Note: Descriptions are shown in the official language in which they were submitted.
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MODIFIED FACTOR IX POLYPEPTIDES AND USES THEREOF
[001] This application claims benefit of U.S. Provisional Application Serial
No. 61/124,567;
filed on April 16, 2008, and U.S. Provisional Application Serial No.
61/045,961; filed on April 17,
2008, the contents of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[002] The invention relates to modified Factor IX polypeptides such as Factor
IX polypeptides
with one or more introduced glycosylation sites. The modified Factor IX
polypeptides may exhibit
increased in vitro or in vivo stability such as a longer plasma half-life. The
invention also relates
to methods of making modified Factor IX polypeptides, and methods of using
modified Factor IX
polypeptides, for example, to treat patients afflicted with hemophilia B.
BACKGROUND OF THE INVENTION
[003] Hemophilia B effects one out of 34,500 males and is caused by various
genetic defects in
the gene encoding coagulation Factor IX (FIX) that result in either low or
undetectable FIX protein
in the blood (Kurachi, et al., Hematol. Oncol. Clin. North Am. 6:991-997,
1992; Lillicrap,
Haemophilia 4:350-357, 1998). Insufficient levels of FIX lead to defective
coagulation and
symptoms that result from uncontrolled bleeding. Hemophilia B is treated
effectively by the
intravenous infusion of either plasma-derived or recombinant FIX protein
either to stop bleeds that
have already initiated or to prevent bleeding from occurring (prophylaxis)
(Dargaud, et al., Expert
Opin. Biol. Ther. 7:651-663; Giangrande, Expert Opin. Pharmacother. 6:1517-
1524, 2005).
Effective prophylaxis requires maintaining a minimum trough level of FIX of
about 1% of normal
levels (Giangrande, Expert Opin. Pharmacother. 6:1517-1524, 2005). Because of
the
approximately 18 to 24 hour half-life of native FIX (either plasma-derived or
recombinant), FIX
levels drop to less than 1% of normal levels within 3 to 4 days following
bolus injection which
necessitates repeat injection on average every three days to achieve effective
prophylaxsis
(Giangrande, Expert Opin. Pharmacother. 6:1517-1524, 2005). Such frequent
intravenous
injection is problematic for patients and is a hurdle for achieving effective
prophylaxsis (Petrini,
Haemophilia 13 Suppl 2:16-22, 2007), especially in children. A FIX protein
with a longer half-life
would enable less frequent administration and thus, be of significant medical
benefit.
SUMMARY OF THE INVENTION
[004] The application provides FIX polypeptides (also referred to as modified
FIX polypeptides,
FIX muteins, or FIX variants) comprising amino acid sequences that have been
modified by
introducing one or more glycosylation sites. In some embodiments, the one or
more glycosylation
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sites may be N-linked glycosylation sites. In some embodiments, the
polypeptides have
coagulation activity. In some embodiments, the modified polypeptides may
comprise at least one
substitution such as, but not limited to R338A and V86A. In some embodiments,
the modified
polypeptides may comprise both the R338A and V86A substitutions.
[005] The application also provides FIX polypeptides comprising amino acid
sequences that
have been modified by introducing one or more glycosylation sites. The FIX
polypeptides may
further comprise a carbohydrate chain attached to the one or more introduced
glycosylation sites.
In some embodiments, the carbohydrate chain may be an N-linked carbohydrate
chain. In some
embodiments, the carbohydrate chain may have a mammalian carbohydrate chain
structure. In
some embodiments, the carbohydrate chain may have a human carbohydrate chain
structure. In
some embodiments, the attachment of a carbohydrate chain at one or more of the
introduced
glycosylation sites may increase serum half-life of the polypeptide by, for
example, at least 30%
relative to the polypeptide lacking the introduced glycosylation sites. In
some embodiments, the
attachment of a carbohydrate chain at one or more of the introduced
glycosylation sites does not
reduce the amount of secreted polypeptide by, for example, more than 50%
relative to the amount
of the secreted polypeptide lacking the introduced glycosylation sites. In
some embodiments, the
attachment of a carbohydrate chain at one or more of the introduced
glycosylation sites does not
inhibit interaction of the polypeptide with at least one of Factor VIII
(FVIII), Factor XI (FXI), or
Factor X (FX) by, for example, more than 50% relative to interaction of the
polypeptide lacking
the carbohydrate chain at the introduced glycosylation sites with FVIII, FXI,
or FX. In some
embodiments, the modified polypeptide may have a specific activity of, for
example, at least 100
units per mg of polypeptide.
[006] The one or more glycosylation sites may be introduced via one or more
amino acid
substitutions. The substitutions may be at surface exposed residues and/or be
limited to
substitutions that do not introduce a mutation known to be associated with
hemophilia B.
Exemplary embodiments include FIX polypeptides comprising one or more
substitutions such as,
but not limited to:
(a) G4T; E33N; E36T; E36N; R37N; F75N; F77T; E83T; D85N; V86A; K91T; A103T;
V107T;
K122N; K122T; S138N; A146N; T148N; F150T; P151N; T159N; A161T; A161N; T169N;
Q170N; T172N; D177N; D177E; F178T; K201N; K201T; K214T; V223N; G226N; Y226T;
K228N; K228T; E239N; E242N; 1251T; A262T; E294N; R338A; R338N; K341N; F353N;
H354V; H3541; E355T; V370N; T371V; T3711; E372T; E374N; M391N; K392V; G393T;
E41ON; K413N; L4141;
(b) YIN and S3T; S3N and K5T; G4N and L6T; K5N and E7T; L6N and E8T; E7N and
F9T; F9N
and Qi 1T; VI ON and G12T; Qi IN and N13T; G12N and L14T; N13 and E15T; L14N
and R16T;
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E15N and E17T; M19N and E21T; E20N and K22T; S24N and E26T; F25N and E27T;
E26N and
A28T; E27N and R29T; A28N and E30T; R29N and V31T; E30N and F32T; V3 IN and
E33T;
F32N and N34T; T35N and R37T; T38N and E40T; T39N and F41T; E40N and W42T;
F41N and
K43T; W42N and Q44T; K43N and Y45T; Q44N and V46T; Y45N and D47T; V46N and
G48T;
E52N and N54T; S53N and P55T; G59N and S61T; K63N and D65T; 166N and S68T;
S68N and
E70T; G76N and E78T; E78N and K80T; E83N and D85T; L84N and V86T; 190N and
N92T;
K100N and S102T; S102N and DI 04T; Al 03N and NI 05T; D104N and K1 06T; K1 06N
and
VI 08T; RI 16N and Al 18T; El 19N and Q121T; Q121N and S123T; A127N and P129T;
V135N
and V137T; S136N and S138T; V137N and Q139T; Q139N and S141T; T140N and K142T;
S141N and L143T; E147N and V149T; TI 48N and F150T; V149N and P151T; P151N and
V153T; D152N and D154T; V153N and Y155T; D154N and V156T; Y1 55N and NI 57T;
VI 56N
and S158T; S158N and E160T; T159N and A161T; El 60N and E162T; E162N and
1164T; T163N
and L1 65T; 1164N and DI 66T; L1 65N and NI 67T; DI 66N and 1168T; 1168N and
Q1 70T; T169N
and S171T; S171N and Q173T; T172N and S174T; Q173N and F175T; S174N and N176T;
GI 84N and DI 86T; El 85N and Al 87T; DI 86N and KI 88T; Al 87N and PI 89T; PI
89N and
Q191T; G200N and V202T; K201N and D203T; V202N and A204T; E224N and G226T;
T225N
and V227T; G226N and K228T; V227N and 1229T; H236N and 1238T; 1238N and E240T;
E240N
and E242T; T241N and H243T; H243N and E245T; K247N and N249T; V250N and R252T;
1251N and 1253T; 1253N and P255T; A261N and 1263T; A262N and N264T; D276N and
P278T;
V280N and N282T; F302N and S304T; S304N and Y306T; R312N and F314T; V313N and
H315T; F314N and K316T; H315N and G317T; K316N and R318T; G317N and S319T;
S319N
and L321T; A320N and V322T; R327N and P329T; P329N and V331T; D332N and A334T;
L337N and S339T; S339N and K341T; T340N and F342T; T343N and Y345T; G352N and
H354T; F353N and E355T; H354N and G356T; E355N and G357T; G356N and R358T;
G357N
and D359T; E372N and E374T; W385N and E387T; G386N and E388T; A390N and K392T;
(c) D85N, K122T, and 1251T; D85N, K122T, and E242N; E125N, P126A, and A127T;
P126N,
V128T, and P129A; T148N, F150T, and P151A; F150N, P151A, and D152T; P151N,
V153T, and
A161N; P151N, V153T, and T172N; V153N, Y155T, and E294N; T172N, G226N, and
K228T;
F353N, H354V, and E355T; F353N, H3541, and E355T; V370N, T371V, and E372T;
V370N,
T3711, and E372T; M391N, K392V, and G393T; D85N, P151N, V153T, and K228N;
D85N,
P151N, V153T, and E242N; K122T, P151N, V153T, and K228N; K122T, P151N, V153T,
and
E242N; K122T, P151N, V153T, and 1251T; T148N, F150T, G226N, and K228T; P151N,
V153T,
T172N, and R338A; P151N, V153T, D177E, and F178T; P151N, V153T, G226N, and
K228T;
T172N, G226N, K228T, and R338A; D85N, K122T, P151N, V153T, and E242N; D85N,
P151N,
V153T, G226N, and K228T; K122T, P151N, V153T, G226N, and K228T; S138N, P151N,
V153T, G226N, and K228T; T148N, F150T, G226N, K228T, and R338A; P151N, V153T,
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T172N, G226N, and K228T; P151N, V153T, D177E, F178T, and R338A; P151N, V153T,
G226N, K228T, and R338A; and P151N, V153T, T172N, G226N, K228T, and R338A; and
any
combination thereof.
[007] The one or more glycosylation sites may be introduced into the catalytic
domain of FIX or
activation peptide. Exemplary embodiments include FIX polypeptides comprising
one or more
substitutions such as, but not limited to: R37N; D85N; K122T; S138N; A146N;
A161N; Q170N;
T172N; D177N; F178T; K201N; K228N; E239N; E242N; 1251T; A262T; E294N; E374N;
and
E410N. Other embodiments may comprise the following FIX polypeptides
comprising one or
more substitutions such as: G59N and S61T; K63N and D65T; G76N and E78T; S102N
and
DI 04T; A103N and N105T; DI 04N and K1 06T; El 19N and Q121T; Q121N and S123T;
S136N
and S138T; Q139N and S141T; T140N and K142T; T148N and F150T; V149N and P151T;
P151N and V153T; D152N and D154T; V153N and Y155T; D154N and V156T; V156N and
S158T; S158N and E160T; E160N and E162T; E162N and 1164T; T163N and L165T;
1164N and
D166T; D166N and 1168T; 1168N and Q170T; S171N and Q173T; T172N and S174T;
Q173N
and F175T; S174N and N176T; K201N and D203T; V202N and A204T; E224N and G226T;
T225N and V227T; G226N and K228T; T241N and H243T; 1251N and 1253T; 1253N and
P255T;
A262N and N264T; V280N and N282T; T343N and Y345T; and E372N and E374T; F150N,
P151A, and D152T. In some embodiments, the modified polypeptides may further
comprise at
least one substitution such as, for example, R338A and V86A. In some
embodiments, the
modified polypeptides may further comprise both the R338A and V86A
substitutions.
[008] The one or more glycosylation sites may be introduced by converting an O-
linked
glycosylation site to an N-linked glycosylation site. Exemplary embodiments
include substitutions
such as, but not limited to T169N; T172N; T148N and F150T; and T159N and
A161T. In some
embodiments, the substitutions maybe T172N; T148N and F150T; and T159N and
A161T. In
some embodiments, the modified polypeptides may further comprise at least one
substitution such
as, but not limited to R338A and V86A. In some embodiments, the modified
polypeptides may
further comprise both the R338A and V86A substitutions.
[009] The one or more glycosylation sites may be introduced by inserting
between 1 and 12
amino acid residues between amino acid residues 160-164. The glycosylation
sites may be
introduced between amino acid residues 160-161, between amino acid residues
161-162, between
amino acid residues 162-163, or between amino acid residues 163-164. In some
embodiments,
SEQ ID NO: 2 may be introduced between amino acid residues 160-161, between
amino acid
residues 161-162, between amino acid residues 162-163, or between amino acid
residues 163-164.
In other embodiments, glycosylation sites may be introduced by adding between
1 and 12 amino
acid residues to the C-terminus of the FIX polypeptide. In some embodiments,
SEQ ID NOs: 4, 5,
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6, or 7 may be introduced to the to the C-terminus of the FIX polypeptide. In
some embodiments,
the polypeptide may further comprise amino acid substitutions D177E and F178T.
In some
embodiments, the polypeptide may further comprise amino acid substitutions
P151N and V153T.
In some embodiments, the polypeptide may further comprise amino acid
substitution T172N. In
some embodiments, the polypeptide may further comprise amino acid
substitutions P151N,
V153T, and T172N. In some embodiments, the polypeptide may further comprise
amino acid
substitutions T148N and F150T. In some embodiments, the polypeptide may
further comprise
amino acid substitutions G226N and K228T. In some embodiments, the modified
polypeptides
may further comprise at least one substitution such as R338A and V86A. In some
embodiments,
the modified polypeptides may further comprise both the R338A and V86A
substitutions.
[010] In exemplary embodiments, modified FIX polypeptides are provided
comprising one or
more substitutions such as: D85N; K122T; S138N; T172N; K201N; K228N; E239N;
E242N;
1251T; A262T; E294N; G59N and S61T; G76N and E78T; S102N and D104T; A103N and
N105T; D104N and K106T; El 19N and Q121T; Q121N and S123T; S136N and S138T;
Q139N
and S141T; T140N and K142T; T148N and F150T; V149N and P151T; P151N and V153T;
D152N and D154T; S158N and E160T; E162N and 1164T; T163N and L165T; T172N and
S174T;
Q173N and F175T; K201N and D203T; T225N and V227T; G226N and K228T; 1253N and
P255T; A262N and N264T; V280N and N282T; E372N and E374T; F150N, P151A, and
D152T;
an insertion of SEQ ID NO:2 between A161 and E162; and G226N, K228T, and an
insertion of
SEQ ID NO:2 between A161 and E162. In some embodiments, the modified
polypeptides may
further comprise at least one substitution such as, for example, R338A and
V86A. In some
embodiments, the modified polypeptides may further comprise both the R33 8A
and V86A
substitutions.
[011] The application also provides FIX polypeptides comprising an R338A
substitution and a
V86A substitution. In some embodiments, the polypeptide may have a specific
activity of at least
700 units per mg of polypeptide.
[012] The application also provides pharmaceutical preparations comprising
modified FIX
polypeptides and a pharmaceutically acceptable carrier.
[013] The application also provides methods for treating hemophilia B
comprising administering
to a subject in need thereof a therapeutically effective amount of the
pharmaceutical preparations
described herein.
[014] The application also provides DNA sequences encoding modified
polypeptides, as well as
eukaryotic host cells transfected with the DNA sequences.
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[015] The application also provides methods for producing modified FIX
polypeptides
comprising (i) modifying the amino acid sequence of the polypeptide by
introducing one or more
glycosylation sites; (ii) expressing the polypeptide in a manner which allows
glycosylation at the
one or more glycosylation sites; and (iii) purifying the polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[016] Figure 1 depicts a multiple sequence alignment of FIX sequences within
the activation
peptide from eight species
[017] Figure 2 depicts Western Blot analysis of media from HKB11 cells
transfected with
glycosylation site muteins of FIX. FIX protein was detected using an anti-FIX-
HRP antibody.
[018] Figure 3 depicts a table of expression and activity of FIX glycosylation
site muteins in
HKB11 cells. Expression was determined by ELISA and activity by aPTT assay.
Specific activity
is calculated as international units per mg of FIX protein. Values were
converted to a percentage
of FIX-R338A run in the same transfection experiment. (none): no change in
mobility, (+):
decreased mobility with the number of + indicating degree, (-): increased
mobility.
[019] Figure 4 depicts expression level, coagulation activity, and specific
activity of FIX
glycosylation site muteins in HKB11 cells. Values are expressed as a
percentage of the FIX-
R338A mutein. Constructs are described in Figure 3.
[020] Figure 5 depicts Western Blot analysis of media from HKB11 cells
transfected with the
G226N, K228T glycosylation site mutein of FIX. FIX protein was detected using
an anti-FIX-
HRP antibody.
DESCRIPTION OF THE INVENTION
[0211 The present application provides FIX polypeptides that include one or
more 0- or N-
linked glycosylation sites. Increased glycosylation of therapeutic proteins
may be used to achieve
1) reduced immunogenicity; 2) less frequent administration of the protein; 3)
increased protein
stability such as increased serum half-life; and 4) reduction in adverse side
effects such as
inflammation.
[022] The application provides variants of human FIX with one or more
additional glycosylation
sites. The modified FIX polypeptides may also have an increased plasma half-
life that would
provide, for example, an extended time of protection against bleeding in
hemophilia B patients.
The modified FIX polypeptides would enable hemophilia B patients to achieve
protection against
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bleeding with fewer injections of FIX than is possible with the currently
available therapy of wild
type FIX protein.
[023] The application provides a number of exemplary variants of FIX in which
functional
glycosylation sites were created in both the catalytic domain and in the
activation peptide.
Moreover, the application demonstrates that these variants may be expressed in
mammalian cells,
have increased apparent molecular weight indicative of increased
glycosylation, and maintain
between 50% and 100% activity in a coagulation assay. Finally, these
modification sites may be
combined with alterations that enhance the specific activity of FIX, including
but not limited to the
R338A substitution and/or the V86A substitution (Chang, et al., J. Biol. Chem.
273:12089-12094,
1998 and Chang, et al., J. Biol. Chem. 277:25393-25399, 2002). The combination
with one or
both of the R338A and V86A substitutions compensates for any reduction in
activity resulting
from the addition of glycosylation sites such that the specific activity of
the modified polypeptides
may be similar to or higher than that of wild type FIX.
[024] Once expressed, wild type FIX is a single chain glycoprotein of about
55,000 Daltons. It
can structurally be considered as having four domains: the Gla or gamma
carboxyglutamate-rich
domain; the EGF-like regions; the activation peptide; and the catalytic domain
containing the
active site (Thomson, Blood 67:565-572, 1986). FIX is synthesized in the liver
as a single chain
polypeptide of 461 amino acids and undergoes extensive posttranslational
modification during
passage through the golgi and endoplasmic reticulum (Nemerson, et al., CRC
Crit. Rev. Biochem.
9:45-48, 1980; Stenflo, et al., Annu. Rev. Biochem. 46:157-172, 1977). Both
the signal sequence
and the propeptide are removed resulting in a mature protein of 415 amino
acids (SEQ ID NO: 1)
(Choo, et al., Nature 299:178-180, 1982; Kurachi, et al., Proc. Natl. Acad.
Sci. USA 79:6461-
6464, 1982). Efficient gamma carboxylation is essential for the coagulation
activity of FIX and in
humans 12 Gla residues are generated within the N terminal Gla domain,
although gamma
carboxylation on G1a36 and Gla40 are not required for function (DiScipio, et
al., Biochemistry
18:899-904, 1979; Gillis, et al., Protein Sci. 6:185-196, 1997). In addition,
FIX contains two N-
linked glycosylation sites (N157, N167), six O-linked glycosylation sites
(S53, S61, T159, T169,
T172, T179), and one site each for Ser phosphorylation (S158), tyrosine
sulfation (Y155) and f3-
hydroxylation (D64) (McMullen, et al., Biochem. Biophys. Res. Comm. 115:8-14,
1983).
[025] Activated Factor VII (FVII) initiates the normal hemostatic process by
forming a complex
with tissue factor (TF), exposed as a result of injury to the vessel wall. The
complex subsequently
activates FIX; the active form referred to as FIXa. The activation peptide of
FIX is removed by
proteolytic cleavage at two sites by either Factor XIa (FXIa) or the tissue
factor (TF)/Factor VIIa
complex to generate the catalytically active molecule, Factor IXa (FIXa). FIXa
and Factor VIIIa
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(FVIIIa) convert FX to Factor Xa (FXa), which in turn converts prothrombin to
thrombin.
Thrombin then converts fibrinogen to fibrin resulting in formation of a fibrin
clot.
[026] As wild-type FIX has numerous post-translational modifications some of
which have been
suggested to play a role in the in vivo pharmacokinetic profile, an ectopic
glycosylation site may
be introduced at a position that does not affect these other modifications.
Once produced, FIX
should retain enzymatic activity and interact with FVIII, FXI, and FX in order
to be an effective
treatment for hemophilia B. The introduced glycosylation site should not
perturb these
interactions and function. The application provides, in part, modifications to
FIX which are likely
to result in an increased number of glycosylation sites with minimal
perturbation of function and
thus have utility for increasing the bioavailability of FIX. Finally, these
modification sites may be
combined with alterations that enhance the specific activity of FIX, including
but not limited to the
R338A substitution and/or the V86A substitution. Alterations that enhance the
specific activity of
FIX may compensate for potential loss of coagulation activity and also
potentially prolong the
efficacy of modified molecules by conferring efficacy at lower levels of
protein.
Modified FIX Polypeptides
[027] The application provides FIX polypeptides comprising one or more
introduced
glycosylation sites, that is, modified FIX polypeptides. "Factor IX" as used
herein refers to a
human plasma FIX glycoprotein that is a member of the intrinsic coagulation
pathway and is
essential to blood coagulation. It is to be understood that this definition
includes native as well as
recombinant forms of the human plasma FIX glycoprotein. Unless otherwise
specified or
indicated, as used herein FIX means any functional human FIX protein molecule
in its normal role
in coagulation, including any fragment, analogue, variant, and derivative
thereof. The terms
"fragment," "derivative," "analogue," "mutein," and "variant," when referring
to the polypeptides
of the application, means fragments, derivatives, analogues, muteins, and
variants of the
polypeptides which retain substantially the same biological function or
activity.
[028] Non-limiting examples of FIX polypeptides include FIX, FIXa, and
truncated versions of
FIX having FIX activity. Biologically active fragments, deletion variants,
substitution variants, or
addition variants of any of the foregoing that maintain at least some degree
of FIX activity can also
serve as a FIX polypeptide. In some embodiments, the FIX polypeptides may
comprise an amino
acid sequence at least about 70, 80, 90, or 95% identical to SEQ ID NO: 1. In
some embodiments,
the modified FIX polypeptides are biologically active. Biological activity can
be determined, for
example, by coagulation assays described herein.
[029] Modified FIX polypeptides may also contain conservative substitutions of
amino acids. A
conservative substitution is recognized in the art as a substitution of one
amino acid for another
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amino acid that has similar properties and include, for example, the changes
of alanine to serine;
arginine to lysine; asparagine to glutamine or histidine; aspartate to
glutamate; cysteine to serine;
glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine
to asparagine or
glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine;
lysine to arginine;
methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or
methionine; serine to
threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan
or phenylalanine; and
valine to isoleucine or leucine. In some embodiments, the FIX polypeptides of
SEQ ID NO: 1
comprise from 1-30, from 1-20, or from 1-10 conservative amino acid
substitutions in addition to
the introduction of one or more glycosylation sites.
[030] The single letter abbreviation for a particular amino acid, its
corresponding amino acid,
and three letter abbreviation are as follows: A, alanine (Ala); C, cysteine
(Cys); D, aspartic acid
(Asp); E, glutamic acid (Glu); F, phenylalanine (Phe); G, glycine (Gly); H,
histidine (His); I,
isoleucine (Ile); K, lysine (Lys); L, leucine (Leu); M, methionine (Met); N,
asparagine (Asn); P,
proline (Pro); Q, glutamine (Gln); R, arginine (Arg); S, serine (Ser); T,
threonine (Thr); V, valine
(Val); W, tryptophan (Trp); Y, tyrosine (Tyr); and norleucine (Nle).
[0311 Glycosylation of polypeptides is typically either N-linked or O-linked.
N-linked refers to
the attachment of a carbohydrate moiety to the side chain of an asparagine
residue. The tripeptide
sequences Asn-X-Ser and Asn-X-Thr, where X is any amino acid except proline,
are the
recognition sequences for enzymatic attachment of the carbohydrate moiety to
the Asn side chain.
Thus, the presence of either of these tripeptide sequences in a polypeptide
creates a potential N-
linked glycosylation site. An exemplary N-linked glycosylation site useful for
the invention may
also be represented as follows XI-Asn-X2-X3-X4; where X1 is optionally Asp,
Val, Glu, Gly, or
Ile; X2 is any amino acid except Pro; X3 is Ser or Thr; and X4 is optionally
Val, Glu, Gly, Gln, or
Ile. In some embodiments, Xl is optionally Asp; X2 is Ser; X3 is Thr; and X4
is Gln. In some
embodiments, X1 is Asp; X2 is Ile; X3 is Thr; and X4 is Gln. Addition of N-
linked glycosylation
sites to a FIX polypeptide is accomplished by altering the amino acid sequence
such that one or
more of the above-described tripeptide sequences is introduced.
[032] O-linked glycosylation refers to the attachment of one of the sugars N-
aceytlgalactosamine, galactose, or xylose to a hydroxyamino acid, most
commonly to serine or
threonine, although attachment to 5-hydroxyproline or 5-hydroxylysine is also
possible. Addition
of O-linked glycosylation sites to a FIX polypeptide may be accomplished by
altering the amino
acid sequence such that one or more Ser or Thr residues are introduced.
[033] Glycosylation sites may be introduced, for example, by deleting one or
more amino acid
residues, substituting one or more endogenous FIX amino acid residues with
another amino
acid(s), or adding one or more amino acid residues. The addition of an amino
acid residue may be
9
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either between two existing amino acid residues or at the N- or C-terminal end
of the native FIX
molecule.
[034] The terminology for amino acid substitutions used is as follows. The
first letter represents
the amino acid residue naturally present at a position of human FIX. The
following number
represents the position in the mature human FIX amino acid sequence (SEQ ID
NO:1). The
second letter represent the different amino acid substituting for
(replacing/substituting) the natural
amino acid. As an example, R338A denotes that the Arg residue at position 338
of SEQ ID NO: 1
has been replaced with an Ala residue. With respect to SEQ ID NO: 26 which
includes an
additional 5' amino acid sequence (46 amino acids) of human FIX polypeptide,
position 338 of
SEQ ID NO: 1 corresponds to position 384 of SEQ ID NO: 26.
[035] The FIX residue number system used herein refers to that of the mature
human FIX
protein in which residue 1 represents the first amino acid of the mature FIX
polypeptide following
removal of both the signal sequence and the propeptide. Native or wild type
FIX is the full length
mature human FIX molecule as shown in SEQ ID NO: 1.
[036] In some embodiments, the glycosylation sites are engineered in FIX at
locations that will
not abolish the function of the protein or its expression in cells. In order
to design FIX
polypeptides comprising one or more introduced glycosylation sites, several
criteria may be
applied. In some embodiments, the introduced glycosylation site is surface
exposed. Surface
exposure can be determined based on the solvent accessible surface area as
determined in Autin, et
al., (J. Thromb. Haemost. 3:2044-56, 2005). In some embodiments, the
introduced glycosylation
site does not introduce a mutation known to be associated with hemophilia B.
Known mutations
can be found on the world wide web at
kcl.ac.uk/ip/petergreen/haemBdatabase.html and in Table
1
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TABLE 1
Residue Mutation Residue Mutation Residue Mutation
No. No. No.
2 N-*D 43 K-*E 82 C-*R
3 S-*P 45 Y--+N 88 C-*R
4 G-*S 46 V-*A 90 I-*T
K-*E 47 D--+N 92 N-*H
6 L-*S 48 G-*R 96 E-*K
7 E-*K 49 D-*Y 97 Q-*K
8 E-*K 51 C-*S 99 C-*R
9 F-*I 55 P-*T 108 V-*A
11 Q-*E 56 C-*S 109 C-*Y
12 G-*R 58 N-*K 110 S-*P
14 L-*P 59 G-*S 111 C-*R
17 E-*K 60 G-*S 114 G-*E
18 C-*R 62 C-*Y 115 Y-*C
20 E-*K 64 D--+N 117 L-*F
21 E-*K 66 I-*N 118 A-*V
23 C-*R 67 N-*K 120 N-*Y
25 F-*S 69 Y--+N 121 Q-*H
26 E-*Q 71 C-*S 123 S-*P
27 E-*K 72 W-*R 124 C-*F
30 E-*K 73 C-*R 127 A-*P
32 F-*S 76 G-*R 128 V-*M
33 E-*K 77 F-*S 131 P-*S
38 T-*A 78 E-*K 132 C-*R
41 F-*V 79 G-*R 133 G-*R
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Residue Mutation Residue Mutation Residue Mutation
No. No. No.
145 R-*S 217 V-*E 268 H-*R
146 A-*P 218 T-*I 269 D--+N
178 F-*L 219 A-*T 270 I-*F
180 R-*G 220 A-*T 271 A-*D
T
181 V-*F 221 H--+Y 272 L-*F
182 V-*L 222 C-*S 273 L-*P
183 G-*S 226 G--+N 274 E-*G
V-*I, G,
184 G-*R 227 E 275 L-*Q
187 A-*D 228 K-*T 276 D-*Y
190 G-*C 231 V-*F 277 E-*K
191 Q-*K 233 V-*T 278 P-*H
192 F-*V 234 G-*S 279 L-*V
193 P-*S 236 H-*R 282 N-*I
194 W-*R 237 N-*D 284 Y-*C
195 Q-*K 238 I-*N 287 P-*T
198 L-*S 245 E-*K 288 I-*T
204 A-*V 246 Q-*K 289 C-*R
206 C-*R 248 R-*G 290 I-*H
207 G-*R 250 V-*M 291 A-*T
208 G-*S 251 I-*N 293 K-*E
210 I-*T 256 H-*R 295 Y-*C
211 V-*F 257 H-*Y 296 T-*A
212 N-*K 259 Y--+N 298 I-*N
215 W-*R 260 N-*S 299 F-*V
216 I-*F 264 N-*Y 300 L-*F
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Residue Mutation Residue Mutation Residue Mutation
No. No. No.
301 K-*I 337 L-*I 366 G-*W
303 G-*E 339 S-*P 367 G-*R
305 G-*D 340 T-*R 368 P-*T
307 V-*L 341 K-*E 369 H-*I
308 S-*R 344 I-*F 370 V--+N
T-*V, E,
309 G-*S 345 Y-*C 371 G, l
310 W-*R 346 N-*D 372 E-*T
311 G-*R 347 N-*I 373 V-*E
312 R-*G 348 M-*V 376 T--+N
313 V-*G 349 F-*L 378 F-*L
314 F-*I 350 C-*R 379 L-*F
316 K-*E 351 A-*P 380 T-*P
317 G-*R 352 G-*S 381 G-*R
320 A-*P 353 F--+N 382 I-*F
H-*V, E,
323 L-*F 354 G, I 384 S-*C
324 Q-*E 355 E-*T 385 W-*R
325 Y-*S 356 G-*R 386 G-*S
328 V-*I 357 G-*C 387 E-*K
329 P-*T 358 R-*T 388 E-*G
330 L-*P 359 D--+N 389 C-*G
331 V-*F 360 S-*L 390 A-*T
332 D-*Y 361 C-*S 391 M--+N
K-*V, E,
333 R-*G 363 G-*R 392 G, I
334 A-*T 364 D--+N 393 G-*T
336 C-*R 365 S-*G 394 K-*E
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Residue Mutation Residue Mutation Residue Mutation
No. No. No.
395 Y-*H 400 K--+N 407 W-*R
396 G-*R 402 S-*Y 408 I-*N
397 I-*L 403 R-*W 412 T-*K
398 Y-*S 404 Y-*H 413 K--+N
L-*V, E,
399 T-*N 405 V-*F 414 G, I
[037] It may be desirable to compare the properties of the modified FIX
polypeptides having one
or more introduced glycosylation sites to a control polypeptide. Properties
for comparison include,
for example, solubility, activity, plasma half-life, glycosylation state, and
binding properties. In
some embodiments, the modified FIX polypeptides may be glycosylated. It is
within the purview
of one skilled in the art to select the most appropriate control polypeptide
for comparison. In some
embodiments, the control polypeptide may be identical to the modified
polypeptide except for the
one or more introduced glycosylation sites. Exemplary polypeptides include
wild-type FIX
polypeptide and FIX polypeptides comprising one or more activating
substitutions, such as R338A
and/or V86A.
[038] One aspect of the application provides modified FIX polypeptides having
increased in
vitro or in vivo stability over a control polypeptide. Enhanced serum half-
life and in vivo stability
may be desirable to reduce the frequency of dosing that is required to achieve
therapeutic
effectiveness. Accordingly, in certain embodiments, the glycosylated FIX
polypeptides have a
serum half-life increased by about 20, 30, 40, 60, 80, 100, 150, 200, 300,
400, 500, 600, 700, 800,
900, or 1000% relative to a control protein. In some embodiments, the modified
FIX polypeptides
may have a serum half-life of at least one, at least two, at least three, at
least four, at least five, at
least ten, or at least twenty days or more.
[039] The term "half-life," as used herein in the context of administering a
polypeptide drug to a
patient, is defined as the time required for plasma concentration of a drug in
a patient to be reduced
by one half. Methods for pharmacokinetic analysis and determination of half-
life and in vivo
stability will be familiar to those skilled in the art. Details may be found
in Kenneth, et al.,
Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in
Peters, et al.,
Pharmacokinetc analysis: A Practical Approach (1996). Reference is also made
to
"Pharmacokinetics," M Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev.
edition
(1982), which describes pharmacokinetic parameters such as t-alpha and t-beta
half lives and area
under the curve (AUC).
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[040] The activity of modified FIX polypeptides may be described either as an
absolute value,
such as in units, or as a percentage of the activity of a control polypeptide.
In some embodiments,
the modified FIX polypeptides may have a specific activity that is not reduced
more than about 10,
20, 30, 40, 50, 60, 70, or 80% relative to a control protein. For example, a
modified FIX
polypeptide may have a specific activity that is not reduced more than about
80% relative to a
control FIX polypeptide, if the modified polypeptide maintains at least about
20% of the specific
activity as compared to the specific activity of the control. FIX specific
activity may be defined as
the ability to function in the coagulation cascade, induce the formation of
FXa via interaction with
FVIIIa on an activated platelet, or support the formation of a blood clot. The
activity may be
assessed in vitro by techniques such as clot analysis, as described in, for
example, McCarthy, et al.,
(Thromb. Haemost. 87:824-830,2002), and other techniques known to those
skilled in the art. The
activity may also be assessed in vivo using one of the several animal lines
that have been
intentionally bred with a genetic mutation for hemophilia B such that an
animal produced from
such a line is deficient for FIX. Such lines are available from a variety of
sources such as, without
limitation, the Division of Laboratories and Research, New York Department of
Public Health,
Albany, N.Y. and the Department of Pathology, University of North Carolina,
Chapel Hill, N.C.
Both of these sources, for example, provide canines suffering from canine
hemophilia B.
Alternatively, mice deficient in FIX are also available (Sabatino, et al.,
Blood 104:2767-2774,
2005). In order to test for FIX activity, a test polypeptide is injected into
the diseased animal, a
small cut made and bleeding time compared to a healthy control.
[041] Human wild-type FIX has a specific activity of around 200 units per mg.
One unit of FIX
has been defined as the amount of FIX present in one millilitre of normal
(pooled) human plasma
(corresponding to a FIX level of 100%). In some embodiments, the modified FIX
polypeptides
may have a specific activity of at least about 100 units per mg of FIX
polypeptide. In some
embodiments, the modified FIX polypeptides may have a specific activity of at
least about 120,
140, 160, 180, 200, 220, 240, 260 units, or more per mg of FIX polypeptide. In
some
embodiments, the specific activity of FIX may be measured using the APTT or
activated partial
thromboplastin time assay (described by, e.g., Proctor, et al., Am. J. Clin.
Pathol. 36:212, 1961 and
see Examples).
[042] When expressed in cells, such as liver or kidney cells, FIX polypeptide
may be
synthesized by the cellular machinery, undergoes posttranslational
modification, and is then
secreted by the cells into the extracellular milieu. The amount of FIX
polypeptide secreted from
cells is therefore dependent on both processes of protein translation and
extracellular secretion. In
some embodiments, the modified FIX polypeptides may be secreted in an amount
that is not
reduced more than about 10, 20, 30, 40, 50, 60, 70, or 80% relative to the
amount secreted of a
control protein. For example, a modified FIX polypeptide may be secreted in an
amount that is not
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WO 2009/137254 PCT/US2009/040813
reduced more than about 80% relative to a control FIX polypeptide, if the
modified polypeptide is
secreted in an amount of at least about 20% as compared to the control. The
amount of FIX
polypeptide secreted may be measured, for example, by determining the protein
levels in the
extracellular medium using any art-known method. Traditional methodologies for
protein
quantification include 2-D gel electrophoresis, mass spectrometry, and
antibody binding.
Exemplary methods for assaying protein levels in a biological sample include
antibody-based
techniques, such as immunoblotting (western blotting), immunohistological
assay, enzyme linked
immunosorbent assay (ELISA), or radioimmunoassay (RIA).
[043] In some embodiments, the modified FIX polypeptides interact with at
least one of FVIII,
FXI, or FX at a level not reduced more than about 40, 50, 60, 70, or 80%
relative to the interaction
of a control protein with at least one of FVIII, FXI, or FX. For example, a
modified FIX
polypeptide may interact with at least one of FVIII, FXI, or FX at a level not
reduced more than
about 80% relative to a control FIX polypeptide, if the modified polypeptide
interacts with at least
one of FVIII, FXI, or FX at a level of at least about 20% as compared to the
control. The binding
of FIX to other members of the coagulation cascade can be determined by any
method known to
one skilled in the art, including for example, the methods described in Chang,
et al., (J. Biol.
Chem. 273:12089-12094, 1998).
[044] As previously described, FIX is composed of four structural domains with
different
biological functions. One aspect of the invention is to provide FIX
polypeptides that are modified
in particular domains, such as in the activation peptide and in the catalytic
domain. The
application provides, in part, FIX polypeptides comprising one more
glycosylation sites introduced
into the activation peptide of FIX. The natural glycosylation sites in FIX are
located in the EGF1
domain (two O-linked sites on Ser53 and Ser61) and in the activation peptide
(four O-linked sites
at Thr159, Thr169, Thr172, and Thr179 and 2 N-linked sites at Asn157 and Asn
167). Thus, six of
the eight natural glycosylation sites are located in the activation peptide.
Introduced glycosylation
sites may have less of a negative impact on activity or expression when
introduced into regions of
FIX that have natural glycosylation sites, such as the activation peptide. In
addition, the activation
peptide is cleaved when FIX is activated and so is not required for the
catalytic activity of FIXa.
However, the activation peptide is present in the zymogen, making the
activation peptide an
attractive region to introduce glycosylation sites to improve the circulating
half-life of the
zymogen.
[045] The application also provides, in part, FIX polypeptides comprising one
or more
glycosylation sites. In some embodiments, FIX polypeptides are provided
comprising one or more
substitutions selected from G4T; E33N; E36T; E36N; R37N; F75N; F77T; E83T;
D85N; V86A;
K91T; A103T; V107T; K122N; K122T; S138N; A146N; T148N; F150T; P151N; T159N;
A161T;
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A161N; T169N; Q170N; T172N; D177N; D177E; F178T; K201N; K201T; K214T; V223N;
G226N; Y226T; K228N; K228T; E239N; E242N; 1251T; A262T; E294N; R338A; R338N;
K341N; F353N; H354V; H3541; E355T; V370N; T371V; T3711; E372T; E374N; M391N;
K392V; G393T; E410N; K413N; L4141; or any combination thereof.
[046] In some embodiments, FIX polypeptides are provided comprising one or
more
substitutions selected from YIN and S3T; S3N and K5T; G4N and L6T; K5N and
E7T; L6N and
E8T; E7N and F9T; F9N and Q1 IT; VI ON and G12T; Q1 IN and N13T; G12N and
L14T; N13
and E15T; L14N and R16T; El 5N and E17T; M19N and E21T; E20N and K22T; S24N
and E26T;
F25N and E27T; E26N and A28T; E27N and R29T; A28N and E30T; R29N and V3 IT;
E30N and
F32T; V3 IN and E33T; F32N and N34T; T35N and R37T; T38N and E40T; T39N and
F41T;
E40N and W42T; F4 IN and K43T; W42N and Q44T; K43N and Y45T; Q44N and V46T;
Y45N
and D47T; V46N and G48T; E52N and N54T; S53N and P55T; G59N and S61T; K63N and
D65T; 166N and S68T; S68N and E70T; G76N and E78T; E78N and K80T; E83N and
D85T;
L84N and V86T; 190N and N92T; K100N and S102T; S102N and D104T; A103N and NI
05T;
D104N and K106T; K1 06N and V108T; RI 16N and Al18T; El19N and Q121T; Q121N
and
S123T; A127N and P129T; V135N and V137T; S136N and S1 38T; V137N and Q139T;
Q139N
and S141T; Tl40N and K142T; S141N and L143T; E147N and V149T; T148N and F150T;
V149N and P151T; P151N and V153T; D152N and D154T; V153N and Y155T; D154N and
V156T; Y155N and N157T; V156N and S158T; S158N and E160T; T159N and A161T;
E160N
and E162T; E162N and I164T; T163N and L165T; I164N and D166T; L165N and N167T;
D166N
and I168T; I168N and Q170T; T169N and S171T; S171N and Q173T; T172N and S174T;
Q173N
and F175T; S174N and N176T; G184N and D186T; E185N and A187T; D186N and K188T;
A187N and P189T; P189N and Q191T; G200N and V202T; K201N and D203T; V202N and
A204T; E224N and G226T; T225N and V227T; G226N and K228T; V227N and 1229T;
H236N
and 1238T; 1238N and E240T; E240N and E242T; T241N and H243T; H243N and E245T;
K247N
and N249T; V250N and R252T; 125 IN and 1253T; 1253N and P255T; A261N and
1263T; A262N
and N264T; D276N and P278T; V280N and N282T; F302N and S304T; S304N and Y306T;
R312N and F314T; V313N and H315T; F314N and K316T; H315N and G317T; K316N and
R318T; G317N and S319T; S319N and L321T; A320N and V322T; R327N and P329T;
P329N
and V331T; D332N and A334T; L337N and S339T; S339N and K341T; T340N and F342T;
T343N and Y345T; G352N and H354T; F353N and E355T; H354N and G356T; E355N and
G357T; G356N and R358T; G357N and D359T; E372N and E374T; W385N and E387T;
G386N
and E388T; A390N and K392T; or any combination thereof
[047] In some embodiments, FIX polypeptides are provided comprising one or
more
substitutions selected from D85N, K122T, and I251T; D85N, K122T, and E242N;
E125N, P126A,
and A127T; P126N, V128T, and P129A; T148N, F150T, and P151A; F150N, P151A, and
D152T;
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P151N, V153T, and A161N; P151N, V153T, and T172N; V153N, Y155T, and E294N;
T172N,
G226N, and K228T; F353N, H354V, and E355T; F353N, H3541, and E355T; V370N,
T371V, and
E372T; V370N, T3711, and E372T; M391N, K392V, and G393T; D85N, P151N, V153T,
and
K228N; D85N, P151N, V153T, and E242N; K122T, P151N, V153T, and K228N; K122T,
P151N,
V153T, and E242N; K122T, P151N, V153T, and 1251T; T148N, F150T, G226N, and
K228T;
P151N, V153T, T172N, and R338A; P151N, V153T, D177E, and F178T; P151N, V153T,
G226N,
and K228T; T172N, G226N, K228T, and R338A; D85N, K122T, P151N, V153T, and
E242N;
D85N, P151N, V153T, G226N, and K228T; K122T, P151N, V153T, G226N, and K228T;
S138N,
P151N, V153T, G226N, and K228T; T148N, F150T, G226N, K228T, and R338A; P151N,
V153T, T172N, G226N, and K228T; P151N, V153T, D177E, F178T, and R338A; P151N,
V153T, G226N, K228T, and R338A; and P151N, V153T, T172N, G226N, K228T, and
R338A; or
any combination thereof
[048] In some embodiments, FIX polypeptides are provided comprising one or
more
substitutions selected from
(a) G4T; E33N; E36T; E36N; R37N; F75N; F77T; E83T; D85N; V86A; K91T; A103T;
V107T;
K122N; K122T; S138N; A146N; T148N; F150T; P151N; T159N; A161T; A161N; T169N;
Q170N; T172N; D177N; D177E; F178T; K201N; K201T; K214T; V223N; G226N; Y226T;
K228N; K228T; E239N; E242N; 1251T; A262T; E294N; R338A; R338N; K341N; F353N;
H354V; H3541; E355T; V370N; T371V; T3711; E372T; E374N; M391N; K392V; G393T;
E41ON; K413N; L4141;
(b) YIN and S3T; S3N and K5T; G4N and L6T; K5N and E7T; L6N and E8T; E7N and
F9T; F9N
and Q1 IT; VI ON and G12T; Q1 IN and N13T; G12N and L14T; N13 and E15T; L14N
and R16T;
El 5N and E17T; M19N and E21T; E20N and K22T; S24N and E26T; F25N and E27T;
E26N and
A28T; E27N and R29T; A28N and E30T; R29N and V3 IT; E30N and F32T; V3 IN and
E33T;
F32N and N34T; T35N and R37T; T38N and E40T; T39N and F41T; E40N and W42T;
F41N and
K43T; W42N and Q44T; K43N and Y45T; Q44N and V46T; Y45N and D47T; V46N and
G48T;
E52N and N54T; S53N and P55T; G59N and S61T; K63N and D65T; 166N and S68T;
S68N and
E70T; G76N and E78T; E78N and K80T; E83N and D85T; L84N and V86T; 190N and
N92T;
K100N and S102T; S1 02N and DI 04T; A103N and N105T; D104N and K1 06T; K1 06N
and
VI 08T; RI 16N and Al 18T; El 19N and Q121T; Q121N and S123T; A127N and P129T;
V135N
and V137T; S136N and S138T; V137N and Q139T; Q139N and S141T; T140N and K142T;
S141N and L143T; E147N and V149T; T148N and F150T; V149N and PI 51T; P15 IN
and
V153T; D152N and D154T; V153N and Y155T; DI 54N and VI 56T; YI 55N and NI 57T;
VI 56N
and S158T; S158N and E160T; T159N and Al 61T; El 60N and El 62T; El 62N and 11
64T; TI 63N
and L165T; I164N and D166T; L165N and N167T; D166N and 11 68T; 11 68N and Q1
70T; T169N
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WO 2009/137254 PCT/US2009/040813
and S171T; S171N and Q173T; T172N and S174T; Q173N and F175T; S174N and N176T;
G184N and D186T; E185N and A187T; D186N and K188T; A187N and P189T; P189N and
Q191T; G200N and V202T; K20 IN and D203T; V202N and A204T; E224N and G226T;
T225N
and V227T; G226N and K228T; V227N and 1229T; H236N and 1238T; 1238N and E240T;
E240N
and E242T; T241N and H243T; H243N and E245T; K247N and N249T; V250N and R252T;
I251N and 1253T; 1253N and P255T; A261N and 1263T; A262N and N264T; D276N and
P278T;
V280N and N282T; F302N and S304T; S304N and Y306T; R312N and F314T; V313N and
H315T; F314N and K316T; H315N and G317T; K316N and R318T; G317N and S319T;
S319N
and L321T; A320N and V322T; R327N and P329T; P329N and V331T; D332N and A334T;
L337N and S339T; S339N and K341T; T340N and F342T; T343N and Y345T; G352N and
H354T; F353N and E355T; H354N and G356T; E355N and G357T; G356N and R358T;
G357N
and D359T; E372N and E374T; W385N and E387T; G386N and E388T; A390N and K392T;
(c) D85N, K122T, and I251T; D85N, K122T, and E242N; E125N, P126A, and A127T;
P126N,
V128T, and P129A; T148N, F150T, and P151A; F150N, P151A, and D152T; P151N,
V153T, and
A161N; P151N, V153T, and T172N; V153N, Y155T, and E294N; T172N, G226N, and
K228T;
F353N, H354V, and E355T; F353N, H3541, and E355T; V370N, T371V, and E372T;
V370N,
T3711, and E372T; M391N, K392V, and G393T; D85N, P151N, V153T, and K228N;
D85N,
P151N, V153T, and E242N; K122T, P151N, V153T, and K228N; K122T, P151N, V153T,
and
E242N; K122T, P151N, V153T, and 1251T; T148N, F150T, G226N, and K228T; P151N,
V153T,
T172N, and R338A; P151N, V153T, D177E, and F178T; P151N, V153T, G226N, and
K228T;
T172N, G226N, K228T, and R338A; D85N, K122T, P151N, V153T, and E242N; D85N,
P151N,
V153T, G226N, and K228T; K122T, P151N, V153T, G226N, and K228T; S138N, P151N,
V153T, G226N, and K228T; T148N, F150T, G226N, K228T, and R338A; P151N, V153T,
T172N, G226N, and K228T; P151N, V153T, D177E, F178T, and R338A; P151N, V153T,
G226N, K228T, and R338A; and P151N, V153T, T172N, G226N, K228T, and R338A; and
any
combination thereof.
[049] The application provides, in part, FIX polypeptides comprising one more
glycosylation
sites introduced by converting an endogenous O-linked glycosylation site to an
N-linked
glycosylation site. It has been reported that N-linked glycosylation sites are
more likely to be
sialylated than O-linked glycosylation sites and there is evidence that higher
sialic acid content
confers increased protein half-life. It is generally believed that the
increased sialic acid content
provided by additional N-linked glycosylation may be responsible for the
increased half-life in
blood (White, et al., Thromb. Haemost. 78:261-265, 1997). Exemplary
embodiments of such
modified FIX polypeptides are as follows. In some embodiments, FIX
polypeptides are provided
comprising a T169N substitution. In some embodiments, FIX polypeptides are
provided
comprising a T172N substitution. In some embodiments, FIX polypeptides are
provided
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comprising a T148N substitution and an F150T substitution. In some
embodiments, FIX
polypeptides are provided comprising a T159N substitution and an A161T
substitution.
[050] Another aspect of the invention provides for the insertion of amino
acids into the
activation peptide in order to generate one or more glycosylation sites. The
application provides,
in part, FIX polypeptides comprising one more glycosylation sites introduced
between amino acid
residues 160 to 164 of human FIX. In some embodiments, the amino acid residues
introduced
include a glycosylation site. In some embodiments, the amino acid residues
introduced form a
glycosylation site in combination with wild-type FIX amino acid residues. The
multiple sequence
alignment of the FIX sequence from 8 species demonstrated that the mouse, rat,
and guinea pig
sequences all have additional amino acids (between 7 and 10 residues) in the
activation peptide
that are not found in other species (human, rhesus, dog, rabbit, pig) (Figure
1). These additional
sequences are located between E160 and E162 of human FIX. This suggests that
insertion of at
least 10 amino acid residues is tolerated in the FIX structure at this site.
This region was targeted
for the introduction of additional amino acids and was found to be a good
location as activity was
not significantly reduced. In some embodiments, up to 30, 25, 20, 18, 16, 14,
or 12 amino acid
residues may be inserted between amino acid residues 160 to 164 of human FIX.
In some
embodiments, up to 10 amino acids may be inserted. In some embodiments, up to
9 amino acid
may be inserted.
[051] Depending upon the criteria used to perform the multiple sequence
alignment between FIX
from the eight species, the apparent site at which the additional amino acids
in rat, mouse, and
guinea pig are found can vary such that the site can be either between E160
and A161, between
A161 and E162, between E162 and T163, or between T163 and 1164 of human FIX.
In some
embodiments, the amino acid sequence inserted between E160 and A161 may be SEQ
ID NO: 2.
In some embodiments, the amino acid sequence inserted between A161 and E162
may be SEQ ID
NO: 2. In some embodiments, the amino acid sequence inserted between E162 and
T163 may be
SEQ ID NO: 2. In some embodiments, the amino acid sequence inserted between
T163 and 1164
may be SEQ ID NO: 2. In some embodiments, one or more amino acids may be
inserted between
E160 and A161 and one or more amino acids may be inserted between A161 and
E162. In some
embodiments, one or more amino acids may be inserted between E160 and A161 and
one or more
amino acids maybe inserted between E162 and T163. In some embodiments, one or
more amino
acids maybe inserted between A161 and E162 and one or more amino acids maybe
inserted
between E162 and T163. In some of the above described embodiments, one or more
amino acids
maybe inserted between T163 and 1164. In some embodiments, up to about 30, 25,
20, 18, 16, 14,
or 12 total amino acid residues may be inserted between amino acid residues
160 to 164 of human
FIX. In some embodiments, up to about 10 total amino acids may be inserted
between amino acid
residues 160 to 164 of human FIX. In some embodiments, up to about 9 total
amino acids maybe
CA 02721683 2010-10-15
WO 2009/137254 PCT/US2009/040813
inserted between amino acid residues 160 to 164 of human FIX. In some
embodiments, one, two,
three, or four glycosylation sites may be introduced.
[052] The application further provides modified FIX polypeptides comprising
more than one of
the introduced glycosylation sites disclosed herein. FIX polypeptides are
provided that comprise
at least one introduced glycosylation site in the catalytic domain and at
least one introduced
glycosylation site in the activation peptide. FIX polypeptides are provided
that comprise at least
two introduced glycosylation sites in the catalytic domain. FIX polypeptides
are provided that
comprise at least two introduced glycosylation sites in the activation
peptide. In some
embodiments, the FIX polypeptides may comprise one or more of the following
substitutions:
R37N; D85N; K122T; S138N; A146N; A161N; Q170N; T172N; D177N; F178T; K201N;
K228N;
E239N; E242N; 1251T; A262T; E294N; E374N; and E410N. Other embodiments may
comprise
one or more of the following substitutions: G59N and S6 IT; K63N and D65T;
G76N and E78T;
S102N and D104T; A103N and N105T; D104N and K106T; E119N and Q121T; Q121N and
S123T; S136N and S138T; Q139N and S141T; T140N and K142T; T148N and F150T;
V149N
and P151T; P151N and V153T; D152N and D154T; V153N and Y155T; D154N and V156T;
V156N and S158T; S158N and E160T; E160N and E162T; E162N and 1164T; T163N and
L165T;
1164N and D166T; D166N and 1168T; 1168N and Q170T; S171N and Q173T; T172N and
S174T;
Q173N and F175T; S174N and N176T; K201N and D203T; V202N and A204T; E224N and
G226T; T225N and V227T; G226N and K228T; T241N and H243T; 1251N and 1253T;
1253N and
P255T; A262N and N264T; V280N and N282T; T343N and Y345T; E372N and E374T;
F150N,
P151A, and D152T; and insertion of SEQ ID NO:2 between A161 and E162. In some
embodiments, the modified polypeptides may further comprise at least one
substitution such as, for
example, R338A and V86A. In some embodiments, the modified polypeptides may
further
comprise both the R338A and V86A substitutions.
[053] The application further provides modified FIX polypeptides that comprise
at least one
introduced glycosylation site at the C-terminus of the FIX polypeptide (i.e.,
following amino acid
residue 415 of the FIX polypeptide). FIX polypeptides are provided that
comprise at least two, at
least three, at least four, or more glycosylation sites at the C-terminus of
the FIX polypeptide. In
some embodiments, the FIX polypeptide may comprise the addition of the amino
acid sequence of
SEQ ID NO: 4 at the C-terminus of the FIX polypeptide. In some embodiments,
the FIX
polypeptide may comprise the addition of the amino acid sequence of SEQ ID NO:
5 at the C-
terminus of the FIX polypeptide. In some embodiments, the FIX polypeptide may
comprise the
addition of the amino acid sequence of SEQ ID NO: 6 at the C-terminus of the
FIX polypeptide.
In some embodiments, the FIX polypeptide may comprise the addition of the
amino acid sequence
of SEQ ID NO: 7 at the C-terminus of the FIX polypeptide.
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[054] One aspect of the application provides modified FIX polypeptides
comprising at least one
or more glycosylation sites and one or more substitutions that increase the
activity of FIX.
Examples of activating substitutions include the R338A and the V86A
substitutions. In some
embodiments, modified FIX polypeptides may comprise the R338A substitution. In
some
embodiments, modified FIX polypeptides may comprise the V86A substitution. In
some
embodiments, modified FIX polypeptides may comprise both the R338A and the
V86A
substitution.
[055] A further aspect of the application provides FIX polypeptides with
increased specific
activity. In some embodiments, FIX polypeptides may comprise an R338A
substitution and a
V86A substitution. In some embodiments, the polypeptides may have a specific
activity of at least
about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1400, 1600,
1800, or 2000 units
per mg of polypeptide. The specific activity can be determined as previously
described, such as,
for example, using the APTT assay. These polypeptides are useful as
therapeutic agents,
particularly in patients afflicted with hemophilia B. These polypeptides may
comprise further
substitutions or modifications, such as the glycosylation sites described
herein.
[056] One aspect of the application provides modified Factor IX polypeptides
comprising the
following amino acid sequence:
YNSGKLEEF VQGNLERECMEEKC SFEEAREVFENTERTTEF WKQYVDGDQCE SNPC
LNGGSCKDDINSYEC W CPFGFEGKNCELX85X86TCNIKNGRCEQFCKNSAX 1 o4NKV V
C SCTEGYRLAENX121 KSCEPAVPFPCGRV SVX138QTSKLTRAEX148VX15oX151X152X153D
YVNSX159EZ1X161Z2EZ3TZ4ILDNIX169QSX172QX174FNX177X178TRVVGGEDAKPGQFPW
QV VLNGKVDAFCGGSIVNEKW IVTAAHCV ETX226 VX228ITV VAGEHNIEETEHTEQK
RNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGY
VSGWGRVFHKGRSALVLQYLRVPLVDRATCLX338STKFTIYNNMFCAGX353X354X355
GGRDSCQGDSGGPHX370X371X372VEGTSFLTGIISWGEECAX391X392X393KYGIYTKVS
RYVNWIKE KTX413X414T (SEQ ID NO: 3);
wherein X85 is selected from D and E;
wherein X86 is selected from A, E, P, S, and V;
wherein X104 is selected from D, N, and T;
wherein X121 is selected from N, Q, and T;
wherein X138 is selected from N, S, and T;
wherein X148 and X150 are selected from:
(i) X148 is T and X150 is F;
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WO 2009/137254 PCT/US2009/040813
(ii) X148 is N and X150 is T; and
(iii) X148 is N and X150 is S;
wherein X151 is selected from A, P, and T;
wherein X151 and X153 are selected from:
(i) X151 is P and X153 is V;
(ii) X151 is N and X153 is T; and
(iii) X151 is N and X153 is S;
wherein Z1, Z2, Z3, and Z4 are independently selected from
(i) zero to twelve amino acid residues and
(ii) SEQ ID NO: 2;
wherein X152 is selected from D, N, and T;
wherein X159 and X161 are selected from:
(i) X159 is T and X161 is A;
(ii) X159 is N and X161 is T; and
(iii) X159 is N and X161 is S;
wherein X169 is selected from T and N;
wherein X172 is selected from T and N;
wherein X174 is selected from S and T;
wherein X177 and X178 are selected from:
(i) X177 is D and X178 is F;
(ii) X177 is E and X178 is T; and
(iii) X177 is E or D and X178 is S;
wherein X226 and X228 are selected from:
(i) X226 is G and X228 is K;
(ii) X226 is N and X228 is T; and
(iii) X226 is N and X228 is S;
wherein X338 is selected from R and A;
wherein X353, X354, and x355 are selected from:
(i) X353 is F, X354 is H, X355 is E;
(ii) X353 is N, X354 is V, X355 is T;
(iii) X353 is N, X354 is 1, X355 is T; and
(iv) X353 is N, X354 is H, V, or 1, X355 is S;
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WO 2009/137254 PCT/US2009/040813
wherein X370, X371, and X372 are selected from:
(i) X370 is V, X371 is T, X372 is E;
(ii) X370 is N, X371 is V, X372 is T;
(iii) X370 is N, X371 is I, X372 is T; and
(iv) X370 is N, X371 is T, V, or I, X372 is S;
wherein X391, X392, and X393 are selected from:
(i) X391 is M, X392 is K, X393 is G;
(ii) X391 is N, X392 is K, X393 is T;
(iii) X391 is N, X392 is V, X393 is T; and
(iv) X391 is N, X392 is V or K, X393 is S;
wherein X413 and X414 are selected from:
(i) X413 is K and X414 is L;
(ii) X413 is N and X414 is L; and
(iii) X413 is N and X414 is I; and
wherein the FIX polypeptide comprises at least one introduced glycosylation
site as
compared to the FIX polypeptide having SEQ ID NO: 1.
[057] The introduction of at least one glycosylation site is the result of a
substitution at least one
of the X positions or an insertion in at least one of the Z positions. In some
embodiments, the
modified polypeptide additionally comprises between about 1-30, 1-20, or 1-10
conservative
amino acid changes and maintains FIX activity. In some embodiments, the
modified polypeptide
is at least about 80, 85, 90, 95, or 99% identical to SEQ ID NO: 1 and
maintains FIX activity.
Production of Modified FIX Polypeptides
[058] Amino acid residues may be inserted, deleted, or substituted in order to
introduce a non-
native glycosylation site. For example, glycosylation sites may be introduced
by altering the
amino acid sequence of FIX. Amino acid sequence alteration may be accomplished
by a variety of
techniques, such as, for example, by modifying the corresponding nucleic acid
sequence by site-
specific mutagenesis. Techniques for site-specific mutagenesis are well known
in the art and are
described in, for example, Zoller et al., (DNA 3:479-488, 1984) or Horton, et
al., (Gene 77:61-68,
1989, pp. 61-68). Thus, using the nucleotide and amino acid sequences of FIX,
one may introduce
the alteration(s) of choice. Likewise, procedures for preparing a DNA
construct using polymerase
chain reaction using specific primers are well known to persons skilled in the
art (see, e.g., PCR
Protocols, 1990, Academic Press, San Diego, California, USA).
[059] The nucleic acid construct encoding the FIX polypeptide may also be
prepared
synthetically by established standard methods, for example, the
phosphoramidite method described
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CA 02721683 2010-10-15
WO 2009/137254 PCT/US2009/040813
by Beaucage, et al., (Gene Amplif. Anal. 3:1-26, 1983). According to the
phosphoamidite method,
oligonucleotides are synthesized, for example, in an automatic DNA
synthesizer, purified,
annealed, ligated, and cloned in suitable vectors. The DNA sequences encoding
the human FIX
polypeptides may also be prepared by polymerase chain reaction using specific
primers, for
example, as described in US Patent No. 4,683,202; or Saiki, et al., (Science
239:487-491, 1988).
Furthermore, the nucleic acid construct may be of mixed synthetic and genomic,
mixed synthetic
and cDNA, or mixed genomic and cDNA origin prepared by ligating fragments of
synthetic,
genomic, or cDNA origin (as appropriate), corresponding to various parts of
the entire nucleic acid
construct, in accordance with standard techniques.
[060] The DNA sequences encoding the FIX polypeptides may be inserted into a
recombinant
vector using recombinant DNA procedures. The choice of vector will often
depend on the host
cell into which the vector is to be introduced. The vector may be an
autonomously replicating
vector or an integrating vector. An autonomously replicating vector exists as
an
extrachromosomal entity and its replication is independent of chromosomal
replication, for
example, a plasmid. An integrating vector is a vector that integrates into the
host cell genome and
replicates together with the chromosome(s) into which it has been integrated.
[061] The vector maybe an expression vector in which the DNA sequence encoding
the
modified FIX is operably linked to additional segments required for
transcription, translation, or
processing of the DNA, such as promoters, terminators, and polyadenylation
sites. In general, the
expression vector may be derived from plasmid or viral DNA, or may contain
elements of both.
The term "operably linked" indicates that the segments are arranged so that
they function in
concert for their intended purposes, for example, transcription initiates in a
promoter and proceeds
through the DNA sequence coding for the polypeptide.
[062] Expression vectors for use in expressing FIX polypeptides may comprise a
promoter
capable of directing the transcription of a cloned gene or cDNA. The promoter
may be any DNA
sequence that shows transcriptional activity in the host cell of choice and
may be derived from
genes encoding proteins either homologous or heterologous to the host cell.
[063] Examples of suitable promoters for directing the transcription of the
DNA encoding the
FIX polypeptides in mammalian cells are, for example, the SV40 promoter
(Subramani, et al.,
Mol. Cell Biol. 1:854-864, 1981), the MT-I (metallothionein gene) promoter
(Palmiter, et al.,
Science 222:809-814, 1983), the CMV promoter (Boshart, et al., Cell 41:521-
530, 1985), or the
adenovirus 2 major late promoter (Kaufnan et al.,, Mol. Cell Biol, 2:1304-
1319, 1982).
[064] The DNA sequences encoding the FIX polypeptide may also, if necessary,
be operably
connected to a suitable terminator, such as the human growth hormone
terminator (Palmiter, et al.,
Science 222:809-814, 1983) or TPII (Alber et al., J. Mol. Appl. Gen. 1:419-
434, 1982) or ADH3
CA 02721683 2010-10-15
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(McKnight, et al., EMBO J. 4:2093-2099, 1985) terminators. The expression
vectors may also
contain a polyadenylation signal located downstream of the insertion site.
Polyadenylation signals
include the early or late polyadenylation signal from SV40, the
polyadenylation signal from the
adenovirus 5 Elb region, the human growth hormone gene terminator (DeNoto, et
al., Nucl. Acids
Res. 9:3719-3730, 1981), or the polyadenylation signal from the human FIX
gene. The expression
vectors may also include enhancer sequences, such as the SV40 enhancer.
[065] To direct the FIX polypeptides of the present invention into the
secretory pathway of the
host cells, the native FIX secretory signal sequence may be used.
Alternatively, a secretory signal
sequence (also known as a leader sequence, prepro sequence, or pre sequence)
may be provided in
the recombinant vector. The secretory signal sequence may be joined to the DNA
sequences
encoding the FIX analogues in the correct reading frame. Secretory signal
sequences are
commonly positioned 5' to the DNA sequence encoding the peptide. Exemplary
signal sequences
include, for example, the MPIF-1 signal sequence and the stanniocalcin signal
sequence.
[066] The procedures used to ligate the DNA sequences coding for the FIX
polypeptides, the
promoter, and optionally the terminator and/or secretory signal sequence and
to insert them into
suitable vectors containing the information necessary for replication, are
well known to persons
skilled in the art (see, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold
Spring Harbor, New York, 1989).
[067] Methods of transfecting mammalian cells and expressing DNA sequences
introduced into
the cells are described in, for example, Kaufinan, et al., (J. Mol. Biol.
159:601-621, 1982);
Southern, et al., (J. Mol. Appl. Genet. 1:327-341, 1982); Loyter, et al.,
(Proc. Natl. Acad. Sci. USA
79:422-426, 1982); Wigler, et al., (Cell 14:725-731, 1978); Corsaro, et al.,
(Somatic Cell Genetics
7:603-616, 1981), Graham, et al., (Virology 52:456-467, 1973); and Neumann, et
al., (EMBO J.
1:841-845, 1982). Cloned DNA sequences may be introduced into cultured
mammalian cells by,
for example, lipofection, DEAE-dextran-mediated transfection, microinj ection,
protoplast fusion,
calcium phosphate precipitation, retroviral delivery, electroporation,
sonoporation, laser
irradiation, magnetofection, natural transformation, and biolistic
transformation (see, e.g., Mehier-
Humbert, et al., Adv. Drug Deliv. Rev. 57:733-753, 2005). To identify and
select cells that
express the exogenous DNA, a gene that confers a selectable phenotype (a
selectable marker) is
generally introduced into cells along with the gene or cDNA of interest.
Selectable markers
include, for example, genes that confer resistance to drugs such as neomycin,
puromycin,
hygromycin, and methotrexate. The selectable marker may be an amplifiable
selectable marker,
which permits the amplification of the marker and the exogenous DNA when the
sequences are
linked. Exemplary amplifiable selectable markers include dihydrofolate
reductase (DHFR) and
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WO 2009/137254 PCT/US2009/040813
adenosine deaminase. It is within the purview of one skilled in the art to
choose suitable selectable
markers (see, e.g., US Patent No. 5,238,820).
[068] After cells have been transfected with DNA, they are grown in an
appropriate growth
medium to express the gene of interest. As used herein the term "appropriate
growth medium"
means a medium containing nutrients and other components required for the
growth of cells and
the expression of the active FIX polypeptides.
[069] Media generally include, for example, a carbon source, a nitrogen
source, essential amino
acids, essential sugars, vitamins, salts, phospholipids, protein, and growth
factors, and in the case
of vitamin K dependent proteins such as FIX, vitamin K may also be provided.
Drug selection is
then applied to select for the growth of cells that are expressing the
selectable marker in a stable
fashion. For cells that have been transfected with an amplifiable selectable
marker the drug
concentration may be increased to select for an increased copy number of the
cloned sequences,
thereby increasing expression levels. Clones of stably transfected cells are
then screened for
expression of the FIX polypeptide.
[070] Examples of mammalian cell lines for use in the present invention are
the COS-1 (ATCC
CRL 1650), baby hamster kidney (BHK), HKB11 cells (Cho, et al., J. Biomed.
Sci, 9:631-638,
2002), and HEK-293 (ATCC CRL 1573; Graham, et al., J. Gen. Virol. 36:59-72,
1977) cell lines.
In addition, a number of other cell lines may be used within the present
invention, including rat
Hep I (rat hepatoma; ATCC CRL 1600), rat Hep II (rat hepatoma; ATCC CRL 1548),
TCMK-1
(ATCC CCL 139), Hep-G2 (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO-Kl (ATCC
CCL 61), and CHO-DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA
77:4216-4220,
1980).
[071] FIX polypeptides may be recovered from cell culture medium and may then
be purified by
a variety of procedures known in the art including, but not limited to,
chromatography (e.g., ion
exchange, affinity, hydrophobic, chromatofocusing, and size exclusion),
electrophoretic
procedures (e.g., preparative isoelectric focusing (IEF), differential
solubility (e.g., ammonium
sulfate precipitation)), extraction (see, e.g., Protein Purification, Janson
and Lars Ryden, editors,
VCH Publishers, New York, 1989), or various combinations thereof. In an
exemplary
embodiment, the polypeptides may be purified by affinity chromatography on an
anti-FIX
antibody column. Additional purification may be achieved by conventional
chemical purification
means, such as high performance liquid chromatography. Other methods of
purification are
known in the art, and maybe applied to the purification of the modified FIX
polypeptides (see,
e.g., Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982).
[072] Generally, "purified" shall refer to a protein or peptide composition
that has been
subjected to fractionation to remove various other components, and which
substantially retains its
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WO 2009/137254 PCT/US2009/040813
expressed biological activity. Where the term "substantially purified" is
used, this designation
shall refer to a composition in which the protein or peptide forms the major
component of the
composition, such as constituting about 50%, about 60%, about 70%, about 80%,
about 90%,
about 95%, about 99%, or more of the proteins in the composition.
[073] Various methods for quantifying the degree of purification of the
polypeptide are known
to those of skill in the art. These include, for example, determining the
specific activity of an
active fraction, or assessing the amount of polypeptides within a fraction by
SDS/PAGE analysis.
An exemplary method for assessing the purity of a fraction is to calculate the
specific activity of
the fraction, compare the activity to the specific activity of the initial
extract, and to thus calculate
the degree of purity, herein assessed by a "-fold purification number." The
actual units used to
represent the amount of activity will, of course, be dependent upon the
particular assay technique.
[074] In some embodiments, FIX polypeptides are recombinantly expressed in
tissue culture
cells and glycosylation is the result of the normal post-translational cell
functioning of the host
cell, such as a mammalian cell.
[075] Alternatively, glycosylation may be achieved through chemical or
enzymatic modification.
FIX polypeptides may be glycosylated by using, for example, an enzyme that
adds alpha-(2,6)-
linked sialic acid to protein. For example, dihydrofolate reductase (DHFR)
deficient CHO cells
are commonly used host cells for recombinant glycoprotein production. CHO
cells do not
endogenously express the enzyme beta-galactoside alpha-2,6 sialyltransferase,
which is used to
add sialic acid in the 2,6 linkage to galactose on the mannose alpha-1,3
branch. To add sialic acid
at this linkage to a protein produced in CHO cells, the CHO cells may be
transfected with a
functional beta-galactosidase alpha-2,6 sialyltransferase gene to allow for
incorporation of sialic
acid in the 2,6 linkage to galactose as desired (see, e.g., Lee, et al., J.
Biol. Chem. 264:13848-
13855,1989).
[076] Similarly, a bisecting N-acetylglucosamine (GIcNAc) may be added to FIX
by transfecting
a host cell that does not endogenously produce this oligosaccharide linkage
with the functional
gene for the enzyme N-acetylglucosaminyltransferase, which has been reported
to catalyze
formation of a bisecting GIcNAc structure. Systems have also been established
to express proteins
in plant cells that produce proteins with mammalian glycosylation patterns
(see, e.g., bryophyte
cells, WO 2004/057002).
[077] For N-linked glycosylation, the final structures of the N-glycans are
typically dependent
upon the organism in which the polypeptide is produced. Generally,
polypeptides produced in
bacteria are completely unglycosylated. Polypeptides expressed in insect cells
contain high
mannose and pauci-mannose N-linked oligosaccharide chains, among others.
Polypeptides
produced in mammalian cell culture are usually glycosylated differently
depending upon, for
28
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WO 2009/137254 PCT/US2009/040813
example, the species and cell culture conditions. Further, polypeptides
produced in plant cells
comprise glycan structures that differ significantly from those produced in
animal cells. The goal
in the art of the production of recombinant polypeptides, particularly when
the polypeptides are to
be used as therapeutic agents, is to be able to generate polypeptides that are
correctly glycosylated,
that is, to be able to generate a polypeptide having a glycan structure that
resembles, or is identical
to that present on the naturally occurring form of the polypeptide.
[078] A variety of methods have been proposed in the art to customize the
glycosylation pattern
of a polypeptide (see, e.g., WO 99/22764; WO 98/58964; WO 99/54342; US
Publication No.
2008/0050772; and US Patent No. 5,047,335). Essentially, many of the enzymes
required for the
in vitro glycosylation of polypeptides have been cloned and sequenced. In some
instances, these
enzymes have been used in vitro to add specific sugars to an incomplete glycan
molecule on a
polypeptide. In other instances, cells have been genetically engineered to
express a combination of
enzymes and desired polypeptides such that addition of a desired sugar moiety
to an expressed
polypeptide occurs within the cell.
[079] For O-linked glycosylation, 0-glycans are linked primarily to serine and
threonine
residues and are formed by the stepwise addition of sugars from nucleotide
sugars (Tanner, et al.,
Biochim. Biophys. Acta. 906:81-99, 1987); Hounsell, et al., Glycoconj. J.
13:19-26, 1996).
Polypeptide function can be affected by the structure of the O-linked glycans
present. For a
review of O-linked glycan structures, see, for example, Schachter and
Brockhausen, The
Biosynthesis of Branched O-Linked Glycans, 1989, Society for Experimental
Biology, pp. 1-26
(Great Britain).
[080] Whether a FIX polypeptide has N-linked and/or O-linked glycosylation may
be
determined using standard techniques (see, e.g., Techniques in Glycobiology,
R. Townsend and A.
Hotchkiss, eds. (1997) Marcel Dekker; and Glycoanalysis Protocols (Methods in
Molecular
Biology, Vol. 76), E. Hounsell, ed. (1998) Humana Press). The change in
electrophoretic mobility
of a protein before and after treatment with chemical or enzymatic
deglycosylation (e.g., using
endoglycosidases and/or exoglycosidases) is routinely used to determine the
glycosylation status
of a protein. Enzymatic deglycosylation may be carried out using any of a
variety of enzymes,
including, but not limited to, peptide-N4-(N-acetyl-beta-D-glycosaminyl)
asparagine amidase
(PNGase F), endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and
the like. For
example, sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
analysis of the
protein, either pre-treated with PNGaseF or untreated with PNGaseF, may be
conducted. A
marked decrease in band width and change in migration position after treatment
with PNGaseF
may be considered diagnostic of N-linked glycosylation. The carbohydrate
content of a
glycosylated protein also can be detected using lectin analysis of protein
blots (e.g., proteins
29
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WO 2009/137254 PCT/US2009/040813
separated by SDS-PAGE and transferred to a support, such as a nylon membrane).
Lectins,
carbohydrate binding proteins from various plant tissues, have both high
affinity and narrow
specificity for a wide range of defined sugar epitopes found on glycoprotein
glycans (Cummings,
Methods Enzymol. 230:66-86, 1994). Lectins maybe labeled (either directly or
indirectly)
allowing detection of binding of lectins to carbohydrates on glycosylated
proteins. For example,
when conjugated with biotin or digoxigenin, a lectin bound to a glycosylated
protein can be easily
identified on membrane blots through a reaction utilizing avidin or anti-
digoxigenin antibodies
conjugated with an enzyme such as alkaline phosphatase, beta-galactosidase,
luciferase, or horse
radish peroxidase, to yield a detectable product. Screening with a panel of
lectins with well-
defined specificity provides considerable information about a glycoprotein's
carbohydrate
complement. The electrophoretic mobility of the modified FIX polypeptide may
also be compared
to the mobility of a reference protein.
[081] The application provides, in part, FIX polypeptides with introduced
glycosylation sites,
wherein the carbohydrate chain attached to the glycosylation site may have a
mammalian
carbohydrate chain structure, that is, a mammalian glycosylation pattern. In
some embodiments,
the carbohydrate chain has a human glycosylation pattern. As used herein, a
pattern of
glycosylation refers to the representation of particular oligosaccharide
structures within a given
population of FIX polypeptides. Non-limiting examples of such patterns include
the relative
proportion of oligosaccharide chains that (i) have at least one sialic acid
residue; (ii) lack any sialic
acid residues (i.e., are neutral in charge); (iii) have at least one terminal
galactose residue; (iv)
have at least one terminal N-acetylgalactosamine residue; (v) have at least
one "uncapped"
antenna, that is, have at least one terminal galactose or N-
acetylgalactosamine residue; or (vi) have
at least one fucose linked alphal ->3 to an antennary N-acetylglucosamine
residue.
[082] The pattern of glycosylation may be determined using any method known in
the art,
including, without limitation: high-performance liquid chromatography (HPLC);
capillary
electrophoresis (CE); nuclear magnetic resonance (NMR); mass spectrometry (MS)
using
ionization techniques such as fast-atom bombardment, electrospray, or matrix-
assisted laser
desorption (MALDI); gas chromatography (GC); and treatment with
exoglycosidases in
conjunction with anion-exchange (AIE)-HPLC, size-exclusion chromatography
(SEC), or MS (see,
e.g., Weber, et al., Anal. Biochem. 225:135-142, 1995; Klausen, et al., J.
Chromatog. 718:195-202,
1995; Morris, et al., in Mass Spectrometry of Biological Materials, McEwen et
al., eds., Marcel
Dekker, (1990), pp 137-167; Conboy et al., Biol. Mass Spectrom. 21:397-407,
1992; Hellerqvist,
Meth. Enzymol. 193:554-573, 1990; Sutton, et al., Anal. Biochem. 218:34-46,
1994; Harvey, et
al., Organic Mass Spectrometry 29:753-766, 1994).
CA 02721683 2010-10-15
WO 2009/137254 PCT/US2009/040813
Pharmaceutical Compositions
[083] Based on well known assays used to determine the efficacy for treatment
of conditions
identified above in mammals, and by comparison of these results with the
results of known
medicaments that are used to treat these conditions, the effective dosage of
the polypeptides of this
invention may readily be determined for treatment of each desired indication.
The amount of the
active ingredient to be administered in the treatment of one of these
conditions can vary widely
according to such considerations as the particular polypeptide and dosage unit
employed, the mode
of administration, the period of treatment, the age and sex of the patient
treated, and the nature and
extent of the condition treated.
[084] The application provides, in part, compositions comprising FIX
polypeptides with one or
more introduced glycosylation sites as described herein. The compositions may
be suitable for in
vivo administration and are pyrogen free. The compositions may also comprise a
pharmaceutically acceptable carrier. The phrase "pharmaceutically or
pharmacologically
acceptable" refers to molecular entities and compositions that do not produce
adverse, allergic, or
other untoward reactions when administered to an animal or a human. As used
herein,
"pharmaceutically acceptable carrier" includes any and all solvents,
dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like. The use
of such media and agents for pharmaceutically active substances is well known
in the art.
Supplementary active ingredients also may be incorporated into the
compositions.
[085] The compositions of the present invention include classic pharmaceutical
preparations.
Administration of these compositions according to the present invention may be
via any common
route. The pharmaceutical compositions may be introduced into the subject by
any conventional
method, for example, by intravenous, intradermal, intramuscular, subcutaneous,
intramammary,
intraperitoneal, intrathecal, retrobulbar, intrapulmonary, oral, sublingual,
nasal, anal, vaginal, or
transdermal delivery, or by surgical implantation at a particular site. The
treatment may consist of
a single dose or a plurality of doses over a period of time.
[086] The active compounds may be prepared for administration as solutions of
free base or
pharmacologically acceptable salts in water, suitably mixed with a surfactant,
such as
hydroxypropylcellulose. Dispersions also may be prepared in glycerol, liquid
polyethylene
glycols, and mixtures thereof, and in oils. Under ordinary conditions of
storage and use, these
preparations contain a preservative to prevent the growth of microorganisms.
[087] The pharmaceutical forms, suitable for injectable use, include sterile
aqueous solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile
injectable solutions
or dispersions. The form should be sterile and should be fluid to the extent
that easy syringability
exists. It should be stable under the conditions of manufacture and storage
and should be
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WO 2009/137254 PCT/US2009/040813
preserved against the contaminating action of microorganisms, such as bacteria
and fungi. The
carrier maybe a solvent or dispersion medium containing, for example, water,
ethanol, polyol
(e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the
like) sucrose, L-histidine,
polysorbate 80, or suitable mixtures thereof, and vegetable oils. The proper
fluidity may be
maintained, for example, by the use of a coating, such as lecithin, by the
maintenance of the
required particle size in the case of dispersion, and by the use of
surfactants. The prevention of the
action of microorganisms may be brought about by various antibacterial an
antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. The injectable
compositions may include isotonic agents, for example, sugars or sodium
chloride. Prolonged
absorption of the injectable compositions may be brought about by the use in
the compositions of
agents delaying absorption, for example, aluminum monostearate and gelatin.
[088] Sterile injectable solutions may be prepared by incorporating the active
compounds (e.g.,
FIX polypeptides) in the required amount in the appropriate solvent with
various of the other
ingredients enumerated above, as required, followed by filtered sterilization.
[089] Generally, dispersions may be prepared by incorporating the various
sterilized active
ingredients into a sterile vehicle that contains the basic dispersion medium
and the required other
ingredients from those enumerated above. In the case of sterile powders for
the preparation of
sterile injectable solutions, methods of preparation include, for example,
vacuum-drying and
freeze-drying techniques that yield a powder of the active ingredient plus any
additional desired
ingredient from a previously sterile-filtered solution thereof.
[090] Upon formulation, solutions may be administered in a manner compatible
with the dosage
formulation and in such amount as is therapeutically effective.
"Therapeutically effective amount"
is used herein to refer to the amount of a polypeptide that is needed to
provide a desired level of
the polypeptide in the bloodstream or in the target tissue. The precise amount
will depend upon
numerous factors, for example, the particular FIX polypeptide, the components
and physical
characteristics of the therapeutic composition, intended patient population,
mode of delivery,
individual patient considerations, and the like, and can readily be determined
by one skilled in the
art, based upon the information provided herein.
[091] The formulations may be easily administered in a variety of dosage
forms, such as
injectable solutions, and the like. For parenteral administration in an
aqueous solution, for
example, the solution should be suitably buffered, if necessary, and the
liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular aqueous solutions
are especially
suitable for intravenous, intramuscular, subcutaneous and intraperitoneal
administration.
[092] Dosages of FIX are normally expressed in units. One unit of FIX per kg
of body weight
may raise plasma levels by 0.01 U/ml, that is, 1%. Otherwise healthy patients
have one unit of
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WO 2009/137254 PCT/US2009/040813
FIX per ml of plasma, that is, 100%. Mild cases of hemophilia B are defined by
FIX plasma
concentrations between 6-60%, moderate cases between 1-5%, and severe cases,
which account
for about half of the hemophilia B cases, have less than 1% FIX. Prophylactic
treatment or
treatment of minor hemorrhaging usually requires raising FIX levels to between
15-30%.
Treatment of moderate hemorrhaging usually requires raising levels to between
30-50%, while
treatment of major trauma may require raising levels from 50 to 100%. The
total number of units
needed to raise a patient's blood level can be determined as follows: 1.0
unit/kg x body weight (kg)
x desired percentage increase (% of normal). Parenteral administration may be
carried out with an
initial bolus followed by continuous infusion to maintain therapeutic
circulating levels of drug
product. In some embodiments, between 15 to 150 units/kg of FIX polypeptide
may be
administered. Those of ordinary skill in the art will readily optimize
effective dosages and
administration regimens as determined by good medical practice and the
clinical condition of the
individual patient.
[093] The frequency of dosing will depend on the pharmacokinetic parameters of
the agents and
the routes of administration. The optimal pharmaceutical formulation may be
determined by one
of skill in the art depending on the route of administration and the desired
dosage (see, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20t''
edition, 2000,
incorporated herein by reference). Such formulations may influence the
physical state, stability,
rate of in vivo release, and rate of in vivo clearance of the administered
agents. Depending on the
route of administration, a suitable dose may be calculated according to body
weight, body surface
area, or organ size. Further refinement of the calculations necessary to
determine the appropriate
treatment dose is routinely made by those of ordinary skill in the art without
undue
experimentation, especially in light of the dosage information and assays
disclosed herein, as well
as the pharmacokinetic data observed in animals or human clinical trials.
Exemplary dosing
schedules include, without limitation, administration five times a day, four
times a day, three times
a day, twice daily, once daily, three times weekly, twice weekly, once weekly,
twice monthly, once
monthly, and any combination thereof.
[094] Appropriate dosages may be ascertained through the use of established
assays for
determining blood clotting levels in conjunction with relevant dose response
data. The final
dosage regimen may be determined by the attending physician, considering
factors that modify the
action of drugs, for example, the drug's specific activity, severity of the
damage, and the
responsiveness of the patient, the age, condition, body weight, sex and diet
of the patient, the
severity of any infection, time of administration, and other clinical factors.
[095] The composition may also include an antimicrobial agent for preventing
or deterring
microbial growth. Non-limiting examples of antimicrobial agents suitable for
the present
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WO 2009/137254 PCT/US2009/040813
invention include benzalkonium chloride, benzethonium chloride, benzyl
alcohol, cetylpyridinium
chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,
thimersol, and
combinations thereof.
[096] An antioxidant may be present in the composition as well. Antioxidants
may be used to
prevent oxidation, thereby preventing the deterioration of the preparation.
Suitable antioxidants
for use in the present invention include, for example, ascorbyl palmitate,
butylated hydroxyanisole,
butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl
gallate, sodium
bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and
combinations thereof.
[097] A surfactant may be present as an excipient. Exemplary surfactants
include: polysorbates
such as Tween -20 (polyoxyethylenesorbitan monolaurate) and Tween -80
(polyoxyethylenesorbitan monooleate) and pluronics such as F68 and F88 (both
of which are
available from BASF, Mount Olive, N.J.); sorbitan esters; lipids such as
phospholipids such as
lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty
acids and fatty esters;
steroids such as cholesterol; and chelating agents such as EDTA, zinc and
other such suitable
cations.
[098] Acids or bases may be present as an excipient in the composition. Non-
limiting examples
of acids that may be used include hydrochloric acid, acetic acid, phosphoric
acid, citric acid, malic
acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric
acid, phosphoric acid,
sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable
bases include, without
limitation, sodium hydroxide, sodium acetate, ammonium hydroxide, potassium
hydroxide,
ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate,
sodium citrate,
sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and
combinations thereof.
[099] The amount of any individual excipient in the composition may vary
depending on the
activity of the excipient and particular needs of the composition. Typically,
the optimal amount of
any individual excipient may be determined through routine experimentation,
that is, by preparing
compositions containing varying amounts of the excipient (ranging from low to
high), examining
the stability and other parameters, and then determining the range at which
optimal performance is
attained with no significant adverse effects. Generally, the excipient may be
present in the
composition in an amount of about I% to about 99% by weight, from about 5% to
about 98% by
weight, from about 15 to about 95% by weight of the excipient, with
concentrations less than 30%
by weight. These foregoing pharmaceutical excipients along with other
excipients are described in
"Remington: The Science & Practice of Pharmacy," 19 ed., Williams & Williams,
(1995); the
"Physician's Desk Reference," 52 ed., Medical Economics, Montvale, N.J.
(1998); and Kibbe, A.
H., Handbook of Pharmaceutical Excipients, 3 Edition, American Pharmaceutical
Association,
Washington, D.C., 2000.
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Exemplary Uses
[100] The compositions described herein may be used to treat any bleeding
disorder associated
with functional defects of FIX or deficiencies of FIX such as a shortened in
vivo half-life of FIX,
altered binding properties of FIX, genetic defects of FIX, and a reduced
plasma concentration of
FIX. Genetic defects of FIX comprise, for example, deletions, additions,
and/or substitution of
bases in the nucleotide sequence encoding FIX. In one embodiment, the bleeding
disorder may be
hemophilia B. Symptoms of such bleeding disorders include, for example, severe
epistaxis, oral
mucosal bleeding, hemarthrosis, hematoma, persistent hematuria,
gastrointestinal bleeding,
retroperitoneal bleeding, tongue/retropharyngeal bleeding, intracranial
bleeding, and trauma-
associated bleeding.
[101] The compositions of the present invention may be used for prophylactic
applications. In
some embodiments, modified FIX polypeptides may be administered to a subject
susceptible to or
otherwise at risk of a disease state or injury to enhance the subject's own
coagulative capability.
Such an amount may be defined to be a "prophylactically effective dose."
Administration of the
modified FIX polypeptides for prophylaxis includes situations where a patient
suffering from
hemophilia B is about to undergo surgery and the polypeptide is administered
between one to four
hours prior to surgery. In addition, the polypeptides are suited for use as a
prophylactic against
uncontrolled bleeding, optionally in patients not suffering from hemophilia.
Thus, for example,
the polypeptide may be administered to a patient at risk for uncontrolled
bleeding prior to surgery.
[102] The polypeptides, materials, compositions, and methods described herein
are intended to
be representative examples of the invention, and it will be understood that
the scope of the
invention is not limited by the scope of the examples. Those skilled in the
art will recognize that
the invention may be practiced with variations on the disclosed polypeptides,
materials,
compositions and methods, and such variations are regarded as within the ambit
of the invention.
[103] The following examples are presented to illustrate the invention
described herein, but
should not be construed as limiting the scope of the invention in any way.
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EXAMPLES
[104] In order that this invention may be better understood, the following
examples are set forth.
These examples are for the purpose of illustration only, and are not to be
construed as limiting the
scope of the invention in any manner. All publications mentioned herein are
incorporated by
reference in their entirety.
Example 1: In Silico Analysis
[105] An in silico analysis of the FIX sequence for solvent accessibility,
known secondary
structure, location of known hemophilia B mutations, proximity to predicted
sites of interaction
with both FVIII and FX, and predicted effect of a given mutation upon FIX
protein stability was
performed in order to identify potential sites for the addition of new N-
glycosylation site
consensus sequences.
[106] Based upon this analysis, five sites were identified within the
catalytic domain for creation
of new N-linked glycosylation sites. The selected sites and the altered amino
acid sequences that
were designed are shown in Table 2.
TABLE 2
Mutein name Native Sequence Mutated sequence
G226N, K228T G1y226-Va1227-Lys228 Asn226-Va1227-Thr228
F353N, H354V, E355T Phe353-His354-G1u355 Asn353-Va1354-Thr355
F353N, H3541, E355T Asn353-Ile354-Thr355
V3 70N, T371V, E372T Va1370-Thr371-0072 Asn370-Va1371-Thr372
V370N, T3711, E372T Asn370-Ile371-Thr372
M391N, G393T Met391-Lys392-G1y393 Asn391- Lys392- Thr393
M391N, K392V, G393T Asn391- Va1392- Thr393
K413N Lys413-Leu414-Thr415 Asn413-Leu414-Thr415
K413N, L4141 Asn413-Ile414-Thr415
Example 2: Sequence Alignment of Activation Peptide
[107] Conserved and non-conserved residues in the activation peptide were
identified by a
multiple species sequence alignment as shown in Figure 1.
[108] The consensus sequence of N-linked glycosylation sites is Asn-X-Ser/Thr
(where X is any
amino acid except proline or perhaps aspartic acid which is also not
favorable). To design new N-
linked glycosylation sites in the activation peptide of FIX, the amino acids
within the activation
peptide that are conserved between species was avoided as well as and
consensus sequences for
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WO 2009/137254 PCT/US2009/040813
other N-linked sites and the consensus sequence for tyrosine sulfation so as
not to disrupt these
posttranslational modifications since these could be important for the
pharmacokinetics of FIX.
Using these criteria, three sites for adding new N-linked glycosylation sites
within the activation
peptide were identified as shown in Table 3.
TABLE 3
Designation Mutein Native sequence Glycosylation site created
HG1 A161N A161-E162-T163 N161-E162-T163
HG2 D177E plus F178T D177-F178-T179 N176-E177-T178
HG3 P151N plus V153T P151-D152-V153 N151-D152-T153
Example 3: Insertion ofAmino Acids to Generate N-linked Glycosylation Sites
[109] The multiple sequence alignment (Figure 1) also revealed that mouse,
rat, and guinea pig
have additional amino acids between residues A 161 and E162 relative to human,
rhesus monkey,
pig, dog, and rabbit FIX. These additional amino acids vary in size from 7 to
10 between the three
species and contain an over representation of Asp and to some extent Ile
residues. This
observation demonstrates that between 7 and 10 additional residues are
tolerated at this site in rat,
mouse, and guinea pig without significant effects on FIX activity. Depending
upon the criteria
used to perform the multiple sequence alignment between FIX from the eight
species, the apparent
site at which the additional amino acids in rat, mouse, and guinea pig are
found can vary such that
the site can be either between E160 and A161, between A161 and E162, between
E162 and T163,
or between T163 and 1164 of human FIX.
[110] Nine extra amino acids with the sequence N-S-T-Q-D-N-I-T-Q (SEQ ID NO:
2) were
inserted in the activation peptide between A161 and E162. The sequences " N-S-
T" and "N-1-T"
are the N-linked consensus sequences from the natural FIX activation peptide.
In both cases, these
are followed by a glutamine (Q) and preceded by an aspartic acid (D) in the
natural FIX sequence.
Therefore, a glutamine as well as an aspartic acid were included in an attempt
to emulate the
natural site. It is envisioned that additional sequences containing between 1
and 3 consensus
sequences for N-glycosylation site (N-X-S/T) may also be inserted between A161
and E162.
[111] Additional examples of modified FIX polypeptides are described in Tables
4a, 4b, and 4c.
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TABLE 4a
Construct Substitution Description Specific Activity
(% of Control)
HG10 D85N Creates 1 new N-linked site in EGF1 99
HG11 K122T Creates 1 new N-linked site in EGF2 137
HG12 S138N Creates 1 new N-linked site in EGF2- 75
AP linker region
HG13 K228N Creates 1 new N-linked site in 67
catalytic domain
HG14 E242N Creates 1 new N-linked site in 183
catalytic domain
HG15 K201T Creates 1 new N-linked site in 21
catalytic domain
HG16 E410N Creates 1 new N-linked consensus in 1040
catalytic domain
HG17 K413N Creates 1 new N-linked consensus in 388
catalytic domain
HG1 8 E83T Creates 1 new N-linked consensus in NT
EGF 1 domain
HG19 K214T Creates 1 new N-linked consensus in NT
catalytic domain
HG20 V223N Creates 1 new N-linked consensus in NT
catalytic domain
HG21 Y266T Creates 1 new N-linked consensus in NT
catalytic domain
HG22 F353N-H354V- Creates 1 new N-linked consensus in NT
E355T catalytic domain
HG23 V370N-T371V- Creates 1 new N-linked consensus in NT
E372T catalytic domain
HG24 M391N-K392V- Creates 1 new N-linked consensus in NT
G393T catalytic domain
HG25 415 - T-N-S-T-T Adds 1 new N-linked consensus 57
(SEQ ID NO: 4) sequence to C terminus
HG26 415 - T-N-S-T- Adds 2 new N-linked consensus 42
Q-N-I-T-T sequences to C terminus
(SEQ ID NO: 5)
HG27 415 - T-N-S-T- Adds 3 new N-linked consensus NT
Q-N-I-T-G-N-D- sequences to C terminus
T-E-K-T
(SEQ ID NO: 6)
HG28 415 - T-N-S-T- Adds 4 new N-linked consensus 45
Q-N-I-T-G-N-D- sequences to C terminus
T-E-N-G-T-K-T
(SEQ ID NO: 7)
Specific activity was calculated by dividing the activity measured in the cell
culture
supernatant by the FIX antigen concentration in the same supernatant and is
expressed as a
percentage of the control FIX molecule lacking the new glycosylation sites.
NT: not
tested.
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TABLE 4b
Construct Residue Substitution Specific Activity Specific Activity
(% of WT) (% of Control)
HG3/9 P151N, V153T, G226N, 270 40
K228T
HG5/9 T172N, G226N, K228T 300 47
HG8/9 Insertion of SEQ ID NO: 350 55
2 between A161 and
E162, G226N, K228T
HG3/9/12 5138N, P151N, VI 53T, 200 26
G226N, K228T
HG3/5/9 P151N, V153T, T172N, 190 38
G226N, K228T
HG3/9/10 D85N, P151N, V153T, 200 26
G226N, K228T
HG3/10/13 D85N, P15 IN, V153T, 200 20
K228N
HG3/11/13 K122T, P151N, V153T, 400 40
K228N
HG3/10/14 D85N, P151N, V153T, 210 21
E242N
HG3/11/14 K122T, P151N, V153T, 800 80
E242N
HG10/11/14 D85N, K122T, E242N 650 65
HG3/11/1251T K122T, P151N, V153T, 540 47
1251 T
HG10/11/1251T D85N, K122T, 1251T 570 50
HG3/10/11/14 D85N, K122T, P151N, 400 40
V153T, E242N
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TABLE 4c
Expression Activity
Construct Substitution (relative to control)* (relative to control)*
Cl YIN, S3T 4 1
C2 G4T 5 1
C3 S3N, K5T 4 1
C4 G4N, L6T 4 1
C5 K5N, E7T 4 1
C6 L6N, E8T 4 1
C7 E7N,F9T 4 1
C8 E8N, V1OT 2 1
C9 F9N, Q1 IT 3 1
C10 V1ON,G12T 3 1
C11 Q1 IN, N13T 3 1
C12 G12N,L14T 4 1
C13 N13,E15T 4 2
C14 L14N, R16T 4 1
C15 E15N, E17T 5 1
C16 M19N, E21T 5 1
C17 E20N,K22T 4 1
C18 S24N,E26T 4 1
C19 F25N,E27T 4 1
C20 E26N, A28T 4 1
C21 E27N,R29T 3 1
C22 A28N,E30T 3 1
C23 R29N, V3 IT 3 1
C24 E30N,F32T 5 1
C25 V31N,E33T 4 1
C26 F32N,N34T 4 1
C27 E33N 4 1
C28 E36T 4 1
C29 T35N,R37T 4 1
C30 E36N 4 1
C31 R37N 4 2
C32 T38N,E40T 4 1
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C33 T39N,F41T 4 1
C34 E40N, W42T 4 1
C35 F41N, K43T 3 1
C36 W42N, Q44T 4 1
C37 K43N,Y45T 3 1
C38 Q44N,V46T 4 2
C39 Y45N,D47T 3 1
C40 V46N, G48T 2 1
C41 D47N,D49T 1 1
C42 G48N,Q50T 2 1
C43 E52N,N54T 1 2
C44 S53N, P55T 1 2
C45 L57N, G59T 1 1
C46 G60T 1 1
C47 G59N,S61T 4 4
C48 K63N, D65T 3 2
C49 D64N,I66T 2 1
C50 D65N, N67T 3 1
C51 166N, S68T 2 1
C52 Y69T 1 1
C53 S68N,E70T 4 1
C54 P74N, G76T 1 1
C55 F75N,F77T 1 2
C56 G76N,E78T 2 4
C57 F77N, G79T 1 1
C58 E78N,K80T 2 1
C59 G79N, N81T 1 1
C60 E83N, D85T 2 2
C61 L84N, V86T 2 1
C62 D85N 3 4
C63 K91T 2 2
C64 190N, N92T 1 1
C65 K91N, G93T 1 1
C66 R94T 1 1
C67 K100N,S102T 2 3
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C68 A103T 2 2
C69 S102N, D104T 3 4
C70 A103N, N105T 2 4
C71 D104N, K106T 3 4
C72 V107T 2 3
C73 K106N, V108T 3 3
C74 RI 16N, Al 18T 1 3
C75 L117N,E119T 1 1
C76 Al 18N, N120T 1 2
C77 El 19N, Q121T 2 3
C78 K122T 1 4
C79 Q121N, S123T 3 4
E125N, P126A,
C80 A127T 2 4
P126N, V128T,
C81 P 129A 2 4
C82 A127N,P129T 2 4
V128N,P129A,
C83 F130T, P131A 1 1
C84 P129N,P131T 1 1
C85 R134N,S136T 1 1
C86 V135N, V137T 1 1
C87 S136N, S138T 2 4
C88 V137N, Q139T 2 4
C89 S138N 4 3
C90 Q139N,S141T 3 3
C91 T140N, K142T 2 3
C92 S141N,L143T 3 2
C93 A146N 4 3
C94 E147N, V149T 4 1
T148N, F150T,
C95 P151A 4 2
C96 V149N,P151T 3 3
F150N, P151A,
C97 D152T 3 3
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C98 P151N, V153T 4 4
C99 D152N,D154T 3 4
C100 V153N, Y155T 4 4
C101 D154N, V156T 3 4
C102 Y1 55N, NI 57T 3 4
C103 V156N,S158T 3 4
C104 N157 3 4
C105 S158N,E160T 3 4
C106 T159N, A161T 3 4
C107 E160N,E162T 3 3
C108 A161N 5 4
C109 E162N,1164T 6 4
C110 T163N, L165T 5 4
C111 I164N, D166T 5 4
C112 L165N, N167T 5 4
C113 D166N, I168T 5 4
C114 N167 5 4
C115 I168N, Q170T 6 4
C116 T169N,S171T 5 3
C117 Q170N 5 3
C118 S171N, Q173T 5 3
C119 T172N,S174T 5 3
C120 Q173N,F175T 5 3
C121 S174N, N176T 4 3
C122 F175N, D177T 4 1
C123 F178T 5 1
C124 D177N 5 3
C125 G184N, D186T 4 1
C126 E185N, A187T 1 2
C127 D186N, K188T 2 2
C128 A187N,P189T 1 1
K188N,P189A,
C129 G190T 1 1
C130 P189N, Q191T 1 1
C131 L198N, G200T 1 1
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C132 K201T 2 2
C133 G200N, V202T 2 1
C134 K201N, D203T 3 3
C135 V202N, A204T 3 2
C136 121 ON, N212T 1 1
C137 V21 IN, E213T 1 1
C138 K214T 1 1
C139 E213N, W215T 1 1
C140 V223N 1 1
C141 E224N, G226T 4 2
C142 T225N, V227T 3 3
C143 G226N, K228T 2 2
C144 V227N, I229T 1 3
C145 K228N 3 3
C146 H236N, I238T 1 1
C147 E239T 2 3
C148 I238N, E240T 1 1
C149 E239N 3 3
C150 E240N,E242T 1 1
C151 T241N, H243T 2 3
C152 E242N 1 2
C153 H243N,E245T 2 2
C154 T244N, Q246T 1 1
C155 E245N, K247T 1 1
C156 Q246N,R248T 1 1
C157 K247N, N249T 2 2
C158 R248N, V250T 1 1
C159 1251T 3 4
C160 V250N, R252T 1 1
C161 1251N, 1253T 1 3
R252N, I254T,
C162 P255A 1 2
C163 I253N, P255T 2 3
I254N, P255A,
C164 H256T 1 1
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C165 P255N, H257T 1 2
C166 H256N, N258T 1 1
C167 H257N, Y259T 1 1
C168 N260T 1 1
C169 Y259N, A26 IT 1 1
C170 A262T 4 3
C171 A261N, I263T 2 3
C172 A262N, N264T 3 3
C173 1263N, K265T 1 1
C174 Y266T 1 1
C175 K265N, N267T 1 1
C176 Y266N, H268T 1 1
C177 E274N, D276T 1 1
L275N, E277T,
C178 P278A 1 1
C179 D276N,P278T 1 2
E277N, P278A,
C180 L279T 1 1
C181 P278N, V280T 1 3
C182 L279N,L281T 1 1
C183 V280N, N282T 2 3
C184 L281N, S283T 1 1
C185 Y284T 1 1
C186 S283N, V285T 1 3
C187 E294N 2 2
C188 Y295N, N297T 1 1
C189 T296N,1298T 1 1
C190 F299T 1 1
C191 I298N, L300T 1 1
C192 F299N, K301T 1 1
C193 L300N,F302T 1 1
C194 K301N, G303T 1 1
C195 F302N, S304T 5 1
C196 G303N, G305T 1 1
C197 S304N, Y306T 5 1
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C198 R312N,F314T 5 1
C199 V313N, H315T 5 1
C200 F314N, K316T 4 1
C201 H315N, G317T 5 1
C202 K316N,R318T 4 1
C203 G317N,S319T 4 1
C204 R318N,A320T 3 1
C205 S319N,L321T 5 1
C206 A320N, V322T 4 1
L326N, V328T,
C207 P329A 1 1
C208 R327N,P329T 4 1
V328N,P329A,
C209 L330T 1 1
C210 P329N, V33 IT 3 1
C211 L330N, D332T 1 1
C212 V331N,R333T 1 1
C213 D332N, A334T 3 1
C214 R333N 1 1
C215 L337N,S339T 2 1
C216 R338N 4 1
C217 S339N, K341T 4 1
C218 T340N,F342T 2 1
C219 K341N 5 1
C220 F342N,1344T 1 1
C221 T343N, Y345T 5 1
C222 1344N, N346T 1 1
C223 Y345N, N347T 2 1
C224 A351N,F353T 1 1
C225 G352N, H354T 1 1
C226 F353N, E355T 2 1
C227 H354N, G356T 4 1
C228 E355N, G357T 4 1
C229 G356N,R358T 4 1
C230 G357N, D359T 5 1
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C231 T371N, V373T 1 1
C232 E372N, E374T 2 3
C233 V373N, G375T 1 1
C234 E374N 5 2
C235 W385N, E387T 3 1
C236 G386N,E388T 3 1
C237 A390N, K392T 3 1
C238 M391N, G393T 3 1
C239 K392N, K394T 4 1
C240 G393N, Y395T 1 1
C241 R403N, V405T 2 1
C242 Y404N, N406T 1 1
C243 V405N, W407T 1 1
C244 1408T 1 1
C245 W407N, K409T 1 1
C246 I408N, E410T 1 1
C247 K409N, K41 IT 1 1
C248 E41ON 2 4
C249 K41 IN, K413T 1 1
C250 T412N,L414T 1 1
C251 K413N, T415 2 1
Expression and activity relative to the wild type control are expressed on a
scale
of 1 to 5 where 1 = <10%; 2 = 10-50%; 3 = 50-100%; 4 = 100-200%; 5 = >200%.
Example 4: Substitutions to Convert O-linked to N-linked Glycosylation Sites
[112] Table 5 shows the substitutions that were designed to convert O-linked
glycosylation sites
to N-linked glycosylation sites.
TABLE 5
Designation Mutein Native Sequence N-linked Glycosylation site
(0-linked site underlined) created
HG4 T169N T169-Q170-S171 N169-Q170-S171
HG5 T172N T172-Q173-S174 N172-Q173-S174
HG6 T148N plus T148-V149-F150 N148- V149-T150
F150T
HG7 T159N plusA161T T159-E160-A161 N159-E160-T161
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Example 5: Expression of FIX Variants in HKB11 Cells
[113] In order to determine if the FIX genes with altered protein sequences
could be expressed
and secreted from mammalian cells and to determine the effect of these
substitutions upon FIX
coagulation activity, expression plasmids encoding these FIX variants were
transfected into
HKB11 cells. HKB11 is a human cell line generated by the fusion of HEK293
cells and a B cell
lymphoma. In this example, each of the glycosylation site substitutions were
combined with a
second substitution, R338A, as shown for HG1 through HG8 in Figure 3. The
R338A substitution
increases the specific activity of FIX by 3- to 4-fold as measured by the aPTT
assay. Additional
combinations of two glycosylation site muteins and R338A were also created and
tested
(Figure 3).
[114] Three days after transfection, the media from the cells was collected
and analyzed for FIX
expression by Western blot using an antibody specific to FIX. The results
demonstrated that the
FIX muteins tested were expressed and secreted into the media at levels
similar to that of wild type
FIX or FIX with only the R338A substitution (Figure 2).
[115] The activity of the FIX muteins was also tested using the aPPT assay.
The results
demonstrated that the Factor IX muteins have activity similar to or increased
compared to that of
wild type FIX (Figures 3 and 4). Compared to R338A, the activity of the
muteins varied between
14% and 109%. In particular, muteins HG1, HG3, HG4, HG5, HG6, HG7, HG8, and
HG3 plus
HG8 had coagulation activity that was at least 55% of that of R33 8A, and at
least 300% of wild
type FIX.
Example 6: Glycosylation of Factor IX variants in HKB11 cells
[116] An initial analysis of muteins HG1 through HG8 (Figure 3) and HG9
(Figure 5)
demonstrated that HG2, HG3, HG5, HG6, HG8, and HG9 had increased glycosylation
as
evidenced by an increase in apparent molecular weight on SDS-PAGE gels.
Additional FIX
polypeptides were created containing various combinations of the substitutions
present in HG2,
HG3, HG5, HG6, and HG8. For example, the following combinations were created:
HG2/HG3,
HG8/HG2, HG8/HG3, HG8/HG2/HG3. Other possible combinations include, for
example,
HG3/HG5, HG5/HG8, HG3/HG5/HG8. HG3/HG6, HG5/HG6, HG8/HG6, HG3/HG5/HG6,
HG3/HG6/HG8, HG5/HG6/HG8, and HG3/HG5/HG6/HG8. Combinations with HG9 may also
be
generated.
[117] Glycosylation increases the molecular weight of a protein and this can
be visualized as
reduced mobility in SDS-PAGE gels. As demonstrated in Figures 2 and 3, the
substitutions made
in HG2, HG3, HG5, HG6, HG8, and HG9 resulted in reduced mobility on a SDS-PAGE
gel.
Among these eight single muteins, HG8 which has two additional N-linked
consensus sequences
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inserted between A161 and E162, exhibited the greatest increase in apparent
molecular weight
suggesting that both N-linked sites were functional. Of the four muteins in
which an O-linked site
was mutated to an N-linked site (HG4 to HG7), HG4 exhibited increased mobility
on the gel
demonstrating that this substitution destroyed the O-linked site (thus
reducing the molecular
weight of the FIX protein), but failed to create a functional N-linked site.
In contrast, HG5 and
HG6 exhibited reduced mobility on the gel demonstrating that the substitutions
in these clones did
create functional N-linked glycosylation sites. Mutein HG7 which also has an O-
linked site
mutated to an N-linked site exhibited no change in mobility on the gel
compared to wild-type FIX
or FIX-R338A. Given the fact that the substitution in HG7 would be expected to
eliminate an 0-
linked site and thus increase the mobility on the gel, the finding that there
was no change in
mobility suggests that the introduced N-linked site may be functional.
[118] The combinations of substitutions present in HG2/HG3, HG8/HG2, HG8/HG3,
and
HG8/HG2/HG3 resulted in more significant reductions in mobility compared to
the individual
muteins, indicating that the various new glycosylation sites present in these
combinations were
functional when combined in a single FIX molecule. The HG8/HG2/HG3 mutein that
contains a
total of 4 new N-linked sites exhibited the largest decrease in mobility.
Example 7: Combination of R338A and V86A Substitutions
[119] Amino acid V86 of FIX was changed to alanine by site directed
mutagenesis either in the
context of wild type FIX (WT-FIX) or FIX-R338A. Expression vectors containing
these
constructs were transfected into HKB11 cells and media was collected 3 days
later and assayed for
FIX protein level by ELISA and for FIX coagulation activity by aPTT assay.
Both muteins were
expressed at similar levels to WT-FIX and FIX-R338A. The data from a single
experiment is
summarized in Table 6a. Table 6b summarizes the average of three experiments.
TABLE 6a
Sample FIX Activity ELISA Specific Activity
(mU/mL) ( g/mL) (IU/mg)
pAGE16-V86A 255 0.97 263
pAGE16-R338A 383 0.73 527
pAGE-R338A-V86A 1237 1.03 1198
PAGE 16-FIX 88 0.89 99
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TABLE 6b
Factor IX construct FIX activity FIX expression FIX specific activity
(% of WT-FIX) (% of WT-FIX) (% of WT-FIX)
WT-Factor IX 100 100 100
V86A 210 120 180
R338A 390 90 450
R338A-V86A 950 105 810
[120] The results demonstrate that the V86A substitution alone results in
about a 1.8-fold
increase in specific activity, while R338A alone resulted in a 4.5-fold
increase in specific activity.
The combination of R338A and V86A resulted in a 8.1-fold increase in specific
activity as
compared to wild type FIX. These results show that the positive effects of the
R338A and V86A
substitutions are additive and result in a Factor IX mutein with an 8-fold
increased specific activity
compared to WT-FIX. The R338A-V86A mutein would have improved therapeutic
benefit for
hemophilia B patients as it would allow a 8-fold lower dose of protein to
achieve the same
therapeutic effect as the currently available recombinant WT-IX. In addition,
the increased
specific activity of R338A-V86A is beneficial when creating glycosylated forms
of FIX in which a
reduction in activity may result from glycosylation.
Example 8: Cloning of Human FIX cDNA
[121] A pair of PCR primers complementary to sequences at the 5' and 3' ends
of the coding
region of the human FIX cDNA were designed from the published cDNA sequence
(NM 000133).
The 5' primer (FIXF1; ATCATAAGCTTGCCACCATGCAGCGCGTGAACATG (SEQ ID NO:
8), start codon of FIX is in bold text) contained the first 18 nucleotides of
the FIX coding region
including the ATG start codon preceded by a consensus Kozak sequence
(underlined) and a
HindIll restriction site. The 3' primer (FIXR3, ATCATAAGCTTGATTAGTTAGTGAGA
GGCCCTG) (SEQ ID NO: 9) contained 22 nucleotides of FIX sequence that lies 45
nucleotides 3'
of the end of the FIX coding region preceded by a HindIll site. Amplification
of first strand
cDNA from normal human liver (Stratagene, San Diego CA) using these primers
and high fidelity
proofreading polymerase (Invitrogen, Carlsbad, CA) resulted in a single band
of the expected size
for human FIX cDNA (1464 bp). After digestion with HindI11, the PCR product
was gel purified
and then cloned into the HindIll site of the plasmid pAGE16. Clones in which
the FIX cDNA was
inserted in the forward orientation relative to the CMV promoter in the vector
were identified by
restriction digest. Double stranded DNA sequencing was performed for the
insert of several
clones and alignment of the derived sequence to the published FIX sequence
demonstrated that the
cDNA encodes human FIX with threonine at amino acid 148 of the mature protein.
This plasmid
was designated as pAGE16-Factor IX (pAGE16-FIX).
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Example 9: Generation of Modified FIX Polypeptides
[122] To change various amino acids within the human FIX sequence, a pair of
primers were
designed using the QuickchangeTM primer design program available from
Stratagene. These
primers were used to generate mutations in the pAGE16-FIX plasmid employing
the
QuickchangeTM II XL site directed mutagenesis kit (Stratagene, San Diego, CA)
according to the
manufacturer's instructions. Clones containing the desired substitution were
identified by DNA
sequencing of the entire FIX coding region. Table 7 below shows the sequence
of the sense strand
oligonucleotide used to create the substitutions.
TABLE 7
Substitution Sense Strand Oligonucleotide Sequence
G226N/K228T CTGCTGCCCACTGTGTTGAAACTAACGTTACCATTACA
GTTGTCGCAGGTGAAC (SEQ ID NO: 10)
F353N/H354V/E355T CACCATCTATAACAACATGTTCTGTGCTGGCAACGTGA
CCGGAGGTAGAGATTCATGTCAAGGAGATAGTG
(SEQ ID NO: 11)
V370N/T371V/E372T GATTCATGTCAAGGAGATAGTGGGGGACCCCATAACG
TGACCGTGGAAGGGACCAGTTTCTTAACTGGAATTA
(SEQ ID NO: 12)
M391N/K392V/G393T GGAATTATTAGCTGGGGTGAAGAGTGTGCAAACGTGA
CCAAATATGGAATATATACCAAGGTATCCCGG
(SEQ ID NO: 13)
K413N TCAACTGGATTAAGGAAAAAACAAACCTCACTTAATG
AAAGATGGATTTC (SEQ ID NO: 14)
A161N CTGATGTGGACTATGTAAATTCTACTGAAAATGAAACC
ATTTTGGATAACATCAC (SEQ ID NO: 15)
D177E/F178T ACTCAAAGCACCCAATCATTTAATGAGACCACTCGGGT
TGTTGGTGG (SEQ ID NO: 16)
P151N/V153T ACTTCTAAGCTCACCCGTGCTGAGACTGTTTTTAATGA
TACGGACTATGTAAATTCTACTG (SEQ ID NO: 17)
T169N GAAGCTGAAACCATTTTGGATAACATCAATCAAAGCA
CCCAATC (SEQ ID NO: 18)
T172N ATTTTGGATAACATCACTCAAAGCAACCAATCATTTAA
TGACTTCAC (SEQ ID NO: 19)
T148N/F150T ACTTCTAAGCTCACCCGTGCTGAGAATGTTACTCCTGA
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TGTGGAC (SEQ ID NO: 20)
T159N/A161T GTTTTTCCTGATGTGGACTATGTAAATTCTAATGAAAC
TGAAACCATTTTGGATAAC (SEQ ID NO: 21)
The underlined residues are those that were changed relative to wild type FIX
to create a
consensus sequence for N-linked glycosylation.
[123] In addition to the substitutions described above, a sequence of nine
amino acids containing
2 consensus sequences for N-glycosylation was inserted into the activation
peptide between
residues A161 and E162. To generate this variant of FIX, unique restriction
sites for SnaB1 and
Xbal at Y155 and 1164 were created without altering the amino acid sequence by
site directed
mutagenesis with primers t8216cg8218a and t8188c (Table 8). The resulting
plasmid was
digested with SnaB1 and Xbal to remove the 27 bp fragment corresponding to
residues V156 to
1164 and then ligated to a double stranded fragment created by annealing of
oligonucleotides
2PrimerF and 2PrimerR (Table 8). The sequence of the resulting plasmid was
determined by
double strand DNA sequencing to have an insertion of 27 bp encoding nine amino
acids with the
sequence NSTQDNITQ (SEQ ID NO: 2) that contains two consensus sequences for N-
linked
glycosylation (NXT).
TABLE 8
Primer name Primer sequence (5' to 3')
T8216c_g821 TTCTACTGAAGCTGAAACCATTCTAGATAACATCACTCAAAGCACCC
2a (SEQ ID NO: 22)
T8188c CTGTTTTTCCTGATGTGGACTACGTAAATTCTACTGAAGCTGAAA
(SEQ ID NO: 23)
2PrimerF GTAAATTCTACTGAAGCTAACTCCACACAGGATAATATCACACAAG
AAACCATT (SEQ ID NO: 24)
2PrimerR CTAGAATGGTTTCTTGTGTGATATTATCCTGTGTGGAGTTAGCTTCA
GTAGAATTTAC (SEQ ID NO: 25)
The bold/underline residues are those that were changed relative to wild type
Factor IX to create
either a new restriction site or the insertion of nine additional amino acids
encoding two consensus
sequences for N-linker glycosylation.
Example 10: Cell Culture and Transient Transfection
[124] HKB11 cells (a hybrid of HEK293 and a Burkitt B cell lymphoma line, 2B8)
were grown
in suspension culture on an orbital shaker (100-125 rpm) in a CO2 (5%)
incubator at 37 C in
serum free media (RF#277) supplemented with 10 ng/mL soluble vitamin K3 (Sigma-
Aldrich, St.
Louis, MO) and maintained at a density between 0.25 and 1.5 x 106 cells/mL.
[125] Cells for transfection were collected by centrifugation at 1000 rpm for
5 minutes then
resuspended in FreeStyleTM 293 Expression Medium (Invitrogen, Carlsbad, CA) at
1.1 x 106
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cells/mL. The cells were seeded in 6 well plates (4.6 mL/well) and incubated
on an orbital rotator
(125 rpm) in a 37 C CO2 incubator. For each well, 5 g plasmid DNA was mixed
with 0.2 mL
Opti-MEM I medium (Invitrogen). For each well, 7 L 293fectinTM reagent
(Invitrogen) was
mixed gently with 0.2 mL Opti-MEM I medium and incubated at room temperature
for
minutes. The diluted 293fectinTM was added to the diluted DNA solution, mixed
gently,
incubated at room temperature for 20-30 minutes and then added to each well
that had been seeded
with 5 x 106 (4.6 mL) HKB11 cells. The cells were then incubated on an orbital
rotator (125 rpm)
in a CO2 incubator at 37 C for 3 days after which the cells were pelleted by
centrifugation at
1000 rpm for 5 minutes, and the supernatant was collected and stored at 4 C.
[126] In some instances, HEK293 cells were transfected with expression
constructs for FIX
variants in 384 well plates using standard lipofection protocols and
commercial reagents. The
cells were cultivated for 72 hours post-transfection at which time the
supernatant was harvested for
further analyses.
Example 11: Western Blot for FIX.
[127] Cell culture supernatant (50 L) was mixed with 20 L 4x SDS-PAGE
loading dye,
heated at 95 C for 5 minutes, loaded on NuPAGE 4-12% SDS PAGE gels and then
transferred to
nitrocellulose membranes. After blocking with 5% milk powder for 30 minutes,
the membranes
were incubated with a HRP-labeled goat polyclonal antibody against human FIX
(US Biological,
Swampscott, Massachusetts, Catalog No. F0017-07B) for 60 minutes at room
temperature. After
washing with phosphate-buffered saline with 0.1% Tween -20 buffer, the signal
from HRP was
detected using SuperSignal Pico (Pierce, Rockford, IL) and exposure to x-ray
film.
Example 12: FIX ELISA
[128] FIX antigen levels in cell culture supernatants were determined using a
FIX ELISA kit
(Hyphen Biomed/Aniara, Mason, OH). Cell culture supernatant was diluted in
sample diluent
buffer (supplied in the kit) to achieve a signal within the range of the
standard curve. FIX protein
purified from human plasma (Hyphen Biomed/Aniara, Catalog No. RK032A, specific
activity
196 U/mg) diluted in sample diluent was used as to create a standard curve
from 100 ng/mL to
0.2 ng/mL. Diluted samples and the standards were added to the ELISA plate
that is pre-coated
with a polyclonal anti-FIX capture antibody. After adding the polyclonal
detection antibody, the
plate was incubated at room temperature for 1 hour, washed extensively, then
developed using
TMB substrate (3,3',5,5'-tetramethylbenzidine) as described by the kit
manufacturer and the signal
is measured at 450 nM using a SpectraMax plate reader (Molecular Devices,
Sunnyvale, CA).
The standard curve was fitted to a 2-component plot and the values of the
unknowns extrapolated
from the curve.
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[129] FIX expression levels were also quantitated using commercially available
FIX ELISA
reagents (Haemochrom Diagnostica GmbH, Essen, Germany) according to the
manufacturer's
instructions. Wheat germ agglutinin (Sigma-Aldrich, St. Louis, MO) was coated
on 384 well
MaxiSorpTM plates (NuncTM, Rochester, NY). The wells were blocked, washed, and
then
supernatant was added. After further washing, detection was carried out using
HRP-coupled
polyclonal anti-FIX antibody (Haemochrom Diagnostica GmbH, Essen, Germany).
Example 13: FIX Coagulation Assay
[130] FIX coagulation activity was determined using an aPTT assay in FIX
deficient human
plasma run on a ElectraTM 18000 automatic coagulation analyzer (Beckman
Coulter, Fullerton,
CA). Briefly, three dilutions of supernatant samples in coagulation diluent
were created by the
instrument, and 100 L was then mixed with 100 L FIX deficient plasma
(Aniara, Mason, OH)
and 100 L automated aPTT reagent (rabbit brain phospholipid and micronized
silica (bioMerieux,
Inc., Durham, NC). After the addition of 100 L 25 mM CaC12 solution, the time
to clot formation
was recorded. A standard curve was generated for each run using serial
dilutions of the same
purified human FIX (Hyphen Biomed/Aniara) used as the standard in the ELISA
assay. The
standard curve was routinely a straight line with a correlation coefficient of
0.95 or better and was
used to determine the FIX activity of the unknown samples.
Example 14: Measurement of Circulating FIX
[131] The circulating half-life of FIX polypeptides is measured using an in
vitro assay. This
assay is based on the ability of FIX in vivo and in vitro to mediate the
accumulation of adenovirus
(Ad) in hepatocytes. Briefly, it has been shown that FIX can bind the Ad fiber
knob domain and
provide a bridge for virus uptake through cell surface heparin sulfate
proteoglycans (HSPG)
(Shayakhmetov, et al., J. Virol 79:7478-7491, 2005). An Adenovirus vector
mutant, Ad5mut,
which contains mutations in the fiber knob domain, does not bind to FIX.
Ad5mut has
significantly reduced ability to infect liver cells and liver toxicity in
vivo, demonstrating that FIX
plays a major role in targeting Ad vectors to hepatic cells (Shayakhmetov, et
al., 2005). The
ability of FIX to target Ad vector to hepatic cells can be blocked by
inhibitors of protein-HSPG
interactions (Shayakhmetov, et al., 2005).
[132] Furthermore, HSPG-mediated uptake of FIX contributes significantly to
FIX clearance
and consequently, interfering with the HSPG interaction is expected to
increase the half-life of
FIX. Therefore, in vitro uptake of FIX and/or FIX variants in hepatocytes is
measured, and
variants with reduced uptake are expected to have increased half-life in vivo.
[133] To measure FIX half-life in vitro, mammalian cells are incubated with
adenovirus in the
presence or absence of FIX or FIX variants. Viral uptake is mediated by wild-
type FIX and
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measured by expression of the reporter gene encoded in viral genome, for
example, green
fluorescent protein (GFP) or luciferase expression. Reduced uptake of
adenovirus in the presence
of FIX variants are measured as reduced reporter gene expression, for example,
reduced GFP
fluorescence or reduced luciferase enzymatic activity as compared to wild-type
FIX.
[134] FIX circulating half-life is measured in vivo using standard techniques
well-known to
those of ordinary skill in the art. Briefly, the respective dose of FIX or FIX
variant is administered
to a subject by intravenous injection. Blood samples are taken at a number of
time points after
injection and the FIX concentration is determined by an appropriate assay
(e.g.. ELISA). To
determine the half-life, that is the time at which the concentration of FIX is
half of the
concentration of FIX immediately after dosing, the FIX concentration at the
various time points is
compared to the FIX concentration expected or measured immediately after
administering the dose
of FIX. A correlation between reduced cellular uptake in the in vitro assay
and increased half-life
in the in vivo assay is expected.
[135] All publications and patents mentioned in the above specification are
incorporated herein
by reference. Various modifications and variations of the described methods of
the invention will
be apparent to those skilled in the art without departing from the scope and
spirit of the invention.
[136] Although the invention has been described in connection with specific
embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the above-described modes for
carrying out the
invention which are obvious to those skilled in the field of biochemistry or
related fields are
intended to be within the scope of the following claims. Those skilled in the
art will recognize, or
be able to ascertain using no more than routine experimentation, many
equivalents to the specific
embodiments of the invention described herein. Such equivalents are intended
to be encompassed
by the following claims.
