEQ ID
NO:64.
[0008] Polypeptides of the invention may incorporate one or more
chemical modifications (e.g., PEGylation). In some embodiments,
polypeptides of the invention may comprise a heterologous peptide
as a fusion protein, which may optionally be fused at the
amino-terminal or the carboxy-terminal end of the polypeptide. Also
provided are polynucleotides encoding the polypeptides of the
invention; vectors containing polynucleotides encoding the
polypeptides; and host cells comprising such vectors.
[0009] The present invention also provides pharmaceutical
compositions comprising the polypeptides of the invention and a
pharmaceutically acceptable carrier. Such compositions can be used
in methods provided herein for treating, ameliorating or preventing
arthritis or joint damage in a patient, where the method comprises
administering to a joint of a patient a therapeutically effective
amount of a pharmaceutical composition of the invention. Examples
of conditions that can benefit from such methods include, but are
not limited to arthritis (e.g., osteoarthritis, traumatic
arthritis), and joint damage (e.g., acute joint injury).
[0010] The present invention further provides methods of treating a
subject comprising administering a therapeutically effectively
amount of a polypeptide of the invention. Provided methods include
treating a subject having or at risk of having joint damage and/or
arthritis, comprising administering to the subject a
therapeutically effective amount of one or more polypeptides of the
invention or a pharmaceutical composition thereof. Still further
provided are methods of inducing differentiation of mesenchymal
stem cells into chondrocytes, comprising contacting mesenchymal
stem cells with an effective amount of a polypeptide of the
invention to induce differentiation of the mesenchymal stem cells
into chondrocytes.
[0011] These and other aspects of the invention, including
additional features, advantages, and embodiments of the invention,
will be described and elucidated in further detail in the following
detailed description and appended claims of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts a schematic of hANGPTL3 proteins engineered
to improve protein stability and enhance proteolytic resistance.
During protein production of wild type protein and peptide
sequences, 100% cleavage was observed between Lys423 and Ser424. To
mitigate proteolysis, various mutant peptides were generated
wherein Lys 423 was mutated to Gln or Ser; or Ser424 was mutated to
Thr; or Lys 423 was deleted.
[0013] FIG. 2A and FIG. 2B depict graphical representations of
expression of cartilage specific proteins in the presence or
absence of ANGPTL3 and engineered constructs. Fixed cells were
stained for FIG. 2A Pro-collagen Type 2A quantification (PIIANP) or
FIG. 2B Type II collagen quantification to determine the % of cells
differentiating into chondrocytes following treatment as described
in the Exemplification.
[0014] FIG. 2C depicts graphical representation of quantification
of angiogenesis assays in the presence or absence of ANGPTL3 or
engineered construct as compared to a positive control protein,
bFGF. Total tube length and number of branch points were
quantitative measurements of angiogenesis. Although others have
reported angiogenic activity in ANGPTL3, and this study confirms
activity as well as that of FGF; the results indicated no
significant activity is retained in a Cterminal ANGPTL3
construct.
[0015] FIG. 3A and FIG. 3B are graphical representations showing an
increase in expression of cartilage specific proteins in the
presence of ANGPTL3 or engineered constructs. FIG. 3A. Cells were
evaluated ten days following treatment using qRT-PCR to measure RNA
expression for cartilage specific proteins following treatment as
described. Lubricin, aggrecan and Sox9 represent cartilage related
proteins; IGF and IFITM1 represent differentiation potential, and
osteocalcin and type X collagen represent bone/fibrosis related
proteins. FIG. 3B. Cells were evaluated three days following
treatment as described. Increased aggrecan expression was seen
following treatment with engineered construct or wild type ANGPTL1
C-terminal region polypeptide.
[0016] FIG. 4A-FIG. 4C depict graphical representations of
chondro-protective activity of ANGPTL3 and engineered constructs.
FIG. 4A: Glycosaminoglycan (GAG) release, an indicator of matrix
damage, was inhibited with increasing amount of ANGPTL3 and mutant
constructs. Ex vivo GAG release (an indicator of matrix damage)
inhibition assays were performed using bovine cartilage treated in
the presence or absence of constructs as described. FIG. 4B and
FIG. 4C: NO release was inhibited with increasing amount of ANGPTL3
and engineered constructs as indicated. Chondrocytes were treated
in the presence or absence of constructs as described followed by
Greiss reaction assays to determine the inhibition of NO release as
an indicator of chondro-protection.
[0017] FIG. 5A and FIG. 5B depict a graphical representation
showing an inhibition of type X collagen expression (an indicator
of fibrotic cartilage formation activity) in the presence of
constructs under hypertrophic conditions. Primary chondrocytes were
treated in the presence of absence of constructs under hypertrophic
conditions as described, followed by determination of type X
collagen expression, assessed by immunofluorescence, as a
measurement of formation of fibrotic and hypertrophic
cartilage/chondrocyte differentiation. FIG. 5A depicts results of
wild type C-terminal ANGPTL3 or engineered construct. FIG. 5B
depicts results of C-terminal ANGPTL3 (WT) or engineered constructs
242KQ or 242Kdel or C-terminal ANGPTL1.
[0018] FIG. 6A and FIG. 6B depict a schematic representation of the
dosing paradigm (FIG. 6A), followed by a graphical representation
(FIG. 6B) of the improvement in joint severity after treatment with
mouse ANGPTL3 (17-460) as measured by cartilage erosion score of
the lateral femoral condyle.
[0019] FIG. 7A and FIG. 7B is a graphical representation of
incapacitance measurements (an indicator of pain) in mice following
surgical induction of cartilage damage and subsequent treatment
with ANGPTL3 constructs once weekly for three weeks (beginning on
day 7). FIG. 7A represents incapacitance measurements on day 35
following surgery; and FIG. 7B represents measurements taken on day
56 following surgery.
[0020] FIG. 8. is a graphical representation of the total joint
severity score and improvement in severity to cartilage damage
induced by collagenase in mice following 3 once weekly treatments
(days 7, 14 and 21) of ANGPTL3 constructs (indicated).
[0021] FIG. 9A and FIG. 9B depict results in a rat meniscal tear
model of joint damage following treatment with engineered ANGPTL3
construct. FIG. 9A is a graphical representation of the
proteoglycan content in joints five weeks following treatment; FIG.
9B is a graphical representation of the femoral joint severity
score five weeks following treatment. Results illustrate
improvement to cartilage damage induced by surgical severing of the
meniscus in rats following 3 once weekly treatments (days 7, 14 and
21) of ANGPTL3 constructs (indicated).
[0022] FIG. 10A and FIG. 10B depict results in a rat meniscal tear
model of joint damage following treatment with engineered ANGPTL3
construct. FIG. 10A is a graphical representation of percent of in
vivo repair as measured by severity, safranin 0 intensity,
cartilage area and cartilage thickness. FIG. 10B is a graphical
representation of of incapacitance measurements (an indicator of
pain) in rats following surgical induction of cartilage damage and
subsequent treatment.
[0023] FIG. 11 is a graphical representation of the total gross
severity score to illustrate improvement of cartilage damage
induced by surgical disruption of the medial meniscus in dogs
following biweekly dosing beginning on day 4 (each of the 1.5
ug/dose or 15 ug/dose) or a single 30 ug dose) given on day 7
only.
[0024] FIG. 12 provides a sequence alignment of the C-terminal
domains of human angiopoeitin like family members
hANGPTL1(271-491)/hANGPTL3(241-460)/hANGPTL4(179-406)
DETAILED DESCRIPTION
[0025] The present invention is based, at least in part, on the
identification of Angiopoietin-like 3 (ANGPTL3) polypeptides that
stimulate chondrocyte differentiation of mesenchymal stem cells and
that are resistant to cleavage by proteases (e.g., trypsin-like
proteases). WO2011/008773, describes ANGPTL3 peptide compositions
and use of peptide compositions for treating or preventing
arthritis and joint injury and for inducing differentiation of
mesenchymal cells into chondrocytes. We found that wild type
ANGPTL3 proteins are subject to protease clipping and instability
and have identified sequence variants to mitigate this effect. The
present invention thus provides improved peptide compositions for
repairing cartilage. In particular, provided are ANGPTL3 peptides
modified in accordance with the present invention to have increased
protease-resistance as compared to a wildtype ANGPTL3 polypeptide.
Also provided are compositions and methods for administration of
ANGPTL3 polypeptides to prevent or ameliorate arthritis or joint
injury by administering a polypeptide of the invention into a
joint, a cartilage tissue or a cartilage proximal tissue, or
systemically. Further, the invention provides compositions and
methods for induction of mesenchymal stem cell differentiation into
chondrocytes.
Definitions
[0026] The term "protease-resistant" as used herein refers to a
polypeptide comprising a modification that renders the polypeptide
less susceptible to cleavage by a trypsin-like protease than a
corresponding non-modified wildtype polypeptide. In specific
embodiments a protease-resistant polypeptide is an ANGPTL3
polypeptide that has an amino acid substitution, relative to a
native wildtype peptide sequence, at an R or a K residue.
[0027] "ANGPTL3" refers to a member of the angoipoietin protein
family. An amino acid sequence of ANGPTL3 (GenBank Accession No.
NP_055310.1) is set forth in SEQ ID NO:1; and the corresponding
polynucleotide sequence of which is set forth as SEQ ID NO: 2 (NCBI
reference sequence number NM014495.2, wherein the ANGPTL3 coding
sequence comprises nt 52-1434 of SEQ ID NO:2). "ANGPTL3
polypeptide" refers to a naturally occurring expressed polypeptide.
For the purposes of the present disclosure, the numbering of an
amino acid is typically determined with reference to the
full-length wildtype human ANGPTL3 polypeptide sequence (SEQ ID
NO:1). Thus, in embodiments in which a polypeptide of the invention
contains only a C-terminal portion of full-length ANGPTL3, but not
the N-terminal portion, although the peptide is less than 460 amino
acids in length, the numbering of the positions is based on SEQ ID
NO:1. For example, reference to position 423 of an ANGPTL3
polypeptide of the invention refers to position 423 of SEQ ID NO:1,
even though the ANGPTL3 polypeptide of the invention itself may
only be 200 amino acids in length. In determining an amino acid in
a sequence of interest that "corresponds to" a position in a
reference sequence, such as SEQ ID NO:1, this is performed by
optimally aligning the sequences, e.g., using the default CLUSTAL
alignment parameters or default BLAST 2 alignment parameters and
comparing the sequences. For example, position 423 in a sequence of
interest that is "determined with reference to SEQ ID NO:1", or an
amino acid that "corresponds to" position 423 of SEQ ID NO:1, means
the amino acid that aligns with position 423 of SEQ ID NO:1 when
the sequence of interest is optimally aligned with SEQ ID NO:1.
[0028] The terms "peptidomimetic" and "mimetic" refer to a
synthetic chemical compound that has substantially the same
functional characteristics of a naturally or non-naturally
occurring polypeptide (e.g., ANGPTL3), but different (though
typically similar) structural characteristics. Peptide analogs are
commonly used in the field as non-peptide active compounds (e.g.,
drugs) with properties analogous to those of a template peptide.
Such non-peptide compounds are termed "peptide mimetics" or
"peptidomimetics" (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber
and Freidinger TINS p. 392 (1985); and Evans et al. J Med. Chem.
30:1229 (1987)). Peptide mimetics that are structurally similar to
therapeutically useful peptides may be used to produce an
equivalent or enhanced therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to a paradigm
polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity), such as found in a polypeptide of
interest, but have one or more peptide linkages optionally replaced
by a linkage selected from the group consisting of, e.g.,
--CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-- (cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--,
and --CH.sub.2SO--. A mimetic can be either entirely composed of
synthetic, non-natural analogues of amino acids, or, is a chimeric
molecule of partly natural peptide amino acids and partly
non-natural analogs of amino acids. A mimetic can also incorporate
any amount of natural amino acid conservative substitutions as long
as such substitutions also do not substantially alter the mimetic's
structure and/or activity. For example, a mimetic composition is
within the scope of the invention if it is capable of chondrogenic
activity of an ANGPTL3 polypeptide.
[0029] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. Polypeptides, peptides, and proteins of the
invention comprise protease resistant ANGPTL3 peptidomimetics
having chondrogenic activity.
[0030] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to naturally occurring
amino acids. Naturally occurring amino acids are those encoded by
the genetic code, as well as those amino acids that are later
modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Naturally encoded amino acids are
the 20 common amino acids (alanine, arginine, asparagine, aspartic
acid, cysteine, glutamine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine, and valine) as well as
pyrrolysine, pyrroline-carboxy-lysine, and selenocysteine.
[0031] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every polypeptide sequence
herein which is encoded by a polynucleotide encompasses every
possible silent variation of the nucleic acid. One of skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine, and TGG, which is
ordinarily the only codon for tryptophan) can be modified to yield
a functionally identical molecule. Accordingly, each silent
variation of a nucleic acid that encodes a polypeptide is implicit
in each described sequence.
[0032] One of skill will recognize that individual substitutions,
deletions or additions to a nucleic acid, peptide, polypeptide, or
protein sequence which alters, adds or deletes a single amino acid
or a small percentage of amino acids with reference to an original
encoded amino acid sequence results in a "conservatively modified
variant" where the alteration produces substitution of an amino
acid with a chemically similar amino acid and/or a polypeptide
sequence that produces a structurally similar protein having
similar functional activity to the original protein. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. Such conservatively modified variants are in
addition to and do not exclude polymorphic variants, interspecies
homologs, and alleles of the invention.
[0033] The term "conservative amino acid substitutions" refers to
the substitution (conceptually or otherwise) of an amino acid from
one such group with a different amino acid from the same group. One
example of substitutions is based on analyzing the normalized
frequencies of amino acid changes between corresponding proteins of
homologous organisms (see, e.g., Schulz, G. E. and R. H. Schirmer,
Principles of Protein Structure, Springer-Verlag). According to
such analyses, groups of amino acids may be defined where amino
acids within a group exchange preferentially with each other and,
therefore, resemble each other most in their impact on the overall
protein structure (see, e.g., Schulz, G. E. and R. H. Schirmer,
Principles of Protein Structure, Springer-Verlag). One example of a
set of amino acid groups defined in this manner include: (i) a
charged group, consisting of Glu and Asp, Lys, Arg and His; (ii) a
positively-charged group, consisting of Lys, Arg and His; (iii) a
negatively-charged group, consisting of Glu and Asp; (iv) an
aromatic group, consisting of Phe, Tyr and Trp; (v) a nitrogen ring
group, consisting of His and Trp; (vi) a large aliphatic nonpolar
group, consisting of Val, Leu and Ile; (vii) a slightly-polar
group, consisting of Met and Cys; (viii) a small-residue group,
consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro; (ix)
an aliphatic group consisting of Val, Leu, Ile, Met and Cys; and
(x) a small hydroxyl group consisting of Ser and Thr. Other
examples of conservative substitutions based on shared physical
properties are the substitutions within the following groups:1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,
Creighton, Proteins (1984)).
[0034] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the amino acid sequence or polynucleotide sequence
in the comparison window may comprise additions or deletions (i.e.,
gaps) as compared to the reference sequence (e.g., a polypeptide of
the invention), which does not comprise additions or deletions, for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0035] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same sequences. Two
sequences are "substantially identical" if two sequences have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 95% identity, optionally 96%, 97%, 98%, or 99%
identity over a specified region, or, when not specified, over the
entire sequence), when compared and aligned for maximum
correspondence over a comparison window, or designated region as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. The invention
provides polypeptides that are substantially identical to the
polypeptides, respectively, exemplified herein (e.g., any of SEQ ID
NOs: 11-42), as well as uses thereof including but not limited to
use for treating or preventing arthritis or joint injury.
Optionally, for nucleic acids, the identity exists over a region
that is at least about 150 nucleotides in length, or more
preferably over a region that is 300 to 450 or 600 or more
nucleotides in length, or the entire length of the reference
sequence. For amino acid sequence, optionally, identity exists over
a region that is at least about 50 amino acids in length, or more
preferably over a region that is 100 to 150 or 200 or more amino
acids in length, or the entire length of the reference
sequence.
[0036] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
entered into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. Default program parameters can be used, or alternative
parameters can be designated. The sequence comparison algorithm
then calculates the percent sequence identities for the test
sequences relative to the reference sequence, based on the program
parameters.
[0037] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 50 to 600, usually about 75 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA), or by manual
alignment and visual inspection (see, e.g., Ausubel et al., Current
Protocols in Molecular Biology (1995 supplement)).
[0038] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0039] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0040] The term "isolated," when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is purified to be
essentially free of other cellular components with which it is
associated in the natural state. It is often in a homogeneous or
nearly homogeneous state. It can be in either a dry or aqueous
solution. Purity and homogeneity may be determined using analytical
chemistry techniques known and used typically in the art, e.g.,
polyacrylamide gel electrophoresis, high performance liquid
chromatography, etc. A protein that is the predominant species
present in a preparation is substantially purified. The term
"purified" in some embodiments denotes that a protein gives rise to
essentially one band in an electrophoretic gel. Typically, it means
that a protein is at least 85% pure, more preferably at least 95%
pure, and most preferably at least 99% pure.
[0041] The term "hyaluronic acid" are used herein to include
derivatives of hyaluronic acid that include esters of hyaluronic
acid, salts of hyaluronic acid and also includes the term
hyaluronan. The designation also includes both low and high
molecular weight forms of hyaluronans and crosslinked hyaluronans
or hylans. Examples of such hyaluronans are Synvisc.TM. (Genzyme
Corp. Cambridge, Mass.), ORTHOVISC.TM. (Anika Therapeutics, Woburn,
Mass.), HYALGAN.TM. (Sanofi-Synthelabo Inc., Malvern, Pa.), and
ProVisc (Alcon/Novartis).
[0042] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise.
Angiopoietin-Like 3 Protease-Resistant Polypeptides
[0043] Angiopoietin-like 3 is a member of the angiopoietin-like
family of secreted factors. It is predominantly expressed in the
liver, and has the characteristic structure of angiopoietins,
consisting of a signal peptide, N-terminal coiled-coil domain (CCD)
and the C-terminal fibrinogen (FBN)-like domain. Angiopoietin-like
3 was shown to bind .alpha.V/.beta.3 integrins and FBN-like domain
alone was sufficient to induce endothelial cell adhesion and in
vivo angiogenesis (Camenisch et al., J. Biol. Chem. 277:
17281-17290, 2002). Endogenous ANGPTL3 is generally cleaved in vivo
into amino-terminal and carboxy-terminal fragments. As summarized
above and further described herein, the present invention
contemplates use of various protease-resistant ANGPTL3 proteins
having chondrogenic activity.
[0044] In some embodiments, an isolated polypeptide comprises an
amino acid sequence that has at least 95% identity, or at least
96%, 97%, 98%, or 99% identity, to an amino acid sequence selected
from any one of the sequences of TABLE 1, wherein the polypeptide
comprises an amino acid that is a polar amino acid other than K or
R at position 423 or the polypeptide comprises a deletion at
position 423, as determined with reference to SEQ ID NO:1. The
polypeptides of the invention have chondrogenic activity. In some
embodiments, a polypeptide comprises the amino acid sequence that
has at least 95% identity, or at least or at least 96%, 97%, 98%,
or 99% identity, to an amino acid sequence selected from any one of
SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65, SEQ ID NO:66,
SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70. wherein
the polypeptide comprises an amino acid that is a polar amino acid
other than K or R at position 423 or the polypeptide comprises a
deletion at position 423, as determined with reference to SEQ ID
NO:1, and the polypeptide has chondrogenic activity. In a further
embodiment, a polypeptide comprises the amino acid sequence that
has at least 95% identity, or at least or at least 96%, 97%, 98%,
or 99% identity, to an amino acid sequence selected from any one of
SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ
ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,
SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ ID NO:59, SEQ ID
NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:64
wherein the polypeptide comprises an amino acid that is a polar
amino acid other than K or R at position 423, as determined with
reference to SEQ ID NO:1, and the polypeptide has chondrogenic
activity.
[0045] In some embodiments, an isolated polypeptide comprises an
amino acid sequence selected from any one of the sequences of TABLE
1, wherein the polypeptide comprises an amino acid that is a polar
amino acid other than K or R at position 423 or the polypeptide
comprises a deletion at position 423, as determined with reference
to SEQ ID NO:1, and the polypeptide has chondrogenic activity. In
some embodiments, a polypeptide comprises an amino acid sequence
selected from any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ
ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,
or SEQ ID NO:70 wherein the polypeptide comprises an amino acid
that is a polar amino acid other than K or R at position 423 or the
polypeptide comprises a deletion at position 423, as determined
with reference to SEQ ID NO:1, and the polypeptide has chondrogenic
activity. In a further embodiment, a polypeptide comprises an amino
acid sequence selected from any one of SEQ ID NO:14, SEQ ID NO:15,
SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ
ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID
NO:62, SEQ ID NO:63, or SEQ ID NO:64 wherein the polypeptide
comprises an amino acid that is a polar amino acid other than K or
R at position 423, as determined with reference to SEQ ID NO:1, and
the polypeptide has chondrogenic activity.
[0046] In some embodiments, an isolated polypeptide has at least
95% identity, or at least 96%, 97%, 98%, or 99% identity, to an
amino acid sequence selected from any one of the sequences of TABLE
1, wherein the polypeptide comprises an amino acid that is a polar
amino acid other than K or R at position 423 or the polypeptide
comprises a deletion at position 423, as determined with reference
to SEQ ID NO:1, and the polypeptide has chondrogenic activity. In
some embodiments, a polypeptide has at least 95% identity, or at
least or at least 96%, 97%, 98%, or 99% identity, to an amino acid
sequence selected from any one of SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ
ID NO:69, or SEQ ID NO:70, wherein the polypeptide comprises an
amino acid that is a polar amino acid other than K or R at position
423 or the polypeptide comprises a deletion at position 423, as
determined with reference to SEQ ID NO:1, and the polypeptide has
chondrogenic activity. In a further embodiment, a polypeptide has
at least 95% identity, or at least or at least 96%, 97%, 98%, or
99% identity, to an amino acid sequence selected from any one of
SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ
ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,
SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ ID NO:59, SEQ ID
NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID NO:64
wherein the polypeptide comprises an amino acid that is a polar
amino acid other than K or R at position 423, as determined with
reference to SEQ ID NO:1, and the polypeptide has chondrogenic
activity.
[0047] In some embodiments, an isolated polypeptide is an amino
acid sequence selected from any one of the sequences of TABLE 1. In
some embodiments, a polypeptide is an amino acid sequence selected
from any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:65,
SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID
NO:70. In a further embodiment, a polypeptide is an amino acid
sequence selected from any one of SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO: 16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, or SEQ ID
NO:64.
TABLE-US-00001 TABLE 1 ANGPTL3 variant constructs SEQ ID Construct
Sequence 14 207KQ
IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAI
RPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGL
EKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNA
IPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPE
RRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 15 207KS
IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAI
RPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGL
EKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNA
IPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPE
RRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 16 225KQ
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI
KSTKMLIHPTDSESFE 17 225KS
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI
KSTKMLIHPTDSESFE 18 225ST
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSI
KSTKMLIHPTDSESFE 19 226KQ
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK
STKMLIHPTDSESFE 20 226KS
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK
STKMLIHPTDSESFE 21 228KQ
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST
KMLIHPTDSESFE 22 228KS
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST
KMLIHPTDSESFE 23 228ST
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSIKST
KMLIHPTDSESFE 24 233KQ
EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 25
233KS EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 26
241KQ GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 27 241KS
GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 28 242KQ
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 29 242KS
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 30 225-455KQ
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD
31 225-455KS
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD
32 226-455KQ
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD
33 226-455KS
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD
34 228-455KQ
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 35
228-455KS FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 36
233-455KQ EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 37
233-455KS EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 38
241-455KQ GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 39 241-455KS
GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 40 242-455KQ
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 41 242-455KS
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 58 207Kdel
IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAI
RPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGL
EKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNA
IPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPER
RRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 59 225Kdel
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK
STKMLIHPTDSESFE 60 226Kdel
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS
TKMLIHPTDSESFE 61 228Kdel
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK
MLIHPTDSESFE 62 233Kdel
EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TDSESFE 63
241Kdel GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 64 242Kdel
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 65 225-
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG 455Kdel
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK STKMLIHPTD
66 226- TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
455Kdel PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS TKMLIHPTD
67 228- FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
455Kdel TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK MLIHPTD 68
233- EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
455Kdel RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TD 69 241-
GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF 455Kdel
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 70 242-
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN 455Kdel
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD
[0048] Modified ANGPTL3 polypeptides of the invention have at least
one substitution in the C-terminal portion of the polypeptide to
render the polypeptide protease resistant. The substitution is at
an R or K residue so that polypeptides have increased resistance,
e.g., to trypsin-like proteases. Any amino acid may be substituted
for an R or K in a protease resistant ANGPTL3 polypeptide of the
invention. In some embodiments, a substitution is a polar amino
acid, e.g., H, N, Q, S, T, A, or Y. In some embodiments, a
substitution is H, N, Q, S, T, or Y. In some embodiments, a
substitution is S or Q. In some embodiments, the substitution is Q.
In some embodiments the substitution is S. In some embodiments, a
protease-resistant peptide has an amino acid at position 423, with
reference to SEQ ID NO:1, that is other than K or R. In some
embodiments, a polypeptide of the invention comprises an amino acid
at position 423 that is a polar amino acid. For example, the amino
acid at position 423 may be Q or S or another polar amino acid. In
certain embodiments a polypeptide of the invention has a Q at
position 423. In other embodiments a polypeptide of the invention
has an S at position 423. In some embodiments, in addition to
substitution at 423, the protease-resistant peptide has a
substitution of another R or K in the C-terminus of SEQ ID NO:1, or
a variant thereof, wherein the substitution is a polar amino acid
other than R or K. In some embodiments, the substitution at
position 423 as determined with reference to SEQ ID NO:1, is Q or
S. In still other embodiments a polypeptide of the invention has a
deletion at position 423 as determined with reference to SEQ ID
NO:1.
[0049] In some embodiments, a polypeptide of the invention is 250
amino acids or less in length and comprises the amino acid sequence
of SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID
NO:70.
[0050] In some embodiments, the invention provides for use of
full-length protease-resistant, chondrogenic ANGPTL3 proteins. In
some embodiments, the invention provides for protease-resistant
ANGPTL3 proteins comprising a C-terminal portion of the ANGPTL3
sequence, or a chondrogenic variant thereof. In certain embodiments
ANGPTL3 proteins lack the the amino-terminal end of the native
protein. In some embodiments, protease-resistant ANGPTL3 proteins
of the invention lack the CCD domain and/or lacks significant CCD
activity. Thus, in some embodiments, the protease-resistant ANGPTL3
proteins of the invention comprise at least a fragment (e.g., at
least 100, 150, 200, 220 or 215 contiguous amino acids) of a human
ANGPTL3 protein carboxy-terminal domain, or a substantially
identical sequence to the human carboxy-terminal ANGPTL3 protein
sequence, wherein the polypeptide and variants thereof retains
chondrogenic activity. In some embodiments, a protease-resistant
polypeptide of the invention lacks at least a portion of the
C-terminal sequence, e.g., lacks 5, 10, 15, or 20 amino acids from
the C-terminal end of SEQ ID NO:1 (i.e., lacks 456-460, 451-460,
446-460 or 441-460 of SEQ ID NO:1).
[0051] In some embodiments, a protease-resistant ANGPTL3
polypeptide of the invention comprises contiguous amino acids
corresponding to the amino acid regions: amino acids 241-455, or
241-460 of SEQ ID NO:1; amino acids 242-455, or 242-460 of SEQ ID
NO:1; amino acids 233-455 or 233-460 of SEQ ID NO:1; amino acids
228-455 or 228-460 of SEQ ID NO: 1, amino acids 226-455- or 226-260
or amino acids 225-455- or 225-260 of SEQ ID NO:1 in which an amino
acid is substituted for an R or K or a single residue is deleted.
In some embodiments, a substitution is at position 423 as
determined with reference to SEQ ID NO:1. In some embodiments a
deletion is at position 423 as determined with reference to SEQ ID
NO:1. In some embodiments, a protease-resistant polypeptide
comprises contiguous amino acids corresponding to the amino acid
regions 207-455 or 207-460 of SEQ ID NO:1 in which an amino acid is
substituted for R or K or a single residue is deleted. In some
embodiments, a substitution or deletion is at position 423. In some
embodiments, a substitution is a polar amino acid, e.g., H, N, Q,
S, T, A, or Y. In some embodiments, a substitution is H, N, Q, S,
T, or Y. In some embodiments, a substitution is S or Q. In some
embodiments, a substitution is Q. In certain embodiments a deletion
at position 423 relative to SEQ ID NO:1 is included.
[0052] The invention additionally provides a protease-resistant
polypeptide, wherein the polypeptide comprises an amino acid
sequence having at least 95% identity, or at least 96%, 97%, 98%,
or 99% identity, to amino acids 240-454 of SEQ ID NO:1, amino acids
241-455 of SEQ ID NO: 1, or amino acids 242-455 of SEQ ID NO:1 with
a substitution or deletion at the amino acid corresponding to
position 423 of SEQ ID NO:1, where the substituted amino acid is
not R, and wherein the polypeptide has chondrogenic activity. In
other embodiments, the polypeptide comprises amino acids 240-454 of
SEQ ID NO:1, amino acids 241-455 of SEQ ID NO:1, or amino acids
242-455 of SEQ ID NO:1, each polypeptide with a substitution or
deletion at the amino acid corresponding to position 423 of SEQ ID
NO:1, where the substituted amino acid is Q or S.
[0053] In some embodiments, a protease-resistant ANGPTL3
polypeptide of the invention comprises an amino acid sequence
having at least 95%, or at least 96%, at least 97%, at least 98%,
or at least 99% identity to amino acids amino acids 242-455 or
242-460 of SEQ ID NO:1; 241-455 or 241-460 of SEQ ID NO: 1; amino
acids 233-455 or 233-460 of SEQ ID NO: 1; amino acids 228-455 or
228-460 of SEQ ID NO:1, amino acids 226-455- or 226-260 of SEQ ID
NO:1, or amino acids 225-455- or 225-260 of SEQ ID NO:1 in which an
amino acid is substituted for an R or K, or an R or K is deleted.
In some embodiments, the substitution or deletion is at position
423. In some embodiments, a substitution is a polar amino acid,
e.g., H, N, Q, S, T, A, or Y. In some embodiments, a substitution
is H, N, Q, S, T, or Y. In some embodiments, the substitution is S
or Q. In some embodiments, the substitution is a Q. In certain
embodiments there is a deleted residue at position 423 relative to
SEQ ID NO:1.
[0054] In some embodiments, a protease-resistant ANGPTL3
polypeptide of the invention is 250 or 240 or fewer amino acids in
length and comprises the amino acid sequence of SEQ ID NO:16, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24,
SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:59, SEQ ID
NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ
ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,
and SEQ ID NO:70. In some embodiments, a protease-resistant ANGPTL3
polypeptide of the invention is 230 or 225 or fewer amino acids in
length and comprises the amino acid sequence of SEQ ID NO:24, SEQ
ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ
ID NO:68, SEQ ID NO:69, or SEQ ID NO:70.
[0055] In some embodiments the protease resistant ANGPTL3 proteins
of the invention comprise an amino acid sequence having at least
95% identity, or at least 96%, 97%, 98%, or 99% identity, to the
C-terminal canine, bovine, or equine ANGPTL3 protein sequence. In
some embodiments, the protease-resistant ANGPTL3 proteins of the
invention comprise at least a fragment (e.g., at least 100, 150,
200, 215 contiguous amino acids) of a native canine (SEQ ID NO:4),
equine (SEQ ID NO:5), or bovine (SEQ ID NO:6) ANGPTL3 protein
sequence, or a substantially identical sequence to the native
canine, bovine, or equine ANGPTL3 protein sequence wherein the
polypeptide comprises an amino acid that is a polar amino acid
other than K or R at position 423 or the polypeptide comprises a
deletion at position 423, as determined with reference to SEQ ID
NO:1, and the polypeptide has chondrogenic activity. In some
embodiments, an isolated polypeptide comprises an amino acid
sequence having at least 95% identity, or at least 96%, 97%, 98%,
or 99% identity, to SEQ ID NO:42 or SEQ ID NO:43, wherein the
polypeptide comprises an amino acid that is a polar amino acid
other than K or R at position 423 or the polypeptide comprises a
deletion at position 423, as determined with reference to SEQ ID
NO:1, and the polypeptide has chondrogenic activity. In some
embodiments, a polypeptide has at least 95% identity, or at least
or at least 96%, 97%, 98%, or 99% identity, to SEQ ID NO:42, or SEQ
ID NO:43 wherein the polypeptide comprises an amino acid that is a
polar amino acid other than K or R at position 423 or the
polypeptide comprises a deletion at position 423, as determined
with reference to SEQ ID NO:1, and the polypeptide has chondrogenic
activity. In certain embodiments a polypeptide comprises SEQ ID
NO:42, or SEQ ID NO:43. In a further embodiment, a polypeptide is
SEQ ID NO:42, or SEQ ID NO:43.
[0056] In some embodiments, a protease-resistant ANGPTL3 of the
invention comprises an amino acid sequence that has at least 95%,
or at least 96%, 97%, 98%, or at least 99% identity to amino acids
232-454 of SEQ ID NO:4, amino acids 240-454 of SEQ ID NO:4, amino
acids 227-454 of SEQ ID NO:4, or amino acids 224-454 of SEQ ID NO:4
in which an amino acid is substituted for an R or K or there is a
deletion of an R or K. In some embodiments, the substitution or
deletion is at position 422 of SEQ ID NO:4, which corresponds to
position 423 of SEQ ID NO:1. In some embodiments, a substitution is
a polar amino acid, e.g., H, N, Q, S, T, A, or Y. In some
embodiments, a substitution is H, N, Q, S, T, or Y. In some
embodiments, the substitution is S or Q. In some embodiments, the
substitution is a Q. In some embodiments an amino acid deletion is
at position 422 of SEQ ID NO:4.
[0057] In some embodiments, a protease-resistant ANGPTL3 of the
invention comprises an amino acid sequence that has at least 95%,
or at least 96%, 97%, 98%, or at least 99% identity to amino acids
233-455 of SEQ ID NO:5, amino acids 241-455 of SEQ ID NO:5, amino
acids 228-455 of SEQ ID NO:5, or amino acids 225-455 of SEQ ID NO:5
in which an amino acid is substituted for an R or K or there is a
deletion of an R or K. In some embodiments, the substitution or
deletion is at position 423 of SEQ ID NO:5, which corresponds to
position 423 of SEQ ID NO:1. In some embodiments, a substitution is
a polar amino acid, e.g., H, N, Q, S, T, A, or Y. In some
embodiments, a substitution is H, N, Q, S, T, or Y. In some
embodiments, the substitution is S or Q. In some embodiments, the
substitution is a Q. In some embodiments an amino acid deletion is
at position 423 of SEQ ID NO:5.
[0058] In some embodiments, a protease-resistant ANGPTL3 of the
invention comprises an amino acid sequence that has at least 95%,
or at least 96%, 97%, 98%, or at least 99% identity to amino acids
233-455 of SEQ ID NO:6, amino acids 241-455 of SEQ ID NO:6, amino
acids 228-455 of SEQ ID NO:6, or amino acids 225-455 of SEQ ID NO:6
in which an amino acid is substituted for an R or K or there is a
deletion of an R or K. In some embodiments, the substitution or
deletion is at position 422 of SEQ ID NO:6, which corresponds to
position 423 of SEQ ID NO:1. In some embodiments, a substitution is
a polar amino acid, e.g., H, N, Q, S, T, A, or Y. In some
embodiments, a substitution is H, N, Q, S, T, or Y. In some
embodiments, the substitution is S or Q. In some embodiments, the
substitution is a Q. In some embodiments an amino acid deletion is
at position 422 of SEQ ID NO:6.
[0059] In some embodiments, a protease-resistant ANGPTL3
polypeptide of the invention comprises contiguous amino acids
corresponding to the amino acid regions: amino acids 240-454 of SEQ
ID NO:4; amino acids 232-454 of SEQ ID NO:4; amino acids 227-454 of
SEQ ID NO:4, or amino acids 224-454 of SEQ ID NO:4 in which an
amino acid is substituted for an R or K or there is a deletion of
an R or K. In some embodiments, the substitution or deletion is at
position 422 of SEQ ID NO:4 (which is position 423 as determined
with reference to SEQ ID NO:1). In some embodiments, a substitution
is a polar amino acid, e.g., H, N, Q, S, T, A, or Y. In some
embodiments, a substitution is H, N, Q, S, T, or Y. In some
embodiments, the substitution is S or Q. In some embodiments, the
substitution is Q. In some embodiments an amino acid deletion is at
position 422 of SEQ ID NO:4.
[0060] In some embodiments, a protease-resistant ANGPTL3
polypeptide of the invention comprises contiguous amino acids
corresponding to the amino acid regions: amino acids 241-455 of SEQ
ID NO:5; amino acids 233-455 of SEQ ID NO:5; amino acids 228-455 of
SEQ ID NO:5, or amino acids 225-455 of SEQ ID NO:5 in which an
amino acid is substituted for an R or K or there is a deletion of
an R or K. In some embodiments, the substitution or deletion is at
position 423 (which corresponds to position 423 as determined with
reference to SEQ ID NO:1). In some embodiments, a substitution is a
polar amino acid, e.g., H, N, Q, S, T, A, or Y. In some
embodiments, a substitution is H, N, Q, S, T, or Y. In some
embodiments, the substitution is S or Q. In some embodiments, the
substitution is Q. In some embodiments an amino acid deletion is at
position 423 of SEQ ID NO:5.
[0061] In some embodiments, a protease-resistant ANGPTL3
polypeptide of the invention comprises contiguous amino acids
corresponding to the amino acid regions: amino acids 241-455 of SEQ
ID NO:6; amino acids 233-455 of SEQ ID NO:6; amino acids 228-455 of
SEQ ID NO:6, or amino acids 225-455 of SEQ ID NO:6 in which an
amino acid is substituted for an R or K or there is a deletion of
an R or K. In some embodiments, the substitution or deletion is at
position 422 of SEQ ID NO:6 (which is position 423 as determined
with reference to SEQ ID NO:1). In some embodiments, a substitution
is a polar amino acid, e.g., H, N, Q, S, T, A, or Y. In some
embodiments, a substitution is H, N, Q, S, T, or Y. In some
embodiments, the substitution is S or Q. In some embodiments, the
substitution is Q. In some embodiments there is a deletion at
position 422 of SEQ ID NO:6.
[0062] The ANGPTL3 proteins of the invention as described above may
include native ANGPTL3 protein sequences flanking the regions
described above. Alternatively, in some embodiments, the ANGPTL3
proteins of the invention can include non-native ANGPTL3 protein
flanking sequences. For example, the chondrogenic active portion of
an ANGPTL3 protein can be fused to one or more fusion partners
and/or heterologous amino acids to form a fusion protein. Fusion
partner sequences can include, but are not limited to, amino acid
tags, non-L (e.g., D-) amino acids or other amino acid mimetics to
extend in vivo half-life and/or protease resistance, targeting
sequences or other sequences.
[0063] In some embodiments, a polypeptide of the invention is
PEGylated. In some embodiments, a polypeptide of the invention is
fused to a heterologous peptide. In certain embodiments a
polypeptide is fused to any one of human serum albumin (HSA), an
immunoglobulin heavy chain constant region (Fc), a polyhistidine, a
glutathione S transferase (GST), a thioredoxin, a protein A, a
protein G, a maltose binding protein (MBP), or a fragment of any of
the foregoing heterologous polypeptide(s). In particular
embodiments a heterologous polypeptide is fused at the
amino-terminal end of the polyptide of the invention. In additional
or alternative embodiments a heterologous polypeptide is fused at
the carboxy-terminal end of the polypeptide of the invention.
[0064] ANGPTL3 proteins of the invention have chondrogenic activity
and are protease-resistant. As defined herein, chondrogenesis or
chondrogenic activity refers to the development of chondrocytes
from MSCs. Indicators of chondrogenic activity include, but are not
limited to, cartilage matrix production. Cartilage matrix
production may be measured by various markers, for example, such as
Sox9, type II collagen, or glycosaminoglycan (GAG) production. In
some embodiments, GAG production is measured as a marker for
cartilage matrix production. In some embodiments, a 3-fold increase
in GAG production with cartilage specific protein expression
indicates positive cartilage matrix production.
[0065] A polypeptide may be evaluated for protease resistance using
any known assay that measures cleavage by a serine protease such as
trypsin. In some embodiments, the protease employed to evaluate
proteolysis susceptibility is the serine protease trypsin. A
polypeptide is considered to be protease-resistant if it has
reduced sensitivity to trypsin when compared to its wild-type
counterpart. An example of an assay is to measure the amount of
cleaved product that is generated when a polypeptide is exposed to
trypsin over a period of time in comparison to a corresponding
native human peptide. Cleavage can be measured using any known
assay, e.g., SDS PAGE or LCMS. An illustrative assay is provided in
the Examples section.
[0066] In an illustrative assay, limited proteolysis by
trypsinolysis is performed by incubating 10 ng of the protein to be
evaluated with trypsin at mass ratio of 8000:1 (Protein:Trypsin)
for 1 hr at room temperature. The trypsinolysis reaction can then
be quenched by addition of acetic acid to bring the reaction to pH
3.0. The quenched samples are then separated analyzed by SDS-PAGE,
e.g., on a 4-12% Tris-Bis gel to identify proteins which are
resistant to cleavage from those that are cleaved by the appearance
of a fragment that is generated by trypsin cleavage. The cleavage
product is absent or reduced in the protease-resistant polypeptides
in comparison to their wildtype counterparts.
[0067] In some embodiments, the ANGPTL3 polypeptides of the
invention will comprise at least one non-naturally encoded amino
acid. In some embodiments, a polypeptide comprises 1, 2, 3, 4, or
more unnatural amino acids. Methods of making and introducing a
non-naturally-occurring amino acid into a protein are known. See,
e.g., U.S. Pat. Nos. 7,083,970; and 7,524,647. The general
principles for the production of orthogonal translation systems
that are suitable for making proteins that comprise one or more
desired unnatural amino acid are known in the art, as are the
general methods for producing orthogonal translation systems. For
example, see International Publication Numbers WO 2002/086075,
entitled "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL
tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN
VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/094593,
entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415,
filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO
2005/007624, filed Jul. 7, 2004; WO 2006/110182, filed Oct. 27,
2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS" and WO 2007/103490, filed
Mar. 7, 2007, entitled "SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL
TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS." For discussion
of orthogonal translation systems that incorporate unnatural amino
acids, and methods for their production and use, see also, Wang and
Schultz, (2005) "Expanding the Genetic Code." Angewandte Chemie Int
Ed 44: 34-66; Xie and Schultz, (2005) "An Expanding Genetic Code."
Methods 36: 227-238; Xie and Schultz, (2005) "Adding Amino Acids to
the Genetic Repertoire." Curr Opinion in Chemical Biology 9:
548-554; and Wang, et al., (2006) "Expanding the Genetic Code."
Annu Rev Biophys Biomol Struct 35: 225-249; Deiters, et al, (2005)
"In vivo incorporation of an alkyne into proteins in Escherichia
coli." Bioorganic & Medicinal Chemistry Letters 15:1521-1524;
Chin, et al., (2002) "Addition of p-Azido-L-phenylalanine to the
Genetic Code of Escherichia coli." J Am Chem Soc 124: 9026-9027;
and International Publication No. WO2006/034332, filed on Sep. 20,
2005. Additional details are found in U.S. Pat. Nos. 7,045,337;
7,083,970; 7,238,510; 7,129,333; 7,262,040; 7,183,082; 7,199,222;
and 7,217,809.
[0068] A "non-naturally encoded amino acid" refers to an amino acid
that is not one of the common amino acids or pyrolysine,
pyrroline-carboxy-lysine, or selenocysteine. Other terms that may
be used synonymously with the term "non-naturally encoded amino
acid" are "non-natural amino acid," "unnatural amino acid,"
"non-naturally-occurring amino acid," and variously hyphenated and
non-hyphenated versions thereof. The term "non-naturally encoded
amino acid" also includes, but is not limited to, amino acids that
occur by modification (e.g. post-translational modifications) of a
naturally encoded amino acid (including but not limited to, the 20
common amino acids or pyrrolysine, pyrroline-carboxy-lysine, and
selenocysteine) but are not themselves naturally incorporated into
a growing polypeptide chain by the translation complex. Examples of
such non-naturally-occurring amino acids include, but are not
limited to, N-acetylglucosaminyl-L-serine,
N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.
[0069] A non-naturally encoded amino acid is typically any
structure having any substituent side chain other than one used in
the twenty natural amino acids. Because the non-naturally encoded
amino acids of the invention typically differ from the natural
amino acids only in the structure of the side chain, the
non-naturally encoded amino acids form amide bonds with other amino
acids, including but not limited to, natural or non-naturally
encoded, in the same manner in which they are formed in naturally
occurring polypeptides. However, the non-naturally encoded amino
acids have side chain groups that distinguish them from the natural
amino acids. For example, R optionally comprises an alkyl-, aryl-,
acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,
hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-,
borate, boronate, phospho, phosphono, phosphine, heterocyclic,
enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino
group, or the like or any combination thereof. Other non-naturally
occurring amino acids of interest that may be suitable for use in
the present invention include, but are not limited to, amino acids
comprising a photoactivatable cross-linker, spin-labeled amino
acids, fluorescent amino acids, metal binding amino acids,
metal-containing amino acids, radioactive amino acids, amino acids
with novel functional groups, amino acids that covalently or
noncovalently interact with other molecules, photocaged and/or
photoisomerizable amino acids, amino acids comprising biotin or a
biotin analogue, glycosylated amino acids such as a sugar
substituted serine, other carbohydrate modified amino acids,
keto-containing amino acids, amino acids comprising polyethylene
glycol or polyether, heavy atom substituted amino acids, chemically
cleavable and/or photocleavable amino acids, amino acids with an
elongated side chains as compared to natural amino acids, including
but not limited to, polyethers or long chain hydrocarbons,
including but not limited to, greater than about 5 or greater than
about 10 carbons, carbon-linked sugar-containing amino acids,
redox-active amino acids, amino thioacid containing amino acids,
and amino acids comprising one or more toxic moiety.
[0070] Exemplary non-naturally encoded amino acids that may be
suitable for use in the present invention and that are useful for
reactions with water soluble polymers include, but are not limited
to, those with carbonyl, aminooxy, hydrazine, hydrazide,
semicarbazide, azide and alkyne reactive groups. In some
embodiments, non-naturally encoded amino acids comprise a
saccharide moiety. Examples of such amino acids include
N-acetyl-L-glucosaminyl-L-serine,
N-acetyl-L-galactosaminyl-L-serine,
N-acetyl-L-glucosaminyl-L-threonine,
N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.
Examples of such amino acids also include examples where the
naturally-occurring N- or O-linkage between the amino acid and the
saccharide is replaced by a covalent linkage not commonly found in
nature--including but not limited to, an alkene, an oxime, a
thioether, an amide and the like. Examples of such amino acids also
include saccharides that are not commonly found in
naturally-occurring proteins such as 2-deoxy-glucose,
2-deoxygalactose and the like.
[0071] Another type of modification that can optionally be
introduced into the ANGPTL3 proteins of the invention (e.g. within
the polypeptide chain or at either the N- or C-terminal), e.g., to
extend in vivo half-life, is PEGylation or incorporation of
long-chain polyethylene glycol polymers (PEG). Introduction of PEG
or long chain polymers of PEG increases the effective molecular
weight of the present polypeptides, for example, to prevent rapid
filtration into the urine. In some embodiments, a Lysine residue in
the ANGPTL3 sequence is conjugated to PEG directly or through a
linker. Such linker can be, for example, a Glu residue or an acyl
residue containing a thiol functional group for linkage to the
appropriately modified PEG chain. An alternative method for
introducing a PEG chain is to first introduce a Cys residue at the
C-terminus or at solvent exposed residues such as replacements for
Arg or Lys residues. This Cys residue is then site-specifically
attached to a PEG chain containing, for example, a maleimide
function. Methods for incorporating PEG or long chain polymers of
PEG are well known in the art (described, for example, in Veronese,
F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R.
B., et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, M.
J., et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents
of which is incorporated herein by reference. Other methods of
polymer conjugations known in the art can also be used in the
present invention. In some embodiments, poly(2-methacryloyloxyethyl
phosphorylcholine) (PMPC) is introduced as a polymer conjugate with
the ANGPTL3 proteins of the invention (see, e.g., WO2008/098930;
Lewis, et al., Bioconjug Chem., 19: 2144-55 (2008)). In some
embodiments, a phosphorylcholine-containing polymer conjugate with
the ANGPTL3 proteins can be used in the present invention. A person
of skill would readily recognize that other biocompatible polymer
conjugates can be utilized.
[0072] A more recently reported alternative approach for
incorporating PEG or PEG polymers through incorporation of
non-natural amino acids (as described above) can be performed with
the present polypeptides. This approach utilizes an evolved
tRNA/tRNA synthetase pair and is coded in the expression plasmid by
the amber suppressor codon (Deiters, A, et al. (2004). Bio-org.
Med. Chem. Lett. 14, 5743-5). For example, p-azidophenylalanine can
be incorporated into the present polypeptides and then reacted with
a PEG polymer having an acetylene moiety in the presence of a
reducing agent and copper ions to facilitate an organic reaction
known as "Huisgen [3+2]cycloaddition."
[0073] In certain embodiments, the present invention also
contemplates specific mutations of the ANGPTL3 proteins so as to
alter the glycosylation of the polypeptide. Such mutations may be
selected so as to introduce or eliminate one or more glycosylation
sites, including but not limited to, O-linked or N-linked
glycosylation sites. In certain embodiments, the ANGPTL3 proteins
of the present invention have glycosylation sites and patterns
unaltered relative to the naturally-occurring ANGPTL3 proteins. In
certain embodiments, a variant of ANGPTL3 proteins includes a
glycosylation variant wherein the number and/or type of
glycosylation sites have been altered relative to the
naturally-occurring ANGPTL3 proteins. In certain embodiments, a
variant of a polypeptide comprises a greater or a lesser number of
N-linked glycosylation sites relative to a native polypeptide. An
N-linked glycosylation site is characterized by the sequence:
Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated
as X may be any amino acid residue except proline. The substitution
of amino acid residues to create this sequence provides a potential
new site for the addition of an N-linked carbohydrate chain.
Alternatively, substitutions which eliminate this sequence will
remove an existing N-linked carbohydrate chain. In certain
embodiments, a rearrangement of N-linked carbohydrate chains is
provided, wherein one or more N-linked glycosylation sites
(typically those that are naturally occurring) are eliminated and
one or more new N-linked sites are created.
[0074] Exemplary ANGPTL3 proteins variants include cysteine
variants wherein one or more cysteine residues are deleted from or
substituted for another amino acid (e.g., serine) relative to the
amino acid sequence of the naturally-occurring ANGPTL3 proteins. In
certain embodiments, cysteine variants may be useful when ANGPTL3
proteins must be refolded into a biologically active conformation
such as after the isolation of insoluble inclusion bodies. In
certain embodiments, cysteine variants have fewer cysteine residues
than the native polypeptide. In certain embodiments, cysteine
variants have an even number of cysteine residues to minimize
interactions resulting from unpaired cysteines.
[0075] In some embodiments, functional variants or modified forms
of the ANGPTL3 proteins include fusion proteins of an ANGPTL3
protein of the invention and one or more fusion domains. Well known
examples of fusion domains include, but are not limited to,
polyhistidine, Glu-Glu, glutathione S transferase (GST),
thioredoxin, protein A, protein G, an immunoglobulin heavy chain
constant region (Fc), maltose binding protein (MBP), and/or human
serum albumin (HSA). A fusion domain or a fragment thereof may be
selected so as to confer a desired property. For example, some
fusion domains are particularly useful for isolation of the fusion
proteins by affinity chromatography. For the purpose of affinity
purification, relevant matrices for affinity chromatography, such
as glutathione-, amylase-, and nickel- or cobalt-conjugated resins
are used. Many of such matrices are available in "kit" form, such
as the Pharmacia GST purification system and the QLAexpress.TM.
system (Qiagen) useful with (HIS.sub.6) fusion partners. As another
example, a fusion domain may be selected so as to facilitate
detection of the ANGPTL3 proteins. Examples of such detection
domains include the various fluorescent proteins (e.g., GFP) as
well as "epitope tags," which are usually short peptide sequences
for which a specific antibody is available. Well known epitope tags
for which specific monoclonal antibodies are readily available
include FLAG, influenza virus haemagglutinin (HA), and c-myc tags.
In some cases, the fusion domains have a protease cleavage site,
such as for Factor Xa or Thrombin, which allows the relevant
protease to partially digest the fusion proteins and thereby
liberate the recombinant proteins therefrom. The liberated proteins
can then be isolated from the fusion domain by subsequent
chromatographic separation. In certain embodiments, an ANGPTL3
protein is fused with a domain that stabilizes the ANGPTL3 protein
in vivo (a "stabilizer" domain). By "stabilizing" is meant anything
that increases serum half life, regardless of whether this is
because of decreased destruction, decreased clearance by the
kidney, or other pharmacokinetic effect. Fusions with the Fc
portion of an immunoglobulin are known to confer desirable
pharmacokinetic properties on a wide range of proteins. Likewise,
fusions to human serum albumin can confer desirable properties.
Other types of fusion domains that may be selected include
multimerizing (e.g., dimerizing, tetramerizing) domains and
functional domains (that confer an additional biological function,
as desired). Fusions may be constructed such that the heterologous
peptide is fused at the amino terminus of a polypeptide of the
invention and/or at the carboxy terminus of a polypeptide of the
invention.
Nucleic Acids Encoding Angiopoietin-Like 3 Protease-Resistant
Polypeptides
[0076] The invention also provides nucleic acids encoding protease
resistant polypeptides of the invention and expression vectors and
host cells for expression of a protease resistant polypeptide. In
other aspects, the invention provides a polynucleotide encoding a
polypeptide of the invention and expression vectors and host cells
comprising such a polynucleotide. In some embodiments, the
polynucleotide is optimized for expression in the host cells. In
some embodiments, the invention provides a method of ameliorating
or preventing arthritis or joint injury in a human patient, the
method comprising: administering to a joint of the patient an
expression vector encoding a polypeptide of the invention whereupon
expression of the polypeptide ameliorates or prevents arthritis or
joint injury in the patient. In some embodiments, the patient has
arthritis or joint injury. In some embodiments, the individual does
not have, but is at risk for, arthritis or joint injury. In some
embodiments, the arthritis is osteoarthritis, trauma arthritis, or
autoimmune arthritis.
[0077] Expressing polypeptides of the invention employs routine
techniques in the field of recombinant genetics. Basic texts
disclosing the general methods of use in this invention include
Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory
Manual, 3rd edition; the series Ausubel et al. eds. (2007 with
updated through 2010) Current Protocols in Molecular Biology, among
others known in the art.
[0078] Expression can employ any appropriate host cells known in
the art, for example, mammalian host cells, bacterial host cells,
yeast host cells, insect host cells, etc. Both prokaryotic and
eukaryotic expression systems are widely available. In some
embodiments, the expression system is a mammalian cell expression,
such as a CHO cell expression system. In some embodiments, a
nucleic acid may be codon-optimized to facilitate expression in a
desired host cell.
[0079] Nonviral vectors and systems include plasmids and episomal
vectors, typically comprising an expression cassette for expressing
a protein or RNA, and human artificial chromosomes (see, e.g.,
Harrington et al., Nat Genet 15:345, 1997). For example, nonviral
vectors useful for expression of the polypeptides of the invention
in mammalian (e.g., human) cells include pThioHis A, B & C,
pcDNA3. I/His, pEBVHis A, B & C (Invitrogen, San Diego,
Calif.), MPSV vectors, and numerous other vectors known in the art
for expressing other proteins. Useful viral vectors include, but
are not limited to, vectors based on adenoviruses, adenoassociated
viruses, herpes viruses, vectors based on SV40, papilloma virus,
HBP Epstein Barr virus, fowpox vectors, vaccinia virus vectors and
Semliki Forest virus (SFV).
[0080] The choice of expression vector depends on the intended host
cells in which the vector is to be expressed. Typically, the
expression vectors contain a promoter and other regulatory
sequences (e.g., enhancers) that are operably linked to the
polynucleotides encoding a polypeptide of the invention. In some
embodiments, an inducible promoter is employed to prevent
expression of inserted sequences except under inducing conditions.
Inducible promoters include, e.g., arabinose, lacZ, a
metallothionein promoter, a glucocorticoid promoters or a heat
shock promoter. In addition, other regulatory elements may also be
incorporated to improve expression of a nucleic acid encoding a
polypeptide of the invention, e.g., enhancers, ribosomal binding
site, transcription termination sequences, and the like.
[0081] In some embodiments, a nucleic acid encoding an polypeptide
of the invention may also include a sequence encoding a secretion
signal sequence so that the polypeptide is secreted from the host
cell. Such a sequence can be provided by the vector, or as part of
the ANGPTL3 nucleic acid that is present in the vector.
[0082] Methods for introducing expression vectors containing the
polynucleotide sequences of interest vary depending on the type of
cellular host. For example, calcium chloride transfection is
commonly utilized for prokaryotic cells, whereas calcium phosphate
treatment or electroporation may be used for other cellular hosts
(see generally Sambrook et al., supra). Other methods include,
e.g., electroporation, calcium phosphate treatment,
liposome-mediated transformation, injection and microinjection,
ballistic methods, virosomes, immunoliposomes, polycation: nucleic
acid conjugates, naked DNA, artificial virions, fusion to the
herpes virus structural protein VP22, agent-enhanced uptake of DNA,
and ex vivo transduction. For long-term, high-yield production of
recombinant proteins, stable expression will often be desired. For
example, cell lines which stably express polypeptides of the
invention can be prepared using expression vectors of the invention
which contain viral origins of replication or endogenous expression
elements and a selectable marker gene.
[0083] In some embodiments, nucleic acids encoding protease
resistant ANGPTL3 polypeptides of the invention can be delivered to
a patient for treatment of a joint-related injury or disease.
Delivery of such nucleic acids can be achieved using any means
known in the art, but is typically performed using direct injection
into the affected joint. In some embodiments, a DNA is delivered as
naked DNA using direct injection into the joint. In some
embodiments, a viral vector is employed, including, but not limited
to, an adenovirus or adenovirus-associated vector, a herpes virus
vector, fowlpox virus, or a vaccinia virus vector.
Methods of Therapeutic Use of Polypeptides, and Indications
[0084] Provided methods of the invention include a method of
treating a subject comprising administering to the subject a
therapeutically effective amount of a polypeptide of the invention,
wherein the subject has or is at risk of joint damage or arthritis.
The invention also provides a method of ameliorating or preventing
arthritis or joint injury in a human patient, the method
comprising: administering to a joint of the patient a composition
comprising an effective amount of a polypeptide of the invention,
thereby ameliorating or preventing arthritis or joint injury in the
patient. In some embodiments, the patient has arthritis or joint
injury. In some embodiments, the individual does not have, but is
at risk for, arthritis or joint injury. In some embodiments, the
arthritis is osteoarthritis, trauma arthritis, or autoimmune
arthritis. In some embodiments, the composition administered to the
further comprises hyaluronic acid.
[0085] In another aspect, the invention provides a method of
inducing differentiation of mesenchymal stem cells into
chondrocytes, the method comprising, contacting mesenchymal stem
cells with a sufficient amount of a polypeptide of the invention to
induce differentiation of the stem cells into chondrocytes. In some
embodiments, the method is performed in vivo and the stem cells are
present in a human patient.
[0086] It is contemplated that polypeptides, compositions, and
methods of the present invention may be used to treat, ameliorate
or prevent any type of articular cartilage damage (e.g., joint
damage or injury) including, for example, damage arising from a
traumatic event or tendon or ligament tear. In some embodiments,
proteins of the invention are administered to prevent or ameliorate
arthritis or joint damage, for example where there is a genetic or
family history of arthritis or joint damage or joint injury or
prior or during joint surgery. In some embodiments polypeptides,
compositions and methods are used to treat joint damage. In
particular embodiments joint damage is traumatic joint injury. In
other embodiments joint damage is damage arising from age or
inactivity. In yet other embodiments joint damage is damage arising
from an autoimmune disorder. In some embodiments of the invention,
polypeptides, compositions, and methods of the present invention
may be used to treat, ameliorate or prevent osteoarthritis. In some
embodiments, the polypeptides, compositions and methods are used to
ameliorate or prevent arthritis in a subject at risk of having or
acquiring arthritis. In some embodiments, the polypeptides,
compositions and methods are used to ameliorate or prevent joint
damage in a subject at risk of having or acquiring joint
damage.
[0087] In some embodiments, polypeptides, compositions, and methods
of the present invention provide a method for stimulating
chondrocyte proliferation and cartilage production in cartilagenous
tissues that have been damaged, e.g., due to traumatic injury or
chondropathy. In particular embodiments polypeptides, compositions,
and methods of the present invention are useful for treatment of
cartilage damage in joints, e.g., at articulated surfaces, e.g.,
spine, shoulder, elbow, wrist, joints of the fingers, hip, knee,
ankle, and joints of the feet. Examples of diseases or disorders
that may benefit from treatment include osteoarthritis, rheumatoid
arthritis, other autoimmune diseases, or osteochondritis dessicans.
In addition, cartilage damage or disruption occurs as a result of
certain genetic or metabolic disorders, cartilage malformation is
often seen in forms of dwarfism in humans, and/or cartilage damage
or disruption is often a result of reconstructive surgery; thus
polypeptides, compositions, and methods would be useful therapy in
these patients, whether alone or in connection with other
approaches.
[0088] It is further contemplated that polypeptides, compositions,
and methods of the present invention may be used to treat,
ameliorate or prevent various cartilagenous disorders and/or
associated symptoms or effects of such conditions. Exemplary
conditions or disorders for treatment, amelioration and/or
prevention with polypeptides, compositions, and methods of the
invention, include, but are not limited to systemic lupus
erythematosis, rheumatoid arthritis, juvenile chronic arthritis,
osteoarthritis, degenerative disc disease, spondyloarthropathies,
Ehlers Danlos syndrome, systemic sclerosis (scleroderma) or tendon
disease. Other conditions or disorders that may benefit from
treatment with polypeptides for amelioration of associated effects
include idiopathic inflammatory myopathies (dermatomyositis,
polymyositis), Sjogren's syndrome, systemic vasculitis,
sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia,
paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia
(idiopathic thrombocytopenic purpura, immune-mediated
thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis), diabetes mellitus, immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis), demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barr syndrome, and chronic inflammatory demyelinating
polyneuropathy, hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis, inflammatory
bowel disease (ulcerative colitis: Crohn's disease),
gluten-sensitive enteropathy, and Whipple's disease, autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis, allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria, immunologic diseases of the lung
such as eosinophilic pneumonias, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis, transplantation associated diseases
including graft rejection and graft-versus-host-disease.
[0089] A "patient" as used herein refers to any subject that is
administered a therapeutic polypeptide of the invention. It is
contemplated that the polypeptides, compositions, and methods of
the present invention may be used to treat a mammal. As used herein
a "subject" refers to any mammal, including humans, domestic and
farm animals, and zoo, sports or pet animals, such as cattle (e.g.
cows), horses, dogs, sheep, pigs, rabbits, goats, cats, etc. In
some embodiments of the invention, the subject is a human. In
certain embodiments, the subject is a horse. In other embodiments
the subject is a dog.
[0090] In some embodiments, the polypeptides of the invention can
be heterologous to the mammal to be treated. For example, a human
ANGPTL3 protein or fragments thereof, a protein or peptide derived
from a human ANGPTL3 protein (e.g., a modified human ANGPTL3
protein, a conservative variant of human ANGPTL3 protein, a
peptidomimetic derived from a human ANGPTL3 protein) are used in
the treatment of an animal such as an equine, bovine or canine. In
some embodiments, a heterologous ANGPTL3 protein can be used to
expand chondrocyte populations in culture for transplantation. In
some embodiments, expanded cultures will then be optionally admixed
with polypeptides and compositions homologous to the mammal to be
treated, and placed in the joint space or directly into the
cartilage defect. Alternatively, polypeptides of the invention are
derived from the same species, i.e., a human ANGPTL3 protein or
fragments thereof, a protein or peptide derived from a human
ANGPTL3 protein (e.g., a modified human ANGPTL3 protein, a
conservative variant of human ANGPTL3 protein, a peptidomimetic
derived from a human ANGPTL3 protein) is used in the treatment of a
human patient. By using a protein derived from the same species of
mammal as is being treated, inadvertent immune responses may be
avoided.
[0091] In some embodiments, polypeptides and compositions of the
present invention are applied by direct injection into the synovial
fluid of a joint, systemic administration (oral or intravenously)
or directly into a cartilage defect, either alone or complexed with
a suitable carrier for extended release of protein. In some
embodiments, polypeptides or compositions are administered in a
biocompatible matrix or scaffold. Polypeptides, compositions, and
methods of the present invention can also be used in conjunction
with a surgical procedure at an affected joint. Administration of a
polypeptide of the invention may occur prior to, during or in
conjunction with, and/or after a surgical procedure. For example,
polypeptides, compositions and methods of the invention can be used
to expand chondrocyte populations in culture for autologous or
allogenic chondrocyte implantation (ACI). Chondrocytes can be
optionally implanted with concurrent treatment consisting of
administration of polypeptides and compositions of the present
invention. In these procedures, for example, chondrocytes can be
harvested arthroscopically from an uninjured minor load-bearing
area of a damaged joint, and can be cultured in vitro, optionally
in the presence of polypeptides and compositions of the present
invention and/or other growth factors to increase the number of
cells prior to transplantation. Expanded cultures are then
optionally admixed with polypeptides and compositions of the
present invention and/or placed in the joint space or directly into
the defect. In certain embodiments, expanded cultures (optionally
with polypeptides of the present invention) are placed in the joint
space suspended in a matrix or membrane. In other embodiments,
polypeptides and compositions of the present invention can be used
in combination with one or more periosteal or perichondrial grafts
that contain cartilage forming cells and/or help to hold the
transplanted chondrocytes or chondrocyte precursor cells in place.
In some embodiments, polypeptides and compositions of the present
invention are used to repair cartilage damage in conjunction with
other procedures, including but not limited to lavage of a joint,
stimulation of bone marrow, abrasion arthroplasty, subchondral
drilling, or microfracture of proximal subchondral bone.
Optionally, following administration of polypeptides and
compositions of the present invention and growth of cartilage,
additional surgical treatment may be beneficial to suitably contour
newly formed cartilage surface(s).
Pharmaceutical Compositions
[0092] Therapeutic compositions comprising provided polypeptides
are within the scope of the present invention, and are specifically
contemplated in light of the identification of several polypeptide
sequences exhibiting enhanced stability and protease resistance.
Thus, in a further aspect, the invention provides a pharmaceutical
composition comprising a therapeutically effective amount of a
polypeptide of the invention. In certain embodiments,
pharmaceutical compositions further comprise a pharmaceutically or
physiologically acceptable carrier. In some embodiments, a
pharmaceutical composition further comprises a hyaluronic acid or a
derivative thereof.
[0093] In addition, the invention provides a method of ameliorating
or preventing arthritis or joint injury in a human patient, the
method comprising: administering to a joint of the patient a
composition comprising an effective amount of a polypeptide of the
invention, thereby ameliorating or preventing arthritis or joint
injury in the patient. In some embodiments, the patient has
arthritis or joint injury. In some embodiments, the individual does
not have, but is at risk for, arthritis or joint injury. In some
embodiments, the arthritis is osteoarthritis, trauma arthritis, or
autoimmune arthritis. In some embodiments, the composition
administered to the further comprises hyaluronic acid.
[0094] In another aspect, the invention provides a method of
inducing differentiation of mesenchymal stem cells into
chondrocytes, the method comprising, contacting mesenchymal stem
cells with a sufficient amount of a polypeptide of the invention to
induce differentiation of the stem cells into chondrocytes. In some
embodiments, the method is performed in vivo, the stem cells are
present in a human patient, and the contacting comprises
administering to a joint of the patient a composition comprising an
effective amount of a polypeptide of the invention, thereby
inducing differentiation of stem cells into chondrocytes, and
generation of cartilage.
[0095] Therapeutic compositions comprising nucleic acids encoding
polypeptides of the invention can be delivered to a patient for
treatment of a joint-related injury or disease, and are also within
the scope of the present invention. In some embodiments,
pharmaceutical compositions comprise naked DNA encoding a
polypeptide of the invention. In some embodiments, a viral vector
is employed to effect delivery and a pharmaceutical composition
comprises a vector encoding a polypeptide of the invention,
including, but not limited to, an adenovirus or
adenovirus-associated vector, a herpes virus vector, fowlpox virus,
or a vaccinia virus vector. Pharmaceutical compositions comprise a
therapeutically effective amount of a nucleic acid encoding a
polypeptide of the invention with a pharmaceutically or
physiologically acceptable carrier.
[0096] In another aspect of the present invention, provided
polypeptides for use as a medicament for treatment of joint damage
is contemplated. In certain embodiments polypeptides of the
invention for use as a medicament for amelioration of arthritis or
joint damage are provided. In some embodiments arthritis is
osteoarthritis, trauma arthritis or autoimmune arthritis. In some
embodiments joint damage is traumatic joint injury, autoimmune
damage, age related damage, or damage related to inactivity. In
other embodiments, nucleic acid encoding a polypeptide of the
invention for use in a medicament is provided.
[0097] Formulations suitable for administration include excipients,
including but not limited to, aqueous and non-aqueous solutions,
isotonic sterile solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. In certain embodiments
pharmaceutical compositions comprise a therapeutically effective
amount of a peptide in admixture with a pharmaceutically acceptable
formulation agent selected for suitability with the mode of
administration, delivery format, and desired dosage. See, e.g.,
Remington's Pharmaceutical Sciences (18.sup.th Ed., A. R. Gennaro,
ed., Mack Publishing Company 1990), and subsequent editions of the
same. The primary vehicle or carrier in a pharmaceutical
composition can be aqueous or non-aqueous in nature. For example, a
suitable vehicle or carrier for injection can be water,
physiological saline solution or artificial cerebrospinal fluid,
optionally supplemented with other materials common in compositions
for parenteral administration. For example, buffers may be used,
e.g., to maintain the composition at physiological pH or at a
slightly lower pH, typically within a range of from about pH 5 to
about pH 8, and may optionally include sorbitol, serum albumin,
detergent, or other additional component. In certain embodiments
pharmaceutical compositions comprising polypeptides or a nucleic
acid encoding a polypeptide of the invention can be prepared for
storage in a lyophilized form using appropriate excipients (e.g.,
sucrose).
[0098] In yet other embodiments formulation with an agent, such as
injectable microshperes, bio-erodable particles, polymeric
compounds, beads, or liposomes or other biocompatible matrix that
provides for controlled or sustained release of the polypeptide or
a nucleic acid encoding a polypeptide of the invention can then be
delivered via a depot injection. For example, polypeptides or
nucleic acid encoding a polypeptide of the invention may be
encapsulated in liposomes, or formulated as microparticles or
microcapsules or may be incorporated into other vehicles, such as
biodegradable polymers, hydrogels, cyclodextrins (see for example
Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et
al., International PCT publication Nos. WO 03/47518 and WO
03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA
microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent
Application Publication No. US 2002130430), biodegradable
nanocapsules, and bioadhesive microspheres, or by proteinaceous
vectors (O'Hare and Normand, International PCT Publication No. WO
00/53722) or by the use of conjugates. Still other suitable
delivery mechanisms include implantable delivery devices.
[0099] The dose of a compound of the present invention for treating
the above-mentioned diseases or disorders varies depending upon the
manner of administration, the age and/or the body weight of the
subject, and the condition of the subject to be treated, and
ultimately will be decided by the attending physician or
veterinarian. The dose administered to a subject, in the context of
the present invention should be sufficient to effect a beneficial
response in the subject over time. Such a dose is a
"therapeutically effective amount". Accordingly, an appropriate
dose may be determined by the efficacy of the particular protein or
a nucleic acid encoding a polypeptide of the invention employed and
the condition of the subject, as well as the body weight or surface
area of the area to be treated. The size of the dose also will be
determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
protein or vector in a particular subject. Administration can be
accomplished via single or divided doses, or as a continuous
infusion via an implantation device or catheter. Frequency of
dosing will depend upon the pharmacokinetic parameters of the
polypeptide or a nucleic acid encoding a polypeptide of the
invention in the formulation used. A clinician may titer dosage
and/or modify administration to achieve the desired therapeutic
effects. A typical dosage ranges from about 0.01 .mu.g/kg to about
100 mg/kg, depending on the factors. In certain embodiments, a
dosage ranges from about 0.1 .mu.g/kg up to about 10 mg/kg; or
about 0.1 .mu.g/kg; about 0.5 .mu.g/kg; about 1 .mu.g/kg; about 2
.mu.g/kg; about 0.5 .mu.g/kg; about 10 .mu.g/kg; about 15 .mu.g/kg;
about 20 .mu.g/kg; about 25 .mu.g/kg; about 30 .mu.g/kg; about 35
.mu.g/kg; about 40 .mu.g/kg; about 45 .mu.g/kg; about 50 .mu.g/kg;
about 55 .mu.g/kg; about 60 .mu.g/kg; about 65 .mu.g/kg; about 75
.mu.g/kg; about 85 .mu.g/kg; about 100 .mu.g/kg. In certain
embodiments a dosage is about 50 .mu.g/kg; about 100 .mu.g/kg;
about 150 .mu.g/kg; about 200 .mu.g/kg; about 250 .mu.g/kg; about
300 .mu.g/kg; about 350 .mu.g/kg; about 400 .mu.g/kg; about 450
.mu.g/kg; about 500 .mu.g/kg; about 550 .mu.g/kg; about 600
.mu.g/kg; about 650 .mu.g/kg; about 700 .mu.g/kg; about 750
.mu.g/kg; about 800 .mu.g/kg; about 850 .mu.g/kg; about 900
.mu.g/kg; about 950 .mu.g/kg; about 1 mg/kg; about 2 mg/kg; about 3
mg/kg; about 4 mg/kg; about 5 mg/kg; about 6 mg/kg; about 7 mg/kg;
about 8 mg/kg; about 9 mg/kg; about 10 mg/kg.
Methods of Administration
[0100] Any method for delivering the proteins or a nucleic acid
encoding a polypeptide of the invention of the invention to an
affected joint can be used. In the practice of this invention,
compositions can be parenterally administered, for example
injected, e.g., intra-articularly (i.e., into a joint),
intravenously, intramuscularly, subcutaneously; infused, or
implanted, e.g., in a membrane, matrix, device, etc. When injected,
infused or implanted, delivery can be directed into the suitable
tissue or joint, and delivery may be direct bolus delivery or
continuous delivery. In some embodiments delivery can be in a
suitable tissue located in close proximity to an affected joint. In
some embodiments delivery may be via diffusion, or via timed
release bolus. In some embodiments, a controlled release system
(e.g., a pump) can be placed in proximity of the therapeutic
target, e.g., the joint to which the polypeptide is administered.
In other embodiments, compositions can be selected for ingestion,
e.g., inhalation or oral delivery.
[0101] The therapeutic polypeptides or a nucleic acid encoding a
polypeptide of the invention of the present invention can also be
used effectively in combination with one or more additional active
agents (e.g., hyaluronic acid or a derivative or salt thereof,
growth factor (e.g., FGF18, BMP7), chondrogenic agent (e.g., oral
salmon calcitonin, SD-6010 (iNOS inhibitor), vitamin D3
(choliecalciferol), collagen hydrolyzate, rusalatide acetate,
avocado soy unsaponifiables (ASU), a compound described in
WO2012/129562, kartogenin), a steroid, a non-steroidal
anti-inflammatory agent (NSAID), etc.) depending on the desired
therapy or effect to improve or enhance the therapeutic effect of
either. This process can involve administering both agents to the
patient at the same time, either as a single composition or
pharmacological formulation that includes both agents, or by
administering two distinct compositions or formulations, wherein
one composition includes a polypeptide or a polynucleotide encoding
a polypeptide of the invention and the other includes the second
agent(s). Administration of a therapeutic composition comprising a
polypeptide or a polynucleotide encoding a polypeptide of the
invention can precede or follow administration of the second agent
by intervals ranging from minutes to weeks.
[0102] Formulations of compounds can be stored in sterile vials as
a solution, suspension, gel, emulsion, solid, or as a dehydrated or
lyophilized powder. Formulations can be presented in unit-dose or
multi-dose sealed containers, such as ampules and vials. In some
embodiments formulations can be presented in single or
multi-chambered pre-filled syringes (e.g., liquid syringes,
lysosyringes). Solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described.
[0103] Also provided are kits comprising the polypeptides or a
nucleic acid encoding a polypeptide of the invention of the
invention. In one embodiment provided are kits for producing a
single dose administration unit. The kit comprises a first
container comprising a dried polypeptide or a nucleic acid encoding
a polypeptide of the invention and a second container having an
aqueous reconstitution formula. In certain embodiments one
container comprises a single chamber pre-filled syringe. In other
embodiments the containers are encompassed as a multi-chambered
pre-filled syringe
EXEMPLIFICATION
[0104] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1: Protease-Resistant Angpt13 Peptide Constructs
[0105] Various N-terminal truncation mutants were constructed to
remove O-linked glycosylations and facilitate biophysical protein
characterization. To identify protease-resistant peptides, amino
acid substitutions were introduced into various positions of human
Angpt13 peptide fragments corresponding to the C-terminal region of
the peptide. FIG. 1 shows positions of mutations in the human
Angpt13. Constructs were initially prepared with His tags. The
mutant proteins were: 225-460 K423Q (225KQ), 225-460 S424T(225ST),
226-460 K423Q (226KQ), 226-460 K423S (226KS), 228-460 K423Q
(228KQ), 228-460 S424T (228ST), 233-460 K423Q (233KQ), 233-460
K423S (233KS), 241-460 K423Q (241KQ), 241-460 K423S (241KS),
241-460 Kdel (241Kdel), 242-460 K423Q (242KQ), 242-460 K423S
(242KS) and 242-460 Kdel (242Kdel).
[0106] His-tagged proteins were expressed in HEK Freestyle.TM.
cells and purified by Ni-NTA column chromatography. Tag-less
C-terminal constructs were also cloned, purified by previously
described method (Gonzalez R et al PNAS 2010). Briefly, target
protein with signal sequence (1-16) was cloned in a mammalian
expression vector with cytomegalovirus promoter. At 96 h after
DNA/PEI transfection in HEK 293 Freestyle (Invitrogen), media
containing secreted target protein were harvested and purified by
Hi-Trap SP column (GE Healthcare). Protein was eluted between 50 mM
MES (pH 6.0), 125 mM NaCl to 50 mM MES (pH 6.0), 150 mM NaCl.
SDS-PAGE confirmed that the purified protein was at least 95%
pure.
[0107] Protease-resistance was assessed as follows. Limited
trypsinolysis was performed by incubating 10 ng of each prepared
protein with trypsin at mass ratio of 8000:1 (Protein:Trypsin) for
1 h r at room temperature. The trypsinolysis reaction was then
quenched by addition of acetic acid to bring the reaction to pH
3.0, and quenched samples were analyzed by LC/MS. A 5 min RP HPLC
peak corresponding to the mass of the C-terminal 43 amino acids
(S424-E460) was evident for the respective wild type protein
constructs. The clip site was at the same site, i.e., between K423
and S424, as observed during full length wild type ANGPLT3 protein
production. This peak was absent when the Lys at the clip site was
mutated to Gln. Each of peptide constructs 225KQ, 228KQ, 233KQ,
233KS, 241KQ, and 242KQ; and the wildtype 225 peptide were prepared
and analyzed. The peak corresponding to the mass of the C-terminal
43 amino acids was absent when the Lys at the clip site was mutated
to Gln or Ser for each of the constructs, or when the Lys at
position 423 was deleted.
Example 2: Integrin Binding Assays
[0108] .alpha.V.beta.3 integrin Prepared peptides 225KQ, 228KQ,
233KQ, 241KQ and 242KQ were tested in vitro for binding to
.alpha.V.beta.3 integrin. Briefly, Maxisorp plates were coated with
2 .mu.g/ml Integrin .alpha.V.beta.3, and various concentrations of
polypeptide construct (indicated) were added. Bound peptide was
detected by the addition of Anti-ANGPTL3 mAb followed by
horseradish peroxidase-conjugated Goat anti-Mouse IgG antibody. All
tested peptides retained or improved integrin binding capacity.
EC.sub.50 for each were determined from the binding data, and
results are shown in TABLE 2.
TABLE-US-00002 TABLE 2 In vitro binding of ANGPTL3 and engineered
polypeptide constructs to Integrins .alpha.5.beta.1 integrin
EC.sub.50 .alpha.V.beta.3 integrin EC.sub.50 WT 3.054 3.245 242KQ
1.566 3.076 241KQ 2.693 4.032 233KQ 13.83 6.636 228KQ 4.26 4.051
225KQ 19.89 11.18
[0109] .alpha.5.beta.1 integrin Prepared peptides 225KQ, 228KQ,
233KQ, 241KQ and 242KQ were tested in vitro for binding to
.alpha.5.beta.1 integrin. Plates were coated with 2 .mu.g/ml as
described above but with Integrin .alpha.5.beta.1, and various
concentrations of polypeptide construct (indicated) were added, and
detection carried out as described above. All tested peptides
retained or improved integrin binding capacity. EC.sub.50 for each
were determined from the binding data, and results are shown in
TABLE 2.
Example 3: Functional Analysis of Constructs
[0110] Cell culture and differentiation. Primary human bone marrow
derived mesenchymal stem cells (hMSCs) were FACS sorted and proven
to be >98% positive for CD29, CD44, CD166 and CD105 and <0.1%
positive for CD45; and cells were used from passages 2-8 for
experiments. Human cartilage resident MSCs (hCR-MSCs) were derived
from human primary articular chondrocytes, which were separated
into single cells, clonally grown in MSCGM and validated as MSCs
through chondrogenic, osteogenic and adipogenic differentiation.
Cells were FACS sorted and proven to be >98% positive for CD166
and CD105. hCR-MSCs were cultured up to 20 passages with no
alteration in the cell profile, growth or differentiation rates
identified.
[0111] Chondrogenesis. Peptide constructs of the invention were
evaluated in physical and functional assays to assess
chondrogenesis activity.
[0112] Engineered constructs provided herein are derived from
ANGPTL3 which belongs to a family of seven identified ANGPTL
proteins that have structural similarity to the angiopoietins, but
lack the ability to bind the Tie2 receptor and thus have distinct
functions. ANGPTL proteins contain an N-terminal coiled-coil domain
(CCD) and a C-terminal fibrinogen-like domain (FLD), and are
believed to be tightly regulated by their microenvironment and
interactions with the extracellular matrix (ECM) such as
fibronectin and integrins. Conklin et al., Genomics 62(3): 477-482
(1999); Goh Y Y, et al., Am J Pathol 177(6): 2791-2803 (2010); Goh
Y Y, et al J Biol Chem 285(43): 32999-33009(2010). Sequences for
ANGPTL family members most closely related to ANGPTL3, ANGPTL1
(full length and C-terminal domain) and ANGPTL4 (full length and
C-terminal domain) are provided in Table 3; and FIG. 12 depicts an
alignment across the C-terminal domains of these family members.
Sequence identities across the extracellular domains and C-terminal
domains ANGPTL1, ANGPTL4, as well as other angiopoietin proteins
ANGPTL7, ANGPT1 and ANGPT2 are provided in Table 5. The C-terminal
domain (CT) of ANGPTL3 shares 37% sequence identity with CT ANGPTL1
and 40% sequence identity with CT ANGPTL4.
[0113] Cell-based 2D chondrogenesis was induced in vitro and
assessed as described previously in Johnson, K., et al., (2012)
Science 336, 717. Briefly, primary human bone marrow derived
mesenchymal stem cells (hMSCs) were plated in growth media then
subsequently changed to a chondrogenic stimulation media with and
without constructs.
[0114] To initially image nodule formation, wells were fixed and
stained with Rhodamine B where the nodules were easily detected by
eye and images captured by light microscopy. To facilitate high
throughput imaged-based detection and quantification, chondrogenic
nodules were stained with Nile red which binds non-specifically to
collagens. Nile Red stained nodules were quantified on an Acumen
eX3 (high content imaging device) by excitation with a 488 laser
for rapid detection of the nodules.
TABLE-US-00003 TABLE 3 ANGPTL Family Sequences SEQ ID Construct
Sequence 71 hANGPTL1
MKTFTWTLGVLFFLLVDTGHCRGGQFKIKKINQRRYPRATDGKEEAKKCA 1-491
YTFLVPEQRITGPICVNTKGQDASTIKDMITRMDLENLKDVLSRQKREID
VLQLVVDVDGNIVNEVKLLRKESRNMNSRVTQLYMQLLHEIIRKRDNSLE
LSQLENKILNVTTEMLKMATRYRELEVKYASLTDLVNNQSVMITLLEEQC
LRIFSRQDTHVSPPLVQVVPQHIPNSQQYTPGLLGGNEIQRDPGYPRDLM
PPPDLATSPTKSPFKIPPVTFINEGPFKDCQQAKEAGHSVSGIYMIKPEN
SNGPMQLWCENSLDPGGWTVIQKRTDGSVNFFRNWENYKKGFGNIDGEYW
LGLENIYMLSNQDNYKLLIELEDWSDKKVYAEYSSFRLEPESEFYRLRLG
TYQGNAGDSMMWHNGKQFTTLDRDKDMYAGNCAHFHKGGWWYNACAHSNL
NGVWYRGGHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPID 72 CT
FINEGPFKDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTV hANGPTL1
IQKRTDGSVNFFRNWENYKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIE 271-491
LEDWSDKKVYAEYSSFRLEPESEFYRLRLGTYQGNAGDSMMWHNGKQFTT
LDRDKDMYAGNCAHFHKGGWWYNACAHSNLNGVWYRGGHYRSKHQDGIFW
AEYRGGSYSLRAVQMMIKPID 73 hANGPTL4
MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQ 1-406
LGQGLREHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPE
VLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHK
HLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSG
LFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGD
PHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAY
SLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWW
FGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPM AAEAAS 74 CT
SRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQ hANGPTL4
RRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLR 179-406
DWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFST
WDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGI
FWKTWRGRYYPLQATTMLIQPMAAEAAS
TABLE-US-00004 TABLE 4 Chondrogenesis of ANGPTL family member
proteins Nodule Induction Genbank Protein Formation activity Type
II collagen Accession Angptl1 Yes Yes NP_004664 Angptl2 No n/a
NP_036230 Angptl3 Yes Yes NP_055310 Angptl4 Yes No NP_647475
Angptl6 No No NP_114123 Angptl7 No No NP_066969 Angpt2 No n/a
NP_001138 Angpt1 No n/a NP_004664
[0115] Cell-based 2D chondrogenesis was induced in vitro and
assessed as described previously in Johnson, K., et al., (2012)
Science 336, 717. Briefly, primary human bone marrow derived
mesenchymal stem cells (hMSCs) were plated in growth media then
subsequently changed to a chondrogenic stimulation media with and
without constructs, and cultured for 7 or 14 days. Cells were then
fixed with formaldehyde, washed and then stained using standard
immuno-cytochemical techniques to detect primary cartilage proteins
Pro-collagen Type 2A (PIIANP) (FIG. 2A) and Type II collagen (FIG.
2B). For detection of type II collagen, cells were digested with
0.2% Collagenase II (Worthington Biochemical, Lakewood, N.J.) which
was added to the permeabilization solution. Immuno-fluorescence for
each protein detected was quantified through high content imaging
(Image Express Ultra (Molecular Devices, Sunnyvale, Calif.)), using
multi-wavelength cell scoring script, and as described previously.
See FIG. 2. Aggrecan expression was monitored by preparing cells as
follows: briefly, primary hMSCs (5000 cells) were plated in a
Griener 384 well plate. After 24 hours the growth media was removed
and replaced with 25 .mu.l of DMEM containing 1% FBS. Protein
constructs were then added to each well at the indicated dose, and
cultures were grown at 37.degree. C. for 3 days. The cells were
fixed with 10% formalin and subjected to immunocytochemical methods
to detect Aggrecan protein expression. Wells were imaged on the
ImageXpress Ultra and quantified with the multi-wavelength cell
scoring script, n=6/protein concentration. Results are exemplified
in FIG. 3B relative to control (cells stimulated without construct,
diluent alone) for WT wild type C-terminal (225-460) ANGPTL3,
engineered construct 242KQ or 242Kdel or full length ANGPTL1, a
related family member of the ANGPTL proteins. Similar results were
seen with experiments using each of 225WT, 225KQ, 226KQ, 228KQ,
233KQ, 241KQ and 242KQ constructs.
[0116] Chondrogenesis assays were carried out using assays and
methods described previously and herein for additional ANGPTL
related family members. Experiments were carried out to examine
whether closely related proteins confer chondrogenic activity, and
if the activity was retained in the C-terminal end of the protein.
ANGPTL1 and ANGPTL4 demonstrated activity in nodule formation
assays; however, only ANGPTL1 showed an induction of type II
collagen in chondrogenesis assays. See Table 4. Results of nodule
formation activity and induction of Type II collagen assays are
summarized in Table 4. Additional characterization of ANGPTL1 is
described herein. See other portions of this Example and FIGS.
3-5.
TABLE-US-00005 TABLE 5 Sequence homology among human angiopoeitin
like family members Sequence identity among human angiopoeitin like
family members (ECD or CTD) Family member Family member % Sequence
Identity hANGPTL3_17-460 hANGPTL4_26-406 32.6 hANGPTL3_17-460
hANGPTL1_24-491 25.7 hANGPTL3_17-460 hANGPTL7_27-346 28.1
hANGPTL3_17-460 hANGPT1_23-498 24.1 hANGPTL3_17-460 hANGPT2_19-496
23.4 hANGPTL3_241-460 hANGPTL4_179-406 40.0 hANGPTL3_241-460
hANGPTL1_271-491 36.8 hANGPTL3_241-460 hANGPTL7_122-343 36.4
hANGPTL3_241-460 hANGPT1_277-497 37.3 hANGPTL3_241-460
hANGPT2_275-495 36.4
[0117] RNA expression analysis was also used to evaluate expression
of cartilage specific proteins. Briefly, qRT-PCR hMSCs were grown
in pellet culture (1.times.10.sup.6 cells/pellet) for 3, 7, 10, 21
days in serum free DMEM, 1.times.ITS plus constructs (as
indicated). Media was replaced every 3 days. Lubricin, Aggrecan,
Sox9, IGF1, IFITM1, Osteocalcin and type X collagen mRNA expression
were quantified using Roche LightCycler (data pooled from 3
experiments performed in duplicate (n=6)). FIG. 3A represents
expression data at Day 10 for 242KQ and 225WT. Gene expression data
was similar for all genes at days 3, 7 and 21.
[0118] Full length ANGPTL3 had been previously shown to have
chondrogenesis activity in both human and mouse mesenchymal stem
cells. Constructs were tested for activity in human, mouse, rat and
canine mesenchymal stem cells to demonstrate the ability of
additional species cross reactivity. CR-MSCs from mouse, rat, human
and dog were cultured with constructs as described above for 18
days. Cultures were fixed and stained using standard
imunnocytochemical techniques to detect the chondrocyte specific
protein type II collagen, and type II collagen positive cells were
quantified using high content imaging. Similar fold increase in the
amount of type II collagen quantified was confirmed for each
species of cells evaluated.
[0119] Chondroprotection. Peptide constructs were evaluated in
functional assays to assess chondroprotective activity.
[0120] An ex vivo glycosaminoglycan (GAG) release inhibition assay
(an indicator of matrix damage) was performed as described in
Johnson, K., et al., (2012) Science 336, 717-721. Briefly, bovine
cartilage was isolated, punched into symmetric circles and put into
organ culture. Slices were treated for 48 hours with 20 ng/ml
TNF.alpha. and 10 ng/ml oncostatin M (OSM) (inflammatory mediators)
to induce degradation of the cartilage matrix in the presence or
absence of protein constructs to identify percent inhibition of
glycosaminoglycan (GAG) release. Results shown in FIG. 4A depict
data pooled from 4 donors, n=12 with the engineered constructs as
indicated and WT 225-460.
[0121] An in vitro nitric oxide (NO) inhibition assay (an indicator
of chondro-protection) was performed as described in Johnson, K.,
et al., (2012) Science 336, 717-721. Briefly, primary chondrocytes
were treated for 48 hrs with protein constructs as indicated.
Greiss reaction was performed, to determine the effect of
constructs on inhibition of NO release as Results shown in FIG. 4B
depict results with the engineered constructs as indicated and WT
Cterminal fragment 225-460. Results shown in FIG. 4C depict results
with wild type Cterminal ANGPTL1, engineered ANGPTL3 242KQ or
control.
[0122] Inhibition of fibrotic cartilage formation. Primary human
articular chondrocytes were cultured as described above with the
addition of ascorbic acid and the presence or absence of constructs
(indicated) for 14 days to induce hypertrophy and type X collagen
expression was assessed by immunoflurescence. Results shown in FIG.
5A depict data with constructs 225WT or 242KQ as indicated. Results
shown in FIG. 5B depict data with wild type C-terminal ANGPTL1,
engineered ANGPTL3 242KQ or 242Kdel or wild type C-terminal ANGPTL3
fragment 225-460 as indicated. The presence of wild type or active
constructs confer an inhibitory effect on formation of fibrotic
cartilage under hypertrophic conditions, as detected by expression
of type X collagen.
[0123] Angiogenesis. The WT C-terminal domain of the ANGPTL3
protein has been reported to have angiogenic activities and
properties in vitro and in vivo in a rat corneal model. See
Camenisch et al., J. Biol. Chem. 277(19): 17281-17290 (2002). To
address the possible risk of inducing new blood vessels following
in vivo administration of C-terminal ANGPTL3, in vitro angiogenic
assays were examined. Briefly, primary human umbilical vein
endothelial cells (HUVECs) were serum starved overnight with basal
endothelial cell media. Cells were then labeled with cell tracker
green and added to pre-coated matrigel plates embedded with protein
construct (indicated). Following culture for 18 hours in the
presence of full length ANGPTL3 (50 ng/mL) or 242KQ (50 ng/mL) or
bFGF (50 ng/mL) which was used as a positive control, the number of
branch points and the total tube length formed was quantified using
high content imaging as a measure of angiogenic activity. In
contrast to the effect seen in the presence of full length ANGPTL3
or positive control, no significant increase in either parameter
was detected when cells were incubated with 242KQ. See FIG. 2C.
[0124] CR-MSCs exist within hyaline articular cartilage and
increase in number in response to injury. Following injury to the
cartilage tissue, these cells have the capacity to participate in
repair processes, but do not sufficiently lead to proper cartilage
repair on their own. Patients are therefore left with articular
cartilage that lacks the proper ability to support painless joint
movements and often require surgical intervention and/or a joint
replacement to maintain their quality of life. We have found
ANGPTL3 and in particular engineered protease resistant ANGPTL3
peptides have the ability to direct the differentiation of human
CR-MSCs into chondrocytes, specifically secreting hyaline articular
cartilage proteins type II collagen and Sox9 while inhibiting the
fibrotic cartilage formation noted by expression of type X
collagen.
[0125] No expression of ANGPTL3 has been reported to our knowledge
nor observed in our studies using western blotting in human
chondrocytes, human MSCs or human synovial fibroblasts. In rodent
joints, little to no expression was found through
immunohistochemistry (IHC). However, in human osteoarthritic
synovial fluid (n=2), low level ANGPTL3 (1.3-6.0 ng/mL) was
detected by enzyme-linked immunosorbent assay (ELISA), suggesting
in a compromised joint, systemically circulating protein can enter
the synovial cavity.
Example 4: In Vivo Analysis of Constructs
[0126] Mouse acute injury surgical model. Surgical transection of
the anterior cruciate ligament (ACL), medial meniscal tibial
ligament (MMTL), and medial collateral ligament (MCL) of the right
knee from C57BL/6 mice (n=12/group) was performed to induce
instability in the knee joint and thus lead to an OA phenotype,
adapted from the previously described model Glasson, S. S., et al.,
Osteoarthritis Cartilage 15, 1061 (2007). To evaluate a potential
therapeutic benefit of ANGPTL3 treatment, 15 weeks following
surgery, mice were dosed intra-articularly as indicated in FIG. 6A
once/per week on weeks 17-19: mANGPTL3 dose=200 ng/knee.
Quantitative assessments of the tibial plateau were made on a 0-4
scale, 0 being normal and 5 being severe osteoarthritis (full
thickness disruption of the cartilage). Two sections from each
mouse were blindly graded by 2 independent observers (FIG. 6B).
[0127] Alleviation of osteoarthritis induced pain for animals was
measured by incapacitance testing, or determining the percentage of
time the mouse stood on a surgically treated leg vs the non-treated
leg using an incapacitance monitoring device. FIG. 7 depicts
results of readouts, representing pain response on days 35 and 56
after surgery were reported as a % weight bearing on the surgical
limb versus the non surgical limb. Treatment depicts results of
animals dosed as described above with full length murine ANGPTL3
(WT17-460) or C-terminal human ANGPTL3 (WT225-460).
[0128] Mouse chronic OA model (collagenase VII induced) Another
widely used animal model of osteoarthritis, the collagenase
VII-induced chronic joint injury model, was used to evaluate in
vivo efficacy of constructs. The model and evaluation was performed
as previously described. See van der Kraan, P. M., et al., Am. J
Pathol. 135, 1001 (1989); and Johnson, K., et al., Science 336, 717
(2012). Briefly, a three (3) day period of inflammation is followed
by collagenase induced destabilization of the joint, resulting in
mild to moderate cartilage destruction. Intra-articular
administration of constructs was carried out following induction in
the knee once/week for three weeks, beginning 3 weeks after
addition of collagenase VII. Forty (42) days following treatment,
joints were collected and sectioned. Histological joint severity
scoring of femoral and tibial plateau allowed quantification of the
tissue repair. The severity of the joint score was determined
through histological scoring as described above. FIG. 8 depicts
repair with 225WT, 225KQ, 228KQ, 233KQ, and 241KQ constructs. To
confirm the presence of protein in the joint (long term
intra-articular retention), tissue was fixed and stained for the
presence of the WT protein construct through immunohistochemistry.
Analysis confirmed the presence of protein indicating
intra-articular retention of ANGPTL3 (with no effects seen on
lipid/triglyceride, assessed using a standard metabolic panel, data
not shown.)
[0129] Histological analysis and grading on Safranin O stained
sections of the medial tibial plateau (for detection of
proteoglycan at the injury site, as described above) revealed
regeneration in cartilage matrix (data not shown). Qualitative
analysis confirmed replacement of proteoglycans similar to levels
seen in a naive mouse, while vehicle controls did not show similar
replacement. Tissue sections were also stained as described above
for type II collagen 8 weeks following injection of the injury.
Qualitative analyses confirmed an increase of type II collagen in
joints treated with construct similar to levels seen in a naive
mouse; while vehicle treated controls did not show similar increase
(data not shown).
[0130] Rat Meniscal Tear Model
[0131] A rat surgical injury model was also used to evaluate in
vivo efficacy of constructs. The model and evaluation was initially
performed as previously described Gerwin N. et al. Osteoarthritis
Cartilage. Suppl 3: S24 (2010). Briefly, skin was shaven over a
knee joint and the medial collateral ligament (MCL) was isolated
through an incision, and the MCL was stabilized and a distal cut of
the meniscus made using a scalpel. On weeks 1, 2 and 3 following
surgery protein construct or vehicle control was injected
intra-articularly, then joints were collected and sectioned at 4
and 6 weeks after surgery. Histological joint severity scoring of
femoral and tibial plateau were performed for quantification of the
tissue repair as described above. Data is shown for the 6 weeks
analyses.
[0132] Healthy hyaline cartilage replaced damage following
treatment. Histological analysis and grading of the lateral tibial
plateau of safranin O stained cartilage were performed as described
above and quantified Results demonstrated animals treated with
242KQ construct revealed regeneration in cartilage matrix and
replacement of proteoglycans similar to levels seen in a naive rat,
while vehicle controls did not show similar replacement. See FIG.
9. Similar results were seen with 225WT.
[0133] A slightly altered surgically induced meniscal tear model
from that described above was used to initiate cartilage damage in
male Lewis rats in order to test the efficacy of 242KQ in promoting
cartilage repair in vivo. Surgery on rats was performed to
completely sever the medial collateral ligament and the medial
meniscus to destabilize the joint so that future weight bearing
would lead to rapid degeneration of the cartilage. An incision was
made to sever the ligament on both sides of the needle, thus
ensuring a complete cut. A scalpel blade was then used to slip
under the patellar ligament into the synovial space and the pointed
tip was used to cut the meniscus. A successful cut was accomplished
when the joint dislocated laterally. One week after surgery, rats
were dosed by intra-articular injection of 242KQ or saline in a
volume of 25 uL into the intra-articular synovial space.
[0134] Twenty eight days after meniscal tear surgery and twenty one
days post intra-articular injection of saline or construct, study
animals were euthanized and affected joints were harvested for
analysis, fixed in 10% formalin in PBS, decalcified with formic
acid, and embedded in paraffin prior to sectioning. Coronal
sections were cut and stained for Safranin O or left unstained for
future immunohistochemical staining. Analysis revealed that the
medial tibial plateau had the greatest amount of cartilage damage
and it was decided to evaluate only this area of the joint for
efficacy of 242KQ. Using the OARSI scoring system, cartilage
severity scores were assigned for six sections across the width of
the tibial cartilage for each animal (N=10) in a blinded manner.
Scoring was done twice at different time-points and the scores were
then averaged to create a score of cartilage damage. Additionally,
objective scoring analyses were performed with a custom script
generated in Matlab. The algorithm identified the articular
cartilage surfaces and objectively quantified additional cartilage
parameters (zonal analyses, safranin O intensity, cartilage area,
cartilage thickness). Results are depicted in FIG. 10A.
[0135] Structural repair of cartilage is not always associated with
relief of pain, at least in humans. Although rodent physiology and
gait are significantly different than humans, 242KQ was evaluated
to determine if there was any improvement in the gait or length of
time spent on the surgical limb after treatment. Incapacitance
monitoring was performed on rats treated with 242KQ. Rats were
subjected to the modified meniscal surgery as described above. One
week following the surgery, 242KQ was injected into the synovial
space. On day 28, the rats were placed on an incapacitance monitor
on their hind limbs and 30 subsequent readings were taken over 10
minutes for each rat to determine the percent of time spent (weight
distribution) on each hind limb. These data give an indication of
the pain-induced weight redistribution It was determined that in
the rat meniscal tear model, treatment with 242KQ one week
following surgery led to a partial restoration of the equal weight
bearing capacity of the rats. See FIG. 10B.
[0136] One of the primary challenges during spontaneous or surgical
cartilage repair is the replacement of hyaline articular cartilage
with fibrotic cartilage. To explore the type of cartilage repair
mediated by ANGPTL3, sections from the rat knees collected from the
rat meniscal tear study performed above were stained for the
presence of type II collagen (to indicate hyaline articular
cartilage) and type X collagen (to indicate fibrotic cartilage).
After a single injection of 20 .mu.g of 242KQ, there was a
qualitative reduction in the amount of type X collagen
expression.
[0137] Long term retention of 242KQ following intravenous and
intra-articular injection into rat knees was determined through
.sup.124I labeling of protein and administration followed by
PET/uCT imaging to monitor retention. See, Gerwin, N., et al.
(2006) Advanced drug delivery reviews 58, 226-242. The mean
residence time (MRT) after IA injection of 242KQ into the joint was
determined to be .about.17.3 h which is significantly increased
over the standard 2-3 h reported (See TABLE 6)
TABLE-US-00006 TABLE 6 Persistence of .sup.124I 242KQ Dose
C.sub.max AUC.sub.0-inf CL Vss MRT T.sub.1/2 Route (.mu.g)
(.mu.g/mL) (hr*.mu.g/mL) (mL/h) (mL) (h) (h) IV 164.2 129.3 22.1
7.4 53.4 7.2 12.4 IA 38.3 0.2 1.9 -- -- 17.3 7.2
[0138] Dog partial menisectomy joint injury model We also evaluated
ANGPTL3 activity in a canine joint injury model. The model was
performed and evaluations performed as described in Connor, J. R.,
et al., Osteoarthritis and cartilage OARS, Osteoarthritis Research
Society 17, 1236-1243 (2009). Briefly, skin was shaven over a knee
joint and the medial collateral ligament (MCL) was isolated through
an incision, and the MCL was stabilized and a distal cut of the
meniscus made using a scalpel. Four (4) days following surgery,
animals received either twice weekly dosing (1.5 ug or 15 ug), or a
single dose (30 ug) of the protein construct (full length canine
ANGPTL3) on day 7 or vehicle control (injected intra-articularly).
Dogs were euthanized on day 28 and the knees were subjected to
histological, sectioning and grading as described above for the rat
and mouse experiments. FIG. 10 depicts the Total gross score of the
repair associated with treatment of canine ANGPTL3. Upon
histological grading and evaluation of the dog joint sections
stained with Safranin 0, areas where the most severe cartilage loss
took place in the saline groups was the portion of the joint that
had the largest reduction in lesion area following a single 30
.mu.g dose of cANGPTL3.
[0139] It is understood that the examples and embodiments described
herein are for illustrative purposes and that various modifications
or changes in light thereof will be suggested to persons skilled in
the art and are to be included within the spirit and purview of
this application and scope of the appended claims.
TABLE-US-00007 SEQUENCES SEQ ID Construct Sequence 1 Human
MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGH ANGPTL3
GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKN
EEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLK
TFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKP
RAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCD
VISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYV
LRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWD
HKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGR
LYSIKSTKMLIHPTDSESFE 2 Human
ttccagaagaaaacagttccacgttgcttgaaattgaaaatcaagataaaaatgt ANGPTL3
tcacaattaagctccttctttttattgttcctctagttatttcctccagaattga REFSEQ
tcaagacaattcatcatttgattctctatctccagagccaaaatcaagatttgct
atgttagacgatgtaaaaattttagccaatggcctccttcagttgggacatggtc
ttaaagactttgtccataagacgaagggccaaattaatgacatatttcaaaaact
caacatatttgatcagtctttttatgatctatcgctgcaaaccagtgaaatcaaa
gaagaagaaaaggaactgagaagaactacatataaactacaagtcaaaaatgaag
aggtaaagaatatgtcacttgaactcaactcaaaacttgaaagcctcctagaaga
aaaaattctacttcaacaaaaagtgaaatatttagaagagcaactaactaactta
attcaaaatcaacctgaaactccagaacacccagaagtaacttcacttaaaactt
ttgtagaaaaacaagataatagcatcaaagaccttctccagaccgtggaagacca
atataaacaattaaaccaacagcatagtcaaataaaagaaatagaaaatcagctc
agaaggactagtattcaagaacccacagaaatttctctatcttccaagccaagag
caccaagaactactccctttcttcagttgaatgaaataagaaatgtaaaacatga
tggcattcctgctgaatgtaccaccatttataacagaggtgaacatacaagtggc
atgtatgccatcagacccagcaactctcaagtttttcatgtctactgtgatgtta
tatcaggtagtccatggacattaattcaacatcgaatagatggatcacaaaactt
caatgaaacgtgggagaactacaaatatggttttgggaggcttgatggagaattt
tggttgggcctagagaagatatactccatagtgaagcaatctaattatgttttac
gaattgagttggaagactggaaagacaacaaacattatattgaatattcttttta
cttgggaaatcacgaaaccaactatacgctacatctagttgcgattactggcaat
gtccccaatgcaatcccggaaaacaaagatttggtgttttctacttgggatcaca
aagcaaaaggacacttcaactgtccagagggttattcaggaggctggtggtggca
tgatgagtgtggagaaaacaacctaaatggtaaatataacaaaccaagagcaaaa
tctaagccagagaggagaagaggattatcttggaagtctcaaaatggaaggttat
actctataaaatcaaccaaaatgttgatccatccaacagattcagaaagctttga
atgaactgaggcaaatttaaaaggcaataatttaaacattaacctcattccaagt
taatgtggtctaataatctggtattaaatccttaagagaaagcttgagaaataga
ttttttttatcttaaagtcactgtctatttaagattaaacatacaatcacataac
cttaaagaataccgtttacatttctcaatcaaaattcttataatactatttgttt
taaattttgtgatgtgggaatcaattttagatggtcacaatctagattataatca
ataggtgaacttattaaataacttttctaaataaaaaatttagagacttttattt
taaaaggcatcatatgagctaatatcacaactttcccagtttaaaaaactagtac
tcttgttaaaactctaaacttgactaaatacagaggactggtaattgtacagttc
ttaaatgttgtagtattaatttcaaaactaaaaatcgtcagcacagagtatgtgt
aaaaatctgtaatacaaatttttaaactgatgcttcattttgctacaaaataatt
tggagtaaatgtttgatatgatttatttatgaaacctaatgaagcagaattaaat
actgtattaaaataagttcgctgtctttaaacaaatggagatgactactaagtca
cattgactttaacatgaggtatcactataccttatt 3 Murine
MHTIKLFLFVVPLVIASRVDPDLSSFDSAPSEPKSRFAMLDDVKILANGLLQLGH ANGPTL3
GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLRTNEIKEEEKELRRTTSTLQVKN
EEVKNMSVELNSKLESLLEEKTALQHKVRALEEQLTNLILSPAGAQEHPEVTSLK
SFVEQQDNSIRELLQSVEEQYKQLSQQHMQIKEIEKQLRKTGIQEPSENSLSSKS
RAPRTTPPLQLNETENTEQDDLPADCSAVYNRGEHTSGVYTIKPRNSQGFNVYCD
TQSGSPWTLIQHRKDGSQDFNETWENYEKGFGRLDGEFWLGLEKIYAIVQQSNYI
LRLELQDWKDSKHYVEYSFHLGSHETNYTLHVAEIAGNIPGALPEHTDLMFSTWN
HRAKGQLYCPESYSGGWWWNDICGENNLNGKYNKPRTKSRPERRRGIYWRPQSRK
LYAIKSSKMMLQPTT 4 Canine
MYTIKLFLFIIPLVISSKIDRDYSSYDSVSPEPKSRFAMLDDVKILANGLLQLGH ANGPTL3
GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTNEIKEEEKELRRTTSKLQVKN
EEVKNMSLELNSKVESLLEEKILLQQKVRYLEKQLTSLIKNQPEIQEHPEVTSLK
TFVEQQDNSIKDLLQTVEEQYRQLNQQHSQIKEIENQLRNVIQESTENSLSSKPR
APRTTPFLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDV
KSGSSWTLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYIL
RIELEDWNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDH
KAKGHVNCPESYSGGWWWHNVCGENNLNGKYNKQRAKTKPERRRGLYWKSQNGRL
YSIKSTKMLIHPIDSESSE 5 Equine
MYTIKLFLVIAPLVISSRIDQDYSSLDSIPPEPKSRFAMLDDVKILANGLLQLGH ANGPTL3
GLKDFVHKTKGQINDIFQKLNIFDQSFYALSLQTNEIKEEEKELRRTTSKLQVKN
EEVKNMSLELNSKLESLLEEKSLLQQKVKYLEEQLTKLIKNQPEIQEHPEVTSLK
TFVEQQDNSIKDLLQTMEEQYRQLNQQHSQIKEIENQLRRTGIQESTENSLSSKP
RAPRTTPSFHLNETKDVEHDDFPADCTTIYNRGEHTSGIYSIKPSNSQVFNVYCD
VISGSSWILIQRRIDGSQNFNETWQNYKYGFGRLDFEFWLGLEKIYSIVKRSNYI
LRIELEDWKDNKHTIEYSFHLGNHETNYTLHLVEITGNVPNALPEHKDLVFSTWD
HKAKGQLNCLESYSGGWWWHDVCGGDNPNGKYNKPRSKTKPERRRGICWKSQNGR
LYTIKSTKMLIHPIDSESFELRQIKKPMN 6 Bovine
MYTIKLFLIIAPLVISSRTDQDYTSLDSISPEPKSRFAMLDDVKILANGLLQLGH ANGPTL3
GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTNEIKEEEKELRRATSKLQVKN
EEVKNMSLELDSKLESLLEEKILLQQKVRYLEDQLTDLIKNQPQIQEYLEVTSLK
TLVEQQDNSIKDLLQIVEEQYRQLNQQQSQIKEIENQLRRTGIKESTEISLSSKP
RAPRTTPSFHSNETKNVEHDDIPADCTIIYNQGKHTSGIYSIRPSNSQVFNVYCD
VKSGSSMTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVMQSNYI
LRIELEDWKDKYYTEYSFHLGDHETNYTLHLAEISGNGPKAFPEHKDLMFSTWDH
KAKGHFNCPESNSGGWWYHDVCGENNLNGKYNKPKAKAKPERKEGICWKSQDGRL
YSIKATKMLIHPSDSENSE 7 207-455WT
IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAI
RPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGL
EKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNA
IPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPE
RRRGLSWKSQNGRLYSIKSTKMLIHPTD 8 225-455WT
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD
9 228-455WT FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 10
233-455WT EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 11
241-455WT GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 12 ANGPTL3KQ
MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGH
GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKN
EEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLK
TFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKP
RAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCD
VISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYV
LRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWD
HKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGR
LYSIKSTKMLIHPTDSESFE 13 ANGPTL3KS
MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGH
GLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKN
EEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLK
TFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKP
RAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCD
VISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYV
LRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWD
HKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGR
LYSIKSTKMLIHPTDSESFE 14 207KQ
IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAI
RPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGL
EKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNA
IPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPE
RRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 15 207KS
IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAI
RPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGL
EKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNA
IPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPE
RRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 16 225KQ
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI
KSTKMLIHPTDSESFE 17 225KS
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI
KSTKMLIHPTDSESFE 18 225ST
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSI
KSTKMLIHPTDSESFE 19 226KQ
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK
STKMLIHPTDSESFE 20 226KS
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK
STKMLIHPTDSESFE 21 228KQ
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST
KMLIHPTDSESFE 22 228KS
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST
KMLIHPTDSESFE 23 228ST
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRAKTKPERRRGLSWKSQNGRLYSIKST
KMLIHPTDSESFE 24 233KQ
EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 25
233KS EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTDSESFE 26
241KQ GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 27 241KS
GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 28 242KQ
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 29 242KS
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 30 225-455KQ
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD
31 225-455KS
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTD
32 226-455KQ
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD
33 226-455KS
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIK STKMLIHPTD
34 228-455KQ
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 35
228-455KS FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKST KMLIHPTD 36
233-455KQ EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 37
233-455KS EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIH PTD 38
241-455KQ GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 39 241-455KS
GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 40 242-455KQ
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRAQSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 41 242-455KS
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 42 Canine
FLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSW 227KQ
TLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELED
WNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHV
NCPESYSGGWWWHNVCGENNLNGKYNKQRAQTKPERRRGLYWKSQNGRLYSIKST
KMLIHPIDSESSE 43 Canine
FLHLNETKNVEHNDIPANCTTIYNRGEHTSGIYSIRPSNSQVFNVYCDVKSGSSW 227KS
TLIQHRIDGSQNFNETWENYRYGFGRLDGEFWLGLEKIYSIVKQSNYILRIELED
WNDNKHYIEYFFHLGNHETNYTLHLVEITGNILNALPEHKDLVFSTWDHKAKGHV
NCPESYSGGWWWHNVCGENNLNGKYNKQRASTKPERRRGLYWKSQNGRLYSIKST
KMLIHPIDSESSE 44 Nucleic
ACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTC acid
CTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGC sequence
CATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGT 225WT
AGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAA
CGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGG
CCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAG
TTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAA
ATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAA
TGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAA
GGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGT
GTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCC
AGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATA
AAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 45 Nucleic
ACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTC acid
CTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGC sequence
CATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGT 225KQ
AGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAA
CGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGG
CCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAG
TTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAA
ATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAA
TGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAA
GGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGT
GTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCC
AGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATA
AAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 46 Nucleic
ACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTC acid
CTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGC sequence
CATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGT 225KS
AGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAA
CGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGG
CCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAG
TTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAA
ATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAA
TGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAA
GGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGT
GTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCC
AGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATA
AAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 47 Nucleic
ACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTG acid
CTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCAT sequence
CAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGT 226KQ
CCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGT
GGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCT
AGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTG
GAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATC
ACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGC
AATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGA
CACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTG
GAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGA
GAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAA
TCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 48 Nucleic
ACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTG acid
CTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCAT sequence
CAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGT 226KS
CCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGT
GGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCT
AGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTG
GAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATC
ACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGC
AATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGA
CACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTG
GAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGA
GAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAA
TCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 49 Nucleic
TTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAAT acid
GTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACC sequence
CAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGG 228KQ
ACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGA
ACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAA
GATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGAC
TGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAA
CCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCC
GGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTC
AACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAA
ACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAG
AAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACC
AAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 50 Nucleic
TTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAAT acid
GTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACC sequence
CAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGG 228KS
ACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGA
ACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAA
GATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGAC
TGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAA
CCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCC
GGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTC
AACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAA
ACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAG
AAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACC
AAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 51 Nucleic
GAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAATGTACCACCATTTATA acid
ACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGT sequence
TTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACAT 233KQ
CGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTT
TTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGT
GAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAA
CATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTAC
ATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTT
GGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGT
TATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTA
AATATAACAAACCAAGAGCACAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTG
GAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCAT
CCAACAGATTCAGAAAGCTTTGAA 52 Nucleic
GAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAATGTACCACCATTTATA acid
ACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGT sequence
TTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACAT 233KS
CGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTT
TTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGT
GAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAA
CATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTAC
ATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTT
GGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGT
TATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTA
AATATAACAAACCAAGAGCAAGCTCTAAGCCAGAGAGGAGAAGAGGATTATCTTG
GAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCAT
CCAACAGATTCAGAAAGCTTTGAA 53 Nucleic
GGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCA acid
TGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTAT sequence
ATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTC 241KQ
AATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTT
GGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACG
AATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTAC
TTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATG
TCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAA
AGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCAT
GATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAAT
CTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATA
CTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 54 Nucleic
GGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCA acid
TGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTAT sequence
ATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTC 241KS
AATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTT
GGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACG
AATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTAC
TTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATG
TCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAA
AGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCAT
GATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCT
CTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATA
CTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 55 Nucleic
ATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGT acid
ATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATC sequence
AGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAAT 242KQ
GAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGT
TGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAAT
TGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTG
GGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCC
CCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGC
AAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGAT
GAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCACAATCTA
AGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTC
TATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 56 Nucleic
ATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGT acid
ATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATC sequence
AGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAAT 242KS
GAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGT
TGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAAT
TGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTG
GGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCC
CCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGC
AAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGAT
GAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAGCTCTA
AGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTC
TATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA 57 Nucleic
TTTTTGCATCTCAACGAAACGAAGAATGTCGAACACAACGACATTCCGGCAAATT acid
GCACAACTATCTACAATAGAGGCGAACATACGTCCGGTATCTACTCCATTAGACC sequence
TTCAAACAGCCAGGTATTCAATGTGTACTGCGATGTAAAGTCAGGATCGTCATGG c227KQ
ACACTGATCCAGCATAGGATCGACGGGTCCCAGAACTTCAACGAGACATGGGAGA
ACTACCGCTATGGATTTGGAAGGCTGGATGGGGAGTTCTGGTTGGGACTTGAGAA
AATCTACAGCATTGTGAAGCAGTCGAACTACATTCTCCGGATTGAACTGGAGGAC
TGGAATGACAACAAACACTACATCGAGTATTTCTTTCATCTCGGCAACCATGAAA
CGAATTACACCTTGCACCTTGTGGAAATCACGGGCAACATTTTGAACGCGCTGCC
AGAACACAAAGACCTGGTGTTTTCGACATGGGATCACAAAGCAAAGGGGCACGTG
AACTGTCCCGAATCATATAGCGGGGGATGGTGGTGGCACAATGTCTGTGGTGAGA
ACAATCTCAACGGGAAATACAATAAGCAGCGAGCTCAGACGAAACCCGAGCGGCG
GAGAGGTCTGTATTGGAAGTCGCAGAATGGACGCCTGTATTCGATCAAATCGACG
AAAATGCTCATCCACCCCATCGACTCCGAATCGTCGGAG 58 207Kdel
IQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAI
RPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGL
EKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNA
IPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPER
RRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 59 225Kdel
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK
STKMLIHPTDSESFE 60 226Kdel
TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS
TKMLIHPTDSESFE 61 228Kdel
FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK
MLIHPTDSESFE 62 233Kdel
EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TDSESFE 63
241Kdel GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 64 242Kdel
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE 65 225-
TTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISG 455Kdel
SPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIE
LEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAK
GHFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIK STKMLIHPTD
66 226- TPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGS
455Kdel PWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIEL
EDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKG
HFNCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKS TKMLIHPTD
67 228- FLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPW
455Kdel TLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELED
WKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHF
NCPEGYSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTK MLIHPTD 68
233- EIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQH
455Kdel RIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNK
HYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEG
YSGGWWWHDECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHP TD 69 241-
GIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNF 455Kdel
NETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFY
LGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWH
DECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 70 242-
IPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFN 455Kdel
ETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYL
GNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHD
ECGENNLNGKYNKPRASKPERRRGLSWKSQNGRLYSIKSTKMLIHPTD 71 hANGPTL1
MKTFTWTLGVLFFLLVDTGHCRGGQFKIKKINQRRYPRATDGKEEAKKCAYTFLVP 1-491
EQRITGPICVNTKGQDASTIKDMITRMDLENLKDVLSRQKREIDVLQLVVDVDGNI
VNEVKLLRKESRNMNSRVTQLYMQLLHEIIRKRDNSLELSQLENKILNVTTEMLKM
ATRYRELEVKYASLTDLVNNQSVMITLLEEQCLRIFSRQDTHVSPPLVQVVPQHIP
NSQQYTPGLLGGNEIQRDPGYPRDLMPPPDLATSPTKSPFKIPPVTFINEGPFKDC
QQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTVIQKRTDGSVNFFRNWE
NYKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIELEDWSDKKVYAEYSSFRLEPES
EFYRLRLGTYQGNAGDSMMWHNGKQFTTLDRDKDMYAGNCAHFHKGGWWYNACAHS
NLNGVWYRGGHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPID 72 CT
FINEGPFKDCQQAKEAGHSVSGIYMIKPENSNGPMQLWCENSLDPGGWTVIQKRTD hANGPTL1
GSVNFFRNWENYKKGFGNIDGEYWLGLENIYMLSNQDNYKLLIELEDWSDKKVYAE 271-491
YSSFRLEPESEFYRLRLGTYQGNAGDSMMWHNGKQFTTLDRDKDMYAGNCAHFHKG
GWWYNACAHSNLNGVWYRGGHYRSKHQDGIFWAEYRGGSYSLRAVQMMIKPID 73 hANGPTL4
MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMNVLAHGLLQLGQGLR 1-406
EHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRVDPEVLHSLQTQLKAQ
NSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEM
AQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGW
TVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDW
DGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLR
RDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQA
TTMLIQPMAAEAAS 74 CT
SRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGS hANGPTL4
VDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFS 179-406
VHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLS
GGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAA EAAS
Sequence CWU 1
1
741460PRTHomo sapiens 1Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val
Pro Leu Val Ile Ser1 5 10 15Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe
Asp Ser Leu Ser Pro Glu 20 25 30Pro Lys Ser Arg Phe Ala Met Leu Asp
Asp Val Lys Ile Leu Ala Asn 35 40 45Gly Leu Leu Gln Leu Gly His Gly
Leu Lys Asp Phe Val His Lys Thr 50 55 60Lys Gly Gln Ile Asn Asp Ile
Phe Gln Lys Leu Asn Ile Phe Asp Gln65 70 75 80Ser Phe Tyr Asp Leu
Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu 85 90 95Lys Glu Leu Arg
Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu 100 105 110Val Lys
Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu 115 120
125Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln
130 135 140Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro Glu His
Pro Glu145 150 155 160Val Thr Ser Leu Lys Thr Phe Val Glu Lys Gln
Asp Asn Ser Ile Lys 165 170 175Asp Leu Leu Gln Thr Val Glu Asp Gln
Tyr Lys Gln Leu Asn Gln Gln 180 185 190His Ser Gln Ile Lys Glu Ile
Glu Asn Gln Leu Arg Arg Thr Ser Ile 195 200 205Gln Glu Pro Thr Glu
Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg 210 215 220Thr Thr Pro
Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp225 230 235
240Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr
245 250 255Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe
His Val 260 265 270Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu
Ile Gln His Arg 275 280 285Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr
Trp Glu Asn Tyr Lys Tyr 290 295 300Gly Phe Gly Arg Leu Asp Gly Glu
Phe Trp Leu Gly Leu Glu Lys Ile305 310 315 320Tyr Ser Ile Val Lys
Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 325 330 335Asp Trp Lys
Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 340 345 350Asn
His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn 355 360
365Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp
370 375 380Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr
Ser Gly385 390 395 400Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn
Asn Leu Asn Gly Lys 405 410 415Tyr Asn Lys Pro Arg Ala Lys Ser Lys
Pro Glu Arg Arg Arg Gly Leu 420 425 430Ser Trp Lys Ser Gln Asn Gly
Arg Leu Tyr Ser Ile Lys Ser Thr Lys 435 440 445Met Leu Ile His Pro
Thr Asp Ser Glu Ser Phe Glu 450 455 46022126DNAHomo sapiens
2ttccagaaga aaacagttcc acgttgcttg aaattgaaaa tcaagataaa aatgttcaca
60attaagctcc ttctttttat tgttcctcta gttatttcct ccagaattga tcaagacaat
120tcatcatttg attctctatc tccagagcca aaatcaagat ttgctatgtt
agacgatgta 180aaaattttag ccaatggcct ccttcagttg ggacatggtc
ttaaagactt tgtccataag 240acgaagggcc aaattaatga catatttcaa
aaactcaaca tatttgatca gtctttttat 300gatctatcgc tgcaaaccag
tgaaatcaaa gaagaagaaa aggaactgag aagaactaca 360tataaactac
aagtcaaaaa tgaagaggta aagaatatgt cacttgaact caactcaaaa
420cttgaaagcc tcctagaaga aaaaattcta cttcaacaaa aagtgaaata
tttagaagag 480caactaacta acttaattca aaatcaacct gaaactccag
aacacccaga agtaacttca 540cttaaaactt ttgtagaaaa acaagataat
agcatcaaag accttctcca gaccgtggaa 600gaccaatata aacaattaaa
ccaacagcat agtcaaataa aagaaataga aaatcagctc 660agaaggacta
gtattcaaga acccacagaa atttctctat cttccaagcc aagagcacca
720agaactactc cctttcttca gttgaatgaa ataagaaatg taaaacatga
tggcattcct 780gctgaatgta ccaccattta taacagaggt gaacatacaa
gtggcatgta tgccatcaga 840cccagcaact ctcaagtttt tcatgtctac
tgtgatgtta tatcaggtag tccatggaca 900ttaattcaac atcgaataga
tggatcacaa aacttcaatg aaacgtggga gaactacaaa 960tatggttttg
ggaggcttga tggagaattt tggttgggcc tagagaagat atactccata
1020gtgaagcaat ctaattatgt tttacgaatt gagttggaag actggaaaga
caacaaacat 1080tatattgaat attcttttta cttgggaaat cacgaaacca
actatacgct acatctagtt 1140gcgattactg gcaatgtccc caatgcaatc
ccggaaaaca aagatttggt gttttctact 1200tgggatcaca aagcaaaagg
acacttcaac tgtccagagg gttattcagg aggctggtgg 1260tggcatgatg
agtgtggaga aaacaaccta aatggtaaat ataacaaacc aagagcaaaa
1320tctaagccag agaggagaag aggattatct tggaagtctc aaaatggaag
gttatactct 1380ataaaatcaa ccaaaatgtt gatccatcca acagattcag
aaagctttga atgaactgag 1440gcaaatttaa aaggcaataa tttaaacatt
aacctcattc caagttaatg tggtctaata 1500atctggtatt aaatccttaa
gagaaagctt gagaaataga ttttttttat cttaaagtca 1560ctgtctattt
aagattaaac atacaatcac ataaccttaa agaataccgt ttacatttct
1620caatcaaaat tcttataata ctatttgttt taaattttgt gatgtgggaa
tcaattttag 1680atggtcacaa tctagattat aatcaatagg tgaacttatt
aaataacttt tctaaataaa 1740aaatttagag acttttattt taaaaggcat
catatgagct aatatcacaa ctttcccagt 1800ttaaaaaact agtactcttg
ttaaaactct aaacttgact aaatacagag gactggtaat 1860tgtacagttc
ttaaatgttg tagtattaat ttcaaaacta aaaatcgtca gcacagagta
1920tgtgtaaaaa tctgtaatac aaatttttaa actgatgctt cattttgcta
caaaataatt 1980tggagtaaat gtttgatatg atttatttat gaaacctaat
gaagcagaat taaatactgt 2040attaaaataa gttcgctgtc tttaaacaaa
tggagatgac tactaagtca cattgacttt 2100aacatgaggt atcactatac cttatt
21263455PRTMus musculus 3Met His Thr Ile Lys Leu Phe Leu Phe Val
Val Pro Leu Val Ile Ala1 5 10 15Ser Arg Val Asp Pro Asp Leu Ser Ser
Phe Asp Ser Ala Pro Ser Glu 20 25 30Pro Lys Ser Arg Phe Ala Met Leu
Asp Asp Val Lys Ile Leu Ala Asn 35 40 45Gly Leu Leu Gln Leu Gly His
Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60Lys Gly Gln Ile Asn Asp
Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln65 70 75 80Ser Phe Tyr Asp
Leu Ser Leu Arg Thr Asn Glu Ile Lys Glu Glu Glu 85 90 95Lys Glu Leu
Arg Arg Thr Thr Ser Thr Leu Gln Val Lys Asn Glu Glu 100 105 110Val
Lys Asn Met Ser Val Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu 115 120
125Glu Glu Lys Thr Ala Leu Gln His Lys Val Arg Ala Leu Glu Glu Gln
130 135 140Leu Thr Asn Leu Ile Leu Ser Pro Ala Gly Ala Gln Glu His
Pro Glu145 150 155 160Val Thr Ser Leu Lys Ser Phe Val Glu Gln Gln
Asp Asn Ser Ile Arg 165 170 175Glu Leu Leu Gln Ser Val Glu Glu Gln
Tyr Lys Gln Leu Ser Gln Gln 180 185 190His Met Gln Ile Lys Glu Ile
Glu Lys Gln Leu Arg Lys Thr Gly Ile 195 200 205Gln Glu Pro Ser Glu
Asn Ser Leu Ser Ser Lys Ser Arg Ala Pro Arg 210 215 220Thr Thr Pro
Pro Leu Gln Leu Asn Glu Thr Glu Asn Thr Glu Gln Asp225 230 235
240Asp Leu Pro Ala Asp Cys Ser Ala Val Tyr Asn Arg Gly Glu His Thr
245 250 255Ser Gly Val Tyr Thr Ile Lys Pro Arg Asn Ser Gln Gly Phe
Asn Val 260 265 270Tyr Cys Asp Thr Gln Ser Gly Ser Pro Trp Thr Leu
Ile Gln His Arg 275 280 285Lys Asp Gly Ser Gln Asp Phe Asn Glu Thr
Trp Glu Asn Tyr Glu Lys 290 295 300Gly Phe Gly Arg Leu Asp Gly Glu
Phe Trp Leu Gly Leu Glu Lys Ile305 310 315 320Tyr Ala Ile Val Gln
Gln Ser Asn Tyr Ile Leu Arg Leu Glu Leu Gln 325 330 335Asp Trp Lys
Asp Ser Lys His Tyr Val Glu Tyr Ser Phe His Leu Gly 340 345 350Ser
His Glu Thr Asn Tyr Thr Leu His Val Ala Glu Ile Ala Gly Asn 355 360
365Ile Pro Gly Ala Leu Pro Glu His Thr Asp Leu Met Phe Ser Thr Trp
370 375 380Asn His Arg Ala Lys Gly Gln Leu Tyr Cys Pro Glu Ser Tyr
Ser Gly385 390 395 400Gly Trp Trp Trp Asn Asp Ile Cys Gly Glu Asn
Asn Leu Asn Gly Lys 405 410 415Tyr Asn Lys Pro Arg Thr Lys Ser Arg
Pro Glu Arg Arg Arg Gly Ile 420 425 430Tyr Trp Arg Pro Gln Ser Arg
Lys Leu Tyr Ala Ile Lys Ser Ser Lys 435 440 445Met Met Leu Gln Pro
Thr Thr 450 4554459PRTCanis familiaris 4Met Tyr Thr Ile Lys Leu Phe
Leu Phe Ile Ile Pro Leu Val Ile Ser1 5 10 15Ser Lys Ile Asp Arg Asp
Tyr Ser Ser Tyr Asp Ser Val Ser Pro Glu 20 25 30Pro Lys Ser Arg Phe
Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 40 45Gly Leu Leu Gln
Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60Lys Gly Gln
Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln65 70 75 80Ser
Phe Tyr Asp Leu Ser Leu Gln Thr Asn Glu Ile Lys Glu Glu Glu 85 90
95Lys Glu Leu Arg Arg Thr Thr Ser Lys Leu Gln Val Lys Asn Glu Glu
100 105 110Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Val Glu Ser
Leu Leu 115 120 125Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Arg Tyr
Leu Glu Lys Gln 130 135 140Leu Thr Ser Leu Ile Lys Asn Gln Pro Glu
Ile Gln Glu His Pro Glu145 150 155 160Val Thr Ser Leu Lys Thr Phe
Val Glu Gln Gln Asp Asn Ser Ile Lys 165 170 175Asp Leu Leu Gln Thr
Val Glu Glu Gln Tyr Arg Gln Leu Asn Gln Gln 180 185 190His Ser Gln
Ile Lys Glu Ile Glu Asn Gln Leu Arg Asn Val Ile Gln 195 200 205Glu
Ser Thr Glu Asn Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg Thr 210 215
220Thr Pro Phe Leu His Leu Asn Glu Thr Lys Asn Val Glu His Asn
Asp225 230 235 240Ile Pro Ala Asn Cys Thr Thr Ile Tyr Asn Arg Gly
Glu His Thr Ser 245 250 255Gly Ile Tyr Ser Ile Arg Pro Ser Asn Ser
Gln Val Phe Asn Val Tyr 260 265 270Cys Asp Val Lys Ser Gly Ser Ser
Trp Thr Leu Ile Gln His Arg Ile 275 280 285Asp Gly Ser Gln Asn Phe
Asn Glu Thr Trp Glu Asn Tyr Arg Tyr Gly 290 295 300Phe Gly Arg Leu
Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr305 310 315 320Ser
Ile Val Lys Gln Ser Asn Tyr Ile Leu Arg Ile Glu Leu Glu Asp 325 330
335Trp Asn Asp Asn Lys His Tyr Ile Glu Tyr Phe Phe His Leu Gly Asn
340 345 350His Glu Thr Asn Tyr Thr Leu His Leu Val Glu Ile Thr Gly
Asn Ile 355 360 365Leu Asn Ala Leu Pro Glu His Lys Asp Leu Val Phe
Ser Thr Trp Asp 370 375 380His Lys Ala Lys Gly His Val Asn Cys Pro
Glu Ser Tyr Ser Gly Gly385 390 395 400Trp Trp Trp His Asn Val Cys
Gly Glu Asn Asn Leu Asn Gly Lys Tyr 405 410 415Asn Lys Gln Arg Ala
Lys Thr Lys Pro Glu Arg Arg Arg Gly Leu Tyr 420 425 430Trp Lys Ser
Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met 435 440 445Leu
Ile His Pro Ile Asp Ser Glu Ser Ser Glu 450 4555469PRTEquus
caballus 5Met Tyr Thr Ile Lys Leu Phe Leu Val Ile Ala Pro Leu Val
Ile Ser1 5 10 15Ser Arg Ile Asp Gln Asp Tyr Ser Ser Leu Asp Ser Ile
Pro Pro Glu 20 25 30Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys
Ile Leu Ala Asn 35 40 45Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp
Phe Val His Lys Thr 50 55 60Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys
Leu Asn Ile Phe Asp Gln65 70 75 80Ser Phe Tyr Ala Leu Ser Leu Gln
Thr Asn Glu Ile Lys Glu Glu Glu 85 90 95Lys Glu Leu Arg Arg Thr Thr
Ser Lys Leu Gln Val Lys Asn Glu Glu 100 105 110Val Lys Asn Met Ser
Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu 115 120 125Glu Glu Lys
Ser Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln 130 135 140Leu
Thr Lys Leu Ile Lys Asn Gln Pro Glu Ile Gln Glu His Pro Glu145 150
155 160Val Thr Ser Leu Lys Thr Phe Val Glu Gln Gln Asp Asn Ser Ile
Lys 165 170 175Asp Leu Leu Gln Thr Met Glu Glu Gln Tyr Arg Gln Leu
Asn Gln Gln 180 185 190His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu
Arg Arg Thr Gly Ile 195 200 205Gln Glu Ser Thr Glu Asn Ser Leu Ser
Ser Lys Pro Arg Ala Pro Arg 210 215 220Thr Thr Pro Ser Phe His Leu
Asn Glu Thr Lys Asp Val Glu His Asp225 230 235 240Asp Phe Pro Ala
Asp Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 245 250 255Ser Gly
Ile Tyr Ser Ile Lys Pro Ser Asn Ser Gln Val Phe Asn Val 260 265
270Tyr Cys Asp Val Ile Ser Gly Ser Ser Trp Ile Leu Ile Gln Arg Arg
275 280 285Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Gln Asn Tyr
Lys Tyr 290 295 300Gly Phe Gly Arg Leu Asp Phe Glu Phe Trp Leu Gly
Leu Glu Lys Ile305 310 315 320Tyr Ser Ile Val Lys Arg Ser Asn Tyr
Ile Leu Arg Ile Glu Leu Glu 325 330 335Asp Trp Lys Asp Asn Lys His
Thr Ile Glu Tyr Ser Phe His Leu Gly 340 345 350Asn His Glu Thr Asn
Tyr Thr Leu His Leu Val Glu Ile Thr Gly Asn 355 360 365Val Pro Asn
Ala Leu Pro Glu His Lys Asp Leu Val Phe Ser Thr Trp 370 375 380Asp
His Lys Ala Lys Gly Gln Leu Asn Cys Leu Glu Ser Tyr Ser Gly385 390
395 400Gly Trp Trp Trp His Asp Val Cys Gly Gly Asp Asn Pro Asn Gly
Lys 405 410 415Tyr Asn Lys Pro Arg Ser Lys Thr Lys Pro Glu Arg Arg
Arg Gly Ile 420 425 430Cys Trp Lys Ser Gln Asn Gly Arg Leu Tyr Thr
Ile Lys Ser Thr Lys 435 440 445Met Leu Ile His Pro Ile Asp Ser Glu
Ser Phe Glu Leu Arg Gln Ile 450 455 460Lys Lys Pro Met
Asn4656459PRTBos taurus 6Met Tyr Thr Ile Lys Leu Phe Leu Ile Ile
Ala Pro Leu Val Ile Ser1 5 10 15Ser Arg Thr Asp Gln Asp Tyr Thr Ser
Leu Asp Ser Ile Ser Pro Glu 20 25 30Pro Lys Ser Arg Phe Ala Met Leu
Asp Asp Val Lys Ile Leu Ala Asn 35 40 45Gly Leu Leu Gln Leu Gly His
Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60Lys Gly Gln Ile Asn Asp
Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln65 70 75 80Ser Phe Tyr Asp
Leu Ser Leu Gln Thr Asn Glu Ile Lys Glu Glu Glu 85 90 95Lys Glu Leu
Arg Arg Ala Thr Ser Lys Leu Gln Val Lys Asn Glu Glu 100 105 110Val
Lys Asn Met Ser Leu Glu Leu Asp Ser Lys Leu Glu Ser Leu Leu 115 120
125Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Arg Tyr Leu Glu Asp Gln
130 135 140Leu Thr Asp Leu Ile Lys Asn Gln Pro Gln Ile Gln Glu Tyr
Leu Glu145 150 155 160Val Thr Ser Leu Lys Thr Leu Val Glu Gln Gln
Asp Asn Ser Ile Lys 165 170 175Asp Leu Leu Gln Ile Val Glu Glu Gln
Tyr Arg Gln Leu Asn Gln Gln 180 185 190Gln Ser Gln Ile Lys Glu Ile
Glu Asn Gln Leu Arg Arg Thr Gly Ile 195 200 205Lys Glu Ser Thr Glu
Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg 210 215 220Thr Thr Pro
Ser Phe His Ser Asn Glu Thr Lys Asn Val Glu His Asp225 230 235
240Asp Ile Pro Ala Asp Cys Thr Ile Ile Tyr Asn Gln Gly Lys His Thr
245 250 255Ser Gly Ile Tyr Ser Ile Arg Pro Ser Asn Ser Gln
Val Phe Asn Val 260 265 270Tyr Cys Asp Val Lys Ser Gly Ser Ser Trp
Thr Leu Ile Gln His Arg 275 280 285Ile Asp Gly Ser Gln Asn Phe Asn
Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295 300Gly Phe Gly Arg Leu Asp
Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile305 310 315 320Tyr Ser Ile
Val Met Gln Ser Asn Tyr Ile Leu Arg Ile Glu Leu Glu 325 330 335Asp
Trp Lys Asp Lys Tyr Tyr Thr Glu Tyr Ser Phe His Leu Gly Asp 340 345
350His Glu Thr Asn Tyr Thr Leu His Leu Ala Glu Ile Ser Gly Asn Gly
355 360 365Pro Lys Ala Phe Pro Glu His Lys Asp Leu Met Phe Ser Thr
Trp Asp 370 375 380His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Ser
Asn Ser Gly Gly385 390 395 400Trp Trp Tyr His Asp Val Cys Gly Glu
Asn Asn Leu Asn Gly Lys Tyr 405 410 415Asn Lys Pro Lys Ala Lys Ala
Lys Pro Glu Arg Lys Glu Gly Ile Cys 420 425 430Trp Lys Ser Gln Asp
Gly Arg Leu Tyr Ser Ile Lys Ala Thr Lys Met 435 440 445Leu Ile His
Pro Ser Asp Ser Glu Asn Ser Glu 450 4557248PRTHomo sapiens 7Ile Gln
Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro1 5 10 15Arg
Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His 20 25
30Asp Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His
35 40 45Thr Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe
His 50 55 60Val Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile
Gln His65 70 75 80Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp
Glu Asn Tyr Lys 85 90 95Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp
Leu Gly Leu Glu Lys 100 105 110Ile Tyr Ser Ile Val Lys Gln Ser Asn
Tyr Val Leu Arg Ile Glu Leu 115 120 125Glu Asp Trp Lys Asp Asn Lys
His Tyr Ile Glu Tyr Ser Phe Tyr Leu 130 135 140Gly Asn His Glu Thr
Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly145 150 155 160Asn Val
Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr 165 170
175Trp Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser
180 185 190Gly Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu
Asn Gly 195 200 205Lys Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu
Arg Arg Arg Gly 210 215 220Leu Ser Trp Lys Ser Gln Asn Gly Arg Leu
Tyr Ser Ile Lys Ser Thr225 230 235 240Lys Met Leu Ile His Pro Thr
Asp 2458231PRTHomo sapiens 8Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile
Arg Asn Val Lys His Asp1 5 10 15Gly Ile Pro Ala Glu Cys Thr Thr Ile
Tyr Asn Arg Gly Glu His Thr 20 25 30Ser Gly Met Tyr Ala Ile Arg Pro
Ser Asn Ser Gln Val Phe His Val 35 40 45Tyr Cys Asp Val Ile Ser Gly
Ser Pro Trp Thr Leu Ile Gln His Arg 50 55 60Ile Asp Gly Ser Gln Asn
Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr65 70 75 80Gly Phe Gly Arg
Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 85 90 95Tyr Ser Ile
Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 100 105 110Asp
Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 115 120
125Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn
130 135 140Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser
Thr Trp145 150 155 160Asp His Lys Ala Lys Gly His Phe Asn Cys Pro
Glu Gly Tyr Ser Gly 165 170 175Gly Trp Trp Trp His Asp Glu Cys Gly
Glu Asn Asn Leu Asn Gly Lys 180 185 190Tyr Asn Lys Pro Arg Ala Lys
Ser Lys Pro Glu Arg Arg Arg Gly Leu 195 200 205Ser Trp Lys Ser Gln
Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 210 215 220Met Leu Ile
His Pro Thr Asp225 2309228PRTHomo sapiens 9Phe Leu Gln Leu Asn Glu
Ile Arg Asn Val Lys His Asp Gly Ile Pro1 5 10 15Ala Glu Cys Thr Thr
Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met 20 25 30Tyr Ala Ile Arg
Pro Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp 35 40 45Val Ile Ser
Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly 50 55 60Ser Gln
Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly65 70 75
80Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile
85 90 95Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp Trp
Lys 100 105 110Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly
Asn His Glu 115 120 125Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr
Gly Asn Val Pro Asn 130 135 140Ala Ile Pro Glu Asn Lys Asp Leu Val
Phe Ser Thr Trp Asp His Lys145 150 155 160Ala Lys Gly His Phe Asn
Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp 165 170 175Trp His Asp Glu
Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys 180 185 190Pro Arg
Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys 195 200
205Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile
210 215 220His Pro Thr Asp22510223PRTHomo sapiens 10Glu Ile Arg Asn
Val Lys His Asp Gly Ile Pro Ala Glu Cys Thr Thr1 5 10 15Ile Tyr Asn
Arg Gly Glu His Thr Ser Gly Met Tyr Ala Ile Arg Pro 20 25 30Ser Asn
Ser Gln Val Phe His Val Tyr Cys Asp Val Ile Ser Gly Ser 35 40 45Pro
Trp Thr Leu Ile Gln His Arg Ile Asp Gly Ser Gln Asn Phe Asn 50 55
60Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu65
70 75 80Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln Ser
Asn 85 90 95Tyr Val Leu Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn Lys
His Tyr 100 105 110Ile Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu Thr
Asn Tyr Thr Leu 115 120 125His Leu Val Ala Ile Thr Gly Asn Val Pro
Asn Ala Ile Pro Glu Asn 130 135 140Lys Asp Leu Val Phe Ser Thr Trp
Asp His Lys Ala Lys Gly His Phe145 150 155 160Asn Cys Pro Glu Gly
Tyr Ser Gly Gly Trp Trp Trp His Asp Glu Cys 165 170 175Gly Glu Asn
Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Ala Lys Ser 180 185 190Lys
Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser Gln Asn Gly Arg 195 200
205Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile His Pro Thr Asp 210
215 22011215PRTHomo sapiens 11Gly Ile Pro Ala Glu Cys Thr Thr Ile
Tyr Asn Arg Gly Glu His Thr1 5 10 15Ser Gly Met Tyr Ala Ile Arg Pro
Ser Asn Ser Gln Val Phe His Val 20 25 30Tyr Cys Asp Val Ile Ser Gly
Ser Pro Trp Thr Leu Ile Gln His Arg 35 40 45Ile Asp Gly Ser Gln Asn
Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 50 55 60Gly Phe Gly Arg Leu
Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile65 70 75 80Tyr Ser Ile
Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 85 90 95Asp Trp
Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 100 105
110Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn
115 120 125Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser
Thr Trp 130 135 140Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu
Gly Tyr Ser Gly145 150 155 160Gly Trp Trp Trp His Asp Glu Cys Gly
Glu Asn Asn Leu Asn Gly Lys 165 170 175Tyr Asn Lys Pro Arg Ala Lys
Ser Lys Pro Glu Arg Arg Arg Gly Leu 180 185 190Ser Trp Lys Ser Gln
Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 195 200 205Met Leu Ile
His Pro Thr Asp 210 21512460PRTArtificial SequenceK423Q 12Met Phe
Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser1 5 10 15Ser
Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 25
30Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn
35 40 45Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys
Thr 50 55 60Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe
Asp Gln65 70 75 80Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile
Lys Glu Glu Glu 85 90 95Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln
Val Lys Asn Glu Glu 100 105 110Val Lys Asn Met Ser Leu Glu Leu Asn
Ser Lys Leu Glu Ser Leu Leu 115 120 125Glu Glu Lys Ile Leu Leu Gln
Gln Lys Val Lys Tyr Leu Glu Glu Gln 130 135 140Leu Thr Asn Leu Ile
Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu145 150 155 160Val Thr
Ser Leu Lys Thr Phe Val Glu Lys Gln Asp Asn Ser Ile Lys 165 170
175Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln
180 185 190His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr
Ser Ile 195 200 205Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro
Arg Ala Pro Arg 210 215 220Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile
Arg Asn Val Lys His Asp225 230 235 240Gly Ile Pro Ala Glu Cys Thr
Thr Ile Tyr Asn Arg Gly Glu His Thr 245 250 255Ser Gly Met Tyr Ala
Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 260 265 270Tyr Cys Asp
Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285Ile
Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295
300Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys
Ile305 310 315 320Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg
Ile Glu Leu Glu 325 330 335Asp Trp Lys Asp Asn Lys His Tyr Ile Glu
Tyr Ser Phe Tyr Leu Gly 340 345 350Asn His Glu Thr Asn Tyr Thr Leu
His Leu Val Ala Ile Thr Gly Asn 355 360 365Val Pro Asn Ala Ile Pro
Glu Asn Lys Asp Leu Val Phe Ser Thr Trp 370 375 380Asp His Lys Ala
Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly385 390 395 400Gly
Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 405 410
415Tyr Asn Lys Pro Arg Ala Gln Ser Lys Pro Glu Arg Arg Arg Gly Leu
420 425 430Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser
Thr Lys 435 440 445Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu
450 455 46013460PRTArtificial SequenceK423S 13Met Phe Thr Ile Lys
Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser1 5 10 15Ser Arg Ile Asp
Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 25 30Pro Lys Ser
Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 40 45Gly Leu
Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60Lys
Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln65 70 75
80Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu
85 90 95Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu
Glu 100 105 110Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu
Ser Leu Leu 115 120 125Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys
Tyr Leu Glu Glu Gln 130 135 140Leu Thr Asn Leu Ile Gln Asn Gln Pro
Glu Thr Pro Glu His Pro Glu145 150 155 160Val Thr Ser Leu Lys Thr
Phe Val Glu Lys Gln Asp Asn Ser Ile Lys 165 170 175Asp Leu Leu Gln
Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln 180 185 190His Ser
Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile 195 200
205Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg
210 215 220Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys
His Asp225 230 235 240Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn
Arg Gly Glu His Thr 245 250 255Ser Gly Met Tyr Ala Ile Arg Pro Ser
Asn Ser Gln Val Phe His Val 260 265 270Tyr Cys Asp Val Ile Ser Gly
Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285Ile Asp Gly Ser Gln
Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295 300Gly Phe Gly
Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile305 310 315
320Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu
325 330 335Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr
Leu Gly 340 345 350Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala
Ile Thr Gly Asn 355 360 365Val Pro Asn Ala Ile Pro Glu Asn Lys Asp
Leu Val Phe Ser Thr Trp 370 375 380Asp His Lys Ala Lys Gly His Phe
Asn Cys Pro Glu Gly Tyr Ser Gly385 390 395 400Gly Trp Trp Trp His
Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 405 410 415Tyr Asn Lys
Pro Arg Ala Ser Ser Lys Pro Glu Arg Arg Arg Gly Leu 420 425 430Ser
Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 435 440
445Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 450 455
46014253PRTArtificial Sequence207-460 K423Q 14Ile Gln Glu Pro Thr
Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro1 5 10 15Arg Thr Thr Pro
Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His 20 25 30Asp Gly Ile
Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His 35 40 45Thr Ser
Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His 50 55 60Val
Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His65 70 75
80Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys
85 90 95Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu
Lys 100 105 110Ile Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg
Ile Glu Leu 115 120 125Glu Asp Trp Lys Asp Asn Lys His Tyr Ile Glu
Tyr Ser Phe Tyr Leu 130 135 140Gly Asn His Glu Thr Asn Tyr Thr Leu
His Leu Val Ala Ile Thr Gly145 150 155 160Asn Val Pro Asn Ala Ile
Pro Glu Asn Lys Asp Leu Val Phe Ser Thr 165 170 175Trp Asp His Lys
Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser 180 185
190Gly Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly
195 200 205Lys Tyr Asn Lys Pro Arg Ala Gln Ser Lys Pro Glu Arg Arg
Arg Gly 210 215 220Leu Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser
Ile Lys Ser Thr225 230 235 240Lys Met Leu Ile His Pro Thr Asp Ser
Glu Ser Phe Glu 245 25015253PRTArtificial Sequence207-460 K423S
15Ile Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro1
5 10 15Arg Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys
His 20 25 30Asp Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly
Glu His 35 40 45Thr Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln
Val Phe His 50 55 60Val Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr
Leu Ile Gln His65 70 75 80Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu
Thr Trp Glu Asn Tyr Lys 85 90 95Tyr Gly Phe Gly Arg Leu Asp Gly Glu
Phe Trp Leu Gly Leu Glu Lys 100 105 110Ile Tyr Ser Ile Val Lys Gln
Ser Asn Tyr Val Leu Arg Ile Glu Leu 115 120 125Glu Asp Trp Lys Asp
Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu 130 135 140Gly Asn His
Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly145 150 155
160Asn Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr
165 170 175Trp Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly
Tyr Ser 180 185 190Gly Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn
Asn Leu Asn Gly 195 200 205Lys Tyr Asn Lys Pro Arg Ala Ser Ser Lys
Pro Glu Arg Arg Arg Gly 210 215 220Leu Ser Trp Lys Ser Gln Asn Gly
Arg Leu Tyr Ser Ile Lys Ser Thr225 230 235 240Lys Met Leu Ile His
Pro Thr Asp Ser Glu Ser Phe Glu 245 25016236PRTArtificial
Sequence225-460 K423Q 16Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg
Asn Val Lys His Asp1 5 10 15Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr
Asn Arg Gly Glu His Thr 20 25 30Ser Gly Met Tyr Ala Ile Arg Pro Ser
Asn Ser Gln Val Phe His Val 35 40 45Tyr Cys Asp Val Ile Ser Gly Ser
Pro Trp Thr Leu Ile Gln His Arg 50 55 60Ile Asp Gly Ser Gln Asn Phe
Asn Glu Thr Trp Glu Asn Tyr Lys Tyr65 70 75 80Gly Phe Gly Arg Leu
Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 85 90 95Tyr Ser Ile Val
Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 100 105 110Asp Trp
Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 115 120
125Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn
130 135 140Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser
Thr Trp145 150 155 160Asp His Lys Ala Lys Gly His Phe Asn Cys Pro
Glu Gly Tyr Ser Gly 165 170 175Gly Trp Trp Trp His Asp Glu Cys Gly
Glu Asn Asn Leu Asn Gly Lys 180 185 190Tyr Asn Lys Pro Arg Ala Gln
Ser Lys Pro Glu Arg Arg Arg Gly Leu 195 200 205Ser Trp Lys Ser Gln
Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 210 215 220Met Leu Ile
His Pro Thr Asp Ser Glu Ser Phe Glu225 230 23517236PRTArtificial
Sequence225-460 K423S 17Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg
Asn Val Lys His Asp1 5 10 15Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr
Asn Arg Gly Glu His Thr 20 25 30Ser Gly Met Tyr Ala Ile Arg Pro Ser
Asn Ser Gln Val Phe His Val 35 40 45Tyr Cys Asp Val Ile Ser Gly Ser
Pro Trp Thr Leu Ile Gln His Arg 50 55 60Ile Asp Gly Ser Gln Asn Phe
Asn Glu Thr Trp Glu Asn Tyr Lys Tyr65 70 75 80Gly Phe Gly Arg Leu
Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 85 90 95Tyr Ser Ile Val
Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 100 105 110Asp Trp
Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 115 120
125Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn
130 135 140Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser
Thr Trp145 150 155 160Asp His Lys Ala Lys Gly His Phe Asn Cys Pro
Glu Gly Tyr Ser Gly 165 170 175Gly Trp Trp Trp His Asp Glu Cys Gly
Glu Asn Asn Leu Asn Gly Lys 180 185 190Tyr Asn Lys Pro Arg Ala Ser
Ser Lys Pro Glu Arg Arg Arg Gly Leu 195 200 205Ser Trp Lys Ser Gln
Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 210 215 220Met Leu Ile
His Pro Thr Asp Ser Glu Ser Phe Glu225 230 23518236PRTArtificial
Sequence225-460 S424T 18Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg
Asn Val Lys His Asp1 5 10 15Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr
Asn Arg Gly Glu His Thr 20 25 30Ser Gly Met Tyr Ala Ile Arg Pro Ser
Asn Ser Gln Val Phe His Val 35 40 45Tyr Cys Asp Val Ile Ser Gly Ser
Pro Trp Thr Leu Ile Gln His Arg 50 55 60Ile Asp Gly Ser Gln Asn Phe
Asn Glu Thr Trp Glu Asn Tyr Lys Tyr65 70 75 80Gly Phe Gly Arg Leu
Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 85 90 95Tyr Ser Ile Val
Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 100 105 110Asp Trp
Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 115 120
125Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn
130 135 140Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser
Thr Trp145 150 155 160Asp His Lys Ala Lys Gly His Phe Asn Cys Pro
Glu Gly Tyr Ser Gly 165 170 175Gly Trp Trp Trp His Asp Glu Cys Gly
Glu Asn Asn Leu Asn Gly Lys 180 185 190Tyr Asn Lys Pro Arg Ala Lys
Thr Lys Pro Glu Arg Arg Arg Gly Leu 195 200 205Ser Trp Lys Ser Gln
Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 210 215 220Met Leu Ile
His Pro Thr Asp Ser Glu Ser Phe Glu225 230 23519235PRTArtificial
Sequence226-460 K423Q 19Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn
Val Lys His Asp Gly1 5 10 15Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn
Arg Gly Glu His Thr Ser 20 25 30Gly Met Tyr Ala Ile Arg Pro Ser Asn
Ser Gln Val Phe His Val Tyr 35 40 45Cys Asp Val Ile Ser Gly Ser Pro
Trp Thr Leu Ile Gln His Arg Ile 50 55 60Asp Gly Ser Gln Asn Phe Asn
Glu Thr Trp Glu Asn Tyr Lys Tyr Gly65 70 75 80Phe Gly Arg Leu Asp
Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr 85 90 95Ser Ile Val Lys
Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp 100 105 110Trp Lys
Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly Asn 115 120
125His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn Val
130 135 140Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr
Trp Asp145 150 155 160His Lys Ala Lys Gly His Phe Asn Cys Pro Glu
Gly Tyr Ser Gly Gly 165 170 175Trp Trp Trp His Asp Glu Cys Gly Glu
Asn Asn Leu Asn Gly Lys Tyr 180 185 190Asn Lys Pro Arg Ala Gln Ser
Lys Pro Glu Arg Arg Arg Gly Leu Ser 195 200 205Trp Lys Ser Gln Asn
Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met 210 215 220Leu Ile His
Pro Thr Asp Ser Glu Ser Phe Glu225 230 23520235PRTArtificial
Sequence226-460 K423S 20Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn
Val Lys His Asp Gly1 5 10 15Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn
Arg Gly Glu His Thr Ser 20 25 30Gly Met Tyr Ala Ile Arg Pro Ser Asn
Ser Gln Val Phe His Val Tyr 35 40 45Cys Asp Val Ile Ser Gly Ser Pro
Trp Thr Leu Ile Gln His Arg Ile 50 55 60Asp Gly Ser Gln Asn Phe Asn
Glu Thr Trp Glu Asn Tyr Lys Tyr Gly65 70 75 80Phe Gly Arg Leu Asp
Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr 85 90 95Ser Ile Val Lys
Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp 100 105 110Trp Lys
Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly Asn 115 120
125His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn Val
130 135 140Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr
Trp Asp145 150 155 160His Lys Ala Lys Gly His Phe Asn Cys Pro Glu
Gly Tyr Ser Gly Gly 165 170 175Trp Trp Trp His Asp Glu Cys Gly Glu
Asn Asn Leu Asn Gly Lys Tyr 180 185 190Asn Lys Pro Arg Ala Ser Ser
Lys Pro Glu Arg Arg Arg Gly Leu Ser 195 200 205Trp Lys Ser Gln Asn
Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met 210 215 220Leu Ile His
Pro Thr Asp Ser Glu Ser Phe Glu225 230 23521233PRTArtificial
Sequence228-460 K423Q 21Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys
His Asp Gly Ile Pro1 5 10 15Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly
Glu His Thr Ser Gly Met 20 25 30Tyr Ala Ile Arg Pro Ser Asn Ser Gln
Val Phe His Val Tyr Cys Asp 35 40 45Val Ile Ser Gly Ser Pro Trp Thr
Leu Ile Gln His Arg Ile Asp Gly 50 55 60Ser Gln Asn Phe Asn Glu Thr
Trp Glu Asn Tyr Lys Tyr Gly Phe Gly65 70 75 80Arg Leu Asp Gly Glu
Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile 85 90 95Val Lys Gln Ser
Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp Trp Lys 100 105 110Asp Asn
Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu 115 120
125Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn Val Pro Asn
130 135 140Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp Asp
His Lys145 150 155 160Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr
Ser Gly Gly Trp Trp 165 170 175Trp His Asp Glu Cys Gly Glu Asn Asn
Leu Asn Gly Lys Tyr Asn Lys 180 185 190Pro Arg Ala Gln Ser Lys Pro
Glu Arg Arg Arg Gly Leu Ser Trp Lys 195 200 205Ser Gln Asn Gly Arg
Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile 210 215 220His Pro Thr
Asp Ser Glu Ser Phe Glu225 23022233PRTArtificial Sequence228-460
K423S 22Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp Gly Ile
Pro1 5 10 15Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser
Gly Met 20 25 30Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val
Tyr Cys Asp 35 40 45Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His
Arg Ile Asp Gly 50 55 60Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr
Lys Tyr Gly Phe Gly65 70 75 80Arg Leu Asp Gly Glu Phe Trp Leu Gly
Leu Glu Lys Ile Tyr Ser Ile 85 90 95Val Lys Gln Ser Asn Tyr Val Leu
Arg Ile Glu Leu Glu Asp Trp Lys 100 105 110Asp Asn Lys His Tyr Ile
Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu 115 120 125Thr Asn Tyr Thr
Leu His Leu Val Ala Ile Thr Gly Asn Val Pro Asn 130 135 140Ala Ile
Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp Asp His Lys145 150 155
160Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp
165 170 175Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr
Asn Lys 180 185 190Pro Arg Ala Ser Ser Lys Pro Glu Arg Arg Arg Gly
Leu Ser Trp Lys 195 200 205Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys
Ser Thr Lys Met Leu Ile 210 215 220His Pro Thr Asp Ser Glu Ser Phe
Glu225 23023233PRTArtificial Sequence228-460 S424T 23Phe Leu Gln
Leu Asn Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro1 5 10 15Ala Glu
Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met 20 25 30Tyr
Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp 35 40
45Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly
50 55 60Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe
Gly65 70 75 80Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile
Tyr Ser Ile 85 90 95Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu Asp Trp Lys 100 105 110Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe
Tyr Leu Gly Asn His Glu 115 120 125Thr Asn Tyr Thr Leu His Leu Val
Ala Ile Thr Gly Asn Val Pro Asn 130 135 140Ala Ile Pro Glu Asn Lys
Asp Leu Val Phe Ser Thr Trp Asp His Lys145 150 155 160Ala Lys Gly
His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp 165 170 175Trp
His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys 180 185
190Pro Arg Ala Lys Thr Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys
195 200 205Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met
Leu Ile 210 215 220His Pro Thr Asp Ser Glu Ser Phe Glu225
23024228PRTArtificial Sequence233-460 K423Q 24Glu Ile Arg Asn Val
Lys His Asp Gly Ile Pro Ala Glu Cys Thr Thr1 5 10 15Ile Tyr Asn Arg
Gly Glu His Thr Ser Gly Met Tyr Ala Ile Arg Pro 20 25 30Ser Asn Ser
Gln Val Phe His Val Tyr Cys Asp Val Ile Ser Gly Ser 35 40 45Pro Trp
Thr Leu Ile Gln His Arg Ile Asp Gly Ser Gln Asn Phe Asn 50 55 60Glu
Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu65 70 75
80Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln Ser Asn
85 90 95Tyr Val Leu Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn Lys His
Tyr 100 105 110Ile Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu Thr Asn
Tyr Thr Leu 115 120 125His Leu Val Ala Ile Thr Gly Asn Val Pro Asn
Ala Ile Pro Glu Asn 130 135 140Lys Asp Leu Val Phe Ser Thr Trp Asp
His Lys Ala Lys Gly His Phe145 150 155 160Asn Cys Pro Glu Gly Tyr
Ser Gly Gly Trp Trp Trp His Asp Glu Cys 165 170 175Gly Glu Asn Asn
Leu Asn Gly Lys Tyr Asn Lys Pro Arg Ala Gln Ser 180 185 190Lys Pro
Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser Gln Asn Gly Arg 195 200
205Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile His Pro Thr Asp Ser
210 215 220Glu Ser Phe Glu22525228PRTArtificial Sequence233-460
K423S 25Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro Ala
Glu Cys Thr Thr1 5 10 15Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met
Tyr Ala Ile Arg Pro 20 25 30Ser Asn Ser Gln Val Phe His Val Tyr Cys
Asp Val Ile Ser Gly Ser 35 40 45Pro Trp Thr Leu Ile Gln His Arg Ile
Asp Gly Ser Gln Asn Phe Asn 50 55 60Glu Thr Trp Glu Asn Tyr Lys Tyr
Gly Phe Gly Arg Leu Asp Gly Glu65 70 75 80Phe Trp Leu Gly Leu Glu
Lys Ile Tyr Ser Ile Val Lys Gln Ser Asn 85 90 95Tyr Val Leu Arg Ile
Glu Leu Glu Asp Trp Lys Asp Asn Lys His Tyr 100 105 110Ile Glu Tyr
Ser Phe Tyr Leu Gly Asn His Glu Thr Asn Tyr Thr Leu 115 120 125His
Leu Val Ala Ile Thr Gly Asn Val Pro Asn Ala Ile Pro Glu Asn 130 135
140Lys Asp Leu Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly His
Phe145 150 155 160Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp
His Asp Glu Cys 165 170 175Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn
Lys Pro Arg Ala Ser Ser 180 185 190Lys Pro Glu Arg Arg Arg Gly Leu
Ser Trp Lys Ser Gln Asn Gly Arg 195 200 205Leu Tyr Ser Ile Lys Ser
Thr Lys Met Leu Ile His Pro Thr Asp Ser 210 215 220Glu Ser Phe
Glu22526220PRTArtificial Sequence241-460 K423Q 26Gly Ile Pro Ala
Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr1 5 10 15Ser Gly Met
Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 20 25 30Tyr Cys
Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 35 40 45Ile
Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 50 55
60Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile65
70 75 80Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu 85 90 95Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr
Leu Gly 100 105 110Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala
Ile Thr Gly Asn 115 120 125Val Pro Asn Ala Ile Pro Glu Asn Lys Asp
Leu Val Phe Ser Thr Trp 130 135 140Asp His Lys Ala Lys Gly His Phe
Asn Cys Pro Glu Gly Tyr Ser Gly145 150 155 160Gly Trp Trp Trp His
Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 165 170 175Tyr Asn Lys
Pro Arg Ala Gln Ser Lys Pro Glu Arg Arg Arg Gly Leu 180 185 190Ser
Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 195 200
205Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 210 215
22027220PRTArtificial Sequence241-460 K423S 27Gly Ile Pro Ala Glu
Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr1 5 10 15Ser Gly Met Tyr
Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 20 25 30Tyr Cys Asp
Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 35 40 45Ile Asp
Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 50 55 60Gly
Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile65 70 75
80Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu
85 90 95Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu
Gly 100 105 110Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile
Thr Gly Asn 115 120 125Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu
Val Phe Ser Thr Trp 130 135 140Asp His Lys Ala Lys Gly His Phe Asn
Cys Pro Glu Gly Tyr Ser Gly145 150 155 160Gly Trp Trp Trp His Asp
Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 165 170 175Tyr Asn Lys Pro
Arg Ala Ser Ser Lys Pro Glu Arg Arg Arg Gly Leu 180 185 190Ser Trp
Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 195 200
205Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 210 215
22028219PRTArtificial Sequence242-460 K423Q 28Ile Pro Ala Glu Cys
Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser1 5 10 15Gly Met Tyr Ala
Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr 20 25 30Cys Asp Val
Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile 35 40 45Asp Gly
Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly 50 55 60Phe
Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr65 70 75
80Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp
85 90 95Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly
Asn 100 105 110His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr
Gly Asn Val 115 120 125Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val
Phe Ser Thr Trp Asp 130 135 140His Lys Ala Lys Gly His Phe Asn Cys
Pro Glu Gly Tyr Ser Gly Gly145 150 155 160Trp Trp Trp His Asp Glu
Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr 165 170 175Asn Lys Pro Arg
Ala Gln Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser 180 185 190Trp Lys
Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met 195 200
205Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 210
21529219PRTArtificial Sequence242-460 K423S 29Ile Pro Ala Glu Cys
Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser1 5 10 15Gly Met Tyr Ala
Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr 20 25 30Cys Asp Val
Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile 35 40 45Asp Gly
Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly 50 55 60Phe
Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr65 70 75
80Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp
85 90 95Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly
Asn 100 105 110His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr
Gly Asn Val 115 120 125Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val
Phe Ser Thr Trp Asp 130 135 140His Lys Ala Lys Gly His Phe Asn Cys
Pro Glu Gly Tyr Ser Gly Gly145 150 155 160Trp Trp Trp His Asp Glu
Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr 165 170 175Asn Lys Pro Arg
Ala Ser Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser 180 185 190Trp Lys
Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met 195 200
205Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 210
21530231PRTArtificial Sequence225-455 K423Q 30Thr Thr Pro Phe Leu
Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp1 5 10 15Gly Ile Pro Ala
Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 20 25 30Ser Gly Met
Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 35 40 45Tyr Cys
Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 50 55 60Ile
Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr65 70 75
80Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile
85 90 95Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu 100 105 110Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe
Tyr Leu Gly 115 120 125Asn His Glu Thr Asn Tyr Thr Leu His Leu Val
Ala Ile Thr Gly Asn 130 135 140Val Pro Asn Ala Ile Pro Glu Asn Lys
Asp Leu Val Phe Ser Thr Trp145 150 155 160Asp His Lys Ala Lys Gly
His Phe Asn Cys Pro Glu Gly Tyr Ser Gly 165 170 175Gly Trp Trp Trp
His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 180 185 190Tyr Asn
Lys Pro Arg Ala Gln Ser Lys Pro Glu Arg Arg Arg Gly Leu 195 200
205Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys
210 215 220Met Leu Ile His Pro Thr Asp225 23031231PRTArtificial
Sequence225-455 K423S 31Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg
Asn Val Lys His Asp1 5 10 15Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr
Asn Arg Gly Glu His Thr 20 25 30Ser Gly Met Tyr Ala Ile Arg Pro Ser
Asn Ser Gln Val Phe His Val 35 40 45Tyr Cys Asp Val Ile Ser Gly Ser
Pro Trp Thr Leu Ile Gln His Arg 50 55 60Ile Asp Gly Ser Gln Asn Phe
Asn Glu Thr Trp Glu Asn Tyr Lys Tyr65 70 75 80Gly Phe Gly Arg Leu
Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 85 90 95Tyr Ser Ile Val
Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 100 105 110Asp Trp
Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 115 120
125Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn
130 135 140Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser
Thr Trp145 150 155 160Asp His Lys Ala Lys Gly His Phe Asn Cys Pro
Glu Gly Tyr Ser Gly 165 170 175Gly Trp Trp Trp His Asp Glu Cys Gly
Glu Asn Asn Leu Asn Gly Lys 180 185 190Tyr Asn Lys Pro Arg Ala Ser
Ser Lys Pro Glu Arg Arg Arg Gly Leu 195 200 205Ser Trp Lys Ser Gln
Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 210 215 220Met Leu Ile
His Pro Thr Asp225 23032230PRTArtificial Sequence226-455 K423Q
32Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp Gly1
5 10 15Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr
Ser 20 25 30Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His
Val Tyr 35 40 45Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln
His Arg Ile 50 55 60Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn
Tyr Lys Tyr Gly65 70 75 80Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu
Gly Leu Glu Lys Ile Tyr 85 90 95Ser Ile Val Lys Gln Ser Asn Tyr Val
Leu Arg Ile Glu Leu Glu Asp 100 105 110Trp Lys Asp Asn Lys His Tyr
Ile Glu Tyr Ser Phe Tyr Leu Gly Asn 115 120 125His Glu Thr Asn Tyr
Thr Leu His Leu Val Ala Ile Thr Gly Asn Val 130 135 140Pro Asn Ala
Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp Asp145 150 155
160His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly
165 170 175Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly
Lys Tyr 180 185 190Asn Lys Pro Arg Ala Gln Ser Lys Pro Glu Arg Arg
Arg Gly Leu Ser 195 200 205Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser
Ile Lys Ser Thr Lys Met 210 215 220Leu Ile His Pro Thr Asp225
23033230PRTArtificial Sequence226-455 K423S 33Thr Pro Phe Leu Gln
Leu Asn Glu Ile Arg Asn Val Lys His Asp Gly1 5 10 15Ile Pro Ala Glu
Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser 20 25 30Gly Met Tyr
Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr 35 40 45Cys Asp
Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile 50 55 60Asp
Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly65 70 75
80Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr
85 90 95Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu
Asp 100 105 110Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr
Leu Gly Asn 115 120 125His Glu Thr Asn Tyr Thr Leu His Leu Val Ala
Ile Thr Gly Asn Val 130 135 140Pro Asn Ala Ile Pro Glu Asn Lys Asp
Leu Val Phe Ser Thr Trp Asp145 150 155 160His Lys Ala Lys Gly His
Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly 165 170 175Trp Trp Trp His
Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr 180 185 190Asn Lys
Pro Arg Ala Ser Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser 195 200
205Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met
210 215 220Leu Ile His Pro Thr Asp225 23034228PRTArtificial
Sequence228-455 K423Q 34Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys
His Asp Gly Ile Pro1 5 10 15Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly
Glu His Thr Ser Gly Met 20 25 30Tyr Ala Ile Arg Pro Ser Asn Ser Gln
Val Phe His Val Tyr Cys Asp 35 40 45Val Ile Ser Gly Ser Pro Trp Thr
Leu Ile Gln His Arg Ile Asp Gly 50 55 60Ser Gln Asn Phe Asn Glu Thr
Trp Glu Asn Tyr Lys Tyr Gly Phe Gly65 70 75 80Arg Leu Asp Gly Glu
Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile 85 90 95Val Lys Gln Ser
Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp Trp Lys 100 105 110Asp Asn
Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu 115 120
125Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn Val Pro Asn
130 135 140Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp Asp
His Lys145 150 155 160Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr
Ser Gly Gly Trp Trp 165 170 175Trp His Asp Glu Cys Gly Glu Asn Asn
Leu Asn Gly Lys Tyr Asn Lys 180 185 190Pro Arg Ala Gln Ser Lys Pro
Glu Arg Arg Arg Gly Leu Ser Trp Lys 195 200 205Ser Gln Asn Gly Arg
Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile 210 215 220His Pro Thr
Asp22535228PRTArtificial Sequence228-455 K423S 35Phe Leu Gln Leu
Asn Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro1 5 10 15Ala Glu Cys
Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met 20 25 30Tyr Ala
Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp 35 40 45Val
Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly 50 55
60Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly65
70 75 80Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser
Ile 85 90 95Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp
Trp Lys 100 105 110Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu
Gly Asn His Glu 115 120 125Thr Asn Tyr Thr Leu His Leu Val Ala Ile
Thr Gly Asn Val Pro Asn 130 135 140Ala Ile Pro Glu Asn Lys Asp Leu
Val Phe Ser Thr Trp Asp His Lys145 150 155 160Ala Lys Gly His Phe
Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp 165 170 175Trp His Asp
Glu Cys Gly Glu
Asn Asn Leu Asn Gly Lys Tyr Asn Lys 180 185 190Pro Arg Ala Ser Ser
Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys 195 200 205Ser Gln Asn
Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile 210 215 220His
Pro Thr Asp22536223PRTArtificial Sequence233-455 K423Q 36Glu Ile
Arg Asn Val Lys His Asp Gly Ile Pro Ala Glu Cys Thr Thr1 5 10 15Ile
Tyr Asn Arg Gly Glu His Thr Ser Gly Met Tyr Ala Ile Arg Pro 20 25
30Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp Val Ile Ser Gly Ser
35 40 45Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly Ser Gln Asn Phe
Asn 50 55 60Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp
Gly Glu65 70 75 80Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile Val
Lys Gln Ser Asn 85 90 95Tyr Val Leu Arg Ile Glu Leu Glu Asp Trp Lys
Asp Asn Lys His Tyr 100 105 110Ile Glu Tyr Ser Phe Tyr Leu Gly Asn
His Glu Thr Asn Tyr Thr Leu 115 120 125His Leu Val Ala Ile Thr Gly
Asn Val Pro Asn Ala Ile Pro Glu Asn 130 135 140Lys Asp Leu Val Phe
Ser Thr Trp Asp His Lys Ala Lys Gly His Phe145 150 155 160Asn Cys
Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp His Asp Glu Cys 165 170
175Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Ala Gln Ser
180 185 190Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser Gln Asn
Gly Arg 195 200 205Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile His
Pro Thr Asp 210 215 22037223PRTArtificial Sequence233-455 K423S
37Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro Ala Glu Cys Thr Thr1
5 10 15Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met Tyr Ala Ile Arg
Pro 20 25 30Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp Val Ile Ser
Gly Ser 35 40 45Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly Ser Gln
Asn Phe Asn 50 55 60Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly Arg
Leu Asp Gly Glu65 70 75 80Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser
Ile Val Lys Gln Ser Asn 85 90 95Tyr Val Leu Arg Ile Glu Leu Glu Asp
Trp Lys Asp Asn Lys His Tyr 100 105 110Ile Glu Tyr Ser Phe Tyr Leu
Gly Asn His Glu Thr Asn Tyr Thr Leu 115 120 125His Leu Val Ala Ile
Thr Gly Asn Val Pro Asn Ala Ile Pro Glu Asn 130 135 140Lys Asp Leu
Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly His Phe145 150 155
160Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp His Asp Glu Cys
165 170 175Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Ala
Ser Ser 180 185 190Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser
Gln Asn Gly Arg 195 200 205Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu
Ile His Pro Thr Asp 210 215 22038215PRTArtificial Sequence241-455
K423Q 38Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His
Thr1 5 10 15Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe
His Val 20 25 30Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile
Gln His Arg 35 40 45Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu
Asn Tyr Lys Tyr 50 55 60Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu
Gly Leu Glu Lys Ile65 70 75 80Tyr Ser Ile Val Lys Gln Ser Asn Tyr
Val Leu Arg Ile Glu Leu Glu 85 90 95Asp Trp Lys Asp Asn Lys His Tyr
Ile Glu Tyr Ser Phe Tyr Leu Gly 100 105 110Asn His Glu Thr Asn Tyr
Thr Leu His Leu Val Ala Ile Thr Gly Asn 115 120 125Val Pro Asn Ala
Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp 130 135 140Asp His
Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly145 150 155
160Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys
165 170 175Tyr Asn Lys Pro Arg Ala Gln Ser Lys Pro Glu Arg Arg Arg
Gly Leu 180 185 190Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile
Lys Ser Thr Lys 195 200 205Met Leu Ile His Pro Thr Asp 210
21539215PRTArtificial Sequence241-455 K423S 39Gly Ile Pro Ala Glu
Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr1 5 10 15Ser Gly Met Tyr
Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 20 25 30Tyr Cys Asp
Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 35 40 45Ile Asp
Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 50 55 60Gly
Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile65 70 75
80Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu
85 90 95Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu
Gly 100 105 110Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile
Thr Gly Asn 115 120 125Val Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu
Val Phe Ser Thr Trp 130 135 140Asp His Lys Ala Lys Gly His Phe Asn
Cys Pro Glu Gly Tyr Ser Gly145 150 155 160Gly Trp Trp Trp His Asp
Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 165 170 175Tyr Asn Lys Pro
Arg Ala Ser Ser Lys Pro Glu Arg Arg Arg Gly Leu 180 185 190Ser Trp
Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 195 200
205Met Leu Ile His Pro Thr Asp 210 21540214PRTArtificial
Sequence242-455 K423Q 40Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg
Gly Glu His Thr Ser1 5 10 15Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser
Gln Val Phe His Val Tyr 20 25 30Cys Asp Val Ile Ser Gly Ser Pro Trp
Thr Leu Ile Gln His Arg Ile 35 40 45Asp Gly Ser Gln Asn Phe Asn Glu
Thr Trp Glu Asn Tyr Lys Tyr Gly 50 55 60Phe Gly Arg Leu Asp Gly Glu
Phe Trp Leu Gly Leu Glu Lys Ile Tyr65 70 75 80Ser Ile Val Lys Gln
Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp 85 90 95Trp Lys Asp Asn
Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly Asn 100 105 110His Glu
Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn Val 115 120
125Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp Asp
130 135 140His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser
Gly Gly145 150 155 160Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn
Leu Asn Gly Lys Tyr 165 170 175Asn Lys Pro Arg Ala Gln Ser Lys Pro
Glu Arg Arg Arg Gly Leu Ser 180 185 190Trp Lys Ser Gln Asn Gly Arg
Leu Tyr Ser Ile Lys Ser Thr Lys Met 195 200 205Leu Ile His Pro Thr
Asp 21041214PRTArtificial Sequence242-455 K423S 41Ile Pro Ala Glu
Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser1 5 10 15Gly Met Tyr
Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr 20 25 30Cys Asp
Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile 35 40 45Asp
Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly 50 55
60Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr65
70 75 80Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu
Asp 85 90 95Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu
Gly Asn 100 105 110His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile
Thr Gly Asn Val 115 120 125Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu
Val Phe Ser Thr Trp Asp 130 135 140His Lys Ala Lys Gly His Phe Asn
Cys Pro Glu Gly Tyr Ser Gly Gly145 150 155 160Trp Trp Trp His Asp
Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr 165 170 175Asn Lys Pro
Arg Ala Ser Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser 180 185 190Trp
Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met 195 200
205Leu Ile His Pro Thr Asp 21042233PRTArtificial Sequencecanine 227
K423Q 42Phe Leu His Leu Asn Glu Thr Lys Asn Val Glu His Asn Asp Ile
Pro1 5 10 15Ala Asn Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser
Gly Ile 20 25 30Tyr Ser Ile Arg Pro Ser Asn Ser Gln Val Phe Asn Val
Tyr Cys Asp 35 40 45Val Lys Ser Gly Ser Ser Trp Thr Leu Ile Gln His
Arg Ile Asp Gly 50 55 60Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr
Arg Tyr Gly Phe Gly65 70 75 80Arg Leu Asp Gly Glu Phe Trp Leu Gly
Leu Glu Lys Ile Tyr Ser Ile 85 90 95Val Lys Gln Ser Asn Tyr Ile Leu
Arg Ile Glu Leu Glu Asp Trp Asn 100 105 110Asp Asn Lys His Tyr Ile
Glu Tyr Phe Phe His Leu Gly Asn His Glu 115 120 125Thr Asn Tyr Thr
Leu His Leu Val Glu Ile Thr Gly Asn Ile Leu Asn 130 135 140Ala Leu
Pro Glu His Lys Asp Leu Val Phe Ser Thr Trp Asp His Lys145 150 155
160Ala Lys Gly His Val Asn Cys Pro Glu Ser Tyr Ser Gly Gly Trp Trp
165 170 175Trp His Asn Val Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr
Asn Lys 180 185 190Gln Arg Ala Gln Thr Lys Pro Glu Arg Arg Arg Gly
Leu Tyr Trp Lys 195 200 205Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys
Ser Thr Lys Met Leu Ile 210 215 220His Pro Ile Asp Ser Glu Ser Ser
Glu225 23043233PRTArtificial Sequencecanine 227 K423S 43Phe Leu His
Leu Asn Glu Thr Lys Asn Val Glu His Asn Asp Ile Pro1 5 10 15Ala Asn
Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Ile 20 25 30Tyr
Ser Ile Arg Pro Ser Asn Ser Gln Val Phe Asn Val Tyr Cys Asp 35 40
45Val Lys Ser Gly Ser Ser Trp Thr Leu Ile Gln His Arg Ile Asp Gly
50 55 60Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Arg Tyr Gly Phe
Gly65 70 75 80Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile
Tyr Ser Ile 85 90 95Val Lys Gln Ser Asn Tyr Ile Leu Arg Ile Glu Leu
Glu Asp Trp Asn 100 105 110Asp Asn Lys His Tyr Ile Glu Tyr Phe Phe
His Leu Gly Asn His Glu 115 120 125Thr Asn Tyr Thr Leu His Leu Val
Glu Ile Thr Gly Asn Ile Leu Asn 130 135 140Ala Leu Pro Glu His Lys
Asp Leu Val Phe Ser Thr Trp Asp His Lys145 150 155 160Ala Lys Gly
His Val Asn Cys Pro Glu Ser Tyr Ser Gly Gly Trp Trp 165 170 175Trp
His Asn Val Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys 180 185
190Gln Arg Ala Ser Thr Lys Pro Glu Arg Arg Arg Gly Leu Tyr Trp Lys
195 200 205Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met
Leu Ile 210 215 220His Pro Ile Asp Ser Glu Ser Ser Glu225
23044708DNAArtificial Sequencenucleic acid encoding 225WT
44actactccct ttcttcagtt gaatgaaata agaaatgtaa aacatgatgg cattcctgct
60gaatgtacca ccatttataa cagaggtgaa catacaagtg gcatgtatgc catcagaccc
120agcaactctc aagtttttca tgtctactgt gatgttatat caggtagtcc
atggacatta 180attcaacatc gaatagatgg atcacaaaac ttcaatgaaa
cgtgggagaa ctacaaatat 240ggttttggga ggcttgatgg agaattttgg
ttgggcctag agaagatata ctccatagtg 300aagcaatcta attatgtttt
acgaattgag ttggaagact ggaaagacaa caaacattat 360attgaatatt
ctttttactt gggaaatcac gaaaccaact atacgctaca tctagttgcg
420attactggca atgtccccaa tgcaatcccg gaaaacaaag atttggtgtt
ttctacttgg 480gatcacaaag caaaaggaca cttcaactgt ccagagggtt
attcaggagg ctggtggtgg 540catgatgagt gtggagaaaa caacctaaat
ggtaaatata acaaaccaag agcaaaatct 600aagccagaga ggagaagagg
attatcttgg aagtctcaaa atggaaggtt atactctata 660aaatcaacca
aaatgttgat ccatccaaca gattcagaaa gctttgaa 70845708DNAArtificial
Sequencenucleic acid encoding 225 K423Q 45actactccct ttcttcagtt
gaatgaaata agaaatgtaa aacatgatgg cattcctgct 60gaatgtacca ccatttataa
cagaggtgaa catacaagtg gcatgtatgc catcagaccc 120agcaactctc
aagtttttca tgtctactgt gatgttatat caggtagtcc atggacatta
180attcaacatc gaatagatgg atcacaaaac ttcaatgaaa cgtgggagaa
ctacaaatat 240ggttttggga ggcttgatgg agaattttgg ttgggcctag
agaagatata ctccatagtg 300aagcaatcta attatgtttt acgaattgag
ttggaagact ggaaagacaa caaacattat 360attgaatatt ctttttactt
gggaaatcac gaaaccaact atacgctaca tctagttgcg 420attactggca
atgtccccaa tgcaatcccg gaaaacaaag atttggtgtt ttctacttgg
480gatcacaaag caaaaggaca cttcaactgt ccagagggtt attcaggagg
ctggtggtgg 540catgatgagt gtggagaaaa caacctaaat ggtaaatata
acaaaccaag agcacaatct 600aagccagaga ggagaagagg attatcttgg
aagtctcaaa atggaaggtt atactctata 660aaatcaacca aaatgttgat
ccatccaaca gattcagaaa gctttgaa 70846708DNAArtificial
Sequencenucleic acid encoding 225 K423S 46actactccct ttcttcagtt
gaatgaaata agaaatgtaa aacatgatgg cattcctgct 60gaatgtacca ccatttataa
cagaggtgaa catacaagtg gcatgtatgc catcagaccc 120agcaactctc
aagtttttca tgtctactgt gatgttatat caggtagtcc atggacatta
180attcaacatc gaatagatgg atcacaaaac ttcaatgaaa cgtgggagaa
ctacaaatat 240ggttttggga ggcttgatgg agaattttgg ttgggcctag
agaagatata ctccatagtg 300aagcaatcta attatgtttt acgaattgag
ttggaagact ggaaagacaa caaacattat 360attgaatatt ctttttactt
gggaaatcac gaaaccaact atacgctaca tctagttgcg 420attactggca
atgtccccaa tgcaatcccg gaaaacaaag atttggtgtt ttctacttgg
480gatcacaaag caaaaggaca cttcaactgt ccagagggtt attcaggagg
ctggtggtgg 540catgatgagt gtggagaaaa caacctaaat ggtaaatata
acaaaccaag agcaagctct 600aagccagaga ggagaagagg attatcttgg
aagtctcaaa atggaaggtt atactctata 660aaatcaacca aaatgttgat
ccatccaaca gattcagaaa gctttgaa 70847705DNAArtificial
Sequencenucleic acid encoding 226 K423Q 47actccctttc ttcagttgaa
tgaaataaga aatgtaaaac atgatggcat tcctgctgaa 60tgtaccacca tttataacag
aggtgaacat acaagtggca tgtatgccat cagacccagc 120aactctcaag
tttttcatgt ctactgtgat gttatatcag gtagtccatg gacattaatt
180caacatcgaa tagatggatc acaaaacttc aatgaaacgt gggagaacta
caaatatggt 240tttgggaggc ttgatggaga attttggttg ggcctagaga
agatatactc catagtgaag 300caatctaatt atgttttacg aattgagttg
gaagactgga aagacaacaa acattatatt 360gaatattctt tttacttggg
aaatcacgaa accaactata cgctacatct agttgcgatt 420actggcaatg
tccccaatgc aatcccggaa aacaaagatt tggtgttttc tacttgggat
480cacaaagcaa aaggacactt caactgtcca gagggttatt caggaggctg
gtggtggcat 540gatgagtgtg gagaaaacaa cctaaatggt aaatataaca
aaccaagagc acaatctaag 600ccagagagga gaagaggatt atcttggaag
tctcaaaatg gaaggttata ctctataaaa 660tcaaccaaaa tgttgatcca
tccaacagat tcagaaagct ttgaa 70548705DNAArtificial Sequencenucleic
acid encoding 226 K423S 48actccctttc ttcagttgaa tgaaataaga
aatgtaaaac atgatggcat tcctgctgaa 60tgtaccacca tttataacag aggtgaacat
acaagtggca tgtatgccat cagacccagc 120aactctcaag tttttcatgt
ctactgtgat gttatatcag gtagtccatg gacattaatt 180caacatcgaa
tagatggatc acaaaacttc aatgaaacgt gggagaacta caaatatggt
240tttgggaggc ttgatggaga attttggttg ggcctagaga agatatactc
catagtgaag 300caatctaatt atgttttacg aattgagttg gaagactgga
aagacaacaa acattatatt 360gaatattctt tttacttggg aaatcacgaa
accaactata cgctacatct agttgcgatt 420actggcaatg tccccaatgc
aatcccggaa aacaaagatt tggtgttttc tacttgggat 480cacaaagcaa
aaggacactt caactgtcca gagggttatt caggaggctg gtggtggcat
540gatgagtgtg gagaaaacaa cctaaatggt aaatataaca aaccaagagc
aagctctaag
600ccagagagga gaagaggatt atcttggaag tctcaaaatg gaaggttata
ctctataaaa 660tcaaccaaaa tgttgatcca tccaacagat tcagaaagct ttgaa
70549699DNAArtificial Sequencenucleic acid encoding 228 K423Q
49tttcttcagt tgaatgaaat aagaaatgta aaacatgatg gcattcctgc tgaatgtacc
60accatttata acagaggtga acatacaagt ggcatgtatg ccatcagacc cagcaactct
120caagtttttc atgtctactg tgatgttata tcaggtagtc catggacatt
aattcaacat 180cgaatagatg gatcacaaaa cttcaatgaa acgtgggaga
actacaaata tggttttggg 240aggcttgatg gagaattttg gttgggccta
gagaagatat actccatagt gaagcaatct 300aattatgttt tacgaattga
gttggaagac tggaaagaca acaaacatta tattgaatat 360tctttttact
tgggaaatca cgaaaccaac tatacgctac atctagttgc gattactggc
420aatgtcccca atgcaatccc ggaaaacaaa gatttggtgt tttctacttg
ggatcacaaa 480gcaaaaggac acttcaactg tccagagggt tattcaggag
gctggtggtg gcatgatgag 540tgtggagaaa acaacctaaa tggtaaatat
aacaaaccaa gagcacaatc taagccagag 600aggagaagag gattatcttg
gaagtctcaa aatggaaggt tatactctat aaaatcaacc 660aaaatgttga
tccatccaac agattcagaa agctttgaa 69950699DNAArtificial
Sequencenucleic acid encoding 228 K423S 50tttcttcagt tgaatgaaat
aagaaatgta aaacatgatg gcattcctgc tgaatgtacc 60accatttata acagaggtga
acatacaagt ggcatgtatg ccatcagacc cagcaactct 120caagtttttc
atgtctactg tgatgttata tcaggtagtc catggacatt aattcaacat
180cgaatagatg gatcacaaaa cttcaatgaa acgtgggaga actacaaata
tggttttggg 240aggcttgatg gagaattttg gttgggccta gagaagatat
actccatagt gaagcaatct 300aattatgttt tacgaattga gttggaagac
tggaaagaca acaaacatta tattgaatat 360tctttttact tgggaaatca
cgaaaccaac tatacgctac atctagttgc gattactggc 420aatgtcccca
atgcaatccc ggaaaacaaa gatttggtgt tttctacttg ggatcacaaa
480gcaaaaggac acttcaactg tccagagggt tattcaggag gctggtggtg
gcatgatgag 540tgtggagaaa acaacctaaa tggtaaatat aacaaaccaa
gagcaagctc taagccagag 600aggagaagag gattatcttg gaagtctcaa
aatggaaggt tatactctat aaaatcaacc 660aaaatgttga tccatccaac
agattcagaa agctttgaa 69951684DNAArtificial Sequencenucleic acid
encoding 233 K423Q 51gaaataagaa atgtaaaaca tgatggcatt cctgctgaat
gtaccaccat ttataacaga 60ggtgaacata caagtggcat gtatgccatc agacccagca
actctcaagt ttttcatgtc 120tactgtgatg ttatatcagg tagtccatgg
acattaattc aacatcgaat agatggatca 180caaaacttca atgaaacgtg
ggagaactac aaatatggtt ttgggaggct tgatggagaa 240ttttggttgg
gcctagagaa gatatactcc atagtgaagc aatctaatta tgttttacga
300attgagttgg aagactggaa agacaacaaa cattatattg aatattcttt
ttacttggga 360aatcacgaaa ccaactatac gctacatcta gttgcgatta
ctggcaatgt ccccaatgca 420atcccggaaa acaaagattt ggtgttttct
acttgggatc acaaagcaaa aggacacttc 480aactgtccag agggttattc
aggaggctgg tggtggcatg atgagtgtgg agaaaacaac 540ctaaatggta
aatataacaa accaagagca caatctaagc cagagaggag aagaggatta
600tcttggaagt ctcaaaatgg aaggttatac tctataaaat caaccaaaat
gttgatccat 660ccaacagatt cagaaagctt tgaa 68452684DNAArtificial
Sequencenucleic acid encoding 233 K423S 52gaaataagaa atgtaaaaca
tgatggcatt cctgctgaat gtaccaccat ttataacaga 60ggtgaacata caagtggcat
gtatgccatc agacccagca actctcaagt ttttcatgtc 120tactgtgatg
ttatatcagg tagtccatgg acattaattc aacatcgaat agatggatca
180caaaacttca atgaaacgtg ggagaactac aaatatggtt ttgggaggct
tgatggagaa 240ttttggttgg gcctagagaa gatatactcc atagtgaagc
aatctaatta tgttttacga 300attgagttgg aagactggaa agacaacaaa
cattatattg aatattcttt ttacttggga 360aatcacgaaa ccaactatac
gctacatcta gttgcgatta ctggcaatgt ccccaatgca 420atcccggaaa
acaaagattt ggtgttttct acttgggatc acaaagcaaa aggacacttc
480aactgtccag agggttattc aggaggctgg tggtggcatg atgagtgtgg
agaaaacaac 540ctaaatggta aatataacaa accaagagca agctctaagc
cagagaggag aagaggatta 600tcttggaagt ctcaaaatgg aaggttatac
tctataaaat caaccaaaat gttgatccat 660ccaacagatt cagaaagctt tgaa
68453660DNAArtificial Sequencenucleic acid encoding 241 K423Q
53ggcattcctg ctgaatgtac caccatttat aacagaggtg aacatacaag tggcatgtat
60gccatcagac ccagcaactc tcaagttttt catgtctact gtgatgttat atcaggtagt
120ccatggacat taattcaaca tcgaatagat ggatcacaaa acttcaatga
aacgtgggag 180aactacaaat atggttttgg gaggcttgat ggagaatttt
ggttgggcct agagaagata 240tactccatag tgaagcaatc taattatgtt
ttacgaattg agttggaaga ctggaaagac 300aacaaacatt atattgaata
ttctttttac ttgggaaatc acgaaaccaa ctatacgcta 360catctagttg
cgattactgg caatgtcccc aatgcaatcc cggaaaacaa agatttggtg
420ttttctactt gggatcacaa agcaaaagga cacttcaact gtccagaggg
ttattcagga 480ggctggtggt ggcatgatga gtgtggagaa aacaacctaa
atggtaaata taacaaacca 540agagcacaat ctaagccaga gaggagaaga
ggattatctt ggaagtctca aaatggaagg 600ttatactcta taaaatcaac
caaaatgttg atccatccaa cagattcaga aagctttgaa 66054660DNAArtificial
Sequencenucleic acid encoding 241 K423S 54ggcattcctg ctgaatgtac
caccatttat aacagaggtg aacatacaag tggcatgtat 60gccatcagac ccagcaactc
tcaagttttt catgtctact gtgatgttat atcaggtagt 120ccatggacat
taattcaaca tcgaatagat ggatcacaaa acttcaatga aacgtgggag
180aactacaaat atggttttgg gaggcttgat ggagaatttt ggttgggcct
agagaagata 240tactccatag tgaagcaatc taattatgtt ttacgaattg
agttggaaga ctggaaagac 300aacaaacatt atattgaata ttctttttac
ttgggaaatc acgaaaccaa ctatacgcta 360catctagttg cgattactgg
caatgtcccc aatgcaatcc cggaaaacaa agatttggtg 420ttttctactt
gggatcacaa agcaaaagga cacttcaact gtccagaggg ttattcagga
480ggctggtggt ggcatgatga gtgtggagaa aacaacctaa atggtaaata
taacaaacca 540agagcaagct ctaagccaga gaggagaaga ggattatctt
ggaagtctca aaatggaagg 600ttatactcta taaaatcaac caaaatgttg
atccatccaa cagattcaga aagctttgaa 66055657DNAArtificial
Sequencenucleic acid encoding 242 K423Q 55attcctgctg aatgtaccac
catttataac agaggtgaac atacaagtgg catgtatgcc 60atcagaccca gcaactctca
agtttttcat gtctactgtg atgttatatc aggtagtcca 120tggacattaa
ttcaacatcg aatagatgga tcacaaaact tcaatgaaac gtgggagaac
180tacaaatatg gttttgggag gcttgatgga gaattttggt tgggcctaga
gaagatatac 240tccatagtga agcaatctaa ttatgtttta cgaattgagt
tggaagactg gaaagacaac 300aaacattata ttgaatattc tttttacttg
ggaaatcacg aaaccaacta tacgctacat 360ctagttgcga ttactggcaa
tgtccccaat gcaatcccgg aaaacaaaga tttggtgttt 420tctacttggg
atcacaaagc aaaaggacac ttcaactgtc cagagggtta ttcaggaggc
480tggtggtggc atgatgagtg tggagaaaac aacctaaatg gtaaatataa
caaaccaaga 540gcacaatcta agccagagag gagaagagga ttatcttgga
agtctcaaaa tggaaggtta 600tactctataa aatcaaccaa aatgttgatc
catccaacag attcagaaag ctttgaa 65756657DNAArtificial Sequencenucleic
acid encoding 242 K423S 56attcctgctg aatgtaccac catttataac
agaggtgaac atacaagtgg catgtatgcc 60atcagaccca gcaactctca agtttttcat
gtctactgtg atgttatatc aggtagtcca 120tggacattaa ttcaacatcg
aatagatgga tcacaaaact tcaatgaaac gtgggagaac 180tacaaatatg
gttttgggag gcttgatgga gaattttggt tgggcctaga gaagatatac
240tccatagtga agcaatctaa ttatgtttta cgaattgagt tggaagactg
gaaagacaac 300aaacattata ttgaatattc tttttacttg ggaaatcacg
aaaccaacta tacgctacat 360ctagttgcga ttactggcaa tgtccccaat
gcaatcccgg aaaacaaaga tttggtgttt 420tctacttggg atcacaaagc
aaaaggacac ttcaactgtc cagagggtta ttcaggaggc 480tggtggtggc
atgatgagtg tggagaaaac aacctaaatg gtaaatataa caaaccaaga
540gcaagctcta agccagagag gagaagagga ttatcttgga agtctcaaaa
tggaaggtta 600tactctataa aatcaaccaa aatgttgatc catccaacag
attcagaaag ctttgaa 65757699DNAArtificial Sequencenucleic acid
sequence encoding canine 227KQ 57tttttgcatc tcaacgaaac gaagaatgtc
gaacacaacg acattccggc aaattgcaca 60actatctaca atagaggcga acatacgtcc
ggtatctact ccattagacc ttcaaacagc 120caggtattca atgtgtactg
cgatgtaaag tcaggatcgt catggacact gatccagcat 180aggatcgacg
ggtcccagaa cttcaacgag acatgggaga actaccgcta tggatttgga
240aggctggatg gggagttctg gttgggactt gagaaaatct acagcattgt
gaagcagtcg 300aactacattc tccggattga actggaggac tggaatgaca
acaaacacta catcgagtat 360ttctttcatc tcggcaacca tgaaacgaat
tacaccttgc accttgtgga aatcacgggc 420aacattttga acgcgctgcc
agaacacaaa gacctggtgt tttcgacatg ggatcacaaa 480gcaaaggggc
acgtgaactg tcccgaatca tatagcgggg gatggtggtg gcacaatgtc
540tgtggtgaga acaatctcaa cgggaaatac aataagcagc gagctcagac
gaaacccgag 600cggcggagag gtctgtattg gaagtcgcag aatggacgcc
tgtattcgat caaatcgacg 660aaaatgctca tccaccccat cgactccgaa tcgtcggag
69958252PRTArtificial Sequence201-460 K423del 58Ile Gln Glu Pro Thr
Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro1 5 10 15Arg Thr Thr Pro
Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His 20 25 30Asp Gly Ile
Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His 35 40 45Thr Ser
Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His 50 55 60Val
Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His65 70 75
80Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys
85 90 95Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu
Lys 100 105 110Ile Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg
Ile Glu Leu 115 120 125Glu Asp Trp Lys Asp Asn Lys His Tyr Ile Glu
Tyr Ser Phe Tyr Leu 130 135 140Gly Asn His Glu Thr Asn Tyr Thr Leu
His Leu Val Ala Ile Thr Gly145 150 155 160Asn Val Pro Asn Ala Ile
Pro Glu Asn Lys Asp Leu Val Phe Ser Thr 165 170 175Trp Asp His Lys
Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser 180 185 190Gly Gly
Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly 195 200
205Lys Tyr Asn Lys Pro Arg Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu
210 215 220Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser
Thr Lys225 230 235 240Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe
Glu 245 25059235PRTArtificial Sequence225-460 K423del 59Thr Thr Pro
Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp1 5 10 15Gly Ile
Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 20 25 30Ser
Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 35 40
45Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg
50 55 60Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys
Tyr65 70 75 80Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu
Glu Lys Ile 85 90 95Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg
Ile Glu Leu Glu 100 105 110Asp Trp Lys Asp Asn Lys His Tyr Ile Glu
Tyr Ser Phe Tyr Leu Gly 115 120 125Asn His Glu Thr Asn Tyr Thr Leu
His Leu Val Ala Ile Thr Gly Asn 130 135 140Val Pro Asn Ala Ile Pro
Glu Asn Lys Asp Leu Val Phe Ser Thr Trp145 150 155 160Asp His Lys
Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly 165 170 175Gly
Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 180 185
190Tyr Asn Lys Pro Arg Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser
195 200 205Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr
Lys Met 210 215 220Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu225
230 23560234PRTArtificial Sequence226-460 K423del 60Thr Pro Phe Leu
Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp Gly1 5 10 15Ile Pro Ala
Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser 20 25 30Gly Met
Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr 35 40 45Cys
Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile 50 55
60Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly65
70 75 80Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile
Tyr 85 90 95Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu Asp 100 105 110Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe
Tyr Leu Gly Asn 115 120 125His Glu Thr Asn Tyr Thr Leu His Leu Val
Ala Ile Thr Gly Asn Val 130 135 140Pro Asn Ala Ile Pro Glu Asn Lys
Asp Leu Val Phe Ser Thr Trp Asp145 150 155 160His Lys Ala Lys Gly
His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly 165 170 175Trp Trp Trp
His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr 180 185 190Asn
Lys Pro Arg Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp 195 200
205Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu
210 215 220Ile His Pro Thr Asp Ser Glu Ser Phe Glu225
23061232PRTArtificial Sequence228-460 K423del 61Phe Leu Gln Leu Asn
Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro1 5 10 15Ala Glu Cys Thr
Thr Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met 20 25 30Tyr Ala Ile
Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp 35 40 45Val Ile
Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly 50 55 60Ser
Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe Gly65 70 75
80Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile
85 90 95Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp Trp
Lys 100 105 110Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly
Asn His Glu 115 120 125Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr
Gly Asn Val Pro Asn 130 135 140Ala Ile Pro Glu Asn Lys Asp Leu Val
Phe Ser Thr Trp Asp His Lys145 150 155 160Ala Lys Gly His Phe Asn
Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp 165 170 175Trp His Asp Glu
Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys 180 185 190Pro Arg
Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser 195 200
205Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile His
210 215 220Pro Thr Asp Ser Glu Ser Phe Glu225 23062227PRTArtificial
Sequence233-460 K423del 62Glu Ile Arg Asn Val Lys His Asp Gly Ile
Pro Ala Glu Cys Thr Thr1 5 10 15Ile Tyr Asn Arg Gly Glu His Thr Ser
Gly Met Tyr Ala Ile Arg Pro 20 25 30Ser Asn Ser Gln Val Phe His Val
Tyr Cys Asp Val Ile Ser Gly Ser 35 40 45Pro Trp Thr Leu Ile Gln His
Arg Ile Asp Gly Ser Gln Asn Phe Asn 50 55 60Glu Thr Trp Glu Asn Tyr
Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu65 70 75 80Phe Trp Leu Gly
Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln Ser Asn 85 90 95Tyr Val Leu
Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn Lys His Tyr 100 105 110Ile
Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu Thr Asn Tyr Thr Leu 115 120
125His Leu Val Ala Ile Thr Gly Asn Val Pro Asn Ala Ile Pro Glu Asn
130 135 140Lys Asp Leu Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly
His Phe145 150 155 160Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp
Trp His Asp Glu Cys 165 170 175Gly Glu Asn Asn Leu Asn Gly Lys Tyr
Asn Lys Pro Arg Ala Ser Lys 180 185 190Pro Glu Arg Arg Arg Gly Leu
Ser Trp Lys Ser Gln Asn Gly Arg Leu 195 200 205Tyr Ser Ile Lys Ser
Thr Lys Met Leu Ile His Pro Thr Asp Ser Glu 210 215 220Ser Phe
Glu22563219PRTArtificial Sequence241-460 K423del 63Gly Ile Pro Ala
Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr1 5 10 15Ser Gly Met
Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 20 25 30Tyr Cys
Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 35 40 45Ile
Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 50 55
60Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile65
70 75 80Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu 85 90 95Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr
Leu Gly 100 105 110Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala
Ile Thr Gly Asn 115 120 125Val Pro Asn Ala Ile Pro Glu
Asn Lys Asp Leu Val Phe Ser Thr Trp 130 135 140Asp His Lys Ala Lys
Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly145 150 155 160Gly Trp
Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 165 170
175Tyr Asn Lys Pro Arg Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser
180 185 190Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr
Lys Met 195 200 205Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 210
21564218PRTArtificial Sequence242-460 K423del 64Ile Pro Ala Glu Cys
Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser1 5 10 15Gly Met Tyr Ala
Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr 20 25 30Cys Asp Val
Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile 35 40 45Asp Gly
Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly 50 55 60Phe
Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr65 70 75
80Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp
85 90 95Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly
Asn 100 105 110His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr
Gly Asn Val 115 120 125Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val
Phe Ser Thr Trp Asp 130 135 140His Lys Ala Lys Gly His Phe Asn Cys
Pro Glu Gly Tyr Ser Gly Gly145 150 155 160Trp Trp Trp His Asp Glu
Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr 165 170 175Asn Lys Pro Arg
Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp 180 185 190Lys Ser
Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu 195 200
205Ile His Pro Thr Asp Ser Glu Ser Phe Glu 210
21565230PRTArtificial Sequence225-455 K423del 65Thr Thr Pro Phe Leu
Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp1 5 10 15Gly Ile Pro Ala
Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 20 25 30Ser Gly Met
Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 35 40 45Tyr Cys
Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 50 55 60Ile
Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr65 70 75
80Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile
85 90 95Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu 100 105 110Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe
Tyr Leu Gly 115 120 125Asn His Glu Thr Asn Tyr Thr Leu His Leu Val
Ala Ile Thr Gly Asn 130 135 140Val Pro Asn Ala Ile Pro Glu Asn Lys
Asp Leu Val Phe Ser Thr Trp145 150 155 160Asp His Lys Ala Lys Gly
His Phe Asn Cys Pro Glu Gly Tyr Ser Gly 165 170 175Gly Trp Trp Trp
His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 180 185 190Tyr Asn
Lys Pro Arg Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser 195 200
205Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met
210 215 220Leu Ile His Pro Thr Asp225 23066229PRTArtificial
Sequence226-455 K423del 66Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg
Asn Val Lys His Asp Gly1 5 10 15Ile Pro Ala Glu Cys Thr Thr Ile Tyr
Asn Arg Gly Glu His Thr Ser 20 25 30Gly Met Tyr Ala Ile Arg Pro Ser
Asn Ser Gln Val Phe His Val Tyr 35 40 45Cys Asp Val Ile Ser Gly Ser
Pro Trp Thr Leu Ile Gln His Arg Ile 50 55 60Asp Gly Ser Gln Asn Phe
Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly65 70 75 80Phe Gly Arg Leu
Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr 85 90 95Ser Ile Val
Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp 100 105 110Trp
Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly Asn 115 120
125His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn Val
130 135 140Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr
Trp Asp145 150 155 160His Lys Ala Lys Gly His Phe Asn Cys Pro Glu
Gly Tyr Ser Gly Gly 165 170 175Trp Trp Trp His Asp Glu Cys Gly Glu
Asn Asn Leu Asn Gly Lys Tyr 180 185 190Asn Lys Pro Arg Ala Ser Lys
Pro Glu Arg Arg Arg Gly Leu Ser Trp 195 200 205Lys Ser Gln Asn Gly
Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu 210 215 220Ile His Pro
Thr Asp22567227PRTArtificial Sequence228-455 K423del 67Phe Leu Gln
Leu Asn Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro1 5 10 15Ala Glu
Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met 20 25 30Tyr
Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp 35 40
45Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly
50 55 60Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe
Gly65 70 75 80Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile
Tyr Ser Ile 85 90 95Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu
Glu Asp Trp Lys 100 105 110Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe
Tyr Leu Gly Asn His Glu 115 120 125Thr Asn Tyr Thr Leu His Leu Val
Ala Ile Thr Gly Asn Val Pro Asn 130 135 140Ala Ile Pro Glu Asn Lys
Asp Leu Val Phe Ser Thr Trp Asp His Lys145 150 155 160Ala Lys Gly
His Phe Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp 165 170 175Trp
His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys 180 185
190Pro Arg Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser
195 200 205Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu
Ile His 210 215 220Pro Thr Asp22568222PRTArtificial Sequence233-455
K423del 68Glu Ile Arg Asn Val Lys His Asp Gly Ile Pro Ala Glu Cys
Thr Thr1 5 10 15Ile Tyr Asn Arg Gly Glu His Thr Ser Gly Met Tyr Ala
Ile Arg Pro 20 25 30Ser Asn Ser Gln Val Phe His Val Tyr Cys Asp Val
Ile Ser Gly Ser 35 40 45Pro Trp Thr Leu Ile Gln His Arg Ile Asp Gly
Ser Gln Asn Phe Asn 50 55 60Glu Thr Trp Glu Asn Tyr Lys Tyr Gly Phe
Gly Arg Leu Asp Gly Glu65 70 75 80Phe Trp Leu Gly Leu Glu Lys Ile
Tyr Ser Ile Val Lys Gln Ser Asn 85 90 95Tyr Val Leu Arg Ile Glu Leu
Glu Asp Trp Lys Asp Asn Lys His Tyr 100 105 110Ile Glu Tyr Ser Phe
Tyr Leu Gly Asn His Glu Thr Asn Tyr Thr Leu 115 120 125His Leu Val
Ala Ile Thr Gly Asn Val Pro Asn Ala Ile Pro Glu Asn 130 135 140Lys
Asp Leu Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly His Phe145 150
155 160Asn Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp His Asp Glu
Cys 165 170 175Gly Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg
Ala Ser Lys 180 185 190Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser
Gln Asn Gly Arg Leu 195 200 205Tyr Ser Ile Lys Ser Thr Lys Met Leu
Ile His Pro Thr Asp 210 215 22069214PRTArtificial Sequence241-455
K423del 69Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu
His Thr1 5 10 15Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val
Phe His Val 20 25 30Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu
Ile Gln His Arg 35 40 45Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp
Glu Asn Tyr Lys Tyr 50 55 60Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp
Leu Gly Leu Glu Lys Ile65 70 75 80Tyr Ser Ile Val Lys Gln Ser Asn
Tyr Val Leu Arg Ile Glu Leu Glu 85 90 95Asp Trp Lys Asp Asn Lys His
Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 100 105 110Asn His Glu Thr Asn
Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn 115 120 125Val Pro Asn
Ala Ile Pro Glu Asn Lys Asp Leu Val Phe Ser Thr Trp 130 135 140Asp
His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly145 150
155 160Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly
Lys 165 170 175Tyr Asn Lys Pro Arg Ala Ser Lys Pro Glu Arg Arg Arg
Gly Leu Ser 180 185 190Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile
Lys Ser Thr Lys Met 195 200 205Leu Ile His Pro Thr Asp
21070213PRTArtificial Sequence242-455 K423del 70Ile Pro Ala Glu Cys
Thr Thr Ile Tyr Asn Arg Gly Glu His Thr Ser1 5 10 15Gly Met Tyr Ala
Ile Arg Pro Ser Asn Ser Gln Val Phe His Val Tyr 20 25 30Cys Asp Val
Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg Ile 35 40 45Asp Gly
Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr Gly 50 55 60Phe
Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile Tyr65 70 75
80Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu Asp
85 90 95Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly
Asn 100 105 110His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr
Gly Asn Val 115 120 125Pro Asn Ala Ile Pro Glu Asn Lys Asp Leu Val
Phe Ser Thr Trp Asp 130 135 140His Lys Ala Lys Gly His Phe Asn Cys
Pro Glu Gly Tyr Ser Gly Gly145 150 155 160Trp Trp Trp His Asp Glu
Cys Gly Glu Asn Asn Leu Asn Gly Lys Tyr 165 170 175Asn Lys Pro Arg
Ala Ser Lys Pro Glu Arg Arg Arg Gly Leu Ser Trp 180 185 190Lys Ser
Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys Met Leu 195 200
205Ile His Pro Thr Asp 21071491PRTHomo sapiens 71Met Lys Thr Phe
Thr Trp Thr Leu Gly Val Leu Phe Phe Leu Leu Val1 5 10 15Asp Thr Gly
His Cys Arg Gly Gly Gln Phe Lys Ile Lys Lys Ile Asn 20 25 30Gln Arg
Arg Tyr Pro Arg Ala Thr Asp Gly Lys Glu Glu Ala Lys Lys 35 40 45Cys
Ala Tyr Thr Phe Leu Val Pro Glu Gln Arg Ile Thr Gly Pro Ile 50 55
60Cys Val Asn Thr Lys Gly Gln Asp Ala Ser Thr Ile Lys Asp Met Ile65
70 75 80Thr Arg Met Asp Leu Glu Asn Leu Lys Asp Val Leu Ser Arg Gln
Lys 85 90 95Arg Glu Ile Asp Val Leu Gln Leu Val Val Asp Val Asp Gly
Asn Ile 100 105 110Val Asn Glu Val Lys Leu Leu Arg Lys Glu Ser Arg
Asn Met Asn Ser 115 120 125Arg Val Thr Gln Leu Tyr Met Gln Leu Leu
His Glu Ile Ile Arg Lys 130 135 140Arg Asp Asn Ser Leu Glu Leu Ser
Gln Leu Glu Asn Lys Ile Leu Asn145 150 155 160Val Thr Thr Glu Met
Leu Lys Met Ala Thr Arg Tyr Arg Glu Leu Glu 165 170 175Val Lys Tyr
Ala Ser Leu Thr Asp Leu Val Asn Asn Gln Ser Val Met 180 185 190Ile
Thr Leu Leu Glu Glu Gln Cys Leu Arg Ile Phe Ser Arg Gln Asp 195 200
205Thr His Val Ser Pro Pro Leu Val Gln Val Val Pro Gln His Ile Pro
210 215 220Asn Ser Gln Gln Tyr Thr Pro Gly Leu Leu Gly Gly Asn Glu
Ile Gln225 230 235 240Arg Asp Pro Gly Tyr Pro Arg Asp Leu Met Pro
Pro Pro Asp Leu Ala 245 250 255Thr Ser Pro Thr Lys Ser Pro Phe Lys
Ile Pro Pro Val Thr Phe Ile 260 265 270Asn Glu Gly Pro Phe Lys Asp
Cys Gln Gln Ala Lys Glu Ala Gly His 275 280 285Ser Val Ser Gly Ile
Tyr Met Ile Lys Pro Glu Asn Ser Asn Gly Pro 290 295 300Met Gln Leu
Trp Cys Glu Asn Ser Leu Asp Pro Gly Gly Trp Thr Val305 310 315
320Ile Gln Lys Arg Thr Asp Gly Ser Val Asn Phe Phe Arg Asn Trp Glu
325 330 335Asn Tyr Lys Lys Gly Phe Gly Asn Ile Asp Gly Glu Tyr Trp
Leu Gly 340 345 350Leu Glu Asn Ile Tyr Met Leu Ser Asn Gln Asp Asn
Tyr Lys Leu Leu 355 360 365Ile Glu Leu Glu Asp Trp Ser Asp Lys Lys
Val Tyr Ala Glu Tyr Ser 370 375 380Ser Phe Arg Leu Glu Pro Glu Ser
Glu Phe Tyr Arg Leu Arg Leu Gly385 390 395 400Thr Tyr Gln Gly Asn
Ala Gly Asp Ser Met Met Trp His Asn Gly Lys 405 410 415Gln Phe Thr
Thr Leu Asp Arg Asp Lys Asp Met Tyr Ala Gly Asn Cys 420 425 430Ala
His Phe His Lys Gly Gly Trp Trp Tyr Asn Ala Cys Ala His Ser 435 440
445Asn Leu Asn Gly Val Trp Tyr Arg Gly Gly His Tyr Arg Ser Lys His
450 455 460Gln Asp Gly Ile Phe Trp Ala Glu Tyr Arg Gly Gly Ser Tyr
Ser Leu465 470 475 480Arg Ala Val Gln Met Met Ile Lys Pro Ile Asp
485 49072221PRTArtificial SequenceCterminal hANGPTL1 271-491 72Phe
Ile Asn Glu Gly Pro Phe Lys Asp Cys Gln Gln Ala Lys Glu Ala1 5 10
15Gly His Ser Val Ser Gly Ile Tyr Met Ile Lys Pro Glu Asn Ser Asn
20 25 30Gly Pro Met Gln Leu Trp Cys Glu Asn Ser Leu Asp Pro Gly Gly
Trp 35 40 45Thr Val Ile Gln Lys Arg Thr Asp Gly Ser Val Asn Phe Phe
Arg Asn 50 55 60Trp Glu Asn Tyr Lys Lys Gly Phe Gly Asn Ile Asp Gly
Glu Tyr Trp65 70 75 80Leu Gly Leu Glu Asn Ile Tyr Met Leu Ser Asn
Gln Asp Asn Tyr Lys 85 90 95Leu Leu Ile Glu Leu Glu Asp Trp Ser Asp
Lys Lys Val Tyr Ala Glu 100 105 110Tyr Ser Ser Phe Arg Leu Glu Pro
Glu Ser Glu Phe Tyr Arg Leu Arg 115 120 125Leu Gly Thr Tyr Gln Gly
Asn Ala Gly Asp Ser Met Met Trp His Asn 130 135 140Gly Lys Gln Phe
Thr Thr Leu Asp Arg Asp Lys Asp Met Tyr Ala Gly145 150 155 160Asn
Cys Ala His Phe His Lys Gly Gly Trp Trp Tyr Asn Ala Cys Ala 165 170
175His Ser Asn Leu Asn Gly Val Trp Tyr Arg Gly Gly His Tyr Arg Ser
180 185 190Lys His Gln Asp Gly Ile Phe Trp Ala Glu Tyr Arg Gly Gly
Ser Tyr 195 200 205Ser Leu Arg Ala Val Gln Met Met Ile Lys Pro Ile
Asp 210 215 22073406PRTHomo sapiens 73Met Ser Gly Ala Pro Thr Ala
Gly Ala Ala Leu Met Leu Cys Ala Ala1 5 10 15Thr Ala Val Leu Leu Ser
Ala Gln Gly Gly Pro Val Gln Ser Lys Ser 20 25 30Pro Arg Phe Ala Ser
Trp Asp Glu Met Asn Val Leu Ala His Gly Leu 35 40 45Leu Gln Leu Gly
Gln Gly Leu Arg Glu His Ala Glu Arg Thr Arg Ser 50 55 60Gln Leu Ser
Ala Leu Glu Arg Arg Leu Ser Ala Cys Gly Ser Ala Cys65 70 75 80Gln
Gly Thr Glu Gly Ser
Thr Asp Leu Pro Leu Ala Pro Glu Ser Arg 85 90 95Val Asp Pro Glu Val
Leu His Ser Leu Gln Thr Gln Leu Lys Ala Gln 100 105 110Asn Ser Arg
Ile Gln Gln Leu Phe His Lys Val Ala Gln Gln Gln Arg 115 120 125His
Leu Glu Lys Gln His Leu Arg Ile Gln His Leu Gln Ser Gln Phe 130 135
140Gly Leu Leu Asp His Lys His Leu Asp His Glu Val Ala Lys Pro
Ala145 150 155 160Arg Arg Lys Arg Leu Pro Glu Met Ala Gln Pro Val
Asp Pro Ala His 165 170 175Asn Val Ser Arg Leu His Arg Leu Pro Arg
Asp Cys Gln Glu Leu Phe 180 185 190Gln Val Gly Glu Arg Gln Ser Gly
Leu Phe Glu Ile Gln Pro Gln Gly 195 200 205Ser Pro Pro Phe Leu Val
Asn Cys Lys Met Thr Ser Asp Gly Gly Trp 210 215 220Thr Val Ile Gln
Arg Arg His Asp Gly Ser Val Asp Phe Asn Arg Pro225 230 235 240Trp
Glu Ala Tyr Lys Ala Gly Phe Gly Asp Pro His Gly Glu Phe Trp 245 250
255Leu Gly Leu Glu Lys Val His Ser Ile Thr Gly Asp Arg Asn Ser Arg
260 265 270Leu Ala Val Gln Leu Arg Asp Trp Asp Gly Asn Ala Glu Leu
Leu Gln 275 280 285Phe Ser Val His Leu Gly Gly Glu Asp Thr Ala Tyr
Ser Leu Gln Leu 290 295 300Thr Ala Pro Val Ala Gly Gln Leu Gly Ala
Thr Thr Val Pro Pro Ser305 310 315 320Gly Leu Ser Val Pro Phe Ser
Thr Trp Asp Gln Asp His Asp Leu Arg 325 330 335Arg Asp Lys Asn Cys
Ala Lys Ser Leu Ser Gly Gly Trp Trp Phe Gly 340 345 350Thr Cys Ser
His Ser Asn Leu Asn Gly Gln Tyr Phe Arg Ser Ile Pro 355 360 365Gln
Gln Arg Gln Lys Leu Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg 370 375
380Gly Arg Tyr Tyr Pro Leu Gln Ala Thr Thr Met Leu Ile Gln Pro
Met385 390 395 400Ala Ala Glu Ala Ala Ser 40574228PRTArtificial
SequenceCterminal hANGPTL4 179-406 74Ser Arg Leu His Arg Leu Pro
Arg Asp Cys Gln Glu Leu Phe Gln Val1 5 10 15Gly Glu Arg Gln Ser Gly
Leu Phe Glu Ile Gln Pro Gln Gly Ser Pro 20 25 30Pro Phe Leu Val Asn
Cys Lys Met Thr Ser Asp Gly Gly Trp Thr Val 35 40 45Ile Gln Arg Arg
His Asp Gly Ser Val Asp Phe Asn Arg Pro Trp Glu 50 55 60Ala Tyr Lys
Ala Gly Phe Gly Asp Pro His Gly Glu Phe Trp Leu Gly65 70 75 80Leu
Glu Lys Val His Ser Ile Thr Gly Asp Arg Asn Ser Arg Leu Ala 85 90
95Val Gln Leu Arg Asp Trp Asp Gly Asn Ala Glu Leu Leu Gln Phe Ser
100 105 110Val His Leu Gly Gly Glu Asp Thr Ala Tyr Ser Leu Gln Leu
Thr Ala 115 120 125Pro Val Ala Gly Gln Leu Gly Ala Thr Thr Val Pro
Pro Ser Gly Leu 130 135 140Ser Val Pro Phe Ser Thr Trp Asp Gln Asp
His Asp Leu Arg Arg Asp145 150 155 160Lys Asn Cys Ala Lys Ser Leu
Ser Gly Gly Trp Trp Phe Gly Thr Cys 165 170 175Ser His Ser Asn Leu
Asn Gly Gln Tyr Phe Arg Ser Ile Pro Gln Gln 180 185 190Arg Gln Lys
Leu Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg Gly Arg 195 200 205Tyr
Tyr Pro Leu Gln Ala Thr Thr Met Leu Ile Gln Pro Met Ala Ala 210 215
220Glu Ala Ala Ser225
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