Note: Descriptions are shown in the official language in which they were submitted.
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GLYCOPEGYLATED FACTOR IX
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional Patent
Application No.
60/527,089, filed on December 3, 2003, which is incorporated herein by
reference in their
entirety for all purposes, U.S. Provisional Patent Application No. 60/539,387,
filed January
26, 2004; U.S. Provisional Patent Application No. 60/592,744, filed July 29,
2004; U.S.
Provisional Patent Application No. 60/614,518, filed September 29, 2004; and
U.S.
Provisional Patent Application No. 60/623,387, filed October 29, 2004 each of
which is
incorporated herein by reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] Vitamin K-dependent proteins (e.g., Factor IX) contain 9 to 13 gamma-
carboxyglutamic acid residues (Gla) in their amino terminal 45 residues. The
Gla residues
are produced by enzymes in the liver that utilize vitamin K to carboxylate the
side chains of
glutamic acid residues in protein precursors. Vitamin K-dependent proteins are
involved in a
number of biological processes, of which the most well described is blood
coagulation
(reviewed in Nelsestuen, Vitam. Horm. 58: 355-389 (2000)). Vitamin K-dependent
proteins
include protein Z, protein S, prothrombin (Factor II), Factor X, Factor IX,
protein C, Factor
VII, Gas6, and matrix GLA protein. Factors VII, IX, X and II function in
procoagulation
processes while protein C, protein S and protein Z serve in anticoagulation
roles. Gash is a
growth arrest hormone encoded by growth arrest-specific gene 6 (gash) and is
related to
protein S. See, Manfioletti et al. Mol. Cell. Biol. 13: 4976-4985 (1993).
Matrix GLA protein
normally is found in bone and is critical to prevention of calcification of
soft tissues in the
circulation. Luo et al. Nature 386: 78-81 (1997).
[0003] The regulation of blood coagulation is a process that presents a number
of leading
health problems, including both the failure to form blood clots as well as
thrombosis, the
formation of unwanted blood clots. Agents that prevent unwanted clots are used
in many
situations and a variety of agents are available. Unfortunately, most current
therapies have
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undesirable side effects. Orally administered anticoagulants such as Warfarin
act by
inhibiting the action of vitamin K in the liver, thereby preventing complete
carboxylation of
glutamic acid residues in the vitamin K-dependent proteins, resulting in a
lowered
concentration of active proteins in the circulatory system and reduced ability
to form clots.
Warfarin therapy is complicated by the competitive nature of the drug with its
target.
Fluctuations of dietary vitamin K can result in an over-dose or under-dose of
Warfarin.
Fluctuations in coagulation activity are an undesirable outcome of this
therapy.
[0004] Injected substances such as heparin, including low molecular weight
heparin, also are
commonly used anticoagulants. Again, these compounds are subject to overdose
and must be
carefully monitored.
[0005] A newer category of anticoagulants includes active-site modified
vitamin K-
dependent clotting factors such as factor VIIa and IXa. The active sites are
blocked by serine
protease inhibitors such as chloromethylketone derivatives of amino acids or
short peptides.
The active site-modified proteins retain the ability to form complexes with
their respective
cofactors, but are inactive, thereby producing no enzyme activity and
preventing complexing
of the cofactor with the respective active enzymes. In short, these proteins
appear to offer the
benefits of anticoagulation therapy without the adverse side effects of other
anticoagulants.
Active site modified factor Xa is another possible anticoagulant in this
group. Its cofactor
protein is factor Va. Active site modified activated protein C (APC) may also
form an
effective inhibitor of coagulation. See, Sorensen et al. J. Biol. ClZern. 272:
11863-11868
(1997). Active site modified APC binds to factor Va and prevents factor Xa
from binding.
[0006] A major inhibition to the use of vitamin K-dependent clotting factors
is cost.
Biosynthesis of vitamin K-dependent proteins is dependent on an intact
glutamic acid
carboxylation system, which is present in a small number of animal cell types.
Overproduction of these proteins is limited by this enzyme system.
Furthermore, the
effective dose of these proteins is high. A common dosage is 1000 ~.g of
peptide/kg body
weight. See, Harker et al. 1997, supra.
[0007] Another phenomena that hampers the use of therapeutic peptides is the
well known
aspect of of protein glycosylation is the relatively short ih vavo half life
exhibited by these
peptides. Overall, the problem of shot ih vivo half life means that
therapeutic glycopeptides
must be administered frequently in high dosages, which ultimately translate to
higher health
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care costs than might be necessary if a more efficient method for making
longer lasting, more
effective glycoprotein therapeutics was available.
[0008] Factor VIIa, for example, illustrates this problem. Factor VII and VIIa
have
circulation half times of about 2-4 hours in the human. That is, within 2-4
hours, the
concentration of the peptide in the serum is reduced by half. When Factor VIIa
is used as a
procoagulant to treat certain forms of hemophilia, the standard protocol is to
inject VIIa every
i
two hours and at high dosages (45 to 90 µg/kg body weight). See, Hedner et
al., Transfus.
Med. Rev. 7: 78-83 (1993)). Thus, use of these proteins as procoagulants or
anticoagulants
(in the case of factor VIIa) requires that the proteins be administered at
frequent intervals and
at high dosages.
[0009] One solution to the problem of providing cost effective glycopeptide
therapeutics has
been to provide peptides with longer in vivo half lives. For example,
glycopeptide
therapeutics with improved pharmacolcinetic properties have been produced by
attaching
synthetic polymers to the peptide backbone. An exemplary polymer that has been
conjugated
to peptides is polyethylene glycol) ("PEG"). The use of PEG to derivatize
peptide
therapeutics has been demonstrated to reduce the immunogenicity of the
peptides. For
example, U.S. Pat. No. 4,179,337 (I~avis et al.) discloses non-immunogenic
polypeptides
such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or
polypropylene glycol. In addition to reduced immunogenicity, the clearance
time in
circulation is prolonged due to the increased size of the PEG-conjugate of the
polypeptides in
question.
[0010] The principal mode of attachment of PEG, and its derivatives, to
peptides is a non-
specific bonding through a peptide amino acid residue (see e.g., U.S. Patent
No. 4,088,538
U.S. Patent No. 4,496,689, U.S. Patent No. 4,414,147, U.S. Patent No.
4,055,635, and PCT
WO 87/00056). Another mode of attaching PEG to peptides is through the non-
specific
oxidation of glycosyl residues on a glycopeptide (see e.g., WO 94/05332).
[0011] In these non-specific methods, poly(ethyleneglycol) is added in a
random, non-
specific manner to reactive 'residues on a peptide backbone. Of course, xandom
addition of
PEG molecules has its drawbacks, including a lack of homogeneity of the final
product, and
the possibility for reduction in the biological or enzymatic activity of the
peptide. Therefore,
for the production of therapeutic peptides, a derivitization strategy that
results in the
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formation of a specifically labeled, readily characterizable, essentially
homogeneous product
is superior. Such methods have been developed.
[0012] Specifically labeled, homogeneous peptide therapeutics can be produced
i~r vitro
through the action of enzymes. Unlike the typical non-specific methods for
attaching a
synthetic polymer or other label to a peptide, enzyme-based syntheses have the
advantages of
regioselectivity and stereoselectivity. Two principal classes of enzymes for
use in the
synthesis of labeled peptides are glycosyltransferases (e.g.,
sialyltransferases,
oligosaccharyltransferases, N-acetylglucosaminyltransferases), and
glycosidases. These
enzymes can be used for the specific attachment of sugars which can be
subsequently
modified to comprise a therapeutic moiety. Alternatively, glycosyltransferases
and modified
glycosidases can be used to directly transfer modified sugars to a peptide
backbone (see e.g.,
U.S. Patent 6,399,336, and U.S. Patent Application Publications 20030040037,
20040132640, 20040137557, 20040126838, and 20040142856, each of which are
incorporated by reference herein). Methods combining both chemical and
enzymatic
synthetic elements are also known (see e.g., Yamamoto et al. Carbohydr. Res.
305: 415-422
(1998) and U.S. Patent Application Publication 20040137557 which is
incorporated herein by
reference).
[0013] Factor IX is an extremely valuable therapeutic peptide. Although
commercially
available forms of Factor IX are in use today, these peptides can be improved
by
modifications that enhance the pharmacokinetics of the resulting isolated
glycoprotein
product. Thus, there remains a need in the art for longer lasting Factor IX
peptides with
improved effectiveness and better pharmacokinetics. Furthermore, to be
effective for the
largest number of individuals, it must be possible to produce, on an
industrial scale, a Factor
IX peptide with improved therapeutic pharmacokinetics that has a predictable,
essentially
homogeneous, structure which can be readily reproduced over, and over again.
[0014] Fortunately, Factor IX peptides with improved pharmacokinetics and
methods for
making them have now been discovered. In addition to Factor IX peptides with
improved
pharmacokinetics, the invention also provides industrially practical and cost
effective
methods for the production of these Factor IX peptides. The Factor IX peptides
of the
invention comprise modifying groups such as PEG moieties, therapeutic
moieties,
biomolecules and the like. The present invention therefore fulfills the need
for Factor IX
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peptides with improved the therapeutic effectiveness and improved
pharmacokinetics for the
treatment of conditions and diseases wherein Factor IX provides effective
therapy.
SUMMARY OF THE INVENTION
[0015] It has now been discovered that the controlled modification of Factor
IX with one or
more polyethylene glycol) moieties affords novel Factor IX derivatives with
improved
pharmacokinetic properties. Furtherrilore, cost effective methods for reliable
production of
the modified Factor IX peptides of the invention have been discovered and
developed.
[0016] In one aspect, the present invention provides a Factor IX peptide that
includes the
moiety:
In the formula above, ID is -OH or R1-L-HN-. The symbol G represents R1-L- or -
C(O)(C1-
Cg)alkyl. R1 is a moiety comprising a straight-chain or branched polyethylene
glycol)
residue; and L is a linker which is a member selected from a bond, substituted
or
unsubstituted alkyl and substituted or unsubstituted heteroalkyl. Generally,
when D is OH, G
is Rl-L-, and when G is -C(O)(C1-C6)alkyl, D is Rl-L-NH-. As will be
appreciated by those
of skill in the art, in the sialic acid analogues set forth herein, COOH also
represents COO' or
a salt thereof.
[0017] In another aspect, the invention provides a method of making a PEG-
ylated Factor IX
comprising the moiety above. The method of the invention includes (a)
contacting a
substrate Factor IX peptide with a PEG-sialic acid donor and an enzyme that
transfers the
PEG-sialic acid onto an amino acid or glycosyl residue of the Factor IX
peptide, under
conditions appropriate for the transfer. An exemplary PEG-sialic acid donor
moiety has the
formula:
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O'
P-
OH
NHZ
[0018] In one embodiment the host is mammalian cell. In other embodiments the
host cell is
an insect cell, plant cell, a bacteria or a fungi.
[0019] In another aspect, the invention provides a method of treating a
condition in a subject
in need thereof, wherein the condition is characterized by compromised
coagulation in the
subject. The method comprises the step of administering to the subject an
amount of the
Factor IX peptide conjugate of the invention effective to ameliorate the
condition in the
subject. An exemplary disease treatable by this method is hemophilia.
[0020] In another aspect, the invention provides a pharmaceutical formulation
comprising the
Factor IX peptide of the invention and a pharmaceutically acceptable carrier.
[0021] Other objects and advantages of the invention will be apparent to those
of skill in the
art from the detailed description that follows.
DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is the structure of Factor IX, showing the presence and location
of potential
glycosylation sites at Asn 157, Asn 167; Ser 53, Ser 61, Thr 159, Thr 169, and
Thr 172.
[0023] FIG. 2 is a scheme showing an exemplary embodiment of the invention in
which a
carbohydrate residue on a Factor IX peptide is remodeled and glycopegylated:
(A) sialic acid
moieties are removed by sialidase and the resulting galactose residues are
glycopegylated
with the sialic acid derivative of FIG. 5; (B) a mannose residue is
glycopegylated with the
sialic acid PEG; (C) a sialic acid moiety of an N-glycan is glycopegylated
with the sialic acid
PEG; (D) a sialic acid moiety is of an O-glycan is glycopegylated with the
sialic acid PEG;
(E) SDS PAGE gel of Factor IX from 2(A); (F) SDS PAGE gel of Factor IX from
the
reaction producing 2(C) and 2(D).
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[0024] FIG. 3 is a plot comparing the in vivo residence lifetimes of
unglycosylated Factor IX
and enzymatically glycopegylated Factor IX.
[0025] FIG. 4 is a table comparing the activities of the species shown in FIG.
3.
[0026] FIG. 5 is the amino acid sequence of Factor IX.
[0027] FIG. 6 is a graphic presentation of the pharmacokinetic properties of
various
glycopegylated Factor IX molecules compared to a non-pegylated Factor IX.
[0028] FIG. 7 is a table of representative modified sugar species of use in
the present
invention.
[0029] FIG. 8 is a table of representative modified sugar species of use in
the present
invention.
[0030] FIG. 9 is a table of sialyl transferases of use to transfer onto an
acceptor a modified
sialic acid moietiy, such as those set forth herein and unmodified sialic acid
moieties.
DETAILED DESCRIPTION OF THE INVENTION AND
THE PREFERRED EMBODIMENTS
Abbreviations
[0031] PEG, poly(ethyleneglycol); PPG, poly(propyleneglycol); Ara, arabinosyl;
Fru,
fructosyl; Fuc, fucosyl; Gal, galactosyl; GaINAc, N-acetylgalactosaminyl; Glc,
glucosyl;
GIcNAc, N-acetylglucosaminyl; Man, mannosyl; ManAc, mannosaminyl acetate; Xyl,
xylosyl; and NeuAc, sialyl (N-acetylneuraminyl); M6P, mannose-6-phosphate;
Sia, sialic
acid, N-acetylneuraminyl, and derivatives and analogues thereof.
Definitions
[0032] Unless defined otherwise, all technical and scientific terms used
herein generally have
the same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Generally, the nomenclature used herein and the laboratory
procedures in
cell culture, molecular genetics, organic chemistry and nucleic acid chemistry
and
hybridization are those well known and commonly employed in the art. Standard
techniques
are used for nucleic acid and peptide synthesis. The techniques and procedures
are generally
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performed according to conventional methods in the art and various general
references (see
gehei°ally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d
ed. (1989)
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is
incorporated
herein by reference), which are provided throughout this document. The
nomenclature used
herein and the laboratory procedures in analytical chemistry, and organic
synthetic described
below are those well known and commonly employed in the art. Standard
techniques, or
modifications thereof, are used for chemical syntheses and chemical analyses.
[0033] All oligosaccharides described herein are described with the name or
abbreviation for
the non-reducing saccharide (i.e., Gal), followed by the configuration of the
glycosidic bond
(a or (3), the ring bond (1 or 2), the ring position of the reducing
saccharide involved in the
bond (2, 3, 4, 6 or 8), and then the name or abbreviation of the reducing
saccharide (i.e.,
GIcNAc). Each saccharide is preferably a pyranose. For a review of standard
glycobiology
nomenclature see, Essentials of Glycobiology Varki et al. eds. CSHL Press
(1999).
[0034] Oligosaccharides are considered to have a reducing end and a non-
reducing end,
whether or not the saccharide at the reducing end is in fact a reducing sugar.
In accordance
with accepted nomenclature, oligosaccharides are depicted herein with the non-
reducing end
on the left and the reducing end on the right.
[0035] The term "sialic acid" refers to any member of a family of nine-carbon
carboxylated
sugars. The most common member of the sialic acid family is N-acetyl-
neuraminic acid (2-
keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onic acid
(often
abbreviated as NeuSAc, NeuAc, or NANA). A second member of the family is N-
glycolyl-
neuraminic acid (NeuSGc or NeuGc), in which the N-acetyl group of NeuAc is
hydroxylated.
A third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (I~DN)
(Nadano et al.
(1986) J. Biol. Chem. 261: 11550-11557; I~anamori et al., J. Biol. Chem. 265:
21811-21819
(1990)). Also included are 9-substituted sialic acids such as a 9-O-Cl-C6 acyl-
NeuSAc like
9-O-lactyl-NeuSAc or 9-O-acetyl-NeuSAc, 9-deoxy-9-fluoro-NeuSAc and 9-azido-9-
deoxy-
NeuSAc. For review of the sialic acid family, see, e.g., Varki, Glycobiology
2: 25-40 (1992);
Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-
Verlag, New
York (1992)). The synthesis and use of sialic acid compounds in a sialylation
procedure is
disclosed in international application WO 92/16640, published October 1, 1992.
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[0036] "Peptide" refers to a polymer in which the monomers are amino acids and
are joined
together through amide bonds, alternatively referred to as a polypeptide.
Additionally,
unnatural amino acids, for example, (3-alanine, phenylglycine and homoarginine
are also
included. Amino acids that are not gene-encoded may also be used in the
present invention.
Furthermore, amino acids that have been modified to include reactive groups,
glycosylation
sites, polymers, therapeutic moieties, biomolecules and the like may also be
used in the
invention. All of the amino acids used in the present invention may be either
the d - or 1-
isomer. The 1-isomer is generally preferred. In addition, other
peptidomimetics are also
useful in the present invention. As used herein, "peptide" refers to both
glycosylated and
unglycosylated peptides. Also included are petides that are incompletely
glycosylated by a
system that expresses the peptide. For a general review, see, Spatola, A. F.,
in Chemistry and
Biochemistry of Amino Acids, Peptides and Proteins, B. Weinstein, eds., Marcel
Dekker,
New York, p. 267 (1983).
[0037] The term "peptide conjugate," refers to species of the invention in
which a peptide is
conjugated with a modified sugar as set forth herein.
[0038] The term "amino acid" refers to naturally occurring and synthetic amino
acids, as well
as amino acid analogs and amino acid mimetics that function in a manner
similar to the
naturally occurring amino acids. Naturally occurring amino acids are those
encoded by the
genetic code, as well as those amino acids that are later modified, e.g.,
hydroxyproline, y-
carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds
that have
the same basic chemical structure as a naturally occurring amino acid, i.e.,
an a carbon that is
bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,
homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs
have modified
R groups (e.g., norleucine) or modified peptide backbones, but retain the same
basic chemical
structure as a naturally occurring amino acid. Amino acid mimetics refers to
chemical
compounds that have a structure that is different from the general chemical
structure of an
amino acid, but that function in a manner similar to a naturally occurring
amino acid. As
used herein, "amino acid," whether it is in a linker or a component of a
peptide sequence
refers to both the D- and L-isomer of the amino acid as well as mixtures of
these two isomers.
[0039] As used herein, the term "modified sugar," refers to a naturally- or
non-naturally-
occurring carbohydrate that is enzymatically added onto an amino acid or a
glycosyl residue
of a peptide in a process of the invention. The modified sugar is selected
from a number of
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enzyme substrates including, but not limited to sugar nucleotides (mono-, di-,
arid tri-
phosphates), activated sugars (e.g., glycosyl halides, glycosyl mesylates) and
sugars that are
neither activated nor nucleotides. The "modified sugar" is covalently
functionalized with a
"modifying group." Useful modifying groups include, but are not limited~to,
PEG moieties,
therapeutic moieties, diagnostic moieties, biomolecules and the like. The
modifying group is
preferably not a naturally occurring, or an unmodified carbohydrate. The locus
of
functionalization with the modifying group is selected such that it does not
prevent the
"modified sugar" from being added enzymatically to a peptide.
[0040] The term "water-soluble" refers to moieties that have some detectable
degree of
solubility in water. Methods to detect and/or quantify water solubility are
well known in the
art. Exemplary water-soluble polymers include peptides, saccharides,
poly(ethers),
poly(amines), poly(carboxylic acids) and the like. Peptides can have mixed
sequences of be
composed of a single amino acid, e.g., poly(lysine). An exemplary
polysaccharide is
poly(sialic acid). An exemplary poly(ether) is polyethylene glycol).
Polyethylene imine) is
an exemplary polyamine, and poly(acrylic) acid is a representative
poly(carboxylic acid).
[0041] The polymer backbone of the water-soluble polymer can be polyethylene
glycol) (i.e.
PEG). However, it should be understood that other related polymers are also
suitable for use
in the practice of this invention and that the use of the term PEG or
polyethylene glycol) is
intended to be inclusive and not exclusive in this respect. The term PEG
includes
polyethylene glycol) in any of its forms, including alkoxy PEG, difunctional
PEG,
multiarmed PEG, forked PEG, branched PEG, pendent PEG (i.e. PEG or related
polymers
having one or more functional groups pendent to the polymer backbone), or PEG
with
degradable linkages therein.
[0042] The polymer backbone can be linear or branched. Branched polymer
backbones are
generally known in the art. Typically, a branched polymer has a central branch
core moiety
and a plurality of linear polymer chains linked,to the central branch core.
PEG is commonly
used in branched forms that can be prepared by addition of ethylene oxide to
various polyols,
such as glycerol, pentaerythritol and sorbitol. The central branch moiety can
also be derived
from several amino acids, such as lysine. The branched polyethylene glycol)
can be
represented in general form as R(-PEG-OH)m in which R represents the core
moiety, such as
glycerol or pentaerythritol, and m represents the number of arms. Mufti-armed
PEG
CA 02549413 2006-06-02
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molecules, such as those described in U.S. Pat. No. 5,932,462, which is
incorporated by
reference herein in its entirety, can also be used as the polymer backbone.
[0043] Many other polymers are also suitable for the invention. Polymer
backbones that are
non-peptidic and water-soluble, with from 2 to about 300 termini, are
particularly useful in
the invention. ,Examples of suitable polymers include, but are not limited to,
other
poly(alkylene glycols), such as polypropylene glycol) ("PPG"); copolymers of
ethylene
glycol and propylene glycol and the like, poly(oxyethylated polyol),
poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(a-hydroxy
acid),
polyvinyl alcohol), polyphosphazene, polyoxazoline, poly(N-
acryloylmorpholine), such as
described in U.S. Pat. No. 5,629,384, which is incorporated by reference
herein in its entirety,
and copolymers, terpolymers, and mixtures thereof. Although the molecular
weight of each
chain of the polymer backbone can vary, it is typically in the range of from
about 100 Da to
about 100,000 Da, often from about 6,000 Da to about 80,000 Da.
[0044] The "area under the curve" or "AUC", as used herein in the context of
administering a
peptide drug to a patient, is defined as total area under the curve that
describes the
concentration of drug in systemic circulation in the patient as a function of
time from zero to
infinity.
[004'5] The term "half life" or "t1/z", as used herein in the context of
administering a peptide
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. There may be more than one half life
associated with the
peptide drug depending on multiple clearance mechanisms, redistribution, and
other
mechanisms well known in the art. Usually, alpha and beta half lives are
defined such that
the alpha phase is associated with redistribution, and the beta phase is
associated with
clearance. However, with protein drugs that are, for the most part, confined
to the
bloodstream, there can be at least two clearance half lives. For some
glycosylated peptides,
rapid beta phase clearance may be mediated via receptors on macrophages, or
endothelial
cells that recognize terminal galactose, N-acetylgalactosamine, N-
acetylglucosamine,
mannose, or fucose. Slower beta phase clearance may occur via renal glomerular
filtration
for molecules with an effective radius < 2 nm (approximately 68 kD) andlor
specific or non-
specific uptake and metabolism in tissues. GlycoPEGylation may cap terminal
sugars (e.g.,
galactose or N-acetylgalactosamine) and thereby block rapid alpha phase
clearance via
receptors that recognize these sugars. It may also confer a larger effective
radius and thereby
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decrease the volume of distribution and tissue uptake, thereby prolonging the
late beta phase.
Thus, the precise impact of glycoPEGylation on alpha phase and beta phase half
lives will
vary depending upon the size, state of glycosylation, and other parameters, as
is well knownn
in the art. Further explanation of "half life" is found in Pharmaceutical
Biotechnology
(1997, DFA Crommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp
101 -
120).
[0046] The term "glycoconjugation," as used herein, refers to the
enzymatically mediated
conjugation of a modified sugar species to an amino acid or glycosyl residue
of a
polypeptide, e.g., an Factor IX peptide substrate. A subgenus of
"glycoconjugation" is
"glycol-PEGylation," in which the modifying group of the modified sugar is
polyethylene
glycol), and alkyl derivative (e.g., m-PEG) or reactive derivative (e.g., H2N-
PEG, HOOC-
PEG) thereof.
[0047] The terms "large-scale" and "industrial-scale" are used interchangeably
and refer to a
reaction cycle that produces at least about 250 mg, preferably at least about
500 mg, and
more preferably at least about 1 gram of glycoconjugate at the completion of a
single reaction
cycle.
[0048] The term, "glycosyl linking group," as used herein refers to a glycosyl
residue to
which a modifying group (e.g., PEG moiety, therapeutic moiety, biomolecule) is
covalently
attached; the glycosyl linking group joins the modifying group to the
remainder of the
conjugate. In the methods of the invention, the "glycosyl linking group"
becomes covalently
attached to a glycosylated or unglycosylated peptide, thereby linleing the
agent to an amino
acid and/or glycosyl residue on the peptide. A "glycosyl linking group" is
generally derived
from a "modified sugar" by the enzymatic attachment of the "modified sugar" to
an amino
acid and/or glycosyl residue of the peptide. The glycosyl linking group can be
a saccharide-
derived structure that is degraded during formation of modifying group-
modified sugar
cassette (e.g., oxidation-~Schiff base formation-reduction), or the glycosyl
linking group
may be intact. An "intact glycosyl linking group" refers to a linking group
that is derived
from a glycosyl moiety in which the saccharide monomer that links the
modifying group and
to the remainder of the conjugate is not degraded, e.g., oxidized, e.g., by
sodium
metaperiodate. "Intact glycosyl linking groups" of the invention may be
derived from a
naturally occurring oligosaccharide by addition of glycosyl units) or removal
of one or more
glycosyl unit from a parent saccharide structure.
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[0049] The term "targeting moiety," as used herein, refers to species that
will selectively
localize in a particular tissue or region of the body. The localization is
mediated by specific
recognition of molecular determinants, molecular size of the targeting agent
or conjugate,
ionic interactions, hydrophobic interactions and the like. Other mechanisms of
targeting an
agent to a particular tissue or region are knovm to those of skill in the art.
Exemplary
targeting moieties include antibodies, antibody fragments, transferrin, HS-
glycoprotein,
coagulation factors, serum proteins, (3-glycoprotein, G-CSF, GM-CSF, M-CSF,
EPO, serum
proteins (e.g., Factors VII, VIIa, VIII, IX, and X) and the like.
[0050] As used herein, "therapeutic moiety" means any agent useful for therapy
including,
but not limited to, antibiotics, anti-inflammatory agents, anti-tumor drugs,
cytotoxins, and
radioactive agents. "Therapeutic moiety" includes prodrugs of bioactive
agents, constructs in
which more than one therapeutic moiety is bound to a carrier, e.g, multivalent
agents.
Therapeutic moiety also includes proteins and constructs that include
proteins. Exemplary
proteins include, but are not limited to, Granulocyte Colony Stimulating
Factor (GCSF),
Granulocyte Macrophage Colony Stimulating Factor (GMCSF), Interferon (e.g.,
Interferon-
a, -(3, -y), Interleukin (e.g., Interleukin II), serum proteins (e.g., Factors
VII, VIIa, VIII, IX,
and X), Human Chorionic Gonadotropin (HCG), Follicle Stimulating Hormone (FSH)
and
Lutenizing Hormone (LH) and antibody fusion proteins (e.g. Tumar Necrosis
Factor
Receptor ((TNFR)/Fc domain fusion protein)).
[0051] As used herein, "pharmaceutically acceptable carrier" includes any
material, which
when combined with the conjugate retains the conjugates' activity and is non-
reactive with
the subject's immune systems. Examples include, but are not limited to, any of
the standard
pharmaceutical carriers such as a phosphate buffered saline solution, water,
emulsions such
as oiliwater emulsion, and various types of wetting agents. Other carriers may
also include
sterile solutions, tablets including coated tablets and capsules. Typically
such carriers contain
excipients such as starch, milk, sugar, certain types of clay, gelatin,
stearic acid or salts
thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums,
glycols, or other
known excipients. Such carriers may also include flavor and color additives or
other
ingredients. Compositions comprising such carriers are formulated by well
known
conventional methods.
[0052] As used herein, "administering," means oral administration,
administration as a
suppository, topical contact, intravenous, intraperitoneal, intramuscular,
intralesional,
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intranasal or subcutaneous administration, or the implantation of a slow-
release device e.g., a
mini-osmotic pump, to the subject. Adminsitration is by any route including
parenteral, and
transmucosal (e.g., oral, nasal, vaginal, rectal, or transdermal). Parenteral
administration
includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal,
subcutaneous,
intraperitoneal, intraventricular, and intracranial. Moreover, where injection
is to treat a
tumor, e.g., induce apoptosis, administration may be directly to the tumor
and/or into tissues
surrounding the tumor. Other modes of delivery include, but are not limited
to, the use of
liposomal formulations, intravenous infusion, transdermal patches, etc.
[0053] The term "ameliorating" or "ameliorate" refers to any indicia of
success in the
treatment of a pathology or condition, including any objective or subjective
parameter such as
abatement, remission or diminishing of symptoms or an improvement in a
patient's physical
or mental well-being. Amelioration of symptoms can be based on objective or
subjective
parameters; including the results of a physical examination and/or a
psychiatric evaluation.
[0054] The term "therapy" refers to"treating" or "treatment" of a disease or
condition
including preventing the disease or condition from occurring in an animal that
may be
predisposed to the disease but does not yet experience or exhibit symptoms of
the disease
(prophylactic treatment), inhibiting the disease (slowing or arresting its
development),
providing relief from the symptoms or side-effects of the disease (including
palliative
treatment), and relieving the disease (causing regression of the disease).
[0055] The term "effective amount" or "an amount effective to"or a
"therapeutically effective
amount" or any gramatically equivalent term means the amount that, when
administered to an
animal for treating a disease, is sufficient to effect treatment for that
disease.
[0056] The term "isolated" refers to a material that is substantially or
essentially free from
components, which are used to produce the material. For peptide conjugates of
the invention,
the term "isolated" refers to material that is substantially or essentially
free from components
which normally accompany the material in the mixture used to prepare the
peptide conjugate.
"Isolated" and "pure" are used interchangeably. Typically, isolated peptide
conjugates of the
invention have a level of purity preferably expressed as a range. The lower
end of the range
of purity for the peptide conjugates is about 60%, about 70% or about 80% and
the upper end
of the range of purity is about 70%, about 80%, about 90% or more than about
90%.
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[0057] When the peptide conjugates are more than about 90% pure, their
purities are also
preferably expressed as a range. The lower end of the range of purity is about
90%, about
92%, about 94%, about 96% or about 98%. The upper end of the range of purity
is about
92%, about 94%, about 96%, about 98% or about 100% purity.
[0058] Purity is determined by any art-recognized method of analysis (e.g.,
band intensity on
a silver stained gel, polyacrylamide gel electrophoresis, HPLC, or a similar
means).
[0059] "Essentially each member of the population," as used herein, describes
a
characteristic of a population of peptide conjugates of the invention in which
a selected
percentage of the modified sugars added to a peptide are added to multiple,
identical acceptor
sites on the peptide. "Essentially each member of the population" speaks to
the
"homogeneity" of the sites on the peptide conjugated to a modified sugar and
refers to
conjugates ofthe invention, which are at least about 80%, preferably at least
about 90% and
more preferably at least about 95% homogenous.
[0060] "Homogeneity," refers to the structural consistency across a population
of acceptor
moieties to which the modified sugars are conjugated. Thus, in a peptide
conjugate of the
invention in which each modified sugar moiety is conjugated to an acceptor
site having the
same structure as the acceptor site to which every other modified sugar is
conjugated, the
peptide conjugate is said to be about 100% homogeneous. Homogeneity is
typically
expressed as a range. The lower end of the range of homogeneity for the
peptide conjugates
is about 60%, about 70% or about 80% and the upper end of the range of purity
is about 70%, .
about 80%, about 90% or more than about 90%.
[0061] When the peptide conjugates are more than or equal to about 90%
homogeneous, their
homogeneity is also preferably expressed as a range. The lower end of the
range of
homogeneity is about 90%, about 92%, about 94%, about 96% or about 98%. The
upper end
of the range of purity is about 92%, about 94%, about 96%, about 98% or about
100%
homogeneity. The homogeneity of the peptide conjugates is typically determined
by one or
more methods known to those of skill in the art, e.g., liquid chromatography-
mass
spectrometry (LC-MS), matrix assisted laser desorption mass time of flight
spectrometry
(MALDITOF), capillary electrophoresis, and the like. The discussion above is
equally
relevant for other O-glycosylation and N-glycosylation sites.
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[0062] "Substantially uniform glycoform" or a "substantially uniform
glycosylation pattern,"
when referring to a glycopeptide species, refers to the percentage of acceptor
moieties that
are glycosylated by the glycosyltransferase of interest (e.g.,
fucosyltransferase). For
example, in the case of a a1,2 fucosyltransferase, a substantially uniform
fucosylation pattern
exists if substantially all (as defined below) of the Gal(31,4-GIcNAc-R and
sialylated
analogues thereof are fucosylated in a peptide conjugate of the invention. It
will be
a
understood by one of skill in the art, that the starting material may contain
glycosylated
acceptor moieties (e.g., fucosylated Gal[i1,4-GIcNAc-R moieties). Thus, the
calculated
percent glycosylation will include acceptor moieties that are glycosylated by
the methods of
the invention, as well as those acceptor moieties already glycasylated in the
starting material.
[0063] The term "substantially" in the above definitions of "substantially
uniform" generally
means at least about 40%, at least about 70%, at least about 80%, or more
preferably at least
about 90%, and still more preferably at least about 95% of the acceptor
moieties for a
particular glycosyltransferase are glycosylated. For example, if a Factor IX
peptide conjugate
includes a Ser linked glycosyl residues, at least about 70%, 80%, 90%, 95%,
97%, 99%,
99.2%, 99.4%, 99.6%, or more preferably 99.8% of the peptides in the
population will have
the same glycosyl residue covalently bound to the same Ser residue.
[0064] Where substituent groups are specified by their conventional chemical
formulae,
written from left to right, they equally encompass the chemically identical
substituents, which
would result from writing the structure from right to left, e.g., -CH20- is
intended to also
recite -OCHz-.
[0065] The term "alkyl," by itself or as part of another substituent means,
unless otherwise
stated, a straight or branched chain, or cyclic hydrocarbon radical, or
combination thereof,
which may be fully saturated, mono- or polyunsaturated and can include di- and
multivalent
radicals, having the number of carbon atoms designated (i.e. C1-CIO means one
to ten
carbons). Examples of saturated hydrocarbon radicals include, but are not
limited to, groups
such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-
butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-
pentyl, n-
hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one
having one or more
double bonds or triple bonds. Examples of unsaturated alkyl groups include,
but are not
limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-
pentadienyl, 3-(1,4-
pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the highex homologs
and isomers.
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The term "alkyl," unless otherwise noted, is also meant to include those
derivatives of alkyl
defined in more detail below, such as "heteroalkyl." Alkyl groups that are
limited to
hydrocarbon groups are termed "homoalkyl". .
[0066] The term "alkylene" by itself or as part of another substituent means a
divalent radical
derived from an alkane, as exemplified, but not limited, by -CH2CH2CH2CH2-,
and further
includes those groups described below as "heteroalkylene." Typically, an alkyl
(or alkylene)
group will have from 1 to 24 carbon atoms, with those groups having 10 or
fewer carbon
atoms being preferred in the present invention. A "lower alkyl" or "lower
alkylene" is a
shorter chain alkyl or alkylene group, generally having eight or fewer carbon
atoms.
[0067] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are
used in their
conventional sense, and refer to those alkyl groups attached to the remainder
of the molecule
via an oxygen atom, an amino group, or a sulfur atom, respectively.
[0068] The term "heteroalkyl," by itself or in combination with another term,
means, unless
otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon
radical, or
combinations thereof, consisting of the stated number of carbon atoms and at
least one
heteroatom selected from the group consisting of O, N, Si and S, and wherein
the nitrogen
and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may
optionally be
quaternized. The heteroatom(s) O, N and S and Si may be placed at any interior
position of
the heteroalkyl group or at the position at which the alkyl group is attached
to the remainder
of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -
CH2-CH2-NH-
CH3, -CH2-CHZ-N(CH3)-CH3, -CH2-S-CH2-CH3, -CHZ-CHZ,-S(O)-CH3, -CHZ-CH2-S(O)2_
CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. Up to
two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -
CHz-O-
Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another
substituent
means a divalent radical derived from heteroalkyl, as exemplified, but not
limited by, -CHZ-
CHZ-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CHZ-. For heteroalkylene groups,
heteroatoms
can also occupy either or both of the chain termini (e.g., alkyleneoxy,
alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and
heteroalkylene
linking groups, no orientation of the linking group is implied by the
direction in which the
formula of the linking group is written. For example, the formula -C(O)2R'-
represents both
-C(O)2R'- and -R' C(O)2-.
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[0069] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in
combination with
other terms, represent, unless otherwise stated, cyclic versions of "alkyl"
and "heteroalkyl",
respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at
which the heterocycle is attached to the remainder of the molecule. Examples
of cycloalkyl
include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-
cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not
limited to, 1-
(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-
morpholinyl, 3-
morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,
tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.
[0070] The terms "halo" or "halogen," by themselves or as part of another
substituent, mean,
mless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
Additionally, terms
such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For
example, the
team "halo(C1-C4)alkyl" is mean to include, but not be limited to,
trifluoromethyl, 2,2,2-
trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.
[0071] The term "aryl" means, unless otherwise stated, a polyunsaturated,
aromatic,
substituent that can be a single ring or multiple rings (preferably from 1 to
3 rings), which are
fused together or linlced covalently. The term "heteroaryl" refers to aryl
groups (or rings) that
contain from one to four heteroatoms selected from N, O, and S, wherein the
nitrogen and
sulfur atoms are optionally oxidized, and the nitrogen atoms) are optionally
quaternized. A
heteroaryl group can be attached to the remainder of the molecule through a
heteroatom.
Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-
naphthyl, 2-naphthyl,
4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-
imidazolyl,,
pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-
isoxazolyl, 4-
isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-
furyl, 2-thienyl, 3-
thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-
benzothiazolyl, purinyl,
2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-
quinoxalinyl, 3-
quinolyl, tetrazolyl, benzo[b]fiuanyl, benzo[b]thienyl, 2,3-
dihydrobenzo[1,4]dioxin-6-yl,
benzo[1,3]dioxol-5-yl and 6-quinolyl. Substituents for each ofthe above noted
aryl and
heteroaryl ring systems are selected from the group of acceptable substituents
described
below.
[0072] For brevity, the term "aryl" when used in combination with other terms
(e.g., aryloxy,
arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined
above. Thus, the
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teen "arylalkyl" is meant to include those radicals in which an aryl group is
attached to an
alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including
those alkyl groups
in which a carbon atom (e.g., a methylene group) has been replaced by, for
example, an
oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,
and the
like).
[0073] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and
"heteroaxyl") is
meant to include both substituted and unsubstituted forms of the indicated
radical. Preferred
substituents for each type of radical are provided below.
[0074] Substituents for the alkyl and heteroalkyl radicals (including those
groups often
referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl,
cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are generically
referred to as "alkyl
group substituents," and they can be one or more of a variety of groups
selected from, but not
limited to: -OR', =O, =NR', N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -
OC(O)R', -
C(O)R', -COZR', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -
NR"C(O)2R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")-NR."', -S(O)R', -S(O)ZR', -
S(O)ZNR'R", -NRSOZR', -GN and NOZ in a number ranging from zero to (2m'+1),
where
m' is the total number of carbon atoms in such radical. R', R", R"' and R""
each preferably
independently refer to hydrogen, substituted or unsubstituted heteroalkyl,
substituted or
unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or
unsubstituted alkyl,
alkoxy or thioallcoxy groups, or arylalkyl groups. When a compound of the
invention
includes more than one R group, for example, each of the R groups is
independently selected
as are each R', R", R"' and R"" groups when more than one of these groups is
present. When
R' and R" are attached to the same nitrogen atom, they can be combined with
the nitrogen
atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to
include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of
substituents,
one of skill in the art will understand that the term "alkyl" is meant to
include groups
including carbon atoms bound to groups other than hydrogen groups, such as
haloalkyl (e.g.,
-CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHzOCH3, and the
like).
[0075] Similar to the substituents described for the alkyl radical,
substituents for the aryl and
heteroaryl groups are generically referred to as "aryl group substituents."
The substituents
are selected from, for example: halogen, -OR', =O, =NR', =N-OR', -NR'R", -SR',
-halogen,
-SiR'R"R"', -OC(O)R', -C(O)R', -C02R', -CONR'R", -OC(O)NR'R", -NR"C(O)R',
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-~~-C(O)s»R»>~ -~»C(O)ZR~~ -~-C(~~R»R»>)-~»»~ -~-C(~~R»)-~»>~ _
S(O)R', -S(O)zR', -S(O)ZNR'R", -NRSOZR', -CN and N02, -R', -N3, -CH(Ph)2,
fluoro(C1-
C4)alkoxy, and fluoro(C1-C~.)alkyl, in a number ranging from zero to the total
number of open
valences on the aromatic ring system; and where R', R", R"' and R"" are
preferably
independently selected from hydrogen, substituted or unsubstituted alkyl,
substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl and substituted
or unsubstituted
heteroaryl. When a compound of the invention includes more than one R group,
for example,
each of the R groups is independently selected as are each R', R", R"' and R""
groups when
more than one of these groups is present. In the schemes that follow, the
symbol X
represents "R" as described above.
[0076] Two of the substituents on adjacent atoms of the aryl or heteroaryl
ring may
optionally be replaced with a substituent of the formula -T-C(O)-(CRR')q-U-,
wherein T and
U are independently NR-, -O-, -CRR'- or a single bond, and q is an integer of
from 0 to 3.
Alternatively, two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may
optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein
A and B are
independently -CRR'-, -O-, -NR-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single
bond, and r is
an integer of from 1 to 4. One of the single bonds of the new ring so formed
may optionally
be replaced with a double bond. Alternatively, two of the substituents on
adjacent atoms of
the aryl or heteroaryl ring may optionally be replaced with a substituent of
the formula -
(CRR')s-X-(CR"R"')d-, where s and d are independently integers of from 0 to 3,
and X is -
O-, -NR'-, -S-, -S(O)-, -S(O)Z-, or-S(O)2NR'-. The substituents R, R', R" and
R"' are
preferably independently selected from hydrogen or substituted or
unsubstituted (C1-
C6)alkyl.
[0077] As used herein, the term "heteroatom" is meant to include oxygen (O),
nitrogen (N),
sulfur (S) and silicon (Si).
Introduction
[0078] As described above, Factor IX is vital in the blood coagulation
cascade. The structure
and sequence of Factor IX is provided in FIG. 1. A deficiency of Factor IX in
the body
characterizes a type of hemophilia (type B). Treatment of this disease is
usually limited to
intravenous tranfusion of human plasma protein concentrates of Factor IX.
However, in
addition to the practical disadvantages of time and expense, transfusion of
blood concentrates
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involves the risk of transmission of viral hepatitis, acquired immune
deficiency syndrome or
thromboembolic diseases to the recipient.
[0079] While Factor IX has demonstrated itself as an important and useful
compound for
therapeutic applications, present methods for the production of Factor IX from
recombinant
cells (U.S. Patent No. 4,770,999) results in a product with a rather short
biological half life
and an inaccurate glycosylation pattern that could potentially lead to
immunogenicity, loss of
function, an increased need for both larger and more frequent doses in order
to achieve the
same effect, and the like.
[0080] To improve the effectiveness of recombinant Factor IX used for
therapeutic purposes,
the present invention provides conjugates of glycosylated and unglycosylated
Factor IX
peptides with polymers, e.g., PEG (m-PEG), PPG (m-PPG), etc. The conjugates
may be
additionally or alternatively modified by further conjugation with diverse
species such as
therapeutic moieties, diagnostic moieties, targeting moieties and the like.
[0081] The conjugates of the invention are formed by the enzymatic attachment
of a
modified sugar to the glycosylated or unglycosylated peptide. Glycosylation
sites and
glycosyl residues provide loci for conjugating modifying groups to the
peptide, e.g., by
glycoconjugation. An exemplary modifying group is a water-soluble polymer,
such as
polyethylene glycol), e.g., methoxy-polyethylene glycol). Modification of the
Factor IX
peptides can improve the stability and retention time of the recombinant
Factor IX in a
patient's circulation, and/or reduce the antigenicity of recombinant Factor
IX.
[0082] The methods of the invention make it possible to assemble peptides and
glycopeptides
that nave a substantially homogeneous derivatization pattern. The enzymes used
in the
invention are generally selective for a particular amino acid residue,
combination of amino
acid residues, or particular glycosyl residues of the peptide. The methods are
also practical
for large-scale production of modified peptides and glycopeptides. Thus, the
methods of the
invention provide a practical means for large-scale preparation of
glycopeptides having
preselected uniform derivatization patterns.
[0083] The present invention also provides conjugates of glycosylated and
unglycosylated
peptides with increased therapeutic half life due to, for example, reduced
clearance rate, or
reduced rate of uptake by the immune or reticuloendothelial system (RES).
Moreover, the
methods of the invention provide a means for masking antigenic determinants on
peptides,
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thus reducing or eliminating a host immune response against the peptide.
Selective
attachment of targeting agents can also be used to target a peptide to a
particular tissue or cell
surface receptor that is specific for the particular targeting agent.
Tlie Conjugates
[0084] In a first aspect, the present invention provides a conjugate between a
selected
modifying group and a Factor IX peptide.
[0085] The link between the peptide and the modifying group includes a
glycosyl linking
group interposed between the peptide and the selected moiety. As discussed
herein, the
selected moiety is essentially any species that can be attached to a
saccharide unit, resulting
in a "modified sugar" that is recognized by an appropriate transferase enzyme,
which appends
the modified sugar onto the peptide. The saccharide component of the modified
sugar, when
interposed between the peptide and a selected moiety, becomes a "glycosyl
linking group,"
e.g., an "intact glycosyl linking group." The glycosyl linking group is formed
from any
mono- or oligo-saccharide that, after modification with the modifying group,
is a substrate for
an enzyme that adds the modified sugar to an amino acid or glycosyl residue of
a peptide.
[0086] The glycosyl linking group can be, or can include, a saccharide moiety
that is
degradatively modified before or during the addition of the modifying group.
For example,
the glycosyl linking group can be derived from a saccharide residue that is
produced by
oxidative degradation of an intact saccharide to the corresponding aldehyde,
e.g., via the
action of metaperiodate, and subsequently converted to a Schiff base with an
appropriate
amine, which is then reduced to the corresponding amine.
[0087] Exemplary conjugates of the invention correspond to the general
structure:
Peptide Sugar Linker Sugar Agent
U s ~' ' ~ t
a b c d
[0088] in which the symbols a, b, c, d and s represent a positive, non-zero
integer; and t is
either 0 or a positive integer. The "agent" is a therapeutic agent, a
bioactive agent, a
detectable label, water-soluble moiety (e.g., PEG, m-PEG, PPG, and m-PPG) or
the like. The
"agent" can be a peptide, e.g., enzyme, antibody, antigen, etc. The linker can
be any of a
22
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WO 2005/055950 PCT/US2004/041070
wide array of linking groups, ihfi°a. Alternatively, the linker may be
a single bond or a "zero
order linker."
[0089] In an exemplary embodiment, the selected modifying group is a water-
soluble
polymer, e.g., m-PEG. The water-soluble polymer is covalently attached to the
peptide via a
glycosyl linking group. The glycosyl linking group is covalently attached to
an amino acid
residue or a glycosyl residue of the peptide. The invention also provides
conjugates in which
an amino acid residue and a glycosyl residue are modified with a glycosyl
linking group.
[0090] An exemplary water-soluble polymer is polyethylene glycol), e.g.,
methoxy-
poly(ethylene glycol). The polyethylene glycol) used in the present invention
is not
restricted to any particular form or molecular weight range. For unbranched
polyethylene
glycol) molecules the molecular weight is preferably between 500 and 100,000.
A molecular
weight of 2,000-60,000 daltons is preferably used and more preferably of from
about 5,000 to
about 30,000 daltons.
[0091] In another embodiment the polyethylene glycol) is a branched PEG having
more than
one PEG moiety attached. Examples of branched PEGs are described in U.S. Pat.
No.
5,932,462; U.S. Pat. No. 5,342,940; U.S. Pat. No. 5,643,575; U.S. Pat. No.
5,919,455; U.S.
Pat. No. 6,113,906; U.S. Pat. No. 5,183,660; WO 02109766; Kodera Y.,
Bioconjugate
Cl~emist~y 5: 283-288 (1994); and Yamasaki et al., Agric. Biol. Chem., 52:
2125-2127, 1998.
Additional useful branched polymer species are set forth herein.
[0092] In a preferred embodiment the molecular weight of each polyethylene
glycol) of the
branched PEG is equal to or greater than about 2,000, 5,000, 10,000, 15,000,
20,000, 40,000,
50,000 and 60,000 daltons.
[0093] In addition to providing conjugates that are formed through an
enzymatically added
glycosyl linking group, the present invention provides conjugates that are
highly homogenous
in their substitution patterns. Using the methods of the invention, it is
possible to form
peptide conjugates in which essentially all of the modified sugar moieties
across a population
of conjugates of the invention are attached to multiple copies of a
structurally identical amino
acid or glycosyl residue. Thus, in a second aspect, the invention provides a
peptide conjugate
having a population of water-soluble polymer moieties, which are covalently
bound to the
peptide through an intact glycosyl linking group. In a preferred conjugate of
the invention,
essentially each member of the population is bound via the glycosyl linking
group to a
23
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WO 2005/055950 PCT/US2004/041070
glycosyl residue of the peptide, and each glycosyl residue of the peptide to
which the
glycosyl linking group is attached has the same structure.
[0094] Also provided is a peptide conjugate having a population of water-
soluble polymer
moieties covalently bound thereto through a glycosyl linking group. In a
preferred
embodiment, essentially every member of the population of water soluble
polymer moieties
is bound to an amino acid residue of the peptide via a glycosyl linking group,
and each amino
acid residue having a glycosyl linking group attached thereto has the same
structure.
[0095] The present invention also provides conjugates analogous to those
described above in
which the peptide is conjugated to a therapeutic moiety, diagnostic moiety,
targeting moiety,
toxin moiety or the like via an intact glycosyl linking group. Each of the
above-recited
moieties can be a small molecule, natural polymer (e.g., polypeptide) or
synthetic polymer.
The peptides of the invention include at least one N-, or O-linked
glycosylation site, which is
glycosylated with a glycosyl residue that includes a PEG moiety. The PEG is
covalently
attached to the Factor IX peptide via an intact glycosyl linking group. The
glycosyl linking
group is covalently attached to either an amino acid residue or a glycosyl
residue of the Facot
IX peptide. Alternatively, the glycosyl linking group is attached to one or
more glycosyl
units of a glycopeptide. The invention also provides conjugates in which the
glycosyl linking
group is attached to both an amino acid residue and a glycosyl residue.
[0096] In an exemplary embodiment, the Factor IX peptide comprises a moiety
having the
formula;
[0097] In the formula above, D is a member selected from -OH and Rl-L-HN-; G
is a
member selected from Rl-L- and -C(O)(C1-C6)alkyl; Rl is a moiety comprising a
member
selected a moiety comprising a straight-chain or branched polyethylene glycol)
residue; and
L is a linker which is a member selected from a bond, substituted or
unsubstituted alkyl and
24
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WO 2005/055950 PCT/US2004/041070
substituted or unsubstituted heteroalkyl,such that when D is OH, G is Rl-L-,
and when G is -
C(O)(Cl-C6)alkyl, D is Rl-L-NH-.
[0100] In one embodiment, a Rl-L has the formula:
R~-HN
a
O
wherein a is an integer from 0 to 20.
[0101] In an exemplary embodiment, Rl has a structure that is a member
selected from:
O O
S-(CHZCHaO)eCH3 , ~ S-(CHZCHaO)eCH3
9
NHC(O)CHZCHz(OCHaCHz)fOCH3 NHC(O)OCHzCHZ(OCHzCHz)fOCH3
O O
q O-(CH2CH20)eCH3 , and ~ q O-(CHzCHzO)BCH3
NHC(O)CHZCHz(OCHZCHZ)~OCH3 ' NHC(O)OCHZCHZ(OCHZCHZ)fOCH3
wherein a and f are integers independently selected from 1 to 2500; and q is
an integer from 1
to 20. In other embodiments Rl has a structure that is a member selected from:
CA 02549413 2006-06-02
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O '
NHC(O)OCH2CH2(OCHaCHZ)eOCH3
9
NHS '
HN
HC(O)OCH2CH2(OCHzCH2)fOCH3
O
O
NHC(O)CHZCH2(OCHZCH~)eOCH3
NHZ
HN
HC(O)CH~CH~(OCHzCH2)fOCH3
0
NHC(0)CHZCHZ(OCH~CHZ)BOCH3
and
NHC(O)CH~CH2(OCH2CHz)fOCH3
~NHC(O)CHZCH2(OCH~CH~)fOCH3
9~
O
NHC(O)OCH2CH~(OCHZCHz)eOCH3
NHC(O)OCHzCH2(OCH2CH~)fOCH3
~NHC(O)OCH2CH2(OCH~CH~)fOCH3
9~
O
wherein e, f and f are integers independently selected from 1 to 2500; and q
and q' are
integers independently selected from 1 to 20.
[0102] In still another embodiment, the invention provides a Factor IX peptide
conjugate
wherein Rl has a structure that is a member selected from:
26
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NHC(O)OCHZCH~(OCHzCH2)e0CH3
' O
NHC(O)OCH2CH2(OCHzCHz)fOCH3
NH
N HZ
~NHC(0)OCHzCH2(OCH2CH~)fOCH3 , and
4
NHC(O)CHZCHZ(OCHZCH~)eOCH3
O
NHC(O)CH2CH~(OCHzCH2)fOCH3
NHJ
NHz q..
HC(O)CHZCH2(OCHzCH~)fOCH3
O
9~
wherein e, f and f are integers independently selected from 1 to 2500; and q,
q' and q"are
integers independently selected from 1 to 20.
[0103] In other embodiments, RI has a structure that is a member selected
from:
-C(O)CH~CHz(OCH2CH~)eOCH3 ; and
-C(O)OCH2CH2(OCH2CH2)fOCH3
wherein a and f are integers independently selected from 1 to 2500.
[0104] In another exemplary embodiment, the invention provides a peptide
comprising a
moiety having the formula:
3al-
The Gal can be attached to an amino acid or to a glycosyl residue that is
directly or indirectly
(e.g., through a glycosyl residue) attached to an amino acid.
[0105] In other embodiments, the moiety has the formula:
27
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-Gal-GaINAc-
The Gal can be attached to an amino acid or to a glycosyl residue that is
directly or indirectly
(e.g., through a glycosyl residue) attached to an amino acid.
[0106] In an exemplary embodiment, this structure is associated with
glycoPEGylation of
an O-glycosylation site on Factor IX (FIG. 2B).
[0107] In a still further exemplary embodiment the peptide comprises a moiety
according
to the formula
3al-GaINAc-AA
wherein AA is an amino acid residue of said peptide and, in each of the above
structures, D
and G are as described herein.
[0108] Exemplary amino acid residues of the peptide at which one or more of
the above
species can be conjugated include serine and threonine, e.g., serine 53 or 61
or threonine 159,
1'62 or 172 of SEQ. ID. NO:1.
[0109] In another exemplary embodiment, the invention provides a Factor IX
conjugate
that includes a glycosyl residue having the formula:
(Fuc); ari ~[GIcNAc-(Gal)a]e (Sia)I - (R)~ ~r.
-~ i ~ ~~[GIcNAc-(Gal)h]f-(Sia)m (R)'~~S
GIcNAc-GIcNAc-Man
I Mari ~[GIcNAc-(Gal)]g (Sia)I- (R),~ ~t
~ ~[GIcNAc-(Gal)d]h (Sia)m (R)~,~u
9
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[0110] wherein a, b, c, d, i, r, s, t, and a are integers independently
selected from 0 and 1.
The index q is 1. The indices e, f, g, and h are independently selected from
the integers from
0 to 6. The indices j, k, l, and m are independently selected from the
integers from 0 and 100.
The indices v, w, x, and y are independently selected from 0 and 1, and at
least one of v, w, x
and y is 1. The symbol AA represents an amino acid residue of the Factor IX
peptide.
[0111] The symbol Sia-(R) represents a~group that has the formula:
D
H
OH
wherein D is selected from -OH and R1-L-HN-. The symbol G is represents R1-L-
or
-C(O)(C1-C6)alkyl. Rl represents a moiety that includes a straight-chain or
branched
poly(ethylene~glycol) residue. L is a linker which is a member selected from a
bond,
substituted or unsubstituted alkyl and substituted or unsubstituted
heteroalkyl. In general,
when D is OH, G is Rl-L-, and when G is -C(O)(C1-C6)alkyl, D is Rl-L-NH-.
[0112] In aaiother exemplary embodiment, the PEG-modified sialic acid moiety
in the
conjugate of the invention has the formula:
OH
I
HOHzC COOH
H0~' N O O
~0
C \ S HO
in which the index "s" represents an integer from 0 to 20, and n is an integer
from 1 to2500.
In a selected embodiment, s is 1, and the PEG is approximately 20 kD.
[0113] In a still further exemplary embodiment, the PEG-modified sialic acid
in has the
formula:
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OH
HOHZC COOH
HO~~.~N O O--5
~L
HO
0 O
~,0/CH3
in which L is a substituted or unsubstituted alkyl or substituted or
unsubstituted heteroalkyl
linker moiety joining the sialic acid moiety and the PEG moiety.
[0114] In an exemplary embodiment, in which the glycosyl residue has the
structure set
forth above, it is conjugated to one or both Asn 157 and Asn 167.
[0115] Factor IX has been cloned and sequenced. Essentially any Factor IX
peptide having
any sequence is of use as the Factor IX peptide component of the conjugates of
the present
invention. In an exemplary embodiment, the peptide has the sequence presented
herein as
SEQ ID NO:1:
YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPC
LNGGSCKDD1NSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCT
EGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQ
STQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETG
VKITVVAGEHNIEETEHTEQKRNVIRIIPHFINYNAAINKYNHDIALLELDEPLVLNSYV
TPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFT
IYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTK
VSRYVNWIKEKTKLT.
[0116] The present invention is in no way limited to the sequence set forth
herein. Factor
IX variants are well known in the art, as described in, for example, U.S.
Patent Nos.
4,770,999, 5,521,070 in which a tyrosine is replaced by an alanine in the
first position, U.S.
Patent No. 6,037,452, in which Factor XI is linked to an alkylene oxide group,
and U.S.
Patent No. 6,046,380, in which the DNA encoding Factor IX is modified in at
least one splice
site. As demonstrated herein, variants of Factor IX axe well known in the art,
and the present
disclosure encompasses those variants known or to be developed or discovered
in the future.
[0117] Methods for determining the activity of a mutant or modified Factor IX
can be
carried out using the methods described in the art, such as a one stage
activated partial
CA 02549413 2006-06-02
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thromboplastin time assay as described in, for example, Biggs (1972, Human
Blood
Coagulation Haemostasis and Thrombosis (Ed. 1), Oxford, Blackwell, Scientific,
pg. 614).
Briefly, to assay the biological activity of a Factor IX molecule developed
according to the
methods of the present invention, the assay can be performed with equal
volumes of
activated partial thromboplastin reagent, Factor IX deficient plasma isolated
from a patient
with hemophilia B using sterile phlebotomy techniques well known in the art,
and normal
pooled plasma as standard, or the sample. In this assay, one unit of activity
is defined as that
amount present in one milliliter of normal pooled plasma. Further, an assay
for biological
activity based on the ability of Factor IX to reduce the clotting time of
plasma from Factor
IX-deficient patients to normal can be performed as described in, for example,
Proctor and
Rapaport (Amef°. J. Clivt. Path. 36: 212 (1961).
[0118] The peptides of the invention include at least one N-linked or O-linked
glycosylation site, at least one of which is conjugated to a glycosyl residue
that includes a
PEG moiety. The PEG is covalently attached to the peptide via an intact
glycosyl linlcing
group. The glycosyl linlcing group is covalently attached to either an amino
acid residue or a
glycosyl residue of the peptide. Alternatively, the glycosyl linlcing group is
attached to one
or more glycosyl units of a glycopeptide. The invention also provides
conjugates in which
the glycosyl linking group is attached to both an amino acid residue and a
glycosyl residue.
[0119] The PEG moiety is attached to an intact glycosyl linker directly, or
via a non-
glycosyl linker, e.g., substituted or unsubstituted alkyl, substituted or
unsubstituted
heteroalkyl.
Modifred Sugars
[0120] The present invention uses modified sugars and modified sugar
nucleotides to form
conjugates of the modified sugars. In modified sugar compomds of the
invention, the sugar
moiety is preferably a saccharide, a deoxy-saccharide, an amino-saccharide, or
an N-acyl
saccharide. The term "saccharide" and its equivalents, "saccharyl," "sugar,"
and "glycosyl"
refer to monomers, dimers, oligomers and polymers. The sugar moiety is also
functionalized
with a modifying group. The modifying group is conjugated to the sugar moiety,
typically,
through conjugation with an amine, sulfhydryl or hydroxyl, e.g., primary
hydroxyl, moiety on
the sugar. In an exemplary embodiment, the modifying group is attached through
an amine
31
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WO 2005/055950 PCT/US2004/041070
moiety on the sugar, e.g., through an amide, a urethane or a urea that is
formed through the
reaction of the amine with a reactive derivative of the modifying group.
j0121] Any sugar can be utilized as the sugar core of the conjugates of the
invention.
Exemplary sugar cores that are useful in forming the compositions of the
invention include,
but are not limited to, glucose, galactose, mannose, fucose, and sialic acid.
Other useful
sugars include amino 'sugars such as glucosamine, galactosamine; mannosamine,
the 5-amine
analogue of sialic acid and the like. The sugar core can be a structure found
in nature or it
can be modified to provide a site for conjugating the modifying group. For
example, in one
embodiment, the invention provides a sialic acid derivative in which the 9-
hydroxy moiety is
replaced with an amine. The amine is readily derivatized with an activated
analogue of a
selected modifying group.
[0122] In an exemplary embodiment, the invention utilizes a modified sugar
amine that has
the formula:
G
NH-L-R~
in which G is a glycosyl moiety, L is a bond or a linker and Rl is the
modifying group.
Exemplary bonds are those that are formed between an NH2 Oll the glycosyl
moiety and a
group of complementary reactivity on the modifying group. Thus, exemplary
bonds include,
but are not limited to NHRI, ORI, SR1 and the like. For example, when Rl
includes a
carboxylic acid moiety, this moiety may be activated and coupled with an NH2
moiety on the
glycosyl residue affording a bond having the structure NHC(O)Rl. Similarly,
the OH and SH
groups can be converted to the corresponding ether or thioether derivatives,
respectively.
[0123] Exemplary linkers include alkyl and heteroalkyl moieties. The linkers
include
linking groups, for example acyl-based linking groups, e.g., -C(O)NH-, -
OC(O)NH-, and the
like. The linking groups are bonds formed between components of the species of
the
invention, e.g., between the glycosyl moiety and the linker (L), or between
the linker and the
modifying group (Rl). Other linking groups are ethers, thioethers and amines.
For example,
in one embodiment, the linker is an amino acid residue, such as a glycine
residue. The
carboxylic acid moiety of the glycine is converted to the corresponding amide
by reaction
with an amine on the glycosyl residue, and the amine of the glycine is
converted to the
32
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corresponding amide or urethane by reaction with an activated carboxylic acid
or carbonate
of the modifying group.
[0124] Another exemplary linker is a PEG moiety or a PEG moiety that is
functionalized
with an amino acid residue. The PEG is to the glycosyl group through the amino
acid residue
at one PEG terminus and bound to Rl through the other PEG terminus.
Alternatively, the
amino acid residue is bound to R~ and the PEG terminus not bound to the amino
acid is
bound to the glycosyl group.
(0125] An exemplary species for NH-L-Rl has the formula:
-NH{C(O)(CH2)aNH}S{C(O)(CH2)b(OCHZCHZ)~O(CH2)aNH}tRl, in which the indices s
and t
are independently 0 or 1. The indices a, b and d are independently integers
from 0 to 20, and
c is an integer from 1 to 2500. Other similar linkers are based on species in
which the -NH
moiety is replaced by another group, for example, -S, -O or -CH2.
[0126] ~ More particularly, the invention utilizes compounds in which NH-L-Rl
is:
NHC(O)(CHZ)$NHC(O)(CH2)b(OCHZCHZ)~O(CH2)aNHRI,
NHC(O)(CHZ)b(OCH2CH2)~O(CI-I2)aNHRI, NHC(O)O(CHz)b(OCH2CH2)~O(CH2)aNHRI,
NH(CH2)aNHC(O)(CH2)b(OCH2CH2)~O(CH2)aNHR.I, NHC(O)(CI-I2)aNHRI,
NH(CH2)aNHRI, and NHRI. In these formulae, the indices a, b and d are
independently
selected from the integers from 0 to 20, preferably from 1 to 5. The index c
is an integer
from 1 to 2500.
[0127] In the discussion that follows the invention is illustrated by
reference to the use of
selected derivatives of sialic acid. Those of skill in the art will recognize
that the focus of the
discussion is for clarity of illustration and that the structures and
compositions set forth are
generally applicable across the genus of saccharide groups, modified
saccharide groups,
activated modified saccharide groups and conjugates of modified saccharide
groups.
[0128] In an illustrative embodiment, G is sialic acid and selected compounds
of use in the
invention have the formulae:
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HOOC 0 CH(OH)CH(OH)CHZOH
HOOC O CH(OH)CH(OH)CH20H HO
HO ~NHC(O)(CHZ)aNHR~
'NHC(0)(CHz)aNHC(O)(CHZ)e(OCHzCHZ)~0(CHZ)dNHR~ .
IOH
HOOC 0 CH(OH)CH(OH)CHzOH
HOOC 0 CH(OH)CH(OH)CHZOH HO
HO NH(CHz)aNHR1
~NHC(O)(CHz)aNHC(O)O(CHZ)ti(OCHzCH2)~0(CH2)dNHR~ ' OH
OH
and
OH
[0129] ~ As those of skill in the art will appreciate, the sialic acid moiety
in the exemplary
compounds above can be replaced with any other amino-saccharide including, but
not limited
to, glucosamine, galactosamine, mannosamine, their N-acetyl derivatives, and
the like.
[0130] In another illustrative embodiment, a primary hydroxyl moiety of the
sugar is
functionalized with the modifying group. For example, the 9-hydroxyl of sialic
acid can be
converted to the corresponding amine and functionalized to provide a compound
according to
the invention. Formulae according to this embodiment include:
HOOC O CH(OH)CH(OH)CH20H HOOC O CH(OH)CH(OH)CHZOH
HO HO
NH(CHZ)aNHC(O)O(CHZ)e(OCHzCHz)~0(CHz)dNHR~ ~NHC(0)(CHa)b(OCHzCHz)~0(CHz)dNHR~
OH IOH
HOOC O CH(OH)CH(OH)CHZOH
HOOC 0 CH(OH)CH(OH)CHZOH '
HO
HO
NHC(O)0(CHa)b(OCHzCHa)~O(CHz)aNHR~
NHC(O)0(CHz)b(OCHZCHz)~0(CHz)dNHRj '
OH
OH
HOOC O CH(OH)CH(OH)CH20H
HO
~NHR~
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HOOC 0 CH(OH)CH(OH)CHzNHC(O)(CHZ)aNHC(O)(CH2)b(OCHZCHZ)~0(CH2)dNHR~
HO
'NHC(0)CH3
IOH
HOOC 0 CH(OH)CH(OH)CHZNHC(0)(CHZ)aNHC(0)0(CH2)b(OCHZCHZ)~O(CH2)dNHR~
HO
'NHC(0)CH3 '
IOH
HOOC 0 CH(OH)CH(OH)CHzNH(CHz)aNHC(0)0(CHz)b(OCHaCHz)~0(CHZ)dNHR~
HO
_NHC(O)CH3
IOH
HOOC 0 CH(OH)CH(OH)CHZNHC(O)(CHZ)aNHR~ HOOC 0 CH(OH)CH(OH)CHZNH(CHz)aNHR~
HO
HO Y 'NHC(O)CH3
'NHC(0)CH3 . ,
OH
OH
HOOC O CH(OH)CH(OH)CH2NHC(0)(CHZ)b(OCHzCHz)~0(CHZ)dNHR~ ,
HO
'NHC(0)CH3 HOOC O CH(OH)CH(OH)CHZNHR~
IOH HO
HOOC 0 CH(OH)CH(OH)CHZNHC(O)0(CH2)b(OCH2CHz)~0(CHz)dNHRj NHC(O)CH3
HO ~ OH
'NHC(0)CH3
OH
[0131] In a further exemplary embodiment, the invention utilizes modified
sugars in which
the 6-hydroxyl position is converted to the corresponding amine moiety, which
bears a linker-
modifying group cassette such as those set forth above. Exemplary saccharyl
groups that can
be used as the core of these modified sugars include Gal, GaINAc, Glc, GIcNAc,
Fuc, Xyl,
Man, and the like. A representative modified sugar according to this
embodiment has the
formula:
Rs
R3
~O
R4 R~
R5
in which R3-RS and R~ are members independently selected from H, OH, C(O)CH3,
NH, and
NH C(O)CH3, R6 is ORI, NHRI or NH-L-Rl, which is as described above.
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i
[0132] Selected conjugates of use in the invention are based on mannose,
galactose or
glucose, or on species having the stereochemistry of mannose, galactose or
glucose. The
general formulae of these conjugates are:
Rs
O
R3um,
OH
R4 R5 _ : and
[0133] In another exemplary embodiment, the invention utilizes compounds as
set forth
above that are activated as the corresponding nucleotide, sugars. Exemplary
sugar nucleotides
that are used in the present invention in their modified form include
nucleotide mono-, di- or
triphosphates or analogs thereof. In a preferred embodiment, the modified
sugar nucleotide is
selected from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Even more
preferably,
the sugar nucleotide portion of the modified sugar nucleotide is selected from
UDP-galactose,
UDP-galactosamine, UDP-glucose, UDP-glucosamine, GDP-mamlose, GDP-fucose, CMP-
sialic acid, or CMP-NeuAc. In an exemplary embodiment, the nucleotide
phosphate is
attached to C-1.
[0134] Thus, in an illustrative embodiment in which the glycosyl moiety is
sialic acid, the
invention utilizes compounds having the formulae:
HOOC O CH(OH)CH(OH)CH20H
H N N w0 ,~ov\O~P~ L-Ri ~ and
o-
N~ ~ OH
\\ OH
O HO
HOOC O CH(OH)CH(OH)CH2NH-L~-R~
~\ / 0 II I
O \ ~
H N ~ N ,~~ ~'~~~\\\O~P ~_ ~NHC(O)CH3
N~ IOH
OH
O HO
in which L-R1 is as discussed above, and L1-R1 represents a linker bound to
the modifying
group. As with L, exemplary linker species according to L1 include a bond,
alkyl or
heteroallcyl moieties. Exemplary modified sugar nucleotide compounds according
to these
embodiments are set forth in FIG. 7 and FIG. 8.
36
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WO 2005/055950 PCT/US2004/041070
[0135] In another exemplary embodiment, the invention provides a conjugate
formed
between a modified sugar of the invention and a substrate Factor IX peptide.
In this
embodiment, the sugar moiety of the modified sugar becomes a glycosyl linking
group
interposed between the substrate and the modifying group. An exemplary
glycosyl linking
group is an intact glycosyl linking group, in which the glycosyl moiety or
moieties forming
the linking group are not degraded by chemical (e.g., sodium metaperiodate) or
enzymatic
processes (e.g., oxidase). Selected conjugates of the invention include a
modifying group
that is attached to the amine moiety of an amino-saccharide, e.g.,
mannosamine, glucosamine,
galactosamine, sialic acid etc. Exemplary modifying group-intact glycosyl
linking group
cassettes according to this motif are based on a sialic acid structure, such
as those having the
formulae:
~ ~OH R~-~~.-HN
H O' Y
R~-~~-NH~ ' ; and ~Hsl
[0136] In the formulae above, Rl and Ll are as described above.
[0137] In still a further exemplary embodiment, the conjugate is formed
between a
substrate Factor IX and a saccharyl moiety in which the modifying group is
attached through
a linker at the 6-carbon position of the saccharyl moiety. Thus, illustrative
conjugates
according to this embodiment have the formulae:
R~ ~/HN O N-L R1
H
and
Rs ~ ~ Rs
r
R4
in which the radicals are as discussed above. Those of skill will appreciate
that the modified
saccharyl moieties set forth above can also be conjugated to a substrate
through an oxygen or
nitrogen atom at the 2, 3, 4, or 5 carbon atoms.
[0138] Illustrative compounds of use in this embodiment include compounds
having the
formulae:
37
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R' O CH2NHC(O)(CHZ)aNHC(0)(CHZ)b(OCHZCH2)~0(CHZ)dNHR~
Rs ~ ~Ra
R°
R7 O CHaNHC(O)(CH2)aNHC(O)0(CHZ)y(OCHZCHZ)~O(CH2)dNHRt
Rs ~ ~Rs
R~
R' O CNZNH(CH2)aNHC(0)0(CHZ)y(OCHZCHZ)~0(CHZ)dNHRi
R
R~
R' O CHzNHG(Oj0(CH2)aNHR~
R~ 0 CHZNHC(O)(CH=)eNHR~
Rs R3
Rs Ra
Rq
R4 R~ O CHZNHRi
R' 0 CHzNHC(O)(CHZ)b(OCH2CHZ)~O(CHa)dNHRt
Rs R3
and
R
Rs R'
R~
R' 0 CHzNHC(O)0(CH2)b(OCH2CHz)~0(CHZ)dNHRi
Rs ~ ~Ra
R~
in which the R groups and the indices are as described above.
[0139] The invention also provides for the use of sugar nucleotides modified
with L-Rl at
the 6-carbon position. Exemplary species according to this embodiment include:
niu_i _a~
R
R~ IP IP O Base
w0 ~ \O
O' O'
Y HO OH
in which the R groups, and L, represent moieties as discussed above. The index
"y" is 0, 1 or
2.
[0140] A further exemplary nucleotide sugar of use in the invention is based
on a species
having the stereochemistry of GDP mannose. Exemplary species according to this
embodiment have the structure:
38
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
NHZ
and
HR1
'- O
R~,, o
NH
Ra .,.~niOw I O O N N~NHz
O' O-
R5
HO~~\ ~'OH
[0141] In a still further exemplary embodiment, the invention provides a
conjugate in
which the modified sugar is base on the stereochemistry of UDP galactose. An
exemplary
nucleotide sugar of use in this invention has the structure:
0
~ ,~ R1
HN' 1 la O
R3
O
Ra ."~~iio~0 O O O N
P~.O~ '~p~
0' O-
HO~~ ~'OH ; and
NHR~ O
R3 HN
O
Ra ,,~~ir~0~0 O O O N
Rs ~ ~O~P~O~.~~
O' O
HO~~~, ~'OH .
[0142] In another exemplary embodiment, the nucleotide sugar is based on the
stereochemistry of glucose. Exemplary species according to this embodiment
have the
formulae:
39
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
R~
O
0
R5 I~o~ I~0
O' O-
H0~' ~°OH ; and
HRH
R ~~''' O
Ra
Rs ~ ~Oi
O'
[0143] The modifying group, R1, is any of a number of species including, but
not limited
to, water-soluble polymers, water-insoluble polymers, therapeutic agents,
,diagnostic agents
and the like. The nature of exemplary modifying groups is discussed in greater
detail
hereinbelow.
Modifying Groups
Water-Soluble Polymers
[0144] Many water-soluble polymers are known to those of skill in the art and
are useful in
practicing the present invention. The term water-soluble polymer encompasses
species such
as saccharides (e.g., dextran, amylose, hyalouronic acid, poly(sialic acid),
heparans, heparins,
etc.); poly (amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);
nucleic acids;
synthetic polymers (e.g., poly(acrylic acid), poly(ethers), e.g.,
poly(ethylene glycol);
peptides, proteins, and the like. The present invention may be practiced with
any water-
soluble polymer with the sole limitation that the polymer must include a point
at which the
remainder of the conjugate can be attached.
[0145] Methods for activation of polymers can also be found in WO 94/17039,
U.S. Pat.
No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No. 5,219,564, U.S. Pat.
No.
5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and more WO 93/15189, and for
conjugation between activated polymers and peptides, e.g. Coagulation Factor
VIII (WO
94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.
4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.
Biochem. Biotech.
11: 141-45 (1985)).
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WO 2005/055950 PCT/US2004/041070
[0146] Preferred water-soluble polymers are those in which a substantial
proportion of the
polymer molecules in a sample of the polymer are of approximately'the same
molecular
weight; such polymers are "homodisperse."
[0147] The present invention is further illustrated by reference to a
polyethylene glycol)
conjugate. Several reviews and monographs on the functionalization and
conjugation of PEG
are available. See, for example, Harris, Macf°onol. Chem. Phys. C25:
325-373 (1985);
Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme
Micr°ob. Technol.
14: 866-874 (1992); Delgado et al., Critical Reviems in Therapeutic
Ds°ug Ca~~ie~° Systeyns 9:
249-304 (1992); Zalipsky, Bioconjugate Chern. 6: 150-165 (1995); and Bhadra,
et al.,
PlzarwZazie, 57:5-29 (2002). Routes for preparing reactive PEG molecules and
forming
conjugates using the reactive molecules are known in the art. For example,
U.S. Patent No.
5,672,662 discloses a water soluble and isolatable conjugate of an active
ester of a polymer
acid selected from linear or branched poly(alkylene oxides), poly(oxyethylated
polyols),
poly(olefinic alcohols), and poly(acrylomorpholine).
[0148] U.S. Patent No. 6,376,604 sets forth a method for preparing a water-
soluble
1-benzotriazolylcarbonate ester of a water-soluble and non-peptidic polymer by
reacting a
terminal hydroxyl of the polymer with di(1-benzotriazoyl)carbonate in an
organic solvent.
The active ester is used to form conjugates with a biologically active agent
such as a protein
or peptide.
[0149] WO 99145964 describes a conjugate comprising a biologically active
agent and an
activated water soluble polymer comprising a polymer backbone having at least
one terminus
linked to the polymer backbone through a stable linkage, wherein at least one
terminus
comprises a branching moiety having proximal reactive groups linked to the
branching
moiety, in which the biologically active agent is linked to at least one of
the proximal reactive
groups. Other branched polyethylene glycols) are described in WO 96/21469,
U.S. Patent
No. 5,932,462 describes a conjugate formed with a branched PEG molecule that
includes a
branched terminus that includes reactive functional groups. The free reactive
groups are
available to react with a biologically active species, such as a protein or
peptide, forming
conjugates between the polyethylene glycol) and the biologically active
species. U.S. Patent
No. 5,446,090 describes a bifunctional PEG linker and its use in forming
conjugates having a
peptide at each of the PEG linker termini.
41
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
[0150] Conjugates that include degradable PEG linkages are described in WO
99134833;
and WO 99/14259, as well as in U.S. Patent No. 6,348,558. Such degradable
linkages are
applicable in the present invention.
[0151] The art-recognized methods of polymer activation set forth above are of
use in the
context of the present invention in the formation of the branched polymers set
forth herein
and also for the conjugation of these branched polymers to other species,
e.g., sugars, sugar
nucleotides and the like.
[0152] Exemplary polyethylene glycol) molecules of use in the invention
include, but are
not limited to, those having the formula:
Y
1~ 8
Z (CH2)b-X(CH2CH20)e(CH2)d-A R
in which R8 is H, OH, NHZ, substituted or unsubstituted alkyl, substituted or
wsubstituted
aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted
heterocycloalkyl,
substituted or unsubstituted heteroalkyl, e.g., acetal, OHC-, H2N-(CH2)q-, HS-
(CH2)q, or
-(CHZ)gC(Y)Zl. The index "e" represents an integer from 1 to 2500. The indices
b, d, and q
independently represent integers from 0 to 20. The symbols Z and Zl
independently
represent OH, NHZ, leaving groups, e.g., imidazole, p-nitrophenyl, HOBT,
tetrazole, halide,
S-R9, the alcohol portion of activated esters; -(CHZ)pC(Yl)V, or -
(CH2)pU(CHz)SC(Yl)~. The
symbol Y represents H(2), =O, =S, =N-Rl°. The symbols X, Y, Yl, Al, and
U independently
represent the moieties O, S, N-R11. The symbol V represents OH, NH2, halogen,
S-R12, the
alcohol component of activated esters, the amine component of activated
amides, sugar-
nucleotides, and proteins. The indices p, q, s and v are members independently
selected from
the integers from 0 to 20. The symbols R9, Rl°, Rl l and R1~
independently represent H,
substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or
unsubstituted aryl, substituted or unsubstituted heterocycloalkyl and
substituted or
unsubstituted heteroaryl.
[0153] In other exemplary embodiments, the polyethylene glycol) molecule is
selected
from the following:
42
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WO 2005/055950 PCT/US2004/041070
Me-(OCH2CH2)e-O~ Z Me-(OCH2CH2)e-OUZ
TOf
O
Me-(OCH2CH2)e-O~ Me-(OCH2CH2)e-N~O~Z
II IIz
0 0 0
Me-(OCH2CHz)e-O Z H O
Me-(OCH2CH2)e~N
_Z
O
Me-(OCH2CH2)e-S-Z
Me-(OCH2CH2)e-N-Z Me-(OCH~CH2)e~ N
O
[0154] The polyethylene glycol) useful in forming the conjugate of the
invention is either
linear or branched. Branched polyethylene glycol) molecules suitable for use
in the
invention include, but are not limited to, those described by the follawing
formula:
R$-A~~(OCH2CH2)e-X~
(CH2)q
R$'-A2~(OCH2CH2)f -X~ ~ Z
~ Y
in which R$ and R$' are members independently selected from the groups defined
for R8,
above. A1 and A2 are members independently selected from the groups defined
for Al,
above. The indices e, f, o, and q are as described above. Z and Y are as
described above. Xl
and Xl' are members independently selected from S, SC(O)NH, HNC(O)S, SC(O)O,
O, NH,
NHC(O), (O)CNH and NHC(O)O, OC(O)NH.
[0155] In other exemplary embodiments, the branched PEG is based upon a
cysteine,
serine or di-lysine core. Thus, further exemplary branched PEGS include:
43
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
0
NHC(O)OCH2CHZ(OCHZCHZ)eOCH3
HO ~ 'r
NHZ
HN
NHC(O)OCHZCHZ(OCHzCH2)~OCH3
O
O
NHC(O)CH2CH2(OCHZCHZ)eOCH3
HO
NHa
HN
NHG(0)CHZCHZ(OCHzCH2)e0GH3
O
O O
HO ~ 'S-(CHZCHZO)eCH3 , HO ~ ~S-(CHaCHzO)BCH3
NHC(O)CHZCHz(OCHZCHZ)~OCH3 NHC(O)OCHzCHZ(OCHaCH2)rOCH3
O O
HO ~ 'O-(GHzCHZO)8CH3 , HO ~ = O-(CHZCH20)BCH3
NHC(0)CHzCHz(OCHZGH~fOCH3 NHC(O)OCHZCH2(OCHZCHz)fOCH3
O O
HO ~ 'O-(CH2CHa0)aCH3 HO ~ ~S-(CHzCH20)BCH3
NHC(O)CHZCHZOCH3 NHC(O)OGH3
and
O
HO ~ 'S-(CHZCHzO)eCH3
NHC(O)CH3
[0156] In yet another embodiment, the branched PEG moiety is based upon a tri-
lysine
peptide. The tri-lysine can be mono-, di-, tri-, or tetra-PEG-ylated.
Exemplary species
according to this embodiment have the formulae:
44
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WO 2005/055950 PCT/US2004/041070
C(O)OCHzCHz(OCHzCH2)eOCH3
'' O
NHC(O)OCHZCHZ(OCH2CHz)fOCH3
NH
NHZ 9"
~NHC(O)OCHzCHz(OCHzCHz)fOCH3 , and
9
(O)CH2GHz(OCHzCNz)e0CH3
O
NHC(O)CHzCHz(OCHZCHz)fOCH3
NH
NHz q~~
HC(O)CH2CH2(OCH2GH2)sOCH3
in which e, f and f are independently selected integers from 1 to 2500; and q,
q' and q" are
independently selected integers from 1 to 20.
[0157] In exemplary embodiments of the invention, the PEG is m-PEG (5 kD, 10
kD,
lSkD, 20kD or 30 kD). An exemplary branched PEG species is a serine- or
cysteine-(m-
PEG)2 in which the m-PEG is a 20 kD m-PEG.
[0158] As will be apparent to those of skill, the branched polymers of use in
the invention
include variations on the themes set forth above. For example the di-lysine-
PEG conjugate
shown above can include three polymeric subunits, the third bonded to the a-
amine shown as
unmodified in the structure above. Similarly, the use of a tri-lysine
functionalized with three
or four polymeric subunits is within the scope of the invention.
[0159] Additional exemplary species of use in the invention include:
Me~O~O~S
OH
HEN
O ;
Me~0~0~0
OH
HZN
o ; and
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WO 2005/055950 PCT/US2004/041070
Me~0~0~0
OH
HN
Me~0~0~0~0 O
'' J''f
and carbonates and active esters of these species, such as:
a ~ 'O
HN
Me~O O O~O
and
Me~0~0
a
H
f
[0160] Other activating, or leaving groups, appropriate for activating linear
PEGs of use in
preparing the compounds set forth herein include, but are not limited to the
species:
0
N
J~. N°~ J~
N~ ~N-o o-~ , ~ ~ N-o o~
SO
_A
N
/ I iH
N-N~ ~ S
N-O"O--5 , \N N\
O
O
O
O
O .-.. ~
N-o~o~
I N o o-~ ,
0
0
F F O HN-NH ~
IIII ~ \N-O"O-
F ~ ~ O~O
S ;and
O
F F
[0161] PEG molecules that are activated with these and other species and
methods of
making the activated PEGS are set forth in WO 04/083259.
[0162] Those of skill in the art will appreciate that one or more of the m-PEG
arms of the
branched polymer can be replaced by a PEG moiety with a different terminus,
e.g., OH,
COOH, NHZ, CZ-Clo-alkyl, etc. Moreover, the structures above are readily
modified by
46
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WO 2005/055950 PCT/US2004/041070
inserting alkyl linkers (or removing carbon atoms) between the a-carbon atom
and the
functional group of the side chain of the "amino acid". Thus, "homo"
derivatives and higher
homologues, as well as lower homologues are useful "amino acid" cores for
branched PEGS
of use in the present invention.
[0163] The branched PEG species set forth herein are readily prepared by
methods such as
that set forth in the scheme below:
NHz
HX.~~OH .~ ~O~('~/O~OTs KOH, MeOH ~O~O~X~OH
O _ ~ O
O
NOZ ~O~O~O~NH
CHzCl2/TEA /O~O~X~~OH
a \1 / IIO
in which Xa is O or S and r is an integer from 1 to 5. The indices a and f are
independently
selected integers from 1 to 2500.
[0164] Thus, according to this scheme, a natural or unnatural amino acid is
contacted with
an activated m-PEG derivative, in this case the tosylate, forming 1 by
alkylating the side-
chain heteroatom Xa. The mono-functionalized m-PEG amino acid is submitted to
N-
acylation conditions with a reactive m-PEG derivative, thereby assembling
branched m-PEG
2. As one of skill will appreciate, the tosylate leaving group can be replaced
with any
suitable leaving group, e.g., halogen, mesylate, triflate, etc. Similarly, the
reactive carbonate
utilized to acylate the amine can be replaced with an active ester, e.g., N-
hydroxysuccinimide, etc., or the acid can be activated i~ situ using a
dehydrating agent such
as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.
[0165] In an exemplary embodiment, the modifying group is a PEG moiety,
however, any
modifying group, e.g., water-soluble polymer, water-insoluble polymer,
therapeutic moiety,
etc., can be incorporated in a glycosyl moiety through an appropriate linkage.
The modified
sugar is formed by enzymatic means, chemical means or a combination thereof,
thereby
producing a modified sugar. In an exemplary embodiment, the sugars are
substituted with an
active amine at any position that allows for the attachment of the modifying
moiety, yet still
47
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WO 2005/055950 PCT/US2004/041070
allows the sugar to function as a substrate for an enzyme capable of coupling
the modified
sugar to the peptide. In an exemplary embodiment, when galactosamine~ is the
modified
sugar, the amine moiety is attached to the carbon atom at the 6-position.
Water-soluble Polymer Modified Species
[0166] Water-soluble polymer modified nucleotide sugar species in which the
sugar moiety
is modified with a water-soluble polymer are of use in the present invention.
An exemplary
modified sugar nucleotide bears a sugar group that is modified through an
amine moiety on
the sugar. Modified sugar nucleotides, e.g., saccharyl-amine derivatives of a
sugar
nucleotide, are also of use in the methods of the invention. For example, a
saccharyl amine
(without the modifying group) can be enzymatically conjugated to a peptide (or
other
species) and the free saccharyl amine moiety subsequently conjugated to a
desired modifying
group. Alternatively, the modified sugar nucleotide,can function as a
substrate for an enzyme
that transfers the modified sugar to a saccharyl acceptor on a substrate,
e.g., a peptide,
glycopeptide, lipid, aglycone, glycolipid, etc.
[0167] In one embodiment in which the saccharide core is galactose or glucose,
RS is
NHC(O)Y.
[0168] In an exemplary embodiment, the modified sugar is based upon a 6-amino-
N-
acetyl-glycosyl moiety. As shown below for N-acetylgalactosamine, the 6-amino-
sugar
moiety is readily prepared by standard methods.
0
OH R
O ~ 'NH
HO ~
AcNH O O O N' 'O
O
O' O-
H0~ OH
O
ar-- R=OH c p /CH3
R=NHZ R= HN~O
O "
R=
FiN~~~'~(O~O~CH3
s n
OII /
a. galactose oOxidase ; NHqOAc, NaBH~CN ; b. A~~~O~ ,CH3
J 'O
c. ~ O ,CH3 s
A~O~ ~O
n
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CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
[0169] In the scheme above, the index n represents an integer from 1 to 2500,
preferably
from 10 to 1500, and more preferably from 10 to 1200. The symbol "A"
represents an
activating group, e.g., a halo, a component of an activated ester (e.g., a N-
hydroxysuccinimide ester), a component of a carbonate (e.g., p-nitrophenyl
carbonate) and
the like. Those of skill in the art will appreciate that other PEG-amide
nucleotide sugars are
readily prepared by this and analogous methods.
[0170] In other exemplary embodiments, the amide moiety is replaced by a group
such as a
urethane or a urea.
[0171] In still further embodiments, Rl is a branched PEG, for example, one of
those
species set forth above. Illustrative compounds according to this embodiment
include:
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HOOC O CH(OH)CH(OH)CHZOH
O
HO
\NHC(O)(CHZ)aNHC(O)(CHZ)e(OCHZCHZ)~0(CHZ)dNH S-(CHaCHZO)aCH3
OH NHC(O)X"CHzCHZ(OCHZCHZ),OCH,
HOOC O CH(OH)CH(OH)CHZOH
HO O
NHC(O)(CHZ)aNH ~S-(CHaCH20)QCH3
OH ~ (p)X°CHZCHa(OCHZCHZ),OCH3
O
HOOC O CH(OH)CH(OH)GH2NH(CHZ)aNH ~ ~S-(CHZCHZO)mCH,
HO NHC(O)X°CHiCH2(OCHaCHa),OCH3
~NHC(O)CH3
OH
O
HOOC O CH(OH)CH(OH)CHZNH(CHI)aNHC(O)O(CHZ)b(OOH2CHz)~O(CHz)dNH ~ ~S-
(CH2CHx0)~CH3
HO NHC(O)X°CHzCHz(OCHzCH2),OCH3
NHC(O)CH3
OH O
HOOC O CH(OH)CH(OH)CHZNH(CHZ)aNHC(O)O(CHa)y(OGHZCHZ)~O(CH~)dNH ~ 'S-
(CH2CH20)oCH3
HO NHC(O)X"CHZCHZ(OCH,CH2),OCH,
'NHC(O)CH3
OH
O
HOOC O CH(OH)CH(OH)CHZNHC(0)0(CHZ)b(OCHzCHz)~O(CHZ)dNH ~ ~S-(CH2CH20)oCH,
HO NHC(O)X°CHZCH2(OCH2CH2),OCH,
'NHC(O)CH3
OH
in Which X4 is a bond or O.
[0172] Moreover, as discussed above, the present invention provides nucleotide
sugars that
are modified with a water-soluble polymer, which is either straight-chain or
branched. For
example, compounds having the formula shown below are within the scope of the
present
invention:
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NOOC O CH(OH)CH(OH)CHzOH
\\ / O 0
H ~o .,~~~~~a~p~ ~ ~I ~
N L~~S-(CH2CH,0)aCH,
0-
N OH NHC(O)X°CH,CH,(OCH,CH~),OCH, ; and
OH
O HO
O
HOOC O CH(OH)CH(OH)CHZNH-L" ~S-(CH~CH20),CH,
IO
_ \\ ~ O NHC(0)X°CH~CH,(OCH,CH,),OCH,
N NHC(O)CH3
HZN ov0 ,°vWO~P O.
N~ OH
\\ OH
0 HO
in which X4 is O or a bond.
[0173] Similarly, the invention provides nucleotide sugars of those modified
sugar species
in which the carbon at the 6-position is modified:
NHC(O)(CH~)aNH g-(CH2CH20)eCH3
NHC(O)X4CH2CH2(OCH2CH2)fOCH3
O
O
~NH
O
O~ l~ N NHZ
IwO
O'
in which X4 is a bond or O.
[0174] Also provided are conjugates of peptides and glycopeptides, lipids and
glycolipids
that include the compositions of the invention. For example, the invention
provides
conjugates having the following formulae:
0
W
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HOOC O CH(OH)CH(OH)CHzOH
O
_NHC(O)(CHZ)aNH S-(CH,CHzO),CH,
IOH NHC(O)CH,CHZ(OCH2CHz),OCH,
O
HOOC O CH(OH)CH(OH)CH2NH(CHZ)aNH ~ ~S-(CHzCHaO)aCH, and
0 NHC(O)CHZCH2(OCHZCH2),OCH,
NHC(O)CH3
OH
O
H00C O CH(OH)CH(OH)CH2NH(CH2),NHC(O)0(CHZ)b(OCHzCHZ)~0(CHz)dNH ~ ~S-
(CHzCHzO)aCH,
O NHC(0)CHZCHZ(OCH,CH,),OCH,
NHC(O)CH3
OH
Water-insoluble Polymers
[0175] In another embodiment, analogous to those discussed above, the modified
sugars
include a water-insoluble polymer, rather than a water-soluble polymer. The
conjugates of
the invention may also include one or more water-insoluble polymers. This
embodiment of
the invention is illustrated by the use of the conjugate as a vehicle with
which to deliver a
therapeutic peptide in a controlled manner. Polymeric drug delivery systems
are known in
the art. S2e, for example, Dunn et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY
SYSTEMS, ACS Symposium Series Vol. 469, American Chemical Society, Washington,
D.C.
1991. Those of skill in the art will appreciate that substantially any known
drug delivery
system is applicable to the conjugates of the present invention.
[0176] Representative water-insoluble polymers include, but are not limited
to,
polyphosphazines, polyvinyl alcohols), polyamides, polycarbonates,
polyallcylenes,
polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyallcylene
terephthalates,
1 S polyvinyl ethers, polyvinyl esters, polyvinyl halides,
polyvinylpyrrolidone, polyglycolides,
polysiloxanes, polyurethanes, poly(methyl methacrylate), poly(ethyl
methacrylate),
poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl
methacrylate),
poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate),
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poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),
poly(octadecyl
acrylate) polyethylene, polypropylene, polyethylene glycol), polyethylene
oxide), poly
(ethylene terephthalate), polyvinyl acetate), polyvinyl chloride, polystyrene,
polyvinyl
pyrrolidone, pluronics and polyvinylphenol and copolymers thereof.
[0177] Synthetically modified natural polymers of use in conjugates of the
invention
include, but axe not limited to, alkyl celluloses, hydroxyalkyl celluloses,
cellulose ethers,
cellulose esters, and nitrocelluloses. Particularly preferred members of the
broad classes of
synthetically modified natural polymers include, but are not limited to,
methyl cellulose,
ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
hydroxybutyl
methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose
acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose
sulfate sodium salt,
and polymers of acrylic and methacrylic esters and alginic acid.
[0178] These and the other polymers discussed herein can be readily obtained
from
commercial sources such as Sigma Chemical Co. (St. Louis, MO.), Polysciences
(Warrenton,
PA.), Aldrich (Milwaukee, WL), Fluka (Ronkonkoma, NY), and BioRad (Richmond,
CA), or
else synthesized from monomers obtained from these suppliers using standard
techniques.
[0179] Representative biodegradable polymers of use in the conjugates of the
invention
include, but are not limited to, polylactides, polyglycolides and copolymers
thereof,
polyethylene terephthalate), poly(butyric acid), poly(valeric acid),
poly(lactide-co-
caprolactone), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters,
blends and
copolymers thereof. Of particular use are compositions that form gels, such as
those
including collagen, pluronics and the like.
[0180] The polymers of use in the invention include "hybrid' polymers that
include water-
insoluble materials having within at least a portion of their structure, a
bioresorbable
molecule. An example of such a polymer is one that includes a water-insoluble
copolymer,
which has a bioresorbable region, a hydrophilic region and a plurality of
crosslinlcable
functional groups per polymer chain.
[011] For purposes of the present invention, "water-insoluble materials"
includes
materials that are substantially insoluble in water or, water-containing
environments. Thus,
although certain regions or segments of the copolymer may be hydrophilic or
even water-
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soluble, the polymer molecule, as a whole, does not to any substantial measure
dissolve in
water.
[0182] For purposes of the present invention, the teen "bioresorbable
molecule" includes a
region that is capable of being metabolized or broken down and resorbed and/or
eliminated
through normal excretory routes by the body. Such metabolites or break down
products are
preferably substantially non-toxic to the body.
[0183] The bioresorbable region may be either hydrophobic or hydrophilic, so
long as the
copolymer composition as a whole is not rendered water-soluble. Thus, the
bioresorbable
region is selected based on the preference that the polymer, as a whole,
remains water-
insoluble. Accordingly, the relative properties, i.e., the kinds of functional
groups contained
by, and the relative proportions of the bioresorbable xegion, and the
hydrophilic region are
selected to ensure that useful bioresorbable compositions remain water-
insoluble.
[0184] Exemplary resorbable polymers include, for example, synthetically
produced
resorbable block copolymers of poly(a-hydroxy-carboxylic
acid)/poly(oxyalkylene, (see,
Color et al., U.S. Patent No. 4,826,945). These copolymers are not crosslinked
and are water-
soluble so that the body can excrete the degraded block copolymer
compositions. See,
Younes et al., JBio~zed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al.,
JBzonZed.
Mater. Res. 22: 993-1009 (1988).
[0185] Presently preferred bioresorbable polymers include one or more
components
selected from poly(esters), poly(hydroxy acids), poly(lactones), poly(amides),
poly(ester-
amides), poly (amino acids), poly(anhydrides), poly(orthoesters),
poly(carbonates),
poly(phosphazines), poly(phosphoesters), poly(thioesters), polysaccharides and
mixtures
thereof. More preferably still, the bioresorbable polymer includes a
poly(hydroxy) acid
component. Of the poly(hydroxy) acids, polylactic acid, polyglycolic acid,
polycaproic acid,
polybutyric acid, polyvaleric acid and copolymers and mixtures thereof are
preferred.
[0186] In addition to forming fragments that are absorbed in vivo
("bioresorbed"),
preferred polymeric coatings for use in the methods of the invention can also
form an
excxetable and/or metabolizable fragment.
[0187] Higher order copolymers can also be used in the present invention. For
example,
Casey et al., U.S. Patent No. 4,438,253, which issued on March 20, 1984,
discloses tri-block
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copolymers produced from the transesterification of poly(glycolic acid) and an
hydroxyl-
ended poly(alkylene glycol). Such compositions are disclosed for use as
resorbable
monofilament sutures. The flexibility of such compositions is controlled by
the incorporation
of an aromatic orthocarbonate, such as tetra-p-tolyl orthocarbonate into the
copolymer
structure.
[0188] Other polymers based on lactic and/or glycolic acids can also be
utilized. For
example, Spinu, U.S. Patent No. 5,202,413, which issued on April 13, 1993,
discloses
biodegradable multi-block copolymers having sequentially ordered blocks of
polylactide
and/or polyglycolide produced by ring-opening polymerization of lactide andlor
glycolide
onto either an oligomeric diol or a diamine residue followed by chain
extension with a di-
functional compound, such as, a diisocyanate, diacylchloride or
dichlorosilane.
[0189] Bioresorbable regions of coatings useful in the present invention can
be designed to
be hydrolytically and/or enzymatically cleavable. For purposes of the present
invention,
"hydrolytically cleavable" refers to the susceptibility of the copolymer,
especially the
bioresorbable region, to hydrolysis in water or a water-containing
environment. Similarly,
"enzymatically cleavable" as used herein refers to the susceptibility of the
copolymer,
especially the bioresorbable region, to cleavage by endogenous or exogenous
enzymes.
[0190] When placed within the body, the hydrophilic region can be processed
into
excretable and/or metabolizable fragments. Thus, the hydrophilic region can
include, for
example, polyethers, polyalkylene oxides, polyols, polyvinyl pyrrolidine),
polyvinyl
alcohol), poly(alkyl oxazolines), polysaccharides, carbohydrates, peptides,
proteins and
copolymers and mixtures thereof. Furthermore, the hydrophilic region can also
be, for
example, a poly(allcylene) oxide. Such poly(allcylene) oxides can include, for
example,
polyethylene) oxide, polypropylene) oxide and mixtures and copolymers thereof.
[0191] Polymers that are components of hydrogels are also useful in the
present invention.
Hydrogels are polymeric materials that are capable of absorbing relatively
large quantities of
water. Examples of hydrogel forming compounds include, but are not limited to,
polyacrylic
acids, sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl
pyrrolidine, gelatin,
carrageenan and other polysaccharides, hydroxyethylenemethacrylic acid (HEMA),
as well as
derivatives thereof, and the like. Hydrogels can be produced that are stable,
biodegradable
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and bioresorbable. Moreover, hydrogel compositions can include subunits that
exhibit one or
more of these properties. .
[0192] Bio-compatible hydrogel compositions whose integrity can be controlled
through
crosslinking are known and are presently preferred for use in the methods of
the invention.
For example, Hubbell et al., U.S. Patent Nos. 5,410,016, which issued on April
25, 1995 and
5,529,914, which issued on June 25, 1996, disclose water-soluble systems,
which are
crosslinked block copolymers having a water-soluble central block segment
sandwiched
between two hydrolytically labile extensions. Such copolymers are further end-
capped with
photopolymerizable acrylate functionalities. When crosslinked, these systems
become
hydrogels. The water soluble central block of such copolymers can include
polyethylene
glycol); whereas, the hydrolytically labile extensions can be a poly(a,-
hydroxy acid), such as
polyglycolic acid or polylactic acid. See, Sawhney et al., Macromolecules 26:
581-587
(1993).
[0193] In another preferred embodiment, the gel is a thermoreversible gel.
Thermoreversible gels including components, such as pluronics, collagen,
gelatin,
hyalouronic acid, polysaccharides, polyurethane hydrogel, polyurethane-urea
hydrogel and
combinations thereof are presently preferred.
j0194] In yet another exemplary embodiment, the conjugate of the invention
includes a
component of a liposome. Liposomes can be prepared according to methods known
to those
skilled in the art, for example, as described in Eppstein et al., U.S. Patent
No. 4,522,811,
which issued on June 11, 1985. For example, liposome formulations may be
prepared by
dissolving appropriate lipids) (such as stearoyl phosphatidyl ethanolamine,
stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an
inorganic
solvent that is then evaporated, leaving behind a thin film of dried lipid on
the surface of the
container. An aqueous solution of the active compound or its pharmaceutically
acceptable
salt is then introduced into the container. The container is then swirled by
hand to free lipid
material from the sides of the container and to disperse lipid aggregates,
thereby forming the
liposomal suspension.
[0195] The above-recited microparticles and methods of preparing the
microparticles are
offered by way of example and they are not intended to define the scope of
microparticles of
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use in the present invention. It will be apparent to those of skill in the art
that an array of
microparticles, fabricated by different methods, are of use in the present
invention.
[0196] The structural formats discussed above in the context of the water-
soluble
polymers, both straight-chain and branched are generally applicable with
respect to the water-
insoluble polymers as well. Thus, for example, the cysteine, serine, dilysine,
and trilysine
branching cores can be functionalized with two water-insoluble polymer
moieties. The
methods used to produce these species are generally closely analogous to those
used to
produce the water-soluble polymers.
[0197] The degree of PEG substitution of the conjugates can be controlled by
choice of
stoichiometry, number of available glycosylation sites, selection of an enzyme
that is
selective for a particular site, and the like (FIG. 2F). The glycoPEGylated
Factor IX species
display enhanced circulatory half life relative to the unlabeled Factor IX
(FIG. 3, FIG. 6).
The Methods
[019] In addition to the conjugates discussed above, the present invention
provides
methods for preparing these and other conjugates. Moreover, the invention
provides methods
of preventing, curing or ameliorating a disease state by administering a
conjugate of the
invention to a subject at risk of developing the disease or a subject that has
the disease.
[0199] Thus, the invention provides a method of forming a covalent conjugate
between a
selected moiety and a Factor IX peptide.
[0200] In exemplary embodiments, the conjugate is formed between a water-
soluble
polymer, a therapeutic moiety, targeting moiety or a biomolecule, and a
glycosylated or non-
glycosylated Factor IX peptide. The polymer, therapeutic moiety or biomolecule
is
conjugated to the peptide via a glycosyl linking group, which is interposed
between, and
covalently linked to both the peptide and the modifying group (e.g., water-
soluble polymer).
The method includes contacting the peptide with a mixture containing a
modified sugar and
an enzyme, e.g., a glycosyltransferase, that conjugates the modified sugar to
the substrate
(e.g., peptide, aglycone, glycolipid). The reaction is conducted under
conditions appropriate
to form a covalent bond between the modified sugar and the Factor IX peptide.
[0201] The acceptor Factor IX peptide is typically synthesized de hovo, or
recombinantly
expressed in a prokaryotic cell (e.g., bacterial cell, such as E. colt) or in
a eukaryotic cell such
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as a mammalian, yeast, insect, fungal or plant cell. The peptide can be either
a full-length
protein or a fragment. Moreover, the peptide can be a wild type or mutated
peptide. In an
exemplary embodiment, the peptide includes a mutation that adds or removed one
or more N-
or O-linked glycosylation sites to the peptide sequence.
[0202] In an exemplary embodiment, Factor IX is O-glycosylated and
functionalized with
a water-soluble polymer in the following manner. The peptide is either
produced with an
available amino acid glycosylation site or, if glycosylated, the glycosyl
moiety is trimmed off
to exposed the amino acid. For example, a serine or threonine is a-1 N-acetyl
amino
galactosylated (GaINAc) and the NAc-galactosylated peptide is sialylated with
a sialic acid-
modifying group cassette using ST6GaINAcTI. Alternatively, the NAc-
galactosylated
peptide is galactosylated using Core-1-GaIT-1 and the product is sialylated
with a sialic acid-
modifying group cassette using ST3Ga1T1. An exemplary conjugate according to
this
method has the following linkages: Thr-a-1-GaINAc-(3-1,3-Gal-a2,3-Sia*, in
which Sia* is
the sialic acid-modifying group cassette.
[0203] In the methods of the invention, such as that set forth above, using
multiple
enzymes and saccharyl donors, the individual glycosylation steps may be
performed
separately, or combined in a "single pot" reaction. For example, in the three
enzyme reaction
set forth above the GaINAc tranferase, GaIT and SiaT and their donors may be
combined in a
single vessel. Alternatively, the GaINAc reaction can be performed alone and
both the GaIT
and SiaT and the appropriate saccharyl donors added as a single step. Another
mode of
running the reactions involves adding each enzyme and an appropriate donor
sequentially and
conducting the reaction in a "single pot" motif. Combinations of each of the
methods set
forth above are of use in preparing the compounds of the invention.
[0204] In the conjugates of the invention, particularly the glycopegylated N-
linked glycans,
the Sia-modifying group cassette can be linked to the Gal in an a-2,6, or a-
2,3 linkage.
[0200] The method of the invention also provides for modification of
incompletely
glycosylated Factor IX peptides that are produced recombinantly. Employing a
modified
sugar in a method of the invention, the peptide can be simultaneously further
glycosylated
and derivatized with, e.g., a water-soluble polymer, therapeutic agent, or the
like. The sugar
moiety of the modified sugar can be the residue that would properly be
conjugated to the
acceptor in a fully glycosylated peptide, or another sugar moiety with
desirable properties.
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[0201] Exemplary methods of modifying peptides of use in the present invention
are set
forth in WO04/099231, WO 03/031464, and the references set forth therein.
[0202] In an exemplary embodiment, the invention provides a method of making a
PEG-
ylated Factor IX comprising the moiety:
H
HO
'OH
O
NH-G
OH
D
wherein D is -OH or R1-L-HN-. The symbol G represents R1-L- or -C(O)(C1-
C6)alkyl. R1 is
a moiety comprising a a straight-chain or branched polyethylene glycol)
residue. The
symbol L represents a linker selected from a bond, substituted or
unsubstituted alkyl and
substituted or unsubstituted heteroalkyl. In general, when D is OH, G is Rl-L-
, and when G
is -C(O)(C1-C6)alkyl, D is Rl-L-NH-. The method of the invention includes, (a)
contacting a
substrate Factor IX peptide with a PEG-sialic acid donor and an enzyme that is
capable of
transferring the PEG-sialic acid moiety from the donor to the substrate Factor
IX peptide.
[0203] An exemplary PEG-sialic acid donor is a nucleotide sugar such as that
having the
formula:
n-
1 S NHz
and an enzyme that transfers the PEG-sialic acid onto an amino acid or
glycosyl residue of
the Factor IX peptide, under conditions appropriate for the transfer.
[0204] In one embodiment the substrate Factor IX peptide is expressed in a
host cell prior to
the formation of the conjugate of the invention. An exemplary host cell is a
mammalian cell.
In other embodiments the host cell is an insect cell, plant cell, a bacteria
or a fungi.
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[0205] The method presented herein is applicable to each of the Factor IX
conjugates set
forth in the sections above.
[0206] Factor IX peptides modified by the methods of the invention can be
synthetic or wild-
type peptides or they can be mutated peptides, produced by methods known in
the art, such as
site-directed mutagenesis. Glycosylation of peptides is typically either N-
linked or O-linked.
An exemplary N-linkage is the attachment of the modified sugar to the side
chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine and
asparagine-X-
threonine, where X is any amino acid except proline, are the recognition
sequences for
enzymatic attachment of a carbohydrate moiety to the asparagine side chain.
Thus, the'
presence of either of these tripeptide sequences in a polypeptide creates a
potential
glycosylation site. O-linked glycosylation refers to the attachment of one
sugar (e.g., N-
acetylgalactosamine, galactose, mannose, GIcNAc, glucose, fucose or xylose) to
the hydroxy
side chain of a hydroxyamino acid, preferably serine or threonine, although
unusual or non-
natural amino acids, e.g., 5-hydroxyproline or 5-hydroxylysine may also be
used.
[0207] Addition of glycosylation sites to a peptide or other structure is
conveniently
accomplished by altering the amino acid sequence such that it contains one or
more
glycosylation sites. The addition may also be made by the incorporation of one
or more
species presenting an -OH group, preferably serine or threonine residues,
within the sequence
of the peptide (for O-linked glycosylation sites). The addition may be made by
mutation or
by full chemical synthesis of the peptide. The peptide amino acid sequence is
preferably
altered through changes at the DNA level, particularly by mutating the DNA
encoding the
peptide at preselected bases such that codons are generated that will
translate into the desired
amino acids. The DNA mutations) are preferably made using methods known in the
art.
[0208] In an exemplary embodiment, the glycosylation site is added by
shuffling
polynucleotides. Polynucleotides encoding a candidate peptide can be modulated
with DNA
shuffling protocols. DNA shuffling is a process of recursive recombination and
mutation,
performed by random fragmentation of a pool of related genes, followed by
reassembly of the
fragments by a polymerase chain reaction-like process. See, e.g., Stemmer,
P~oc. Natl. Acad.
Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); and U.S.
Patent
Nos. 5,605,793, 5,837,458, 5,830,721 and 5,811,238.
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[0209] Exemplary methods of adding or removing glycosylation sites, and adding
or
removing glycosyl structures or substructures are described in detail in
W004/099231,
W003/031464 and related U.S. and PCT applications.
[0210] The present invention also utilizes means of adding (or removing) one
or more
selected glycosyl residues to a Factor IX peptide, after which a modified
sugar is conjugated
to at least one of the selected glycosyl residues of the peptide. Such
techniques are useful, for
example, when it is desired to conjugate the modified sugar to a selected
glycosyl residue that
is either not present on a Factor IX peptide or is not present in a desired
amount. Thus, prior
to coupling a modified sugar to a peptide, the selected glycosyl residue is
conjugated to the
peptide by enzymatic or chemical coupling. In another embodiment, the
glycosylation
pattern of a glycopeptide is altered prior to the conjugation of the modified
sugar by the
removal of a carbohydrate residue from the glycopeptide. See, for example WO
98/31826.
For example, sialic acid groups can be removed from Factor IX, forming asialo-
Factor IX,
prior to glycoPEGylating using a PEG modified sialic acid (FIG. 2E).
[0211] Exemplary attachment points for selected glycosyl residue include, but
are not limited
to: (a) consensus sites for N-linked glycosylation, and sites for O-linked
glycosylation; (b)
terminal glycosyl moieties that are acceptors for a glycosyltransferase; (c)
arginine,
asparagine and histidine; (d) free carboxyl groups; (e) free sulfliydryl
groups such as those of
cysteine; (f) free hydroxyl groups such as those of serine, threonine, or
hydroxyproline; (g)
aromatic. residues such as those of phenylalanine, tyrosine, or tryptophan; or
(h) the amide
group of glutamine. Exemplary methods of use in the present invention are
described in WO
87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC CRIT. REV.
BI~CHEM., pp.
259-306 (1981).
[0212] The PEG modified sugars are conjugated to a glycosylated or non-
glycosylated
peptide using an appropriate enzyme to mediate the conjugation. Preferably,
the
concentrations of the modified donor sugar(s), enzymes) and acceptor peptides)
are selected
such that glycosylation proceeds until the acceptor is consumed. The
considerations
discussed below, while set forth in the context of a sialyltransferase, are
generally applicable
to other glycosyltransferase reactions.
[0213] A number of methods of using glycosyltransferases to synthesize desired
oligosaccharide structures are known and are generally applicable to the
instant invention.
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Exemplary methods are described, for instance, WO 96/32491, Ito et al., Pure
Appl. Chem.
65: 753 (1993), U.S. Pat. Nos. 5,352,670, 5,374,541, 5,545,553, and commonly
owned U.S.
Pat. Nos. 6,399,336, and 6,440,703 which are incorporated herein by reference.
[0214] The present invention is practiced using a single glycosyltransferase
or a combination
of glycosyltransferases. For example, one can use a combination of a
sialyltransferase and a
galactosyltransferase. In those embodiments using more than one enzyme, the
enzymes and
substrates are preferably combined in an initial reaction mixture, or the
enzymes and reagents
for a second enzymatic reaction are added to the reaction medium once the
first enzymatic
reaction is complete or nearly complete. By conducting two enzymatic reactions
in sequence
in a single vessel, overall yields are improved over procedures in which an
intermediate
species is isolated. Moreover, cleanup and disposal of extra solvents and by-
products is
reduced.
[0215] In a preferred embodiment, each of the first and second enzyme is a
glycosyltransferase. In another preferred embodiment, one enzyme is an
endoglycosidase. In
an additional preferred embodiment, more than two enzymes are used to assemble
the
modified glycoprotein of the invention. The enzymes are used to alter a
saccharide structure
on the peptide at any point either before or after the addition of the
modified sugar to the
peptide.
[0216] In another embodiment, the method makes use of one or more exo- or
endoglycosidase. The glycosidase is typically a mutant, which is engineered to
form glycosyl
bonds rather than rupture them. The mutant glycanase typically includes a
substitution of an
amino acid residue for an active site acidic amino acid residue. For example,
when the
endoglycanase is endo-H, the substituted active site residues will typically
be Asp at position
130, Glu at position 132 or a combination thereof. The amino acids are
generally replaced
with serine, alanine, asparagine, or glutamine.
[0217] The mutant enzyme catalyzes the reaction, usually by a synthesis step
that is
analogous to the reverse reaction of the endoglycanase hydrolysis step. In
these
embodiments, the glycosyl donor molecule (e.g., a desired oligo- or mono-
saccharide
structure) contains a leaving group and the reaction proceeds with the
addition of the donor
molecule to a GIcNAc residue on the protein. For example, the leaving group
can be a
halogen, such as fluoride. In other embodiments, the leaving group is a Asn,
or a Asn-
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peptide moiety. In yet further embodiments, the GIcNAc residue on the glycosyl
donor
molecule is modifted. For example, the GIcNAc residue may comprise a 1,2
oxazoline
moiety.
[0218] In a preferred embodiment, each of the enzymes utilized to produce a
conjugate of the
invention are present in a catalytic amount. The catalytic amount of a
particular enzyme
varies according to the concentration of that enzyme's substrate as well as to
reaction
conditions such as temperature, time and pH value. Means for determining the
catalytic
amount for a given enzyme under preselected substrate concentrations and
reaction
conditions are well known to those of skill in the art.
[0219] The temperature at which an above process is carried out can range from
just above
freezing to the temperature at which the most sensitive enzyme denatures.
Preferred
temperature ranges are about 0 °C to about 55 °C, and more
preferably about 20 ° C to about
37 °C. In another exemplary embodiment, one or more components of the
present method
are conducted at an elevated temperature using a thermophilic enzyme.
[0220] The reaction mixture is maintained for a period of time sufficient for
the acceptor to
be glycosylated, thereby forming the desired conjugate. Some of the conjugate
can often be
detected after a few hours, with recoverable amounts usually being obtained
within 24 hours
or less. Those of skill in the art understand that the rate of reaction is
dependent on a number
of variable factors (e.g, enzyme concentration, donor concentration, acceptor
concentration,
temperature, solvent volume), which are optimized for a selected system.
[0221] The present invention also provides for the industrial-scale production
of modified
peptides. As used herein, an industrial scale generally produces at least 250
mg, preferably at
least S00 mg and more preferably, at least one gram of finished, purified
conjugate.
[0222] In the discussion that follows, the invention is exemplified by the
conjugation of
modified sialic acid moieties to a glycosylated peptide. The exemplary
modified sialic acid is
labeled with PEG. The focus of the following discussion on the use of PEG-
modified sialic
acid and glycosylated peptides is for clarity of illustration and is not
intended to imply that
the invention is limited to the conjugation of these two partners. One of
skill understands that
the discussion is generally applicable to the additions of modified glycosyl
moieties other
than sialic acid. Moreover, the discussion is equally applicable to the
modification of a
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WO 2005/055950 PCT/US2004/041070
glycosyl unit with agents other than PEG including other PEG moieties,
therapeutic moieties,
and biomolecules.
[0223] An enzymatic approach can be used for the selective introduction of
PEGylated or
PPGylated carbohydrates onto a peptide or glycopeptide. The method utilizes
modified
sugars containing PEG, PPG, or a maslced reactive functional group, and is
combined with
the appropriate glycosyltransferase or glycosynthase. By selecting the
glycosyltransferase
that will make the desired carbohydrate linkage and utilizing the modified
sugar as the donor
substrate, the PEG or PPG can be introduced directly onto the peptide
backbone, onto
existing sugar residues of a glycopeptide or onto sugar residues that have
been added to a
peptide.
[0224] An acceptor for the sialyltransferase is present on the peptide to be
modified by the
methods of the present invention either as a naturally occurring structure or
one placed there
recombinantly, enzymatically or chemically. Suitable acceptors, include, for
example,
galactosyl acceptors such as Gal[i1,4G1cNAc, Gal(31,4GalNAc, Gal(31,3Ga1NAc,
lacto-N-
tetraose, Gal(31,3G1cNAc, Gal(31,3Ara, Gal(31,6G1cNAc, Gal(31,4G1c (lactose),
and other
acceptors known to those of skill in the art (see, e.g., Paulson et al., J.
Biol. Chem. 253: 5617-
5624 (1978)).
[0225] In one embodiment, an acceptor for the sialyltransferase is present on
the
glycopeptide to be modified upon in vivo synthesis of the glycopeptide. Such
glycopeptides
can be sialylated using the claimed methods without prior modification of the
glycosylation
pattern of the glycopeptide. Alternatively, the methods of the invention can
be used to
sialylate a peptide that does not include a suitable acceptor; one first
modifies the peptide to
include an acceptor by methods known to those of skill in the art. In an
exemplary
embodiment, a GaINAc residue is added by the action of a GaINAc transferase.
[0226] In an exemplary embodiment, the galactosyl acceptor is assembled by
attaching a
galactose residue to an appropriate acceptor linked to the peptide, e.g., a
GIcNAc. The .
method includes incubating the peptide to be modified with a reaction mixture
that contains a
suitable amount of a galactosyltransferase (e.g., gal[i 1,3 or gal(31,4), and
a suitable galactosyl
donor (e.g., UDP-galactose). The reaction is allowed to proceed substantially
to completion
or, alternatively, the reaction is terminated when a preselected amount of the
galactose
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residue is added. Other methods of assembling a selected saccharide acceptor
will be
apparent to those of skill in the art.
[0227] In yet another embodiment, glycopeptide-linked oligosaccharides are
first "trimmed,"
either in whole or in part, to expose either an acceptor for the
sialyltransferase or a moiety to
which one or more appropriate residues can be added to obtain a suitable
acceptor. Enzymes
such as glycosyltransferases and endoglycosidases (see, for example U.S.
Patent No.
5,716,812) are useful for the attaching and trimming reactions.
[022] In the discussion that follows, the method of the invention is
exemplified by the use of
modified sugars having a PEG moiety attached thereto. The focus of the
discussion is for
clarity of illustration. Those of skill will appreciate that the discussion is
equally relevant to
those embodiments in which the modified sugar bears a therapeutic moiety,
biomolecule or
the like.
[0229] In an exemplary embodiment of the invention in which a carbohydrate
residue is
"trimmed" prior to the addition of the modified sugar high mannose is trimmed
back to the
first generation biantennary structure. A modified sugar bearing a PEG moiety
is conjugated
to one or more of the sugar residues exposed by the "trimming back." In one
example, a PEG
moiety is added via a GIcNAc moiety conjugated to the PEG moiety. The modified
GIcNAc
is attached to one or both of the terminal mannose residues of the biantennary
structure.
Alternatively, an unmodified GIcNAc can be added to one or both of the termini
of the
branched species.
[0230] In another exemplary embodiment, a PEG moiety is added to one or both
of the
terminal mannose residues of the biantennary structure via a modified sugar
having a
galactose residue, which is conjugated to a GIcNAc residue added onto the
terminal mannose
residues. Alternatively, an unmodified Gal can be added to one or both
terminal GIcNAc
residues.
[0231] In yet a further example, a PEG moiety is added onto a Gal residue
using a modified
sialic acid.
[0232] In another exemplary embodiment, a high mamlose structure is "trimmed
back" to the
mannose from which the biantennary structure branches. In one example, a PEG
moiety is
added via a GIcNAc modified with the polymer. Alternatively, an unmodified
GIcNAc is
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
added to the mannose, followed by a Gal with an attached PEG moiety. In yet
another
embodiment, unmodified GIcNAc and Gal residues are sequentially added to the
mannose,
followed by a sialic acid moiety modified with a PEG moiety.
[0233] In a further exemplary embodiment, high mannose is "trimmed back" to
the GIcNAc
to which the first mannose is attached. The GIcNAc is conjugated to a Gal
residue bearing a
PEG moiety. Alternatively, an unmodified Gal is added to the GIcNAc, followed
by the
addition of a sialic acid modified with a water-soluble sugar. In yet a
further example, the
terminal GIcNAc is conjugated with Gal and the GIcNAc is subsequently
fucosylated with a
modified fucose bearing a PEG moiety.
[0234] High mamiose may also be trimmed back to the first GIcNAc attached to
the Asn of
the peptide. In one example, the GIcNAc of the GIcNAc-(Fuc)a residue is
conjugated wit ha
GIcNAc bearing a water soluble polymer. In another example, the GIcNAc of the
GIcNAc-(Fuc)a residue is modified with Gal, which bears a water soluble
polymer. In a still
further embodiment, the GIcNAc is modified with Gal, followed by conjugation
to the Gal of
a sialic acid modified with a PEG moiety.
[0235] Other exemplary embodiments are set forth in commonly owned U.S. Patent
application Publications: 20040132640; 20040063911; 20040137557; U.S. Patent
application
Nos: 101369,979; 10/410,913; 10/360,770; 10/410,945 and PCT/US02/32263 each of
which
is incorporated herein by reference.
[0236] The examples set forth above provide an illustration of the power of
the methods set
forth herein. Using the methods described herein, it is possible to "trim
back" and build up a
carbohydrate residue of substantially any desired structure. The modified
sugar can be added
to the termini of the carbohydrate moiety as set forth above, or it can be
intemnediate between
the peptide core and the terminus of the carbohydrate.
[0237] In an exemplary embodiment, an existing sialic acid is removed from a
Factor IX
glycopeptide using a sialidase, thereby unmasking all or most of the
underlying galactosyl
residues. Alternatively, a peptide or glycopeptide is labeled with galactose
residues, or an
oligosaccharide residue that terminates in a galactose unit. Following the
exposure of or
addition of the galactose residues, an appropriate sialyltransferase is used
to add a modified
sialic acid. The approach is summarized in Scheme 1.
66
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
Scheme 1
NH2
O I \ N Gal Glycoprotein
a ~-~o Gal
O
HO pH O_+N~ G81
HO 0' ~--O Na HO OH
PEG or PPG~N~NH OH O
H IoI Sialyltransferase
CMP-SA-5-NHCOCHZNH-PEG(PPG)
SA-5-NHCOCHZNH-PEG
Glycoprotein Gal
Gal-SA-5-NHCOCH2NH-PEG
Gal
SA-5-NHCOCH2NH-PEG
[0238] In yet a further approach, summarized in Scheme 2, a masked reactive
functionality is
present on the sialic acid. The masked reactive group is preferably unaffected
by the
conditions used to attach the modified sialic acid to the Factor IX. After the
covalent
attachment of the modified sialic acid to the peptide, the mask is removed and
the peptide is
conjugated with an agent such as PEG. The agent is conjugated to the peptide
in a specific
manner by its reaction with the unmasked reactive group on the modified sugar
residue.
67
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
Scheme 2
Gal Glycoprotein
NHS Gal
. N w SA-5-NHCOCH2S-SEt
~.l,o Gal
o-P-o o Gal
HO OH O_+N f-1
Ho ~ o o'+Na HO OH Sialyltransferase Gal-SA-5-NHCOCH2S-SEt
EtS~S~NH off o i al
0
SA-5-NHCOCH2S-SEt
SA-5-N HCOCH2S-PEG
Glycoprotein Gal
1. dithiothreitol
Gal=SA-5-NHCOCH2S-PEG 2. PEG-halide or PPG halide
Gal
SA-5-NHCOCH2S-PEG
[0239] Any modified sugar set forth herein can be used with its appropriate
glycosyltransferase, depending on the terminal sugars of the oligosaccharide
side chains of
the glycopeptide (Table 1). As discussed above, the terminal sugar of the
glycopeptide
required for introduction of the PEGylated structure can be introduced
naturally during
expression or it can be produced post expression using the appropriate
glycosidase(s),
glycosyltransferase(s) or mix of glycosidase(s) and glycosyltransferase(s).
68
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
Table 1
Q Ra_Y I X_R~
R3-Y ~ X-R~
O , O O
R2 Z ' ~NH
Rz Z R4_A u° ~ .~O R~ A O.~P~.O-p-O''~ O ~N~O
O-O + Or-O_ N~ O_+Na O_+Na \ j
H lO~IOH
HO OH UDP-galactosamine-derivatives
UDP-galactose-derivatives (when A = NH, R4 may be acetyl)
Q~ X_Ra Q. X_R~
Ra_Y o o Rs_Y o
Rz-Z NH
Rz_Z R _A o o ~-~ R4_A o o ~-~o
4 _ _ II_ N O O-P-~ -P
O O +Na ~- N~ O~+Na O- Oa
a H\O~~IOH HO OH
UDP-Glucose-derivatives UDP-Glucosamine-derivatives
(when A = NH, Rq may be acetyl)
o
0
°
R - II ~ N N NHZ
N NH O-'P~p-P-p O
o_+Na ~_+Na
II N N NHZ
o-P-o~ R~-X ° A_R4 Ho off
la ~_+Na
Z-R3 GDP-fucose-derivatives
Ho off Rz_Y
GDP-Maimose-derivatives
X = 0, NH, S, CH2, N-(Rl-5)2~
Y=X; Z=X; A=X; B=X.
Q = H~, O, S, NH, N-R.
R, Rl-q = H, Linker-M, M.
M =PEG, e.g., m-PEG
[0240] In a further exemplary embodiment, UDP-galactose-PEG is reacted with
bovine milk
[i1,4-galactosyltransferase, thereby transferring the modified galactose to
the appropriate
terminal N-acetylglucosamine structure. The terminal GIcNAc residues on the
glycopeptide
may be produced during expression, as may occur in such expression systems as
mammalian,
insect, plant or fungus, but also can be produced by treating the glycopeptide
with a sialidase
and/or glycosidase andlor glycosyltransferase, as required.
69
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WO 2005/055950 PCT/US2004/041070
[0241] In another exemplary embodiment, a GIcNAc transferase, such as GNT1-5,
is utilized
to transfer PEGylated-GIcN to a terminal mannose residue on a glycopeptide. In
a still
further exemplary embodiment, an the N- and/or O-linked glycan structures are
enzymatically removed from a glycopeptide to expose an amino acid or a
terminal glycosyl
residue that is subsequently conjugated with the modified sugar. For example,
an
endoglycanase is used to remove the N-linked structures of a glycopeptide to
expose a
terminal GIcNAc as a GIcNAc-linked-Asn on the glycopeptide. UDP-Gal-PEG and
the
appropriate galactosyltransferase is used to introduce the PEG-galactose
functionality onto
the exposed GIcNAc.
[0242] In an alternative embodiment, the modified sugar is added directly to
the peptide
backbone using a glycosyltransferase known to transfer sugar residues to the
peptide
baclcbone. This exemplary embodiment is set forth in Scheme 3. Exemplary
glycosyltransferases useful in practicing the present invention include, but
are not limited to,
GaINAc transferases (GaINAc T1-14), GIcNAc transferases, fucosyltransferases,
glucosyltransferases, xylosyltransferases, mannosyltransferases and the like.
Use of this
approach allows the direct addition of modified sugars onto peptides that lack
any
carbohydrates or, alternatively, onto existing glycopeptides. In both cases,
the addition of the
modified sugar occurs at specific positions on the peptide backbone as defined
by the
substrate specificity of the glycosyltransferase and not in a random manner as
occurs during
modification of a protein's peptide backbone using chemical methods. An array
of agents
can be introduced into proteins or glycopeptides that lack the
glycosyltransferase substrate
peptide sequence by engineering the appropriate amino acid sequence into the
polypeptide
chain.
Scheme 3
HO OH
_ O O Protein or Glycoprotein
HO
o NH o o ~ ~ GaINH-CO(CH~)4NH-PEG
p-P~.~-p_o p~
O Na O-"Na N
Ho off GaINAc Transferase
(GaINAc T3) GaINH-CO(CH2)4NH-PEG
NH
PEG
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
[0243] In each of the exemplary embodiments set forth above, one or more
additional
chemical or enzymatic modification steps can be utilized following the
conjugation of the
modified sugar to the peptide. In an exemplary embodiment, an enzyme (e.g.,
fucosyltransferase) is used to append a glycosyl unit (e.g., fucose) onto the
terminal modified
sugar attached to the peptide. In another example, an enzymatic reaction is
utilized to "cap"
sites to which the modified sugar failed to conjugate. Alternatively, a
chemical reaction is
utilized to alter the structure of the conjugated modified sugar. For example,
the conjugated
modified sugar is reacted with agents that stabilize or destabilize its
linkage with the peptide
component to which the modified sugar is attached. In another example, a
component of the
modified sugar is deprotected following its conjugation to the peptide. One of
skill will
appreciate that there is an array of enzymatic and chemical procedures that
are useful in the
methods of the invention at a stage after the modified sugar is conjugated to
the peptide.
Further elaboration of the modified sugar-peptide conjugate is within the
scope of the
invention.
Es~zv~zes
[0244] In addition to the enzymes discussed above in the context of forming
the acyl-linked
conjugate, the glycosylation pattern of the conjugate and the starting
substrates (e.g.,
peptides, lipids) can be elaborated, trimmed back or otherwise modified by
methods utilizing
other enzymes. The methods of remodeling peptides and lipids using enzymes
that transfer a
sugar donor to an acceptor are discussed in great detail in DeFrees, WO
03/031464 A2,
published April 17, 2003. A brief summary of selected enzymes of use in the
present method
is set forth below.
Glycosyltt~~rnsfe~~ases
[0245] Glycosyltransferases catalyze the addition of activated sugars (donor
NDP- or NMP-
sugars), in a step-wise fashion, to a protein, glycopeptide, lipid or
glycolipid or to the non-
reducing end of a growing oligosaccharide. N-linked glycopeptides are
synthesized via a
transferase and a lipid-linlced oligosaccharide donor Dol-PP-NAGzGIc3Man9 in
an en block
transfer followed by trimming of the core. In this case the nature of the
"core" saccharide is
somewhat different from subsequent attachments. A very large number of
glycosyltransferases are known in the art.
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CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
[0246] The glycosyltransferase to be used in the present invention may be any
as long as it
can utilize the modified sugar as a sugar donor. Examples of such enzymes
include Leloir
pathway glycosyltransferase, such as galactosyltransferase, N-
acetylglucosaminyltransferase,
N-acetylgalactosaminyltransferase, fucosyltransferase, sialyltransferase,
mannosyltransferase,
xylosyltransferase, glucurononyltransferase and the like.
[0247] For enzymatic saccharide syntheses that involve glycosyltransferase
reactions,
glycosyltransferase can be cloned, or isolated from any source. Many cloned
glycosyltransferases are lcnomn, as are their polynucleotide sequences. See,
e.g., "The WWW
Guide To Cloned Glycosyltransferases," (http://www.vei.co.ul</TGN/g_t
~uide.htm).
Glycosyltransferase amino acid sequences and nucleotide sequences encoding
glycosyltransferases from which the amino acid sequences can be deduced are
also found in
various publicly available databases, including GenBank, Swiss-Prot, EMBL, and
others.
[0248] Glycosyltransferases that can be employed in the methods of the
invention include,
but are not limited to, galactosyltransferases, fucosyltransferases,
glucosyltransferases, N-
acetylgalactosaminyltransferases, N-acetylglucosaminyltransferases,
glucuronyltransferases,
sialyltransferases, mannosyltransferases, glucuronic acid transferases,
galacturonic acid
transferases, and oligosaccharyltransferases. Suitable glycosyltransferases
include those
obtained from eukaryotes, as well as from prokaryotes.
[0249] DNA encoding glycosyltransferases may be obtained by chemical
synthesis, by
screening reverse transcripts of mRNA from appropriate cells or cell line
cultures, by
screening genomic libraries from appropriate cells, or by combinations of
these procedures.
Screening of mRNA or genomic DNA may be carried out with oligonucleotide
probes
generated from the glycosyltransferases gene sequence. Probes may be labeled
with a
detectable group such as a fluorescent group, a radioactive atom or a
chemiluminescent group
in accordance with knovtm procedures and used in conventional hybridization
assays. In the
alternative, glycosyltransferases gene sequences may be obtained by use of the
polymerase
chain reaction (PCR) procedure, with the PCR oligonucleotide primers being
produced from
the glycosyltransferases gene sequence. See, U.S. Pat. No. 4,683,195 to Mullis
et al. and U.S.
Pat. No. 4,683,202 to Mullis.
[0250] The glycosyltransferase may be synthesized in host cells transformed
with vectors
containing DNA encoding the glycosyltransferases enzyme. Vectors are used
either to
72
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
amplify DNA encoding the glycosyltransferases enzyme andlor to express DNA
which
encodes the glycosyltransferases enzyme. An expression vector is a replicable
DNA
construct in which a DNA sequence encoding the glycosyltransferases enzyme is
operably
linked to suitable control sequences capable of effecting the expression of
the
glycosyltransferases enzyme in a suitable host. The need for such control
sequences will
vary depending upon the host selected and the transformation method chosen.
Generally,
control sequences include a transcriptional promoter, an optional operator
sequence to control
transcription, a sequence encoding suitable mRNA ribosomal binding sites, and
sequences
which control the termination of transcription and translation. Amplification
vectors do not
require expression control domains. All that is needed is the ability to
replicate in a host,
usually conferred by an origin of replication, and a selection gene to
facilitate recognition of
transformants.
[0251] In an exemplary embodiment, the invention utilizes a prokaryotic
enzyme. Such
glycosyltransferases include enzymes involved in synthesis of
lipooligosaccharides (LOS),
which are produced by many gram negative bacteria (Preston et al., Critical
Reviews in
Microbiology 23(3): 139-180 (1996)). Such enzymes include, but are not limited
to, the
proteins of the r~fa operons of species such as E. coli and Salmonella
typhirnur~iuna, which
include a (31,6 galactosyltransferase and a (31,3 galactosyltransferase (see,
e.g., EMBL
Accession Nos. M80599 and M86935 (E. coli); EMBL Accession No. 556361 (S.
typhimuf~ium)), a glucosyltransferase (Swiss-Prot Accession No. P25740 (E.
coli), an (31,2-
glucosyltransferase (r faJ)(Swiss-Prot Accession No. P27129 (E. coli) and
Swiss-Prot
Accession No. P19817 (S. yphimur~ium)), and an (31,2-N-
acetylglucosaminyltransferase
(r faK)(EMBL Accession No. U00039 (E. cola). Other glycosyltransferases for
which amino
acid sequences are known include those that are encoded by operons such as
r~faB, which
have been characterized in organisms such as Klebsiella pneumoniae, E. coli,
Salmonella
typhimur~ium, Salmonella enter~ica, Yer~sinia enter~ocolitiea, Mycobacter~iurn
lepr~osum, and the
r~hl operon of Pseudonaonas aer~uginosa.
[0252] Also suitable for use in the present invention are glycosyltransferases
that are
involved in producing structures containing lacto-N-neotetraose, D-galactosyl-
(3-1,4-N-
acetyl-D-glucosaminyl-(3-1,3-D-galactosyl-(3-1,4-D-glucose, and the Pk blood
group
trisaccharide sequence, D-galactosyl-a-1,4-D-galactosyl-(3-1,4-D-glucose,
which have been
identified in the LOS of the mucosal pathogens Neisser~ia gonnor~hoeae and N.
meningitidis
73
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
(Scholten et al., J. Med. Micr~obiol. 41: 236-243 (1994)). The genes from N.
menihgitidis and
N. gohorrhoeae that encode the glycosyltransferases involved in the
biosynthesis of these
structures have been identified from N. meningatidis immunotypes L3 and L1
(Jennings et al.,
Mol. Microbiol. 18: 729-740 (1995)) and the N. gono~rhoeae mutant F62
(Gotshlich, .I. Exp.
Med. 180: 2181-2190 (1994)). In N. menzngitidis, a locus consisting of three
genes, lgtA,
lgtB and lg E, encodes the glycosyltransferase enzymes required for addition
of the last three
of the sugars in the lacto-N neotetraose chain (Wakarchuk et al., J. Biol.
Chem. 271: 19166-
73 (1996)). Recently the enzymatic activity of the lgtB and lgtA gene product
was
demonstrated, providing the first direct evidence for their proposed
glycosyltransferase
function (Wakarchuk et al., J. Biol. Clzem. 271(45): 28271-276 (1996)). In N.
gono~y~hoeae,
there are two additional genes, lgtD which adds (3-D-GaINAc to the 3 position
of the terminal
galactose of the facto-N-neotetraose structure and lgtC which adds a terniinal
a-D-Gal to the
lactose element of a truncated LOS, thus creating the Pk blood group antigen
structure
(Gotshlich (1994), supra.). In N. mer~i~gitidis, a separate immmotype L1 also
expresses the
Pk blood group antigen and has been shown to carry an lgtC gene (Jennings et
al., (1995),
supf~a.). Neisseria glycosyltransferases and associated genes are also
described in USPN
5,545,553 (Gotschlich). Genes for a,1,2-fucosyltransferase and a,1,3-
fucosyltransferase from
Helicobacter pylof°i has also been characterized (Martin et al., .I.
Biol. Chem. 272: 21349-
21356 (1997)). Also of use in the present invention are the
glycosyltransferases of
Campylobacter jejuna (see, for example, http://afmb.cnrs-
mrs.fr/~pedro/CAZY/gtf 42.htm1).
Fucosyltf~ansferases
[0253] In some embodiments, a glycosyltransferase used in the method of the
invention is a
fucosyltransferase. Fucosyltransferases are known to those of slcill in the
art. Exemplary
fucosyltransferases include enzymes, which transfer L-fucose from GDP-fucose
to a hydroxy
position of an acceptor sugar. Fucosyltransferases that transfer non-
nucleotide sugars to an
acceptor are also of use in the present invention.
[0254] In some embodiments, the acceptor sugar is, for example, the GIcNAc in
a
Gal(3(1-a3,4)GIcNAc(3- group in an oligosaccharide glycoside. Suitable
fucosyltransferases
for this reaction include the Gal(3(1~3,4)GIcNAc(31-oc(1-
~3,4)fucosyltransferase (FTIII E.C.
No. 2.4.1.65), which was first characterized from human milk (see, Palcic, et
al.,
Ca~bohyd~°ate Res. 190: 1-11 (1989); Prieels, et al., J. Biol. Chern.
256: 10456-10463 (1981);
74
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
and Nunez, et al., Can. J. ChenZ. 59: 2086-2095 (1981)) and the
Gal(3(1~4)GIcNAc(3-
afucosyltransferases (FTIV, FTV, FTVI) which are found in human serum. FTVII
(E.C. No.
2.4.1.65), a sialyl a(2-~3)Gal(3((1-~3)GIcNAc(3 fucosyltransferase, has also
been
characterized. A recombinant form of the Gal(3(1-X3,4) GIcNAc(3-
all ~3,4)fucosyltransferase has also been characterized (see, Dumas, et al.,
Bioof°g. Med.
Letters 1: 425-428 (1991) and I~ukowska-Latallo, et al., Genes and Development
4: 1288-
1303 (1990)). Other exemplary fucosyltransferases include, for example, a1,2
fucosyltransferase (E.C. No. 2.4.1.69). Enzymatic fucosylation can be carried
out by the
methods described in Mollicone, et al., Eur. J. Biochem. 191: 169-176 (1990)
or U.S. Patent
No. 5,374,655. Cells that are used to produce a fucosyltransferase will also
include an
enzymatic system for synthesizing GDP-fucose.
Galactosyltransfe~ases
[0255] In another group of embodiments, the glycosyltransferase is a
galactosyltransferase.
Exemplary galactosyltransferases include a(1,3) galactosyltransferases (E.C.
No. 2.4.1.151,
see, e.g., Dabkowski et al., Ti°ansplant Proc. 25:2921 (19.93) and
Yamamoto et al. Nature
345: 229-233 (1990), bovine (GenBank j04989, Joziasse et al., J. Biol. ClaenZ.
264: 14290-
14297 (1989)), murine (GenBank m26925; Larsen et al., P~oc. Nat'l. Acad. Sci.
ZISA 86:
8227-8231 (1989)), porcine (GenBank L36152; Strahan et al., Immunogenetics 41:
101-105
(1995)). Another suitable a1,3 galactosyltransferase is that which is involved
in synthesis of
the blood group B antigen (EC 2.4.1.37, Yamamoto et al., J. Biol. Chena. 265:
1146-1151
(1990) (human)). Yet a further exemplary galactosyltransferase is core Gal-T1.
[0256] Also suitable for use in the methods of the invention are (3(1,4)
galactosyltransferases,,
which include, for example, EC 2.4.1.90 (LacNAc synthetase) and EC 2.4.1.22
(lactose
synthetase) (bovine (D'Agostaro et al., Eur~. J. Biochem. 183: 211-217
(1989)), human (Masri
et al., Biochem. Biophys. Res. Commun. 157: 657-663 (1988)), murine (Nalcazawa
et al., J.
Biochem. 104: 165-168 (1988)), as well as E.C. 2.4.1.38 and the ceramide
galactosyltransferase (EC 2.4.1.45, Stahl et al., J. Neur~osci. Res. 38: 234-
242 (1994)). Other
suitable galactosyltransferases include, for example, a1,2
galactosyltransferases (from e.g.,
Scl7izosaccharomyces pombe, Chapell et al., Mol. Biol. Cell 5: 519-528
(1994)).
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Sialyltt~ansferases
[0257] Sialyltransferases are another type of glycosyltransferase that is
useful in the
recombinant cells and reaction mixtures of the invention. Cells that produce
recombinant
sialyltransferases will also produce CMP-sialic acid, which is a sialic acid
donor for
sialyltransferases. Examples of sialyltransferases that are suitable for use
in the present
invention include ST3Ga1 III (e.g., a rat or human ST3Ga1 III), ST3Ga1 IV,
ST3Gal I,
ST3GalII, ST6Ga1 I, ST3Gal V, ST6Gal II, ST6GaINAc I, ST6GaINAc II, and
ST6GaINAc
III (the sialyltransferase nomenclature used herein is as described in Tsuji
et al.,
Glycobiology 6: v-xiv (1996)). An exemplary a(2,3)sialyltransferase referred
to as
a(2,3)sialyltransferase (EC 2.4.99.6) transfers sialic acid to the non-
reducing terminal Gal of
a Gal(31-~3Glc disaccharide or glycoside. See, Van den Eijnden et al., J.
Biol. Chem. 256:
3159 (1981), Weinstein et al., J. Biol. Chem. 257: 13845 (1982) and Wen et
al., J. Biol.
Claem. 267: 21011 (1992). Another exemplary a2,3-sialyltransferase (EC
2.4.99.4) transfers
sialic acid to the non-reducing terminal Gal of the disaccharide or glycoside.
see, Rearick et
al., J. Biol. Chem. 254: 4444 (1979) and Gillespie et al., J. Biol. ClZem.
267: 21004 (1992).
Further exemplary enzymes include Gal-(3-1,4-GIcNAc a-2,6 sialyltransferase
(See,
I~urosawa et al. Euf°. J. Biochena. 219: 375-381 (1994)).
[0258] Preferably, for glycosylation of carbohydrates of glycopeptides the
sialyltransferase
will be able to transfer sialic acid to the sequence Gal[31,4G1cNAc-, the most
common
penultimate sequence underlying the terminal sialic acid on fully sialylated
carbohydrate
structures (see, Table 2).
[0259] Table 2: Sialyltransferases which use the Gal[31,4G1cNAc sequence as an
acceptor
substrate
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Sialyltransferase Source ~ Sequence(s) formed Ref.
ST6GalI Mammalian NeuAca2,6Ga1[31,4G1cNAc-1
ST3GalIII Mammalian NeuAca2,3Ga1(31,4G1cNAc-1
NeuAca2,3Ga1(31,3G1cNAc-
ST3GalIV Mammalian NeuAca2,3Ga1(31,4G1cNAc-1
NeuAca2,3Ga1(31,3G1cNAc-
ST6GalII Mammalian NeuAca2,6Ga1(31,4G1cNAc
ST6GaIII photobacterium NeuAca2,6Ga1[31,4G1cNAc-2
ST3Ga1 V N. meningitidesNeuAca2,3Ga1(31,4G1cNAc-3
N. gono~y~hoeae
1) Goochee et al., BiolTechnology 9: 1347-1355 (19H1)
2) Yamamoto et al., J. Biochem. 120: 104-110 (1996)
3) Gilbert et al., .I. Biol. Chem. 271: 28271-28276 (1996)
[0260] Other sialyltransferases of use in the present invention include those
set forth in the
table of FIG. 4. The sialyltransferases can be used to transfer a PEGylated
sialic acid moiety
from a PEGylated sialic acid donor species onto an N-linked glycosyl residue
of a peptide
(FIG. 2C) or an O-linked glycosyl residue of Factor IX (FIG. 2D).
[0261] An example of a sialyltransferase that is useful in the claimed methods
is ST3Gal III,
which is also referred to as a(2,3)sialyltransferase (EC 2.4.99.6). This
enzyme catalyzes the
transfer of sialic acid to the Gal of a Gal(31,3G1cNAc or Gal[i1,4G1cNAc
glycoside (see, e.g.,
Wen et al., J. Biol. Chem. 267: 21011 (1992); Van den Eijnden et al., J. Biol.
Chern. 256:
3159 (1991)) and is responsible for sialylation of asparagine-linlced
oligosaccharides in
glycopeptides. The sialic acid is linked to a Gal with the formation of an a-
linkage between
the two saccharides. Bonding (linkage) between the saccharides is between the
2-position of
NeuAc and the 3-position of Gal. This particular enzyme can be isolated from
rat liver
(Weinstein et al., J. Biol. Chem. 257: 13845 (1982)); the human cDNA (Sasaki
et al. (1993)
J. Baol. Chem. 268: 22782-22787; Kitagawa & Paulson (1994) .l. Biol. Chem.
269: 1394-
1401) and genomic (Kitagawa et al. (1996) J. Biol. Clzenz. 271: 931-938) DNA
sequences are
known, facilitating production of this enzyme by recombinant expression. In a
preferred
embodiment, the claimed sialylation methods use a rat ST3Ga1 III.
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[0262] Other exemplary sialyltransferases of use in the present invention
include those
isolated from Campylobacter jejuni, including the a,(2,3). See, e.g,
WO99149051.
[0263] Sialyltransferases other those listed in Table 2, are also useful in an
economic and
efficient large-scale process for sialylation of commercially important
glycopeptides. As a
simple test to find out the utility of these other enzymes, various amounts of
each enzyme
(1-100 mU/mg protein) are reacted with asialo-al AGP (at 1-10 mg/ml) to
compare the
ability of the sialyltransferase of interest to sialylate glycopeptides
relative to either bovine
ST6Ga1 I, ST3Ga1 III or both sialyltransferases. Alternatively, other
glycopeptides or
glycopeptides, or N-linked oligosaccharides enzymatically released from the
peptide
backbone can be used in place of asialo-al AGP for this evaluation.
Sialyltransferases with
the ability to sialylate N-linked oligosaccharides of glycopeptides more
efficiently than
ST6Ga1 I are useful in a practical large-scale process for peptide
sialylation.
GaINAe tf~ayisfef~ases
[0264] N-acetylgalactosaminyltransferases are of use in practicing the present
invention,
particularly for binding a GaINAc moiety to an amino acid of the O-linked
glycosylation site
of the peptide. Suitable N-acetylgalactosaminyltransferases include, but are
not limited to,
a(1,3) N-acetylgalactosaminyltransferase, (3(1,4) N-
acetylgalactosaminyltransferases (Nagata
et al., J. Biol. ClZena. 267: 12082-12089 (1992) and Smith et al., J. Biol
Chem. 269: 15162
(1994)) and polypeptide N-acetylgalactosaminyltransferase (Homa et al., J.
Biol. Chem. 268:
12609 (1993)).
[0265] Production of proteins such as the enzyme GaINAc TI_xx from cloned
genes by
genetic engineering is well known. See, eg., U.S. Pat. No. 4,761,371. One
method involves
collection of sufficient samples, then the amino acid sequence of the enzyme
is determined
by N-terminal sequencing. This information is then used to isolate a cDNA
clone encoding a
full-length (membrane bound) transferase which upon expression in the insect
cell line Sf~
resulted in the synthesis of a fully active enzyme. The acceptor specificity
of the enzyme is
then determined using a semiquantitative analysis of the amino acids
surrounding known
glycosylation sites in 16 different proteins followed by in vitro
glycosylation studies of
synthetic peptides. This work has demonstrated that certain amino acid
residues are
overrepresented in glycosylated peptide segments and that residues in specific
positions
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surrounding glycosylated serine and threonine residues may have a more marked
influence on
acceptor efficiency than other amino acid moieties.
Cell Bound Glyeosyltfansfefases
[0266] In another embodiment, the enzymes utilized in the method of the
invention are cell-
s bound glycosyltransferases. Although many soluble glycosyltransferases are
known (see, for
example, U.S. Pat. No. 5,032,519), glycosyltransferases are generally in
membrane-bound
form when associated with cells. Many of the membrane-bound enzymes studied
thus far are
considered to be intrinsic proteins; that is, they are not released from the
membranes by
sonication and require detergents for solubilization. Surface
glycosyltransferases have been
identified on the surfaces of vertebrate and invertebrate cells, and it has
also been recognized
that these surface transferases maintain catalytic activity under
physiological conditions.
However, the more recognized function of cell surface glycosyltransferases is
for intercellular
recognition (Roth, MOLECULAR APPR~ACHES to SUPRACELLULAR PHENOMENA, 1990).
[0267] Methods have been developed to alter the glycosyltransferases expressed
by cells.
For example, Larsen et al., P~oc. Natl. Acad. Scz. USA 86: 8227-8231 (1989),
report a genetic
approach to isolate cloned cDNA sequences that determine expression of cell
surface
oligosaccharide structures and their cognate glycosyltransferases. A cDNA
library generated
from mRNA isolated from a marine cell line known to express UDP-galactose:.(3.-
D-
galactosyl-1,4-N-acetyl-D-glucosaminide a-1,3-galactosyltransferase was
transfected into
COS-1 cells. The transfected cells were then cultured and assayed for a 1-3
ga'lactosyltransferase activity.
[0268] Francisco et al., Pr°oc. Natl. Acad. Sci. USA 89: 2713-2717
(1992), disclose a method
of anchoring (3-lactamase to the external surface of Escher°ichia coli.
A tripartite fusion
consisting of (i) a signal sequence of an outer membrane protein, (ii) a
membrane-spanning
section of an outer membrane protein, and (iii) a complete mature (3-lactamase
sequence is
produced resulting in an active surface bound (3-lactamase molecule. However,
the Francisco
method is limited only to procaryotic cell systems and as recognized by the
authors, requires
the complete tripartite fusion for proper functioning.
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Sulfotransferases
[0269] The invention also provides methods for producing peptides that include
sulfated
molecules, including, for example sulfated polysaccharides such as heparin,
heparan sulfate,
carragenen, and related compounds. Suitable sulfotransferases include, for
example,
chondroitin-6-sulphotransferase (chicken cDNA described by Fukuta et al., J.
Biol. Chern.
270: 18575-18580 (1995); GenBank Accession No. D49915), glycosaminoglycan N-
acetylglucosamine N-deacetylase/N-sulphotransferase 1 (Dixon et al., Genomics
26: 239-241
(1995); UL18918), and glycosaminoglycan N-acetylglucosamine N-deacetylase/N-
sulphotransferase 2 (marine cDNA described in Orellana et al., J. Biol. Chena.
269: 2270-
2276 (1994) and Eriksson et al., J. Biol. Cherry. 269: 10438-10443 (1994);
human cDNA
described in GenBank Accession No. U2304).
Glycosidases
[0270] This invention also encompasses the use of wild-type and mutant
glycosidases.
Mutant ~-galactosidase enzymes have been demonstrated to catalyze the
formation of
disaccharides through the coupling of an a-glycosyl fluoride to a galactosyl
acceptor
molecule. (Withers, U.S. Pat. No. 6,284,494; issued Sept. 4, 2001). Other
glycosidases of
use in this invention include, for example, (3-glucosidases, (3-
galactosidases, (3-mannosidases,
(3-acetyl glucosaminidases, (3-N-acetyl galactosaminidases, a-xylosidases, a-
fucosidases,
cellulases, xylanases, galactanases, mannanases, hemicellulases, amylases,
glucoamylases, a-
glucosidases, a-galactosidases, a-mannosidases, a-N-acetyl glucosaminidases, a-
N-acetyl
galactose-aminidases, a-xylosidases, a-fucosidases, and
neuraminidases/sialidases. In an
exemplary embodiment, a sialidase is used to remove sialic acid from an N-
glycan of Factor
IX (FIG. 2A) prior to glycoPEGylating. The invention also provides a method
that does not
require the prior removal of sialic acid. Thus, a method that incorporates a
sialic acid
exchange reaction using a modified sialic acid moiety and ST3Ga13 is of use in
the present
invention.
Iuirrzobilized Enzymes
[0271] The present invention also provides for the use of enzymes that are
immobilized on a
solid and/or soluble support. In an exemplary embodiment, there is provided a
glycosyltransferase that is conjugated to a PEG via an intact glycosyl linker
according to the
methods of the invention. The PEG-linker-enzyme conjugate' is optionally
attached to solid
CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
support. The use of solid supported enzymes in the methods of the invention
simplifies the
work up of the reaction mixture and purification of the reaction product, and
also enables the
facile recovery of the enzyme. The glycosyltransferase conjugate is utilized
in the methods
of the invention. Other combinations of enzymes and supports will be apparent
to those of
skill in the art.
Fusion Proteins
[0272] In other exemplary embodiments, the methods of the invention utilize
fusion proteins
that have more than one enzymatic activity that is involved in synthesis of a
desired
glycopeptide conjugate. The fusion polypeptides can be composed of, for
example, a
catalytically active domain of a glycosyltransferase that is joined to a
catalytically active
domain of an accessory enzyme. The accessory enzyme catalytic domain can, for
example,
catalyze a step in the formation of a nucleotide sugar that is a donor for the
glycosyltransferase, or catalyze a reaction involved in a glycosyltransferase
cycle. For
example, a polynucleotide that encodes a glycosyltransferase can be joined, in-
frame, to a
polynucleotide that encodes an enzyme involved in nucleotide sugar synthesis.
The resulting
fusion protein can then catalyze not only the synthesis of the nucleotide
sugar, but also the
transfer of the sugar moiety to the acceptor molecule. The fusion protein can
be two or more
cycle enzymes linked into one expressible nucleotide sequence. In other
embodiments the
fusion protein includes the catalytically active domains of two or more
glycosyltransferases.
See, for example, 5,641,668. The modified glycopeptides of the present
invention can be
readily designed and manufactured utilizing various suitable fusion proteins
(see, for
example, PCT Patent Application PCT/CA98/01180, which was published as WO
99/31224
on June 24, 1999.)
Preparation of Modified Sugars
[0273] In general, the sugar moiety or sugar moiety-linker cassette and the
PEG or PEG-
linker cassette groups are linked together through the use of reactive groups,
which are
typically transformed by the linking process into a new organic functional
group or
unreactive species. The sugar reactive functional group(s), is located at any
position on the
sugar moiety. Reactive groups and classes of reactions useful in practicing
the present
invention are generally those that are well known in the art of bioconjugate
chemistry.
Currently favored classes of reactions available with reactive sugar moieties
are those, which
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CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
proceed under relatively mild conditions. These include, but are not limited
to nucleophilic
substitutions (e.g., reactions of amines and alcohols with acyl halides,
active esters),
electrophilic substitutions (e.g., enamine reactions) and additions to carbon-
carbon and
carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder
addition). These and
other useful reactions are discussed in, for example, March, ADVANCED ORGANIC
CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE
TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., M~DIFICATI~N
OF
PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society,
Washington, D.C., 1982.
[0274] Useful reactive functional groups pendent from a sugar nucleus or
modifying group
include, but axe not limited to:
(a) carboxyl groups and various derivatives thereof including, but not limited
to,
N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl
imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and
aromatic
esters;
(b) hydroxyl groups, which can be converted to, e.g., esters, ethers,
aldehydes, etc.
(c) haloalkyl groups, wherein the halide can be later displaced with a
nucleophilic
group such as, for example, an amine, a carboxylate anion, thiol anion,
carbanion, or
an allcoxide ion, thereby r esulting in the covalent attachment of a new group
~at the
functional group of the halogen atom;
(d) dienophile groups, which are capable of participating in Diels-Alder
reactions
such as, for example, maleimido groups;
(e) aldehyde or lcetone groups, such that subsequent derivatization is
possible via
formation of carbonyl derivatives such as, for example, imines, hydrazones,
semicarbazones or oximes, or via such mechanisms as Grignard addition or
alkyllithium addition;
(f) sulfonyl halide groups for subsequent reaction with amines, for example,
to form
sulfonamides;
(g) thiol groups, which can be, for example, converted to disulfides or
reacted with
acyl halides;
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WO 2005/055950 PCT/US2004/041070
(h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated
or
oxidized;
(i) alkenes, which can undergo, for example, cycloadditions, acylation,
Michael
addition, etc; and
(j) epoxides, which can react with, for example, amines and hydroxyl
compounds.
[0275] The reactive functional groups can be chosen such that they do not
participate in, or
interfere with, the reactions necessary to assemble the reactive sugar nucleus
or modifying
group. Alternatively, a reactive functional group can be protected from
participating in the
reaction by the presence of a protecting group. Those of skill in the art
understand how to
protect a particular functional group such that it does not interfere with a
chosen set of
reaction conditions. For examples of useful protecting groups, see, for
example, Greene et
al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York,
1991.
[0276] In the discussion that follows, a number of specific examples of
modified sugars that
are useful in practicing the present invention are set forth. In the exemplary
embodiments, a
sialic acid derivative is utilized as the sugar nucleus to which the modifying
group is
attached. The focus of the discussion on sialic acid derivatives is for
clarity of illustration
only and should not be co~lstrued to limit the scope of the invention. Those
of skill in the art
will appreciate that a variety of other sugar moieties can be activated and
derivatized in a
manner analogous to that set forth using sialic acid as an example. For
example, numerous
methods are available for modifying galactose, glucose, N-acetylgalactosamine
and fucose to
name a few sugar substrates, which are readily modified by art recognized
methods. See, for
example, E111alabi et al., Cu~~. Med. Che~n. 6: 93 (1999); and Schafer et al.,
J. Of~g. Che~~.
65: 24 (2000)).
[0277] In an exemplary embodiment, the peptide that is modified by a method of
the
invention is a glycopeptide that is produced in mammalian cells (e.g., CHO
cells) or in a
transgenic animal and thus, contains N- and/or O-linked oligosaccharide
chains, which are
incompletely sialylated. The oligosaccharide chains of the glycopeptide
lacking a sialic acid
and containing a terminal galactose residue can be PEGylated, PPGylated or
otherwise
modified with a modified sialic acid.
[0278] In Scheme 4, the amino glycoside 1, is treated with the active ester of
a protected
amino acid (e.g., glycine) derivative, converting the ,sugar amine residue
into the
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WO 2005/055950 PCT/US2004/041070
corresponding protected amino acid amide adduct. The adduct is treated with an
aldolase to
form a,-hydroxy carboxylate 2. Compound 2 is converted to the corresponding
CMP
derivative by the action of CMP-SA synthetase, followed by catalytic
hydrogenation of the
CMP derivative to produce compound 3. The amine introduced via formation of
the glycine
adduct is utilized as a locus of PEG attaclunent by reacting compound 3 with
an activated
PEG or PPG derivative (e.g., PEG-C(O)NHS, PEG-OC(O)O-p-nitrophenyl), producing
species such as 4 or 5, respectively.
Scheme 4
OH 1. CMP-SA synthetase, CTP
HO NH 1. Z-Glycine-NHS HO 0H 2. Hz/Pd/C
HO 2 2. NeuAc Aldolase, pyruvate HO ~ O O-~Na
HO O Z~ - OH 0
OH ~ o NH
1 Z NHS
NHZ 'N
O ~ '~ O
_O O N O 0
HO ~ + ~ PEG-~-NHS HO OH 0' Na
p OH O Na ~ HO ; O O-+Na HO OH
HO - 0 O- Na HO OH O
PEG-C~ ~NH off 0 HZN~NH OH
N 4 ~ 0
H O
O
CMP-SA-5-NHCOCHZNH-PEG pEG-~C(O)0-pNPC CMP-SA-5-NHCOCHZNH2
CMP-SA-5-NHCOCHZNH-C(Q)O-PEG
[0279] Table 3 sets forth representative examples of sugar monophosphates that
are
derivatized with a PEG moiety. Certain of the compounds of Table 3 are
prepared by the
method of Scheme 4. Other derivatives are prepared by art-recognized methods.
See, for
example, Keppler et al., Glycobiology 11: 11R (2001); and Charter et al.,
Glycobiology 10:
1049 (2000)). Other amine reactive PEG and PPG analogues are commercially
available, or
they can be prepared by methods readily accessible to those of skill in the
art.
[0280] Table 3
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WO 2005/055950 PCT/US2004/041070
NHZ NHa
O I N ~O O I N ~O
O.-P_O O O-P_O O
HO OH ~_+N~ HO H ~_+N
HO ~ 0' ~p-+Na H~O--O~H R-O ~ O' ~-O'+Na HO OH
R-NH ' OH O AcNH OH 0
CMP-SA-5 ~NH-R CMP-NeuAc-9-O-R
NHZ . NHZ
0
O-rP'O 0 N O -P_O 0 N O
HO OH ~_+N~ H ~_+N f-l
HO ~ O~ ~-O'+Na H ~O--O( H R-NH ~ O~ ~--O-+Na HO OH
R-O ' OH 0 AcNH OH 0
CMP-NeuAc-9-NH-R NHZ
CMP-KDN-5-0-R NHZ 0 I 'N
w N Ii N'~O
R-NH o o- N~a p
R-O 0 _ ~ ~J HO O O +Na Hp pH
O Na N
HO ~H p O-+Na HO OH ACNH ' OH 0
AcNH ' o
off CMP-NeuAc-8-NH-R
CMP-NeuAc-8-0-R NH2
NHZ 0 I ~ N
O I \~ 0-P-0 O Nk0
0'~ o ~N o HO NH-R o_+N~
HO O-R p-~+Na N HO O O'+Na HO OH
HO O O'+Na HO OH ACNH OH O
AcNH o
OH
CMP-NeuAc-7-NH-R NHa
CMP-NeuAc-7-O-R ~N O
O I N~0 O-IPI-0 O N~O
HO OH o~o' N~ HO OH o +N~
HO O O- Na HO OH
HO O O'+Na HO OH AONH ' O
AcNH ~-R o NH-R
CMP-NeuAc-4-O-R CMP-NeuAc-4-NH-R
[0281] The modified sugar phosphates of use in practicing the present
invention can be
substituted in other positions as well as those set forth above. Presently
preferred
substitutions of sialic acid are set forth in the formula below:
NH2
~~N
O ~~O
0-P_O O
R2 Y __X R~ ° +N" 1-
R3-B O O +Na HO OH
R4-A O
Z-R5
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in which X is a linking group, which is preferably selected from -O-, -N(H)-, -
S, CH2-, and -
N(R)Z, in which each R is a member independently selected from Rl-R5. The
symbols Y, Z,
A and B each represent a group that is selected from the group set forth above
for the identity
of X. X, Y, Z, A and B are each independently selected and, therefore, they
can be the same
or different. The symbols R~, RZ, R3, R4 and RS represent H, a PEG moiety,
therapeutic
moiety, biomolecule or other moiety. Alternatively, these symbols represent a
linker that is
bound to a PEG moiety, therapeutic moiety, biomolecule or other moiety.
[0282] Exemplary moieties attached to the conjugates disclosed herein include,
but are not
limited to, PEG derivatives (e.g., acyl-PEG, acyl-alkyl-PEG, alkyl-acyl-PEG
carbamoyl-
PEG, aryl-PEG), PPG derivatives (e.g., acyl-PPG, acyl-alkyl-PPG, alkyl-acyl-
PPG
carbamoyl-PPG, aryl-PPG), therapeutic moieties, diagnostic moieties, mamlose-6-
phosphate,
heparin, heparan, SLeX, mannose, mannose-6-phosphate, Sialyl Lewis X, FGF,
VFGF,
proteins, chondroitin, keratan, dermatan, albumin, integrins, antennary
oligosaccharides,
peptides and the like. Methods of conjugating the various modifying groups to
a saccharide
moiety are readily accessible to those of skill in the art (POLY (ETHYLENE
GLYCOL
CHEMISTRY : BIOTECHNICAL AND BIOMEDICAL APPLICATIONS, J. Milton Harris, Ed.,
Plenum
Pub. Corp., 1992; POLY (ETHYLENE GLYCOL) CHEMICAL AND BIOLOGICAL APPLICATIONS,
J.
Milton Harris, Ed., ACS Symposium Series No. 680, American Chemical Society,
1997;
Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Dunn
et al.,
Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium Series Vol. 469,
American Chemical Society, Washington, D.C. 1991).
Linker Groups (Cross-linking Groups)
[0283] Preparation of the modified sugar for use in the methods of the present
invention
includes attachment of a PEG moiety to a sugar residue and preferably, forming
a stable
adduct, which is a substrate for a glycosyltransferase. Thus, it is often
preferred to use a
linker, e.g., one formed by reaction of the PEG and sugar moiety with a cross-
linking agent to
conjugate the PEG and the sugar. Exemplary bifunctional compounds which can be
used for
attaching modifying groups to carbohydrate moieties include, but are not
limited to,
bifunctional poly(ethyleneglycols), polyamides, polyethers, polyesters and the
like. General
approaches for linlcing carbohydrates to other molecules are known in the
literature. See, for
example, Lee et al., Biochemistry 28: 1856 (1989); Bhatia et al., Anal.
Biochena. 178: 408
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CA 02549413 2006-06-02
WO 2005/055950 PCT/US2004/041070
(1989); Janda et al., J. Am. Chem. Soc. 112: 8886 (1990) and Bednarski et al.,
WO 92/18135.
In the discussion that follows, the reactive groups are treated as benign on
the sugar moiety of
the nascent modified sugar. The focus of the discussion is for clarity of
illustration. Those of
skill in the art will appreciate that the discussion is relevant to reactive
groups on the
modifying group as well.
[0284] A variety of reagents are used to modify the components of the modified
sugar with
intramolecular chemical crosslinks (for reviews of crosslinking reagents and
crosslinking
procedures see: Wold, F., Meth. Enzymol. 25: 623-651, 1972; Weetall, H. H.,
and Gooney, D.
A., In: ENZYMES as DRUGS. (Holcenberg, and Roberts, eds.) pp. 395-442, Wiley,
New York,
1981; Ji, T. H., Meth. Enzyrnol. 91: 580-609, 1983; Mattson et al., Mol. Biol.
Rep. 17: 167-
183, 1993, all of which are incorporated herein by reference). Preferred
crosslinking reagents
are derived from various zero-length, homo-bifunctional, and hetero-
bifunctional crosslinking
reagents. Zero-length crosslinking reagents include direct conjugation of two
intrinsic
chemical groups with no introduction of extrinsic material. Agents that
catalyze formation of
a disulfide bond belong to this category. Another example is reagents that
induce
condensation of a carboxyl and a primary amino group to form an amide bond
such as
carbodiimides, ethylchlorofonnate, Woodward's reagent K (2-ethyl-5-
phenylisoxazolium-3'-
sulfonate), and carbonyldiimidazole. In addition to these chemical reagents,
the enzyme
transglutaminase (glutamyl-peptide y-glutamyltransferase; EG 2.3.2.13) may be
used as zero-
length crosslinking reagent. This enzyme catalyzes acyl transfer reactions at
carboxamide
groups of protein-bound glutaminyl residues, usually with a primary amino
group as
substrate. Preferred homo- and hetero-bifunctional reagents contain two
identical or two
dissimilar sites, respectively, which may be reactive for amino, sulflzydryl,
guanidino, indole,
or nonspecific groups.
Purification of Factor IX Conjugates
[0285] The products produced by the above processes can be used without
purification.
However, it is usually preferred to recover the product. Standard, well-known
techniques for
recovery of glycosylated saccharides such as thin or thick layer
chromatography, column
chromatography, ion exchange chromatography, or membrane filtration can be
used. It is
preferred to use membrane filtration, more preferably utilizing a reverse
osmotic membrane,
or one or more column chromatographic techniques for the recovery as is
discussed
hereinafter and in the literature cited herein. For instance, membrane
filtration wherein the
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membranes have molecular weight cutoff of about 3000 to about 10,000 can be
used to
remove proteins such as glycosyl transferases. Nanofiltration or reverse
osmosis can then be
used to remove salts and/or purify the product saccharides (see, e.g., WO
98/15581).
Nanofilter membranes are a class of reverse osmosis membranes that pass
monovalent salts
but retain polyvalent salts and uncharged solutes larger than about 100 to
about 2,000
Daltons, depending upon the membrane used. Thus, in a typical application,
saccharides
prepared by the methods of the present invention will be retained in the
membrane and
contaminating salts will pass through.
[0286] If the modified glycoprotein is produced intracellularly, as a first
step, the particulate
debris, either host cells or lysed fragments, is removed, for example, by
centrifugation or
ultrafiltration; optionally, the protein may be concentrated with a
commercially available
protein concentration filter, followed by separating the polypeptide variant
from other
impurities by one or more steps selected from immunoaffinity chromatography,
ion-exchange
column fractionation (e.g., on diethylaminoethyl (DEAF) or matrices containing
carboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose, CM
Blue-
Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose, WGA-Sepharose, Con A-
Sepharose, Ether Toyopearl, Butyl Toyopearl, Phenyl Toyopearl, or protein A
Sepharose,
SDS-PAGE chromatography, silica chromatography, chromatofocusing, reverse
phase HPLC
(e.g., silica gel with appended aliphatic groups), gel filtration using, e.g.,
Sephadex molecular
sieve or size-exclusion chromatography, chromatography on columns that
selectively bind
the polypeptide, and ethanol or ammonium sulfate precipitation.
[0287] Modified glycopeptides produced in culture are usually isolated by
initial extraction
from cells, enzymes, etc., followed by one or more concentration, salting-out,
aqueous ion-
exchange, or size-exclusion chromatography steps. Additionally, the modified
glycoprotein
may be purified by affinity chromatography. Finally, HPLC may be employed for
final
purification steps.
[0288] A protease inhibitor, e.g., methylsulfonylfluoride (PMSF) may be
included in any of
the foregoing steps to inhibit proteolysis and antibiotics may be included to
prevent the
growth of adventitious contaminants.
[0289] Within another embodiment, supernatants from systems which produce the
modified
glycopeptide of the invention are first concentrated using a commercially
available protein
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concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit.
Following the concentration step, the concentrate may be applied to a suitable
purification
matrix. For example, a suitable affinity matrix may comprise a ligand for the
peptide, a lectin
or antibody molecule bound to a suitable support. Alternatively, an anion-
exchange resin
may be employed, for example, a matrix or substrate having pendant DEAE
groups. Suitable
matrices include acryhamide, agarose, dextran, cellulose, or other types
commonly employed
in protein purification. Alternatively, a ration-exchange step may be
employed. Suitable
ration exchangers include various insoluble matrices comprising sulfopropyl or
carboxymethyl groups. Sulfopropyl groups are particularly preferred.
[0290] Finally, one or more RP-HPLC steps employing hydrophobic RP-HPLC media,
e.g.,
silica gel having pendant methyl or other aliphatic groups, may be employed to
fiuther purify
a pohypeptide variant composition. Some or all of the foregoing purification
steps, in various
combinations, can also be employed to provide a homogeneous modified
glycoprotein.
[0291] The modified glycopeptide of the invention resulting from a large-scale
fermentation
may be purified by methods analogous to those disclosed by Urdal et al., J.
Chromatog. 296:
171 (1984). This reference describes two sequential,1ZP-HPLC steps for
purification of
recombinant human IL-2 on a preparative HPLC column. Alternatively, techniques
such as
affinity chromatography may be utilized to purify the modified glycoprotein.
Pharmaceutical Compositions
[0292] In another aspect, the invention provides a pharmaceutical composition.
The
pharmaceutical composition includes a pharmaceutically acceptable diluent and
a covalent
conjugate between a non-naturally-occurring, PEG moiety, therapeutic moiety or
biomohecule and a glycosylated or non-ghycosylated Factor IX peptide. The
polymer,
therapeutic moiety or biomolecuhe is conjugated to the peptide via an intact
ghycosyl linking
group interposed between and covalenthy linked to both the peptide and the
polymer,
therapeutic moiety or biomolecule.
[0293] Pharmaceutical compositions of the invention are suitable for use in a
variety of drug
delivery systems. Suitable formulations for use in the present invention are
found in
Remington's Pharynaceutical Sciences, Mace Publishing Company, Philadelphia,
PA, 17th
ed. (1985). For a brief review of methods for drug delivery, see, Langer,
Science 249:1527-
1533 (1990).
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[0294] The pharmaceutical compositions may be formulated for any appropriate
manner of
administration, including for example, topical, oral, nasal, intravenous,
intracranial,
intraperitoneal, subcutaneous or intramuscular administration. For parenteral
administration,
such as subcutaneous injection, the carrier preferably comprises water,
saline, alcohol, a fat, a
wax or a buffer. For oral administration, any of the above carriers or a solid
carrier, such as
mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum,
cellulose, glucose,
sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres
(e.g.,
polylactate polyglycolate) may also be employed as carriers for the
pharmaceutical
compositions of this invention. Suitable biodegradable microspheres are
disclosed; for
example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
[0295] Commonly, the pharmaceutical compositions are administered
parenterally, e.g.,
intravenously. Thus, the invention provides compositions for parenteral
administration which
comprise the compound dissolved or suspended in an acceptable carrier,
preferably an
aqueous carrier, e.g., water, buffered water, saline, PBS and the like. The
compositions may
contain pharmaceutically acceptable auxiliary substances as required to
approximate
physiological conditions, such as pH adjusting and buffering agents, tonicity
adjusting agents,
wetting agents, detergents and the like.
[0296] These compositions may be sterilized by conventional sterilization
techniques, or may
be sterile filtered. The resulting aqueous solutions may be packaged for use
as is, or
lyophilized, the lyophilized preparation being combined with a sterile aqueous
carrier prior to
administration. The pH of the preparations typically will be between 3 and 1
l, more
preferably from 5 to 9 and most preferably from 7 and 8.
[0297] In some embodiments the glycopeptides of the invention can be
incorporated into
liposomes formed from standard vesicle-forming lipids. ~ A variety of methods
are available
for preparing liposomes, as described in, e.g., Szoka et al., AyZh. Rev.
Biophys. Bioe~cg. 9: 467
(1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of
liposomes using
a variety of targeting agents (e.g., the sialyl galactosides of the invention)
is well knomn in
the art (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044).
[0298] Standard methods for coupling targeting agents to liposomes can be
used. These
methods generally involve incorporation into liposomes of lipid components,
such as
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phosphatidylethanolamine, which can be activated for attachment of targeting
agents, or
derivatized lipophilic compounds, such as lipid-derivatized glycopeptides of
the invention.
[0299] Targeting mechanisms generally require that the targeting agents be
positioned on the
surface of the liposome in such a manner that the target moieties are
available for interaction
with the target, for example, a cell surface receptor. The carbohydrates of
the invention may
be attached to a lipid molecule before the liposome is formed using methods
known to those
of skill in the art (e.g., alkylation or acylation of a hydroxyl group present
on the
carbohydrate with a long chain alkyl halide or with a fatty acid,
respectively). Alternatively,
the liposome may be fashioned in such a way that a connector portion is first
incorporated
into the membrane at the time of forming the membrane. The connector portion
must have a
lipophilic portion, which is firmly embedded and anchored in the membrane. It
must also
have a reactive portion, which is chemically available on the aqueous surface
of the liposome.
The reactive portion is selected so that it will be chemically suitable to
form a stable chemical
bond with the targeting agent or carbohydrate, which is added later. In some
cases it is
possible to attach the target agent to the connector molecule directly, but in
most instances it
is more suitable to use a third molecule to act as a chemical bridge, thus
linking the connector
molecule which is in the membrane with the target agent or carbohydrate which
is extended,
three dimensionally, off of the vesicle surface.
[0300] The compounds prepared by the methods of the invention may also find
use as
diagnostic reagents. For example, labeled compounds can be used to locate
areas of
inflammation or tumor metastasis in a patient suspected of having an
inflammation. For this
use, the compounds can be labeled with lash 14C, or tritium. '
[0301] The active ingredient used in the pharmaceutical compositions of the
present
invention is glycopegylated Factor IX and its derivatives having the
biological properties of
participating in the blood coagulation cascade. The liposomal dispersion of
the present
invention is useful as a parenteral formulation in treating coagulation
disorders characterized
by low or defective coagulation such as various forms of hemophilia.
Preferably, the Factor
IX composition of the present invention is administered parenterally (e.g. IV,
IM, SC or IP).
Effective dosages are expected to vary considerably depending on the condition
being treated
and the route of administration but are expected to be in the range of about
0.1 to 000 ~.glkg
body weight of the active material. Preferable doses for treatment of
coagulation disorders
are about 50 to about 3000 wg fkg three times a week. More preferrabley, about
500 to about
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2000 ~.g /kg three times a weelc. More preferrably, about 750 to about 1500 ~g
/kg three
times a week, and more preferrably about 1000 ~.g /kg three times a week.
Because the
present invention provides a Factor IX with an enhanced i~ vivo residence
time, the stated
dosages are optionally lowered when a composition of the invention is
administered. [Are
these dosages appropriate?]
[0302] The following examples are provided to illustrate the conjugates, and
methods and
of the present invention, but not to limit the claimed invention.
EXAMPLES
Example 1
Preparation of UDP-GaINAc-6'-CHO
[0303] UDP-GaINAc (200 mg, 0.30 mmoles) was dissolved in a 1 mM CuSO4 solution
(20
mL) and a 25 mM NaH2P04 solution (pH 6.0; 20 mL). Galactose oxidase (240 U;
240 ~L)
and catalase (13000 U; 130 ~L) were then added, the reaction system equipped
with a balloon
filled with oxygen and stirred at room temperature for seven days. The
reaction mixture was
then filtered (spin cartridge; MWCO SK) and the filtrate (~40 mL) was stored
at 4° C until
required. TLC (silica; EtOH/water (7/2); Rf= 0.77; visualized with
anisaldehyde stain).
Example 2
Preparation of UDP-GaINAc-6'-NH2
[0304] Ammonium acetate (15 mg, 0.194 mmoles) and NaBH3CN (1M THF solution;
0.17
mL, 0.17 mmoles) were added to the UDP-GaINAc-6'-CHO solution from above (2 mL
or
20 mg) at 0°C and allowed to warm to room temperature overnight. The
reaction was filtered
through a G-10 column with water and the product collected. The appropriate
fractions were
freeze-dried and stored frozen. TLC (silica; ethanol/water (7/2); Rf= 0.72;
visualized with
ninhydrin reagent).
Example 3
Preparation of UDP-GaINAc-6-NHCO(CH2)2-O-PEG-OMe (1 KDa)
[0305] The galactosaminyl-1-phosphate-2-NHCO(CH2)a-O-PEG-OMe (1 KDa) (SS mg,
0.045 mmoles) was dissolved in DMF (G mL ) and pyridine (1.2 mL). UMP-
morpholidate
(60 mg, 0.15 mmoles) was then added and the resulting mixture stiiTed at
70°C for 4~ h. The
solvent was removed by bubbling nitrogen through the reaction mixture and the
residue
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purified by reversed phase chromatography (C-18 silica, step gradient between
10 to 80%,
methanol/water). The desired fractions were collected and dried at reduced
pressure to yield
50 mg (70%) of a white solid. TLC (silica, propanol/HZO/NH40H, (30/20/2), Rf=
0.54). MS
(MALDI): Observed, 1485, 1529, 1618, 1706.
Example 4
Preparation of Cysteine-PEG2 (2)
NHa
HS~OH + ~O~O~OTs KOH, MeOH ~ ~O~ NHZ
n O'~ ~ 7'n~ ~S~OH
O O
O
/ NO~ ~O~O~O~NH
n
CH2Clz/TEA /O~O~S~OH
/ IIn
O
4.1 Synthesis of (1)
[0306] Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was added to a
solution of
L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20 mL) under argon. The
mixture
was stined at room temperature for 30 min, and then mPEG-O-tosylate of
molecular mass 20
kilodalton (Ts; 1.0 g, 0.05 mmol) was added in several portions over 2 hours.
The mixture
was stirred at room temperature for 5 days, and concentrated by rotary
evaporation. The
residue was diluted with water (30 mL), and stirred at room temperature for 2
hours to
destroy any excess 20 lcilodalton mPEG- O-tosylate. The solution was then
neutralized with
acetic acid, the pH adjusted to pH 5.0 and loaded onto a reverse phase
chromatography (C-18
silica) column. The column was eluted with a gradient of methanol/water (the
product elutes
at about 70% methanol), product elution monitored by evaporative light
scattering, and the
appropriate fractions collected and diluted with water (500 mL). This solution
was
chromatographed (ion exchange, XK 50 Q, BIG Beads, 300 mL, hydroxide form;
gradient of
water to water/acetic acid-0.75N) and the pH of the appropriate fractions
lowered to 6.0 with
acetic acid. This solution was then captured on a reversed phase column (C-18
silica) and
eluted with a gradient of methanol/water as described above. The product
fractions were
pooled, concentrated, redissolved in water and freeze-dried to afford 453 mg
(44%) of a
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white solid (1). Structural data for the compound were as follows: 1H-NMR (500
MHz;
D20) 8 2.83 (t, 2H, O-C-CH2-S), 3.05 (q, 1H, S-CHH-CHN), 3.18 (q, 1H, (q, 1H,'
S-CHH-
CHN); 3.38 (s, 3H, CH30), 3.7 (t, OCH CH O), 3.95 (q, 1H, CHN). The purity of
the
product was confirmed by SDS PAGE.
4.2 Synthesis of (2)
[0307] Triethylamine (~0.5 mL) was added dropwise to a solution of 1 (440 mg,
22 ~mol)
dissolved in aWydrous CHZC12 (30 mL) until the solution was basic. A solution
of 20
kilodalton mPEG-O-p-nitrophenyl carbonate (660 mg, 33 qmol) and N-
hydroxysuccinimide
(3.6 mg, 30.8 ~mol) in CHZCIz (20 mL) was added in several portions over 1 h
at room
temperature. The reaction mixture was stirred at room temperature for 24 h.
The solvent was
then removed by rotary evaporation,~the residue was dissolved in water (100
mL), and the pH
adjusted to 9.5 with 1.0 N NaOH. The basic solution was stirred at room
temperature for 2 h
and was then neutralized with acetic acid to a pH 7Ø The solution was then
loaded onto a
reversed phase chromatography (C-18 silica) column. The column was eluted with
a gradient
of methanol/water (the product elutes at about 70% methanol), product elution
monitored by
evaporative light scattering, and the appropriate fractions collected and
diluted with water
(500 mL). This solution was chromatographed (ion exchange, XK 50 Q, BIG Beads,
300
mL, hydroxide form; gradient of water to water/acetic acid-0.75N) and the pH
of the
appxopriate fractions lowered to 6.0 with acetic acid. This solution was then
captured on a
reversed phase column (C-18 silica) and eluted with a gradient of
methanol/water as
described above. The product fractions were pooled, concentrated, redissolved
in water and
freeze-dried to afford 575 mg (70 %) of a white solid (2). Structural data for
the compound
were as follows: 1H-NMR (500 MHz; D2O) 8 2.83 (t, 2H, O-C-CH -S), 2.95 (t, 2H,
O-C-
CH - _ -S), 3.12 (q, 1H, S-CHH-CHN), 3.39 (s, 3H CH30), 3.71 (t, OCHZCH O).
The purity of
the product was confirmed by SDS PAGE.
Example 5
Preparation of UDP-GaINAc-6-NHCO(CHZ)2-O-PEG-OMe (1 KDa).
[0308] Galactosaminyl-1-phosphate-2-NHCO(CH2)2-O-PEG-OMe (1 kilodalton) (58
mg,
0.045 mmoles) was dissolved in DMF (6 mL ) and pyridine (1.2 mL). UMP-
morpholidate
(60 mg, 0.15 mmoles) was then added and the resulting mixture stirred at
70°C for 48 h. The
solvent was removed by bubbling nitrogen through the reaction mixture and the
residue
purified by reversed phase chromatography (C-18 silica, step gradient between
10 to 80%,
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WO 2005/055950 PCT/US2004/041070
methanol/water). The desired fractions were collected and dried at reduced
pressure to yield
50 mg (70%) of a white solid. TLC (silica, propanollHZO/NH40H, (30/20/2), Rf=
0.54). MS
(MALDI): Observed, 1485, 1529, 1618, 1706.
Example 6
GlycoPEGylation of Factor IX produced in CHO cells
[0309] This example sets forth the preparation of asialoFactor IX and its
sialylation with
CMP-sialic acid-PEG.
6.1 Desialylatioh of ~°Facto~~ IX
[0310] A recombinant form of Coagulation Factor IX (rFactor IX ) was made in
CHO
cells. 6000 IU of rFactor IX were dissolved in a total of 12 mL USP H20. This
solution was,
transferred to a Centricon Plus 20, PL-10 centrifugal filter with another 6 mL
USP H20. The
solution was concentrated to 2 mL and then diluted with 15 mL 50 mM Tris-HCl
pH 7.4,
0.15 M NaCI, 5 mM CaCl2, 0.05% NaN3 and then reconcentrated. The
dilution/concentration
was repeated 4 times to effectively change the buffer to a final volume of 3.0
mL. Of this
solution, 2.9 mL (about 29 mg of rFactor IX) was transferred to a small
plastic tube and to it
was added 530 mU a2-3,6,8-Neuraminidase- agarose conjugate (Vibrio eholerae,
Calbiochem, 450 ~,L). The reaction mixture was rotated gently for 26.5 hours
at 32 °C. The
mixture was centrifuged 2 minutes at 10,000 rpm and the supernatant was
collected. The
agarose beads (containing neuraminidase) were washed 6 times with 0.5 mL 50 mM
Tris-HCl
pH 7.12, 1 M NaCI, 0.05% NaN3. The pooled washings and supernatants were
centrifuged
again for 2 minutes at 10,000 rpm to remove any residual agarose resin. The
pooled,
desialylated protein solution was diluted to 19 mL with the same buffer and
concentrated
down to ~ 2 mL in a Gentricon Plus 20 PL-10 centrifugal filter. The solution
was twice
diluted with 15 mL of 50 mM Txis-HCl pH 7.4, 0.15 M NaCI, 0.05% NaN3 and
reconcentrated to 2 mL. The final desialyated rFactor IX solution was diluted
to 3 mL final
volume (~10 mg/mL) with the Tris Buffer. Native and desialylated rFactor IX
samples were
analyzed by IEF-Electrophoresis. Isoelectric Focusing Gels (pH 3-7) were run
using 1.5 ~.L
(15 ~.g) samples first diluted with 10 ~.L Tris buffer and mixed with 12 ~.L
sample loading
buffer. Gels were loaded, run and fixed using standard procedures. Gels were
stained with
Colloidal Blue Stain (Figure 154), showing a band for desialylated Factor IX.
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Example 7
Preparation of PEG (1 kDa and lO kDa)-SA-Factor IX
[0311] Desialylated rFactor-IX (29 mg, 3 mL) was divided into two 1.5 mL (14.5
mg)
samples in two 15 mL centrifuge tubes. Each solution was diluted with 12.67 mL
50 mM
Tris-HGl pH 7.4, 0.15 M NaCI, 0.05% NaN3 and either CMP-SA-PEG-lk or lOk (7.25
~.mol)
was added. The tubes were inverted gently to mix and 2.9 U ST3Ga13 (326 ~.L)
was added
(total volume 14.5 mL). The tubes were inverted again and rotated gently for
65 hours at 32
°C. The reactions were stopped by freezing at -20 °C. 10 ~,g
samples of the reactions were
analyzed by SDS-PAGE. The PEGylated proteins were purified on a Toso Haas
Biosep
G3000SW (21.5 x 30 cm, 13 um) HPLC column with Dulbecco's Phosphate Buffered
Saline,
pH 7.1 (Gibco), 6 mL/min. The reaction and purification were monitored using
SDS Page
and IEF gels. Novex Tris-Glycine 4-20% 1 mm gels were loaded with 10 pL (10
wg) of
samples after dilution with 2 p.L of 50 mM Tris-HCI, pH 7.4, 150 mM NaCI,
0.05% NaN3
buffer and mixing with 12 ~L sample loading buffer and 1 ~,L 0.5 M DTT and
heated for 6
minutes at 85 °G. Gels were stained with Colloidal Blue Stain (Figure
155) showing a band
for PEG (1 kDa and 10 kDa)-SA-Factor IX.
Example 8
Direct Sialyl-GlycoPEGylation of Factor IX
[0312] This example sets forth the preparation of sialyl-PEGylation of Factor
IX without
prior sialidase treatment.
8.1 Sialyl-PEGylatiov~ of Factor'-IX with CMP-SA-PEG-(10 KDa)
[0313] Factor IX (1100 IU), which was expressed in CHO cells and was fully
sialylated,
was dissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2% sucrose, 0.05%
NaN3 and
0.01% polysorbate 80, pH 5Ø The CMP-SA-PEG-(10 kDa) (27 mg, 2.5 pmol) was
then
dissolved in the solution and 1 U of ST3Ga13 was added. The reaction was
complete after
gently mixing for 28 hours at 32°C. The reaction was analyzed by SDS-
PAGE as described
by Invitrogen. The product protein was purified on an Amersham Superdex 200
(10 x 300
mm, 13 ~,m) HPLC column with phosphate buffered saline, pH 7.0 (PBS), 1
mL/min. Rt =
9.5 min.
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Example 9
Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(20 kDa)
[0314] Factor IX (1100 IU), which was expressed in CHO cells and was fully
sialylated,
was dissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2% sucrose, 0.05%
NaN3 and
0.01 % polysorbate 80, pH 5Ø The CMP-SA-PEG-(20 kDa) (50 mg, 2.3 ~,mol) was
then
dissolved in the solution and CST-II was added. The reaction mixture was
complete after
gently mixing for 42 hours at 32°C. The reaction was analyzed by SDS-
PAGE as described
by Invitrogen.
[0315] The product protein was purified on an Amersham Superdex 200 (10 x 300
mm, 13
Vim) HPLC column with phosphate buffered saline, pH 7.0 (Fisher), 1 mL/min. Rt
= 8.6 min.
Example 10
Sialic Acid Capping of GlycoPEGylated Factor IX
[0316] This examples sets forth the procedure for sialic acid capping of
sialyl-
glycoPEGylated peptides. Here, Factor-IX is the exemplary peptide.
10.1 Sialic acid capping ofN liked ahd O-linked Glycans ofFactor-I~f SA-PEG
(10 kDa)
[0317] Purified r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated in a
Centricon° Plus
PL-10 (Millipore Corp., Bedford, MA) centrifugal filter and the buffer was
changed to 50
mM Tris-HCl pH 7.2, 0.15 M NaCI, 0.05% NaN3 to a final volume of 1.85 mL. The
protein
solution was diluted with 372 ~L of the same Tris buffer and 7.4 mg CMP-SA (12
~.mol) was
20 added as a solid. The solution was inverted gently to mix and 0.1 U ST3Gall
and 0.1 U
ST3Gal3 were added. The reaction mixture was rotated gently for 42 hours at 32
°C.
[0318] A 10 ~.g sample of the reaction was analyzed by SDS-PAGE. Novex Tris-
Glycine
4-12% 1 mm gels were performed and stained using Colloidal Blue as described
by
Invitrogen. Briefly, samples, 10 ~,L (10 ~.g), were mixed with 12 ~,L sample
loading buffer
and 1 ~.L 0.5 M DTT and heated for 6 minutes at 85 °C (Figure 156, lane
4).
Example 11
Glycopegylated Factor IX Pharmacokinetic Study
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[0319] Four glycoPEGylated FIX variants (PEG-9 variants) were tested in a PK
study in
normal mice. The activity of the four compounds had previously been
established in vitro by
clot, endogenous thrombin potential (ETP), and thromboelastograph (TEG)
assays. The
activity results are summarized in Table I.
Compound Clot activityETP TEG
(% of lasma)(relative s ~ecific(relative s ecific
activity activity
BeneFIX 45% 1.0 1.0
PEG-9-2K (LS) 27% 0.3 0.2
PEG-9-2K (HS) 20% 0.2 0.1
PEG-9-lOK 11% 0.6 0.3
PEG-9-30K 14% 0.9 0.4
[0320] To assess the prolongation of activity of the four PEG-9 compounds in
circulation,
a PK study was designed and performed. Non-hemophilic mice were used, 2 animal
per time
point, 3 samples per animal. Sampling time points were 0, 0.08, 0.17, 0.33, 1,
3, 5, 8, 16, 24,
30, 48, 64, 72, and 96 h post compound administration. Blood samples were
centrifuged and
stored in two aliquots; one for clot analysis and one for ELISA. Due,to
material restrictions,
the PEG-9 compounds were dosed in different amounts: BeneFIX 250 U/kg; 2K(low
substitution: "LS" (1-2 PEG substitutions per peptide molecule) 200 U/kg;
2K(high
substitution: "HS" (3-4 PEG substitutions per peptide molecule) 200 U/kg; l OK
100 U/kg;
30K 100 U/kg. All doses were based on measuxed clotting assay units.
[0321] The results are outlined in FIG. 6 and Table II.
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Table II
Compound Dose Cmax AUC CL
(U/kg) (U/mL) (h-U/mL (mL/h/kg)
BeneFIX 250 0.745 1.34 187
PEG-9-2K (LS) 200 0.953 4.69 42.7
PEG-9-2K (HS) 200 0.960 9.05 22.1
PEG-9-l OK 100 0.350 2.80 35.7
PEG-9-30K 100 1.40 8.83 11.3
[0322] The results demonstrate a trend towards prolongation for all the PEG-9
compounds.
The values of AUC and Cmax were not compared directly. However, clearance (CL)
was
compared and CL is lower for the PEG-9 compounds compared to BeneFIX,
indicating a
longer residence time in the mice. The time for the last detectable clot
activity is increased
for the PEG-9 compounds compared to BeneFIX, even though BeneFIX was
admiilistered at
the highest dose.
Example 12
Preparation of LS and HS GlycoPEGylated Factor IX
[0323] GlycoPEGylated Factor IX with a low degree of substitution with PEG
were
prepared from native Factor IX by an exchange reaction catalyzed by ST3Ga1-
III. The
reactions were performed in a buffer of 10 mM histidine, 260 mM glycine, 1%
sucrose and
0.02% Tween 80, pH 7.2. For PEGylation with CMPSA-PEG (2 kD and 10 kD), Factor
IX
(0.5 mg/mL) was incubated with ST3GalIII (50 mU/mL) and CMP-SA-PEG (0.5 mM)
for 16
h at 32°C. For PEGylation with CMP-SA-PEG 30 lcD, the concentration of
Factor IX was
increased to 1.0 mg/mL, and the concentration of CMP-SA-PEG was decreased to
0.17 mM.
Under these conditions, more than 90% of the Factor IX molecules were
substituted with at
least one PEG moiety.
[0324] GlycoPEGylated Factor IX with a high degree of substitution with PEG
were
prepared by enzymatic desialylation of native Factor IX. The Factor IX peptide
was buffer
exchanged into 50 mM mES, pH 6.0, using a PD10 column, adjusted to a
concentration of
0.66 mg/mL and treated with AUS sialidase (5 mU/mL) for 16 h at 32°C.
Desialylation was
verified by SDS-PAGE, HPLC and MALDI glycan analysis. Asialo Factor IX was
purified
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on Q Sepharose FF to remove the sialidase. The CaCl2 fraction was concentrated
using an
Ultral5 concentrator and buffer exchanged into MES, pH 6.0 using a PD10
column.
[0325] 2kD and 10 kD PEGylation of asialo-Factor IX (0.5 mg/mL) was carried
out by
incubation with ST3Ga1-III (50 mUlmL) and CMP-SA-PEG (0.5 mM) at 32°C
for 16h. For
PEGylation with CMPSA-PEG-30kD, the concentration of Factor IX was increased
to 1.0
mg/mL and the concentration of CMP-SA-PEG was decreased to 0.17 mM. After 16 h
of
PEGylation, glycans with terminal galactose were capped with sialic acid by
adding 1 mM
CMP-SA and continuing the incubation for an additional 8 h at 32°C.
Under these conditions,
more than 90% of the Factor IX molecules were substituted with at least one
PEG moiety.
Factor IX produced by this method has a higher apparent molecular weight in
SDS-PAGE.
Example 13
Preparation of O-GlycoPEGylated Factor IX
[0326] O-glycan chains were introduced de novo into native Factor IX (1 mg/mL)
by
incubation of the peptide with GaINAcT-II (25mU/mL) and 1 mM UDP-GaINAc at
32°C.
After 4 h of incubation, the PEGylation reaction was initiated by adding CMPSA-
PEG (2Kd
or lOI~d at 0.5 mM or 30 kDd at 0.17 mM) and ST6GalNAc-I (25 mU/mL) and
incubating
for an additional 20 h.
[0327] It is understood that the examples and embodiments described herein are
for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of
this application and scope of the appended claims. All publications, patents,
and patent
applications cited herein ar a hereby incorporated by reference in their
entirety for all
purposes.
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